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- Table of Contents
Volume 35 (2013), Issue 2, Pages 67–72
Early Complication in Sickle Cell Anemia Children due to A(TA)nTAA Polymorphism at the Promoter of UGT1A1 Gene
1Laboratoire d’Hématologie Moléculaire et Cellulaire, Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia
2Université de Tunis El Manar, Tunis, Tunisia
3Département de Biologie Clinique, Institut Salah Azaiez de Cancer, Université de Tunis El Manar, Tunis, Tunisia
4Département de Biochimie, Hôpital de Traumatologie et des Grands Brulés, Université de Tunis El Manar, Ben Arous, Tunisia
Received 7 November 2012; Accepted 13 April 2013
Academic Editor: Sudhir Srivastava
Copyright © 2013 Leila Chaouch et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aim. To determine the implication of the polymorphism, namely, A(TA)nTAA of UGT1A1 in lithogenesis for the first time in Tunisia among sickle cell anemia (SCA) children patients. Material and Methods. Our study was performed in 2010 and it involved 76 subjects chosen as control group characterized with normal hemoglobin status and presence of cholelithiasis and 102 SCA pediatric patients among whom 52 have cholelithiasis. We analyzed the polymorphism A(TA)nTAA at the UGT1A1 promoter and the relationships between the various A(TA)nTAA genotypes and alleles and bilirubin levels and occurrence of cholelithiasis. Results and Discussion. The repartition of genotypes found according to serum bilirubin level shows a significant association between genotypes carrying variant (TA)7 and hyperbilirubinemia (). We demonstrated the association of two genotypes with gallstones formation among SCA children patients: (TA)7/(TA)7 and (TA)7/(TA)8 with and , respectively. (TA)7 and (TA)8 allele variants act as a risk factor for early gallstones formation in SCA patients with and , respectively. As for the control group only the genotype (TA)7/(TA)7 presented a risk factor for gallstones formation. Conclusion. The novelty of this report is that it is the first time that a similar study was made on the Tunisian children sickle cell population and that the results show a clear association of (TA)7 variant in early gallstones formation in Tunisian SCA children. Interestingly our findings highlighted the association of (TA)8 variant as well, which was not found in previous studies.
SCA is a heterogeneous monogenic disease due to a single mutation A/T at the sixth codon of the β-globin gene (βS) . The clinical complications arising from sickle cell disease include vasoo-cclusive crisis and its outcomes . As a result of chronic hemolysis, hyperbilirubinemia is often observed leading to the formation of pigment cholelithiasis which could be busted by the presence of UGT1A1 defects. Indeed UGT1A1 gene encodes the uridine diphosphate glucuronosyltransferase 1A1, enzyme responsible for bilirubin glucuronidation . The UGT1A1 gene is located in chromosome 2q37 . Various UGT1A1 gene defects and polymorphisms have been described so far at the origin of reduced enzyme activity . Among these, a variation in the number of TA repeat at the A(TA)6TAA nucleotide sequence in the promoter region, considered as the wild type. In fact, the addition of an extra (TA) at this sequence leads to a variant A(TA)7TAA which was described to cause reduced glucuronidation and hyperbilirubinemia associated with the Gilbert syndrome [2, 5]. This variation at the promoter seems to interfere with binding of the transcription factor IID which is responsible for the transcription of UGT1A1 gene. In fact, The A(TA)nTAA element is the binding site for transcription factor IID, which is one of the factors responsible for the initiation of transcription and the presence of this longer A(TA)nTAA element in the promoter region of the gene for bilirubin UDP-glucuronosyltransferase 1 resulting in reduced expression of bilirubin-UGT1 (30% of normal) and hence causing unconjugated hyperbilirubinemia . Studies of a possible association between polymorphisms of candidate genes related to the modulation of clinical complications of SCA have shown that sickle cell patients who carry the variation (TA)7 are favorable for gallstone formation [4–11]. Besides, other studies have shown the correlation of cholelithiasis and A(TA)7TAA variant of UGT1A1 promoter with chronic hemolytic diseases such as thalassemia minor, which represent a risk factor for cholelithiasis and the Gilbert mutation further increases this risk [12–16]. The prevalence of cholelithiasis observed in SCA children is about 30% reported for different ethnical groups (United States, Guadeloupe) [17, 18].
