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
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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.
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