Date of Award

2001

Degree Type

Thesis

Degree Name

Doctor of Philosophy

Faculty

Faculty of Communications, Health and Science

First Advisor

Associate Professor Nigel Laing

Second Advisor

Associate Professor Nigel Laing

Abstract

This thesis contributes to the Human Genome Project by adding detail to the physical and genetic maps of the human genome, and by identifying a strong candidate gene for a form of distal myopathy. Genomic clones for the human skeletal muscle genes slow troponin (TNN/1), alpha actin (ACTA1), and (3-tropomyosin (TPM2) were isolated for use in the fluorescent in situ hybridisation localisation of these genes on the cytogenetic map of the human genome. The localisation of these genes made them potential candidates for inherited skeletal muscle diseases, including the muscular dystrophies investigated here. Microsatellite, VNTR and RFLP markers were used in a search for linkage to a novel form of distal myopathy segregating in a Western Australian family. The decadic logarithm of the likelihood ratio, or 'lod score' method, was used to determine linkage between markers and this distal myopathy gene. A 22.4 cM candidate region was identified at 14q11.2. This was the first localisation of a distal myopathy gene. The Human Genome Organisation Nomenclature Committee reserved MPD1, 'myopathy, distal 1 ', for this form of distal myopathy, now known as Laing myopathy. The MPD1 candidate region was excluded as the disease gene location for two other forms of distal myopathy. Silburn myopathy in 1994, which established the genetic heterogeneity of distal myopathy, and Felice myopathy in 1996. The exclusion of the MPD1 and French-Canadian OPMD candidate regions as disease gene locations for a putative-OPMD segregating in a Western Australian family, proved that this disease gene did not lie at 14q11.2. Testing an MPD1 muscle-specific candidate gene for the Laing myopathy mutation, the myosin heavy polypeptide 7 gene (MYH7), identified seven base changes between the MPD1 proband sequence and the published MYH7 eDNA sequence. All of these base changes were found in eight unrelated, unaffected Western Australians, therefore none of them were the Laing myopathy mutation. Two further differences to the published MYH7 sequence segregated exclusively with the MPD1 proband. One of these, the MYH7 G5073C (cDNA)/G23628C (gDNA) base change, caused a critical change to the MYH7 13-myosin heavy chain polypeptide product (13-MyHC). An A 1663P 13-MyHC substitution. G23628/C 23628 segregated with Laing myopathy in the Western Australian distal myopathy family. This segregation was confirmed by a single-strand conformation polymorphism test, then used to test 256 unaffected chromosomes. None possessed MYH7C23628. Two patients from European distal myopathy families phenotypically similar to Laing myopathy, the Voit and Scoppetta families, were tested for the presence of MYH7 gDNA G23628/C23628 heterozygosity. Both were homozygous MYH7 G23628. One of these patients (Voit) was also tested for MYH7 eDNA G5073/C5073 heterozygosity. She was homozygous MYH7 G5073. An analysis of the effect of the 13-MyHC A 1663P substitution at various levels of protein structure strengthened the candidature of MYH7 G5073C as the Laing myopathy mutation. It demonstrated the extreme rarity of the 13-MyHC A 1663P substitution; it showed that this substitution did have a detrimental effect on coiled-coil formation; and it identified ways in which the 13-MyHC A 1663P substitution could disrupt myofibrillogenesis or contractility. Future research directions are identified and the contribution of this work to evolving concepts in muscular dystrophy is evaluated.

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