In this paper, we intend to study the impact of A(TA)nTAA variation at the UGT1A1 gene promoter on hyperbilirubinemia and on the occurrence of cholelithiasis for the first time among SCA Tunisian children. SCA is the second sickle cell hemoglobinopathy after β-thalassemia in Tunisia, representing a real public health problem. The average frequency of the trait in our country is 1.89% . The organization of care of sickle cell disease children in Tunisia is the National Center of Bone Marrow Transplantation.
76 subjects with cholelithiasis and 102 sickle cell patients were involved in this study performed in 2010. Patients were selected on the basis of homozygosity for β-globin gene from National Center of Bone Marrow Transplantation, Tunis, Tunisia. All SCA patients are children (less than 16 years old) and were characterized by hyperbilirubinemia and 52 of them have cholelithiasis.
2.2.1. Clinical Events Analyzed
Liver/biliary ultrasound scans were performed annually to detect cholelithiasis only in SCA patients over the age of three years. Cholelithiasis was diagnosed for all patients on the basis of echodense images within the gallbladder with acoustic shadowing or gravitational change in position.
2.2.2. Laboratory Methods
Diagnosis of sickle cell patient is performed using cation-exchange high-performance liquid chromatography (HPLC) (D10 Biorad) and further confirmation by means of molecular diagnosis by restriction fragment length polymorphism (RFLP) using DdeI as previously described by Bachir 2000 . Biochemical data were averaged for each patient in steady state (at least three values). We determined total and fetal hemoglobin (HbF) concentrations (D10, Biorad) and reticulocyte count and other hematologic parameters using (ABX pentra 60c+). Total unconjugated and conjugated bilirubin concentrations in serum were determined by a standardized colorimetric procedure (Cobras Integra, Meylan, France).
2.2.3. A(TA)nTAA Genotyping
Genomic DNA was isolated from white blood cells of total blood using standard method (phenol/chloroform). A(TA)nTAA sequences were genotyped by polymerase chain reaction (PCR) using a couple of primers, namely, TAF: 5′-TCGTCCTTCTTCCTCTCTGG-3′ and TAR: 5′-TCCTGCTCCTGCCAGAGGTT-3′. Polymerase chain reaction was performed in 25 μL reaction volumes containing 100 ng of genomic DNA, 0.2 mmol/L of each dNTP, 50 mmol/L KCl, 15 mmol/L Tris-HCl PH 8.0, 2.5 mmol/L MgCl2, 0.5 U AmpliTaq polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA), and 10 pmol of each forward and reverse primers. The PCR cycling conditions included an initial denaturation of 10 min at 96°C followed by 35 cycles of 96°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min. The run was ended by a final extension at 72°C for 7 min.
PCR products were then purified and doubly sequenced (forward and reverse) by ABI PRISM Big Dye Terminator on Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) and an ABI 310 DNA sequencer (PEApplied Biosystems, Foster City, USA).
2.3. Data Analysis
The sample of SCA patients was divided into two groups according to the presence or absence of cholelithiasis. 76 patients with normal hemoglobin (AA) and presented cholelithiasis were enrolled in the analysis. We compared demographic and hematological and clinical data between the groups of patients. As for A(TA)nTAA polymorphism genetic differences between the groups were evaluated. We defined two intervals of total bilirubin levels. The first includes total bilirubin value <35 μmol/L which is the critical value of total bilirubin associated with the Gilbert syndrome. The second interval includes bilirubin value higher than the cutting point 35 μmol/L. We investigated the relationships between genotypes found and these intervals.
2.4. Statistical Analysis
The demographic and hematologic data are normally distributed, so we used means and standard deviations. The bilirubin data are not normally distributed, so we used medians. For each variable (demographic, hematological, and biochemical) differences between cases and controls were evaluated applying the t-test or the nonparametric Mann-Whitney test as appropriate using SPSS (version 18). The Hardy-Weinberg equilibrium was tested using the software package Arlequin (version 3.01). Genetic differences between cases and controls were evaluated applying exact tests to genotypic or allelic contingency tables using compare 2 (version 1.02). The relationships between genotypes found and total bilirubin level were evaluated applying Fisher’s exact test using compare 2 (version 1.02). We calculated values for the entire tests and Fisher’s exact test and chi-squared test were used as appropriate.
