Case Reports in Genetics

Volume 2013 (2013), Article ID 941684, 7 pages

http://dx.doi.org/10.1155/2013/941684

Case Report

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

Piotr K. Janicki,¹ Sonia Vaida,¹ and Hamid A. B. AL-Mondhiry²

¹Laboratory of Perioperative Genomics, Department of Anesthesiology, Penn State University College of Medicine, MS Hershey Medical Center, H187, 500 University Dr, Hershey, PA 17033, USA

²Division of Hematology-Oncology, Penn State University College of Medicine, MS Hershey Medical Center, Hershey, PA 17033, USA

Received 29 January 2013; Accepted 19 February 2013

Academic Editors: S.-C. Chae, P. D. Cotter, A. Sazci, and C. Yapijakis

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.

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).

Figure 1: Segregation of missense variants 2102 R/H (exon 23), 513 R/K (exon 10) and 107 D/H (exon 3). Half-filled symbols indicate heterozygotes for investigated mutatons. Completely filled symbols indicate homozygous for all investigated mutations (i.e., proband). Open symbols indicate no carrier of investigated mutations. Question marks indicate that genotyping has not been performed.

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).

Table 1: Summary of coverage for analyzed samples by next generation sequencing method.

Sample
Average target coverage
Total bases
Total reads
Mapped reads
Bottleneck score

Original proband DNA sample
547
583,280,200
5,832,802
4,964,887
31.81

Duplicate
534
549,426,400
5,494,264
4,720,781
32.81

Table 2: Mutations observed in coding sequences (CDS) of gene in the investigated proband and her blood relatives.

Patient
Bleeding phenotype
FV activity (%)
Variants in CDS of gene
Mature FV protein variants
SNPs (ID)

Female proband
Epistaxis multiple miscarriages
0–4%
Homozygote for nonsynonymous variants:

169486641 G > C
2102 R/H (exon 23)
No dbSNP ID

169519112 C > T
513 R/K (exon 10)
rs6020

169541513 C > G
107 D/H (exon 3)
rs6019

Homozygote for synonymous variants:

169529973 C > T
135 A/A (exon 4)
rs6029

169529826 C > A
184 S/S (exon 4)
rs6022

Newborn son of proband
No bleeding
36%
Heterozygote for nonsynonymous variants:

169486641 G > C
2102 R/H (exon 23)
No dbSNP ID

169519112 C > T
513 R/K (exon 10)
rs6020

169541513 C > G
107 D/H (exon 3)
rs6019

Heterozygote for synonymous variants:

169529973 C > T
135 A/A (exon 4)
rs6029

169529826 C > A
184 S/S (exon 4)
rs6022

Mother of proband
No bleeding
Reported normal
Heterozygote for nonsynonymous variants:

169486641 G > C
2102 R/H (exon 23)
No dbSNP ID

169519112 C > T
513 R/K (exon 10)
rs6020

169541513 C > G
107 D/H (exon 3)
rs6019

Heterozygote for synonymous variants:

169529973 C > T
135 A/A (exon 4)
rs6029

169529826 C > A
184 S/S (exon 4)
rs6022

Father of proband
No bleeding
Reported normal
Heterozygote for nonsynonymous variants:

169486641 G > C
2102 R/H (exon 23)
No dbSNP ID

169519112 C > T
513 R/K (exon 10)
rs6020

169541513 C > G
107 D/H (exon 3)
rs6019

Heterozygote for synonymous variants:

169529973 C > T
135 A/A (exon 4)
rs6029

169529826 C > A
184 S/S (exon 4)
rs6022

Proband’s husband
No bleeding
Reported normal
No above CDS variants detected in
—
—

Table 3: Summary of observed variants in noncoding sequences of gene in investigated proband.

Type of variant
Type of carrier
Position-R
Position-L
Reference base
Variant base
SNPs (ID)

