Two novel mutations in severe factor VII deficiency

Authors


Dr John H. McVey, Haemostasis and Thrombosis, MRC Clinical Sciences Centre, The Faculty of Medicine, Imperial College, Du Cane Road, London W12 0NN, UK. E-mail: john.mcvey@csc.mrc.ac.uk

Summary

We have characterized the molecular defect in two families with severe factor VII (FVII) deficiency. In family I, the proband was found to be homozygous for a novel 18 bp deletion in exon  8 (g.10896-10913del) resulting in the in-frame deletion of six amino acids in the serine protease domain. Molecular modelling suggests the deletion is likely to disrupt folding of the FVII molecule. The reduced FVII antigen (21 U/dl) and negligible activity (0·4 U/dl) in the patient's plasma indicated that the deletion affected both the secretion/stability and function of the mutant protein. In family II, the proband was found to be a compound heterozygote for a novel missense mutation (g.7884G>A; FVII G117R) in exon  5 encoding the EGF2 domain of FVII and a nonsense mutation (g.8960C>T; FVII R152X) in exon  6. Extensive sequence comparison in a wide evolutionary context suggested that the Gly117 residue is critical for structure of FVII. The grossly reduced FVII antigen (1·1 U/dl) and activity (0·4 U/dl) plasma values indicate the mutation primarily affected the folding/secretion or stability of the protein.

Hereditary factor VII (FVII) deficiency is a rare, autosomal recessive bleeding disorder (FVII mutation database: http://europium.csc.mrc.ac.uk/) (McVey et al, 2001; Perry, 2002), first recognized more than 50 years ago (Alexander et al, 1951). Although the original index patient showed a severe bleeding phenotype, subsequent reported cases have displayed a highly variable bleeding tendency (McVey et al, 2001). Some individuals experience mild mucous membrane bleeding, menorrhagia and postsurgical bleeding but more significant events such as haemarthroses, soft tissue bleeds and life-threatening gastrointestinal and central nervous system bleeds are well recognized. Postpartum central nervous system bleeds are characteristic of severely affected individuals. The potential severity of the clinical phenotype of FVII deficiency reflects the pivotal role of FVII in the initiation of coagulation.

Methods

Coagulation tests

Factor VII coagulant activity (FVII:C) was determined by a prothrombin time-based one-stage assay using human placental thromboplastin (Thromborel-S, Behring; Sysmex, Milton Keynes, UK) and FVII deficient human plasma (Diagnostic Reagents, Thame, UK). FVII antigen (FVII:Ag) was measured by an enzyme-linked immunosorbent assay (Asserachrom VII:Ag, Diagnostica Stago; Axis-Shield, Dundee, UK).

Molecular genetic analysis

DNA was extracted from whole blood samples and from a chorionic villus biopsy (CVB) sample using a commercial kit according to the manufacturer's instructions (Nucleon BACC2; Thistle Scientific, Uddingston, UK). Exons  1a, 1b, 2–8 and their flanking exon–intron boundaries were amplified by polymerase chain reaction (PCR). Oligonucleotide primer sequences and PCR conditions are available at the FVII mutation database (http://europium.csc.mrc.ac.uk/). PCR fragments were analysed by direct DNA sequencing using an ABI Prism 3700 DNA analyzer (Applied Biosystems, Warrington, UK). All nucleotide substitutions were confirmed by direct DNA sequence analysis performed in both forward and reverse directions. The nucleotide substitutions and amino acid substitutions were designated as recommended by a nomenclature working group (Antonarakis, 1998) and numbered using the sequence with accession number J02933.

Molecular modelling

Insight II (Accelrys, Cambridge, UK) was used with the crystal co-ordinates of FVIIa (Kemball-Cook et al, 1999; 1CVW) and the tissue factor (TF)-FVIIa complex (Banner et al, 1996; 1DAN) available in the Protein Data Bank (http://www.rcsb.org/pdb/).

Sequence alignment

Alignments were generated using the ClustalW (version 1.7) multiple sequence alignment program (Thompson et al, 1994).

