BioMed Research International: Genetics The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . All rights reserved. Evaluating LRRK2 Genetic Variants with Unclear Pathogenicity Mon, 02 Mar 2015 08:28:44 +0000 Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been known to be a major genetic component affecting Parkinson’s disease (PD). However, the pathogenicity of many of the LRRK2 variants is unclear because they have been detected in single patients or also in patients and controls. Here, we selected 5 exonic variants (L1165P, T1410M, M1646T, L2063X, and Y2189C) from each of the protein domain of LRRK2 and analysed their possible association with pathogenicity using in vitro functional assays. Point mutations representing each of these variants were incorporated into the LRRK2 gene, and functional aspects such as the percentage of cell survival upon application of stress and kinase activity were measured. Our results showed that all 5 variants had a significantly negative effect on the survival of cells, in both presence and absence of stress, as compared to the wild-type. In addition, there was also a slight increase in kinase activity in most of the variants in comparison to the wild-type. A negative correlation between cell survival and kinase activity was observed. These data suggest that most of the variants despite being located in different domains of LRRK2 appear to exert a potential pathogenic effect possibly through an increased kinase activity, supporting a gain of function mechanism. Fathima Shaffra Refai, Shin Hui Ng, and Eng-King Tan Copyright © 2015 Fathima Shaffra Refai et al. All rights reserved. Ethnic-Specific Genetic Association of Variants in the Corticotropin-Releasing Hormone Receptor 1 Gene with Nicotine Dependence Mon, 23 Feb 2015 06:37:08 +0000 Twin and family studies indicate that smoking addiction is highly influenced by genetic factors. Variants in the corticotropin-releasing hormone receptor 1 (CRHR1) gene have been associated with alcoholism and depression. In this study, we tested five single nucleotide polymorphisms (SNPs) in CRHR1 for their association with ND, which was assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI), and the Fagerström test for ND (FTND) in 2,037 subjects from 602 families of either European American (EA) or African American (AA) ancestry. Association analysis of the five SNPs revealed a significant association of rs171440 with SQ in the AA sample and with SQ and FTND in the pooled AA and EA samples. Haplotype-based association analysis indicated significant association of haplotypes C-C (56.9%) and T-C (38.9%), formed by SNPs rs171440 and rs1396862, with SQ in the AA sample, C-C-G (47.6%) with SQ, and T-C-G (42.3%), formed by SNPs rs171440, rs1396862, and rs878886, with SQ and FTND in the pooled AA and EA samples. However, none of these associations remained significant after correction for multiple testing. Together, our results provide suggestive evidence for the involvement of CRHR1 in ND, which warrants further investigation using larger independent samples. Xiujun Tang, Shumin Zhan, Liping Yang, Wenyan Cui, Jennie Z. Ma, Thomas J. Payne, and Ming D. Li Copyright © 2015 Xiujun Tang et al. All rights reserved. Sudden Sensorineural Hearing Loss and Polymorphisms in Iron Homeostasis Genes: New Insights from a Case-Control Study Wed, 18 Feb 2015 13:45:49 +0000 Background. Even if various pathophysiological events have been proposed as explanations, the putative cause of sudden hearing loss remains unclear. Objectives. To investigate and to reveal associations (if any) between the main iron-related gene variants and idiopathic sudden sensorineural hearing loss. Study Design. Case-control study. Materials and Methods. A total of 200 sudden sensorineural hearing loss patients (median age 63.65 years; range 10–92) were compared with 400 healthy control subjects. The following genetic variants were investigated: the polymorphism c.−8CG in the promoter of the ferroportin gene (FPN1; SLC40A1), the two isoforms C1 and C2 (p.P570S) of the transferrin protein (TF), the amino acidic substitutions p.H63D and p.C282Y in the hereditary hemochromatosis protein (HFE), and the polymorphism c.–582AG in the promoter of the HEPC gene, which encodes the protein hepcidin (HAMP). Results. The homozygous genotype c.−8GG of the SLC40A1 gene revealed an OR for ISSNHL risk of 4.27 (CI 95%, 2.65–6.89; ), being overrepresented among cases. Conclusions. Our study indicates that the homozygous genotype FPN1 −8GG was significantly associated with increased risk of developing sudden hearing loss. These findings suggest new research should be conducted in the field of iron homeostasis in the inner ear. Alessandro Castiglione, Andrea Ciorba, Claudia Aimoni, Elisa Orioli, Giulia Zeri, Marco Vigliano, and Donato Gemmati Copyright © 2015 Alessandro Castiglione et al. All rights reserved. Association of Polymorphisms of the Receptor for Advanced Glycation End Products Gene and Susceptibility to Sporadic Abdominal Aortic Aneurysm Wed, 18 Feb 2015 06:39:12 +0000 Accumulating evidence has suggested that receptor for advanced glycation end products (RAGE) is involved in the development and progression of human abdominal aortic aneurysms (AAAs). However, the association between RAGE gene polymorphisms and AAA has not yet been determined. The present study was aimed at analyzing the potential association between the RAGE gene polymorphisms and AAAs. A cohort of 381 patients and 436 age-matched healthy controls were genotyped to detect the three RAGE polymorphisms (−374 T/A, −429 T/C, and G82S) using SNaPshot. Our study demonstrated a significant difference in the genotype and allele frequencies of the RAGE G82S polymorphism between the AAA patients and the controls. Further stratification by gender and smoking status revealed that the presence of the RAGE 82S allele confers a higher risk for developing AAA in men and smokers. Moreover, AAA patients with the variant 82S allele of RAGE presented with reduced serum soluble RAGE (sRAGE) production, and this decrease was more significant in men and smokers with AAA. Our study provides preliminary evidence that the 82S allele of RAGE is a risk factor for AAA. This new piece of knowledge regarding RAGE may be clinically important for the prevention and therapy of AAAs. Ye Yao, Junli Zhuang, You Li, Bao Jing, Hali Li, Jingbo Li, Changgang Shao, Keshen Li, and Haiyang Wang Copyright © 2015 Ye Yao et al. All rights reserved. Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Thu, 12 Feb 2015 09:39:43 +0000 Several studies have revealed that aquaporins play a role in tumor progression and invasion. In breast carcinomas, high levels of aquaporin 5 (AQP5), a membrane protein involved in water transport, have been linked to increased cell proliferation and migration, thus facilitating tumor progression. Despite the potential role of AQP5 in mammary oncogenesis, the mechanisms controlling mammary AQP5 expression are poorly understood. In other tissues, AQP5 expression has been correlated with its promoter methylation, yet, very little is known about AQP5 promoter methylation in the mammary gland. In this work, we used the mouse mammary gland cell line EpH4, in which we controlled AQP5 expression via the steroid hormone dexamethasone (Dex) to further investigate mechanisms regulating AQP5 expression. In this system, we observed a rapid drop of AQP5 mRNA levels with a delay of several hours in AQP5 protein, suggesting transcriptional control of AQP5 levels. Yet, AQP5 expression was independent of its promoter methylation, or to the presence of negative glucocorticoid receptor elements (nGREs) in its imminent promoter region, but was rather influenced by the cell proliferative state or cell density. We conclude that AQP5 promoter methylation is not a universal mechanism for AQP5 regulation and varies on cell and tissue type. Barbara Arbeithuber, Roland Thuenauer, Yasmin Gravogl, Zsolt Balogi, Winfried Römer, Alois Sonnleitner, and Irene Tiemann-Boege Copyright © 2015 Barbara Arbeithuber et al. All rights reserved. A Potential Epigenetic Marker Mediating Serum Folate and Vitamin B12 Levels Contributes to the Risk of Ischemic Stroke Sun, 01 Feb 2015 07:00:25 +0000 Stroke is a multifactorial disease that may be associated with aberrant DNA methylation profiles. We investigated epigenetic dysregulation for the methylenetetrahydrofolate reductase (MTHFR) gene among ischemic stroke patients. Cases and controls were recruited after obtaining signed written informed consents following a screening process against the inclusion/exclusion criteria. Serum vitamin profiles (folate, vitamin B12, and homocysteine) were determined using immunoassays. Methylation profiles for CpGs A and B in the MTHFR gene were determined using a bisulfite-pyrosequencing method. Methylation of MTHFR significantly increased the susceptibility risk for ischemic stroke. In particular, CpG A outperformed CpG B in mediating serum folate and vitamin B12 levels to increase ischemic stroke susceptibility risks by 4.73-fold. However, both CpGs A and B were not associated with serum homocysteine levels or ischemic stroke severity. CpG A is a potential epigenetic marker in mediating serum folate and vitamin B12 to contribute to ischemic stroke. Loo Keat Wei, Heidi Sutherland, Anthony Au, Emily Camilleri, Larisa M. Haupt, Siew Hua Gan, and Lyn R. Griffiths Copyright © 2015 Loo Keat Wei et al. All rights reserved. Is the Experience of Thermal Pain Genetics Dependent? Wed, 28 Jan 2015 08:53:47 +0000 It is suggested that genetic variations explain a significant portion of the variability in pain perception; therefore, increased understanding of pain-related genetic influences may identify new targets for therapies and treatments. The relative contribution of the different genes to the variance in clinical and experimental pain responses remains unknown. It is suggested that the genetic contributions to pain perception vary across pain modalities. For example, it has been suggested that more than 60% of the variance in cold pressor responses can be explained by genetic factors; in comparison, only 26% of the variance in heat pain responses is explained by these variations. Thus, the selection of pain model might markedly influence the magnitude of the association between the pain phenotype and genetic variability. Thermal pain sensation is complex with multiple molecular and cellular mechanisms operating alone and in combination within the peripheral and central nervous system. It is thus highly probable that the thermal pain experience is affected by genetic variants in one or more of the pathways involved in the thermal pain signaling. This review aims to present and discuss some of the genetic variations that have previously been associated with different experimental thermal pain models. Emilia Horjales-Araujo and Joergen B. Dahl Copyright © 2015 Emilia Horjales-Araujo and Joergen B. Dahl. All rights reserved. Structure-Function Based Molecular Relationships in Ewing’s Sarcoma Thu, 22 Jan 2015 14:17:38 +0000 Ewing’s Sarcoma Oncogene (ews) on chromosome 22q12 is encoding a ubiquitously expressed RNA-binding protein (EWS) with unknown function that is target of tumor-specific chromosomal translocations in Ewing’s sarcoma family of tumors. A model of transcription complex was proposed in which the heterodimer Rpb4/7 binds to EAD, connecting it to Core RNA Pol II. The DNA-binding domain, provided by EFP, is bound to the promoter. Rpb4/7 binds RNA, stabilizing the transcription complex. The complex Rpb4/7 can stabilize the preinitiation complexes by converting the conformation of RNA Pol II. EWS may change its conformation, so that NTD becomes accessible. Two different mechanisms of interaction between EWS and RNA Pol II are proposed: (I) an intermolecular EWS-EWS interaction between two molecules, pushing conformation from “closed” to “open” state, or (II) an intramolecular interaction inside the molecule of EWS, pushing conformation of the molecule from “closed” to “open” state. The modified forms of EWS may interact with Pol II subunits hsRpb5 and hsRpb7. The EWS and EFPs binding partners are described schematically in a model, an attempt to link the transcription with the splicing. The proposed model helps to understand the functional molecular interactions in cancer, to find new partners and ways to treat cancer. Roumiana Todorova Copyright © 2015 Roumiana Todorova. All rights reserved. The Correlation between miRNA and Lymph Node Metastasis in Gastric Cancer Thu, 22 Jan 2015 14:15:17 +0000 Lymph node metastasis (LNM) in gastric cancer is associated with higher rate of cancer recurrence and poor prognosis. As a result, a reliable biomarker for the prediction of LNM is important and would be valuable in the clinical practice. MiRNA microarray revealed that ten miRNAs were expressed significantly different among patients with or without LNM. A total of 46 gastric cancer patients were enrolled and divided into two groups (23 in each group) according to the presence or absence of LNM. RT-PCR of these 10 miRNAs was investigated and compared between the two groups. MiR-1207-5p was significantly upregulated in gastric cancer patients without LNM compared with those with LNM. Patients with upregulated miR-1207-5p had less scirrhous stromal reaction, less lymphovascular invasion, and earlier pathological T category, N category, and TNM stage, compared with those with downregulated or unchanged miR-1207-5p. Multivariate analysis showed that stromal reaction type, lymphovascular invasion, pathological T category and TNM stage, and expression of miR-1207-5p were independent risk factors of LNM. MiR-1207-5p could serve as a useful biomarker in the prediction of LNM in gastric cancer. Kuo-Hung Huang, Yuan-Tzu Lan, Wen-Liang Fang, Jen-Hao Chen, Su-Shun Lo, Anna Fen-Yau Li, Shih-Hwa Chiou, Chew-Wun Wu, and Yi-Ming Shyr Copyright © 2015 Kuo-Hung Huang et al. All rights reserved. Correlation of CCNA1 Promoter Methylation with Malignant Tumors: A Meta-Analysis Introduction Thu, 15 Jan 2015 07:53:11 +0000 Epigenetic silencing of tumor suppressor genes by promoter methylation plays vital roles in the process of carcinogenesis. The purpose of this meta-analysis was to determine whether the aberrant methylation of cyclin A1 (CCNA1) may be of great significance to human malignant tumors. By searching both English and Chinese language-based electronic databases carefully, we tabulated and analyzed parameters from each study. All human-associated case-control studies were included providing available data for CCNA1 methylation and reporting the adjusted odds ratios (ORs) and 95% confidence intervals (CI) conducted with the use of Version 12.0 STATA software. A total of 10 case-control studies (619 patients with cancers and 292 healthy controls) were included for the following statistical analysis. Pooled OR values from all articles revealed that the frequency of CCNA1 methylation in cancer tissues was significantly higher than those of normal tissues . Further ethnicity indicated that the frequency of CCNA1 methylation was correlated with the development of malignant tumors among all those included experimental subgroups (all ). These data from results indicated a significant connection of CCNA1 methylation with poor progression in human malignant tumors among both Caucasian and Asian populations. Bin Yang, Shuai Miao, Le-Ning Zhang, Hong-Bin Sun, Zhe-Nan Xu, and Chun-Shan Han Copyright © 2015 Bin Yang et al. All rights reserved. Copy Number Variations in a Population-Based Study of Charcot-Marie-Tooth Disease Thu, 08 Jan 2015 09:24:59 +0000 Copy number variations (CNVs) are important in relation to diversity and evolution but can sometimes cause disease. The most common genetic cause of the inherited peripheral neuropathy Charcot-Marie-Tooth disease is the PMP22 duplication; otherwise, CNVs have been considered rare. We investigated CNVs in a population-based sample of Charcot-Marie-Tooth (CMT) families. The 81 CMT families had previously been screened for the PMP22 duplication and point mutations in 51 peripheral neuropathy genes, and a genetic cause was identified in 37 CMT families (46%). Index patients from the 44 CMT families with an unknown genetic diagnosis were analysed by whole-genome array comparative genomic hybridization to investigate the entire genome for larger CNVs and multiplex ligation-dependent probe amplification to detect smaller intragenomic CNVs in MFN2 and MPZ. One patient had the pathogenic PMP22 duplication not detected by previous methods. Three patients had potentially pathogenic CNVs in the CNTNAP2, LAMA2, or SEMA5A, that is, genes related to neuromuscular or neurodevelopmental disease. Genotype and phenotype correlation indicated likely pathogenicity for the LAMA2 CNV, whereas the CNTNAP2 and SEMA5A CNVs remained potentially pathogenic. Except the PMP22 duplication, disease causing CNVs are rare but may cause CMT in about 1% (95% CI 0–7%) of the Norwegian CMT families. Helle Høyer, Geir J. Braathen, Anette K. Eek, Gry B. N. Nordang, Camilla F. Skjelbred, and Michael B. Russell Copyright © 2015 Helle Høyer et al. All rights reserved. Association between Genetic Variants on Chromosome 15q25 Locus and Several Nicotine Dependence Traits in Polish Population: A Case-Control Study Tue, 06 Jan 2015 06:58:16 +0000 Tobacco smoking continues to be a leading cause of disease and mortality. Recent research has confirmed the important role of nicotinic acetylcholine receptor (nAChR) gene cluster on chromosome 15q 24-25 in nicotine dependence and smoking. In this study we tested the association of smoking initiation, age at onset of daily smoking, and heaviness of smoking with five single nucleotide polymorphisms (SNPs) within the CHRNA5-CHRNA3-CHRNB4 cluster. The group of 389 adult subjects of European ancestry from the north of Poland, including 212 ever (140 current and 72 former) and 177 never smokers with mean age 49.26, was genotyped for rs16969868, rs1051730, rs588765, rs6495308, and rs578776 polymorphisms. Distributions of genotypes for rs16969868 and rs1051730 were identical so they were analyzed together. Further analysis revealed the association between rs16969868-1051730 (OR = 2.66; 95% CI: 1.30–5.42) and number of cigarettes smoked per day (CPD) with heaviness of nicotine addiction measured by the Fagerström Test for Nicotine Dependence (FTND) (OR = 2.60; 95% CI: 1.24–5.43). No association between these polymorphisms and other phenotypes was found. Similarly, the association between rs588765, rs6495308, rs578776, and analyzed phenotypes was not confirmed. This study provides strong evidence for the role of the CHRNA5-CHRNA3-CHRNB4 cluster in heaviness of nicotine addiction. Krzysztof Buczkowski, Alicja Sieminska, Katarzyna Linkowska, Slawomir Czachowski, Grzegorz Przybylski, Ewa Jassem, and Tomasz Grzybowski Copyright © 2015 Krzysztof Buczkowski et al. All rights reserved. Common Polymorphism in the LRP5 Gene May Increase the Risk of Bone Fracture and Osteoporosis Sun, 14 Dec 2014 12:34:06 +0000 The low-density lipoprotein receptor-related protein 5 gene (LRP5) was identified to be linked to the variation in bone mineral density and types of bone diseases. The present study was aimed at examining the association of LRP5 rs3736228 C>T gene with bone fracture and osteoporosis by meta-analysis. A systematic electronic search of literature was conducted to identify all published studies in English or Chinese on the association of the LRP5 gene with bone fracture and osteoporosis risks. All analyses were calculated using the Version 12.0 STATA software. Odds ratios (ORs) and their corresponding 95% confidence interval (95% CI) were calculated. An updated meta-analysis was currently performed, including seven independent case-control studies. Results identified that carriers of rs3736228 C>T variant in the LRP5 gene were associated with an increased risk of developing osteoporosis and fractures under 4 genetic models but not under the dominant model (OR = 1.19, 95% CI = 0.97~1.46, and ). Ethnicity-subgroup analysis implied that LRP5 rs3736228 C>T mutation was more likely to develop osteoporosis and fractures among Asians and Caucasians in majority of subgroups. These results suggest that there is a modest effect of the LRP5 rs3736228 C>T on the increased susceptibility of bone fracture and osteoporosis. Guang-Yue Xu, Yong Qiu, and Hai-Jun Mao Copyright © 2014 Guang-Yue Xu et al. All rights reserved. Genetic Multipartitions Based on D-Loop Sequences and Chromosomal Patterns in Brown Chromis, Chromis multilineata (Pomacentridae), in the Western Atlantic Sun, 19 Oct 2014 11:45:01 +0000 Connectivity levels among Brazilian reef fish fauna populations have attracted growing interest, mainly between mainland shores and oceanic islands. The Pomacentridae, whose phylogeographic patterns are largely unknown in the Atlantic, are a family of dominant fish in reef regions. We present data on the variability and population structure of damselfish Chromis multilineata in different areas along the northeast coast of Brazil and in the waters around the oceanic islands of Fernando de Noronha (FNA) and Saint Peter and Saint Paul Archipelago (SPSPA) through analysis of the HVR1 mtDNA sequence of the control region. The remote SPSPA exhibits the highest level of genetic divergence among populations. Conventional and molecular cytogenetic analysis showed similar karyotype patterns (2n = 48 acrocentrics) between these insular areas. Our estimates reveal three genetically different population groups of C. multilineata on the Brazilian coast. The level of genetic structure is higher than previous data suggested, indicating complex panel of interactions between the oceanic island and coastal populations of Brazil. Inailson Márcio Costa da Cunha, Allyson Santos de Souza, Eurico Azevedo Dias Jr., Karlla Danielle Jorge Amorim, Rodrigo Xavier Soares, Gideão Wagner Werneck Félix da Costa, Erik García-Machado, Pedro Manoel Galetti Jr., and Wagner Franco Molina Copyright © 2014 Inailson Márcio Costa da Cunha et al. All rights reserved. Gender-Dependent Effect of GSTM1 Genotype on Childhood Asthma Associated with Prenatal Tobacco Smoke Exposure Thu, 18 Sep 2014 12:38:05 +0000 It remains unclear whether the GSTM1 genotype interacts with tobacco smoke exposure (TSE) in asthma development. This study aimed to investigate the interactions among GSTM1 genotype, gender, and prenatal TSE with regard to childhood asthma development. In a longitudinal birth cohort in Taiwan, 756 newborns completed a 6-year follow-up, and 591 children with DNA samples available for GSTM1 genotyping were included in the study, and the interactive influences of gender-GSTM1 genotyping-prenatal TSE on childhood asthma development were analyzed. Among these 591 children, 138 (23.4%) had physician-diagnosed asthma at 6 years of age, and 347 (58.7%) were null-GSTM1. Prenatal TSE significantly increased the prevalence of childhood asthma in null-GSTM1 children relative to those with positive GSTM1. Further analysis showed that prenatal TSE significantly increased the risk of childhood asthma in girls with null-GSTM1. Furthermore, among the children without prenatal TSE, girls with null-GSTM1 had a significantly lower risk of developing childhood asthma and a lower total IgE level at 6 years of age than those with positive GSTM1. This study demonstrates that the GSTM1 null genotype presents a protective effect against asthma development in girls, but the risk of asthma development increases significantly under prenatal TSE. Chih-Chiang Wu, Chia-Yu Ou, Jen-Chieh Chang, Te-Yao Hsu, Ho-Chang Kuo, Chieh-An Liu, Chih-Lu Wang, Chia-Ju Chuang, Hau Chuang, Hsiu-Mei Liang, and Kuender D. Yang Copyright © 2014 Chih-Chiang Wu et al. All rights reserved. Therapeutic Use of MicroRNAs in Lung Cancer Tue, 16 Sep 2014 12:41:15 +0000 Lung cancer is a leading cause of cancer deaths worldwide. Although the molecular pathways of lung cancer have been partly known, the high mortality rate is not markedly changed. MicroRNAs (miRNAs) are small noncoding RNAs that actively modulate cell physiological processes as apoptosis, cell-cycle control, cell proliferation, DNA repair, and metabolism. Several studies demonstrated that miRNAs are involved in the pathogenesis of lung diseases including lung cancer and they negatively regulate gene and protein expression by acting as oncogenes or tumor suppressors. In this review we summarize the current knowledge on the role of miRNAs and their target genes in lung tumorigenesis and evaluate their potential use as therapeutic agents in lung cancer. In particular, we describe methodological approaches such as inhibition of oncogenic miRNAs or replacement of tumor suppressor miRNAs, both in in vitro and in vivo assays. Furthermore we discuss new strategies to achieve in vivo tissue specific delivery, potential off-target effects, and safety of miRNAs systemic delivery. Orazio Fortunato, Mattia Boeri, Carla Verri, Massimo Moro, and Gabriella Sozzi Copyright © 2014 Orazio Fortunato et al. All rights reserved. MicroRNA as New Tools for Prostate Cancer Risk Assessment and Therapeutic Intervention: Results from Clinical Data Set and Patients’ Samples Tue, 16 Sep 2014 08:37:56 +0000 Prostate cancer (PCa) is one of the leading causes of cancer-related death in men. Despite considerable advances in prostate cancer early detection and clinical management, validation of new biomarkers able to predict the natural history of tumor progression is still necessary in order to reduce overtreatment and to guide therapeutic decisions. MicroRNAs are endogenous noncoding RNAs which offer a fast fine-tuning and energy-saving mechanism for posttranscriptional control of protein expression. Growing evidence indicate that these RNAs are able to regulate basic cell functions and their aberrant expression has been significantly correlated with cancer development. Therefore, detection of microRNAs in tumor tissues and body fluids represents a new tool for early diagnosis and patient prognosis prediction. In this review, we summarize current knowledge about microRNA deregulation in prostate cancer mainly focusing on the different clinical aspects of the disease. We also highlight the potential roles of microRNAs in PCa management, while also discussing several current challenges and needed future research. Alessio Cannistraci, Anna Laura Di Pace, Ruggero De Maria, and Désirée Bonci Copyright © 2014 Alessio Cannistraci et al. All rights reserved. Levels of Soluble E-Cadherin in Breast, Gastric, and Colorectal Cancers Tue, 16 Sep 2014 07:32:18 +0000 Soluble E-cadherin is a 80 kDa protein fragment coming from the proteolytic cleavage of the extracellular domain of the full length epithelial cadherin, a molecule involved in cell adhesion/polarity and tissue morphogenesis. In comparison with normal epithelia, cancer cells show a decreased cadherin-mediated intercellular adhesion, and sE-cad levels normally increase in body fluids (blood and urine). This review focuses on soluble E-cadherin in sera of patients affected by three solid cancers (breast, gastric, and colorectal cancers) and how its levels correlate or not with some cancer parameters (e.g., dimension, progression, and localisation). We will describe the main proteomics approaches adopted to measure sE-cad both in vivo and in vitro and the most important findings about its behaviour in cancer dynamics. Ombretta Repetto, Paolo De Paoli, Valli De Re, Vincenzo Canzonieri, and Renato Cannizzaro Copyright © 2014 Ombretta Repetto et al. All rights reserved. The Role of MicroRNAs in Ovarian Cancer Wed, 10 Sep 2014 13:40:15 +0000 Ovarian cancer is the most lethal of malignant gynecological tumors. Its lethality may be due to difficulties in detecting it at an early stage and lack of effective treatments for patients with an advanced or recurrent status. Therefore, there is a strong need for prognostic and predictive markers to diagnose it early and to help optimize and personalize treatment. MicroRNAs are noncoding RNAs that regulate target genes posttranscriptionally. They are involved in carcinogenesis, cell cycle, apoptosis, proliferation, invasion, metastasis, and chemoresistance. The dysregulation of microRNAs is involved in the initiation and progression of human cancers including ovarian cancer, and strong evidence that microRNAs can act as oncogenes or tumor suppressor genes has emerged. Several microRNA signatures that are unique to ovarian cancer have been proposed, and serum-circulating microRNAs have the potential to be useful diagnostic and prognostic biomarkers. Various microRNAs such as those in the miR-200 family, the miR-199/214 cluster, or the let-7 paralogs have potential as therapeutic targets for disseminated or chemoresistant ovarian tumors. Although many obstacles need to be overcome, microRNA therapy could be a powerful tool for ovarian cancer prevention and treatment. In this review, we discuss the emerging roles of microRNAs in various aspects of ovarian cancer. Yasuto Kinose, Kenjiro Sawada, Koji Nakamura, and Tadashi Kimura Copyright © 2014 Yasuto Kinose et al. All rights reserved. Association of vWA and TPOX Polymorphisms with Venous Thrombosis in Mexican Mestizos Sun, 31 Aug 2014 11:10:24 +0000 Objective. Venous thromboembolism (VTE) is a multifactorial disorder and, worldwide, the most important cause of morbidity and mortality. Genetic factors play a critical role in its aetiology. Microsatellites are the most important source of human genetic variation having more phenotypic effect than many single nucleotide polymorphisms. Hence, we evaluate a possible relationship between VTE and the genetic variants in von Willebrand factor, human alpha fibrinogen, and human thyroid peroxidase microsatellites to identify possible diagnostic markers. Methods. Genotypes were obtained from 177 patients with VTE and 531 nonrelated individuals using validated genotyping methods. The allelic frequencies were compared; Bayesian methods were used to correct population stratification to avoid spurious associations. Results. The vWA-18, TPOX-9, and TPOX-12 alleles were significantly associated with VTE. Moreover, subjects bearing the combination vWA-18/TPOX-12 loci exhibited doubled risk for VTE (95% CI = 1.02–3.64), whereas the combination vWA-18/TPOX-9 showed an OR = 10 (95% CI = 4.93–21.49). Conclusions. The vWA and TPOX microsatellites are good candidate biomarkers in venous thromboembolism diseases and could help to elucidate their origins. Additionally, these polymorphisms could become useful markers for genetic studies of VTE in the Mexican population; however, further studies should be done owing that this data only show preliminary evidence. Marco Antonio Meraz-Ríos, Abraham Majluf-Cruz, Carla Santana, Gino Noris, Rafael Camacho-Mejorado, Leonor C. Acosta-Saavedra, Emma S. Calderón-Aranda, Jesús Hernández-Juárez, Jonathan J. Magaña, and Rocío Gómez Copyright © 2014 Marco Antonio Meraz-Ríos et al. All rights reserved. Molecular Mechanisms Underlying the Role of MicroRNAs in the Chemoresistance of Pancreatic Cancer Thu, 28 Aug 2014 12:14:55 +0000 Pancreatic ductal adenocarcinoma (PDAC) is an extremely severe disease where the mortality and incidence rates are almost identical. This is mainly due to late diagnosis and limited response to current treatments. The tumor macroenvironment/microenvironment have been frequently reported as the major contributors to chemoresistance in PDAC, preventing the drugs from reaching their intended site of action (i.e., the malignant duct cells). However, the recent discovery of microRNAs (miRNAs) has provided new directions for research on mechanisms underlying response to chemotherapy. Due to their tissue-/disease-specific expression and high stability in tissues and biofluids, miRNAs represent new promising diagnostic and prognostic/predictive biomarkers and therapeutic targets. Furthermore, several studies have documented that selected miRNAs, such as miR-21 and miR-34a, may influence response to chemotherapy in several tumor types, including PDAC. In this review, we summarize the current knowledge on the role of miRNAs in PDAC and recent advances in understanding their role in chemoresistance through multiple molecular mechanisms. Ingrid Garajová, Tessa Y. Le Large, Adam E. Frampton, Christian Rolfo, Johannes Voortman, and Elisa Giovannetti Copyright © 2014 Ingrid Garajová et al. All rights reserved. The Potential of MicroRNAs in Personalized Medicine against Cancers Thu, 28 Aug 2014 00:00:00 +0000 MicroRNAs orchestrate the expression of the genome and impact many, if not all, cellular processes. Their deregulation is thus often causative of human malignancies, including cancers. Numerous studies have implicated microRNAs in the different steps of tumorigenesis including initiation, progression, metastasis, and resistance to chemo/radiotherapies. Thus, microRNAs constitute appealing targets for novel anticancer therapeutic strategies aimed at restoring their expression or function. As microRNAs are present in a variety of human cancer types, microRNA profiles can be used as tumor-specific signatures to detect various cancers (diagnosis), to predict their outcome (prognosis), and to monitor their treatment (theranosis). In this review, we present the different aspects of microRNA biology that make them remarkable molecules in the emerging field of personalized medicine against cancers and provide several examples of their industrial exploitation. Anne Saumet, Anthony Mathelier, and Charles-Henri Lecellier Copyright © 2014 Anne Saumet et al. All rights reserved. Association of a miRNA-137 Polymorphism with Schizophrenia in a Southern Chinese Han Population Wed, 27 Aug 2014 12:05:45 +0000 Both genome wide association study (GWAS) and biochemical studies of Caucasian populations indicate a robust association between the miR-137 genetic variant rs1625579 and schizophrenia, but inconsistent results have been reported. To assay the association between this variant and schizophrenia, we genotyped 611 schizophrenic patients from Southern Chinese Han population for the risk single nucleotide polymorphism (SNP) rs1625579 using the SNaPshot technique and compared the clinical profiles of different genotypes. Additionally, a meta-analysis was performed using the combined sample groups from five case-control publications and the present study. Both the genotype and allele distributions of the rs1625579 SNP were significantly different between patients and controls ( and 0.026, SNP). TT genotype carriers showed slightly lower Brief Assessment of Cognition in Schizophrenia- (BACS-) derived working memory performance than G carriers (15.58 ± 9.56 versus 19.71 ± 8.18, ). In the meta-analysis, we observed a significant association between rs1625579 and schizophrenia under different genetic models (all ). The results of our study and meta-analysis provide convincing evidence