Homozygous nonsense mutation (Cys72→stop) in the human F7 gene: a not life-threatening mutation despite the absence of circulating factor VII

Abstract

Inherited factor (F)VII deficiency is a rare bleeding disorder (estimated prevalence 1 : 500 000), transmitted as an autosomal recessive trait expressed clinically only in homozygotes. In the Algerian population where consanguineous marriages are still frequent, the prevalence of such a recessive coagulation disorder is higher with a clinical and social impact. We herein report a novel mutation Cys72→stop associated with a moderate bleeding tendency, identified in two unrelated Algerian families: in a young child (family I), in the homozygous state (FVII:C and FVII:Ag < 1%) with severe quantitative FVII deficiency; and in an adult (family II), compound heterozygous, associated with a Cys310→Phe mutation, already reported (FVII:C 3% and FVII:Ag 34%). In family I, the proband, a girl, presented at birth with umbilical hemorrhage and when 7 months old with bilateral epistaxis. The FVII deficiency was diagnosed when she was 18 months old, suffering from recurrent epistaxis, mouth bleeding, spontaneous hematoma after minor trauma. Her parents, first cousins, had no family history of bleeding diathesis. In family II, the proband, a 41-year-old man, had suffered from spontaneous hematoma and extensive bleeding since childhood but the FVII deficiency was diagnosed in adulthood during a preoperative screening for a polypectomy. The patient's daugthers and son had FVII:C and FVII:Ag in the normal range or slightly reduced and were asymptomatic. In both families, liver disease and vitamin K deficiency were ruled out as well as thrombophilia (factor V Leiden and antithrombin, protein C or protein S deficiency). Informed consent was obtained in family I from the parents and in family II from the patient and his children. DNA extraction, design of the polymerase chain reaction (PCR) primers derived from the F7 gene sequence and PCR protocols were performed as previously described [1Borensztajn K. Chafa O. Alhenc-Gelas M. Salha S. Reghis A. Fischer A.-.M. Tapon-Bretaudiere J. Characterization of two novel splice site mutations in human factor VII gene causing severe plasma factor VII deficiency and bleeding diathesis.Br J Haematol. 2002; 117: 168-71Crossref PubMed Scopus (0) Google Scholar]. Mutations were detected by sequencing the coding regions, the exon–intron boundaries and the 5′ flanking region. In family I, a C→A mutation at nucleotide 6036 resulted in the substitution of a cysteine codon in position 72, for a stop codon, was found in the homozygous state for the proband, both her parents being heterozygous for the same mutation. In family II, the same mutation, cys72→stop, was found as well as a Cys310→Phe mutation, both in the heterozygous state in the proband. The Cys310→Phe mutation was also present in a heterozygous condition in one daughter and one son whereas the Cys72→stop mutation was present in a heterozygous condition in three daughters. More than 140 F7 gene lesions have been reported in the literature. Among them only eight nonsense mutations have been characterized [2McVey J.H. Boswell E. Mumford A.D. Kemball-Cook G. Tuddemham E.G.D. Factor VII deficiency and the FVII mutation database.Hum Mutat. 2001; 17: 3-17Crossref PubMed Scopus (0) Google Scholar, 3Perry D.J. Factor VII deficiency.Br J Haematol. 2002; 118: 689-700Crossref PubMed Scopus (0) Google Scholar], which means that they are rare compared with the 90 missense mutations found in the F7 gene. Among them, mutation Ser52→stop is the only nonsense mutation found so far in the homozygous state [4Giansily-Blaizot M. Aguilar Martinez P. Briquel M.E. D'Oiron R. De Maistre E. Epelbaum S. Schved J.F. Two novel cases of cerebral haemorrhages at neonatal period, associated with inherited FVII deficiency, one of them revealing a new nonsense mutation (Ser52→Stop).Blood Coagul Fibrinolysis. 2003; 14: 217-20Crossref PubMed Scopus (0) Google Scholar]. The absence of cysteine at position 72 prevents the formation of a disulfide bond with the cysteine 61 in the EGF1 domain. Morover, the C→A substitution at nt 6036 in exon 4 generates a premature termination codon in exon 4, leading to a truncated protein devoid of procoagulant activity. Severe bleeding is expected, for the novel Cys72→stop mutation as FVII plays a crucial role in blood coagulation. Our homozygous patient suffered from a moderate but not life-threatening bleeding tendency. In contrast, the other homozygous nonsense mutation (Ser52→stop) producing a short inactive polypeptide [4Giansily-Blaizot M. Aguilar Martinez P. Briquel M.E. D'Oiron R. De Maistre E. Epelbaum S. Schved J.F. Two novel cases of cerebral haemorrhages at neonatal period, associated with inherited FVII deficiency, one of them revealing a new nonsense mutation (Ser52→Stop).Blood Coagul Fibrinolysis. 2003; 14: 217-20Crossref PubMed Scopus (0) Google Scholar] belongs to the homozygous life-threatening FVII deficiencies revealed by intracranial and gastrointestinal hemorrhages in childhood. These severely affected patients have severe gene defects such as stop codons [4Giansily-Blaizot M. Aguilar Martinez P. Briquel M.E. D'Oiron R. De Maistre E. Epelbaum S. Schved J.F. Two novel cases of cerebral haemorrhages at neonatal period, associated with inherited FVII deficiency, one of them revealing a new nonsense mutation (Ser52→Stop).Blood Coagul Fibrinolysis. 