Prenatal diagnosis of severe factor vii deficiency using mutation detection and linkage analysis

Field: Medicine (Health Sciences)

Severe inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder of blood coagulation. Both clinical expression and mutational spectrum are highly variable with a weak correlation between the FVII coagulant activity and the clinical bleeding tendency (Cooper et al, 1997). About 15% of homozygous patients present with cerebral haemorrhages occurring shortly after birth, accounting for a high mortality rate. Here, we report on a family whose first child was affected with a life-threatening bleeding diathesis related to a severe FVII deficiency. The combination of mutational analysis of FVII gene and linkage analysis allowed accurate prenatal diagnosis to be performed in a subsequent pregnancy. The propositus, a 2-year-old girl, presented with soft tissue haematoma during the early days of life and an intracranial haemorrhage before 2 months of age. Investigations documented very low levels of FVII activity (FVIIc < 2%) and FVII antigen (FVIIAg 7%). Both parents are asymptomatic but express reduced FVII levels (Father: FVIIc, 37%; FVIIAg, 26%; Mother: FVIIc, 48%; FVII Ag, 50%). Mutation screening was performed on the nine exons and the boundaries of the proband's FVII gene using a combination of denaturing gradient gel electrophoresis (DGGE) and direct sequencing (Fig 1) (Giansily et al, 1999). The patient was shown to have a compound heterozygous genotype with two nucleotide changes: a 6070 + 1G→A and a 8909T→C. The 6070 + 1G→A mutation occurs in the invariant GT dinucleotide at the 5′ donor splice site of intron 4 and is associated with exon 4 skipping, as previously described (McVey et al, 1998). The 8909T→C nucleotide change, localized within exon 6, induces a 135Cys→Arg substitution and disrupts the single disulphide bridge formed by Cys135 and Cys262 that links the light and heavy chains of the activated FVII molecule (Cooper et al, 1997). Thus, the 135Cys→Arg substitution is probably a deleterious mutation (Peyvandi et al, 1998). Both the 6070 + 1G→A and the 135Cys→Arg mutations eliminate a restriction site for the MluI and NlaIII enzymes, respectively, allowing easy polymerase chain reaction (PCR)-based detection assays to be developed. The mother is heterozygous for the 6070 + 1G→A mutation while the father is heterozygous for the 135Cys→Arg mutation. In addition, intron 7 of the FVII gene contains a highly polymorphic locus (Marchetti et al, 1992) characterized by a variable number of a 37-bp monomer repeat (VNTR), with the corresponding H6 and H7 alleles depending on a six- or a seven-repeat content. In this family, both parents were heterozygous and the H6 allele was associated with the mutation in both cases. A and B illustrate the mutation-specific detection assays (the 6070 + 1G→A mutation in A and the 135Cys→Arg mutation in B), while C shows the linkage analysis. In A, B and C, the DNA samples in lanes 1–4 are from the fetus, the affected sister, the father and the mother respectively (M: molecular weight marker; UD: undigested PCR product). Note that the maternally derived 6070 + 1G→A mutation (A, lane 4) removes the unique MluI restriction site in one allele resulting in a 386-bp fragment, while the normal digested allele gives two fragments of 262 bp and 124 bp respectively. Similarly, the paternally derived 135Cys→Arg mutation (B, lane 3) destroys the first NlaIII restriction site in one allele resulting in an apparent three bands pattern (a 279-bp, a 186-bp and two unresolved 101- to 93-bp fragments). The linkage analysis (C) further demonstrates that the fetus (lane 1) is homozygous for the H7 allele and did not inherit the mutation-bearing alleles present in the affected sister (lane 2). At 14 weeks of a subsequent pregnancy, a prenatal diagnosis was performed using amniocentesis. Fetal DNA was extracted directly from amniotic cells using conventional methods, followed by PCR amplification and digestion of the PCR fragments using the MluI and NlaIII enzymes, according to the manufacturer's recommendations. The fetus was homozygous for the wild-type FVII alleles, as well as for the H7 allele of intron 7 associated with the normal genotype in this family. Therefore, we concluded that the fetus was unaffected. The newborn was clinically normal and the FVIIC level determined at 6 months of age was 71%. To our knowledge, our observation is the third report in the literature of early molecular prenatal diagnosis of FVII deficiency (Millar et al, 1992; McVey et al, 1998) and emphasizes the reliability of mutation-specific detection assays in such families affected with a severe bleeding diathesis. Because complete mutation screening may be fastidious and not always available for families affected with rare disorders, our report also illustrates that prenatal diagnosis can be successfully performed by linkage analysis using highly polymorphic intragenic loci, and that the combination of mutation analysis and linkage analysis is not restricted to research settings, but is currently becoming available in routine clinical laboratories. The authors are grateful to Mrs Sandrine Geffroy and Valerie Baijot for technical assistance.