that rs1625579 is significantly associated with schizophrenia. Furthermore, the miR-137 polymorphism influences the working memory performance of schizophrenic patients in a Chinese Han population. Guoda Ma, Jingwen Yin, Jiawu Fu, Xudong Luo, Haihong Zhou, Hua Tao, Lili Cui, You Li, Zhixiong Lin, Bin Zhao, Zheng Li, Juda Lin, and Keshen Li Copyright © 2014 Guoda Ma et al. All rights reserved. MicroRNAs: Promising New Antiangiogenic Targets in Cancer Thu, 14 Aug 2014 09:06:23 +0000 MicroRNAs are one class of small, endogenous, non-coding RNAs that are approximately 22 nucleotides in length; they are very numerous, have been phylogenetically conserved, and involved in biological processes such as development, differentiation, cell proliferation, and apoptosis. MicroRNAs contribute to modulating the expression levels of specific proteins based on sequence complementarity with their target mRNA molecules and so they play a key role in both health and disease. Angiogenesis is the process of new blood vessel formation from preexisting ones, which is particularly relevant to cancer and its progression. Over the last few years, microRNAs have emerged as critical regulators of signalling pathways in multiple cell types including endothelial and perivascular cells. This review summarises the role of miRNAs in tumour angiogenesis and their potential implications as therapeutic targets in cancer. Sandra Gallach, Silvia Calabuig-Fariñas, Eloisa Jantus-Lewintre, and Carlos Camps Copyright © 2014 Sandra Gallach et al. All rights reserved. Transcription Regulation of E-Cadherin by Zinc Finger E-Box Binding Homeobox Proteins in Solid Tumors Wed, 13 Aug 2014 08:10:00 +0000 Downregulation of E-cadherin in solid tumors with regional migration and systematic metastasis is well recognized. In view of its significance in tumorigenesis and solid cancer progression, studies on the regulatory mechanisms are important for the development of target treatment and prediction of clinical behavior for cancer patients. The vertebrate zinc finger E-box binding homeobox (ZEB) protein family comprises 2 major members: ZEB1 and ZEB2. Both contain the motif for specific binding to multiple enhancer boxes (E-boxes) located within the short-range transcription regulatory regions of the E-cadherin gene. Binding of ZEB1 and ZEB2 to the spaced E-cadherin E-boxes has been implicated in the regulation of E-cadherin expression in multiple human cancers. The widespread functions of ZEB proteins in human malignancies indicate their significance. Given the significance of E-cadherin in the solid tumors, a deeper understanding of the functional role of ZEB proteins in solid tumors could provide insights in the design of target therapy against the migratory nature of solid cancers. Thian-Sze Wong, Wei Gao, and Jimmy Yu-Wai Chan Copyright © 2014 Thian-Sze Wong et al. All rights reserved. E-Cadherin and Gastric Cancer: Cause, Consequence, and Applications Tue, 12 Aug 2014 11:10:30 +0000 E-cadherin (epithelial-cadherin), encoded by the CDH1 gene, is a transmembrane glycoprotein playing a crucial role in maintaining cell-cell adhesion. E-cadherin has been reported to be a tumor suppressor and to be down regulated in gastric cancer. Besides genetic mutations in CDH1 gene to induce hereditary diffuse gastric cancer (HDGC), epigenetic factors such as DNA hypermethylation also contribute to the reduction of E-cadherin in gastric carcinogenesis. In addition, expression of E-cadherin could be mediated by infectious agents such as H. pylori (Helicobacter pylori). As E-cadherin is vitally involved in signaling pathways modulating cell proliferation, survival, invasion, and migration, dysregulation of E-cadherin leads to dysfunction of gastric epithelial cells and contributes to gastric cancer development. Moreover, changes in its expression could reflect pathological conditions of gastric mucosa, making its role in gastric cancer complicated. In this review, we summarize the functions of E-cadherin and the signaling pathways it regulates. We aim to provide comprehensive perspectives in the molecular mechanism of E-cadherin and its involvement in gastric cancer initiation and progression. We also focus on its applications for early diagnosis, prognosis, and therapy in gastric cancer in order to open new avenues in this field. Xin Liu and Kent-Man Chu Copyright © 2014 Xin Liu and Kent-Man Chu. All rights reserved. Cytogenetic as an Important Tool for Diagnosis and Prognosis for Patients with Hypocellular Primary Myelodysplastic Syndrome Mon, 11 Aug 2014 08:49:56 +0000 We analyzed cytogenetically 105 patients with hypocellular primary MDS and their clinical implications. The main chromosomal abnormalities found were del(5q)/−5, del(6q)/+6, del(7q)/−7, del(11q), and del(17p). Pediatric patients had a higher frequency of abnormal karyotypes compared with adult patients ( < 0,05). From our patients, 18% showed evolution of the disease. The chromosomal abnormalities presented in the diagnosis of patients who evolved to AML included numerical (−7, +8) and structural del(6q), del(7q), i(7q), t(7;9), i(9q), and del(11q) abnormalities and complex karyotypes. Although the frequency of evolution from hypocellular MDS to AML is low, our results suggest that some chromosomal alterations may play a critical role during this process. We applied the IPSS in our patients because this score system has been proved to be useful for predicting evolution of disease. When we considered the patients according to group 1 (intermediate-1) and group 2 (intermediate-2 and high risk), we showed that group 2 had a high association with respect to the frequency of abnormal karyotypes ( < 0,0001), evolution of disease ( < 0,0001), and mortality ( < 0,001). In fact, the cytogenetic analysis for patients with hypocellular primary MDS is an important tool for diagnosis, prognosis, in clinical decision-making and in follow-up. Daiane Corrêa de Souza, Cecília de Souza Fernandez, Adriana Camargo, Alexandre Gustavo Apa, Elaine Sobral da Costa, Luis Fernando Bouzas, Eliana Abdelhay, and Teresa de Souza Fernandez Copyright © 2014 Daiane Corrêa de Souza et al. All rights reserved. Evaluation and Integration of Genetic Signature for Prediction Risk of Nasopharyngeal Carcinoma in Southern China Sun, 10 Aug 2014 05:55:52 +0000 Genetic factors, as well as environmental factors, play a role in development of nasopharyngeal carcinoma (NPC). A number of single nucleotide polymorphisms (SNPs) have been reported to be associated with NPC. To confirm these genetic associations with NPC, two independent case-control studies from Southern China comprising 1166 NPC cases and 2340 controls were conducted. Seven SNPs in ITGA9 at 3p21.3 and 9 SNPs within the 6p21.3 HLA region were genotyped. To explore the potential clinical application of these genetic markers in NPC, we further evaluate the predictive/diagnostic role of significant SNPs by calculating the area under the curve (AUC). Results. The reported associations between ITGA9 variants and NPC were not replicated. Multiple loci of GABBR1, HLA-F, HLA-A, and HCG9 were statistically significant in both cohorts ( range from 5.96 × 10−17 to 0.02). We show for the first time that these factors influence NPC development independent of environmental risk factors. This study also indicated that the SNP alone cannot serve as a predictive/diagnostic marker for NPC. Integrating the most significant SNP with IgA antibodies status to EBV, which is presently used as screening/diagnostic marker for NPC in Chinese populations, did not improve the AUC estimate for diagnosis of NPC. Xiuchan Guo, Cheryl A. Winkler, Ji Li, Li Guan, Minzhong Tang, Jian Liao, Hong Deng, Guy de Thé, Yi Zeng, and Stephen J. O’Brien Copyright © 2014 Xiuchan Guo et al. All rights reserved. Endothelial Nitric Oxide Synthase Gene Polymorphisms and the Risk of Hypertension in an Indian Population Wed, 06 Aug 2014 10:57:59 +0000 Genetic variants of eNOS gene play a significant role in the pathogenesis of hypertension. Many environmental factors have, also, been implicated in the aetiology of hypertension. We carried out an age-matched case-control study among adults. Hypertension was defined according to JNC-VII criteria and eNOS gene polymorphisms were determined by PCR and PCR followed by PCR-RFLP. eNOS intron 4 aa genotype (adjusted OR 6.81; 95% CI 2.29–20.25) and eNOS 894TT genotype (adjusted OR 7.84; 95% CI 2.57–23.96) were associated with the risk of hypertension. Tobacco users (either smoking/chewing or both) with eNOS intron 4 aa genotype (OR 14.00: 95% CI 1.20–163.37), eNOS 894GG genotype (OR 5.56: 95% CI 3.72–8.31), and eNOS T-786C CC genotype (OR 9.00: 95% CI 1.14–71.04) were at an increased risk of hypertension. Similarly a significant gene-environment interaction was observed between individuals consuming alcohol with eNOS intron 4 aa genotype (OR 12.00: 95% CI 1.20–143.73) and eNOS 894GG genotype (OR 1.95: 95% CI 1.35–2.81). The present study identified few susceptible genotypes of the eNOS gene with the risk of hypertension. Moreover, the interactive effects between the environmental factors and the risk of hypertension were dependent on the eNOS genotypes. Priyanka Shankarishan, Prasanta Kumar Borah, Giasuddin Ahmed, and Jagadish Mahanta Copyright © 2014 Priyanka Shankarishan et al. All rights reserved. Interactions between E-Cadherin and MicroRNA Deregulation in Head and Neck Cancers: The Potential Interplay Mon, 04 Aug 2014 11:30:18 +0000 E-cadherin expression in the head and neck epithelium is essential for the morphogenesis and homeostasis of epithelial tissues. The cadherin-mediated cell-cell contacts are required for the anchorage-dependent growth of epithelial cells. Further, survival and proliferation require physical tethering created by proper cell-cell adhesion. Otherwise, the squamous epithelial cells will undergo programmed cell death. Head and neck cancers can escape from anoikis and enter into the epithelial-mesenchymal transition stages via the modulation of E-cadherin expression with epigenetic mechanisms. At epigenetic level, gene expression control is not dependent on the DNA sequence. In the context of E-cadherin regulation in head and neck cancers, 2 major mechanisms including de novo promoter hypermethylation and microRNA dysregulation are most extensively studied. Both of them control E-cadherin expression at transcription level and subsequently hinder the overall E-cadherin protein level in the head and neck cancer cells. Increasing evidence suggested that microRNA mediated E-cadherin expression in the head and neck cancers by directly/indirectly targeting the transcription suppressors of E-cadherin, ZEB1 and ZEB2. Thian-Sze Wong, Wei Gao, and Jimmy Yu-Wai Chan Copyright © 2014 Thian-Sze Wong et al. All rights reserved. MicroRNAs in Soft Tissue Sarcomas: Overview of the Accumulating Evidence and Importance as Novel Biomarkers Mon, 04 Aug 2014 09:17:45 +0000 Sarcomas are distinctly heterogeneous tumors and a variety of subtypes have been described. Although several diagnostic explorations in the past three decades, such as identification of chromosomal translocation, have greatly improved the diagnosis of soft tissue sarcomas, the unsolved issues, including the limited useful biomarkers, remain. Emerging reports on miRNAs in soft tissue sarcomas have provided clues to solving these problems. Evidence of circulating miRNAs in patients with soft tissue sarcomas and healthy individuals has been accumulated and is accelerating their potential to develop into clinical applications. Moreover, miRNAs that function as novel prognostic factors have been identified, thereby facilitating their use in miRNA-targeted therapy. In this review, we provide an overview of the current knowledge on miRNA deregulation in soft tissue sarcomas, and discuss their potential as novel biomarkers and therapeutics. Tomohiro Fujiwara, Toshiyuki Kunisada, Ken Takeda, Koji Uotani, Aki Yoshida, Takahiro Ochiya, and Toshifumi Ozaki Copyright © 2014 Tomohiro Fujiwara et al. All rights reserved. The Clinicopathological Significance of MicroRNA-155 in Breast Cancer: A Meta-Analysis Sun, 03 Aug 2014 08:40:31 +0000 Objective. Previous studies demonstrated that the associations between expression level of microRNA-155 (miR-155) and clinicopathological significance of breast cancer remained inconsistent. Therefore, we performed a meta-analysis based on eligible studies to summarize the possible associations. Methods. We identified eligible studies published up to May 2014 by a comprehensive search of PubMed, EMBASE, CNKI, and VIP databases. The analysis was performed with RevMan. 5.0 software. Results. A total of 15 studies were included. The results of meta-analysis showed that miR-155 was positively correlated with breast cancer with standardized mean difference (SMD) = 1.22. Elevated miR-155 was found in Her-2 positive or lymph node metastasis positive, or p53 mutant type breast cancer. But the result showed to be insignificant in TNM comparison. With respect to estrogen receptor alpha (ER) and progesterone receptor (PR) status, both of them showed significant associations with SMD = −1.2 and −1.85, respectively. Conclusion. MiR-155 detection might have a diagnostic value in breast cancer patients. It might be used as an auxiliary biomarker for different clinicopathological breast cancer. Hui Zeng, Cheng Fang, Seungyoon Nam, Qing Cai, and Xinghua Long Copyright © 2014 Hui Zeng et al. All rights reserved. Rho GTPase-Activating Protein 35 rs1052667 Polymorphism and Osteosarcoma Risk and Prognosis Sun, 20 Jul 2014 00:00:00 +0000 The Rho GTPase-activating protein 35 (ARHGAP35), an important Rho family GTPase-activating protein, may be associated with tumorigenesis of some tumors. Here, we investigated the relationship between an important polymorphic variant at 3′-UTR of this gene (rs1052667) and osteosarcoma risk and prognosis. This hospital-based case-control study, including 247 osteosarcoma patients and 428 age-, sex-, and race-matched healthy controls, was conducted in Guangxi population. Genotypes were tested using TaqMan PCR technique. We found a significant difference in the frequency of rs1052667 genotypes between cases and controls. Compared with the homozygote of rs1052667 C alleles (rs1052667-CC), the genotypes with rs1052667 T alleles (namely, rs1052667-CT or -TT) increased osteosarcoma risk (odds ratios: 2.41 and 7.35, resp.). Moreover, rs1052667 polymorphism was correlated with such pathological features of osteosarcoma as tumor size, tumor grade, and tumor metastasis. Additionally, this polymorphism also modified the overall survival and recurrence-free survival of osteosarcoma cases. Like tumor grade, ARHGAP35 rs1052667 polymorphism was an independent prognostic factor influencing the survival of osteosarcoma. These results suggest that ARHGAP35 rs1052667 polymorphism may be associated with osteosarcoma risk and prognosis. Jinmin Zhao, Hua Xu, Maolin He, Zhe Wang, and Yang Wu Copyright © 2014 Jinmin Zhao et al. All rights reserved. Novel Hypoxanthine Guanine Phosphoribosyltransferase Gene Mutations in Saudi Arabian Hyperuricemia Patients Wed, 09 Jul 2014 08:54:41 +0000 Over the past decade, a steady increase in the incidence of HPRT-related hyperuricemia (HRH) has been observed in Saudi Arabia. We examined all the nine exons of HPRT gene for mutations in ten biochemically confirmed hyperuricemia patients, including one female and three normal controls. In all, we identified 13 novel mutations in Saudi Arabian HPRT-related hyperuricemia patients manifesting different levels of uric acid. The Lys103Met alteration was highly recurrent and was observed in 50% of the cases, while Ala160Thr and Lys158Asn substitutions were found in two patients. Moreover, in 70% of the patients ≥2 mutations were detected concurrently in the HPRT gene. Interestingly, one of the patients that harbored Lys103Met substitution along with two frameshift mutations at codons 85 and 160 resulting in shortened protein demonstrated unusually high serum uric acid level of 738 μmol/L. Two of the seven point mutations that resulted in amino acid change (Lys103Met and Val160Gly) were predicted to be damaging by SIFT and Polyphen and were further analyzed for their protein stability and function by molecular dynamics simulation. The identified novel mutations in the HPRT gene may prove useful in the prenatal diagnosis and genetic counseling. Mohammed Alanazi, Abdulrahman Saud Al-Arfaj, Zainularifeen Abduljaleel, Hussein Fahad Al-Arfaj, Narasimha Reddy Parine, Jilani Purusottapatnam Shaik, Zahid Khan, and Akbar Ali Khan Pathan Copyright © 2014 Mohammed Alanazi et al. All rights reserved. A Novel COL4A5 Mutation Identified in a Chinese Han Family Using Exome Sequencing Sun, 06 Jul 2014 09:59:01 +0000 Alport syndrome (AS) is a monogenic disease of the basement membrane (BM), resulting in progressive renal failure due to glomerulonephropathy, variable sensorineural hearing loss, and ocular anomalies. It is caused by mutations in the collagen type IV alpha-3 gene (COL4A3), the collagen type IV alpha-4 gene (COL4A4), and the collagen type IV alpha-5 gene (COL4A5), which encodes type IV collagen α3, α4, and α5 chains, respectively. To explore the disease-related gene in a four-generation Chinese Han pedigree of AS, exome sequencing was conducted on the proband, and a novel deletion mutation c.499delC (p.Pro167Gln36) in the COL4A5 gene was identified. This mutation, absent in 1,000 genomes project, HapMap, dbSNP132, YH1 databases, and 100 normal controls, cosegregated with patients in the family. Neither sensorineural hearing loss nor typical COL4A5-related ocular abnormalities (dot-and-fleck retinopathy, anterior lenticonus, and the rare posterior polymorphous corneal dystrophy) were present in patients of this family. The phenotypes of patients in this AS family were characterized by early onset-age and rapidly developing into end-stage renal disease (ESRD). Our discovery broadens the mutation spectrum in the COL4A5 gene associated with AS, which may also shed new light on genetic counseling for AS. Xiaofei Xiu, Jinzhong Yuan, Xiong Deng, Jingjing Xiao, Hongbo Xu, Zhaoyang Zeng, Liping Guan, Fengping Xu, and Sheng Deng Copyright © 2014 Xiaofei Xiu et al. All rights reserved. Analysis of Genotype 1b Hepatitis C Virus IRES in Serum and Peripheral Blood Mononuclear Cells in Patients Treated with Interferon and Ribavirin Thu, 03 Jul 2014 10:04:55 +0000 Hepatitis C virus (HCV) highly conserved IRES (internal ribosome entry site) sequence, localized within the 5′-untranslated region (5′UTR), may determine viral properties like replication efficiency and cell tropism. The aim of the present study was to characterize newly emerging 5′UTR variants in serum and peripheral blood mononuclear cells (PBMC) in chronic hepatitis C patients treated with interferon (IFN) and ribavirin and to identify their effect on IRES secondary structures. The study group consisted of 87 patients infected with genotype 1b from whom serum and PBMC samples were collected at 9 time points (before, during, and after treatment). New 5′UTR variants developed in 9 patients. Out of the overall 14 new variants, 9 (64%) were found in PBMC. HCV variants with decreased thermodynamic stability were identified only in PBMC and C183U mutation was the most common one in this compartment. In conclusion, antiviral treatment may favor emergence of new 5′UTR variants both in blood and in PBMC compartments. However, variants developing in the latter compartment were predicted to have lower thermodynamic stability of the IRES secondary structures compared to serum strains. C-U change in position 183, which has not been described previously, might indicate viral adaptation to lymphoid cells. Iwona Bukowska-Ośko, Kamila Caraballo Cortés, Agnieszka Pawełczyk, Rafał Płoski, Maria Fic, Karol Perlejewski, Urszula Demkow, Hanna Berak, Andrzej Horban, Tomasz Laskus, and Marek Radkowski Copyright © 2014 Iwona Bukowska-Ośko et al. All rights reserved. MTHFR Gene Polymorphism and Age of Onset of Schizophrenia and Bipolar Disorder Thu, 03 Jul 2014 00:00:00 +0000 Objective. Several studies with contradictory results from different cultures about association of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in schizophrenia and bipolar disorders. Little is known about this association in Arab culture and Egypt. So the present study aimed to assess the association of MTHFR C677T polymorphism in bipolar disorder (BD) and schizophrenia in comparison to control group. The association between MTHFR C677T polymorphism and the age at onset in schizophrenia or BD was also studied. Methods. Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) were used to examine the genotype and allele frequencies of MTHFR C677T polymorphism in 149 healthy subjects and 134 bipolar and 103 schizophrenia patients. Results. In BD and schizophrenia, there was a higher prevalence of MTHFR C677T polymorphism than healthy subjects. Earlier age at onset was found in patients with BD, carrying one copy of the T allele or CT genotypes but not in patients with schizophrenia. Conclusion. The present findings suggest that the MTHFR C677T polymorphisms are likely to be associated with the risk of developing BD and schizophrenia and influence the age at onset of BD but not the age at onset of schizophrenia. Mohamed A. El-Hadidy, Hanaa M. Abdeen, Sherin M. Abd El-Aziz, and Mohammad Al-Harrass Copyright © 2014 Mohamed A. El-Hadidy et al. All rights reserved. Whole Exome Sequencing Reveals Genetic Predisposition in a Large Family with Retinitis Pigmentosa Mon, 30 Jun 2014 00:00:00 +0000 Next-generation sequencing has become more widely used to reveal genetic defect in monogenic disorders. Retinitis pigmentosa (RP), the leading cause of hereditary blindness worldwide, has been attributed to more than 67 disease-causing genes. Due to the extreme genetic heterogeneity, using general molecular screening alone is inadequate for identifying genetic predispositions in susceptible individuals. In order to identify underlying mutation rapidly, we utilized next-generation sequencing in a four-generation Chinese family with RP. Two affected patients and an unaffected sibling were subjected to whole exome sequencing. Through bioinformatics analysis and direct sequencing confirmation, we identified p.R135W transition in the rhodopsin gene. The mutation was subsequently confirmed to cosegregate with the disease in the family. In this study, our results suggest that whole exome sequencing is a robust method in diagnosing familial hereditary disease. Juan Wu, Lijia Chen, Oi Sin Tam, Xiu-Feng Huang, Chi-Pui Pang, and Zi-Bing Jin Copyright © 2014 Juan Wu et al. All rights reserved. Genetic Testing in Hereditary Breast and Ovarian Cancer Using Massive Parallel Sequencing Thu, 26 Jun 2014 09:24:21 +0000 High throughput methods such as next generation sequencing are increasingly used in molecular diagnosis. The aim of this study was to develop a workflow for the detection of BRCA1 and BRCA2 mutations using massive parallel sequencing in a 454 GS Junior bench top sequencer. Our approach was first validated in a panel of 23 patients containing 62 unique variants that had been previously Sanger sequenced. Subsequently, 101 patients with familial breast and ovarian cancer were studied. BRCA1 and BRCA2 exon enrichment has been performed by PCR amplification using the BRCA MASTR kit (Multiplicom). Bioinformatic analysis of reads is performed with the AVA software v2.7 (Roche). In total, all 62 variants were detected resulting in a sensitivity of 100%. 71 false positives were called resulting in a specificity of 97.35%. All of them correspond to deletions located in homopolymeric stretches. The analysis of the homopolymers stretches of 6 bp or longer using the BRCA HP kit (Multiplicom) increased the specificity of the detection of BRCA1 and BRCA2 mutations to 99.99%. We show here that massive parallel pyrosequencing can be used as a diagnostic strategy to test for BRCA1 and BRCA2 mutations meeting very stringent sensitivity and specificity parameters replacing traditional Sanger sequencing with a lower cost. Anna Ruiz, Gemma Llort, Carmen Yagüe, Neus Baena, Marina Viñas, Montse Torra, Anna Brunet, Miquel A. Seguí, Eugeni Saigí, and Miriam Guitart Copyright © 2014 Anna Ruiz et al. All rights reserved. Prevalence of Catalase (-21 A/T) Gene Variant in South Indian (Tamil) Population Thu, 26 Jun 2014 00:00:00 +0000 Catalase, an endogenous antioxidant enzyme, is responsible for regulating reactive species levels. Several epidemiologic studies have suggested that single nucleotide polymorphism in catalase gene may be associated with many diseases. The genotype of CAT (-21 A/T) point mutation in promoter region of catalase gene was determined by polymerase chain based restriction fragment length polymorphism analysis in the DNA of 100 healthy volunteers. The frequency of CAT (-21 A/T) gene polymorphism AA, AT, and TT genotypes was found to be 7, 23, and 70 percent, respectively. The mutant “T” allele frequency was found to be 0.82 among the south Indian (Tamil) population. Chi square analysis showed that the study population lies within the Hardy-Weinberg equilibrium. The wild type genotype (AA) was found to be very low (7%) and the mutant genotype (AT/TT) was found to be more prevalent (93%) among the south Indian population. This suggests that the high prevalence of mutant genotype may increase the susceptibility to oxidative stress associated diseases. A. Lourdhu Mary, K. Nithya, W. Isabel, and T. Angeline Copyright © 2014 A. Lourdhu Mary et al. All rights reserved. The Gene-Gene Interaction of INSIG-SCAP-SREBP Pathway on the Risk of Obesity in Chinese Children Tue, 17 Jun 2014 06:22:02 +0000 Background. Childhood obesity has become a global public health problem in recent years. This study aimed to explore the association of genetic variants in INSIG-SCAP-SREBP pathway with obesity in Chinese children. Methods. A case-control study was conducted, including 705 obese cases and 1,325 nonobese controls. We genotyped 15 single nucleotide polymorphisms (SNPs) of five genes in INSIG-SCAP-SREBP pathway, including insulin induced gene 1 (INSIG1), insulin induced gene 2 (INSIG2), SREBP cleavage-activating protein gene (SCAP), sterol regulatory element binding protein gene 1 (SREBP1), and sterol regulatory element binding protein gene 2 (SREBP2). We used generalized multifactor dimensionality reduction (GMDR) and logistic regression to investigate gene-gene interactions. Results. Single polymorphism analyses showed that SCAP rs12487736 and rs12490383 were nominally associated with obesity. We identified a 3-locus interaction on obesity in GMDR analyses , involving 3 genetic variants of INSIG2, SCAP, and SREBP2. The individuals in high-risk group of the 3-locus combinations had a 79.9% increased risk of obesity compared with those in low-risk group (, 95% CI: 1.475–2.193, ). Conclusion. We identified interaction of three genes in INSIG-SCAP-SREBP pathway on risk of obesity, revealing that these genes affect obesity more likely through a complex interaction pattern than single gene effect. Fang-Hong Liu, Jie-Yun Song, Xiao-Rui Shang, Xiang-Rui Meng, Jun Ma, and Hai-Jun Wang Copyright © 2014 Fang-Hong Liu et al. All rights reserved. Genetic Diagnosis of Charcot-Marie-Tooth Disease in a Population by Next-Generation Sequencing Mon, 16 Jun 2014 00:00:00 +0000 Charcot-Marie-Tooth (CMT) disease is the most prevalent inherited neuropathy. Today more than 40 CMT genes have been identified. Diagnosing heterogeneous diseases by conventional Sanger sequencing is time consuming and expensive. Thus, more efficient and less costly methods are needed in clinical diagnostics. We included a population based sample of 81 CMT families. Gene mutations had previously been identified in 22 families; the remaining 59 families were analysed by next-generation sequencing. Thirty-two CMT genes and 19 genes causing other inherited neuropathies were included in a custom panel. Variants were classified into five pathogenicity classes by genotype-phenotype correlations and bioinformatics tools. Gene mutations, classified certainly or likely pathogenic, were identified in 37 (46%) of the 81 families. Point mutations in known CMT genes were identified in 21 families (26%), whereas four families (5%) had point mutations in other neuropathy genes, ARHGEF10, POLG, SETX, and SOD1. Eleven families (14%) carried the PMP22 duplication and one family carried a MPZ duplication (1%). Most mutations were identified not only in known CMT genes but also in other neuropathy genes, emphasising that genetic analysis should not be restricted to CMT genes only. Next-generation sequencing is a cost-effective tool in diagnosis of CMT improving diagnostic precision and time efficiency. Helle Høyer, Geir J. Braathen, Øyvind L. Busk, Øystein L. Holla, Marit Svendsen, Hilde T. Hilmarsen, Linda Strand, Camilla F. Skjelbred, and Michael B. Russell Copyright © 2014 Helle Høyer et al. All rights reserved. The Relationship between Interleukin-18 Polymorphisms and Allergic Disease: A Meta-Analysis Thu, 05 Jun 2014 10:50:42 +0000 Recent studies have suggested that IL-18 −607C/A and −137G/C polymorphisms may be associated with the risk of allergic disease; however, individually published results are inconclusive. Therefore, we performed a meta-analysis to clarify whether IL-18 −607C/A and −137G/C polymorphisms were associated with the risk of allergic disease. A total of 21 studies including 5,331 cases and 9,658 controls were involved in this meta-analysis. In the overall analysis and the subgroup analysis according to ethnicity, we did not find significant association between IL-18 −607C/A or −137G/C polymorphism and the risk of allergic disease (all ). However, in a stratified analysis by type of allergic disease, our results indicated that IL-18 −607C/A polymorphism was associated with a significantly decreased risk of allergic asthma in heterozygous comparison and IL-18 −137G/C was associated with a significantly decreased risk of allergic dermatitis in recessive model and homozygous comparison. In the stratified analysis by source of control, IL-18−607C/A showed significantly reduced risk in population-based subgroup, and for IL-18 −137G/C only significantly decreased risk was found in the hospital-based subgroup. Our meta-analysis suggests that IL-18 −607C/A and −137G/C polymorphisms may be protective factors for the risk of allergic asthma and allergic dermatitis, respectively. Daye Cheng, Yiwen Hao, Wenling Zhou, and Yiran Ma Copyright © 2014 Daye Cheng et al. All rights reserved. Molecular Testing for Fragile X: Analysis of 5062 Tests from 1105 Fragile X Families—Performed in 12 Clinical Laboratories in Spain Wed, 28 May 2014 10:56:46 +0000 Fragile X syndrome is the most common inherited form of intellectual disability. Here we report on a study based on a collaborative registry, involving 12 Spanish centres, of molecular diagnostic tests in 1105 fragile X families comprising 5062 individuals, of whom, 1655 carried a full mutation or were mosaic, three cases had deletions, 1840 had a premutation, and 102 had intermediate alleles. Two patients with the full mutation also had Klinefelter syndrome. We have used this registry to assess the risk of expansion from parents to children. From mothers with premutation, the overall rate of allele expansion to full mutation is 52.5%, and we found that this rate is higher for male than female offspring (63.6% versus 45.6%; ). Furthermore, in mothers with intermediate alleles (45–54 repeats), there were 10 cases of expansion to a premutation allele, and for the smallest premutation alleles (55–59 repeats), there was a 6.4% risk of expansion to a full mutation, with 56 repeats being the smallest allele that expanded to a full mutation allele in a single meiosis. Hence, in our series the risk for alleles of 59 repeats is somewhat higher than in other published series. These findings are important for genetic counselling. María-Isabel Tejada, Guillermo Glover, Francisco Martínez, Miriam Guitart, Yolanda de Diego-Otero, Isabel Fernández-Carvajal, Feliciano J. Ramos, Concepción Hernández-Chico, Elizabet Pintado, Jordi Rosell, María-Teresa Calvo, Carmen Ayuso, María-Antonia Ramos-Arroyo, Hiart Maortua, and Montserrat Milà Copyright © 2014 María-Isabel Tejada et al. All rights reserved. Omics Technologies and Neovascular Ocular Disorders Mon, 26 May 2014 07:06:36 +0000 Daniel Petrovič, Quan Dong Nguyen, Borut Peterlin, and Goran Petrovski Copyright © 2014 Daniel Petrovič et al. All rights reserved. Genetic Variability, Character Association, and Path Analysis for Economic Traits in Menthofuran Rich Half-Sib Seed Progeny of Mentha piperita L. Thu, 22 May 2014 11:38:05 +0000 Menthofuran rich eight half-sib seed progeny of Mentha piperita (MPS-36) were studied for various genetic parameters, namely, coefficient of variation, heritability, genetic advance, correlation, and path of various plant and oil attributes, namely, plant height, L : S ratio, herb yield, β-myrcene, limonene, 1,8-cineole, menthone, menthofuran, neomenthone, pulegone, and menthol. Maximum genotypic coefficient of variation and genetic advance as percentage of mean were recorded for pulegone, followed by menthofuran and 1,8-cineole. The genotypic correlation in general was higher than phenotypic; positive significant correlation was recorded for limonene with 1,8-cineole and menthone, β-myrcene with limonene, and 1,8-cineole and menthofuran with neomenthol. A high direct positive effect on menthofuran was of neomenthol. Birendra Kumar, Himanshi Mali, and Ekta Gupta Copyright © 2014 Birendra Kumar et al. All rights reserved. iSS-PseDNC: Identifying Splicing Sites Using Pseudo Dinucleotide Composition Wed, 21 May 2014 14:19:08 +0000 In eukaryotic genes, exons are generally interrupted by introns. Accurately removing introns and joining exons together are essential processes in eukaryotic gene expression. