Case Reports in Genetics

Case Reports in Genetics / 2013 / Article

Case Report | Open Access

Volume 2013 |Article ID 941684 | https://doi.org/10.1155/2013/941684

Piotr K. Janicki, Sonia Vaida, Hamid A. B. AL-Mondhiry, "Targeted Next-Generation Resequencing of Gene Identifies Novel Multiple Variants Pattern in Severe Hereditary Factor V Deficiency", Case Reports in Genetics, vol. 2013, Article ID 941684, 7 pages, 2013. https://doi.org/10.1155/2013/941684

Targeted Next-Generation Resequencing of Gene Identifies Novel Multiple Variants Pattern in Severe Hereditary Factor V Deficiency

Academic Editor: A. Sazci
Received29 Jan 2013
Accepted19 Feb 2013
Published15 Apr 2013

Abstract

The present study investigated the genetic defects underlying severe Factor V deficiency in a 26-year-old Columbian (South America) female and her immediate family (both parents and newborn child) by next generation sequencing (NGS) of the entire gene locus. Five mutations in the coding sequence of , including three missense single-nucleotide variants (R2102H, R513K, D107H) and two synonymous variants (A135A , S184S), were identified and confirmed by the Sanger sequencing in the investigated proband (homozygote for all detected mutations), her parents, and her newborn child (all heterozygotic carriers for identified mutations). Each of the three missense variants was previously associated with separate phenotypes, including Factor V deficiency (R2102H), thrombosis (R513K) and frequent miscarriages (D107H). In addition, at least 75 additional single-nucleotide variants (including six novels) were identified in untranslated region of .

1. Introduction

Coagulation Factor V is a large 330-kD glycoprotein which consists of 2224 amino acid residues including a 28-residue leader peptide, which is structurally and functionally homologous to coagulation Factor VIII [1, 2]. The human Factor V gene (official name ) maps to chromosome 1q23 and contains 25 exons (8). Factor V deficiency is a rare autosomal recessive disorder (incidence < 1 in 1 million), characterized by low levels of antigen and activity [3]. At present, more than one hundred deficiency-causing mutations in the locus have been described, and although most of them are private, a few are common, being found in several individuals of both European, Middle-Eastern and Asiatic descents. In the present study, we used for the first time DNA next-generation sequence (NGS) analysis to detect mutation pattern in the entire locus of a 26-year-old Hispanic parturient with severe Factor V deficiency, as well as in her asymptomatic parents and newborn baby. The relationship between combinations of mutations and clinical phenotypes was evaluated.

2. Case Presentation

The study protocol was approved by IRB at PSU Hershey Medical College. It was performed in adherence to the tenets of the declaration of Helsinki. Written informed consent was obtained from all participants. The investigated patient was 26-year-old Hispanic (born in Columbia, South America) parturient (G4P1) with several bleeding episodes before and during present pregnancy, multiple fresh frozen plasma (FFP) transfusions, and a history of three miscarriages in the past. The patient was previously diagnosed clinically to have severe Factor V deficiency on the basis of several previous bleeding episodes and laboratory studies demonstrating coagulopathy with moderate to severe decrease in Factor V activity. The remaining past medical history was unremarkable. The family history revealed that both biological parents had no history of bleeding or other coagulation symptoms and had reported normal Factor V activity. In addition, she has two siblings, apparently without any clinical signs of coagulation disorders. In the course of the current pregnancy the patient delivered the healthy male newborn, who has not displayed, at the time of this analysis, any signs of coagulation abnormalities, besides decreased (36%) level of Factor V. For the purpose of this investigation, we collected the samples of blood (proband and newborn son) and saliva (both biological parents and proband’s husband) for DNA analysis (Figure 1).

