Haddad, Lema; (1998) New approaches to co-segregation studies and mutation detection in familial hypercholesterolaemia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In most patients with heterozygous familial hypercholesterolaemia (FH) the disorder is caused by a mutation in the LDL receptor (LDLR) gene or the apolipoprotein B (APOB) gene. However, in a number of patients no mutation is detected in either gene. This suggests either the presence of variations in another gene(s) causing a phenotype identical to that of FH, or insensitivity of the technique used for the detection of all mutations. To explore the reasons why using the current techniques no mutation is detected in the LDLR gene in a proportion of patients with a clinical diagnosis of heterozygous FH, two different approaches were undertaken. A de novo mutation scanning method was developed, termed programmable melting display (PMD). This technique was required to be highly sensitive, capable of detecting all base changes and enabling the rapid analysis of a large number of samples. Analysis of a set of heterozygous FH samples demonstrated that PMD was more sensitive than SSCP under the conditions used for the detection of mutations in exon 3 of the LDLR gene. High throughput single stranded conformational polymorphism (SSCP) analysis was used to screen a set of 791 apparent heterozygous FH patients for mutations in the LDLR gene. In a sub-set of these in whom no mutation was found by SSCP analysis, a microsatellite-based approach to co-segregation studies of FH kindreds was developed. The CEPH (Centre d'Etude Polymorphism Humaine) reference families in addition to a well defined physical map of the region aided in the selection of markers. Two markers, D19S394 and D19S221, proved to be very valuable for this study. The applicability of co-segregation analysis using these microsatellites for prenatal diagnosis of a homozygous FH kindred was demonstrated, and the relationship between microsatellite allele size, linkage disequilibrium and FH causing mutations (e.g. R329X, E80K, V408M and G528D) in different population groups was also explored. Co-segregation analysis of 27 FH kindreds (166 individuals) in whom no mutation had been found, showed strong evidence for the involvement of the LDLR locus and the phenotype of hypercholesterolaemia. In two families where non-co-segregation was evident, the hypercholesterolaemia was not due to a defect in the LDLR gene, but to mutations in other genes causing familial defective apolipoprotein B (FDB) and familial combined hyperlipidaemia (FCH). In one large family however, with clear-cut autosomal dominant hypercholesterolaemia, there were numerous exclusions of co-segregation with the LDLR and APOB (apolipoprotein B) locus. This data provides evidence for the presence of a third locus causing a phenotype identical to that seen in FH due to LDLR (or APOB) gene defects.

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