Sequence analysis of codon 117 in the normal population

Polymerase chain reaction amplicons spanning the mutation in exon  5 of the F7 gene were generated from genomic DNA obtained from 53 individuals of Indian origin using the oligonucleotide primers 5′-biotin-CTGACCCCCAGAAGCCCCTCTC-3′, 5′-GCCTGGTCACCTGTGGGTGTGCAG-3′ and the following PCR conditions: 94°C, 3 min; eight cycles of: 94°C, 1 min; 71·5°C (decreasing by 0·5°C per cycle), 45 s; 72°C, 1 min; 20 cycles of: 94°C, 1 min; 67·5°C, 45 s; 72°C, 1 min; 72°C, 10 min. The biotinylated amplicons were immobilized on streptavidin-coated sepharose HP beads according to the manufacturer's instructions (Amersham Pharmacia, Little Chalfont, UK) and the strands were separated using 0·2 mol/l NaOH. The pyrosequencing primer 5′-GCCAGCAGAGAGTACC-3′ was annealed to the captured strand. The primed single-stranded DNA templates were then transferred to the microtitre plate-based PSQ 96 Pyrosequencer (Pyrosequencing, Uppsala, Sweden). Analysis of the sequence surrounding codon 117 was performed with the PSQ 96 SNP Reagent kit (Pyrosequencing) used in accordance with the manufacturer's instructions.

Results

Clinical history

Family I. A female infant (Fig 1A, II.1) born to Saudi Arabian parents was noted to have haemorrhagic problems from birth. The parents were first cousins and, although there were two other consanguineous marriages in the same family, there was no family history of bleeding. The infant was diagnosed with severe FVII deficiency and suffered a fatal intracranial haemorrhage at the age of 6 weeks.

Figure 1.

Laboratory data and F7 molecular genetic analysis for families I and II (panels A and B). Details of the polymorphisms (VNTR in intron 7, His115, Arg353 and Ser333) are available on the FVII mutation database (http://europium.csc.mrc.ac.uk).

A year later a male infant (Fig 1A, II.2) was born to the same parents. He was born at term by normal vaginal delivery. FVII deficiency was diagnosed at birth (FVII:C 0·4 U/dl). On day 2 of life, he had haematemesis and was treated with fresh-frozen plasma and prothrombin complex concentrate. On day 4, he was noted to be oozing from an umbilical venous catheter and had multiple bruises. Immediate treatment with prophylactic recombinant FVIIa (NovoSeven; Novo Nordisk, Crawley, UK) was started at a dose of 30–40 μg/kg twice daily, subsequently this was changed to 60 μg/kg once daily. No significant bleeding problems have been reported under this regime, with no evidence of anti-FVII inhibitor antibody development based on continued effective treatment over a 3-year period.

Family II. A male infant (Fig 1B, II.1) was born to apparently unrelated Indian parents with no family history of bleeding. The child was well until 2·5 years old, when he developed a spontaneous knee haemarthrosis. Investigations revealed severe FVII deficiency (FVII:C 0·4 U/dl). The initial bleed responded to FVII concentrate (Bio Products Laboratory, Elstree, UK), but over the next year, there were recurrent bleeds into both knees and ankles, one of which became a target joint. The patient is now maintained on prophylactic recombinant FVIIa (NovoSeven) 30–40 μg/kg on alternate days with very good effect and with no evidence of inhibitor antibody formation based on continued effective treatment over an 18-month period.

Molecular genetic analysis of the F7 gene

The F7 genes of families I and II were analysed by PCR amplification of the exons and their flanking exon–intron boundaries, followed by direct sequencing of the PCR products to identify the cause of FVII deficiency. In family I (Fig 1A, II.2), FVII deficiency was associated with a novel 18 bp in-frame deletion in exon  8 (g.10896-10913del; ‘g.’ denotes that the nucleotide numbering is from a genomic reference sequence) of the F7 gene, deleting the nucleotides encoding amino acid residues 327–332 (MetPheCysAlaGlyTyr) in the serine protease domain. The proband was homozygous for this deletion while both parents were found to be heterozygous.

In family II, the affected individual (Fig 1B, II.1) was found to be a compound heterozygote for two substitutions in the F7 gene, a missense mutation (g.7884G>A, FVII G117R) in exon 5 and a nonsense mutation (g.8960C>T, FVII R152X) in exon 6. The father was found to be heterozygous for the G117R mutation and the mother heterozygous for the R152X mutation. The nonsense mutation (g.8960C>T, FVII R152X) has been previously described in a compound heterozygous individual with two further mutations (g.10798C>T, FVII A294V and g.11128delC) on the other allele (Wulff & Herrmann, 2000). The missense substitution (g.7884G>A, FVII G117R) has not previously been reported. To determine the population frequency of the g.7884G>A substitution, the nucleotide sequence of F7 exon  5 was studied by pyrosequence analysis of genomic DNA obtained from 53 individuals of Indian origin. The 106 alleles studied showed exclusively the wild-type sequence.