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapid and effective detection of splice sites that play important roles in gene structure annotation and even in RNA splicing. Although a series of computational methods were proposed for splice site identification, most of them neglected the intrinsic local structural properties. In the present study, a predictor called “iSS-PseDNC” was developed for identifying splice sites. In the new predictor, the sequences were formulated by a novel feature-vector called “pseudo dinucleotide composition” (PseDNC) into which six DNA local structural properties were incorporated. It was observed by the rigorous cross-validation tests on two benchmark datasets that the overall success rates achieved by iSS-PseDNC in identifying splice donor site and splice acceptor site were 85.45% and 87.73%, respectively. It is anticipated that iSS-PseDNC may become a useful tool for identifying splice sites and that the six DNA local structural properties described in this paper may provide novel insights for in-depth investigations into the mechanism of RNA splicing. Wei Chen, Peng-Mian Feng, Hao Lin, and Kuo-Chen Chou Copyright © 2014 Wei Chen et al. All rights reserved. The Association of HLA-Class I and Class II with Hodgkin’s Lymphoma in Iranian Patients Wed, 21 May 2014 08:51:16 +0000 The Hodgkin’s lymphoma disease (HD) is a common malignant neoplasm with germinal centre B-cell origin. It has been suggested that the HLA class I and class II regions have susceptibility effects on HD. In different ethnic groups, different HLA class I and class II alleles affect HD. As a result, there is no consensus which of the different HLA alleles confers susceptibility to HD. In this study, we aimed to ascertain the role of HLA class I and class II alleles in association with Hodgkin’s lymphoma in Iranian patients. We performed a case-control genotyping study in 85 Iranian HD patients which were selected from the Bone Marrow Transplantation Department of Taleghani Hospital and 150 controls using the SSP-PCR. Our results demonstrated that the 68, 51, and 15 alleles were significantly more frequent in HD patients in comparison to controls (; OR = 6.188, ; OR = 2.86, ; OR = 5.315, resp.) and they have significant susceptibility effects on HD in Iranian population. There are reports of other populations with regard to consistency and inconsistency to our results. Further studies with large sample size or the meta-analysis are needed to explain the exact associations of HLA gene with HD. Arezou Sayad, Mohammad Taghi Akbari, Mahshid Mehdizadeh, Abolfazl Movafagh, and Abbas Hajifathali Copyright © 2014 Arezou Sayad et al. All rights reserved. Genetic and Environmental Influences on the Allocation of Adolescent Leisure Time Activities Tue, 20 May 2014 08:05:40 +0000 There is a growing recognition of the importance of the out-of-school activities in which adolescents choose to participate. Youth activities vary widely in terms of specific activities and in time devoted to them but can generally be grouped by the type and total duration spent per type. We collected leisure time information using a 17-item leisure time questionnaire in a large sample of same- and opposite-sex adolescent twin pairs . Using both univariate and multivariate genetic models, we sought to determine the type and magnitude of genetic and environmental influences on the allocation of time toward different leisure times. Results indicated that both genetic and shared and nonshared environmental influences were important contributors to individual differences in physical, social, intellectual, family, and passive activities such as watching television. The magnitude of these influences differed between males and females. Environmental influences were the primary factors contributing to the covariation of different leisure time activities. Our results suggest the importance of heritable influences on the allocation of leisure time activity by adolescents and highlight the importance of environmental experiences in these choices. Brett C. Haberstick, Joanna S. Zeiger, and Robin P. Corley Copyright © 2014 Brett C. Haberstick et al. All rights reserved. The (G>A) rs11573191 Polymorphism of PLA2G5 Gene Is Associated with Premature Coronary Artery Disease in the Mexican Mestizo Population: The Genetics of Atherosclerotic Disease Mexican Study Sun, 18 May 2014 16:03:38 +0000 Coronary artery disease (CAD) is a multifactorial disorder that results from an excessive inflammatory response. Secretory phospholipase A2-V (sPLA2-V) encoded by PLA2G5 gene promotes diverse proinflammatory processes. The aim of the present study was to analyze if PLA2G5 gene polymorphisms are associated with premature CAD. Three PLA2G5 polymorphisms (rs11573187, rs2148911, and rs11573191) were analyzed in 707 patients with premature CAD and 749 healthy controls. Haplotypes were constructed after linkage disequilibrium analysis. Under dominant, recessive, and additive models, the rs11573191 polymorphism was associated with increased risk of premature CAD (OR = 1.51, Pdom = 3.5 × 10−3; OR = 2.95, Prec = 0.023; OR = 1.51, Padd = 1.2 × 10−3). According to the informatics software, this polymorphism had a functional effect modifying the affinity of the sequence by the MZF1 transcription factor. PLA2G5 polymorphisms were in linkage disequilibrium and the CGA haplotype was associated with increased risk of premature CAD (OR = 1.49, P = 0.0023) and with hypertension in these patients (OR = 1.75, P = 0.0072). Our results demonstrate the association of the PLA2G5 rs11573191 polymorphism with premature CAD. In our study, it was possible to distinguish one haplotype associated with increased risk of premature CAD and hypertension. Gilberto Vargas-Alarcón, Carlos Posadas-Romero, Teresa Villarreal-Molina, Edith Alvarez-León, Javier Angeles-Martinez, María Elena Soto, Irma Monroy-Muñoz, Juan Gabriel Juárez, Carlos Jerges Sánchez-Ramírez, Julian Ramirez-Bello, Silvestre Ramírez-Fuentes, José Manuel Fragoso, and José Manuel Rodríguez-Pérez Copyright © 2014 Gilberto Vargas-Alarcón et al. All rights reserved. Prospective Evaluation of Whole Genome MicroRNA Expression Profiling in Childhood Acute Lymphoblastic Leukemia Wed, 14 May 2014 10:48:18 +0000 Dysregulation of microRNA (miRNA) expression contributes to the pathogenesis of several clinical conditions. The aim of this study is to evaluate the associations between miRNAs and childhood acute lymphoblastic leukemia (ALL) to discover their role in the course of the disease. Forty-three children with ALL and 14 age-matched healthy controls were included in the study. MicroRNA microarray expression profiling was used for peripheral blood and bone marrow samples. Aberrant miRNA expressions associated with the diagnosis and outcome were prospectively evaluated. Confirmation analysis was performed by real time RT-PCR. miR-128, miR-146a, miR-155, miR-181a, and miR-195 were significantly dysregulated in ALL patients at day 0. Following a six-month treatment period, the change in miRNA levels was determined by real time RT-PCR and expression of miR-146a, miR-155, miR-181a, and miR-195 significantly decreased. To conclude, these miRNAs not only may be used as biomarkers in diagnosis of ALL and monitoring the disease but also provide new insights into the potential roles of them in leukemogenesis. Muhterem Duyu, Burak Durmaz, Cumhur Gunduz, Canan Vergin, Deniz Yilmaz Karapinar, Serap Aksoylar, Kaan Kavakli, Nazan Cetingul, Gulersu Irken, Yontem Yaman, Ferda Ozkinay, and Ozgur Cogulu Copyright © 2014 Muhterem Duyu et al. All rights reserved. The Analysis of Genetic Aberrations in Children with Inherited Neurometabolic and Neurodevelopmental Disorders Tue, 13 May 2014 16:58:30 +0000 Inherited encephalopathies include a broad spectrum of heterogeneous disorders. To provide a correct diagnosis, an integrated approach including genetic testing is warranted. We report seven patients with difficult to diagnose inborn paediatric encephalopathies. The diagnosis could not be attained only by means of clinical and laboratory investigations and MRI. Additional genetic testing was required. Cytogenetics, PCR based tests, and array-based comparative genome hybridization were performed. In 4 patients with impaired language abilities we found the presence of microduplication in the region 16q23.1 affecting two dose-sensitive genes: WWOX (OMIM 605131) and MAF (OMIM 177075) (1 case), an interstitial deletion of the 17p11.2 region (2 patients further diagnosed as Smith-Magenis syndrome), and deletion encompassing first three exons of Myocyte Enhancer Factor gene 2MEF2C (1 case). The two other cases represented progressing dystonia. Characteristic GAG deletion in DYT1 consistently with the diagnosis of torsion dystonia was confirmed in 1 case. Last enrolled patient presented with clinical picture consistent with Krabbe disease confirmed by finding of two pathogenic variants of GALC gene and the absence of mutations in PSAP. The integrated diagnostic approach including genetic testing in selected examples of complicated hereditary diseases of the brain is largely discussed in this paper. Krystyna Szymańska, Krzysztof Szczałuba, Agnieszka Ługowska, Ewa Obersztyn, Marek Radkowski, Beata A. Nowakowska, Katarzyna Kuśmierska, Jolanta Tryfon, and Urszula Demkow Copyright © 2014 Krystyna Szymańska et al. All rights reserved. Structure-Function Correlation Analysis of Connexin50 Missense Mutations Causing Congenital Cataract: Electrostatic Potential Alteration Could Determine Intracellular Trafficking Fate of Mutants Tue, 06 May 2014 12:17:36 +0000 Connexin50 (Cx50) mutations are reported to cause congenital cataract probably through the disruption of intercellular transport in the lens. Cx50 mutants that undergo mistrafficking have generally been associated with failure to form functional gap junction channels; however, sometimes even properly trafficked mutants were found to undergo similar consequences. We hereby wanted to elucidate any structural bases of the varied functional consequences of Cx50 missense mutations through in silico approach. Computational studies have been done based on a Cx50 homology model to assess conservation, solvent accessibility, and 3-dimensional localization of mutated residues as well as mutation-induced changes in surface electrostatic potential, H-bonding, and steric clash. This was supplemented with meta-analysis of published literature on the functional properties of connexin missense mutations. Analyses revealed that the mutation-induced critical alterations of surface electrostatic potential in Cx50 mutants could determine their fate in intracellular trafficking. A similar pattern was observed in case of mutations involving corresponding conserved residues in other connexins also. Based on these results the trafficking fates of 10 uncharacterized Cx50 mutations have been predicted. Further experimental analyses are needed to validate the observed correlation. Devroop Sarkar, Kunal Ray, and Mainak Sengupta Copyright © 2014 Devroop Sarkar et al. All rights reserved. Experience of Preimplantation Genetic Diagnosis with HLA Matching at the University Hospital Virgen del Rocío in Spain: Technical and Clinical Overview Thu, 24 Apr 2014 12:39:43 +0000 Preimplantation genetic diagnosis (PGD) of genetic diseases, combined with HLA matching (PGD-HLA), is an option for couples at risk of transmitting a genetic disease to select unaffected embryos of an HLA tissue type compatible with that of an existing affected child. Here we present the results of our PGD-HLA program at the Department of Genetics, Reproduction and Fetal Medicine of the University Hospital Virgen del Rocío in Seville. Seven couples have participated in our program because of different indications. Overall, 26 cycles were performed, providing a total of 202 embryos. A conclusive molecular diagnosis and HLA-typing could be assured in 96% of the embryos. The percentage of transfers per cycle was 26.9% and the birth rate per cycle was 7.7% per transfer. Our PGD-HLA program resulted in the birth of 2 healthy babies, HLA-identical to their affected siblings, with successful subsequent haematopoietic stem cell (HSC) transplantations. Both HSC-transplanted children are currently doing well 48 and 21 months following transplantation, respectively. All the procedures, including HSCs umbilical cord transplantation, were performed in our hospital. Raquel María Fernández, Ana Peciña, Maria Dolores Lozano-Arana, Beatriz Sánchez, Jordi Guardiola, Juan Carlos García-Lozano, Salud Borrego, and Guillermo Antiñolo Copyright © 2014 Raquel María Fernández et al. All rights reserved. Gender Specific Association of RAS Gene Polymorphism with Essential Hypertension: A Case-Control Study Thu, 17 Apr 2014 06:31:39 +0000 Renin-angiotensin system (RAS) polymorphisms have been studied as candidate risk factors for hypertension with inconsistent results, possibly due to heterogeneity among various genetic and environmental factors. A case-control association study was conducted to investigate a possible involvement of polymorphisms of three RAS genes: AGT M235T (rs699), ACE I/D (rs4340) and G2350A (rs4343), and AGTR1 A1166C (rs5186) in essential hypertensive patients. A total of 211 cases and 211 controls were recruited for this study. Genotyping was performed using PCR-RFLP method. The genotype and allele distribution of the M235T variant differed significantly in hypertensives and normotensives (OR-CI = 2.62 (1.24–5.76), ; OR-CI = 0.699 (0.518–0.943), ), respectively. When the samples were segregated based on sex, the 235TT genotype and T allele were predominant in the female patients (OR-CI = 5.68 (1.60-25.10), ; OR-CI = 0.522 (0.330–0.826), ) as compare to the male patients (OR-CI = 1.54 (1.24–5.76), ; OR-CI = 0.874 (0.330–0.826), ), respectively. For ACE DD variant, we found overrepresentation of “I”-allele (homozygous II and heterozygous ID) in unaffected males which suggest its protective role in studied population (OR-CI = 0.401 (0.224–0.718); ). The M235T variant of the AGT is significantly associated with female hypertensives and ACE DD variant could be a risk allele for essential hypertension in south India. Kh. Dhanachandra Singh, Ajay Jajodia, Harpreet Kaur, Ritushree Kukreti, and Muthusamy Karthikeyan Copyright © 2014 Kh. Dhanachandra Singh et al. All rights reserved. A Study of Sedentary Behaviour in the Older Finnish Twin Cohort: A Cross Sectional Analysis Tue, 15 Apr 2014 14:10:27 +0000 The aim of the study was to investigate the effects of age, sex, and body mass index (BMI) on total sitting time among the Finnish twin cohort. Also, heritability and environmental factors were analysed. The final sample included 6713 twin individuals 53–67 years of age (46% men). Among them there were 1940 complete twin pairs (732 monozygotic [MZ] and 1208 dizygotic [DZ] twin pairs). Sedentary behaviour was queried with a self-reported questionnaire with multiple-choice questions about sitting time at different domains. The mean total sitting time per day was 6 hours 41 minutes (standard deviation: 2 hours 41 minutes). The total sitting time was less in women than in men (). Older age was associated with less total sitting time (). Those with higher body mass index had higher total sitting time in age and sex adjusted analysis (). MZ pairs were more similar for sitting time than DZ pairs, with initial estimates of heritability for the total sitting time of 35%.The influence of shared environmental factors was negligible (1%), while most (64%) of the variation could be ascribed to unique environmental factors, the latter including measurement error. Maarit Piirtola, Jaakko Kaprio, and Annina Ropponen Copyright © 2014 Maarit Piirtola et al. All rights reserved. A Large French Case-Control Study Emphasizes the Role of Rare Mc1R Variants in Melanoma Risk Thu, 10 Apr 2014 10:13:47 +0000 Background. The MC1R gene implicated in melanogenesis and skin pigmentation is highly polymorphic. Several alleles are associated with red hair and fair skin phenotypes and contribute to melanoma risk. Objective. This work aims to assess the effect of different classes of MC1R variants, notably rare variants, on melanoma risk. Methods. MC1R coding region was sequenced in 1131 melanoma patients and 869 healthy controls. MC1R variants were classified as RHC (R) and non-RHC (r). Rare variants (frequency < 1%) were subdivided into two subgroups, predicted to be damaging (D) or not (nD). Results. Both R and r alleles were associated with melanoma (OR = 2.66 [2.20–3.23] and 1.51 [1.32–1.73]) and had similar population attributable risks (15.8% and 16.6%). We also identified 69 rare variants, of which 25 were novel. D variants were strongly associated with melanoma (OR = 2.38 [1.38–4.15]) and clustered in the same MC1R domains as R alleles (intracellular 2, transmembrane 2 and 7). Conclusion. This work confirms the role of R and r alleles in melanoma risk in the French population and proposes a novel class of rare D variants as important melanoma risk factors. These findings may improve the definition of high-risk subjects that could be targeted for melanoma prevention and screening. Hui-Han Hu, Mériem Benfodda, Nicolas Dumaz, Steven Gazal, Vincent Descamps, Agnès Bourillon, Nicole Basset-Seguin, Angélique Riffault, Khaled Ezzedine, Martine Bagot, Armand Bensussan, Philippe Saiag, Bernard Grandchamp, and Nadem Soufir Copyright © 2014 Hui-Han Hu et al. All rights reserved. Regenerative Medicine Wed, 09 Apr 2014 07:17:34 +0000 Ryuichi Morishita Copyright © 2014 Ryuichi Morishita. All rights reserved. Factors behind Leisure-Time Physical Activity Behavior Based on Finnish Twin Studies: The Role of Genetic and Environmental Influences and the Role of Motives Tue, 08 Apr 2014 00:00:00 +0000 Different approaches are being taken to clarify the role of various factors in the development of physical activity behaviors. Genetic studies are a new area of physical activity research and also the motives for physical activity have been widely studied. The purpose of this paper is to review the findings emerging from the longitudinal genetic studies on leisure-time physical activity and to evaluate the associations between motivational factors and leisure-time physical activity. The focus is to review recent findings of longitudinal Finnish twin studies. The results of the latest longitudinal Finnish twin studies point to the existence of age-specific genetic and environmental influences on leisure-time physical activity. Variations in environmental factors seem to explain the observed deterioration in leisure-time physical activity levels. A decline in genetic influences is seen first from adolescence to young adulthood and again from the age of thirty to the mid-thirties. In the Finnish twin participants, mastery, physical fitness, and psychological state were the major motivation factors associated with consistent leisure-time physical activity behavior. The results also indicate that intrinsic motivation factors may be important for engagement in leisure-time physical activity. Sari Aaltonen, Urho M. Kujala, and Jaakko Kaprio Copyright © 2014 Sari Aaltonen et al. All rights reserved. Genotype by Energy Expenditure Interaction and Body Composition Traits: The Portuguese Healthy Family Study Tue, 25 Mar 2014 12:58:02 +0000 Background and Aims. Energy expenditure has been negatively correlated with fat accumulation. However, this association is highly variable. In the present study we applied a genotype by environment interaction method to examine the presence of Genotype x by Total Daily Energy Expenditure and Genotype x by Daily Energy Expenditure interactions in the expression of different body composition traits. Methods and Results. A total of 958 subjects from 294 families of The Portuguese Healthy Family Study were included in the analysis. TDEE and DEE were assessed using a physical activity recall. Body fat percentages were measured with a bioelectrical impedance scale. GxTDEE and GxDEE examinations were performed using SOLAR 4.0 software. All BC traits were significantly heritable, with heritabilities ranging from 21% to 34%. The GxTDEE and GxDEE interaction models fitted the data better than the polygenic model for all traits. For all traits, a significant GxTDEE and GxDEE interaction was due to variance heterogeneity among distinct levels of TDEE and DEE. For WC, GxTDEE was also significant due to the genetic correlation function. Conclusions. TDEE and DEE are environmental constraints associated with the expression of individuals’ BC genotypes, leading to variability in the phenotypic expression of BC traits. D. M. Santos, P. T. Katzmarzyk, V. P. Diego, T. N. Gomes, F. K. Santos, J. Blangero, and J. A. Maia Copyright © 2014 D. M. Santos et al. All rights reserved. A 3’UTR Polymorphism of IL-6R Is Associated with Chinese Pediatric Tuberculosis Wed, 19 Mar 2014 11:32:30 +0000 Background. IL-6 is a proinflammatory cytokine that plays a critical role in host defense against tuberculosis (TB). Genetic polymorphisms of IL-6 and its receptor IL-6R had been discussed in adult TB recently. However, their role in pediatric TB is still unclear. Due to the obvious differences in TB pathophysiology in children, which may also reflect differences in genetic background, further association studies in pediatric populations are needed. Methods. A case-control study was carried out in a Chinese pediatric population including 353 TB patients and 400 healthy controls. Tag-SNPs of IL-6 and IL-6R genes were selected by Haploview software, genotyped using MassArray, and analyzed statistically. Results. One polymorphism, rs2229238, in the 3’UTR region of IL-6R was observed to be associated with increased resistance to TB (adjusted P = 0.03). The rs2229238 T allele contributed to a reduced risk to TB in recessive heritable model (OR, 0.53; 95% CI, 0.35–0.78). Conclusions. By tag-SNP genotyping based case-control study, we identified a genetic polymorphism in the IL-6R 3’UTR that regulates host resistance to pediatric TB in a Chinese population. Chen Shen, Hui Qi, Lin Sun, Jing Xiao, Qing-qin Yin, Wei-wei Jiao, Xi-rong Wu, Jian-ling Tian, Rui Han, and A-dong Shen Copyright © 2014 Chen Shen et al. All rights reserved. The Dopaminergic Reward System and Leisure Time Exercise Behavior: A Candidate Allele Study Sun, 09 Mar 2014 08:34:58 +0000 Purpose. Twin studies provide evidence that genetic influences contribute strongly to individual differences in exercise behavior. We hypothesize that part of this heritability is explained by genetic variation in the dopaminergic reward system. Eight single nucleotide polymorphisms (SNPs in DRD1: rs265981, DRD2: rs6275, rs1800497, DRD3: rs6280, DRD4: rs1800955, DBH: rs1611115, rs2519152, and in COMT: rs4680) and three variable number of tandem repeats (VNTRs in DRD4, upstream of DRD5, and in DAT1) were investigated for an association with regular leisure time exercise behavior. Materials and Methods. Data on exercise activities and at least one SNP/VNTR were available for 8,768 individuals aged 7 to 50 years old that were part of the Netherlands Twin Register. Exercise behavior was quantified as weekly metabolic equivalents of task (MET) spent on exercise activities. Mixed models were fitted in SPSS with genetic relatedness as a random effect. Results. None of the genetic variants were associated with exercise behavior (), despite sufficient power to detect small effects. Discussion and Conclusions. We did not confirm that allelic variants involved in dopaminergic function play a role in creating individual differences in exercise behavior. A plea is made for large genome-wide association studies to unravel the genetic pathways that affect this health-enhancing behavior. Charlotte Huppertz, Meike Bartels, Maria M. Groen-Blokhuis, Conor V. Dolan, Marleen H. M. de Moor, Abdel Abdellaoui, Catharina E. M. van Beijsterveldt, Erik A. Ehli, Jouke-Jan Hottenga, Gonneke Willemsen, Xiangjun Xiao, Paul Scheet, Gareth E. Davies, Dorret I. Boomsma, James J. Hudziak, and Eco J. C. de Geus Copyright © 2014 Charlotte Huppertz et al. All rights reserved. The Role of Vitamin D Deficiency and Vitamin D Receptor Genotypes on the Degree of Collateralization in Patients with Suspected Coronary Artery Disease Thu, 06 Mar 2014 13:59:56 +0000 We determined the association of vitamin D deficiency and the FokI polymorphism of the vitamin D receptor (VDR) gene in 760 patients who underwent angiography due to suspected coronary artery disease (CAD). Angiography and the Rentrop scoring system were used to classify the severity of CAD in each patient and to grade the extent of collateral development, respectively. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the FokI VDR gene polymorphism. The prevalence of severe vitamin D deficiency (serum 25(OH)D < 10 ng/mL) was significantly higher in patients with at least one stenotic coronary artery compared to those without any stenotic coronary arteries. Severe vitamin D deficiency was not independently associated with collateralization, but it was significantly associated with the VDR genotypes. In turn, VDR genotype was independently associated with the degree of collateralization; the Rentrop scores were the highest in FF, intermediate in Ff, and the lowest in the ff genotype. The results show that FokI polymorphism is independently associated with collateralization. Additionally, vitamin D deficiency is more prevalent in patients with CAD that may result from FokI polymorphism. Therefore, maintaining a normal vitamin D status should be a high priority for patients with CAD. Arash Hossein-Nezhad, Seyede Mahdieh Eshaghi, Zhila Maghbooli, Khadijeh Mirzaei, Mahmood Shirzad, Bryon Curletto, and Tai C. Chen Copyright © 2014 Arash Hossein-Nezhad et al. All rights reserved. Role of the Vasa Vasorum and Vascular Resident Stem Cells in Atherosclerosis Wed, 05 Mar 2014 00:00:00 +0000 Atherosclerosis is considered an “inside-out” response, that begins with the dysfunction of intimal endothelial cells and leads to neointimal plaque formation. The adventitia of large blood vessels has been recognized as an active part of the vessel wall that is involved in the process of atherosclerosis. There are characteristic changes in the adventitial vasa vasorum that are associated with the development of atheromatous plaques. However, whether vasa vasorum plays a causative or merely reactive role in the atherosclerotic process is not completely clear. Recent studies report that the vascular wall contains a number of stem/progenitor cells that may contribute to vascular remodeling. Microvessels serve as the vascular niche that maintains the resident stem/progenitor cells of the tissue. Therefore, the vasa vasorum may contribute to vascular remodeling through not only its conventional function as a blood conducting tube, but also its new conceptual function as a stem cell reservoir. This brief review highlights the recent advances contributing to our understanding of the role of the adventitial vasa vasorum in the atherosclerosis and discusses new concept that involves vascular-resident factors, the vasa vasorum and its associated vascular-resident stem cells, in the atherosclerotic process. Jun-ichi Kawabe and Naoyuki Hasebe Copyright © 2014 Jun-ichi Kawabe and Naoyuki Hasebe. All rights reserved. Detection of Genetic Variations in Coagulopathy-Related Genes Using Ramified Rolling Circle Amplification Sun, 02 Mar 2014 00:00:00 +0000 We evaluated single nucleotide polymorphism (SNP) detection via a target-capture, C-probe ligation, and RAM assay in a single-blind comparison to clinical samples that had been tested with FDA-cleared tests for up to 4 different vascular disease-related SNPs. In the RAM assay circulizable linear probes (C- or padlock probes) were annealed directly to genomic DNA, processed on a largely automated platform, and ligated C-probes were amplified by real-time RAM. After allele determinations were made with the experimental system, the sample genotypes were unblinded and the experimentally determined genotypes were found to be completely consistent with the FDA-cleared test results. The methods and results presented here show that a combination of C-probes, automated sample processing, and isothermal RAM provides a robust, and specific, nucleic acid detection platform that is compatible with automated DNA sample preparation and the throughput requirements of the clinical laboratory. James H. Smith, Miao Cui, David Y. Zhang, Thomas P. Beals, and Fei Ye Copyright © 2014 James H. Smith et al. All rights reserved. Comparison of Gene and Protein Expressions in Rats Residing in Standard Cages with Those Having Access to an Exercise Wheel Tue, 25 Feb 2014 13:40:13 +0000 Lifelong physical inactivity is associated with morbidity in adulthood, possibly influenced by changes in gene and protein expressions occurring earlier in life. mRNA (Affymetrix gene array) and proteomic (2D-DIGE MALDI-TOF/MS) analyses were determined in cardiac tissue of young (3 months) and old (16 months) Sprague-Dawley rats housed with no access to physical activity (SED) versus an exercise wheel (EX). Unfavorable phenotypes for body weight, dyslipidemia, and tumorogenesis appeared more often in adult SED versus EX. No differentially expressed genes (DEGs) occurred between groups at 3 or 16 months. Within groups, SED and EX shared 215 age-associated DEGs. In SED, ten unique DEGs occurred with age; three had cell adhesion functions (fn1, lgals3, ncam2). In EX, five unique DEGs occurred with age; two involved hypothalamic, pituitary, and gonadal hormone axis (nrob2, xpnpep2). Protein expression involved in binding, sugar metabolic processes, and vascular regulation declined with age in SED (KNT1, ALBU, GPX1, PYGB, LDHB, G3P, PYGM, PGM1, ENOB). Protein expression increased with age in EX for ATP metabolic processes (MYH6, MYH7, ATP5J, ATPA) and vascular function (KNT1, ALBU, GPX1). Differences in select gene and protein expressions within sedentary and active animals occurred with age and contributed to distinct health-related phenotypes in adulthood. Helaine M. Alessio, Hayden Ansinelli, Caitlyn Threadgill, and Ann E. Hagerman Copyright © 2014 Helaine M. Alessio et al. All rights reserved. Investigation of NF-κB1 and NF-κBIA Gene Polymorphism in Non-Small Cell Lung Cancer Sun, 23 Feb 2014 13:05:19 +0000 Lung cancer is a complex, multifactorial disease which is the leading cause of cancer death in both men and women. NF-κB is a transcription factor which is known to affect the expression of more than 150 genes related to inflammation, lymphocyte activation, cell proliferation, differentiation, and apoptosis, as well as contributing to cell apoptosis and survival. However, NF-κBIA (IκBα) is the inhibitor of the transcription factor. The -94ins/delATTG polymorphism of the NF-κB1 gene promoter region which causes a functional effect and NF-κBIA 3′UTR A → G polymorphism has been shown to be related to various inflammatory diseases and cancer. Ninety-five NSCLC patients and 99 healthy controls were included in study. The NF-κB1 -94ins/delATTG and NF-κBIA 3′UTR A → G polymorphism have been studied by using PCR-RFLP method. It was found that the NF-κB1 -94ins/delATTG DD genotype and D allele frequencies were higher in patients than healthy controls and the presence of the DD genotype has a 3.5-fold increased risk of the disease (P: 0.014). This study is the first to investigate the NF-κB1 -94ins/delATTG and NF-κBIA 3′UTR A → G polymorphism together in the Turkish population. According to the results, the NF-κB1 -94ins/del ATTG promoter polymorphism may have a role in lung carcinogenesis and prognosis. Y. M. Oltulu, E. Coskunpinar, G. Ozkan, E. Aynaci, P. Yildiz, T. Isbir, and I. Yaylim Copyright © 2014 Y. M. Oltulu et al. All rights reserved. Oxidative Stress, Hypoxia, and Autophagy in the Neovascular Processes of Age-Related Macular Degeneration Sun, 23 Feb 2014 00:00:00 +0000 Age-related macular degeneration (AMD) is the leading cause of severe and irreversible loss of vision in the elderly in developed countries. AMD is a complex chronic neurodegenerative disease associated with many environmental, lifestyle, and genetic factors. Oxidative stress and the production of reactive oxygen species (ROS) seem to play a pivotal role in AMD pathogenesis. It is known that the macula receives the highest blood flow of any tissue in the body when related to size, and anything that can reduce the rich blood supply can cause hypoxia, malfunction, or disease. Oxidative stress can affect both the lipid rich retinal outer segment structure and the light processing in the macula. The response to oxidative stress involves several cellular defense reactions, for example, increases in antioxidant production and proteolysis of damaged proteins. The imbalance between production of damaged cellular components and degradation leads to the accumulation of detrimental products, for example, intracellular lipofuscin and extracellular drusen. Autophagy is a central lysosomal clearance system that may play an important role in AMD development. There are many anatomical changes in retinal pigment epithelium (RPE), Bruch’s membrane, and choriocapillaris in response to chronic oxidative stress, hypoxia, and disturbed autophagy and these are estimated to be crucial components in the pathology of neovascular processes in AMD. Janusz Blasiak, Goran Petrovski, Zoltán Veréb, Andrea Facskó, and Kai Kaarniranta Copyright © 2014 Janusz Blasiak et al. All rights reserved. Identification of Key Genes in the Response to Salmonella enterica Enteritidis, Salmonella enterica Pullorum, and Poly(I:C) in Chicken Spleen and Caecum Sun, 23 Feb 2014 00:00:00 +0000 Salmonella enterica Enteritidis (S. Enteritidis) and Salmonella enterica Pullorum (S. pullorum) are regarded as a threat to poultry production. This study’s aim is to characterize the expression profiles in response to three different challenges and to identify infection-related genes in the chicken spleen and caecum. Groups of the Chinese chicken breed Langshan were challenged with either S. Enteritidis, S. pullorum, or poly(I:C). The concentrations of cytokines and antibodies and the Salmonella colonization level of the caecum and liver were detected in each group at 7 days postinfection. Expression microarray experiments were conducted using mRNA isolated from both spleen and caecum. Crucial differentially expressed genes (DEGs) associated with immunity were identified. Four DEGs were identified in spleen of all three challenge groups (RBM16, FAH, SOX5, and RBM9) and different four genes in caecum (SOUL, FCN2, ANLN, and ACSL1). Expression profiles were clearly different among the three challenged groups. Genes enriched in the spleen of birds infected with S. pullorum were enriched in lymphocyte proliferation related pathways, but the enriched genes in the caecum of the same group were primarily enriched in innate immunity or antibacterial responses. The DEGs that appear across all three challenge groups might represent global response factors for different pathogens. Teng Ma, Guobin Chang, Rong Chen, Zhongwei Sheng, Aiqin Dai, Fei Zhai, Jianchao Li, Mingxiu Xia, Dengke Hua, Lu Xu, Hongzhi Wang, Jing Chen, Lu Liu, and Guohong Chen Copyright © 2014 Teng Ma et al. All rights reserved. Genetic Control of Immune Response and Susceptibility to Infectious Diseases Thu, 13 Feb 2014 12:43:15 +0000 Enrique Medina-Acosta, Helder Takashi Imoto Nakaya, Alessandra Pontillo, and Regina Célia de Souza Campos Fernandes Copyright © 2014 Enrique Medina-Acosta et al. All rights reserved. Evaluation of Bax and Bak Gene Mutations and Expression in Breast Cancer Sun, 09 Feb 2014 11:18:20 +0000 Genetic analyses have provided evidence to suggest that Bax and Bak are the essential genes for apoptosis in mammalians cells. This study aimed to search for biomarkers in breast