3. Mutation Analysis

Genomic DNA (gDNA) was extracted from venous EDTA-whole blood sample (proband) or cord blood (newborn child) employing membrane ultrafiltration method (FujiFilm Life Sciences distributed by Autogene, Holliston, MA, USA), according to the manufacturer recommendations. The saliva samples were collected from both parents and proband/s husband into Oragene container (DNA Genotek, Canada) and extracted according to the manufacturer recommendations. Subsequently two gDNA samples from the proband were submitted to Otogenetics Corporation (Norcross, GA, USA) for target capture and sequencing. Briefly, gDNA was subjected to agarose gel and optical density ratio tests to confirm the purity and concentration prior to Covaris (Covaris, Inc., Woburn, MA, USA) fragmentation. Fragmented gDNAs were tested for size distribution and concentration using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and Nanodrop (Thermo Fisher Scientific, Wilmington DE, USA). Illumina libraries were made from qualified fragmented gDNA using NEBNext reagents (New England Biolabs, Ipswich, MA, USA) and the resulting libraries were subjected to exome enrichment using custom probes targeting 75 kb target on chromosome 1 (169, 481, 192–169, 555, 469 by GRCh 37, Hg19). The resultant libraries were tested for enrichment by qPCR and for size distribution and concentration by an Agilent Bioanalyzer 2100. The samples were then sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA, USA) which generated paired-end reads of 90 or 100 nucleotides. Data was analyzed for data quality, exome coverage, and exome-wide SNP/InDel using the platform provided by DNAnexus (DNAnexus, Inc, Mountain View, CA, USA). The detected polymorphisms in the coding sequence of the locus were subsequently verified using classical Sanger sequencing using exon primers described by van Wijk et al. [4]. This analysis was performed by direct DNA sequencing using ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA).

By NGS, we generated about 549 million bases of sequence as pair-end 90 or 100 nucleotide reads, 86% of which was able to align to human reference sequence. A total of 23% of these sequences mapped to the targeted region corresponding to 75 kb sequence of the locus (NM_0001304.4), with 540-fold mean coverage (Table 1). At this depth of coverage, more than 95% of the target bases were covered to pass quality control filtering based on the PHRED score threshold of calling variants (PHRED > 30). Eighty high-confidence variants were annotated in the target region (3 nonsynonymous single-nucleotide variants and 2 synonymous single-nucleotide variants in the coding region of the target (Table 2), as well as 75 single-nucleotide variants in the noncoding sequence of the target (Table 3). The missense variants were located in the exon 3 (D107H), 10 (R513K), and 23 (R2102H) of the locus (Figure 1), and the investigated patient was a homozygous carrier for all these variants. These variants were additionally confirmed by the Sanger sequencing and showed perfect match in two duplicate samples. The additional verification of the DNA samples from the proband’s newborn son and our patient’s biological parents performed using the Sanger sequencing revealed that all of them were heterozygous carriers for all 3 missense variants (Figure 2). No presence of these variants was shown in the husband of the patient (i.e., biological father of the newborn child).


SampleAverage target coverageTotal bases Total reads Mapped readsBottleneck score

Original proband DNA sample 547 583,280,200 5,832,802 4,964,887 31.81
Duplicate534 549,426,400 5,494,264 4,720,781 32.81


PatientBleeding phenotypeFV activity (%)Variants in CDS of geneMature FV protein variantsSNPs (ID)

Female probandEpistaxis multiple miscarriages 0–4%Homozygote for nonsynonymous variants:
169486641 G > C2102 R/H (exon 23)No dbSNP ID
169519112 C > T513 R/K (exon 10)rs6020
169541513 C > G107 D/H (exon 3)rs6019
Homozygote for synonymous variants:
169529973 C > T135 A/A (exon 4)rs6029
169529826 C > A184 S/S (exon 4)rs6022

Newborn son of probandNo bleeding 36%Heterozygote for nonsynonymous variants:
169486641 G > C2102 R/H (exon 23)No dbSNP ID
169519112 C > T513 R/K (exon 10)rs6020
169541513 C > G107 D/H (exon 3)rs6019
Heterozygote for synonymous variants:
169529973 C > T135 A/A (exon 4)rs6029
169529826 C > A184 S/S (exon 4)rs6022

