Gene expression profiles at the myotonic dystrophy 1 locus : Possible role in disease mechanisms

Myotonic dystrophy 1 (DM1) is the commonest adult onset muscular dystrophy, (prevalence 1/8000). The disorder is characterized by anticipation (earlier onset in successive generations, which is accompanied in this disorder by an increase in severity of symptoms) and variable multisystemic symptom presentation. The mutation is caused by a trinucleotide CTGn expansion in the 3´untranslated region of the myotonic dystrophy protein kinase gene (DMPK) on chromosome 19q13.3 (DM1- locus). The repeat is unstable and in almost all transmissions it expands, providing the molecular explanation for anticipation. The mechanisms by which the repeat leads to the complex symptomatology remain controversial. The purpose of this thesis has been to investigate the expression pattern of the DM1-locus genes as: (a) the repeat is located in a cluster of genes and the expanded triplet could cause changes in the chromatin structures which could alter the expression of the nearby genes; (b) the repeat is located in the 3´UTR of DMPK and expanded transcripts have been shown to be sequestered in the nucleus, but genuine haploinsufficiency of the DMPK gene product has never been demonstrated. When quantifying the DM1-locus genes in skeletal muscle biopsies from DM1 patients and controls our results showed a wide spread of expression and overlap between our groups (DM1, non-DM1 myopathic disorders, and unaffected). However we detected an inverse correlation between the expression of DMPK and DMWD with repeat length measured in peripheral blood leucocytes. This indicated that the repeat expansion might have a modifying effect on the expression of these two genes in skeletal muscle and was the first report to suggest that DMWD expression may be influenced by the expansion. This work was then extended to investigate if the wide spread of expression we observed was due to the heterogenous tissue samples which were used in the quantification experiments. We microdissected single fibres from freeze dried skeletal muscle biopsies from DM1 patients and controls. Fibres from each biopsy sample were pooled according to their staining characteristic for different myosin heavy chains, and quantification was performed with real-time quantitative RT-PCR. The results showed downregulation of DMPK expression in type IIA fibres of DM1 patients compared with unaffected controls, supporting a DMPK haploinsufficiency model for this disorder. This was the first report of DM1-locus gene expression in which mixed cell populations or tissue culture artifacts have been excluded as confounding variables. If gene dosage at the DM 1 -locus contributes to the development of the phenotype, its effects might vary in different tissues as a consequence of the basal expression of each gene. We have quantified the expression pattern of the DM 1 locus genes in a range of different tissues and ages of wildtype mice. Our data shows that there is extensive postnatal regulation of the DM I locus genes in different tissues, and the DM I -locus genes are not co-ordinately regulated. A candidate gene's expression pattern may yield information about its possible involvement in a disorder. The expression pattern of Dmwd follows the DM1 phenotype very well, with the highest expression in lens, testis and skeletal muscle, tissues commonly affected in DM1, and lowest levels in tissues not affected in DM1. If this has any pathophysiological relevance is still highly speculative, as the function of D~ is not known, but this might indicate that DMWD could be more involved in some of the aspects of the phenotype than has generally been recognized. If the expansion does change the adjacent chromatin structure this could result in a field effect leading to abberrant expression of nearby genes. It is therefore of interest to extend our information about this chromosomal locus. We have identified a fourth gene at the DM 1 locus, the radial spokehead protein like I-gene (RSHL1). This gene is located only 2kb upstream of Dmwd and is the first identified radial spokehead protein in mammals. In lower organisms the protein is important for normal flagellar and ciliary movement, and sperm mobility in sea urchin. We have detected testis specific expression of this gene on Northern blots from human and mouse. Whilst downregulation of RSHL1 could perhaps be involved in the male infertility observed in DM1 patients, it may be a much stronger candidate gene for primary cilia dyskinesia, a heterogeneous disorder characterised by defects in sperm mobility, which in some families has been linked to chromosome 19q13.3