3.1. Demographic, Hematological, and Biochemical Analysis
The distribution of each continuous variable was performed using the nonparametric Mann-Whitney test. Our results show that there is no significant difference between the two groups of SCA patient according to the presence or the absence of cholelithiasis (), whereas, the comparison of total conjugated and unconjugated bilirubin concentrations between the two groups of SS children patients shows a significant difference with . Our findings show a significant difference between SCA patients and patients with cholelithiasis considered as control group with (Table 1).
3.2. Analysis of the A(TA)nTAA Polymorphism
All samples were found to be in Hardy-Weinberg equilibrium () for A(TA)nTAA polymorphism. Our results show the presence of seven genotypes, namely, (TA)5/(TA)6, (TA)6/(TA)6, (TA)6/(TA)7, (TA)7/(TA)7, (TA)5/(TA)7, (TA)7/(TA)8, and (TA)8/(TA)8. The distribution of genotypes between children with gallstones and who are without gallstones and the control group are shown in Table 2. The comparison of these genotypes according to the number of (TA) reported that the genotypes (TA)7/(TA)7 and (TA)7/(TA)8 were significantly associated with SCA patients with gallstones (). Our results show a significant association between genotype (TA)7/(TA)7 and gallstones in the control group. Moreover, (TA)7 and (TA)8 allelic variants are found to be associated with gallstones in SCA children patients () Table 2. Association is not found in the control group.
3.3. Relationship between Total Bilirubin Levels and A(TA)nTAA Polymorphism
The repartition of different genotypes depending on two intervals representing bilirubin level in SCA children shows that genotypes carrying (TA)7 and (TA)8 alleles are associated with increased bilirubin level. Similar results were found in the control group (Tables 3(a) and 3(b)).
In the current study we tested 102 SCA Tunisian children patients among whom 52 have cholelithiasis and 76 subjects were chosen as control group characterized with normal hemoglobin status and presence of cholelithiasis and analyzed the polymorphism at the promoter and the relationships between the various UGT1A1 promoter genotypes and alleles and bilirubin levels. In a previous study we were interested to determine the frequency of A(TA)nTAA and Gly71Arg of UGT1A1 in a healthy population . The polymorphism A(TA)nTAA showed that genotype (TA)7/(TA)7 described as being associated with Gilbert’s syndrome was encountered in 11% of the population studied. This percentage is close to the value described in the Caucasian population, estimated at 10% [22, 23]. Concerning the polymorphism Gly71Arg, our results show that the mutated allele is encountered in 15.7% of our studied population. This frequency differs greatly from that reported for Caucasians and Afro-Americans but it is similar to that perceived at the Japanese population [24–28]. All these results suggest that the Tunisian population appears to be heterogeneous for UGT1A1 gene mutation status. The heterogeneity of Tunisian population for SCA haplotypes has been reported previously by Imen et al., 2011 . The authors have demonstrated the predominance of the Benin haplotype in SCA patients suggests that the βS mutation present in Tunisia may have originated from the Benin region and was brought to Tunisia along the slave trade routes. However, they have reported the presence of another atypical haplotype that could be considered as specific to Tunisian chromosome βS. In a previous study we have been interested in the implication of the polymorphism A(TA)nTAA of UGT1A1 in occurrence of cholelithiasis among Tunisian patients with normal hemoglobin status. Our findings have showed that subjects with (TA)7 or (TA)8 variant in their genotypes are associated with high bilirubin level. Furthermore, we have demonstrated that (TA)6/(TA)7 and (TA)7/(TA)7 genotypes and (TA)7 and (TA)8 alleles were significantly associated with an increased risk of gallstone diseases , , and , respectively . In this study, our results show