cancer to be used as prognostic markers for the disease. The Bak and Bax genes expressions were analyzed in 23 breast cancer patients by RT-PCR technique. SSCP technique was used to detect the mobility of the abnormal fragment in Bak exon 4. PCR for Bax promoter was digested with Tau 1 restriction enzyme to identify a single polymorphism G(-248)A. The expression of Bak gene is related to several clinical factors of breast cancer. The analysis of Bax RNA showed 4 isoforms of Bax with different distributions in the normal and tumor tissues. These isoforms were Bax α, d, δ, and ζ. Exon 4 had a normal pattern in all cases of breast cancer. There was a statistically significant difference in the frequency distribution of the G(-248)A genotypes in the breast cancer tissues with grade 3+high, T2 stage, lobular +other, and PR −ve subgroups. In this study, Bak expression seems to lead to development of breast cancer and affects the disease progression. Also, Bax d and Bax δ could be used as risk factor and biomarker for breast cancer with the distribution of G284A. Naglaa Mohamed Kholoussi, Sobhy E. H. El-Nabi, Nora Nassef Esmaiel, Naser Mohamed Abd El-Bary, and Ahmed F. El-Kased Copyright © 2014 Naglaa Mohamed Kholoussi et al. All rights reserved. The 14 bp Del/Ins HLA-G Polymorphism Is Related with High Blood Pressure in Acute Coronary Syndrome and Type 2 Diabetes Mellitus Thu, 06 Feb 2014 09:11:03 +0000 Immunologic and inflammatory processes are involved in the pathogenesis of acute coronary syndrome (ACS) and type 2 diabetes mellitus (DM2). Human leukocyte antigen-G (HLA-G) is a negative regulator of the immune response. This study evaluates the 14 bp Del/Ins HLA-G polymorphism in ACS and DM2. Three hundred and seventy individuals from Western Mexico were recruited and categorized into three groups: ACS (86), DM2 without coronary complications (70), and healthy subjects (214). Genotyping of the 14 bp Del/Ins HLA-G polymorphism was performed by PCR and Native-PAGE. The most common risk factors were hypertension and overweight in ACS and DM2, respectively. The genetic distribution of the 14 bp Del/Ins HLA-G polymorphism showed no significant differences between groups (). Nonetheless, the Ins/Ins genotype was associated with high blood pressure (HBP) in the DM2 group ( = 1.65, ). The genetic recessive model showed similar findings ( = 3.03, ). No association was found in ACS, with a of 0.05; nevertheless, the prevalence of Ins/Ins carriers was quite similar to that found in the DM2-HBP group. The 14 bp Del/Ins HLA-G polymorphism was not a susceptibility factor for ACS or DM2; however, the Ins/Ins genotype might have contributed to the development of HBP in the studied groups. Ilian Janet García-González, Yeminia Valle, Fernando Rivas, Luis Eduardo Figuera-Villanueva, José Francisco Muñoz-Valle, Hector Enrique Flores-Salinas, Bianca Ethel Gutiérrez-Amavizca, Nory Omayra Dávalos-Rodríguez, and Jorge Ramón Padilla-Gutiérrez Copyright © 2014 Ilian Janet García-González et al. All rights reserved. Differential Gene Expression in High- and Low-Active Inbred Mice Thu, 16 Jan 2014 13:13:08 +0000 Numerous candidate genes have been suggested in the recent literature with proposed roles in regulation of voluntary physical activity, with little evidence of these genes’ functional roles. This study compared the haplotype structure and expression profile in skeletal muscle and brain of inherently high- (C57L/J) and low- (C3H/HeJ) active mice. Expression of nine candidate genes [Actn2, Actn3, Casq1, Drd2, Lepr, Mc4r, Mstn, Papss2, and Glut4 (a.k.a. Slc2a4)] was evaluated via RT-qPCR. SNPs were observed in regions of Actn2, Casq1, Drd2, Lepr, and Papss2; however, no SNPs were located in coding sequences or associated with any known regulatory sequences. In mice exposed to a running wheel, Casq1 () and Mstn () transcript levels in the soleus were higher in the low-active mice. However, when these genes were evaluated in naïve animals, differential expression was not observed, demonstrating a training effect. Among naïve mice, no genes in either tissue exhibited differential expression between strains. Considering that no obvious SNP mechanisms were determined or differential expression was observed, our results indicate that genomic structural variation or gene expression data alone is not adequate to establish any of these genes’ candidacy or causality in relation to regulation of physical activity. Michelle Dawes, Trudy Moore-Harrison, Alicia T. Hamilton, Tyrone Ceaser, Kelli J. Kochan, Penny K. Riggs, and J. Timothy Lightfoot Copyright © 2014 Michelle Dawes et al. All rights reserved. The Association Study of Calmodulin 1 Gene Polymorphisms with Susceptibility to Adolescent Idiopathic Scoliosis Thu, 16 Jan 2014 06:37:56 +0000 Objective. Idiopathic scoliosis is the most common pediatric spinal deformity affecting 1% to 3% of the population, and adolescent idiopathic scoliosis (AIS) accounts for approximately 80% of these cases; however, the etiology and pathogenesis of AIS are still uncertain. The current study aims to identify the relationship between calmodulin 1 (CALM1) gene and AIS predisposition, to identify the relationship between the genotypes of the SNPs and the clinical phenotypes of AIS. Methods. 146 AIS patients and 146 healthy controls were enrolled into this case-control study. 12 single nucleotide polymorphisms (SNPs) candidates in CALM1 gene were selected to determine the relationship between CALM1 gene and AIS predisposition. Case-only study was performed to determine the effects of these variants on the severity of the condition. Results. Three SNPs from 12 candidates were found to be associated with AIS predisposition. The ORs were observed as 0.549 (95% CI 0.3519–0.8579, ), 0.549 (95% CI 0.3519–0.8579, ), and 1.6139 (95% CI 1.0576–2.4634, ) for rs2300496, rs2300500, and rs3231718, respectively. There was no statistical difference between main curve, severity, and genotype distributions of all of 12 SNPs. Conclusion. Genetic variants of CALM1 gene are associated with AIS susceptibility. Yu Zhang, Zuchao Gu, and Guixing Qiu Copyright © 2014 Yu Zhang et al. All rights reserved. Mesenchymal Stem Cells for Regenerative Therapy: Optimization of Cell Preparation Protocols Mon, 06 Jan 2014 07:46:54 +0000 Administration of bone marrow-derived mesenchymal stem cells (MSCs) is an innovative approach for the treatment of a range of diseases that are not curable by current therapies including heart failure. A number of clinical trials have been completed and many others are ongoing; more than 2,000 patients worldwide have been administered with culture-expanded allogeneic or autologous MSCs for the treatment of various diseases, showing feasibility and safety (and some efficacy) of this approach. However, protocols for isolation and expansion of donor MSCs vary widely between these trials, which could affect the efficacy of the therapy. It is therefore important to develop international standards of MSC production, which should be evidence-based, regulatory authority-compliant, of good medical practice grade, cost-effective, and clinically practical, so that this innovative approach becomes an established widely adopted treatment. This review article summarizes protocols to isolate and expand bone marrow-derived MSCs in 47 recent clinical trials of MSC-based therapy, which were published after 2007 onwards and provided sufficient methodological information. Identified issues and possible solutions associated with the MSC production methods, including materials and protocols for isolation and expansion, are discussed with reference to relevant experimental evidence with aim of future clinical success of MSC-based therapy. Chiho Ikebe and Ken Suzuki Copyright © 2014 Chiho Ikebe and Ken Suzuki. All rights reserved. Do Telomeres Adapt to Physiological Stress? Exploring the Effect of Exercise on Telomere Length and Telomere-Related Proteins Tue, 24 Dec 2013 16:41:29 +0000 Aging is associated with a tissue degeneration phenotype marked by a loss of tissue regenerative capacity. Regenerative capacity is dictated by environmental and genetic factors that govern the balance between damage and repair. The age-associated changes in the ability of tissues to replace lost or damaged cells is partly the cause of many age-related diseases such as Alzheimer's disease, cardiovascular disease, type II diabetes, and sarcopenia. A well-established marker of the aging process is the length of the protective cap at the ends of chromosomes, called telomeres. Telomeres shorten with each cell division and with increasing chronological age and short telomeres have been associated with a range of age-related diseases. Several studies have shown that chronic exposure to exercise (i.e., exercise training) is associated with telomere length maintenance; however, recent evidence points out several controversial issues concerning tissue-specific telomere length responses. The goals of the review are to familiarize the reader with the current telomere dogma, review the literature exploring the interactions of exercise with telomere phenotypes, discuss the mechanistic research relating telomere dynamics to exercise stimuli, and finally propose future directions for work related to telomeres and physiological stress. Andrew T. Ludlow, Lindsay W. Ludlow, and Stephen M. Roth Copyright © 2013 Andrew T. Ludlow et al. All rights reserved. Why Control Activity? Evolutionary Selection Pressures Affecting the Development of Physical Activity Genetic and Biological Regulation Tue, 24 Dec 2013 15:30:17 +0000 The literature strongly suggests that daily physical activity is genetically and biologically regulated. Potential identities of the responsible mechanisms are unclear, but little has been written concerning the possible evolutionary selection pressures leading to the development of genetic/biological controls of physical activity. Given the weak relationship between exercise endurance and activity levels and the differential genomic locations associated with the regulation of endurance and activity, it is probable that regulation of endurance and activity evolved separately. This hypothesis paper considers energy expenditures and duration of activity in hunter/gatherers, pretechnology farmers, and modern Western societies and considers the potential of each to selectively influence the development of activity regulation. Food availability is also considered given the known linkage of caloric restriction on physical activity as well as early data relating food oversupply to physical inactivity. Elucidating the selection pressures responsible for the genetic/biological control of activity will allow further consideration of these pressures on activity in today’s society, especially the linkages between food and activity. Further, current food abundance is removing the cues for activity that were present for the first 40,000 years of human evolution, and thus future research should investigate the effects of this abundance upon the mechanisms regulating activity. J. Timothy Lightfoot Copyright © 2013 J. Timothy Lightfoot. All rights reserved. Alterations in Hair Follicle Dynamics in Women Tue, 24 Dec 2013 13:43:27 +0000 Endocrine changes supervening after parturition and menopause participate in the control of sebum production and hair growth modulation. The ensuing conditions include some peculiar aspects of hair loss (effluvium), alopecia, and facial hirsutism. The hair cycling is of major clinical relevance because most hair growth disorders result from disturbances in this chronobiological feature. Of note, any correlation between a biologic abnormality and hair cycling disturbance does not prove a relationship of causality. The proportion of postmenopausal women is rising in the overall population. Therefore, the prevalence of these hair follicle disturbances is globally on the rise. Current therapies aim at correcting the underlying hormonal imbalances, and at improving the overall cosmetic appearance. However, in absence of pathogenic diagnosis and causality criteria, chances are low that a treatment given by the whims of fate will adequately control hair effluvium. The risk and frequency of therapeutic inertia are further increased. When the hair loss is not controlled and/or compensated by growth of new hairs, several clinical aspects of alopecia inexorably develop. Currently, there is little evidence supporting any specific treatment for these endocrine hair disorders in post-partum and postmenopausal women. Current hair treatment strategies are symptomatic and nonspecific so current researchers aim at developing new, targeted methods. Claudine Piérard-Franchimont and Gérald E. Piérard Copyright © 2013 Claudine Piérard-Franchimont and Gérald E. Piérard. All rights reserved. Highlights from the Functional Single Nucleotide Polymorphisms Associated with Human Muscle Size and Strength or FAMuSS Study Mon, 23 Dec 2013 13:42:46 +0000 The purpose of the Functional Single Nucleotide Polymorphisms Associated with Human Muscle Size and Strength study or FAMuSS was to identify genetic factors that dictated the response of health-related fitness phenotypes to resistance exercise training (RT). The phenotypes examined were baseline muscle strength and muscle, fat, and bone volume and their response to RT. FAMuSS participants were 1300 young (24 years), healthy men (42%) and women (58%) that were primarily of European-American descent. They were genotyped for ~500 polymorphisms and completed the Paffenbarger Physical Activity Questionnaire to assess energy expenditure and time spent in light, moderate, and vigorous intensity habitual physical activity and sitting. Subjects then performed a 12-week progressive, unilateral RT program of the nondominant arm with the dominant arm used as a comparison. Before and after RT, muscle strength was measured with the maximum voluntary contraction and one repetition maximum, while MRI measured muscle, fat, and bone volume. We will discuss the history of how FAMuSS originated, provide a brief overview of the FAMuSS methods, and summarize our major findings regarding genotype associations with muscle strength and size, body composition, cardiometabolic biomarkers, and physical activity. Linda S. Pescatello, Joseph M. Devaney, Monica J. Hubal, Paul D. Thompson, and Eric P. Hoffman Copyright © 2013 Linda S. Pescatello et al. All rights reserved. Regenerative Medicine for the Cornea Tue, 17 Dec 2013 09:25:32 +0000 Regenerative medicine for the cornea provides a novel treatment strategy for patients with corneal diseases instead of conventional keratoplasty. Limbal transplantation has been performed in patients with a limbal stem cell deficiency. This procedure requires long-term immunosuppression that involves high risks of serious eye and systemic complications, including infection, glaucoma, and liver dysfunction. To solve these problems, ocular surface reconstruction using cultured limbal or oral mucosal epithelial stem cells has been successfully applied to patients. However, cell sheets must be fabricated in a cell processing center (CPC) under good manufacturing practice conditions for clinical use, and the expenses of maintaining a CPC are too high for all hospitals to cover. Therefore, several hospitals should share one CPC to standardize and spread the application of regenerative therapy using tissue-engineered oral mucosal epithelial cell sheets. Consequently, we developed a cell transportation technique for clinical trial to bridge hospitals. This paper reviews the current status of regenerative medicine for the cornea. Yoshinori Oie and Kohji Nishida Copyright © 2013 Yoshinori Oie and Kohji Nishida. All rights reserved. Genetic Variations of α-Methylacyl-CoA Racemase Are Associated with Sporadic Prostate Cancer Risk in Ethnically Homogenous Koreans Sat, 07 Dec 2013 14:04:55 +0000 Background. To assess if the variants of (R)-alpha-methyl-CoA racemase (AMACR) gene would be associated with the risk of sporadic prostate cancer in ethnically homogenous Koreans. Materials and Methods. We enrolled 194 patients with prostate cancer and 169 healthy controls. A total of 17 single nucleotide polymorphisms of the AMACR gene were selected. The distribution of each genotype and haplotype was analyzed and their association with the incidence of prostate cancer was evaluated. Further, we detected AMACR expression in tumor with immunohistochemistry and analyzed its association with genotype regarding prostate cancer risk. Results. AG or GG genotype of rs2278008 (E277K) tended to lower prostate cancer risk. The minor G allele was found to be a significant allele that decreased the risk of prostate cancer (adjusted OR, 0.57; 95% CI, 0.35–0.93, value = 0.025). In patients expression AMACR, AG or GG genotype was also significant genotype in terms of prostate cancer risk (adjusted OR, 0.47; 95% CI, 0.26–0.87, value = 0.017). Further, [GGCGG] haplotype consisted of five coding SNPs of rs2278008, rs34677, rs2287939, rs10941112, and rs3195676 which decreased the risk of prostate cancer ( value = 0.047). Conclusions. Genetic variations of AMACR are associated with the risk of sporadic prostate cancer that underwent radical prostatectomy in Koreans. Sang-Jin Lee, Jae Young Joung, Hyekyoung Yoon, Jeong Eun Kim, Weon Seo Park, Ho Kyung Seo, Jinsoo Chung, Jung-Ah Hwang, Seung-Hyun Hong, Seungyoon Nam, Sohee Park, Jeongseon Kim, Kang Hyun Lee, and Yeon-Su Lee Copyright © 2013 Sang-Jin Lee et al. All rights reserved. Present and Future Perspectives on Cell Sheet-Based Myocardial Regeneration Therapy Wed, 04 Dec 2013 12:07:46 +0000 Heart failure is a life-threatening disorder worldwide and many papers reported about myocardial regeneration through surgical method induced by LVAD, cellular cardiomyoplasty (cell injection), tissue cardiomyoplasty (bioengineered cardiac graft implantation), in situ engineering (scaffold implantation), and LV restrictive devices. Some of these innovated technologies have been introduced to clinical settings. Especially, cell sheet technology has been developed and has already been introduced to clinical situation. As the first step in development of cell sheet, neonatal cardiomyocyte sheets were established and these sheets showed electrical and histological homogeneous heart-like tissue with contractile ability in vitro and worked as functional heart muscle which has electrical communication with recipient myocardium in small animal heart failure model. Next, as a preclinical study, noncontractile myoblast sheets have been established and these sheets have proved to secrete multiple cytokines such as HGF or VEGF in vitro study. Moreover, in vivo studies using large and small animal heart failure model have been done and myoblast sheets could improve diastolic and systolic performance by cytokine paracrine effect such as angiogenesis, antifibrosis, and stem cell migration. Recently evidenced by these preclinical results, clinical trials using autologous myoblast sheets have been started in ICM and DCM patients and some patients showed LV reverse remodelling, improved symptoms, and exercise tolerance. Recent works demonstrated that iPS cell-derived cardiomyocyte sheet were developed and showed electrical and microstructural homogeneity of heart tissue in vitro, leading to the establishment of proof of concept in small and large animal heart failure model. Yoshiki Sawa and Shigeru Miyagawa Copyright © 2013 Yoshiki Sawa and Shigeru Miyagawa. All rights reserved. The Variety of Vertebrate Mechanisms of Sex Determination Wed, 04 Dec 2013 11:39:01 +0000 The review deals with features of sex determination in vertebrates. The mechanisms of sex determination are compared between fishes, amphibians, reptilians, birds, and mammals. We focus on structural and functional differences in the role of sex-determining genes in different vertebrates. Special attention is paid to the role of estrogens in sex determination in nonmammalian vertebrates. Antonina V. Trukhina, Natalia A. Lukina, Natalia D. Wackerow-Kouzova, and Alexander F. Smirnov Copyright © 2013 Antonina V. Trukhina et al. All rights reserved. Virosome Presents Multimodel Cancer Therapy without Viral Replication Wed, 04 Dec 2013 09:23:37 +0000 A virosome is an artificial envelope that includes viral surface proteins and lacks the ability to produce progeny virus. Virosomes are able to introduce an encapsulated macromolecule into the cytoplasm of cells using their viral envelope fusion ability. Moreover, virus-derived factors have an adjuvant effect for immune stimulation. Therefore, many virosomes have been utilized as drug delivery vectors and adjuvants for cancer therapy. This paper introduces the application of virosomes for cancer treatment. In Particular, we focus on virosomes derived from the influenza and Sendai viruses which have been widely used for cancer therapy. Influenza virosomes have been mainly applied as drug delivery vectors and adjuvants. By contrast, the Sendai virosomes have been mainly applied as anticancer immune activators and apoptosis inducers. Kotaro Saga and Yasufumi Kaneda Copyright © 2013 Kotaro Saga and Yasufumi Kaneda. All rights reserved. Polymorphism of the Transferrin Gene in Eye Diseases: Keratoconus and Fuchs Endothelial Corneal Dystrophy Sun, 24 Nov 2013 15:53:39 +0000 Oxidative stress may play a role in the pathogenesis of keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Iron may promote the stress by the Fenton reaction, so its homeostasis should be strictly controlled. Transferrin is essential for iron homeostasis because it transports iron from plasma into cells. The malfunction of transferrin, which may be caused by variation in its gene (TF) variation, may contribute to oxidative stress and change KC and FECD risk. To verify this hypothesis we investigated the association between three polymorphisms of the TF gene, g.3296G>A (rs8177178), g.3481A>G (rs8177179), and c.–2G>A (rs1130459), and KC and FECD occurrence. Genotyping was performed in blood lymphocytes in 216 patients with KC, 130 patients with FECD and 228 controls by PCR-RFLP. We studied also the influence of other risk factors. The A/A genotype and the A allele of the g.3296G>A polymorphism were associated with KC occurrence, while the G allele was negatively correlated with it. We observed a decrease in KC occurrence associated with the A/G genotype of the g.3481A>G polymorphism. We did not find any association between the c.–2G>A polymorphism and KC. No association was found between all three polymorphisms and FECD occurrence. Katarzyna A. Wójcik, Ewelina Synowiec, Manuel P. Jiménez-García, Anna Kaminska, Piotr Polakowski, Janusz Blasiak, Jerzy Szaflik, and Jacek P. Szaflik Copyright © 2013 Katarzyna A. Wójcik et al. All rights reserved. Integration of Data from Omic Studies with the Literature-Based Discovery towards Identification of Novel Treatments for Neovascularization in Diabetic Retinopathy Sun, 24 Nov 2013 08:35:35 +0000 Diabetic retinopathy (DR) is a secondary complication of diabetes associated with retinal neovascularization and represents the leading cause of blindness in the adult population in the developed world. Despite research efforts, the nature of pathogenetic processes leading to DR is still unknown, making development of novel effective treatments difficult. Advances in omic technologies now offer unprecedented insight into global molecular alterations in DR, but identification of novel treatments based on massive amounts of data generated in omic studies still represents a considerable challenge. For this reason, we attempted to facilitate discovery of novel treatments for DR by complementing the interpretation of omic results using the vast body of information existing in the published literature with the literature-based discovery (LBD) approaches. To achieve this, we collected data from transcriptomic studies performed on retinal tissue from animal models of DR, performed a meta-analysis of these datasets and identified altered genes and pathways. Using the SemBT LBD framework, we have determined which therapies could regulate perturbed pathways or that could stabilize the gene expression alterations in DR. We show that by using this approach, we not only could reidentify drugs currently in use or in clinical trials, but also could indicate novel treatment directions for ameliorating neovascularization processes in DR. Ales Maver, Dimitar Hristovski, Thomas C. Rindflesch, and Borut Peterlin Copyright © 2013 Ales Maver et al. All rights reserved. HLA-B*40 Allele Plays a Role in the Development of Acute Leukemia in Mexican Population: A Case-Control Study Wed, 13 Nov 2013 16:04:36 +0000 Among oncohematological diseases, acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) are characterized by the uncontrolled production and accumulation of blasts that can lead to death. Although the physiopathology of these diseases is multifactorial, a genetic factor seems to be at play. Several studies worldwide have shown association of ALL and AML with several alleles of the major histocompatibility complex (MHC). Objective. To determine gene frequencies of HLA-B alleles in Mexicans (individuals with Native American genetic background admixed with European descent) with ALL and AML. Methods. We compared the HLA-B alleles in 213 patients with ALL and 85 patients with AML to those present in 731 umbilical cord blood (UCB) samples as a control group; this was done by means of the PCR-SSP technique. Results. We found an increased frequency of the HLA-B*40 allele in ALL patients as compared to the control group (14.5% versus 9.84%, , OR = 1.67); this was particularly evident in a subgroup of young (less than 18 years old) ALL patients (, OR = 1.76); likewise, a decreased frequency of HLA-B*40 allele in AML patients was observed as compared to the control group (4.70% versus 9.84%, , OR = 0.42). Conclusions. These results might suggest opposing effects of the HLA-B*40 in the genetic susceptibility to develop ALL or AML and offer the possibility to study further the molecular mechanisms of cell differentiation within the bone marrow lineage. Javier Fernández-Torres, Denhi Flores-Jiménez, Antonio Arroyo-Pérez, Julio Granados, and Alberto López-Reyes Copyright © 2013 Javier Fernández-Torres et al. All rights reserved. Therapies for Neovascular Age-Related Macular Degeneration: Current Approaches and Pharmacologic Agents in Development Mon, 11 Nov 2013 14:25:31 +0000 As one of the leading causes of blindness, age-related macular degeneration (AMD) has remained at the epicenter of clinical research in ophthalmology. During the past decade, focus of researchers has ranged from understanding the role of vascular endothelial growth factor (VEGF) in the angiogenic cascades to developing new therapies for retinal vascular diseases. Anti-VEGF agents such as ranibizumab and aflibercept are becoming increasingly well-established therapies and have replaced earlier approaches such as laser photocoagulation or photodynamic therapy. Many other new therapeutic agents, which are in the early phase clinical trials, have shown promising results. The purpose of this paper is to briefly review the available treatment modalities for neovascular AMD and then focus on promising new therapies that are currently in various stages of development. Mostafa Hanout, Daniel Ferraz, Mehreen Ansari, Natasha Maqsood, Saleema Kherani, Yasir J. Sepah, Nithya Rajagopalan, Mohamed Ibrahim, Diana V. Do, and Quan Dong Nguyen Copyright © 2013 Mostafa Hanout et al. All rights reserved. Gene Therapy and Cell-Based Therapies for Therapeutic Angiogenesis in Peripheral Artery Disease Sun, 03 Nov 2013 16:06:21 +0000 Gene therapy and cell-based therapy have emerged as novel therapies to promote therapeutic angiogenesis in critical limb ischemia (CLI) caused by peripheral artery disease (PAD). Although researchers initially focused on gene therapy using proangiogenic factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factors (HGF), cell therapy using bone marrow mononuclear cells (BMMNCs), mesenchymal stem cells (BMMSCs), G-CSF-mobilized peripheral blood mononuclear cells (M-PBMNCs), and endothelial progenitor cells (EPCs) have also been extensively studied. Based on the elaborate studies and favorable results of basic research, some clinical phase I/II trials have been performed, and the results demonstrate the safety of these approaches and their potential for symptomatic improvement in CLI. However, the phase 3 clinical trials have thus far been limited to gene therapy using the HGF gene. Further studies using well-designed larger placebo-controlled and long-term randomized control trials (RCTs) will clarify the effectiveness of gene therapy and cell-based therapy for the treatment of CLI. Furthermore, the development of efficient gene transfer systems and effective methods for keeping transplanted cells healthy will make these novel therapies more effective and ease the symptoms of CLI. Munehisa Shimamura, Hironori Nakagami, Hiroshi Koriyama, and Ryuichi Morishita Copyright © 2013 Munehisa Shimamura et al. All rights reserved. Does the Adult Human Ciliary Body Epithelium Contain “True” Retinal Stem Cells? Mon, 28 Oct 2013 17:05:02 +0000 Recent reports of retinal stem cells being present in several locations of the adult eye have sparked great hopes that they may be used to treat the millions of people worldwide who suffer from blindness as a result of retinal disease or injury. A population of proliferative cells derived from the ciliary body epithelium (CE) has been considered one of the prime stem cell candidates, and as such they have received much attention in recent years. However, the true nature of these cells in the adult human eye has still not been fully elucidated, and the stem cell claim has become increasingly controversial in light of new and conflicting reports. In this paper, we will try to answer the question of whether the available evidence is strong enough for the research community to conclude that the adult human CE indeed harbors stem cells. Rebecca Frøen, Erik O. Johnsen, Bjørn Nicolaissen, Andrea Facskó, Goran Petrovski, and Morten C. Moe Copyright © 2013 Rebecca Frøen et al. All rights reserved. Epigenetic Modifications and Diabetic Retinopathy Mon, 28 Oct 2013 14:34:38 +0000 Diabetic retinopathy remains one of the most debilitating chronic complications, but despite extensive research in the field, the exact mechanism(s) responsible for how retina is damaged in diabetes remains ambiguous. Many metabolic pathways have been implicated in its development, and genes associated with these pathways are altered. Diabetic environment also facilitates epigenetics modifications, which can alter the gene expression without permanent changes in DNA sequence. The role of epigenetics in diabetic retinopathy is now an emerging area, and recent work has shown that genes encoding mitochondrial superoxide dismutase (Sod2) and matrix metalloproteinase-9 (MMP-9) are epigenetically modified, activates of epigenetic modification enzymes, histone lysine demethylase 1 (LSD1), and DNA methyltransferase are increased, and the micro RNAs responsible for regulating nuclear transcriptional factor and VEGF are upregulated. With the growing evidence of epigenetic modifications in diabetic retinopathy, better understanding of these modifications has potential to identify novel targets to inhibit this devastating disease. Fortunately, the inhibitors and mimics targeted towards histone modification, DNA methylation, and miRNAs are now being tried for cancer and other chronic diseases, and better understanding of the role of epigenetics in diabetic retinopathy will open the door for their possible use in combating this blinding disease. Renu A. Kowluru, Julia M. Santos, and Manish Mishra Copyright © 2013 Renu A. Kowluru et al. All rights reserved. The forensiX Evidence Collection Tube and Its Impact on DNA Preservation and Recovery Mon, 28 Oct 2013 13:03:01 +0000 Biological samples are vulnerable to degradation from the time they are collected until they are analysed at the laboratory. Biological contaminants, such as bacteria, fungi, and enzymes, as well as environmental factors, such as sunlight, heat, and humidity, can increase the rate of DNA degradation. Currently, DNA samples are normally dried or frozen to limit their degradation prior to their arrival at the laboratory. In this study, the effect of the sample drying rate on DNA preservation was investigated, as well as a comparison between drying and freezing methods. The drying performances of two commercially available DNA collection tools (swab and drying tube) with different drying rates were evaluated. The swabs were used to collect human saliva, placed into the drying tubes, and stored in a controlled environment at 25°C and 60% relative humidity, or frozen at −20°C, for 2 weeks. Swabs that were stored in fast sample drying tubes yielded 95% recoverable DNA, whereas swabs stored in tubes with slower sample drying rates yielded only 12% recoverable DNA; saliva stored in a microtube at −20°C was used as a control. Thus, DNA sampling tools that offer rapid drying can significantly improve the preservation of DNA collected on a swab, increasing the quantity of DNA available for subsequent analysis. Alex M. Garvin, Ralf Holzinger, Florian Berner, Walter Krebs, Bernhard Hostettler, Elges Lardi, Christian Hertli, Roy Quartermaine, and Christoph Stamm Copyright © 2013 Alex M. Garvin et al. All rights reserved. A Flow Cytometric Analysis of Vitreous Inflammatory Cells in Patients with Proliferative Diabetic Retinopathy Sun, 27 Oct 2013 14:46:04 +0000 The purpose of this study was to investigate inflammatory cells in vitreous from patients with proliferative diabetic retinopathy (PDR) using flow cytometric analysis. Twenty-eight patients with PDR requiring vitrectomy because of macular traction or tractional retinal detachment were enrolled in the study (), and 6 patients with macular hole (MH) formed the control group. Samples of vitreous and peripheral venous blood were obtained at the beginning of vitrectomy. T lymphocytes were found in vitreous from patients with PDR, and CD4/CD8 ratio was higher in vitreous (median 4.3) compared to blood (median 1.9; ). No B lymphocytes were detected in vitreous. The percentage of histiocytes/macrophages was significantly higher in vitreous (median 62.1) in comparison with blood (median 5.5; ). No lymphocytes were detected in vitreous of the control group. There were more T lymphocytes in vitreous from patients with active PDR. No association between cells in the vitreous and visual acuity improvement after surgery was found. In conclusion, T lymphocytes are found in vitreous from patients with PDR and reflect the activity of PDR but do not seem to predict visual prognosis. Higher CD4/CD8 ratio in vitreous compared to blood from patients with PDR is consistent with local inflammatory response in