Variants in 5′-UTR

SNP
Hom
169556050
169556051
c
t
rs2269648

SNP
Hom
169556152
169556153
g
t
New variant

INS
Het
169556812
169556812

a
rs58897818

Intronic variants

SNP
Hom
169486641
169486642
g
c
rs9332666

SNP
Hom
169490392
169490393
g
c
rs2420370

SNP
Hom
169491555
169491556
g
a
rs2420371

SNP
Hom
169496536
169496537
g
c
New variant

SNP
Hom
169498056
169498057
a
g
rs2420372

SNP
Hom
169516507
169516508
g
a
rs12046953

SNP
Hom
169517159
169517160
c
t
rs12026997

SNP
Hom
169517385
169517386
a
g
rs12044669

SNP
Hom
169517904
169517905
g
a
rs2420374

SNP
Hom
169518703
169518704
t
c
rs58875232

DEL
Het
169518885
169518886
a

rs55717622

SNP
Hom
169519417
169519418
g
a
rs7537742

SNP
Hom
169519765
169519766
a
g
rs13306331

SNP
Hom
169520098
169520099
a
g
rs10800456

SNP
Hom
169521553
169521554
g
a
rs2213868

SNP
Hom
169521733
169521734
c
t
rs9332582

SNP
Hom
169523346
169523347
a
g
rs7555832

SNP
Hom
169523389
169523390
t
c
rs11577059

SNP
Het
169523395
169523396
a
t
New variant

SNP
Het
169523396
169523397
a
g
New variant

DEL
Hom
169523397
169523398
t

rs116132528

INS
Het
169524860
169524860

cac
New variant

SNP
Het
169524865
169524866
a
g
New variant

SNP
Hom
169525312
169525313
c
t
rs9332579

INS
Het
169525558
169525558

ctctggc
rs16684

SNP
Het
169525680
169525681
c
a
rs115199761

SNP
Hom
169525766
169525767
t
c
rs2239853

INS
Hom
169526266
169526266

a
rs9332577

SNP
Hom
169526300
169526301
c
t
rs4656688

SNP
Hom
169526367
169526368
a
g
rs4656689

SNP
Hom
169526425
169526426
g
t
rs4656188

SNP
Hom
169526601
169526602
g
c
rs1894697

SNP
Hom
169526646
169526647
a
g
rs1894698

SNP
Hom
169526950
169526951
c
g
rs1894699

SNP
Hom
169527226
169527227
a
g
rs1981491

DEL
Hom
169527470
169527471
a

rs3835454

SNP
Hom
169528075
169528076
c
g
rs9332570

INS
Het
169528255
169528255

aaa
rs58738850

SNP
Hom
169528580
169528581
c
t
rs6012

SNP
Hom
169528722
169528723
c
t
rs6427201

SNP
Hom
169528830
169528831
c
t
rs6427202

SNP
Hom
169529031
169529032
a
c
rs6427203

SNP
Hom
169529132
169529133
c
t
rs6699691

SNP
Hom
169529138
169529139
c
t
rs58931047

SNP
Hom
169530070
169530071
a
c
rs7545236

SNP
Hom
169530077
169530078
g
t
rs7523043

SNP
Hom
169530093
169530094
t
c
rs7534848

SNP
Hom
169530176
169530177
c
g
rs7522982

INS
Hom
169530532
169530532

a
rs5778621; rs77192101

SNP
Hom
169530586
169530587
t
c
rs1894701

SNP
Hom
169531442
169531443
t
c
rs7540556

SNP
Hom
169531571
169531572
t
c
rs4656690

SNP
Hom
169533266
169533267
g
a
rs6678795

SNP
Hom
169533744
169533745
t
a
rs724509

SNP
Hom
169534028
169534029
t
c
rs724507

SNP
Hom
169534966
169534967
t
a
rs2040443

SNP
Hom
169535353
169535354
c
g
rs6685578

SNP
Hom
169536167
169536168
t
c
rs2213869

SNP
Hom
169536650
169536651
g
a
rs2187955

DEL
Het
169536796
169536798
tt
t
New variant

SNP
Hom
169537678
169537679
a
g
rs6670678

SNP
Hom
169538466
169538467
g
a
rs9287095

SNP
Hom
169538544
169538545
c
t
rs2298908

SNP
Hom
169538603
169538604
t
g
rs2298906

SNP
Hom
169539348
169539349
t
g
rs6663533

SNP
Hom
169543263
169543264
c
a
rs10800457

SNP
Hom
169545413
169545414
a
g
rs6677374

SNP
Hom
169549775
169549776
t
c
rs10753787

INS
Het
169551560
169551560

a
rs56901113

SNP
Hom
169554058
169554059
c
g
rs3753305

Variants in 3′-UTR

SNP
Hom
169479974
169479975
c
t
rs970740

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

Figure 2: Amino acid sequence of human factor V deducted from the DNA sequence of proband. Red fonts indicate missense mutations, and blue fonts indicate synonymous mutations. The sequence of first 28 amino acid fragments is given in italics, and the consecutive 25 exons are either underlined or not underlined (pair wise).

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 [6–8] 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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