that total bilirubin level increased with the genotypes (TA)6/(TA)7, (TA)7/(TA)7, and (TA)7/(TA)8 (; and ), respectively. In fact, the addition of an extra (TA) at the TATA box seems to interfere with binding of the transcription factor IID which is responsible for the transcription of UGT1A1 gene. This interference leads to the reduced expression of UGT1A1 and hence in the expression of bilirubin-UGT1 (30% of normal) . Furthermore, our findings show a significant association between genotypes (TA)7/(TA)7 and (TA)7/(TA)8 and cholelithiasis in SCA children patients with a value of and , respectively. (TA)7 and (TA)8 allele variants are found to be associated with cholelithiasis among SCA children with and , respectively. In order to interpret the effect of the A(TA)nTAA promoter polymorphism on the lithogenesis in SCA patients our results were compared against those of a control group. We demonstrated that the genotype (TA)7/(TA)7 was associated with cholelithiasis in SCA and in the control group by cons (TA)7/(TA)8 was associated only among SCA children patients. Our results are similar with those of Chaar et al. on Guadeloupe SCA patients , where they demonstrated that frequency of cholelithiasis was significantly higher in both adult and children patients with (TA)7/(TA)7 and (TA)7/(TA)8 genotypes compared to those with other genotypes. Our results are similar to those of previous studies concerning (TA)7 variant which presents an excess risk for gallstone occurring in children patients with SCA and thalassemia (minor, intermedia, and β0) [4–16]. Outside of hemolytic disease, (TA)7 variant has been reported by many studies to be associated with both hyperbilirubinemia and cholelithiasis [25, 26, 28, 30, 31]. Herein, we demonstrated the association of the genotype (TA)7/(TA)7 with cholelithiasis in SCA patients and in the control group. Our study is the first findings about the implication of A(TA)nTAA of UGT1A1 in lithogenesis among SCA children patients in Tunisia. Our data confirmed the role of (TA)7 variant and highlighted the role of (TA)8 in early gallstones formation; association is not found in previous studies. As future directions of our research, we will focus on the Gly71Arg polymorphism in the first exon of the UGT1A1 gene reported to be associated with the same phenotypes [2, 25, 27, 32, 33]. Also we will focus on other candidate genes which can be associated with both hyperbilirubinemia and cholelithiasis in SCA such as SLCO1BI and SLCO1A2 .
The novelty of this report is that it is the first time that a similar study was made on the Tunisian children sickle cell population and that the results show a clear association of (TA)7/(TA)7 and (TA)7/(TA)8 genotypes as well as the (TA)7 allele with the cholelithiasis and hyperbilirubinemia. Interestingly our findings show the association of (TA)8 allele with the cholelithiasis, association not described previously in other population.
|:||Index of significance|
|SCA:||Sickle cell anemia|
|UGT1A1:||Uridine diphosphoglucuronosyltransferase 1A1.|
- R. H. Tukey and C. P. Strassburg, “Human UDP-glucuronosyltransferases: metabolism, expression, and disease,” Annual Review of Pharmacology and Toxicology, vol. 40, pp. 581–616, 2000.
- P. J. Bosma, J. R. Chowdhury, C. Bakker et al., “The genetic basis of the reduced expression of bilirubin UDP- glucuronosyltransferase 1 in Gilbert's syndrome,” The New England Journal of Medicine, vol. 333, no. 18, pp. 1171–1175, 1995.
- E. Beutler, T. Gelbart, and A. Demina, “Racial variability in the UDP-glucuronosyltransferase 1 (UGT1A1) promoter: a balanced polymorphism for regulation of bilirubin metabolism?” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 14, pp. 8170–8174, 1998.
- M. Imen, B. M. M. Ikbel, C. Leila et al., “Restriction mapping of locus among tunisian sickle-cell patients,” American Journal of Human Biology, vol. 23, no. 6, pp. 815–819, 2011.
- E. V. Haverfield, C. A. McKenzie, T. Forrester et al., “UGT1A1 variation and gallstone formation in sickle cell disease,” Blood, vol. 105, no. 3, pp. 968–972, 2005.