PDR. Mojca Urbančič, Veronika Kloboves Prevodnik, Daniel Petrovič, and Mojca Globočnik Petrovič Copyright © 2013 Mojca Urbančič et al. All rights reserved. Screening of 50 Cypriot Patients with Autism Spectrum Disorders or Autistic Features Using 400K Custom Array-CGH Thu, 24 Oct 2013 15:37:55 +0000 Autism spectrum disorders (ASDs) comprise a distinct entity of neurodevelopmental disorders with a strong genetic component. Despite the identification of several candidate genes and causative genomic copy number variations (CNVs), the majority of ASD cases still remain unresolved. We have applied microarray-based comparative genomic hybridization (array-CGH) using Agilent 400K custom array in the first Cyprus population screening for identification of ASD-associated CNVs. A cohort of 50 ASD patients (G1), their parents (G2), 50 ethnically matched normal controls (G3), and 80 normal individuals having children with various developmental and neurological conditions (G4) were tested. As a result, 14 patients were found to carry 20 potentially causative aberrations, two of which were de novo. Comparison of the four population groups revealed an increased rate of rare disease-associated variants in normal parents of children with autism. The above data provided additional evidence, supporting the complexity of ASD aetiology in comparison to other developmental disorders involving cognitive impairment. Furthermore, we have demonstrated the rationale of a more targeted approach combining accurate clinical description with high-resolution population-oriented genomic screening for defining the role of CNVs in autism and identifying meaningful associations on the molecular level. Ludmila Kousoulidou, Maria Moutafi, Paola Nicolaides, Stavros Hadjiloizou, Christos Christofi, Anna Paradesiotou, Violetta Anastasiadou, Carolina Sismani, and Philippos C. Patsalis Copyright © 2013 Ludmila Kousoulidou et al. All rights reserved. Association Study of Estrogen Receptor Alpha Gene Polymorphisms with Spontaneous Abortion: Is This a Possible Reason for Unexplained Spontaneous Abortion? Sun, 20 Oct 2013 14:43:31 +0000 Estrogen plays a crucial role in fetal and placental development through estrogen receptors. Association of estrogen receptor alpha gene (ESR1) polymorphisms with spontaneous abortion has been shown in some studies. Our main goal was to study the potential association of spontaneous abortion with the ESR1 gene variations (PvuII and XbaI) in fetal tissue. Totally, 161 samples were recruited including 80 samples of formalin-fixed paraffin-embedded fetal tissue from spontaneous abortion and 81 samples of normal term placental tissue. The restriction fragment length polymorphism (RFLP) method was performed for genotyping the rs2234693 (A/G XbaI) and rs9340799 (T/C PvuII) single nucleotide polymorphisms located in intron 1 of ESR1. The results have been confirmed by DNA sequencing analysis. The different genotypes distribution was detected in two study groups. Haplotype analysis indicated that ppxx is protective genotype against spontaneous abortion (P = 0.01). In conclusion, the potential role of ESR1 genetic variation in spontaneous abortion might be valuable in high-risk subjects, and that needs to be confirmed with future studies. Negin Anousha, Arash Hossein-Nezhad, Firouzeh Biramijamal, Ali Rahmani, Zhila Maghbooli, Elahe Aghababaei, and Shahram Nemati Copyright © 2013 Negin Anousha et al. All rights reserved. SNP rs2073618 of the Osteoprotegerin Gene Is Associated with Diabetic Retinopathy in Slovenian Patients with Type 2 Diabetes Sun, 20 Oct 2013 09:28:30 +0000 Recent studies indicate that osteoprotegerin (OPG) acts as an important regulatory molecule in the vasculature. Also, a strong association was observed between circulation OPG and microvascular complication. By considering the possible role of OPG in diabetic retinopathy (DR) we examined two of the most studied polymorphisms of the OPG genes rs2073618 (located in exon I) and rs3134069 (located in the promoter region) and their relation to DR in Slovenian patients with type 2 diabetes. Logistic regression analysis demonstrated that the carriers of the CC genotype had a 2.2 higher risk for DR than those with either the CG genotype or the GG genotype (codominant model for rs2073618). Furthermore, the combined effect of single nucleotide polymorphisms (SNPs) rs2073618 and rs3134069 on the DR was stronger than that of each SNP alone. The odds ratio (OR) for individuals with CC genotype (rs2073618) and AA genotype (rs3134069) compared with carriers of CG/GG (rs2073618) + AA (rs3134069) was 2.54 (95% CI = 1.26–5.13, ). To conclude, these results indicate that SNPs in the OPG gene may be implicated in the pathogenesis of DR. Sara Mankoč Ramuš, Tina Kumše, Mojca Globočnik Petrovič, Daniel Petrovič, and Ines Cilenšek Copyright © 2013 Sara Mankoč Ramuš et al. All rights reserved. Lymphedema and Therapeutic Lymphangiogenesis Wed, 09 Oct 2013 10:49:51 +0000 Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return and swelling of the extremities. Lymphedema is divided into primary and secondary forms based on the underlying etiology. Despite substantial advances in both surgical and conservative techniques, therapeutic options for the management of lymphedema are limited. Although rarely lethal, lymphedema is a disfiguring and disabling condition with an associated decrease in the quality of life. The recent impressive expansion of knowledge on the molecular mechanisms governing lymphangiogenesis provides new possibilities for the treatment of lymphedema. This review highlights the lymphatic biology, the pathophysiology of lymphedema, and the therapeutic lymphangiogenesis using hepatocyte growth factor. Yukihiro Saito, Hironori Nakagami, Yasufumi Kaneda, and Ryuichi Morishita Copyright © 2013 Yukihiro Saito et al. All rights reserved. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma Mon, 07 Oct 2013 17:52:46 +0000 Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe. Elena Ordoñez, Laura Rueda, M. Paz Cañadas, Carme Fuster, and Vincenzo Cirigliano Copyright © 2013 Elena Ordoñez et al. All rights reserved. Functional and Molecular Characterization of Ex Vivo Cultured Epiretinal Membrane Cells from Human Proliferative Diabetic Retinopathy Tue, 01 Oct 2013 08:57:34 +0000 Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNFα activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFRβ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNFα activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo. Zoltán Veréb, Xhevat Lumi, Sofija Andjelic, Mojca Globocnik-Petrovic, Mojca Urbancic, Marko Hawlina, Andrea Facskó, and Goran Petrovski Copyright © 2013 Zoltán Veréb et al. All rights reserved. Familial Aggregation of Metabolic Syndrome Indicators in Portuguese Families Sat, 21 Sep 2013 15:03:06 +0000 Background and Aims. Family studies are well suited to investigate the genetic architecture underlying the metabolic syndrome (MetS). The purposes of this paper were (i) to estimate heritabilities for each of the MetS indicators, and (ii) to test the significance of familial intratrait and cross-trait correlations in MetS markers. Methods and Results. This study included 1,363 individuals from 515 Portuguese families in which five MetS components, including waist circumference (WC), blood pressure (BP), HDL-cholesterol, triglycerides (TG), and glucose (GLU), were measured. Intratrait and cross-trait familial correlations of these five components were estimated using Generalized Estimating Equations. Each MetS component was significantly heritable ( ranged from 0.12 to 0.60) and exhibited strong familial resemblance with correlations between biological relatives of similar magnitude to those observed between spouses. With respect to cross-trait correlations, familial resemblance was very weak except for the HDL-TG pair. Conclusions. The present findings confirm the idea of familial aggregation in MetS traits. Spousal correlations were, in general, of the same magnitude as the biological relatives' correlations suggesting that most of the phenotypic variance in MetS traits could be explained by shared environment. D. M. Santos, P. T. Katzmarzyk, D.-A. Trégouet, T. N. Gomes, F. K. Santos, and J. A. Maia Copyright © 2013 D. M. Santos et al. All rights reserved. Catecholaminergic Gene Variants: Contribution in ADHD and Associated Comorbid Attributes in the Eastern Indian Probands Thu, 19 Sep 2013 16:51:09 +0000 Contribution of genes in attention deficit hyperactivity disorder (ADHD) has been explored in various populations, and several genes were speculated to contribute small but additive effects. We have assessed variants in four genes, DDC (rs3837091 and rs3735273), DRD2 (rs1800496, rs1801028, and rs1799732), DRD4 (rs4646984 and rs4646983), and COMT (rs165599 and rs740603) in Indian ADHD subjects with comorbid attributes. Cases were recruited following the Diagnostic and Statistical Manual for Mental Disorders-IV-TR after obtaining informed written consent. DNA isolated from peripheral blood leukocytes of ADHD probands (), their parents (), and ethnically matched controls () was used for genotyping followed by population- and family-based analyses by the UNPHASED program. DRD4 sites showed significant difference in allelic frequencies by case-control analysis, while DDC and COMT exhibited bias in familial transmission (). rs3837091 “AGAG,” rs3735273 “A,” rs1799732 “C,” rs740603 “G,” rs165599 “G” and single repeat alleles of rs4646984/rs4646983 showed positive correlation with co-morbid characteristics (). Multi dimensionality reduction analysis of case-control data revealed significant interactive effects of all four genes (), while family-based data showed interaction between DDC and DRD2 (). This first study on these gene variants in Indo-Caucasoid ADHD probands and associated co-morbid conditions indicates altered dopaminergic neurotransmission in ADHD. Paramita Ghosh, Kanyakumarika Sarkar, Nipa Bhaduri, Anirban Ray, Keka Sarkar, Swagata Sinha, and Kanchan Mukhopadhyay Copyright © 2013 Paramita Ghosh et al. All rights reserved. DNA Damage and Oxidative Stress in Human Disease Thu, 19 Sep 2013 09:27:38 +0000 Sharbel Weidner Maluf, Norma Possa Marroni, Vanina D. Heuser, and Daniel Prá Copyright © 2013 Sharbel Weidner Maluf et al. All rights reserved. Superoxide-Dismutase Deficient Mutants in Common Beans (Phaseolus vulgaris L.): Genetic Control, Differential Expressions of Isozymes, and Sensitivity to Arsenic Wed, 28 Aug 2013 15:56:12 +0000 Two common bean (Phaseolus vulgaris L.) mutants, sodPv 1 and sodPv 2, exhibiting foliar superoxide dismutase (SOD) activity of only 25% and 40% of their mother control (MC) cv. VL 63 were isolated in EMS-mutagenized (0.15%, 8 h) M2 progeny. Native-PAGE analysis revealed occurrence of Mn SOD, Fe SOD, Cu/Zn SOD I and Cu/Zn SOD II isozymes in MC, while Fe SOD, and Mn SOD were not formed in sodPv 1 and sodPv 2 leaves, respectively. In-gel activity of individual isozymes differed significantly among the parents. SOD deficiency is inherited as recessive mutations, controlled by two different nonallelic loci. Gene expressions using qRT PCR confirmed higher expressions of Cu/Zn SOD transcripts in both mutants and the absence of Fe SOD in sodPv 1 and Mn SOD in sodPv 2. In 50 M arsenic, Cu/Zn SODs genes were further upregulated but other isoforms downregulated in the two mutants, maintaining SOD activity in its control level. In an F2 double mutants of sodPv 1 × sodPv 2, no Fe SOD, and Mn SOD expressions were detectable, while both Cu/Zn SODs are down-regulated and arsenic-induced leaf necrosis appeared. In contrast to both mutants, ROS-imaging study revealed overaccumulation of both superoxides and H2O2 in leaves of double mutant. Dibyendu Talukdar and Tulika Talukdar Copyright © 2013 Dibyendu Talukdar and Tulika Talukdar. All rights reserved. Genetic Susceptibility to Chagas Disease: An Overview about the Infection and about the Association between Disease and the Immune Response Genes Wed, 28 Aug 2013 14:56:02 +0000 Chagas disease, which is caused by the flagellate parasite Trypanosoma cruzi, affects 8–10 million people in Latin America. The disease is endemic and is characterised by acute and chronic phases that develop in the indeterminate, cardiac, and/or gastrointestinal forms. The immune response during human T. cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Polymorphisms in genes involved in the innate and specific immune response are being widely studied in order to clarify their possible role in the occurrence or severity of disease. Here we review the role of classic and nonclassic MHC, KIR, and cytokine host genetic factors on the infection by T. cruzi and the clinical course of Chagas disease. Christiane Maria Ayo, Márcia Machado de Oliveira Dalalio, Jeane Eliete Laguila Visentainer, Pâmela Guimarães Reis, Emília Ângela Sippert, Luciana Ribeiro Jarduli, Hugo Vicentin Alves, and Ana Maria Sell Copyright © 2013 Christiane Maria Ayo et al. All rights reserved. Candidate Genes for Proliferative Diabetic Retinopathy Tue, 27 Aug 2013 13:41:05 +0000 Several candidate genes have been so far implicated in the pathogenesis of proliferative diabetic retinopathy (PDR) in subjects with type 2 diabetes. Since the principal pathogenetic mechanisms for diabetic retinopathy (DR) and PDR are different, the main pathogenetic mechanism in DR is increased vascular permeability, whereas in PDR the crucial pathogenetic mechanisms are fibrosis and neoangiogenesis. Due to that fact, different candidate genes are expected to be involved in the development of either DR or PDR. None of the candidate genes, however, can be fully and solely responsible for the development of PDR and for DR progression into PDR. Epigenetic mechanisms are expected to be involved in the pathogenesis of PDR as well. Gene polymorphisms responsible for PDR and epigenetic mechanisms responsible for PDR are reviewed in this paper. Daniel Petrovič Copyright © 2013 Daniel Petrovič. All rights reserved. Disease Modifying Therapies for Alzheimer's Disease Targeting Aβ Oligomers: Implications for Therapeutic Mechanisms Mon, 26 Aug 2013 08:58:48 +0000 Several lines of evidence indicate that amyloid β (Aβ), particularly Aβ oligomers (AβOs), plays a causative role in Alzheimer's disease. However, the mechanisms underlying the action of an anti-AβO antibody to clarify the toxic action of AβOs remain elusive. Here, we showed that the anti-AβO antibody (monoclonal 72D9) can modify the Aβ aggregation pathway. We also found that 72D9 directly sequesters both extracellular and intraneuronal AβOs in a nontoxic state. Thus, therapeutic intervention targeting AβOs is a promising strategy for neuronal protection in Alzheimer's disease. Etsuro Matsubara, Ayumi Takamura, Yasuhide Okamoto, Hideto Oono, Takashi Nakata, Yasuhito Wakasaya, Takeshi Kawarabayashi, and Mikio Shoji Copyright © 2013 Etsuro Matsubara et al. All rights reserved. Association between Interleukin-1 Gene Single Nucleotide Polymorphisms and Ischemic Stroke Classified by TOAST Criteria in the Han Population of Northern China Mon, 26 Aug 2013 08:37:10 +0000 Increasing evidence suggests that IL-1β (C-511T) and IL-1α (C-889T) genes polymorphisms are associated with the susceptibility to cardiocerebral vascular disease. In this paper, we investigated the relationships between these polymorphisms and the risk of ischemic stroke (IS) classified by TOAST criteria in the north Chinese Han population. 440 cases of IS and 486 age- and gender-matched controls of Chinese Han population were enrolled. Association study showed that the TT genotype and T allele of IL-1α-889 C/T were significantly associated with IS of a large artery atherosclerosis (LAA) (TT: OR = 2.01, 95% CI = 1.34–3.0, and ; T: OR = 1.44, 95% CI = 1.18–1.78, and ). However, there was no significant difference in the distribution of IL-1α-889 C/T genotypes and allele frequencies between the two subgroups (small-artery occlusion (SVD) and cardioembolism (CE)) of IS and control groups. No significant association was also found between the IL-1β-511 TT genotype and T allele (TT: OR = 0.79, 95% CI = 0.56–1.11, and ; T: OR = 0.83, 95% CI = 0.68–1.01, and ) and IS as well as subgroups of CE and SVD. Our results implicated that IL-1α-889 C/T gene polymorphism might be associated with the susceptibility to IS, especially to IS with LAA, in a north Chinese Han population. Zheng Zhang, Li-Jun Liu, Chen Zhang, and Yong-Peng Yu Copyright © 2013 Zheng Zhang et al. All rights reserved. Meta-Analysis of Cytokine Gene Polymorphisms and Outcome of Heart Transplantation Tue, 20 Aug 2013 10:09:41 +0000 We performed a systematic review and meta-analysis with the aim of assessing the association between cytokine gene polymorphisms and graft rejection in heart transplantation. We identified relevant studies from Medline and Embase using PubMed and Ovid search engines, respectively. Allele frequencies and allele and genotypic effects were pooled. Heterogeneity and publication bias were explored. Four to 5 studies were included in pooling of 3 gene polymorphisms. The prevalences of the minor alleles for TNFα-308, TGFβ1-c10, and TGFβ1-c25 were 0.166 (95% CI: 0.129, 0.203), 0.413 (95% CI: 0.363, 0.462), and 0.082 (95% CI: 0.054, 0.111) in the control groups, respectively. Carrying the A allele for the TNFα-308 had 18% (95% CI of OR: 0.46, 3.01) increased risk, but this was not significant for developing graft rejection than the G allele. Conversely, carrying the minor alleles for both TGFβ1-c10 and c25 had nonsignificantly lower odds of graft rejection than major alleles, with the pooled ORs of 0.87 (95% CI: 0.65, 1.18) and 0.70 (95% CI: 0.40, 1.23), respectively. There was no evidence of publication bias for all poolings. An updated meta-analysis is required when more studies are published to increase the power of detection for the association between these polymorphisms and allograft rejection. Sasitorn Yongcharoen, Sasivimol Rattanasiri, D. Olga McDaniel, Mark McEvoy, Chukiat Viwatwongkaseam, Piangchan Rojanavipart, and Ammarin Thakkinstian Copyright © 2013 Sasitorn Yongcharoen et al. All rights reserved. No Association of IFNG+874T/A SNP and NOS2A-954G/C SNP Variants with Nitric Oxide Radical Serum Levels or Susceptibility to Tuberculosis in a Brazilian Population Subset Sun, 18 Aug 2013 09:58:25 +0000 Tuberculosis (TB) is one of the most common infectious diseases in the world. Mycobacterium tuberculosis infection leads to pulmonary active disease in approximately 5–10% of exposed individuals. Both bacteria- and host-related characteristics influence latent infection and disease. Host genetic predisposition to develop TB may involve multiple genes and their polymorphisms. It was reported previously that interferon gamma (IFN-γ) and nitric oxide synthase 2 (NOS2) are expressed on alveolar macrophages from TB patients and are responsible for bacilli control; thus, we aimed this study at genotyping single nucleotide polymorphisms IFNG+874T/A SNP and NOS2A-954G/C SNP to estimate their role on TB susceptibility and determine whether these polymorphisms influence serum nitrite and production. This case-control study enrolled 172 TB patients and 179 healthy controls. Neither polymorphism was associated with susceptibility to TB. NOS2A-954G/C SNP was not associated with serum levels of nitrite and . These results indicate that variants of IFNG+874T/A SNP and NOS2A-954G/C SNP do not influence TB susceptibility or the secretion of nitric oxide radicals in the study population. Ana Cristina C. S. Leandro, Márcia Andrade Rocha, Andreia Lamoglia-Souza, John L. VandeBerg, Valeria Cavalcanti Rolla, and Maria da Gloria Bonecini-Almeida Copyright © 2013 Ana Cristina C. S. Leandro et al. All rights reserved. A Novel Splicing Mutation of KIT Results in Piebaldism and Auburn Hair Color in a Chinese Family Tue, 13 Aug 2013 09:54:30 +0000 Piebaldism is a rare autosomal dominant disorder of melanocyte development, which is mostly caused by KIT gene. The key characteristics of piebaldism include localized poliosis, congenital leukoderma, and other variable manifestations. The previous study has illustrated that the homogeneous MC1R (a gene which is associated with the hair color) variant (p.I120T) coordinating with KIT mutation may lead to auburn hair color and piebaldism. In this study, we have investigated a Chinese family with piebaldism and auburn hair color; the mutation screening of KIT and MC1R genes identified that only a splicing mutation (c. 2484+1G>A) of KIT gene cosegregated with the auburn hair color and piebaldism. The data of this study and others suggests that the KIT mutation may causes of the auburn hair color in the piebaldism patients. Yong-jia Yang, Rui Zhao, Xin-yu He, Li-ping Li, Ke-wei Wang, Liu Zhao, Ming Tu, Jin-song Tang, Zhi-guo Xie, and Yi-min Zhu Copyright © 2013 Yong-jia Yang et al. All rights reserved. The Influence of Gene-Gene and Gene-Environment Interactions on the Risk of Asbestosis Thu, 25 Jul 2013 14:19:00 +0000 This study investigated the influence of gene-gene and gene-environment interactions on the risk of developing asbestosis. The study comprised 262 cases with asbestosis and 265 controls with no asbestos-related disease previously studied for MnSOD, ECSOD, CAT, GSTT1, GSTM1, GSTP1, and iNOS polymorphisms. Data on cumulative asbestos and smoking were available for all subjects. To assess gene-gene and gene-environmental interactions, logistic regression was used. The associations between MnSOD Ala −9Val polymorphism and the risk of asbestosis and between iNOS genotypes and asbestosis were modified by CAT –262 C > T polymorphism (). A strong interaction was found between GSTM1-null polymorphism and smoking (), iNOS (CCTTT)n polymorphism and smoking (), and between iNOS (CCTTT)n polymorphism and cumulative asbestos exposure (). The findings of this study suggest that the interactions between different genotypes, genotypes and smoking, and between genotypes and asbestos exposure have an important influence on the development of asbestosis and should be seriously considered in future research on occupational/environmental asbestos-related diseases. A. Franko, V. Dolžan, N. Arnerić, and M. Dodič-Fikfak Copyright © 2013 A. Franko et al. All rights reserved. Vitamin C Intake Reduces the Cytotoxicity Associated with Hyperglycemia in Prediabetes and Type 2 Diabetes Thu, 25 Jul 2013 08:42:49 +0000 Hyperglycemia leads to the formation of free radicals and advanced glycation end-products (AGEs). Antioxidants can reduce the level of protein glycation and DNA damage. In this study, we compared the levels of vitamin C intake, which is among the most abundant antioxidants obtained from diet, with the levels of fasting plasma glucose (FPG), glycated hemoglobin (A1C), DNA damage, and cytotoxicity in prediabetic subjects and type 2 diabetic subjects. Our results indicated that there was no significant correlation between FPG or A1C and DNA damage parameters (micronuclei, nucleoplasmic bridges, and nuclear buds). FPG and A1C correlated with necrosis (; and ; , resp.). Vitamin C intake correlated negatively with necrosis and apoptosis (; , and ; , resp.). The lack of a correlation between the FPG and A1C and DNA damage could be explained, at least in part, by the elimination of cells with DNA damage by either necrosis or apoptosis (cytotoxicity). Vitamin C appeared to improve cell survival by reducing cytotoxicity. Therefore, the present results indicate the need for clinical studies to evaluate the effect of low-dose vitamin C supplementation in type 2 diabetes. Silvia Isabel Rech Franke, Luiza Louzada Müller, Maria Carolina Santos, Arcênio Fishborn, Liziane Hermes, Patrícia Molz, Camila Schreiner Pereira, Francisca Maria Assmann Wichmann, Jorge André Horta, Sharbel Weidner Maluf, and Daniel Prá Copyright © 2013 Silvia Isabel Rech Franke et al. All rights reserved. Laboratory Genetic Testing in Clinical Practice Sun, 14 Jul 2013 07:52:49 +0000 Ozgur Cogulu, Yasemin Alanay, and Gokce A. Toruner Copyright © 2013 Ozgur Cogulu et al. All rights reserved. Association of Toll-Like Receptor 4 Polymorphisms with Diabetic Foot Ulcers and Application of Artificial Neural Network in DFU Risk Assessment in Type 2 Diabetes Patients Thu, 11 Jul 2013 13:42:58 +0000 The Toll-Like receptor 4 (TLR4) plays an important role in immunity, tissue repair, and regeneration. The objective of the present work was to evaluate the association of TLR4 single nucleotide polymorphisms (SNPs) rs4986790, rs4986791, rs11536858 (merged into rs10759931), rs1927911, and rs1927914 with increased diabetic foot ulcer (DFU) risk in patients with type 2 diabetes mellitus (T2DM). PCR-RFLP was used for genotyping TLR4 SNPs in 125 T2DM patients with DFU and 130 controls. The haplotypes and linkage disequilibrium between the SNPs were determined using Haploview software. Multivariate linear regression (MLR) and artificial neural network (ANN) modeling was done to observe their predictability for the risk of DFU in T2DM patients. Risk genotypes of all SNPs except rs1927914 were significantly associated with DFU. Haplotype ACATC ( value = ) showed strong association with DFU risk. Two haplotypes ATATC ( value = 0.0119) and ATGTT ( value = 0.0087) were found to be protective against DFU. In conclusion TLR4 SNPs and their haplotypes may increase the risk of impairment of wound healing in T2DM patients. ANN model (83%) is found to be better than the MLR model (76%) and can be used as a tool for the DFU risk assessment in T2DM patients. Kanhaiya Singh, Vivek Kumar Singh, Neeraj K. Agrawal, Sanjeev K. Gupta, and Kiran Singh Copyright © 2013 Kanhaiya Singh et al. All rights reserved. Chromosome Instability and Oxidative Stress Markers in Patients with Ataxia Telangiectasia and Their Parents Tue, 09 Jul 2013 16:04:24 +0000 Ataxia telangiectasia (AT) is a rare neurodegenerative disorder, inherited in an autosomal recessive manner. Total blood samples were collected from 20 patients with AT, 13 parents of patients, and 17 healthy volunteers. This study aimed at evaluating the frequency of chromosomal breaks in spontaneous cultures, induced by bleomycin and ionizing radiation, and further evaluated the rates of oxidative stress in AT patients and in their parents, compared to a control group. Three cell cultures were performed to each individual: the first culture did not receive induction to chromosomal instability, the second was exposed to bleomycin, and the last culture was exposed to ionizing radiation. To evaluate the rates of oxidative stress, the markers superoxide dismutase (SOD), catalase (CAT), and thiobarbituric acid (TBARS) were utilized. Significant differences were observed between the three kinds of culture treatments (spontaneous, bleomycin, and radiation induced) and the breaks and chromosomal aberrations in the different groups. The oxidative stress showed no significant differences between the markers. This study showed that techniques of chromosomal instability after the induction of ionizing radiation and bleomycin are efficient in the identification of syndrome patients, with the ionizing radiation being the most effective. Luciane Bitelo Ludwig, Victor Hugo Valiati, Roberta Passos Palazzo, Laura Bannach Jardim, Darlan Pase da Rosa, Silvia Bona, Graziela Rodrigues, Norma Possa Marroni, Daniel Prá, and Sharbel Weidner Maluf Copyright © 2013 Luciane Bitelo Ludwig et al. All rights reserved. Role of HLA, KIR, MICA, and Cytokines Genes in Leprosy Mon, 08 Jul 2013 15:22:21 +0000 Many genes including HLA, KIR, and MICA genes, as well as polymorphisms in cytokines have been investigated for their role in infectious disease. HLA alleles may influence not only susceptibility or resistance to leprosy, but also the course of the disease. Some combinations of HLA and KIR may result in negative as well as positive interactions between NK cells and infected host cells with M. leprae, resulting in activation or inhibition of NK cells and, consequently, in death of bacillus. In addition, studies have demonstrated the influence of MICA genes in the pathogenesis of leprosy. Specifically, they may play a role in the interaction between NK cells and infected cells. Finally, pro- and anti-inflammatory cytokines have been influencing the clinical course of leprosy. Data from a wide variety of sources support the existence of genetic factors influencing the leprosy pathogenesis. These sources include twin studies, segregation analyses, family-based linkage and association studies, candidate gene association studies, and, most recently, genome-wide association studies (GWAS). The purpose of this brief review was to highlight the importance of some immune response genes and their correlation with the clinical forms of leprosy, as well as their implications for disease resistance and susceptibility. Luciana Ribeiro Jarduli, Ana Maria Sell, Pâmela Guimarães Reis, Emília Ângela Sippert, Christiane Maria Ayo, Priscila Saamara Mazini, Hugo Vicentin Alves, Jorge Juarez Vieira Teixeira, and Jeane Eliete Laguila Visentainer Copyright © 2013 Luciana Ribeiro Jarduli et al. All rights reserved. The Association of the Immune Response Genes to Human Papillomavirus-Related Cervical Disease in a Brazilian Population Mon, 08 Jul 2013 13:40:18 +0000 The genetic variability of the host contributes to the risk of human papillomavirus (HPV)-related cervical disease. Immune response genes to HPV must be investigated to define patients with the highest risk of developing malignant disease. The aim of this study was to investigate the association of polymorphic immune response genes, namely KIR, HLA class I and II, and single-nucleotide polymorphisms (SNPs) of cytokines with HPV-related cervical disease. We selected 79 non-related, admixed Brazilian women from the state of Paraná, southern region of Brazil, who were infected with high carcinogenic risk HPV and present cervical intraepithelial neoplasia grade 3 (CIN3), and 150 HPV-negative women from the same region matched for ethnicity. KIR genes were genotyped using an in-house PCR-SSP. HLA alleles were typed using a reverse sequence-specific oligonucleotide technique. SNPs of TNF −308G>A, IL6 −174G>C, IFNG +874T>A, TGFB1 +869T>C +915G>C, and IL10 −592C>A −819C>T −1082G>A were evaluated using PCR-SSP. The KIR genes were not associated with HPV, although some pairs of i(inhibitory)KIR-ligands occurred more frequently in patients, supporting a role for NK in detrimental chronic inflammatory and carcinogenesis. Some HLA haplotypes were associated with HPV. The associations of INFG and IL10 SNPs potentially reflect impaired or invalid responses in advanced lesions. Amanda Vansan Marangon, Gláucia Andreia Soares Guelsin, Jeane Eliete Laguila Visentainer, Sueli Donizete Borelli, Maria Angélica Ehara Watanabe, Márcia Edilaine Lopes Consolaro, Katiany Rizzieri Caleffi-Ferracioli, Cristiane Conceição Chagas Rudnick, and Ana Maria Sell Copyright © 2013 Amanda Vansan Marangon et al. All rights reserved. Haplogroup T Is an Obesity Risk Factor: Mitochondrial DNA Haplotyping in a Morbid Obese Population from Southern Italy Tue, 02 Jul 2013 11:21:24 +0000 Mitochondrial DNA (mtDNA) haplogroups have been associated with the expression of mitochondrial-related diseases and with metabolic alterations, but their role has not yet been investigated in morbid obese Caucasian subjects. Therefore, we investigated the association between mitochondrial haplogroups and morbid obesity in patients from southern Italy. The mtDNA D-loop of morbid obese patients (; BMI > 40 kg/m2) and controls (; BMI < 25 kg/m2) was sequenced to determine the mtDNA haplogroups. The T and J haplogroup frequencies were higher and lower, respectively, in obese subjects than in controls. Women bearing haplogroup T or J had twice or half the risk of obesity. Binomial logistic regression analysis showed that haplogroup T and systolic blood pressure are risk factors for a high degree of morbid obesity, namely, BMI > 45 kg/m2 and in fact together account for 8% of the BMI. In conclusion, our finding that haplogroup T increases the risk of obesity by about two-fold, suggests that, besides nuclear genome variations and environmental factors, the T haplogroup plays a role in morbid obesity in our study population from southern Italy. Carmela Nardelli, Giuseppe Labruna, Rosario Liguori, Cristina Mazzaccara, Maddalena Ferrigno, Valentina Capobianco, Massimo Pezzuti, Giuseppe Castaldo, Eduardo Farinaro, Franco Contaldo, Pasqualina Buono, Lucia Sacchetti, and Fabrizio Pasanisi Copyright © 2013 Carmela Nardelli et al. All rights reserved. Single Nucleotide Polymorphism in the Promoter of the Human Interleukin-13 Gene Is Associated with Asthma in Malaysian Adults Tue, 25 Jun 2013 09:14:01 +0000 Asthma susceptibility genes are mapped to a region on human chromosome 5q31-q33, which contains a cluster of proinflammatory cytokine genes such as interleukin-13 (IL-13), which is associated with asthma. This study investigated the allele frequencies of two single nucleotide polymorphisms (SNPs) (−1111C>T and 4257C>A) in the IL-13 gene between asthmatics and healthy volunteers as well as the relationship between these SNPs and IL-13 production. DNA extracted from buffy coat of asthmatic and control subjects was genotyped using the PCR-RFLP method. Amount of IL-13 produced by mitogen-stimulated peripheral blood leucocytes PBLs (PBLs) was determined by ELISA. The frequencies of the −1111C and 4257G wild-type alleles were 0.52 and 0.55 in asthmatics and were 0.67 and 0.56 in controls. A significant () association was found between genotype and allele frequencies of SNP at position −1111C>T between asthmatic and control groups (OR, 1.810; 95% CI = 1.184 to 2.767; ). The mitogen-stimulated PBLs from asthmatics produced higher amounts of IL-13 production (). The 4257GA heterozygous and 4257AA homozygous mutant alleles were associated with higher IL-13 production in asthmatics (). Our results show that the −1111T mutant allele are associated with asthma and the 4257A mutant alleles are associated with elevated IL-13 production. Ammu Kutty Radhakrishnan, Vijaya Lechimi Raj, Lee-Keng Tan, and Chong-Kin Liam Copyright © 2013 Ammu Kutty Radhakrishnan et al. All rights reserved. The Role of Cytidine Deaminases on Innate Immune Responses against Human Viral Infections Tue, 25 Jun 2013 08:32:47 +0000 The APOBEC family of proteins comprises deaminase enzymes that edit DNA and/or RNA sequences. The APOBEC3 subgroup plays an important role on the innate immune system, acting on host defense against exogenous viruses and endogenous retroelements. The role of APOBEC3 proteins in the inhibition of viral infection was firstly described for HIV-1. However, in the past few years many studies have also shown evidence of APOBEC3 action on other viruses associated with human diseases, including HTLV, HCV, HBV, HPV, HSV-1, and EBV. APOBEC3 inhibits these viruses through a series of editing-dependent and