Molecular Genetic Analysis of the C1-Inhibitor Gene in Hereditary Angio-Oedema

[摘要] The development of recombinant DNA technology has enabled the molecular genetic basis of a number of inherited diseases to be established. This thesis describes the application of this technology to the study of hereditary Cl-inhibitor deficiency or hereditary angio-oedema (HAE). In addition, the findings of a clinical survey of HAE in Scotland are presented. Hereditary angio-oedema is an autosomal dominant disease in which there is either a quantitative (Type I HAE) or functional (Type II HAE) deficiency of Cl-inhibitor, this protein being an important inhibitor of complement system activation and an inhibitor of other serine protease dependent plasma mediator systems. In Type I HAE, which constitutes approximately 85% of cases, functionally normal Cl-inhibitor is present in the serum but at low levels. This contrasts with Type II HAE in which there are normal or sometimes elevated levels of serum Cl-inhibitor as measured by immunoassay, though most of this is in the form of a functionally inactive molecule. Clinically Type I and Type II disease cannot be distinguished since both present with recurrent attacks of subcutaneous or submucous oedema. In this study restriction fragment length polymorphism (RFLP) analysis of the Cl-inhibitor gene was carried out on genomic DNA isolated from 25 normal, unrelated individuals and on genomic DNA samples from 12 Type I HAE kindred and two Type II HAE kindred. A total of 38 restriction endonucleases were used to digest each DNA sample followed by agarose gel electrophoresis and Southern blotting of each sample onto nylon hybridisation membranes. Each digested DNA sample was then probed with a radiolabelled exon 2-8 Cl-inhibitor cDNA which represents the full protein coding sequence of the Cl-inhibitor gene. Four of the 12 Type I HAE kindred were shown to have unique disease-specific RFLPs affecting one allele of the Cl-inhibitor gene. Localisation studies clearly demonstrated that the gene mutations responsible for each RFLP affected exon 4, the 3' exon/ intron boundary of exon 6, exon 7 and exon 8 of the Cl-inhibitor gene. Family studies showed that each mutation co-segregated with the disease. Mutations affecting exon 6 and exon 8 had not been reported previously in Type I HAE. Both these mutations were small, possibly point mutations and it is highly likely that the mutation at the extreme 3' boundary of exon 6 results in loss of the donor splice site for excision of the sixth intron during RNA processing. The effect of the small exon 8 mutation is less certain. Nucleotide sequence analysis of both these mutated areas is currently being undertaken. This should help to establish their likely effects. The remaining two mutations, which were shown to be a complete exon 4 deletion and a complete exon 7 deletion, had recently been documented in Type I HAE by other investigators. No RFLPs were identified in the two Type II HAE kindred. Detection of a disease-specific RFLP in 33% of the Type I HAE kindred tested represents a significantly higher detection rate than other published studies in which 16% was the previous maximum. This increase appears to reflect the greater number of restriction enzymes employed since no other methodological differences were apparent. These results suggest that Type I HAE is likely to be due to a multiplicity of gene mutations as is seen in other genetic diseases including B-thalassaemia, haemophilia A and haemophilia B. Cl-inhibitor gene RFLPs were generated by the restriction enzymes Kpn I and Hgi AI in the normal population. The Kpn I RFLP had not been reported previously and was shown to be due to a mutation that lay approximately 10kb downstream of the Cl-inhibitor gene. The Hgi AI RFLP had already been observed in normal individuals from North America and arose due to a point mutation within the Cl-inhibitor gene itself. Both RFLPs fulfill criteria which are likely to make them useful as indirect genetic markers of the mutant Cl-inhibitor allele in those HAE families who lack a disease-specific RFLP. The clinical survey of HAE in Scotland identified a total of 46 patients which represents a disease incidence of approximately one patient per 110,000 of the population. This is the first documented attempt to calculate disease incidence in Scotland. It became evident from the survey that despite the development of more effective treatment regimes in recent years, significant disease-associated morbidity and mortality still existed within the relatively young patient population. This observation provided ample justification for studying the molecular genetics of HAE since an understanding of this aspect of the disease is central to future disease prevention in the form of prenatal diagnosis.

[发布机构] University:University of Glasgow

[关键词] Genetics, Molecular biology