Developmental Timing and the Role of Cis and Trans Acting Modifiers on CTG Repeat Instability in Murine Models

Myotonic dystrophy type 1 (DM1), is one of a number of genetic diseases whose phenotype is associated with the expansion of a CTG.CAG repeat. In the human population the DM1 triplet repeat locus demonstrates moderate variations in repeat length, the normal range being between 5 and 37 repeats. However, repeat lengths present in patients are far larger, beyond 50 and into the thousands, with variation in a patient, within gametes and somatic mosaicism between and within tissues. The repeat instability level in DM1 patients is effected by several factors including the initial size of the progenitor allele and the patients age. Expansion over time and mutation rate variation between the somatic tissues of an individual is thought to contribute to the tissue specificity and progressive nature of the symptoms, whilst the size of the repeat inherited tends to expand through successive generations. This mechanism correlates with the incidence of 'anticipation' observed in affected families, that is the disease becomes more severe in each successive generation. To investigate the mechanism of repeat expansion murine models have been developed containing the Dmt-162 transgene, which consists of ~162 CTG repeats derived from the human DM1 locus. Previous studies had shown some repeat instability in the somatic tissues of young (male) mice of each line. Dmt- 162 mice have already shown parent-of-origin-effects, with paternal repeat transmissions prone to expansion and maternal repeat transmissions prone to deletion. Further pedigree data analysis confirmed this effect and allowed the repeat transmission patterns to be analysed in more depth. The effects of the parents age on the transmitted repeat size was examined but was found not be significant in either sex. Initially the repeat instability in a number of somatic tissues from mature mice in four of the lines generated was investigated using small pool PCR. Whilst minimal instability was observed in three of the lines, the fourth, Dmt-D demonstrated expansion biased instability which varied greatly between tissues. Investigation at earlier timepoints revealed the mosaicism to increase with age. Microdissection of the brain and the kidney revealed the repeat instability patterns to be region specific. Examination of the repeat mosaicism presented by the regions of the kidney observed dramatic repeat instability within the cortex. This in turn lead to the investigation of a candidate cell type for the dramatic repeat instability, the glomereli. Investigation of transgenic transcription using reverse transcriptase PCR has identified tissue specific transcription of the repeat in three of the investigated lines and ubiquitous transcription in the fourth. The orientation of the transcribed CTG.CAG repeat has also been determined. Finally to investigate the effect of an incomplete mismatch repair complex (MMR), Dmt transgenic mice were crossed on to a background deficient in the MMR protein Pms2 and the effect on repeat transmission and somatic instability studied