2003; 14: 217-20Crossref PubMed Scopus (0) Google Scholar, 5Takamiya O. Okimoto Y. Severe factor VII deficiency with recurrent intracranial haemorrhages owing to double heterozygosity for a splice site mutation of an IVS4 and a novel nonsense mutation in exon 8 (Gln211→term).Br J Haematol. 2001; 84: 669-74Google Scholar], frameshifts [6Millar D.S. Cooper D.N. Kakkar V.V. Schwartz M. Scheibel E. Prenatal exclusion of severe factor VII deficiency by DNA sequencing.Lancet. 1992; 339: 1359Abstract PubMed Google Scholar] and splice site mutations [5Takamiya O. Okimoto Y. Severe factor VII deficiency with recurrent intracranial haemorrhages owing to double heterozygosity for a splice site mutation of an IVS4 and a novel nonsense mutation in exon 8 (Gln211→term).Br J Haematol. 2001; 84: 669-74Google Scholar, 7MacVey J.H. Boswell E.J. Takamya O. Tamagnini G. Valente V. Fidaldo T. Layton M. Tuddenham E.G. Exclusion of the first EGF domain of factor VII by a splice site mutation causes lethal factor VII deficiency.Blood. 1998; 92: 920-6Crossref Google Scholar, 8Tamary H. Fromovich-Amit Y. Shalmon L. Zaizov R. Yaniv I. Klar A. Peretz H. Brenner B. Lanir N. Zivelin A. Seligsohn U. Molecular characterization of four novel mutations causing factor VII deficiency.Hematol J. 2000; 1: 382-9Crossref PubMed Scopus (0) Google Scholar, 9Boresztajn K. Sobrier M.-.L. Fischer A.-.M. Chafa O. Amselem S. Tapon-Bretaudiere J. Factor VII gene intronic mutation in a lethal Factor VII deficiency: effects on splice-site selection.Blood. 2003; 102: 561-3Crossref Scopus (9) Google Scholar] leading to the absence of circulating FVII. These data are consistent with F7 gene knockout experiments in mice: F7–/– mice develop normally to term but die at or shortly after birth from abdominal and intracranial hemorrhage [10Rosen E. Chan J.C.Y. Idusogie E. Clotman F. Vlasuk G. Luther T. Jalbert L.R. Albrecht S. Zhong L. Lissens A. Schoonjans L. Collen D. Costellino F.J. Carmeliet P. Mice lacking Factor VII develop normally but suffer fatal perinatal bleeding.Nature. 1997; 390: 290Crossref PubMed Scopus (184) Google Scholar]. However, Peyvandi et al. [11Peyvandi F. Mannucci P.M. Jenkins P.V. Lee A. Cappola R. Perry D.J. Homozygous 2 pb deletion in the human factor VII gene: a non lethal mutation that is associated with a complete absence of circulating factor VII.Thromb Haemost. 2000; 84: 635-7Crossref Scopus (16) Google Scholar] reported the case of a 5-year-old boy with an homozygous 2 pb deletion in the prepropeptide of FVII: he suffered from gastrointestinal bleeding at the age of 8 months but the diagnosis of FVII deficiency was made later when he was 3 years old, indicating a bleeding tendency less severe than expected. Even if the mutant Cys72→stop protein is translated, the absence of both EGF domains and the catalytic domain cannot produce an active protein. In these conditions, it is puzzling to have no more than mild bleeding symptoms in the affected 18-month-old girl, despite the absence of circulating FVII. Conversly, the other homozygous nonsense mutation cited previously (Ser52→stop), which interrupts the translation 20 amino acids upstream, was revealed by a lethal intracranial hemorrhage. The other mutation found in family II, Cys→310Phe, already reported in the literature, is associated in the homozygous state with a severe qualitative FVII deficiency and a mild bleeding tendency [12Marechetti G. Patracchini P. Gemmati D. De Rosa V. Pinotti M. Rodorigo G. Casonato A. Girolami A. Bernardi F. Detection of two missense mutations and characterization of a repeat polymorphism in the factor VII gene.Hum Genet. 1992; 89: 497-502Google Scholar]. Cys310 is strictly conserved among serine proteases. Modelling of this mutant showed that the loop formed by the disulfide bridge between Cys310 and Cys329 was considerably loosened, inducing a destabilization of the ternary structure of the protein [13Bernardi F. Liney D.L. Patracchini P. Gemmati D. Legnani C. Arcieri P. Pinotti M. Redaelli R. Ballerini G. Pemberton S. Wacey A.I. Mariani G. Tuddenham E.G.D. Marchetti G. Molecular defects in CRM+ factor VII deficiencies: modelling of missenses mutations in the catalytic domain of factor VII.Br J Haematol. 1994; 86: 610-8Crossref PubMed Google Scholar] and possibly a less efficient interaction of the mutant protein with tissue factor. For the compound heterozygous patient in family II, it can be hypothesized that the Cys310→Phe mutation allows production of a partially effective FVII which is sufficient to trigger the tissue factor blood coagulation pathway, but no explanation can be given for the homozygous Cys72→stop mutation with no circulating FVII. This case illustrates the difficulty of predicting the bleeding risk in FVII-deficient patients, based upon the phenotype–genotype relationship. The search for an alternative mechanism able to compensate partially for the absence of circulating FVII is still active. The collaboration with O.C. and A.R. in Alger was supported by a grant from INSERM-DRS.