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Short title: Genetic basis of MH
Full title: Molecular basis of malignant hyperthermia in the South African population
Malignant hyperthermia (MH) is an autosomal dominant disorder that is due to dysregulation of calcium ions in skeletal muscle. Individuals who inherit one of the causative MH mutations are sensitive to certain inhalation anaesthetic gases and muscle depolarizing agents. MH is one of the major causes of death under anaesthesia and has a prevalence of 1:40 000 in adults, but 1:12 000 in children. The reason for this difference is not currently understood, but children are clearly more susceptible than adults. During previous phases of our study we identified two novel MH loci in the human genome. However, the genes and specific mutations at these loci are not yet known. Our current study focuses on whether reported (or novel) mutations within the ryanodine gene on chromosome 19q contribute to the South African MH phenotype. Aims of our long term study include: exclusion of currently reported mutations as the reason for MH in our population, identification of novel genes involved in MH and the characterization of novel mutations in these genes.
Short title: FSHD in South Africa
Full title: Molecular analysis of the D4Z4 locus in the South African FSHD population
Facioscapulohumeral muscular dystrophy (FSHD) is a dominantly inherited, degenerative neuromuscular disorder. Patients initially present with atrophy of the facial musculature, accompanied by weakening of the upper arms and shoulder girdle. The disorder, however, expresses clinically within an extremely broad spectrum. This is clear when comparing patients at age 70 presenting with neither facial nor shoulder girdle weakness, to cases where the lower limb involvement may be observed in early childhood. The disorder is diagnosed on a molecular level via detection of deletion fragments originating from chromosome 4q35. This study proposes to verify the presence or absence of deletion fragments in the South African FSHD population. DNA will be extracted from blood samples of 25 selected individuals with a family history of FSHD, and analysed via standardised molecular procedures. A probe DNA molecule, referred to as p13E‑11, will be utilised to detect DNA fragments originating from chromosome 4q35. The detection procedure will confirm whether the fragments have undergone a reduction in repeat array number or not. Family members harbouring fragments smaller than 35 kilobases i.e. less than 10 copies of the repeat array, are FSHD positive, whereas those with more than 10 copies, FHSD negative. This study will contribute to further characterising the molecular pathogenesis of FSHD in South Africa.
Short title: Metallothionein gene expression in muscle disorders
Full title: Analysis of metallothionein gene expression in patients with muscle disorders
During the production of energy via the mitochondrion, compounds that are harmful to cells are produced. The respective cells thus respond to this altered physiological state by activating the expression of genes that encode proteins which function as antioxidants. Hence, these proteins serve as indicators of cells that are exposed to reactive oxygen species (ROS). This process of ROS production has been observed in muscle degenerative disorders, mitochondrial myopathies and various metabolic diseases. The early detection of these antioxidants will thus serve as a means to identify these cells prior to the onset of the disorder or presentation of symptoms. Due to the high energy demand of muscle cells, it is the aim of this study to investigate the expression profile of metallothioneins in cells of individuals affected with muscle disorders such as facioscapulohumeral muscular dystrophy, malignant hyperthermia as well as mitochondrial myopathies. Patient cell lines for each disorder will be cultured under normal and experimental conditions, and the metallothionein gene expression profile determined utilizing molecular techniques. It is expected that individuals with muscle disorders will present with higher levels of expression of the specific antioxidants than the control individuals. This study will aid in the early detection of cellular oxidative stress thus early detection of an altered metabolic state.
Short title: An improved DMD service
Full title: Towards a comprehensive molecular genetic service to all South African families with DMD / BMD
Duchenne and Becker muscular dystrophy are caused by defects in the DMD gene and only the identification of a mutation gives absolute certainty of diagnosis, accurate carrier detection and prenatal diagnosis in these families. Partial deletions and duplications are the types of mutations detected in about two thirds of patients. While the detection of deletions is relatively simple, the elucidation of point mutations is laborious due to the large size of the DMD gene (79 exons). Carrier detection in non-deletion boys relies on linkage analysis and creatine kinase (CK) levels, methods often not completely reliable. Optimal carrier ascertainment in all families, therefore, relies on the detection of all mutations.
Point mutation scanning findings produced over the period 1999-2001, in the South African cohort of patients, allowed for the identification of alterations in 5% of non-deletion boys. Therefore at the start of 2002, the establishment of an additional more sensitive mutation scanning method was proposed. The method of choice is Transgenomics WAVE machine, a denaturing high performance liquid chromatography system (DHPLC).
The second part of the project involves carrier status determination in female relatives of deletion positive boys, which is complicated by the normal X chromosome in the deletion assay. In order to overcome this problem, the computer-assisted laser densitometry (CALD) technique, which uses the automated fluorescent fragment analysis automated sequencer is being established and its efficacy assessed.
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