independent mechanisms. Many viruses have evolved mechanisms to counteract APOBEC effects, and strategies that enhance APOBEC3 activity constitute a new approach for antiviral drug development. On the other hand, novel evidence that editing by APOBEC3 constitutes a source for viral genetic diversification and evolution has emerged. Furthermore, a possible role in cancer development has been shown for these host enzymes. Therefore, understanding the role of deaminases on the immune response against infectious agents, as well as their role in human disease, has become pivotal. This review summarizes the state-of-the-art knowledge of the impact of APOBEC enzymes on human viruses of distinct families and harboring disparate replication strategies. Valdimara C. Vieira and Marcelo A. Soares Copyright © 2013 Valdimara C. Vieira and Marcelo A. Soares. All rights reserved. HLA-G/C, miRNAs, and Their Role in HIV Infection and Replication Thu, 13 Jun 2013 12:01:10 +0000 In recent years, a number of different mechanisms regulating gene expressions, either in normal or in pathological conditions, have been discovered. This review aims to highlight some of the regulatory pathways involved during the HIV-1 infection and disease progression, focusing on the novel discovered microRNAs (miRNAs) and their relation with immune system’s agents. Human leukocyte antigen (HLA) family of proteins plays a key role because it is a crucial modulator of the immune response; here we will examine recent findings, centering especially on HLA-C and -G, novel players lately discovered to engage in modulation of immune system. We hope to provide novel perspectives useful to find out original therapeutic roads against HIV-1 infection and AIDS progression. Fulvio Celsi, Eulalia Catamo, Giulio Kleiner, Paola Maura Tricarico, Josef Vuch, and Sergio Crovella Copyright © 2013 Fulvio Celsi et al. All rights reserved. The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability Mon, 27 May 2013 14:54:11 +0000 Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed. Ana Lúcia Vargas Arigony, Iuri Marques de Oliveira, Miriana Machado, Diana Lilian Bordin, Lothar Bergter, Daniel Prá, and João Antonio Pêgas Henriques Copyright © 2013 Ana Lúcia Vargas Arigony et al. All rights reserved. Translational Potential into Health Care of Basic Genomic and Genetic Findings for Human Immunodeficiency Virus, Chlamydia trachomatis, and Human Papilloma Virus Thu, 23 May 2013 15:09:30 +0000 Individual variations in susceptibility to an infection as well as in the clinical course of the infection can be explained by pathogen related factors, environmental factors, and host genetic differences. In this paper we review the state-of-the-art basic host genomic and genetic findings’ translational potential of human immunodeficiency virus (HIV), Chlamydia trachomatis (CT), and Human Papilloma Virus (HPV) into applications in public health, especially in diagnosis, treatment, and prevention of complications of these infectious diseases. There is a significant amount of knowledge about genetic variants having a positive or negative influence on the course and outcome of HIV infection. In the field of Chlamydia trachomatis, genomic advances hold the promise of a more accurate subfertility prediction test based on single nucleotide polymorphisms (SNPs). In HPV research, recent developments in early diagnosis of infection-induced cervical cancer are based on methylation tests. Indeed, triage based on methylation markers might be a step forward in a more effective stratification of women at risk for cervical cancer. Our review found an imbalance between the number of host genetic variants with a role in modulating the immune response and the number of practical genomic applications developed thanks to this knowledge. Jelena Malogajski, Ivan Brankovic, Stephan P. Verweij, Elena Ambrosino, Michiel A. van Agtmael, Angela Brand, Sander Ouburg, and Servaas A. Morré Copyright © 2013 Jelena Malogajski et al. All rights reserved. Oxidative Stress in the Pathogenesis of Colorectal Cancer: Cause or Consequence? Tue, 14 May 2013 15:24:58 +0000 There is a growing support for the concept that reactive oxygen species, which are known to be implicated in a range of diseases, may be important progenitors in carcinogenesis, including colorectal cancer (CRC). CRC is one of the most common cancers worldwide, with the highest incidence rates in western countries. Sporadic human CRC may be attributable to various environmental and lifestyle factors, such as dietary habits, obesity, and physical inactivity. In the last decades, association between oxidative stress and CRC has been intensively studied. Recently, numerous genetic and lifestyle factors that can affect an individual's ability to respond to oxidative stress have been identified. The aim of this paper is to review evidence linking oxidative stress to CRC and to provide essential background information for accurate interpretation of future research on oxidative stress and CRC risk. Brief introduction of different endogenous and exogenous factors that may influence oxidative status and modulate the ability of gut epithelial cells to cope with damaging metabolic challenges is also provided. Martina Perše Copyright © 2013 Martina Perše. All rights reserved. Identification and Characterization of DM1 Patients by a New Diagnostic Certified Assay: Neuromuscular and Cardiac Assessments Thu, 09 May 2013 11:45:36 +0000 The expansion of the specific trinucleotide sequence, [CTG], is the molecular pathological mechanism responsible for the clinical manifestations of DM1. Many studies have described different molecular genetic techniques to detect DM1, but as yet there is no data on the analytical performances of techniques used so far in this disease. We therefore developed and validated a molecular method, “Myotonic Dystrophy SB kit,” to better characterize our DM1 population. 113 patients were examined: 20 DM1-positive, 11 DM1/DM2-negative, and13 DM1-negative/DM2-positive, who had a previous molecular diagnosis, while 69 were new cases. This assay correctly identified 113/113 patients, and all were confirmed by different homemade assays. Comparative analysis revealed that the sensitivity and the specificity of the new kit were very high (>99%). Same results were obtained using several extraction procedures and different concentrations of DNA. The distribution of pathologic alleles showed a prevalence of the “classical” form, while of the 96 nonexpanded alleles 19 different allelic types were observed. Cardiac and neuromuscular parameters were used to clinically characterize our patients and support the new genetic analysis. Our findings suggest that this assay appears to be a very robust and reliable molecular test, showing high reproducibility and giving an unambiguous interpretation of results. Rea Valaperta, Valeria Sansone, Fortunata Lombardi, Chiara Verdelli, Alessio Colombo, Massimiliano Valisi, Elisa Brigonzi, Elena Costa, and Giovanni Meola Copyright © 2013 Rea Valaperta et al. All rights reserved. Genetic and Functional Profiling of Crohn's Disease: Autophagy Mechanism and Susceptibility to Infectious Diseases Wed, 08 May 2013 15:01:43 +0000 Crohn's disease is a complex disease in which genome, microbiome, and environment interact to produce the immunological background of the disease. Disease in childhood is more extensive and characterized by a rapid progression, leading to severe repercussions in the course of the disorder. Several genetic variations have been associated with an increased risk of developing the disease and most of these are also implicated in other autoimmune disorders. The gut has many tiers of defense against incursion by luminal microbes, including the epithelial barrier and the innate and adaptive immune responses. Moreover, recent evidence shows that bacterial and viral infections, as well as inflammasome genes and genes involved in the autophagy process, are implicated in Crohn's disease pathogenesis. The aim of this review is to establish how much the diagnostic system can improve, thus increasing the success of Crohn's disease diagnosis. The major expectation for the near future is to be able to anticipate the possible consequences of the disease already in childhood, thus preventing associated complications, and to choose the best treatment for each patient. Annalisa Marcuzzi, Anna Monica Bianco, Martina Girardelli, Alberto Tommasini, Stefano Martelossi, Lorenzo Monasta, and Sergio Crovella Copyright © 2013 Annalisa Marcuzzi et al. All rights reserved. Ultradeep Pyrosequencing of Hepatitis C Virus Hypervariable Region 1 in Quasispecies Analysis Sun, 28 Apr 2013 08:35:08 +0000 Genetic variability of hepatitis C virus (HCV) determines pathogenesis of infection, including viral persistence and resistance to treatment. The aim of the present study was to characterize HCV genetic heterogeneity within a hypervariable region 1 (HVR1) of a chronically infected patient by ultradeep 454 sequencing strategy. Three independent sequencing error correction methods were applied. First correction method (Method I) implemented cut-off for genetic variants present in less than 1%. In the second method (Method II), a condition to call a variant was bidirectional coverage of sequencing reads. Third method (Method III) used Short Read Assembly into Haplotypes (ShoRAH) program. After the application of these three different algorithms, HVR1 population consisted of 8, 40, and 186 genetic haplotypes. The most sensitive method was ShoRAH, allowing to reconstruct haplotypes constituting as little as 0.013% of the population. The most abundant genetic variant constituted only 10.5%. Seventeen haplotypes were present in a frequency above 1%, and there was wide dispersion of the population into very sparse haplotypes. Our results indicate that HCV HVR1 heterogeneity and quasispecies population structure may be reconstructed by ultradeep sequencing. However, credible analysis requires proper reconstruction methods, which would distinguish sequencing error from real variability in vivo. Kamila Caraballo Cortés, Osvaldo Zagordi, Tomasz Laskus, Rafał Płoski, Iwona Bukowska-Ośko, Agnieszka Pawełczyk, Hanna Berak, and Marek Radkowski Copyright © 2013 Kamila Caraballo Cortés et al. All rights reserved. Genetic Dissection of New Genotypes of Drumstick Tree (Moringa oleifera Lam.) Using Random Amplified Polymorphic DNA Marker Tue, 23 Apr 2013 09:44:10 +0000 The knowledge of genetic diversity of tree crop is very important for breeding and improvement program for the purpose of improving the yield and quality of its produce. Genetic diversity study and analysis of genetic relationship among 20 Moringa oleifera were carried out with the aid of twelve primers from, random amplified polymorphic DNA marker. The seeds of twenty M. oleifera genotypes from various origins were collected and germinated and raised in nursery before transplanting to the field at University Agricultural Park (TPU). Genetic diversity parameter, such as Shannon's information index and expected heterozygosity, revealed the presence of high genetic divergence with value of 1.80 and 0.13 for Malaysian population and 0.30 and 0.19 for the international population, respectively. Mean of Nei's gene diversity index for the two populations was estimated to be 0.20. In addition, a dendrogram constructed, using UPGMA cluster analysis based on Nei's genetic distance, grouped the twenty M. oleifera into five distinct clusters. The study revealed a great extent of variation which is essential for successful breeding and improvement program. From this study, M. oleifera genotypes of wide genetic origin, such as T-01, T-06, M-01, and M-02, are recommended to be used as parent in future breeding program. Shamsuddeen Rufai, M. M. Hanafi, M. Y. Rafii, S. Ahmad, I. W. Arolu, and Jannatul Ferdous Copyright © 2013 Shamsuddeen Rufai et al. All rights reserved. The Vitamin D Receptor (VDR) Gene Polymorphisms in Turkish Brain Cancer Patients Wed, 17 Apr 2013 17:30:38 +0000 Objective. It has been stated that brain cancers are an increasingly serious issue in many parts of the world. The aim of our study was to determine a possible relationship between Vitamin D receptor (VDR) gene polymorphisms and the risk of glioma and meningioma. Methods. We investigated the VDR Taq-I and VDR Fok-I gene polymorphisms in 100 brain cancer patients (including 44 meningioma cases and 56 glioma cases) and 122 age-matched healthy control subjects. This study was performed by polymerase chain reaction-based restriction fragment length polymorphism (RF LP). Results. VDR Fok-I ff genotype was significantly increased in meningioma patients (15.9%) compared with controls (2.5%), and carriers of Fok-I ff genotype had a 6.47-fold increased risk for meningioma cases. There was no significant difference between patients and controls for VDR Taq-I genotypes and alleles. Conclusions. We suggest that VDR Fok-I genotypes might affect the development of meningioma. Bahar Toptaş, Ali Metin Kafadar, Canan Cacina, Saime Turan, Leman Melis Yurdum, Nihal Yiğitbaşı, Muhammed Oğuz Gökçe, Ümit Zeybek, and Ilhan Yaylım Copyright © 2013 Bahar Toptaş et al. All rights reserved. Self-Eating: Friend or Foe? The Emerging Role of Autophagy in Idiopathic Pulmonary Fibrosis Wed, 10 Apr 2013 17:37:39 +0000 Idiopathic pulmonary fibrosis is the most common and severe form of idiopathic interstitial pneumonias. Despite an exponential increase in our understanding of potentially important mediators and mechanisms, the pathogenesis remains elusive, and little therapeutic progress has been made in the last few years. Mortality in 3–5 years is still 50%. Autophagy, a highly conserved homeostatic mechanism necessary for cell survival, has been recently implicated in the pathogenesis of pulmonary disorders. In this paper we aim to highlight some key issues regarding the process of autophagy and its possible association with the pathogenesis of idiopathic pulmonary fibrosis. George A. Margaritopoulos, Eliza Tsitoura, Nikos Tzanakis, Demetrios A. Spandidos, Nikos M. Siafakas, George Sourvinos, and Katerina M. Antoniou Copyright © 2013 George A. Margaritopoulos et al. All rights reserved. Therapeutic Time Window for Edaravone Treatment of Traumatic Brain Injury in Mice Wed, 10 Apr 2013 08:39:31 +0000 Traumatic brain injury (TBI) is a major cause of death and disability in young people. No effective therapy is available to ameliorate its damaging effects. Our aim was to investigate the optimal therapeutic time window of edaravone, a free radical scavenger which is currently used in Japan. We also determined the temporal profile of reactive oxygen species (ROS) production, oxidative stress, and neuronal death. Male C57Bl/6 mice were subjected to a controlled cortical impact (CCI). Edaravone (3.0 mg/kg), or vehicle, was administered intravenously at 0, 3, or 6 hours following CCI. The production of superoxide radicals () as a marker of ROS, of nitrotyrosine (NT) as an indicator of oxidative stress, and neuronal death were measured for 24 hours following CCI. Superoxide radical production was clearly evident 3 hours after CCI, with oxidative stress and neuronal cell death becoming apparent after 6 hours. Edaravone administration after CCI resulted in a significant reduction in the injury volume and oxidative stress, particularly at the 3-hour time point. Moreover, the greatest decrease in levels was observed when edaravone was administered 3 hours following CCI. These findings suggest that edaravone could prove clinically useful to ameliorate the devastating effects of TBI. Kazuyuki Miyamoto, Hirokazu Ohtaki, Kenji Dohi, Tomomi Tsumuraya, Dandan Song, Keisuke Kiriyama, Kazue Satoh, Ai Shimizu, Tohru Aruga, and Seiji Shioda Copyright © 2013 Kazuyuki Miyamoto et al. All rights reserved. Expression Pattern of Genes of RLR-Mediated Antiviral Pathway in Different-Breed Chicken Response to Marek’s Disease Virus Infection Mon, 08 Apr 2013 19:14:37 +0000 It has been known that the chicken’s resistance to disease was affected by chicken’s genetic background. And RLR-mediated antiviral pathway plays an important role in detection of viral RNA. However, little is known about the interaction of genetic background with RLR-mediated antiviral pathway in chicken against MDV infection. In this study, we adopted economic line-AA broilers and native Erlang mountainous chickens for being infected with MDV. Upon infection with MDV, the expression of MDA-5 was upregulated in two-breed chickens at 4, 7, and 21 d.p.i. It is indicated that MDA-5 might be involved in detecting MDV in chicken. Interestingly, the expression of IRF-3 and IFN-β genes was decreased in spleen and thymus of broilers at 21 d.p.i, but it was upregulated in immune tissues of Erlang mountainous chickens. And the genome load of MDV in spleen of broiler is significantly higher than that in Erlang mountainous chickens. Meanwhile, we observed that the death of broiler mainly also occurred in this phase. Collectively, these present results demonstrated that the expression patters of IRF-3 and IFN-β genes in chicken against MDV infection might be affected by the genetic background which sequently influence the resistance of chicken response to MDV. Ze-Qing Feng, Ting Lian, Yong Huang, Qing Zhu, and Yi-Ping Liu Copyright © 2013 Ze-Qing Feng et al. All rights reserved. Artificial Box C/D RNAs Affect Pre-mRNA Maturation in Human Cells Sun, 31 Mar 2013 10:03:11 +0000 Box C/D small nucleolar RNAs (snoRNAs) are known to guide the -O-ribose methylation of nucleotides in eukaryotic ribosomal RNAs and small nuclear RNAs. Recently snoRNAs are predicted to regulate posttranscriptional modifications of pre-mRNA. To expand understanding of the role of snoRNAs in control of gene expression, in this study we tested the ability of artificial box C/D RNAs to affect the maturation of target pre-mRNA. We found that transfection of artificial box C/D snoRNA analogues directed to HSPA8 pre-mRNAs into human cells induced suppression of the target mRNA expression in a time- and dose-dependent manner. The artificial box C/D RNA directed to the branch point adenosine of the second intron, as well as the analogue directed to the last nucleotide of the second exon of the HSPA8 pre-mRNA caused the most prominent influence on the level of HSPA8 mRNAs. Neither box D nor the ability to direct -O-methylation of nucleotides in target RNA was essential for the knockdown activity of artificial snoRNAs. Inasmuch as artificial box C/D RNAs decreased viability of transfected human cells, we propose that natural snoRNAs as well as their artificial analogues can influence the maturation of complementary pre-mRNA and can be effective regulators of vital cellular processes. Grigoriy A. Stepanov, Dmitry V. Semenov, Anna V. Savelyeva, Elena V. Kuligina, Olga A. Koval, Igor V. Rabinov, and Vladimir A. Richter Copyright © 2013 Grigoriy A. Stepanov et al. All rights reserved. Feasibility of a Microarray-Based Point-of-Care CYP2C19 Genotyping Test for Predicting Clopidogrel On-Treatment Platelet Reactivity Thu, 28 Mar 2013 18:00:41 +0000 Clopidogrel is a prodrug which is converted into active metabolite by cytochrome P450 isoenzyme, CYP2C19. Numerous polymorphisms of CYP2C19 are reported, and a strong link exists between loss-of-function (LOF) or gain-of-function polymorphisms, clopidogrel metabolism, and clinical outcome. Hence, a fully automated point-of-care CYP2C19 genotyping assay is more likely to bring personalized antiplatelet therapy into real practice. We assessed the feasibility of the Verigene 2C19/CBS Nucleic Acid Test, a fully automated microarray-based assay, compared to bidirectional sequencing, and performed VerifyNow P2Y12 assay to evaluate the effect of CYP2C19 polymorphisms on on-treatment platelet reactivity in 57 Korean patients treated with clopidogrel after percutaneous coronary intervention. The Verigene 2C19/CBS assay identified *2, *3, and *17 polymorphisms with 100% concordance to bidirectional sequencing in 180 minutes with little hands-on time. Patients were classified into 4 groups: extensive (*1/*1; , 21.1%), intermediate (*1/*2, *1/*3; , 57.9%), poor (*2/*2, *2/*3, and *3/*3; , 19.3%), and ultrarapid metabolizers (*1/*17; , 1.8%). The prevalence of the CYP2C19  *2, *3, and *17 alleles was 36.0%, 12.3%, and 0.9%. Platelet reactivity showed gene dose response according to the number of CYP2C19 LOF allele. In conclusion, the Verigene 2C19/CBS assay gave accurate CYP2C19 genotype results which were in well match with the differing on-treatment platelet reactivity. Hyojin Chae, Myungshin Kim, Yoon-Seok Koh, Byung-Hee Hwang, Min-Kyu Kang, Yonggoo Kim, Hae-il Park, and Kiyuk Chang Copyright © 2013 Hyojin Chae et al. All rights reserved. FISH Detection of PML-RARA Fusion in ins(15;17) Acute Promyelocytic Leukaemia Depends on Probe Size Thu, 28 Mar 2013 13:22:04 +0000 The diagnosis of acute promyelocytic leukaemia (APL) is usually confirmed by cytogenetics showing the characteristic t(15;17), but a minority of patients have a masked PML/RARA fusion. We report ten patients with APL and no evidence of the t(15;17), in whom the insertion of RARA into PML could not be demonstrated by initial FISH studies using a standard dual fusion probe but was readily identified using smaller probes. Given the need for rapid diagnosis of APL, it is important to be aware of the false negative rate for large PML/RARA FISH probes in the setting of masked rearrangements. Lynda J. Campbell, Paul Oei, Ross Brookwell, Jake Shortt, Nicola Eaddy, Ashley Ng, Edward Chew, and Peter Browett Copyright © 2013 Lynda J. Campbell et al. All rights reserved. Using the Developmental Gene Bicoid to Identify Species of Forensically Important Blowflies (Diptera: Calliphoridae) Mon, 18 Mar 2013 11:00:15 +0000 Identifying species of insects used to estimate postmortem interval (PMI) is a major subject in forensic entomology. Because forensic insect specimens are morphologically uniform and are obtained at various developmental stages, DNA markers are greatly needed. To develop new autosomal DNA markers to identify species, partial genomic sequences of the bicoid (bcd) genes, containing the homeobox and its flanking sequences, from 12 blowfly species (Aldrichina grahami, Calliphora vicina, Calliphora lata, Triceratopyga calliphoroides, Chrysomya megacephala, Chrysomya pinguis, Phormia regina, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Hemipyrellia ligurriens and Lucilia sericata; Calliphoridae: Diptera) were determined and analyzed. This study first sequenced the ten blowfly species other than C. vicina and L. sericata. Based on the bcd sequences of these 12 blowfly species, a phylogenetic tree was constructed that discriminates the subfamilies of Calliphoridae (Luciliinae, Chrysomyinae, and Calliphorinae) and most blowfly species. Even partial genomic sequences of about 500 bp can distinguish most blowfly species. The short intron 2 and coding sequences downstream of the bcd homeobox in exon 3 could be utilized to develop DNA markers for forensic applications. These gene sequences are important in the evolution of insect developmental biology and are potentially useful for identifying insect species in forensic science. Seong Hwan Park, Chung Hyun Park, Yong Zhang, Huguo Piao, Ukhee Chung, Seong Yoon Kim, Kwang Soo Ko, Cheong-Ho Yi, Tae-Ho Jo, and Juck-Joon Hwang Copyright © 2013 Seong Hwan Park et al. All rights reserved. Molecular Genetics and Genetic Testing in Myotonic Dystrophy Type 1 Mon, 18 Mar 2013 08:35:52 +0000 Myotonic dystrophy type 1 (DM1) is the most common adult onset muscular dystrophy, presenting as a multisystemic disorder with extremely variable clinical manifestation, from asymptomatic adults to severely affected neonates. A striking anticipation and parental-gender effect upon transmission are distinguishing genetic features in DM1 pedigrees. It is an autosomal dominant hereditary disease associated with an unstable expansion of CTG repeats in the 3′-UTR of the DMPK gene, with the number of repeats ranging from 50 to several thousand. The number of CTG repeats broadly correlates with both the age-at-onset and overall severity of the disease. Expanded DM1 alleles are characterized by a remarkable expansion-biased and gender-specific germline instability, and tissue-specific, expansion-biased, age-dependent, and individual-specific somatic instability. Mutational dynamics in male and female germline account for observed anticipation and parental-gender effect in DM1 pedigrees, while mutational dynamics in somatic tissues contribute toward the tissue-specificity and progressive nature of the disease. Genetic test is routinely used in diagnostic procedure for DM1 for symptomatic, asymptomatic, and prenatal testing, accompanied with appropriate genetic counseling and, as recommended, without predictive information about the disease course. We review molecular genetics of DM1 with focus on those issues important for genetic testing and counseling. Dušanka Savić Pavićević, Jelena Miladinović, Miloš Brkušanin, Saša Šviković, Svetlana Djurica, Goran Brajušković, and Stanka Romac Copyright © 2013 Dušanka Savić Pavićević et al. All rights reserved. TNNT2 Gene Polymorphisms Are Associated with Susceptibility to Idiopathic Dilated Cardiomyopathy in the Han Chinese Population Sun, 17 Mar 2013 15:45:29 +0000 Background. Idiopathic dilated cardiomyopathy (DCM) is characterized by ventricular chamber enlargement and systolic dysfunction. The pathogenesis of DCM remains uncertain, and the TNNT2 gene is potentially associated with DCM. To assess the role of TNNT2 in DCM, we examined 10 tagging single nucleotide polymorphisms (SNPs) in the patients. Methods. A total of 97 DCM patients and 189 control subjects were included in the study, and all SNPs were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results. In the TNNT2 gene, there was a significant association between DCM and genotype for the tagging SNPs rs3729547 (, , , and 95% CI = 0.453–0.934) and rs3729843 (, , , and 95% CI = 1.265–2.890) in the Chinese Han population. Linkage disequilibrium (LD) analysis showed that the SNPs rs7521796, rs2275862, rs3729547, rs10800775, and rs1892028, which are approximately 6 kb apart, were in high LD () in the DCM patients. Conclusion. These results suggest that the TNNT2 polymorphisms might play an important role in susceptibility to DCM in the Chinese Han population. Xiaoping Li, Huan Wang, Rong Luo, Haiyong Gu, Channa Zhang, Yu Zhang, Rutai Hui, Xiushan Wu, and Wei Hua Copyright © 2013 Xiaoping Li et al. All rights reserved. Molecular Characterization of TP53 Gene in Human Populations Exposed to Low-Dose Ionizing Radiation Sun, 17 Mar 2013 15:08:43 +0000 Ionizing radiation, such as that emitted by uranium, may cause mutations and consequently lead to neoplasia in human cells. The TP53 gene acts to maintain genomic integrity and constitutes an important biomarker of susceptibility. The present study investigated the main alterations observed in exons 4, 5, 6, 7, and 8 of the TP53 gene and adjacent introns in Amazonian populations exposed to radioactivity. Samples were collected from 163 individuals. Occurrence of the following alterations was observed: (i) a missense exchange in exon 4 (Arg72Pro); (ii) 2 synonymous exchanges, 1 in exon 5 (His179His), and another in exon 6 (Arg213Arg); (iii) 4 intronic exchanges, 3 in intron 7 (C → T at position 13.436; C → T at position 13.491; T → G at position 13.511) and 1 in intron 8 (T → G at position 13.958). Alteration of codon 72 was found to be an important risk factor for cancer development (; ; CI: 1.29–32.64) when adjusted for age and smoking. Thus, TP53 gene may be an important biomarker for carcinogenesis susceptibility in human populations exposed to ionizing radiation. Igor Brasil-Costa, Dayse O. Alencar, Milene Raiol-Moraes, Igor A. Pessoa, Alexandre W. M. Brito, Schneyder R. Jati, Sidney E. B. Santos, Rommel M. R. Burbano, and Ândrea K. C. Ribeiro-dos-Santos Copyright © 2013 Igor Brasil-Costa et al. All rights reserved. Improvement of Hydrodynamics-Based Gene Transfer of Nonviral DNA Targeted to Murine Hepatocytes Sun, 17 Mar 2013 14:23:20 +0000 The liver is an important organ for supporting the life of an individual. Gene transfer toward this organ has been attempted in many laboratories to date; however, there have been few reports on improved liver-targeted gene delivery by using a nonviral vector. In this study, we examined the effect of various types of gene delivery carriers on enhancing the uptake and gene expression of exogenous DNA in murine hepatocytes when a hydrodynamics-based gene delivery (HGD) is performed via tail-vein injection. Mice were singly injected with a large amount of phosphate-buffered saline containing reporter plasmid DNA and/or with a gene delivery carrier. One day after the gene delivery, the animals' livers were dissected and subjected to biochemical, histochemical, and molecular biological analyses. The strongest signal from the reporter plasmid DNA was observed when the DNA was mixed with a polyethylenimine- (PEI-) based reagent. Coinjection with pCRTEIL (a loxP-floxed reporter construct) and pTR/NCre (a liver-specific Cre expression vector) resulted in the liver-specific recombination of pCRTEIL. The combination of PEI with HGD would thus be a valuable tool for liver-specific manipulation to examine the function of a gene of interest in the liver and for creating liver disease models. Shingo Nakamura, Tadaaki Maehara, Satoshi Watanabe, Masayuki Ishihara, and Masahiro Sato Copyright © 2013 Shingo Nakamura et al. All rights reserved. Molecular and Survival Differences between Familial and Sporadic Gastric Cancers Tue, 05 Mar 2013 09:17:33 +0000 Mismatch repair (MMR) and germline E-cadherin (CDH1) mutations are two of the major pathways of carcinogenesis in familial gastric cancer (GC). A total of 260 sporadic and 66 familial GC patients were enrolled and molecular and survival differences were compared. Familial GC patients had earlier onset and were diagnosed at an earlier stage and had both a better 5-year overall survival rate and 3-year disease-free survival rate compared with sporadic GC patients. Only in diffuse type GC, the MSI-H phenotype and abnormal MMR protein expression were significantly higher in familial GC than in sporadic GC. In MSI-H GC, MLH1 promoter methylation was slightly higher in sporadic GC than familial GC (50% versus 23.1%), while the frequency of MMR gene mutation was slightly higher in familial GC than in sporadic GC (15.4% versus 3.1%). All of the patients with MMR gene mutation had diffuse type GC. Among familial GC patients with CDH1 mutation, most patients (72.3%) had diffuse type GC. In summary, for familial GC patients, we recommend screening of MSI status and CDH1 mutation especially for diffuse type GC. Because of the low incidence, mutation analysis of MMR gene might be considered in MSI-H familial GC with diffuse type only. Wen-Liang Fang, Shih-Ching Chang, Yuan-Tzu Lan, Kuo-Hung Huang, Su-Shun Lo, Anna Fen-Yau Li, Chin-Wen Chi, Chew-Wun Wu, and Shih-Hwa Chiou Copyright © 2013 Wen-Liang Fang et al. All rights reserved. Implementation of High Resolution Whole Genome Array CGH in the Prenatal Clinical Setting: Advantages, Challenges, and Review of the Literature Mon, 04 Mar 2013 15:58:26 +0000 Array Comparative Genomic Hybridization analysis is replacing postnatal chromosomal analysis in cases of intellectual disabilities, and it has been postulated that it might also become the first-tier test in prenatal diagnosis. In this study, array CGH was applied in 64 prenatal samples with whole genome oligonucleotide arrays (BlueGnome, Ltd.) on DNA extracted from chorionic villi, amniotic fluid, foetal blood, and skin samples. Results were confirmed with Fluorescence In Situ Hybridization or Real-Time PCR. Fifty-three cases had normal karyotype and abnormal ultrasound findings, and seven samples had balanced rearrangements, five of which also had ultrasound findings. The value of array CGH in the characterization of previously known aberrations in five samples is also presented. Seventeen out of 64 samples carried copy number alterations giving a detection rate of 26.5%. Ten of these represent benign or variables of unknown significance, giving a diagnostic capacity of the method to be 10.9%. If karyotype is performed the additional diagnostic capacity of the method is 5.1% (3/59). This study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate. In addition a thorough review of the literature is presented. Paola Evangelidou, Angelos Alexandrou, Maria Moutafi, Marios Ioannides, Pavlos Antoniou, George Koumbaris, Ioannis Kallikas, Voula Velissariou, Carolina Sismani, and Philippos C. Patsalis Copyright © 2013 Paola Evangelidou et al. All rights reserved. Role of Melanin in Melanocyte Dysregulation of Reactive Oxygen Species Thu, 28 Feb 2013 10:45:16 +0000 We have recently reported a potential alternative tumor suppressor function for p16 relating to its capacity to regulate oxidative stress and observed that oxidative dysregulation in p16-depleted cells was most profound in melanocytes, compared to keratinocytes or fibroblasts. Moreover, in the absence of p16 depletion or exogenous oxidative insult, melanocytes exhibited significantly higher basal levels of reactive oxygen species (ROS) than these other epidermal cell types. Given the role of oxidative stress in melanoma development, we speculated that this increased susceptibility of melanocytes to oxidative stress (and greater reliance on p16 for suppression of ROS) may explain why genetic compromise of p16 is more commonly associated with predisposition to melanoma rather than other cancers. Here we show that the presence of melanin accounts for this differential oxidative stress in normal and p16-depleted melanocytes. Thus the presence of melanin in the skin appears to be a double-edged sword: it protects melanocytes as well as neighboring keratinocytes in the skin through its capacity to absorb UV radiation, but its synthesis in melanocytes results in higher levels of intracellular ROS that may increase melanoma susceptibility. Noah C. Jenkins and Douglas Grossman Copyright © 2013 Noah C. Jenkins and Douglas Grossman. All rights reserved. Detection of C. trachomatis in the Serum of the Patients with Urogenital Chlamydiosis Wed, 13 Feb 2013 11:25:02 +0000 Extragenital chlamydial complications may be associated with systemic spread of infection, but haematogenous route for C. trachomatis dissemination has not been clearly demonstrated. Here we report that serum specimens obtained from patients with chlamydiosis contain elementary bodies of C. trachomatis shown by culture and immunogold electron microscopy. We have found that 31 of the 52 patients had serum precipitates which were infective to McCoy cells. Immunostaining revealed very small inclusions resembling those reported during persistent