- S. L. Carpenter, S. Lieff, T. A. Howard, B. Eggleston, and R. E. Ware, “UGT1A1 promoter polymorphisms and the development of hyperbilirubinemia and gallbladder disease in children with sickle cell anemia,” American Journal of Hematology, vol. 83, no. 10, pp. 800–803, 2008.
- V. Chaar, L. Kéclard, J. P. Diara et al., “Association of UGT1A1 polymorphism with prevalence and age at onset of cholelithiasis in sickle cell anemia,” Haematologica, vol. 90, no. 2, pp. 188–193, 2005.
- A. Driss, K. O. Asare, J. M. Hibbert, B. E. Gee, T. V. Adamkiewicz, and J. K. Stiles, “Sickle cell disease in the post genomic era: a monogenic disease with a polygenic phenotype,” Genomics Insights, vol. 2, pp. 23–48, 2009.
- K. Y. Fertrin, M. B. Melo, Â. M. Assis, S. T. O. Saad, and F. F. Costa, “UDP-glucuronosyltransferase 1 gene promoter polymorphism is associated with increased serum bilirubin levels and cholecystectomy in patients with sickle cell anemia,” Clinical Genetics, vol. 64, no. 2, pp. 160–162, 2003.
- R. Martins, A. Morais, A. Dias et al., “Early modification of sickle cell disease clinical course by UDP-glucuronosyltransferase 1A1 gene promoter polymorphism,” Journal of Human Genetics, vol. 53, no. 6, pp. 524–528, 2008.
- J. N. Milton, P. Sebastiani, N. Solovieff et al., “A genome-wide association study of total Bilirubin and Cholelithiasis risk in sickle cell anemia,” PLoS One, vol. 7, no. 4, Article ID e34741, 2012.
- C. Borgna-Pignatti, F. Rigon, L. Merlo et al., “Thalassemia minor, the Gilbert mutation, and the risk of gallstones,” Haematologica, vol. 88, no. 10, pp. 1106–1109, 2003.
- R. Galanello, S. Piras, S. Barella et al., “Cholelithiasis and Gilbert's syndrome in homozygous β-thalassaemia,” British Journal of Haematology, vol. 115, no. 4, pp. 926–928, 2001.
- V. Kalotychou, K. Antonatou, R. Tzanetea, E. Terpos, D. Loukopoulos, and Y. Rombos, “Analysis of the A(TA)nTAA configuration in the promoter region of the UGT1 A1 gene in Greek patients with thalassemia intermedia and sickle cell disease,” Blood Cells, Molecules, and Diseases, vol. 31, no. 1, pp. 38–42, 2003.
- G. Lahr, J. Brintrup, S. Over, G. E. Feurle, K.-M. Debatin, and E. Kohne, “Codon 104(-G), a dominant β°-thalassemia-like phenotype in a German Caucasian family is associated with mild chronic hemolytic anemia but influenced in severity by co-inherited genetic factors,” Haematologica, vol. 92, no. 9, pp. 1264–1265, 2007.
- R. G. Passon, T. A. Howard, S. A. Zimmerman, W. H. Schultz, and R. E. Ware, “Influence of bilirubin uridine diphosphate-glucuronosyltransferase 1A promoter polymorphisms on serum bilirubin levels and cholelithiasis in children with sickle cell anemia,” Journal of Pediatric Hematology/Oncology, vol. 23, no. 7, pp. 448–451, 2001.
- B. S. Lachman, J. Lazerson, and R. J. Starshak, “The prevalence of cholelithiasis in sickle cell disease as diagnosed by ultrasound and cholecystography,” Pediatrics, vol. 64, no. 5, pp. 601–603, 1979.
- A. Premawardhena, C. A. Fisher, F. Fathiu et al., “Genetic determinants of jaundice and gallstones in haemoglobin E β thalassaemia,” The Lancet, vol. 357, no. 9272, pp. 1945–1946, 2001.
- S. Fattoum, “Les hémoglobinopathies en Tunisie: revue actualisée des données épidémiologiques et moléculaires,” Tunisie Médicale, vol. 84, pp. 687–696, 2006.
- D. Bachir, “La drépanocytose,” Revue Française des Laboratoires, vol. 324, pp. 29–35, 2000.