Mother of probandNo bleedingReported normalHeterozygote for nonsynonymous variants:
169486641 G > C2102 R/H (exon 23)No dbSNP ID
169519112 C > T513 R/K (exon 10)rs6020
169541513 C > G107 D/H (exon 3)rs6019
Heterozygote for synonymous variants:
169529973 C > T135 A/A (exon 4)rs6029
169529826 C > A184 S/S (exon 4)rs6022

Father of probandNo bleedingReported normalHeterozygote for nonsynonymous variants:
169486641 G > C2102 R/H (exon 23)No dbSNP ID
169519112 C > T513 R/K (exon 10)rs6020
169541513 C > G107 D/H (exon 3)rs6019
Heterozygote for synonymous variants:
169529973 C > T135 A/A (exon 4)rs6029
169529826 C > A184 S/S (exon 4)rs6022

Proband’s husband No bleedingReported normalNo above CDS variants detected in


Type of variantType of carrierPosition-RPosition-LReference baseVariant baseSNPs (ID)

Variants in 5′-UTR

SNPHom169556050169556051ctrs2269648
SNPHom169556152169556153gtNew variant
INSHet169556812169556812ars58897818

Intronic variants

SNPHom169486641169486642gcrs9332666
SNPHom169490392169490393gcrs2420370
SNPHom169491555169491556gars2420371
SNPHom169496536169496537gcNew variant
SNPHom169498056169498057agrs2420372
SNPHom169516507169516508gars12046953
SNPHom169517159169517160ctrs12026997
SNPHom169517385169517386agrs12044669
SNPHom169517904169517905gars2420374
SNPHom169518703169518704tcrs58875232
DELHet169518885169518886ars55717622
SNPHom169519417169519418gars7537742
SNPHom169519765169519766agrs13306331
SNPHom169520098169520099agrs10800456
SNPHom169521553169521554gars2213868
SNPHom169521733169521734ctrs9332582
SNPHom169523346169523347agrs7555832
SNPHom169523389169523390tcrs11577059
SNPHet169523395169523396atNew variant
SNPHet169523396169523397agNew variant
DELHom169523397169523398trs116132528
INSHet169524860169524860cacNew variant
SNPHet169524865169524866agNew variant
SNPHom169525312169525313ctrs9332579
INSHet169525558169525558ctctggcrs16684
SNPHet169525680169525681cars115199761
SNPHom169525766169525767tcrs2239853
INSHom169526266169526266ars9332577
SNPHom169526300169526301ctrs4656688
SNPHom169526367169526368agrs4656689
SNPHom169526425169526426gtrs4656188
SNPHom169526601169526602gcrs1894697
SNPHom169526646169526647agrs1894698
SNPHom169526950169526951cgrs1894699
SNPHom169527226169527227agrs1981491
DELHom169527470169527471ars3835454
SNPHom169528075169528076cgrs9332570
INSHet169528255169528255aaars58738850
SNPHom169528580169528581ctrs6012
SNPHom169528722169528723ctrs6427201
SNPHom169528830169528831ctrs6427202
SNPHom169529031169529032acrs6427203
SNPHom169529132169529133ctrs6699691
SNPHom169529138169529139ctrs58931047
SNPHom169530070169530071acrs7545236
SNPHom169530077169530078gtrs7523043
SNPHom169530093169530094tcrs7534848
SNPHom169530176169530177cgrs7522982
INSHom169530532169530532ars5778621; rs77192101
SNPHom169530586169530587tcrs1894701
SNPHom169531442169531443tcrs7540556
SNPHom169531571169531572tcrs4656690
SNPHom169533266169533267gars6678795
SNPHom169533744169533745tars724509
SNPHom169534028169534029tcrs724507
SNPHom169534966169534967tars2040443
SNPHom169535353169535354cgrs6685578
SNPHom169536167169536168tcrs2213869
SNPHom169536650169536651gars2187955
DELHet169536796169536798tttNew variant
SNPHom169537678169537679agrs6670678
SNPHom169538466169538467gars9287095
SNPHom169538544169538545ctrs2298908
SNPHom169538603169538604tgrs2298906
SNPHom169539348169539349tgrs6663533
SNPHom169543263169543264cars10800457
SNPHom169545413169545414agrs6677374
SNPHom169549775169549776tcrs10753787
INSHet169551560169551560ars56901113
SNPHom169554058169554059cgrs3753305

