Molecular-based diagnostics of inherited disorders : multiple exon and trinucleotide repeat expansion analysis

Abstract

The subject of this thesis concerns two main issues. First, the development of appropriate technology for the efficient mutation screening of multiple exons. Secondly, the analysis of trinucleotide repeat disorders in the context of PCR amplification of repetitive DNA for diagnostic purposes. This latter observation has a significant impact on the molecular confirmation of a clinical diagnosis. The development of a microtitre format and direct sequencing of PCR amplified products for multiple exons was found to provide the twin advantages of high throughput and sensitivity for the analysis of mutations in the BRCA1 and BRCA2 genes. The translation of this technical platform to the analysis of mutations in the genetically and clinically heterogeneous disorder of Limb Girdle Muscular Dystrophy (LGMD) was also undertaken. The outcomes of this research were enhanced diagnostics and the identification of novel mutations in the disease-causing genes. As an ancillary aspect of the above research, the identification of carrier females with deletion events in the dystrophin gene was attempted to complement a direct sequencing approach for the identification of point mutations. A rapid and sensitive means of deletion detection was determined, which was compared with the more comprehensive analysis of fluorescently labeled amplification products. The analysis of trinucleotide repeat disorders concerned Huntington disease (HD) and Fragile X syndrome. Analysis for both relies on the accurate sizing of the trinucleotide repeats. This work incorporated the analysis of Huntington disease patients with null alleles and the subsequent identification of mutation events in these patients that led to the incorrect conclusion of apparent homozygosity for trinuclebotide repeats expansions. HD patients were also analysed with intermediate sized CAG repeats to determine if they exhibited instability in the GAG repeat number in other disease loci implicated in neurodegenerative disorders. Two case studies are presented to highlight the difficulties associated with molecular-based diagnostics of Fragile X syndrome. The first case was one of a rare compound heterozygote female, while the second concerned the finding of a microdeletion at the FRAXA locus in a male with a suspected diagnosis of Fragile X syndrome. This male was analysed further at the protein level, which cast doubt on the causative role of the microdeletion in his clinical phenotype.