Molecular Analysis of Duchenne Muscular Dystrophy and Other XP Mutations Using Cloned DNA Sequences

Genetics

Gillard, Elizabeth Frances

University:University of Glasgow

关键词: Genetics;   

Others  :  http://theses.gla.ac.uk/77662/1/10997934.pdf

来源: University of Glasgow

【 摘 要 】

Duchenne muscular dystrophy (DMD) is the most common, lethal X-linked mutation in the United Kingdom, affecting one in every three thousand male livebirths. Affected males have progressive muscle weakness and die as a result of respiratory or cardiac involvement in their late teens or early twenties. Whilst the X-linked nature of DMD has been recognised for many years, the regional assignment of DMD to Xp21 is relatively recent. This was first proposed on the basis of X;autosome translocations and more recently has been supported by linkage analysis using polymorphic DNA probes from within and around the DMD locus. X-linked ichthyosis (XLI) is the most common cause of early onset scaly skin (ichthyosis), affecting one in every five to six thousand male livebirths and is due to a deficiency of steroid sulphatase (STS). Although the X-linked nature of one form of ichthyosis had been recognised for many years, deficiency of STS in association with this condition was a relatively recent discovery. STS was assigned to Xp22 to Xpter on the basis of somatic cell hybrid mapping and to Xp22.3 to Xpter on the basis of a male with an XY translocation. The STS locus is of particular interest as it is one of the few known X-linked loci which escape inactivation in females. The main aim of this project was to isolate DNA sequences from the short arm of the X-chromosome (Xp) which could be used to study either DMD or XLI. In order to achieve this, an x-chromosome specific EcoRI library was constructed from flow-sorted chromosomes. In the initial screening of this library, one hundred and twenty plaques were selected for further study of which twenty eight had human inserts. Sixteen of these were single-copy or low-copy sequences with homology to the human x-chromosome, four were sequences with homology to the autosomes, six had homology to repeat sequences and a further recombinant had homology to multiple sequences in both man and mouse. The X-specific inserts were further characterised by a somatic cell hybrid mapping panel and four were assigned to xp; GMGX9(DXS237) to Xp22.3-pter, GMGX10(DXS238) to Xp21-cen and GMGX1KDXS239) and GMGX12(DXS240) to Xp21-Xp22.3. The ability of these four probes to detect restriction fragment length polymorphisms (RFLPs) was studied using panels of twelve to fourteen X-chromosomes digested with each of thirteen restriction enzymes. Only GMGX9(DXS237) detected an RFLP with HindiII. This had alleles of 4kb and 2.5kb + 1.5kb, which occurred at frequencies of 0.67 and 0.33 respectively, and a Pic value of 0.44. These four probes were also tested in eleven individuals with DMD, BMD or contiguous gene syndromes including DMD, who all had deletions of pERT87(DXS164) and/or XJ(DXS206) (which are deleted in 6-10% of males with DMD/BMD). GMGXll(DXS239) was deleted in one individual with adrenal hypoplasia (AHC), glycerol kinase deficiency (GK) and DMD, whilst GMGX10(DXS238) was deleted in three individuals with large deletions. These eleven individuals were also examined with twenty seven other Xp probes. Only two of these eleven deletions could not be resolved from the others by these probes and there was no smallest region of overlap. The deletion in one boy (pedigree number 5097) with BMD, apparently encompassed that of a second (5265) with DMD, thus excluding the notion of an unique BMD domain in Xp21. Deletion studies in these eleven individuals and in somatic cell hybrids mapped GMGX10(DXS238) proximal to pERT84(DXS142), GMGX11(DXS239) between JBir(DXS270) and Ll-4(DXS68) and GMGX12(PXS240) between C7(DXS28) or B24(DXS67) and GMGX9(DXS239). GMGX10(DXS238), GMGXll(DXS239), GMGX12(DXS240), p20(DXS269), JBir(DXS270) and J66HI(DXS268) were used to screen one hundred and three boys with DMD or BMD, including nine of the eleven deletions described above. GMGX10(DXS238) and GMGX12(DXS240) detected no additional deletions. Thirty six additional deletions or altered fragment sizes were detected with GMGXll(DXS239), p20(DXS269), JBir(DXS270) and J66HI(DXS268). Thus 46% of boys in this group had deletions or altered restriction fragment lengths visualised with DNA probes.(Abstract shortened by ProQuest.).