Homozygous factor V splice site mutation associated with severe factor V deficiency

Abstract

We investigated a family whose proband has a severe bleeding disorder and factor V antigenic and functional levels of 8% and less than 1% of control values, respectively. Molecular analysis of the factor V gene revealed a novel homozygous mutation in the last nucleotide of exon 10. 1701G>T causes activation of a cryptic exonic splice site in exon 10, which encodes part of the factor V heavy chain (A2 domain). This leads to the deletion of 35 nucleotides and results in a frameshift with a premature stop codon at amino acid position 498. The G1701 and corresponding Gln509 are conserved in murine, bovine, and porcine factor V and in human factor VIII. Few factor V deficiency mutations have been identified as yet.Several are present in the heterozygous form in combination with factor V Leiden (Arg506Gln). This is the first reported homozygous splice site mutation in a patient with factor V deficiency. IntroductionAutosomal recessive factor V deficiency is a rare coagulation defect, equally affecting both sexes and diagnosed in multiple ethnic backgrounds. 1 In most patients, the phenotype is clinically benign and is characterized by mild bleeding only. [1][2][3][4] The factor V gene is located on chromosome 1q23, 5 spans more than 80 kb, and contains 25 exons. 6 The 5Ј untranslated sequence, a signal peptide, and the factor V heavy chain (protein domains A1 and A2) are encoded by exons 1 to 12. Exon 13 encodes the B domain, which is posttranslationally removed during activation by thrombin. The light chain (protein domains A3, C1, and C2) and the 3Ј untranslated region are encompassed by exons 14 to 25. 6 Few mutations associated with factor V deficiency have been reported. 7 We investigated the genetic basis of factor V deficiency in a patient with umbilical bleeding at birth, gingival bleeding, and repeated spontaneous central nervous system hemorrhage. She requires weekly prophylactic fresh frozen plasma transfusions. The parents may be distantly related. Both parents and the patient's siblings are clinically unaffected. Study designFactor V activity and antigen assays Factor V coagulant activity was determined in the proband and 5 family members ( Figure 1) by a clotting assay using an MLA 1200 instrument (Medical Laboratory Automation, Pleasantville, NY). Factor V antigen concentrations were investigated with a paired antibody enzyme-linked immunosorbent assay as recommended by the manufacturer (Cedarlane Laboratories, Hornby, BC, Canada). Polymerase chain reaction amplification and sequencingGenomic DNA was isolated from the index patient and 5 relatives. Amplification of 24 factor V exons was achieved by the use of intronic primers. Exon 13 was amplified in 7 overlapping segments because of its large size. All primers and polymerase chain reaction (PCR) conditions are available on www.stanford.edu/ϳzehnder.After identification of the 1701GϾT mutation, only RNA from the proband was available for further studies. RNA was extracted from peripheral blood mononuclear cells according to standard procedures. Revers...

Arg2074Cys missense mutation in the C2 domain of factor V causing moderately severe factor V deficiency: molecular characterization by expression of the recombinant protein

“…[8][9][10][11][12][13] A possible explanation for the recurrence of the Arg2074Cys mutation is the involvement of a CpG mutation hot spot. On the other hand, the presence of a different mutation not involving a CpG dinucleotide at the same residue in FV (Arg2074His), as well as of several hemophilia A-causing mutations in the corresponding residue of FVIII, points to the important role of this amino acid for FV synthesis and secretion.…”

“…5 The genetic basis of severe or moderately severe type I FV deficiency is still poorly explored (up to now, a total of 12 mutations scattered through F5 have been identified). [8][9][10][11][12][13] Most mutations are nonsense, frameshift, or splicing mutations, giving rise to null alleles, whereas only 3 missense mutations have been identified. An additional 8 mutations causing FV deficiency have been reported in the heterozygous state, 5 of which were found in patients who carried the FV Leiden mutation on the second allele, leading to the "pseudohomozygous APC-resistance" condition.…”

Evaluation of factor V mRNA to define the residual factor V expression levels in severe factor V deficiency

“…Patients with severe factor V (FV) deficiency have been found to be homozygous for FV gene small deletions, nonsense and missense mutations, 1 or substitutions affecting invariant bases in the consensus splicing sequences, [2][3][4] which indicate that very low FV levels are compatible with life. On the other hand, the absence of extended deletions, which cannot be corrected by ribosomal slippage or somatic reversion events, might indicate that traces of FV activity are also essential in humans.…”

Molecular characterization and subcellular localization of Tyr478del: a pathogenic in‐frame deletion in coagulation factor V

“…There is no clear relationship between the genotypes or the FVII:C levels and severity of the bleeding diathesis. Hemorrhage in the central nervous system occurs in approximately 4% of affected patients [1] and is considered the most common cause of death in newborns with FVII deficiency.…”

“…Genotyping and sequencing of the FV gene was performed as previously described [1], with annotations as per Jenny et al [2]. The proband is a compound heterozygote with a B-domain frameshift mutation (T2952del) on the paternal allele, leading to a premature termination codon and a 3 bp deletion in exon 10 (nucleotides 1606-1608) on the maternal allele.…”