Prenatal diagnosis

During the molecular genetic analyses described above both mothers became pregnant and requested prenatal diagnosis. To enable rapid diagnosis of the mutation in family I, PCR primers were designed to anneal to either the 18 bp sequence present in the normal allele but deleted in the mutant allele, or the novel sequence spanning the deletion break point that is specific for the deleted allele. PCR analysis comparing the amplification products generated using exon  8 primers (amplifying a product on both wild-type and mutant alleles), wild-type-specific primers (amplifying a product only from a wild-type allele) and deletion-specific primers (amplifying a product only from an allele with the deletion) enabled us to differentiate between wild-type and mutant alleles (Fig 2A). However, the family declined prenatal diagnosis, electing to continue the pregnancy and a third affected child was born.

Figure 2.

(A) PCR analysis of the 18 bp deletion in family I. Left panels: cartoon representation of the PCR products – filled arrows indicate the location of the primers; filled rectangle indicates the sequence 5′ of the deletion; an unfilled rectangle indicates the deleted sequence; and a hatched rectangle indicates the sequence 3′ of the deletion. The deleted sequence string and the encoded amino acids are shown. Right panels: agarose gel electrophoresis of PCR fragments generated using the appropriate primer set to amplify DNA isolated from affected and unaffected individuals are shown. (i) exon  8 primers; (ii) wild-type-specific primers; (iii) deletion-specific primers. (B) Direct DNA sequence analysis of exons  5 and 6 in family II. The wild-type and mutant sequences are shown, and the missense (FVII G117R) and nonsense codons (FVII R152X) indicated.

The single base substitutions found in the F7 genes of family II did not create or destroy any known restriction endonuclease enzyme recognition sites, and therefore the diagnosis relied on direct sequencing of PCR products spanning the two mutations (g.7884G>A and g.8960C>T). Analysis of a CVB sample taken at 10-week gestation indicated the fetus was wild type at both sites (Fig 2B) and subsequently, a healthy child was born (Fig 1B, II.2).

Discussion

Severe FVII deficiency is a rare bleeding disorder, which often leads to life-threatening bleeds in early life. Replacement therapy with plasma-derived products has previously been the mainstay of treatment; however, the availability of recombinant FVIIa (NovoSeven) offers an effective if expensive alternative. In the present study, we have studied two families with severe FVII deficiency; we have identified three molecular defects, two of which have not previously been described.

In family I, a novel 18 bp deletion in exon  8 (g.10896-10913del) affects the nucleotides encoding amino acids 327–332 (MetPheCysAlaGlyTyr) in the serine protease domain. Inspection of a molecular model based on the X-ray crystal structure of the TF-FVIIa complex shows that these amino acids form a β-pleated sheet that runs through the core of the protease domain, with Cys329 forming a disulphide bond with Cys310 (Fig 3A). Deletion of these residues and loss of a disulphide bridge (Cys329–Cys310) is predicted to disrupt the folding of the FVII molecule leading to impaired secretion and/or stability. It is perhaps surprising to find detectable levels of FVII antigen as high as 21 U/dl in plasma from the individual homozygous for the deletion (Fig 1A; II.2); however, this is consistent with the absence of inhibitory antibodies to the replacement therapy, based on continued effective treatment. This individual is homozygous for a polymorphism (FVII R353Q; M2) known to be associated with reduced levels of plasma FVII:Ag (Bernardi et al, 1996); however, it is not possible to predict the effect of this polymorphism on plasma FVII levels in the context of the 18 bp deletion. The deleted amino acid residues are highly conserved in all known vertebrate Gla-EGF1-EGF2-serine protease protein sequences (Fig 3B) (Davidson et al, 2003) suggesting important structural or functional roles for these residues. Indeed the homozygous missense mutations Met327Ile, Phe328Ser and Cys329Gly are associated with extremely low FVII activity (<1, <1 and 3 U/dl respectively) and reduced FVII antigen levels (47, 38 and 55 U/dl respectively) (FVII mutation database; http://europium.csc.mrc.ac.uk/). The molecular defect in FVII g.10896-10913del is clearly twofold. Substantially, reduced levels of detectable circulating FVII antigen (21 U/dl) in this homozygous patient indicate a defect in protein folding, secretion or stability. In addition, the negligible FVII activity (0·4 U/dl) associated with the FVII antigen (21 U/dl) suggests a further severe functional defect in the circulating variant FVII molecules.

Figure 3.