C. trachomatis infection in vitro. DNA specimens from 49 (out of 52) patients with chlamydiosis gave positive PCR readings. The viability of the pathogen present in the sera was confirmed by chlamydial RNA detection in the cell monolayer inoculated by the serum precipitates. By using DNA isolation protocol from 1 mL of serum and quantitative TaqMan PCR, it was estimated that bacterial load in patients’ sera was  GE/mL. These findings for the first time demonstrated that C. trachomatis can be disseminated directly by the plasma, independently from blood cell, which may represent a new possible pathway of the chronic infection development. Therefore, new methodological approaches for detection of C. trachomatis in the serum of patients with complicated and chronic chlamydiosis could be important in the diagnosis of the infection regardless of its anatomical localization. Naylia A. Zigangirova, Yulia P. Rumyantseva, Elena Y. Morgunova, Lidia N. Kapotina, Lubov V. Didenko, Elena A. Kost, Ekaterina A. Koroleva, Yuriy K. Bashmakov, and Ivan M. Petyaev Copyright © 2013 Naylia A. Zigangirova et al. All rights reserved. Diagnosis of Familial Wolf-Hirschhorn Syndrome due to a Paternal Cryptic Chromosomal Rearrangement by Conventional and Molecular Cytogenetic Techniques Sun, 03 Feb 2013 14:35:21 +0000 The use of conventional cytogenetic techniques in combination with fluorescent in situ hybridization (FISH) and single-nucleotide polymorphism (SNP) microarrays is necessary for the identification of cryptic rearrangements in the diagnosis of chromosomal syndromes. We report two siblings, a boy of 9 years and 9 months of age and his 7-years- and 5-month-old sister, with the classic Wolf-Hirschhorn syndrome (WHS) phenotype. Using high-resolution GTG- and NOR-banding karyotypes, as well as FISH analysis, we characterized a pure 4p deletion in both sibs and a balanced rearrangement in their father, consisting in an insertion of 4p material within a nucleolar organizing region of chromosome 15. Copy number variant (CNV) analysis using SNP arrays showed that both siblings have a similar size of 4p deletion (~6.5 Mb). Our results strongly support the need for conventional cytogenetic and FISH analysis, as well as high-density microarray mapping for the optimal characterization of the genetic imbalance in patients with WHS; parents must always be studied for recognizing cryptic balanced chromosomal rearrangements for an adequate genetic counseling. Carlos A. Venegas-Vega, Fernando Fernández-Ramírez, Luis M. Zepeda, Karem Nieto-Martínez, Laura Gómez-Laguna, Luz M. Garduño-Zarazúa, Jaime Berumen, Susana Kofman, and Alicia Cervantes Copyright © 2013 Carlos A. Venegas-Vega et al. All rights reserved. The Italian National External Quality Assessment Program in Molecular Genetic Testing: Results of the VII Round (2010-2011) Tue, 29 Jan 2013 15:41:36 +0000 Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes. F. Censi, F. Tosto, G. Floridia, M. Marra, M. Salvatore, A. M. Baffico, M. Grasso, M. A. Melis, E. Pelo, P. Radice, A. Ravani, C. Rosatelli, N. Resta, S. Russo, M. Seia, L. Varesco, V. Falbo, and D. Taruscio Copyright © 2013 F. Censi et al. All rights reserved. Association of Apolipoprotein A5 Gene 1131TC Polymorphism with the Risk of Metabolic Syndrome in Korean Subjects Mon, 28 Jan 2013 14:19:09 +0000 We assessed the associations between the APOA51131TC polymorphism and lipid parameters and other risk factors of the metabolic syndrome in Korean subjects. A total of 2,901 participants from 20 oriental medical hospitals in Korea were enrolled between 2006 and 2011. According to the modified National Cholesterol Education Program Adult Treatment Panel III definitions, subjects were classified into the metabolic syndrome group and control group. The APOA51131TC genotype was significantly associated with serum high-density lipoprotein cholesterol levels (effect = − 1.700 mg/dL, -E07) in the total study population after adjustment for differences in age and gender. The association of the APOA51131TC genotype with serum log-transformed triglyceride was also significant in an additive genetic model (effect = 0.056 mg/dL, E-19). After adjustment for age and gender, we determined that the odds ratio for the occurrence of the metabolic syndrome was 1.322 for C-allele carriers in the additive model (95% CI = [1.165 − 1.501], E-05). In the current study, we demonstrated that the APOA51131TC polymorphism is associated with the metabolic syndrome because of its remarkable effect on serum triglyceride levels in Korean subjects. Kwang Hoon Song, Seongwon Cha, Sung-Gon Yu, Hyunjoo Yu, Soo A. Oh, and Nam-Sik Kang Copyright © 2013 Kwang Hoon Song et al. All rights reserved. Targeting the Immunogenetic Diseases with the Appropriate HLA Molecular Typing: Critical Appraisal on 2666 Patients Typed in One Single Centre Mon, 21 Jan 2013 09:06:52 +0000 We compared the immunogenetic data from 2666 patients affected by HLA-related autoimmune diseases with those from 4389 ethnically matched controls (3157 cord blood donors CBD, 1232 adult bone marrow donors BMD), to verify the appropriateness of HLA typing requests received in the past decade. The frequency of HLA-B*27 phenotype was 10.50% in 724 ankylosing spondylitis, 16.80% in 125 uveitis (3.41% BMD, 4.24% CBD, ); HLA-B*51 allele was 15.57% in 212 Behçet’s disease (12.91% BMD, 9.88% CBD, ); the HLA-DRB1-rheumatoid arthritis (RA) shared epitope was 13.72% in 554 RA (10.85% BMD, 13.48% CBD, ); the carriers of almost one of HLA-DQB1 susceptibility alleles were 84.91% in 795 celiac disease (CD) and 59.37% in 256 insulin-dependent diabetes mellitus (IDDM) (46.06% in 875 CBD, 42.75% in 662 BMD ). Overall, our results show that the HLA marker frequencies were higher in patients than controls, but lower than expected from the literature data (excluding CD and IDDM) and demonstrate that, in complex immunogenetic conditions, a substantial number of genetic analyses are redundant and inappropriate, burdening to the public health costs. For this reason, we suggest the Italian Scientific Society of Immunogenetics to establish guidelines to improve the appropriateness of typing requests. M. Guarene, C. Capittini, A. De Silvestri, A. Pasi, C. Badulli, I. Sbarsi, A. L. Cremaschi, F. Garlaschelli, C. Pizzochero, M. C. Monti, C. Montecucco, G. R. Corazza, D. Larizza, P. E. Bianchi, L. Salvaneschi, and M. Martinetti Copyright © 2013 M. Guarene et al. All rights reserved. Clinical Genetic Testing of Periodic Fever Syndromes Tue, 01 Jan 2013 08:20:04 +0000 Periodic fever syndromes (PFSs) are a wide group of autoinflammatory diseases. Due to some clinical overlap between different PFSs, differential diagnosis can be a difficult challenge. Nowadays, there are no universally agreed recommendations for most PFSs, and near half of patients may remain without a genetic diagnosis even after performing multiple-gene analyses. Molecular analysis of periodic fevers’ causative genes can improve patient quality of life by providing early and accurate diagnosis and allowing the administration of appropriate treatment. In this paper we focus our discussion on effective usefulness of genetic diagnosis of PFSs. The aim of this paper is to establish how much can the diagnostic system improve, in order to increase the success of PFS diagnosis. The mayor expectation in the near future will be addressed to the so-called next generation sequencing approach. Although the application of bioinformatics to high-throughput genetic analysis could allow the identification of complex genotypes, the complexity of this definition will hardly result in a clear contribution for the physician. In our opinion, however, to obtain the best from this new development a rule should always be kept well in mind: use genetics only to answer specific clinical questions. Annalisa Marcuzzi, Elisa Piscianz, Giulio Kleiner, Alberto Tommasini, Giovanni Maria Severini, Lorenzo Monasta, and Sergio Crovella Copyright © 2013 Annalisa Marcuzzi et al. All rights reserved. 1Click1View: Interactive Visualization Methodology for RNAi Cell-Based Microscopic Screening Thu, 27 Dec 2012 14:22:24 +0000 Technological advancements are constantly increasing the size and complexity of data resulting from large-scale RNA interference screens. This fact has led biologists to ask complex questions, which the existing, fully automated analyses are often not adequate to answer. We present a concept of 1Click1View (1C1V) as a methodology for interactive analytic software tools. 1C1V can be applied for two-dimensional visualization of image-based screening data sets from High Content Screening (HCS). Through an easy-to-use interface, one-click, one-view concept, and workflow based architecture, visualization method facilitates the linking of image data with numeric data. Such method utilizes state-of-the-art interactive visualization tools optimized for fast visualization of large scale image data sets. We demonstrate our method on an HCS dataset consisting of multiple cell features from two screening assays. Lukasz Zwolinski, Marta Kozak, and Karol Kozak Copyright © 2013 Lukasz Zwolinski et al. All rights reserved. HOXA4 Gene Promoter Hypermethylation as an Epigenetic Mechanism Mediating Resistance to Imatinib Mesylate in Chronic Myeloid Leukemia Patients Wed, 26 Dec 2012 15:24:34 +0000 Development of resistance to imatinib mesylate (IM) in chronic myeloid leukemia (CML) patients has emerged as a significant clinical problem. The observation that increased epigenetic silencing of potential tumor suppressor genes correlates with disease progression in some CML patients treated with IM suggests a relationship between epigenetic silencing and resistance development. We hypothesize that promoter hypermethylation of HOXA4 could be an epigenetic mechanism mediating IM resistance in CML patients. Thus a study was undertaken to investigate the promoter hypermethylation status of HOXA4 in CML patients on IM treatment and to determine its role in mediating resistance to IM. Genomic DNA was extracted from peripheral blood samples of 95 CML patients (38 good responders and 57 resistant) and 12 normal controls. All samples were bisulfite treated and analysed by methylation-specific high-resolution melt analysis. Compared to the good responders, the HOXA4 hypermethylation level was significantly higher () in IM-resistant CML patients. On comparing the risk, HOXA4 hypermethylation was associated with a higher risk for IM resistance (OR 4.658; 95% CI, 1.673–12.971; ). Thus, it is reasonable to suggest that promoter hypermethylation of HOXA4 gene could be an epigenetic mechanism mediating IM resistance in CML patients. Marjanu Hikmah Elias, Abdul Aziz Baba, Azlan Husin, Sarina Sulong, Rosline Hassan, Goh Ai Sim, S. Fadilah Abdul Wahid, and Ravindran Ankathil Copyright © 2013 Marjanu Hikmah Elias et al. All rights reserved. IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies Wed, 26 Dec 2012 08:54:45 +0000 The molecular diagnosis of retinal dystrophies is difficult because of the very important number of genes implicated and is rarely helped by genotype-phenotype correlations. This prompted us to develop IROme, a custom designed in solution-based targeted exon capture assay (SeqCap EZ Choice library, Roche NimbleGen) for 60 retinitis pigmentosa-linked genes and three candidate genes (942 exons). Pyrosequencing was performed on a Roche 454 GS Junior benchtop high-throughput sequencing platform. In total, 23 patients affected by retinitis pigmentosa were analyzed. Per patient, 39.6 Mb were generated, and 1111 sequence variants were detected on average, at a median coverage of 17-fold. After data filtering and sequence variant prioritization, disease-causing mutations were identified in ABCA4, CNGB1, GUCY2D, PROM1, PRPF8, PRPF31, PRPH2, RHO, RP2, and TULP1 for twelve patients (55%), ten mutations having never been reported previously. Potential mutations were identified in 5 additional patients, and in only 6 patients no molecular diagnosis could be established (26%). In conclusion, targeted exon capture and next-generation sequencing are a valuable and efficient approach to identify disease-causing sequence variants in retinal dystrophies. Daniel F. Schorderet, Alexandra Iouranova, Tatiana Favez, Leila Tiab, and Pascal Escher Copyright © 2013 Daniel F. Schorderet et al. All rights reserved. How to Deal with the Upcoming Challenges in GMO Detection in Food and Feed Sun, 21 Oct 2012 08:49:43 +0000 Biotech crops are the fastest adopted crop technology in the history of modern agriculture. The commercialisation of GMO is in many countries strictly regulated laying down the need for traceability and labelling. To comply with these legislations, detection methods are needed. To date, GM events have been developed by the introduction of a transgenic insert (i.e., promoter, coding sequence, terminator) into the plant genome and real-time PCR is the detection method of choice. However, new types of genetic elements will be used to construct new GMO and new crops will be transformed. Additionally, the presence of unauthorised GMO in food and feed samples might increase in the near future. To enable enforcement laboratories to continue detecting all GM events and to obtain an idea of the possible presence of unauthorised GMO in a food and feed sample, an intensive screening will become necessary. A pragmatic, cost-effective, and time-saving approach is presented here together with an overview of the evolution of the GMO and the upcoming needs. Sylvia R. M. Broeders, Sigrid C. J. De Keersmaecker, and Nancy H. C. Roosens Copyright © 2012 Sylvia R. M. Broeders et al. All rights reserved. Evaluation of DNA Single and Double Strand Breaks in Women with Cervical Neoplasia Based on Alkaline and Neutral Comet Assay Techniques Wed, 03 Oct 2012 11:40:42 +0000 A hospital-based unmatched case-control study was performed in order to determine the relation of DNA single (ssb) and double (dsb) strand breaks in women with and without cervical neoplasia. Cervical epithelial cells of 30 women: 10 with low grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 without cervical lesions were evaluated using alkaline and neutral comet assays. A significant increase in global DNA damage (ssb + dsb) and dsb was observed in patients with HG-SIL (48.90 ± 12.87 and 23.50 ± 13.91), patients with LG-SIL (33.60 ± 14.96 and 11.20 ± 5.71), and controls (21.70 ± 11.87 and 5.30 ± 5.38; resp.). Pearson correlation coefficient reveled a strong relation between the levels ssb and dsb (𝑟2=0.99, 𝑃=0.03, and 𝑟2=0.94, 𝑃=0.16, resp.) and progression of neoplasia. The increase of dsb damage in patients with HG-SIL was confirmed by DNA breakage detection-FISH (DBD-FISH) on neutral comets. Our results argue in favor of a real genomic instability in women with cervical neoplasia, which was strengthened by our finding of a higher proportion of DNA dsb. Elva I. Cortés-Gutiérrez, Fernando Hernández-Garza, Jorge O. García-Pérez, Martha I. Dávila-Rodríguez, Miguel E. Aguado-Barrera, and Ricardo M. Cerda-Flores Copyright © 2012 Elva I. Cortés-Gutiérrez et al. All rights reserved. Profiling of Age-Related Changes in the Tibialis Anterior Muscle Proteome of the mdx Mouse Model of Dystrophinopathy Wed, 03 Oct 2012 10:13:09 +0000 X-linked muscular dystrophy is a highly progressive disease of childhood and characterized by primary genetic abnormalities in the dystrophin gene. Senescent mdx specimens were used for a large-scale survey of potential age-related alterations in the dystrophic phenotype, because the established mdx animal model of dystrophinopathy exhibits progressive deterioration of muscle tissue with age. Since the mdx tibialis anterior muscle is a frequently used model system in muscular dystrophy research, we employed this particular muscle to determine global changes in the dystrophic skeletal muscle proteome. The comparison of mdx mice aged 8 weeks versus 22 months by mass-spectrometry-based proteomics revealed altered expression levels in 8 distinct protein species. Increased levels were shown for carbonic anhydrase, aldolase, and electron transferring flavoprotein, while the expressions of pyruvate kinase, myosin, tropomyosin, and the small heat shock protein Hsp27 were found to be reduced in aged muscle. Immunoblotting confirmed age-dependent changes in the density of key muscle proteins in mdx muscle. Thus, segmental necrosis in mdx tibialis anterior muscle appears to trigger age-related protein perturbations due to dystrophin deficiency. The identification of novel indicators of progressive muscular dystrophy might be useful for the establishment of a muscle subtype-specific biomarker signature of dystrophinopathy. Steven Carberry, Margit Zweyer, Dieter Swandulla, and Kay Ohlendieck Copyright © 2012 Steven Carberry et al. All rights reserved. Codon Preference Optimization Increases Prokaryotic Cystatin C Expression Wed, 03 Oct 2012 10:11:32 +0000 Gene expression is closely related to optimal vector-host system pairing in many prokaryotes. Redesign of the human cystatin C (cysC) gene using the preferred codons of the prokaryotic system may significantly increase cysC expression in Escherichia coli (E. coli). Specifically, cysC expression may be increased by removing unstable sequences and optimizing GC content. According to E. coli expression system codon preferences, the gene sequence was optimized while the amino acid sequence was maintained. The codon-optimized cysC (co-cysC) and wild-type cysC (wt-cysC) were expressed by cloning the genes into a pET-30a plasmid, thus transforming the recombinant plasmid into E. coli BL21. Before and after the optimization process, the prokaryotic expression vector and host bacteria were examined for protein expression and biological activation of CysC. The recombinant proteins in the lysate of the transformed bacteria were purified using Ni2+-NTA resin. Recombinant protein expression increased from 10% to 46% based on total protein expression after codon optimization. Recombinant CysC purity was above 95%. The significant increase in cysC expression in E. coli expression produced by codon optimization techniques may be applicable to commercial production systems. Qing Wang, Cui Mei, Honghua Zhen, and Jess Zhu Copyright © 2012 Qing Wang et al. All rights reserved. Evaluation of Bovine Embryo Biopsy Techniques according to Their Ability to Preserve Embryo Viability Tue, 02 Oct 2012 13:19:58 +0000 The purpose of this research was to evaluate three embryo biopsy techniques used for preimplantation genetic diagnosis (PGD) in cattle and to recommend the least invasive one for current use, especially when PGD is followed by embryo cryopreservation. Three hundred bovine embryos were biopsied by either one of the needle, aspiration or microblade method, and then checked for viability by freezing/thawing and transplantation to recipient cows. The number of pregnancies obtained after the transfer of biopsied frozen/thawed embryos was assessed 30 days later using ultrasounds. The results were significantly different between the three biopsy methods: the pregnancy rate was of 57% in cows that received embryos biopsied by needle, 43% in cows that received embryos biopsied by aspiration, and 31% in cows that received embryos biopsied by microblade. Choosing an adequate biopsy method is therefore of great importance in embryos that will undergo subsequent cryopreservation, as it significantly influences their viability after thawing. M. Cenariu, E. Pall, C. Cernea, and I. Groza Copyright © 2012 M. Cenariu et al. All rights reserved. Alterations and Chromosomal Variants in the Ecuadorian Population Tue, 02 Oct 2012 12:13:29 +0000 Medical genetics is a field marked by fast progress. Even though it was at one point confined to a group of relatively rare diseases, today it has become a central component in the understanding of disorders and it is the subject of interest for all medical specialties. This paper, shares data on the chromosomal alterations and variations that have been diagnosed in Ecuadorian patients since 1998. A total of 2,636 individual cases have been analyzed by G-banding technique until February 2012. The present work shows this collection of data and the important findings that have appeared throughout these years in hopes that it can contribute to have a deeper understanding of the incidence of chromosomal aberrations and alterations in the Ecuadorian population. César Paz-y-Miño, Nadia Cumbal, Santiago Araujo, and Ma. Eugenia Sánchez Copyright © 2012 César Paz-y-Miño et al. All rights reserved. Utilization of Super BAC Pools and Fluidigm Access Array Platform for High-Throughput BAC Clone Identification: Proof of Concept Sun, 15 Jul 2012 12:38:52 +0000 Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooled Amaranthus hypochondriacus BAC library. Ninety-six SNP loci, spanning the length of A. hypochondriacus linkage groups 1, 2, and 15, were simultaneously tested for clone identification from four BAC super pools, corresponding to 28 384-well plates, using a single Fluidigm integrated fluidic chip (IFC). Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone. PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification. Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of ~$0.05 per reaction. Peter J. Maughan, Scott M. Smith, and Joshua A. Raney Copyright © 2012 Peter J. Maughan et al. All rights reserved. Control and Augmentation of Long-Term Plasmid Transgene Expression In Vivo in Murine Muscle Tissue and Ex Vivo in Patient Mesenchymal Tissue Sun, 01 Jul 2012 09:23:12 +0000 Purpose. In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues. Methods. Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadriceps in vivo was examined. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue, and applied in ex vivo human tissue. Results. In vivo plasmid-based transgene expression did not silence in murine muscle, unlike in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. The inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. Expression was re-induced to a similar level on a temporal basis. Luciferase expression was readily detected ex vivo in human muscle and tendon. Conclusions. Plasmid constructs resulted in long-term in vivo gene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time. David Morrissey, Jan P. van Pijkeren, Simon Rajendran, Sara A. Collins, Garrett Casey, Gerald C. O'Sullivan, and Mark Tangney Copyright © 2012 David Morrissey et al. All rights reserved. Construction of a Llama Bacterial Artificial Chromosome Library with Approximately 9-Fold Genome Equivalent Coverage Thu, 28 Jun 2012 13:27:26 +0000 The Ilama is an important agricultural livestock in much of South America. The llama is increasing in popularity in the United States as a companion animal. Little work has been done to improve llama production using modern technology. A paucity of information is available regarding the llama genome. We report the construction of a llama bacterial artificial chromosome (BAC) library of about 196,224 clones in the vector pECBAC1. Using flow cytometry and bovine, human, mouse, and chicken as controls, we determined the llama genome size to be 2.4×109 bp. The average insert size of the library is 137.8 kb corresponding to approximately 9-fold genome coverage. Further studies are needed to further characterize the library and llama genome. We anticipate that this new library will help facilitate future genomic studies in the llama. K. W. Airmet, J. D. Hinckley, L. T. Tree, M. Moss, S. Blumell, K. Ulicny, A. K. Gustafson, M. Weed, R. Theodosis, M. Lehnardt, J. Genho, M. R. Stevens, and D. L. Kooyman Copyright © 2012 K. W. Airmet et al. All rights reserved. cDNA-SRAP and Its Application in Differential Gene Expression Analysis: A Case Study in Erianthus arundinaceum Tue, 19 Jun 2012 10:14:14 +0000 Erianthus arundinaceum is a wild relative species of sugarcane. The aim of this research was to demonstrate the feasibility of cDNA-SRAP for differential gene expression and to explore the molecular mechanism of drought resistance in E. arundinaceum. cDNA-SRAP technique, for the first time, was applied in the analysis of differential gene expression in E. arundinaceum under drought stress. In total, eight differentially expressed genes with length of 185–427 bp were successfully isolated (GenBank Accession numbers: EU071770, EU071772, EU071774, EU071776, EU071777, EU071779, EU071780, and EU071781). Based on their homologies with genes in GenBank, these genes were assumed to encode ribonuclease III, vacuolar protein, ethylene insensitive protein, aerobactin biosynthesis protein, photosystem II protein, glucose transporter, leucine-rich repeat protein, and ammonia monooxygenase. Real-time PCR analysis on the expression profiling of gene (EU071774) encoding ethylene-insensitive protein and gene (EU071781) encoding ammonia monooxygenase revealed that the expression of these two genes was upregulated both by PEG and ABA treatments, suggesting that they may involve in the drought resistance of E. arundinaceum. This study constitutes the first report of genes activated in E. arundinaceum by drought stress and opens up the application of cDNA-SRAP in differential gene expression analysis in E. arundinaceum under certain stress conditions. Youxiong Que, Liping Xu, Jianwei Lin, Jun Luo, Jingsheng Xu, Jin Zheng, and Rukai Chen Copyright © 2012 Youxiong Que et al. All rights reserved. Effect of Heat Exposure on Gene Expression of Feed Intake Regulatory Peptides in Laying Hens Mon, 07 May 2012 14:45:06 +0000 The aim of this paper was to investigate the effect of heat stress on the regulation of appetite-associated genes in laying hens. Forty eight laying hens were randomly divided into two circumstances: high (31 ± 1.5°C; relative humidity, 82.0 ± 2.2%) or normal (20 ± 2°C, control; relative humidity, 60.1 ± 4.5%) ambient environment. Heat stress decreased body weight gain (𝑃<0.01), feed intake (𝑃<0.01), laying rate (𝑃<0.05), average egg mass (𝑃<0.01), egg production (𝑃<0.01), shell thickness (𝑃<0.01), and feed efficiency (𝑃<0.05). High ambient temperature decreased plasma uric acid (𝑃<0.05). Heat stress significantly increased mRNA levels of ghrelin and cocaine- and amphetamine-regulated transcript (𝑃<0.05) and decreased mRNA levels of cholecystokinin (𝑃<0.05) in the hypothalamus. Heat stress significantly increased (𝑃<0.05) mRNA levels of ghrelin in the glandular stomach and jejunum but significantly decreased (𝑃<0.05) mRNA levels of cholecystokinin in the duodenum and jejunum. In conclusion, heat stress plays a unique role in some special neuropeptides (e.g., ghrelin, cocaine- and amphetamine-regulated transcript, and cholecystokinin), which might participate in the regulation of feed intake in laying hens under high ambient temperature. Zhigang Song, Lei Liu, Ardashir Sheikhahmadi, Hongchao Jiao, and Hai Lin Copyright © 2012 Zhigang Song et al. All rights reserved. Efficiency of Manual Scanning in Recovering Rare Cellular Events Identified by Fluorescence In Situ Hybridization: Simulation of the Detection of Fetal Cells in Maternal Blood Thu, 08 Mar 2012 10:36:51 +0000 Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1–10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning. Ahmed Emad, Seemi Ayub, Oumar Samassékou, Marie-Chantal Grégoire, Macoura Gadji, Aimé Ntwari, Josée Lamoureux, Francis Hemmings, Triantafyllos Tafas, Michael W. Kilpatrick, Kada Krabchi, and Régen Drouin Copyright © 2012 Ahmed Emad et al. All rights reserved. Fenretinide (4-HPR): A Preventive Chance for Women at Genetic and Familial Risk? Mon, 05 Mar 2012 13:40:24 +0000 The incidence and mortality of breast cancer have been recently influenced by several new therapeutic strategies. In particular our knowledge on cancer precursors, risk biomarkers, and genetics has considerably increased, and prevention strategies are being successfully explored. Since their discovery, retinoids, the natural and synthetic derivatives of vitamin A, have been known to play a crucial role in cell and tissue differentiation and their ability to inhibit carcinogenesis has made them the ideal chemopreventive agents studied in several preclinical and clinical trials. Fenretinide (4-HPR) is the most studied retinoid in breast cancer chemoprevention clinical trials due to its selective accumulation in breast tissue and its favorable toxicological profile. This agent showed a significative reduction of the incidence of second breast tumors in premenopausal women confirmed after 15-year followups. Considering Fenretinide protective action, a similar trend on ovarian cancer, this drug warrants reevaluations as a preventive agent for high-risk young women, such as BRCA-1 and 2 mutation carriers or with a high familial risk. This favorable effect therefore provides a strong rationale for a primary prevention trial in these unaffected cohort of women. Massimiliano Cazzaniga, Clara Varricchio, Chiara Montefrancesco, Irene Feroce, and Aliana Guerrieri-Gonzaga Copyright © 2012 Massimiliano Cazzaniga et al. All rights reserved. Characterization, Tissue Expression, and Imprinting Analysis of the Porcine CDKN1C and NAP1L4 Genes Sun, 04 Mar 2012 13:59:20 +0000 CDKN1C and NAP1L4 in human CDKN1C/KCNQ1OT1 imprinted domain are two key candidate genes responsible for BWS (Beckwith-Wiedemann syndrome) and cancer. In order to increase understanding of these genes in pigs, their cDNAs are characterized in this paper. By the IMpRH panel, porcine CDKN1C and NAP1L4 genes were assigned to porcine chromosome 2, closely linked with IMpRH06175 and with LOD of 15.78 and 17.94, respectively. By real-time quantitative RT-PCR and polymorphism-based method, tissue and allelic expression of both genes were determined using F1 pigs of Rongchang and Landrace reciprocal crosses. The transcription levels of porcine CDKN1C and NAP1L4 were significantly higher in placenta than in other neonatal tissues (𝑃<0.01) although both genes showed the highest expression levels in the lung and kidney of one-month pigs (𝑃<0.01). Imprinting analysis demonstrated that in pigs, CDKN1C was maternally expressed in neonatal heart, tongue, bladder, ovary, spleen, liver, skeletal muscle, stomach, small intestine, and placenta and biallelically expressed in lung and kidney, while NAP1L4 was biallelically expressed in the 12 neonatal tissues examined. It is concluded that imprinting of CDKN1C is conservative in mammals but has tissue specificity in pigs, and imprinting of NAP1L4 is controversial in mammalian species. Shun Li, Juan Li, Jiawei Tian, Ranran Dong, Jin Wei, Xiaoyan Qiu, and Caode Jiang Copyright © 2012 Shun Li et al. All rights reserved. