Egd Tuddenham

Haemophilia A diagnosis by analysis of a hypervariable dinucleotide repeat within the factor VIII gene

The diagnosis of haemophilia A and the identification of carriers has greatly improved with knowledge of the structure of the gene for factor VIII. This has permitted the defect to be tracked in families by the study of restriction fragment length polymorphisms (RFLPs), irrespective of the nature of the molecular defect. However, this approach is time-consuming and the information yielded falls away as more polymorphisms are added. Within the factor VIII gene lies another source of polymorphism, a dinucleotide repeat sequence of varying length known as (CA)n. Conventional mapping localised this (CA)n repeat to intron 13. The polymerase chain reaction, used to examine (CA)n variability in genomic DNA from 25 males and 67 females, revealed eight allelic bands between n = 16 and n = 24. 91% of females were heterozygous for this repeat, and family studies showed X-linked mendelian inheritance with allelic frequencies ranging from 1% to 45%. The intron 13 (CA)n repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. The analysis requires DNA from other family members, and relatives of sporadic cases of haemophilia A are only amenable to exclusion. Nonetheless, this intron 13 (CA)n repeat provides the most highly informative marker so far available for factor VIII gene tracking studies in haemophilia A kindreds and a result can be available within a day.

450 million years of hemostasis

Summary.  In mammalian blood coagulation, five proteases (factor VII [FVII]; factor IX [FIX]; factor X [FX]; protein C [PC] and prothrombin [PT]) act with five cofactors (tissue factor [TF]; factor V [FV]; factor VIII [FVIII]; thrombomodulin and protein S) to control the generation of fibrin. Biochemical evidence, molecular cloning data and comparative sequence analysis support the existence of all components of this network in all jawed vertebrates, and strongly suggest that it evolved before the divergence of teleosts over 430 million years ago. Phylogenetic analysis of the amino acid sequences of the Gla–EGF1–EGF2–SP domain serine proteases (FVII, FIX, FX, PC) and the A domain‐containing cofactors (FV and FVIII) strongly supports the evolution of the blood coagulation network through two rounds of gene duplication, and supports the hypothesis that vertebrate evolution benefited from two global genome duplications. The jawless vertebrates (hagfish and lamprey) that diverged over 450 million years ago have a blood coagulation network involving TF, PT and fibrinogen. Preliminary evidence indicates that they may have a smaller complement of Gla–EGF1–EGF2–SP domain proteins, suggesting the existence of a ‘primitive’ coagulation system in jawless vertebrates.

Factor V I359T: a novel mutation associated with thrombosis and resistance to activated protein C

Summary. We report a kindred in which two siblings suffered spontaneous venous thromboses in the second decade of life. Further investigation showed reduced coagulation factor V (FV) activity and activated protein C resistance (APCR) ratio but no other thrombophilic abnormalities. The reduction in APCR ratio persisted in a modified APCR assay in which FV activity was normalized between test and control plasmas. Analysis of the FV gene showed that the thrombotic individuals had a complex genotype that included two novel point mutations c.529G>T and c.1250T>C resulting in FV E119X and FV I359T substitutions inherited on different alleles. Individuals in the kindred with FV E119X or FV I359T substitutions alone were asymptomatic. We suggest that the FV I359T substitution confers pro‐thrombotic risk and APCR, but that this is only clinically manifest when co‐inherited with the FV E119X allele. The FV I359T substitution creates a new consensus sequence for N‐linked glycosylation within the FV heavy chain and we speculate that this abnormal glycosylation may disrupt activated protein C‐mediated proteolysis of the variant FV and FVa.

Identification of two novel mutations in non-Jewish factor XI deficiency.

Summary. We have studied two heterozygous unrelated CRM non‐Jewish FXI‐deficient patients. Neither of the patients carries a previously described mutation. Their FXI genes were screened by SSCP analysis following PCR amplification of each exon and the flanking intronic sequences. DNA fragments showing aberrant mobility were cloned and sequenced. The following mutations were identified: in case 1, a T to G transition in exon 12 results in the substitution of Phe‐442 by Val (FXI‐F442V); in case 2 a C to A transition in exon 5 results in the substitution of Cys‐128 by a nonsense codon (FXI‐C128X). The missense mutation results in a substitution within the protease domain of FXI. Molecular modelling locates this residue in a structurally conserved region of the protease domain and the amino acid substitution may therefore interfere with either chain folding and subsequent secretion or the stability of the protein in plasma. We conclude that the mutations which we have identified are responsible for the inherited abnormality in these patients.

Factor VIIShinjo: A Dysfunctional Factor VII Variant Homozygous for the Substitution Gln for Arg at Position 79

We report a factor VII (FVII) variant, FVIIShinjo, characterized by normal FVII antigen levels and variable procoagulant activity using tissue thromboplastin from different sources. Normal FVII activity is obtained using human placenta thromboplastin but low activity using rabbit or bovine brain thromboplastin. Exons 2-8 and the intron-exon junctions of the FVII genes of the propositus were amplified by PCR from DNA extracted from peripheral white blood cells, and screened by single-strand conformational polymorphism (SSCP) analysis. DNA fragments showing aberrant mobility were cloned and sequenced. We detected a single-point mutation, a homozygous G to A transition at nucleotide position 6,055 in exon 4, which results in the substitution of Arg 79 by Gln in the first EGF-like domain. This mutation results in a loss of a site for the restriction endonuclease MspI. The Msp I digestion pattern of the PCR-amplified exon 3+4 fragments from each member of the family was determined. The Msp I haplotypes were consistent with this G to A transition being associated with reduced FVII activity as detected using thromboplastins from various species. We conclude that the Arg 79 to Gln substitution in the first EGF-like domain of FVII identified in the propositus is responsible for the inherited FVII abnormality in this Japanese family. We postulate that one of the sites of interaction between FVII and tissue thromboplastin includes Arg 79 in the first EGF-like domain of factor VII.

CRM+ haemophilia A due to a missense mutation (372→Cys) at the internal heavy chain thrombin cleavage site

We have used the polymerase chain reaction (PCR) and differential oligonucleotide melting to screen for mutations in selected CpG dinucleotides in the factor VIII genes of haemophilia A patients. By this means we have identified and confirmed by sequencing a novel point mutation in codon 372 (CGC) of the factor VIII gene of a moderately severe CRM+ haemophiliac. The first C of this codon has been substituted by T resulting in the non‐conservative substitution of cysteine for arginine at an essential thrombin cleavage site in factor VIII. Analysis of three intragenic restriction fragment length polymorphisms was uninformative in the patients family. However, DNA analysis for the specific mutation shows one sister and the patients mother to be carriers, and the other sister to be normal. This DNA analysis confirmed the results of phenotype analysis by factor VIII coagulant to von Willebrand factor antigen ratios for the females at risk. The two carrier females had low factor VIII coagulant activity and excess VIII antigen as predicted but the non‐carrier sister also had anomalously high VIII antigen in her plasma. When feasible, mutation specific DNA analysis is able to resolve the difficulties posed by variable phenotype data and unknown level of mutation in sporadic haemophilia A.