(A) Molecular model of the serine protease (grey) and EGF2 (red) domains of human FVIIa in complex with tissue factor (yellow) (Banner et al, 1996). The location of the six amino acids deleted in family I is indicated in blue. The disulphide bonds Cys135–Cys262 and Cys310–Cys329 are shown in stick representation. The active site serine (Ser344) and Gly117 mutated in family II are represented as CPK spheres. Atoms are coloured by element: carbon, green; oxygen, red; nitrogen, blue; sulphur, yellow. (B) Amino acid sequence alignments of Gla-EGF1-EGF2-serine protease sequences from man, chicken and Fugu generated using ClustalW and edited in BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html/). Predicted amino acid sequences of chicken and Fugu Gla-EGF1-EGF2-serine proteases are taken from Davidson et al (2003) and http://europium.csc.mrc.ac.uk/. White text on black background indicates identity with the consensus; black text on grey background indicates similarity with the consensus; black text on white background indicates no similarity. (i) Amino acid sequence surrounding the six amino acid deletion 327–322 found in family I. (ii) Amino acid sequence surrounding Gly117 in EGF2 mutated to Arg in family II.

In family II, we identified two mutations associated with FVII deficiency; a missense mutation in exon  5 (g.7884G>A, FVII G117R) and a nonsense mutation in exon  6 (g.8960C>T, FVII R152X). The missense mutation has not previously been reported and genotype analysis of 53 individuals of similar ethnic origin revealed only the wild-type sequence at this position, suggesting it is not a common polymorphism. The substitution results in the replacement of a hydrogen atom with a large, positively charged, side chain in the second EGF-like domain of FVII. Inspection of the X-ray crystal structure of FVIIa shows this Gly residue to be solvent-exposed and molecular modelling of the replacement of Gly117 by Arg does not reveal any obvious topological problems. However, any displacement of the neighbouring Tyr118 residue would affect the loop Pro134-Cys135-Gly136-Lys137. In FVII, the Cys135 residue forms one half of the disulphide bridge to Cys262 in the heavy chain as shown (Fig 3A), enabling the formation of disulphide-linked two-chain FVIIa molecule after cleavage at Arg152. Amino acid sequence alignments of all known mammalian FVII sequences reveal complete conservation of Gly at this position (Fig 3B). Inspection of the haemophilia B mutation database (http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html) identified 10 individuals with missense mutations (FIX G114R, G114E, G114A, G114V) in the equivalent residue (Gly114) in FIX. Unfortunately, phenotypic data are not available for the FIX G114R mutation; however, the FIX G114E, G114A and G114V mutations are associated with severely reduced activity and antigen levels. Furthermore, Gly at this position is conserved in all known vertebrate Gla-EGF1-EGF2-serine proteases including birds and bony fish. As chicken and the bony fish Fugu rubripes last shared a common ancestor with man 350 and 430 million years ago respectively, it suggests this residue is vital for maintenance of a correctly folded structure. Consistent with this hypothesis are the extremely low levels of FVII antigen (1·1 U/dl) and activity (0·4 U/dl) detected in the patient's plasma; indicating the primary defect lies in folding, secretion or stability of the mutant FVII molecule.

The identification of the causative mutations in these families enabled us to design sensitive and specific diagnostic tools to offer prenatal diagnosis in subsequent pregnancies, although ultimately only one of the two families elected to use the service. CVB at 10–12-week gestation followed by DNA analysis may provide crucial information to enable both clinicians and parents to manage the pregnancy.

In severe FVII deficiency, data on the genetic status of the fetus are invaluable to allow appropriate clinical management of the delivery and perinatal treatment, as postpartum central nervous system bleeds are a frequent complication in affected individuals during this period. In cases in which no functional FVII is produced, these bleeds are often fatal (Chaing et al, 1994; Arbini et al, 1997; McVey et al, 1998; Herrmann et al, 2000; Tamary et al, 2000; Giansily-Blaizot et al, 2001; Takamiya & Okimoto, 2001; Borensztajn et al, 2002; Ariffin et al, 2003; Giansily-Blaizot et al, 2003; this study) (Fig 1A).

Prenatal diagnosis for a single gene disorder also allows the parents the choice of having an unaffected child, as the pregnancy can be terminated if the fetus is affected. However, many people see aborting a fetus as ethically unacceptable. This ethical dilemma is particularly difficult for a genetic disease such as severe FVII deficiency for which effective treatment exists, although the socio-economic costs of treating severe FVII deficiency can be significant. The high cost of prophylactic recombinant therapy and the frequency with which treatment is required compared with haemophilia A and B makes this a challenging disorder for clinicians, patients and their families and ultimately liver transplantation may have to be considered for severely affected individuals.

In summary, we have studied two families with severe FVII deficiency, identifying three mutations in the F7 gene; allowing prenatal diagnosis in one case. Two of the mutations are novel. Our understanding of the consequences of the missense mutation FVII G117R was greatly enhanced by sequence comparisons of recently identified and characterized orthologous and paralogous sequences.

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