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones Thu, 01 Mar 2012 10:33:27 +0000 Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning. Kalpana Dulal, Benjamin Silver, and Hua Zhu Copyright © 2012 Kalpana Dulal et al. All rights reserved. Aberrant Expression of N-Methylpurine-DNA Glycosylase Influences Patient Survival in Malignant Gliomas Tue, 28 Feb 2012 08:15:20 +0000 Aim. To examine the expression of N-methylpurine-DNA glycosylase (MPG) gene and protein in glioma samples with different WHO grades and its association with patients’ survival. Methods. Immunohistochemistry assay, quantitative real-time PCR and Western blot analysis were carried out to investigate the expression of MPG gene and protein in 128 glioma and 10 non-neoplastic brain tissues. Results. MPG gene expression level in glioma tissues was significantly higher than that in non-neoplastic brain tissues (𝑃<0.001). Immunohistochemistry also showed that MPG protein was over-expressed in glioma tissues, which was consistent with the resutls of Western blot analysis. Additionally, the expression levels of MPG gene and protein both increase from grade I to grade IV glioma according to the results of real-time PCR, immunohistochemistry and western blot analysis. Moreover, the survival rate of MPG-positive patients was significantly lower than that of MPG-negative patients (𝑃<0.001). We further confirmed that the over-expression of MPG was a significant and independent prognostic indicator in glioma by multivariate analysis (𝑃<0.001). Conclusions. Our data showed the over-expression of MPG gene and protein in human gliomas, and also suggested for the first time that MPG be an unfavorable independent prognostic indicator for glioma patients. Ce Liu, Yanyang Tu, Jun Yuan, Xinggang Mao, Shiming He, Liang Wang, Guoqiang Fu, Jianhai Zong, and Yongsheng Zhang Copyright © 2012 Ce Liu et al. All rights reserved. In Vivo Testing of MicroRNA-Mediated Gene Knockdown in Zebrafish Mon, 27 Feb 2012 15:25:56 +0000 The zebrafish (Danio rerio) has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology) or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis). The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome. Ivone Un San Leong, Chuan-Ching Lan, Jonathan R. Skinner, Andrew N. Shelling, and Donald R. Love Copyright © 2012 Ivone Un San Leong et al. All rights reserved. Advances in BAC-Based Physical Mapping and Map Integration Strategies in Plants Mon, 27 Feb 2012 11:18:42 +0000 In the advent of next-generation sequencing (NGS) platforms, map-based sequencing strategy has been recently suppressed being too expensive and laborious. The detailed studies on NGS drafts alone indicated these assemblies remain far from gold standard reference quality, especially when applied on complex genomes. In this context the conventional BAC-based physical mapping has been identified as an important intermediate layer in current hybrid sequencing strategy. BAC-based physical map construction and its integration with high-density genetic maps have benefited from NGS and high-throughput array platforms. This paper addresses the current advancements of BAC-based physical mapping and high-throughput map integration strategies to obtain densely anchored well-ordered physical maps. The resulted maps are of immediate utility while providing a template to harness the maximum benefits of the current NGS platforms. Ruvini Ariyadasa and Nils Stein Copyright © 2012 Ruvini Ariyadasa and Nils Stein. All rights reserved. Effective Silencing of Sry Gene with RNA Interference in Developing Mouse Embryos Resulted in Feminization of XY Gonad Wed, 08 Feb 2012 08:41:24 +0000 Delivering siRNA or shRNA into the developing embryos is still a main challenge to use of RNAi in mammalian systems. Here we analyze several factors influencing RNAi-mediated silencing of Sry gene, which is a tightly controlled spatiotemporal expressed gene and only shortly expressed in developing mouse embryo gonad. A Sry gene-specific shRNAs expression vector (pSilencer4.1/Sry565) was constructed. The shRNA constructs were mixed with polyethylenimines (PEIs) to form a complex and then injected into pregnant mice though tail vein. Our results showed that Sry gene was downregulated significantly in developing embryos. Further study revealed that knocking-down of Sry expression resulted in feminization of gonad development in mouse embryos and the expression level of Sox9 and Wt1 gene was also significantly changed by downregulation of Sry. The transfection efficiency is associated with the amount of plasmid DNA injection, injection time, injection speed, and volume. Our studies suggest that transplacental RNAi could be implemented by tail vein injection of plasmid vector into pregnant mice. Ning Wu, Ai-Bing Yu, Hua-Bin Zhu, and Xiu-Kun Lin Copyright © 2012 Ning Wu et al. All rights reserved. Copy-Number Variations Observed in a Japanese Population by BAC Array CGH: Summary of Relatively Rare CNVs Tue, 24 Jan 2012 10:04:29 +0000 Copy-number variations (CNVs) may contribute to genetic variation in humans. Reports regarding existence and characteristics of CNVs in a large apparently healthy Japanese cohort are quite limited. We report the data from a screening of 213 unrelated Japanese individuals using comparative genomic hybridization based on a bacterial artificial chromosome microarray (BAC aCGH). In a previous paper, we summarized the data by focusing on highly polymorphic CNVs (in ≥5.0 % of the individuals). However, rare variations have recently received attention from scientists who espouse a hypothesis called “common disease and rare variants.” Here, we report CNVs identified in fewer than 10 individuals in our study population. We found a total of 126 CNVs at 52 different BAC regions in the genome. The CNVs observed at 27 of the 52 BAC-regions were found in only one unrelated individual. The majority of CNVs found in this study were not identified in the Japanese who were examined in the other studies. Family studies were conducted, and the results demonstrated that the CNVs were inherited from one parent in the families. Yasunari Satoh, Keiko Sasaki, Yuko Shimoichi, Keiko Sugita, Hiroaki Katayama, and Norio Takahashi Copyright © 2012 Yasunari Satoh et al. All rights reserved. Optimal Control of Gene Mutation in DNA Replication Sun, 22 Jan 2012 15:02:00 +0000 We propose a molecular-level control system view of the gene mutations in DNA replication from the finite field concept. By treating DNA sequences as state variables, chemical mutagens and radiation as control inputs, one cell cycle as a step increment, and the measurements of the resulting DNA sequence as outputs, we derive system equations for both deterministic and stochastic discrete-time, finite-state systems of different scales. Defining the cost function as a summation of the costs of applying mutagens and the off-trajectory penalty, we solve the deterministic and stochastic optimal control problems by dynamic programming algorithm. In addition, given that the system is completely controllable, we find that the global optimum of both base-to-base and codon-to-codon deterministic mutations can always be achieved within a finite number of steps. Juanyi Yu, Jr-Shin Li, and Tzyh-Jong Tarn Copyright © 2012 Juanyi Yu et al. All rights reserved. Expression Pattern of the Alpha-Kafirin Promoter Coupled with a Signal Peptide from Sorghum bicolor L. Moench Tue, 17 Jan 2012 10:48:17 +0000 Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP. Norazlina Ahmad, Rajnesh Sant, Milovan Bokan, Kathryn J. Steadman, and Ian D. Godwin Copyright © 2012 Norazlina Ahmad et al. All rights reserved. ENU Mutagenesis Screen to Establish Motor Phenotypes in Wild-Type Mice and Modifiers of a Pre-Existing Motor Phenotype in Tau Mutant Mice Thu, 15 Dec 2011 18:17:46 +0000 Modifier screening is a powerful genetic tool. While not widely used in the vertebrate system, we applied these tools to transgenic mouse strains that recapitulate key aspects of Alzheimer’s disease (AD), such as tau-expressing mice. These are characterized by a robust pathology including both motor and memory impairment. The phenotype can be modulated by ENU mutagenesis, which results in novel mutant mouse strains and allows identifying the underlying gene/mutation. Here we discuss this strategy in detail. We firstly obtained pedigrees that modify the tau-related motor phenotype, with mapping ongoing. We further obtained transgene-independent motor pedigrees: (i) hyperactive, circling ENU 37 mice with a causal mutation in the Tbx1 gene—the complete knock-out of Tbx1 models DiGeorge Syndrome; (ii) ENU12/301 mice that show sudden jerky movements and tremor constantly; they have a causal mutation in the Kcnq1 gene, modelling aspects of the Romano-Ward and Jervell and Lange-Nielsen syndromes; and (iii) ENU16/069 mice with tremor and hypermetric gait that have a causal mutation in the Mpz (Myelin Protein Zero) gene, modelling Charcot-Marie-Tooth disease type 1 (CMT1B). Together, we provide evidence for a real potential of an ENU mutagenesis to dissect motor functions in wild-type and tau mutant mice. Xin Liu, Michael Dobbie, Rob Tunningley, Belinda Whittle, Yafei Zhang, Lars M. Ittner, and Jürgen Götz Copyright © 2011 Xin Liu et al. All rights reserved. Bacterial Cells Carrying Synthetic Dual-Function Operon Survived Starvation Mon, 28 Nov 2011 11:52:50 +0000 A synthetic dual-function operon with a bistable structure was designed and successfully integrated into the bacterial genome. Bistability was generated by the mutual inhibitory structure comprised of the promoters and and the repressors LacI and TetR. Dual function essential for cell growth was introduced by replacing the genes (i.e., hisC and leuB) encoding proteins involved in the biosynthesis of histidine and leucine from their native chromosomal locations to the synthetic operon. Both colony formation and population dynamics of the cells carrying this operon showed that the cells survived starvation and the newly formed population transited between the two stable states, representing the induced hisC and leuB levels, in accordance with the nutritional status. The results strongly suggested that the synthetic design of proto-operons sensitive to external perturbations is practical and functional in native cells. Yuki Matsumoto, Yoichiro Ito, Saburo Tsuru, Bei-Wen Ying, and Tetsuya Yomo Copyright © 2011 Yuki Matsumoto et al. All rights reserved. Differential Gene Expression in Sugarcane in Response to Challenge by Fungal Pathogen Ustilago scitaminea Revealed by cDNA-AFLP Thu, 14 Jul 2011 11:09:58 +0000 Differential gene expression in sugarcane during sugarcane-Ustilago scitaminea interaction was conducted in a smut-resistant genotype. Using cDNA-AFLP along with silver staining, a total of 136 transcript-derived fragments (TDFs) were found to be differentially expressed in response to challenge by U. scitaminea. Forty TDFs, 34 newly induced plus six with obvious upregulated expression after infection, were sequenced and validated by RT-PCR analysis. These results demonstrated that the expression of 37 out of these TDFs in RT-PCR analysis was consistent with that in cDNA-AFLP analysis. Based on BlastX in NCBI, 28 TDFs were assumed to function in sugarcane under U. scitaminea stress. Analysis of expression profile of three TDFs revealed that they responded differently after infection with U. scitaminea, and the transcription was significantly enhanced. The response of two TDFs, SUC06 and SUC09, occurred before that of SUC10. This study enriches our knowledge of the molecular basis for sugarcane response to U. scitaminea infection. Que You-Xiong, Lin Jian-Wei, Song Xian-Xian, Xu Li-Ping, and Chen Ru-Kai Copyright © 2011 Que You-Xiong et al. All rights reserved. Construction of Papaya Male and Female BAC Libraries and Application in Physical Mapping of the Sex Chromosomes Tue, 05 Jul 2011 08:42:38 +0000 Papaya is a major fruit crop in the tropics and has recently evolved sex chromosomes. Towards sequencing the papaya sex chromosomes, two bacterial artificial chromosome (BAC) libraries were constructed from papaya male and female genomic DNA. The female BAC library was constructed using restriction enzyme BstY I and consists of 36,864 clones with an average insert size of 104 kb, providing 10.3x genome equivalents. The male BAC library was constructed using restriction enzyme EcoR I and consists of 55,296 clones with an average insert size of 101 kb, providing 15.0x genome equivalents. The male BAC library was used in constructing the physical map of the male-specific region of the male Y chromosome (MSY) and in filling gaps and extending the physical map of the hermaphrodite-specific region of the Yh chromosome (HSY) and the X chromosome physical map. The female BAC library was used to extend the X physical map gap. The MSY, HSY, and X physical maps offer a unique opportunity to study chromosomal rearrangements, Y chromosome degeneration, and dosage compensation of the papaya nascent sex chromosomes. Andrea R. Gschwend, Qingyi Yu, Paul Moore, Christopher Saski, Cuixia Chen, Jianping Wang, Jong-Kuk Na, and Ray Ming Copyright © 2011 Andrea R. Gschwend et al. All rights reserved. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells Tue, 17 May 2011 11:25:00 +0000 By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. Anita Collavoli, Laura Comelli, Tiziana Cervelli, and Alvaro Galli Copyright © 2011 Anita Collavoli et al. All rights reserved. Chromosome Mapping of Repetitive Sequences in Rachycentron canadum (Perciformes: Rachycentridae): Implications for Karyotypic Evolution and Perspectives for Biotechnological Uses Wed, 30 Mar 2011 13:41:50 +0000 The cobia, Rachycentron canadum, a species of marine fish, has been increasingly used in aquaculture worldwide. It is the only member of the family Rachycentridae (Perciformes) showing wide geographic distribution and phylogenetic patterns still not fully understood. In this study, the species was cytogenetically analyzed by different methodologies, including Ag-NOR and chromomycin A3 (CMA3)/DAPI staining, C-banding, early replication banding (RGB), and in situ fluorescent hybridization with probes for 18S and 5S ribosomal genes and for telomeric sequences (TTAGGG)n. The results obtained allow a detailed chromosomal characterization of the Atlantic population. The chromosome diversification found in the karyotype of the cobia is apparently related to pericentric inversions, the main mechanism associated to the karyotypic evolution of Perciformes. The differential heterochromatin replication patterns found were in part associated to functional genes. Despite maintaining conservative chromosomal characteristics in relation to the basal pattern established for Perciformes, some chromosome pairs in the analyzed population exhibit markers that may be important for cytotaxonomic, population, and biodiversity studies as well as for monitoring the species in question. Uedson Pereira Jacobina, Marcelo de Bello Cioffi, Luiz Gustavo Rodrigues Souza, Leonardo Luiz Calado, Manoel Tavares, João Manzella Jr., Luiz Antonio Carlos Bertollo, and Wagner Franco Molina Copyright © 2011 Uedson Pereira Jacobina et al. All rights reserved. Genetics, Cytogenetics, and Epigenetics of Colorectal Cancer Mon, 14 Feb 2011 12:17:42 +0000 Most of the colorectal cancer (CRC) cases are sporadic, only 25% of the patients have a family history of the disease, and major genes causing syndromes predisposing to CRC only account for 5-6% of the total cases. The following subtypes can be recognized: MIN (microsatellite instability), CIN (chromosomal instability), and CIMP (CpG island methylator phenotype). CIN occurs in 80–85% of CRC. Chromosomal instability proceeds through two major mechanisms, missegregation that results in aneuploidy through the gain or loss of whole chromosomes, and unbalanced structural rearrangements that lead to the loss and/or gain of chromosomal regions. The loss of heterozygosity that occur in the first phases of the CRC cancerogenesis (in particular for the genes on 18q) as well as the alteration of methylation pattern of multiple key genes can drive the development of colorectal cancer by facilitating the acquisition of multiple tumor-associated mutations and the instability phenotype. Lucia Migliore, Francesca Migheli, Roberto Spisni, and Fabio Coppedè Copyright © 2011 Lucia Migliore et al. All rights reserved. Trisomy 14 as a Sole Chromosome Abnormality Is Associated with Older Age, a Heterogenous Group of Myeloid Neoplasms with Dysplasia, and a Wide Spectrum of Disease Progression Thu, 20 Jan 2011 08:19:53 +0000 Trisomy 14 is a rare recurrent cytogenetic abnormality in myeloid neoplasms; however, its clinicopathologic features have not been well described. We report the clinicopathologic, immunophenotypic, and molecular genetic features of 16 cases of myeloid neoplasms with isolated trisomy 14. Our results show that cases with isolated trisomy 14 encompass a heterogenous group of myeloid neoplasms including myelodysplastic syndrome (MDS, 44%), myelodysplastic/myeloproliferative neoplasms (31%), and acute myeloid leukemia (25%). The patients are usually elder (median age 71 years), and there is a male predominance (82%). Multilineage dysplasia is noted in all cases. Oncogenic mutations of genes involved in cell proliferation and/or survival rarely occur. Compared with cases of MDS with diploid karyotype, patients of MDS with isolated trisomy 14 demonstrate a similar overall survival and rate of leukemia transformation. Wei Cui, Carlos E. Bueso-Ramos, C. Cameron Yin, Jianlan Sun, Su Chen, Ramya Muddasani, and Gary Lu Copyright © 2010 Wei Cui et al. All rights reserved. TP53 Mutations in Nonsmall Cell Lung Cancer Tue, 18 Jan 2011 10:46:59 +0000 The tumor suppressor gene TP53 is frequently mutated in human cancers. Abnormality of the TP53 gene is one of the most significant events in lung cancers and plays an important role in the tumorigenesis of lung epithelial cells. Human lung cancers are classified into two major types, small cell lung cancer (SCLC) and nonsmall cell lung cancer (NSCLC). The latter accounts for approximately 80% of all primary lung cancers, and the incidence of NSCLC is increasing yearly. Most clinical studies suggest that NSCLC with TP53 alterations carries a worse prognosis and may be relatively more resistant to chemotherapy and radiation. A deep understanding of the role of TP53 in lung carcinogenesis may lead to a more reasonably targeted clinical approach, which should be exploited to enhance the survival rates of patients with lung cancer. This paper will focus on the role of TP53 in the molecular pathogenesis, epidemiology, and therapeutic strategies of TP53 mutation in NSCLC. Akira Mogi and Hiroyuki Kuwano Copyright © 2011 Akira Mogi and Hiroyuki Kuwano. All rights reserved. Cytogenetics of Premature Ovarian Failure: An Investigation on 269 Affected Women Mon, 17 Jan 2011 09:33:04 +0000 The importance of X chromosome in the aetiology of premature ovarian failure (POF) is well-known but in many cases POF still remains idiopathic. Chromosome aneuploidy increase is a physiological phenomenon related to aging, but the role of low-level sex chromosome mosaicism in ovarian function is still undiscovered. Standard cytogenetic analysis was carried out in a total of 269 patients affected by POF: 27 chromosomal abnormalities were identified, including X chromosome and autosomal structural and numerical abnormalities. In 47 patients with 46,XX karyotype we performed interphase FISH using X alpha-satellite probe in order to identify X chromosome mosaicism rate. Aneuploidy rate in the patient group was significantly higher than the general population group. These findings underline the importance of X chromosome in the aetiology of POF and highlight the potential role of low-level sex chromosome mosaicism in ovarian aging that may lead to a premature onset of menopause. Simona Baronchelli, Donatella Conconi, Elena Panzeri, Angela Bentivegna, Serena Redaelli, Sara Lissoni, Fabiana Saccheri, Nicoletta Villa, Francesca Crosti, Elena Sala, Emanuela Martinoli, Marinella Volontè, Anna Marozzi, and Leda Dalprà Copyright © 2011 Simona Baronchelli et al. All rights reserved. Using Bacterial Artificial Chromosomes in Leukemia Research: The Experience at the University Cytogenetics Laboratory in Brest, France Tue, 11 Jan 2011 14:52:56 +0000 The development of the bacterial artificial chromosome (BAC) system was driven in part by the human genome project in order to construct genomic DNA libraries and physical maps for genomic sequencing. The availability of BAC clones has become a valuable tool for identifying cancer genes. We report here our experience in identifying genes located at breakpoints of chromosomal rearrangements and in defining the size and boundaries of deletions in hematological diseases. The methodology used in our laboratory consists of a three-step approach using conventional cytogenetics followed by FISH with commercial probes, then BAC clones. One limitation to the BAC system is that it can only accommodate inserts of up to 300 kb. As a consequence, analyzing the extent of deletions requires a large amount of material. Array comparative genomic hybridization (array-CGH) using a BAC/PAC system can be an alternative. However, this technique has limitations also, and it cannot be used to identify candidate genes at breakpoints of chromosomal rearrangements such as translocations, insertions, and inversions. Etienne De Braekeleer, Nathalie Douet-Guilbert, Audrey Basinko, Frédéric Morel, Marie-Josée Le Bris, Claude Férec, and Marc De Braekeleer Copyright © 2011 Etienne De Braekeleer et al. All rights reserved. Contributions of Cytogenetics and Molecular Cytogenetics to the Diagnosis of Adipocytic Tumors Tue, 11 Jan 2011 14:44:36 +0000 Over the last 20 years, a number of tumor-specific chromosomal translocations and associated fusion genes have been identified for mesenchymal neoplasms including adipocytic tumors. The addition of molecular cytogenetic techniques, especially fluorescence in situ hybridization (FISH), has further enhanced the sensitivity and accuracy of detecting nonrandom chromosomal translocations and/or other rearrangements in adipocytic tumors. Indeed, most resent molecular cytogenetic analysis has demonstrated a translocation t(11;16)(q13;p13) that produces a C11orf95-MKL2 fusion gene in chondroid lipoma. Additionally, it is well recognized that supernumerary ring and/or giant rod chromosomes are characteristic for atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma, and amplification of 12q13–15 involving the MDM2, CDK4, and CPM genes is shown by FISH in these tumors. Moreover, myxoid/round cell liposarcoma is characterized by a translocation t(12;16)(q13;p11) that fuses the DDIT3 and FUS genes. This paper provides an overview of the role of conventional cytogenetics and molecular cytogenetics in the diagnosis of adipocytic tumors. Jun Nishio Copyright © 2011 Jun Nishio. All rights reserved. Reverse Genetics Modification of Cytomegalovirus Antigenicity and Immunogenicity by CD8 T-Cell Epitope Deletion and Insertion Sun, 26 Dec 2010 20:08:41 +0000 The advent of cloning herpesviral genomes as bacterial artificial chromosomes (BACs) has made herpesviruses accessible to bacterial genetics and has thus revolutionised their mutagenesis. This opened all possibilities of reverse genetics to ask scientific questions by introducing precisely accurate mutations into the viral genome for testing their influence on the phenotype under study or to create phenotypes of interest. Here, we report on our experience with using BAC technology for a designed modulation of viral antigenicity and immunogenicity with focus on the CD8 T-cell response. One approach is replacing an intrinsic antigenic peptide in a viral carrier protein with a foreign antigenic sequence, a strategy that we have termed “orthotopic peptide swap”. Another approach is the functional deletion of an antigenic peptide by point mutation of its C-terminal MHC class-I anchor residue. We discuss the concepts and summarize recently published major scientific results obtained with immunological mutants of murine cytomegalovirus. Niels A. W. Lemmermann, Kai A. Kropp, Christof K. Seckert, Natascha K. A. Grzimek, and Matthias J. Reddehase Copyright © 2011 Niels A. W. Lemmermann et al. All rights reserved. The Selection and Use of Sorghum (Sorghum propinquum) Bacterial Artificial Chromosomes as Cytogenetic FISH Probes for Maize (Zea mays L.) Wed, 22 Dec 2010 12:33:17 +0000 The integration of genetic and physical maps of maize is progressing rapidly, but the cytogenetic maps lag behind, with the exception of the pachytene fluorescence in situ hybridization (FISH) maps of maize chromosome 9. We sought to produce integrated FISH maps of other maize chromosomes using Core Bin Marker loci. Because these 1 Kb restriction fragment length polymorphism (RFLP) probes are below the FISH detection limit, we used BACs from sorghum, a small-genome relative of maize, as surrogate clones for FISH mapping. We sequenced 151 maize RFLP probes and compared in silico BAC selection methods to that of library filter hybridization and found the latter to be the best. BAC library screening, clone verification, and single-clone selection criteria are presented along with an example of transgenomic BAC FISH mapping. This strategy has been used to facilitate the integration of RFLP and FISH maps in other large-genome species. Debbie M. Figueroa, James D. Davis, Cornelia Strobel, Maria S. Conejo, Katherine D. Beckham, Brian C. Ring, and Hank W. Bass Copyright © 2011 Debbie M. Figueroa et al. All rights reserved. Characteristics of Highly Polymorphic Segmental Copy-Number Variations Observed in Japanese by BAC-Array-CGH Sun, 19 Dec 2010 12:05:51 +0000 Segmental copy-number variations (CNVs) may contribute to genetic variation in humans. Reports of the existence and characteristics of CNVs in a large Japanese cohort are quite limited. We report the data from a large Japanese population. We conducted population screening for 213 unrelated Japanese individuals using comparative genomic hybridization based on a bacterial artificial chromosome microarray (BAC-aCGH). We summarize the data by focusing on highly polymorphic CNVs in ≥5.0% of the individual, since they may be informative for demonstrating the relationships between genotypes and their phenotypes. We found a total of 680 CNVs at 16 different BAC-regions in the genome. The majority of the polymorphic CNVs presented on BAC-clones that overlapped with regions of segmental duplication, and the majority of the polymorphic CNVs observed in this population had been previously reported in other publications. Some of the CNVs contained genes which might be related to phenotypic heterogeneity among individuals. Norio Takahashi, Yasunari Satoh, Keiko Sasaki, Yuko Shimoichi, Keiko Sugita, and Hiroaki Katayama Copyright © 2011 Norio Takahashi et al. All rights reserved. BACs as Tools for the Study of Genomic Imprinting Mon, 13 Dec 2010 09:02:38 +0000 Genomic imprinting in mammals results in the expression of genes from only one parental allele. Imprinting occurs as a consequence of epigenetic marks set down either in the father's or the mother's germ line and affects a very specific category of mammalian gene. A greater understanding of this distinctive phenomenon can be gained from studies using large genomic clones, called bacterial artificial chromosomes (BACs). Here, we review the important applications of BACs to imprinting research, covering physical mapping studies and the use of BACs as transgenes in mice to study gene expression patterns, to identify imprinting centres, and to isolate the consequences of altered gene dosage. We also highlight the significant and unique advantages that rapid BAC engineering brings to genomic imprinting research. S. J. Tunster, M. Van De Pette, and R. M. John Copyright © 2011 S. J. Tunster et al. All rights reserved. Bacterial Artificial Chromosome Mutagenesis Using Recombineering Thu, 09 Dec 2010 13:33:26 +0000 Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo. Kumaran Narayanan and Qingwen Chen Copyright © 2011 Kumaran Narayanan and Qingwen Chen. All rights reserved. Sperm DNA Integrity and Meiotic Behavior Assessment in an Infertile Male Carrier of a 9qh+++ Polymorphism Thu, 09 Dec 2010 11:58:17 +0000 Although several reports on male infertility suggest a relationship between chromosome 9 polymorphisms and infertility, the effects on the phenotype have not been extensively reported. In this study, an infertile patient was found to carry a 9qh+++ chromosome. The flow cytometric TUNEL assay and SCD test have been applied to characterize sperm DNA integrity. In order to assess its meiotic behaviour, synapsis, recombination, and aneuploidy, analyses have been also performed. Sperm DNA fragmentation (SDF) was 77.81% and 87% for the TUNEL and SCD tests, respectively. Ninety-two percent of pachytene cells analyzed showed meiotic abnormalities. The mean number of MLH1 foci per pachytene in the control group was higher (49) than the mean found in the 9qh+++ patient (38) (𝑃<.0001). In spermatozoa, significant increases of disomy rates were observed for chromosome 18 and for the sex chromosomes (𝑃<.0001). These disturbances could be present in other male carriers of a less marked 9qh+. A. García-Peiró, M. Oliver-Bonet, J. Navarro, C. Abad, M. Guitart, M. J. Amengual, and J. Benet Copyright © 2011 A. García-Peiró et al. All rights reserved. Genetic Variation of VKORC1 and CYP4F2 Genes Related to Warfarin Maintenance Dose in Patients with Myocardial Infarction Wed, 24 Nov 2010 15:49:07 +0000 The aim of this study was to investigate whether the VKORC1*3 (rs7294/9041 G > A), VKORC1*4 (rs17708472/6009 C > T), and CYP4F2 (rs2108622/1347 C > T) polymorphisms were associated with elevated warfarin maintenance dose requirements in patients with myocardial infarction () from the Warfarin Aspirin Reinfarction Study (WARIS-II). We found significant associations between elevated warfarin dose requirements and VKORC1*3 and VKORC1*4 polymorphisms ( and , resp.), whereas CYP4F2 (1347 C > T) showed a weak association on higher warfarin dose requirements (). However, analysing these variant alleles in a regression analysis together with our previously reported data on VKORC1*2, CYP2C9*2 and CYP2C9*3 polymorphisms, gave no significant associations for neither VKORC1*3, VKORC1*4 nor CYP4F2 (1347 C > T). In conclusion, in patients with myocardial infarction, the individual contribution to warfarin dose requirements from VKORC1*3, VKORC1*4, and CYP4F2 (1347 C > T) polymorphisms was negligible. Our results indicate that pharmacogenetic testing for VKORC1*2, CYP2C9*2 and CYP2C9*3 is more informative regarding warfarin dose requirements than testing for VKORC1*3, VKORC1*4, and CYP4F2 (1347 C > T) polymorphisms. Marianne K. Kringen, Kari Bente Foss Haug, Runa M. Grimholt, Camilla Stormo, Sigrid Narum, Mimi S. Opdal, Jan Toralf Fosen, Armin P. Piehler, Per W. Johansen, Ingebjørg Seljeflot, Jens Petter Berg, and Odd Brørs Copyright © 2011 Marianne K. Kringen et al. All rights reserved. A Comparative BAC Map for the Gilthead Sea Bream (Sparus aurata L.) Wed, 13 Oct 2010 13:39:30 +0000 This study presents the first comparative BAC map of the gilthead sea bream (Sparus aurata), a highly valuated marine aquaculture fish species in the Mediterranean. High-throughput end sequencing of a BAC library yielded 92,468 reads (60.6 Mbp). Comparative mapping was achieved by anchoring BAC end sequences to the three-spined stickleback (Gasterosteus aculeatus) genome. BACs that were consistently ordered along the stickleback chromosomes accounted for 14,265 clones. A fraction of 5,249 BACs constituted a minimal tiling path that covers 73.5% of the stickleback chromosomes and 70.2% of the genes that have been annotated. The N50 size of 1,485 “BACtigs” consisting of redundant BACs is 337,253 bp. The largest BACtig covers 2.15 Mbp in the stickleback genome. According to the insert size distribution of mapped BACs the sea bream genome is 1.71-fold larger than the stickleback genome. These results represent a valuable tool to researchers in the field and may support future projects to elucidate the whole sea bream genome. Heiner Kuhl, Elena Sarropoulou, Mbaye Tine, Georgios Kotoulas, Antonios Magoulas, and Richard Reinhardt Copyright © 2011 Heiner Kuhl et al. All rights reserved. Sex Chromosome Evolution in Amniotes: Applications for Bacterial Artificial Chromosome Libraries Tue, 12 Oct 2010 11:38:49 +0000 Variability among sex chromosome pairs in amniotes denotes a dynamic history. Since amniotes diverged from a common ancestor, their sex chromosome pairs and, more broadly, sex-determining mechanisms have changed reversibly and frequently. These changes have been studied and characterized through the use of many tools and experimental approaches but perhaps most effectively through applications for bacterial artificial chromosome (BAC) libraries. Individual BAC clones carry 100–200 kb of sequence from one individual of a target species that can be isolated by screening, mapped onto karyotypes, and sequenced. With these techniques, researchers have identified differences and similarities in sex chromosome content and organization across amniotes and have addressed hypotheses regarding the frequency and direction of past changes. Here, we review studies of sex chromosome evolution in amniotes and the ways in which the field of research has been affected by the advent of BAC libraries. Daniel E. Janes, Nicole Valenzuela, Tariq Ezaz, Chris Amemiya, and Scott V. Edwards Copyright © 2011 Daniel E. Janes et al. All rights reserved. High Prevalence of Alpha- and Beta-Thalassemia in the Kadazandusuns in East Malaysia: Challenges in Providing Effective Health Care for an Indigenous Group Sun, 05 Sep 2010 09:05:32 +0000 Thalassemia can lead to severe transfusion-dependent anemia, and it is the most common genetic disorder in Malaysia. This paper aims to determine the prevalence of thalassemia in the Kadazandusuns, the largest indigenous group in Sabah, East Malaysia. - and -thalassemia were confirmed in 33.6% and 12.8%, of the individuals studied respectively. The high prevalence of - and -thalassemia in the Kadazandusuns indicates that thalassemia screening, genetic counseling, and prenatal diagnosis should be included as part of their healthcare system. This preliminary paper serves as a baseline for further investigations into the health and genetic defects of the major indigenous population in Sabah, East Malaysia. Jin-Ai Mary Anne Tan, Ping-Chin Lee, Yong-Chui Wee, Kim-Lian Tan, Noor Fadzlin Mahali, Elizabeth George, and Kek-Heng Chua Copyright © 2010 Jin-Ai Mary Anne Tan et al. All rights reserved. Cytogenetic Instability in Childhood Acute Lymphoblastic Leukemia Survivors Tue, 31 Aug 2010 11:03:51 +0000 Contemporary anticancer therapies have largely improved the outcome for children with cancer, especially for Acute Lymphoblastic Leukemia (ALL). Actually, between 78% and 85% of patients achieve complete remission and are alive after 5 years of therapy completion. However, as cure rates increase, new concerns about the late effects of genotoxic treatment emerge, being the risk of developing secondary neoplasias, the most serious life-threatening rising problem. In the present paper, we describe and review the cytogenetic findings in peripheral lymphocytes from ALL survivors, and discuss aspects associated to the occurrence of increased chromosome rearrangements in this growing cohort. María Sol Brassesco, Danilo Jordão Xavier, Marjori Leiva Camparoto, Ana Paula Montaldi, Paulo Roberto D'Auria Vieira de Godoy, Carlos Alberto Scrideli, Luiz Gonzaga Tone, and Elza Tiemi Sakamoto-Hojo Copyright © 2011 María Sol Brassesco et al. All rights reserved. Prospects for the Use of Artificial Chromosomes and Minichromosome-Like Episomes in Gene Therapy Tue, 24 Aug 2010 10:50:07 +0000 Artificial chromosomes and minichromosome-like episomes are large DNA molecules capable of containing whole genomic loci, and be maintained as nonintegrating, replicating molecules in proliferating human somatic cells. Authentic human artificial chromosomes are very difficult to engineer because of the difficulties associated with centromere structure, so they are not widely used for gene-therapy applications. However, OriP/EBNA1-based episomes, which they lack true centromeres, can be maintained stably in dividing cells as they bind to mitotic chromosomes and segregate into daughter cells. These episomes are more easily engineered than true human artificial chromosomes and can carry entire genes along with all their regulatory sequences. Thus, these constructs may facilitate the long-term persistence and physiological regulation of the expression of therapeutic genes, which is crucial for some gene therapy applications. In particular, they are promising vectors for gene therapy in inherited diseases that are caused by recessive mutations, for example haemophilia A and Friedreich's ataxia. Interestingly, the episome carrying the frataxin gene (deficient in Friedreich's ataxia) has been demonstrated to rescue the susceptibility to oxidative stress which is typical of fibroblasts from Friedreich's ataxia patients. This provides evidence of their potential to treat genetic diseases linked to recessive mutations through gene therapy. Sara Pérez-Luz and Javier Díaz-Nido Copyright © 2010 Sara Pérez-Luz and Javier Díaz-Nido. All rights reserved. Analysis of Horse Myostatin Gene and Identification of Single Nucleotide Polymorphisms in Breeds of Different Morphological Types Wed, 14 Jul 2010 11:51:20 +0000 Myostatin (MSTN) is a negative modulator of muscle mass. We characterized the horse (Equus caballus) MSTN gene and identified and analysed single nucleotide polymorphisms (SNPs) in breeds of different morphological types. Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2. The SNPs of the promoter (GQ183900:g.26T>C and GQ183900:g.156T>C, the latter located within a conserved TATA-box like motif) were screened in 396 horses from 16 breeds. The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type) than in light breeds (dolichomorphic type such as Italian Trotter breed). The significant difference of allele frequencies for the SNPs at the promoter and analysis of molecular variance (AMOVA) on haplotypes indicates that these polymorphisms could be associated with variability of morphology traits in horse breeds. Stefania Dall'Olio, Luca Fontanesi, Leonardo Nanni Costa, Marco Tassinari, Laura Minieri, and Adalberto Falaschini Copyright © 2010 Stefania Dall'Olio et al. All rights reserved. Gene Transfer into the Lung by Nanoparticle Dextran-Spermine/Plasmid DNA Complexes Wed, 30 Jun 2010 15:46:17 +0000 A novel cationic polymer, dextran-spermine (D-SPM), has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA) in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range. Syahril Abdullah, Wai Yeng Wendy-Yeo, Hossein Hosseinkhani, Mohsen Hosseinkhani, Ehab Masrawa, Rajesh Ramasamy, Rozita Rosli, Sabariah A. Rahman, and Abraham J. Domb Copyright © 2010 Syahril Abdullah et al. All rights reserved.