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Molecular analysis of myotonic dystrophy type 1 patients with an unusual molecular diagnosis

Braida, Claudia (2008) Molecular analysis of myotonic dystrophy type 1 patients with an unusual molecular diagnosis. PhD thesis, University of Glasgow.

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Abstract

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterised by multiple tissue involvement and caused by an expansion of a (CTG)n repeat within the 3’-UTR of the DMPK gene (19q13.3). Normal individuals contain between 5 and 35 CTG repeats, whereas the repeats in DM1 patients expand in the range of 50 to several thousands. Longer alleles are very unstable and generally always increase in size when transmitted from parent to child, explaining the phenomenon of anticipation defined by earlier age of onset and an increase in the severity of the symptoms. Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous, hereditary motor and sensory neuropathy of the peripheral nervous system. To date, 30 different loci have been mapped and mutations have been identified in more than 20 different genes. The DM1+CMT++ family is a very unusual three generation family in which all patients co-segregate both DM1 and CMT (LOD score = 7.03). It was postulated that either a single or two closely linked mutations near the APOC2 marker must be the cause of DM1 and CMT. Southern blot analysis of restriction digested genomic DNA revealed a fragment equivalent to a small CTG expansion (~200-400) at the DM1 locus, but an expanded allele could not be amplified by PCR. We postulated that the expanded repeats may have predisposed the repeat tract and the flanking regions to further DNA instability, leading to a secondary deletion, insertion and/or rearrangement. These novel mutations might modify the expression of DMPK and/or nearby genes explaining the unusual clinical presentation. To identify the lesion in the DM1+CMT++ family, a variety of molecular approaches was performed. The molecular lesion identified was an insertion of a GC rich region within the CTG repeats. The allele was comprised of a variable number of CTGs at the 5'-end followed by (GGC)3 G (CCG)20 (CCGCTG)14 (CTG)35. Analysis of single molecule separated alleles revealed 3 that the interrupted 3'-end of the array was stable, while the CTG repeats at the 5'-end were unstable. Postulated mechanisms to explain the DM1 and CMT symptoms in the family were: a novel RNA gain-of-function, and/or a novel effect on the downstream genes. Finding an imperfect CTG repeat allele in the DM1+CMT++ family led us to suggest that imperfect CTG repeat alleles may not be unique events and other DM1 patients may also contain similar alleles. To investigate this DNA samples from 14 DM1 patients with an unusual molecular diagnosis were analysed. The majority of these patients presented with an imperfect CTG repeat allele containing CCGCTG hexamers and/or CCG repeats. Five patients contained two or three higher order repeats containing between 18 and 30 bp such as ((CTG)5 (CCG)5), ((CTG)2 (CCGCTG)4) and ((CTG)5 (CCG)2 (CCGCTG)). These findings further suggest that imperfect CTG repeat alleles might not be as rare as was previously believed. The results of this project point out the importance of performing a more detailed molecular characterisation of the DM1 patients, which could lead to the provision of more accurate prognoses and the development of effective therapies.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: myotonic dystrophy, Charcot-Marie-Tooth disease, interruptions, novel repeats.
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology > Molecular Cell and Systems Biology
Supervisor's Name: Monckton, Dr. Darren
Date of Award: 2008
Depositing User: Dr Claudia Braida
Unique ID: glathesis:2008-359
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 Oct 2008
Last Modified: 10 Dec 2012 13:18
URI: http://theses.gla.ac.uk/id/eprint/359

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