Variants in 3′-UTR

SNPHom169479974169479975ctrs970740

Hom: homozygote, Het: heterozygote, SNP: single-nucleotide polymorphism, Del: deletion, and Ins: insertion

4. Discussion

The current study presents several novel findings: (i) it employs NGS approach for molecular diagnosis of FV deficiency, including sequencing of both translated and untranslated regions of locus; (ii) it diagnoses the inherited FV deficiency in the S. American Caucasian family of Hispanic ethnicity; and (iii) it depicts detailed genetic evidence for multiple, coinherited mutations in the locus which may be responsible for opposite phenotype characteristics (i.e., increasing and decreasing thrombosis process).

The results of the current study indicate that the investigated proband was a homozygotic carrier of three separate missense mutations in the coding sequence of . Surprisingly, however, only one of the 3 detected missense mutations (R2102H) was described previously (and called at that time, R2074H, based on different amino acid number order) by Schijver et al. (2002) in the context of the deficiency of the Factor V [5]. This substitution results in the replacement of an arginine (R) by a histidine (H) in amino acid position 2102 located in the C2-domain of Factor V and several lines of evidence reported previously support the notion that this sequence variant is causative for Factor V deficiency phenotype. Interestingly the remaining two detected missense mutations were reported to be associated mostly with either thrombosis [68] or increased risk of preterm delivery (D107H) [9]. It is noteworthy that two missense point mutations which were detected in the current proband were previously described in different populations (R2102H in Tunisian population and R513K in Thai, Chinese and Sub-Saharan populations). Although Factor V deficiency and its causative mutations were reported previously from European Caucasians, it is, to our knowledge, the first detailed description of causative mutation in locus in a family from South America and/or Hispanic ethnicity.

In addition, we established that the investigated proband was a homozygous carrier for two synonymous single-nucleotide variants: A135A (rs6029) and S184S (rs6022) previously reported in the online SNP Medline database. There is no information about the potential phenotypic significance of these mutations. In addition to the previously described single-point mutations in the coding region of gene, the analysis of the NGS data from the proband established a presence of additional 75 polymorphisms in the untranslated sequence of the locus (3 in 5′-UTR part, 71 in the intronic part, and 1 in the 3′-UTR part of the gene) (Table 3). Six of these variants represent newly discovered variants, not presented previously in the SNP Medline database. The types of detected variants include single-nucleotide variants (4 insertions and 4 deletions), as well as 3 additional short indels. The phenotypic significance of these polymorphisms for the Factor V function remains unknown.

The most recently (Oct 2012) accessed Human Genome Mutation database (www.hgmd.org) lists 145 missense mutations in the locus associated with altered function of Factor V. From this list, 94 mutations represent single-point mutations, from which approximately 80 have been strongly associated with Factor V deficiency. In this respect, the present study does not add new mutations to this list but confirms the fact of previously described coinheritance of several separate mutations in the locus [10, 11], and more importantly provides an example of co-inheritance of mutations with presumably opposite phenotypic coagulation characteristics. Similar situation (i.e., coinheritance of polymorphic variants from which one is associated with decreased Factor V activity and other with increased thrombosis) was described previously for much more frequent prothrombotic Leiden mutations, or more recently, promoter −426 G/A polymorphism [12, 13]. Our finding confirms that other prothrombotic mutations in the gene locus may be independently inherited in one patient.

The present study exemplifies the use of NGS approach for detailed diagnosis of the specific clinical pathology and identification of causative mutations for rare genetic disorder. This approach has been recently advocated for both diagnosis and therapy [14]. The obtained genetic data for this cases were successfully used clinically for the peripartum anesthetic management of the previously described patient [15]. Comprehensive genetic analysis through NGS based approaches will increasingly be helpful in establishing the diagnosis of Factor V deficiency (or other genetic coagulation disorders) and thereby improve patient management.

Conflict of Interests

The authors declare that there is no conflict of interests.

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Copyright © 2013 Piotr K. Janicki 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.


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