- L. Chaouch, I. Mahjoubi, I. Louati et al., “Polymorphismes alléliques du gène UDP-glucuronosyltransférase1A1 dans une population Tunisienne,” Les Archives de l’Institut Pasteur de Tunis, vol. 88, pp. 1–4, 2011.
- B. Ostanek, D. Furlan, T. Mavec, and J. Lukac-Bajalo, “UGT1A1(TA)n promoter polymorphism—a new case of a (TA)8 allele in Caucasians,” Blood Cells, Molecules, and Diseases, vol. 38, no. 2, pp. 78–82, 2007.
- B. L. Bihan-Levaufre, J. Francoual, J. Chalas et al., “Prévalence génotypique de la maladie de Gilbert en France,” Gastroentérologie Clinique et Biologique, vol. 25, no. 5, pp. 557–558, 2001.
- C.-S. Huang, G.-A. Luo, M.-J. Huang, S.-C. Yu, and S.-S. Yang, “Variations of the bilirubin uridine-diphosphoglucuronosyl transferase 1A1 gene in healthy Taiwanese,” Pharmacogenetics, vol. 10, no. 6, pp. 539–544, 2000.
- A. Kadakol, S. S. Ghosh, B. S. Sappal, G. Sharma, J. R. Chowdhury, and N. R. Chowdhury, “Genetic lesions of bilirubin-diphosphoglucuronate glucosyltransferase (UGT1A1) causing Crigler-Najjar Gilbert syndromes: correlation of genotype to phenotype,” Human Mutation, vol. 4, pp. 297–306, 2000.
- N. Kaniwa, K. Kurose, H. Jinno et al., “Racial variability in haplotype frequencies of UGT1A1 and glucuronidation activity of a novel single nucleotide polymorphism 686C>T (P229L) found in an African-American,” Drug Metabolism and Disposition, vol. 33, no. 3, pp. 458–465, 2005.
- O. Koiwai, M. Nishizawa, K. Hasada et al., “Gilbert's syndrome is caused by a heterozygous missense mutation in the gene for bilirubin UDP-glucuronosyltransferase,” Human Molecular Genetics, vol. 4, no. 7, pp. 1183–1186, 1995.
- K. Sai, M. Saeki, Y. Saito et al., “UGT1A1 haplotypes associated with reduced glucuronidation and increased serum bilirubin in irinotecan-administered Japanese patients with cancer,” Clinical Pharmacology and Therapeutics, vol. 75, no. 6, pp. 501–515, 2004.
- L. Chaouch, Y. Said, I. Moumni et al., “Implication of genetic variation at the promoter and exon1 of UGT1A1 in occurrence of cholelithiasis in Tunisia,” Annales de Biologie Clinique, vol. 70, no. 6, pp. 702–706, 2012.
- M. Krawczyk, D. Q.-H. Wang, P. Portincasa, and F. Lammert, “Dissecting the genetic heterogeneity of gallbladder stone formation,” Seminars in Liver Disease, vol. 31, no. 2, pp. 157–172, 2011.
- A. Tsezou, M. Tzetis, E. Giannatou et al., “Gilbert syndrome as a predisposing factor for cholelithiasis risk in the Greek adult population,” Genetic Testing and Molecular Biomarkers, vol. 13, no. 1, pp. 143–146, 2009.
- K. Akaba, T. Kimura, A. Sasaki et al., “Neonatal hyperbilirubinemia and mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene: a common missense mutation among Japanese, Koreans and Chinese,” Biochemistry and Molecular Biology International, vol. 46, no. 1, pp. 21–26, 1998.
- S. Aono, Y. Adachi, E. Uyama et al., “Analysis of genes for bilirubin UDP-glucuronosyltransferase in Gilbert's syndrome,” The Lancet, vol. 345, no. 8955, pp. 958–959, 1995.
- S. Buch, C. Schafmayer, H. Vlzke et al., “Loci from a genome-wide analysis of bilirubin levels are associated with gallstone risk and composition,” Gastroenterology, vol. 139, no. 6, pp. 1942–1951, 2010.