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.

The molecular genetic analysis of hemophilia A: a directed search strategy for the detection of point mutations in the human factor VIII gene.

A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA----TGA), 372 (CGC----TGC), 372 (CGC----CAC), and 1689 (CGC----TGC). These are functionally important cleavage sites for either activated protein C or thrombin. Further novel C----T transitions were identified in the remaining arginine codons screened (-5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking.

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 patient's family. However, DNA analysis for the specific mutation shows one sister and the patient's 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.

Purification and characterization of factor VIII 372-Cys: a hypofunctional cofactor from a patient with moderately severe hemophilia A.

We have purified factor VIII from a patient with moderately severe hemophilia A (FVIII, 4 U/dL; FVIII:Ag, 110 U/dL) and subjected the protein to Western blot analysis after time course activation with thrombin. The cross reacting material-positive (CRM+) FVIII has the normal distribution of heavy and light chains before thrombin activation, and, after incubation with the enzyme, appropriate cleavages are made at positions 740 and 1689. However, the normal thrombin cleavage at position 372 in the heavy chain of this molecule does not occur. This result is consistent with the demonstration in the patient's leukocyte DNA of a C to T transition in codon 372, leading to the substitution of a cysteine for an arginine residue at the heavy chain internal cleavage site. The severely impaired functional activity of this molecule confirms that the heavy chain of FVIII must be proteolysed in order to effect full cofactor activation in vivo. However, a threefold activation was detected when this protein was incubated with thrombin. No evidence of thrombin-mediated cleavage at position 336 in the heavy chain was detected, in contrast to the variant recombinant B domainless-molecule, FVIII 372-Ile, described by Pittman and Kaufman (Proc Natl Acad Sci USA 85:2429, 1988). Using gel permeation studies of the FVIII/von Willebrand factor (vWF) complex before and after thrombin activation, we have demonstrated that the 40 Kd A2 domain of wild type FVIII dissociates from vWF after cleavage by the enzyme. In contrast, incomplete dissociation was detected in the case of FVIII 372-Cys. We conclude that the functional defect in FVIII 372-Cys is a consequence of the resistance to proteolysis of the internal scissile bond in the heavy chain.

Biopsy of human preimplantation embryos and sexing by DNA amplification.

A single cell was removed, through a hole made in the zona pellucida, from each of 30 human embryos at the 6-10 cell cleavage stage three days after in-vitro fertilisation. A normal proportion of the embryos (37%) developed to the blastocyst stage by day six in culture and 6 hatched from the zona. Each male embryo was sexed from the DNA by amplification of a repeated sequence specific for the Y chromosome. In 15 embryos with the normal two pronuclei the sex was determined also by in-situ hybridisation with a Y specific probe or fluorescent chromosome staining to detect metaphase Y chromosomes; the results of Y specific amplification were confirmed. This approach may be valuable for couples at risk of transmitting X-linked disease.

Haemorrhagic cystitis in bone marrow transplantation patients: possible increased risk associated with prior busulphan therapy.

Following bone marrow transplantation employing conditioning including 'high-dose' cyclophosphamide, 65 patients were studied for the subsequent development of symptomatic haemorrhagic cystitis. There was no protection from the urothelial toxicity of cyclophosphamide metabolites afforded by the concurrent administration of 2-mercaptoethane sodium sulphonate (mesna) if timing errors in administration were made. Other factors which might increase the risk of haemorrhagic cystitis due to cyclophosphamide administration include the prior administration of busulphan to patients with chronic granulocytic leukaemia, in whom the incidence of haemorrhagic cystitis was 36% compared with 4% in all other patients. We have also investigated the use of intravesical prostaglandin E2 as a treatment for haemorrhagic cystitis in eight patients, two of whom appeared to obtain major benefit.

Pulmonary cryptosporidiosis occurring in a bone marrow transplant patient.

We describe what is believed to be the first bone marrow transplant patient, a 32-year-old man, in whom pulmonary cryptosporidiosis was associated with terminal respiratory failure. The diagnosis, treatment and postmortem histology are discussed together with a brief review of the literature.