Genomic studies of expanded trinucleotide repeats : focus on neuropsychiatric disorders

In a number of neurological and neuropsychiatric disorders, a worsening of the disease phenotype from one generation to the next has been linked to expanded trinucleotide repeat sequences that increase in size upon transmission. The clinical phenomenon of an earlier age of onset or a more severe phenotype in later generations of a family has been termed anticipation. The focus of this thesis has been to study expanded repeat sequences at the genomic level and to identify new expanded repeats that may cause disease. The first method developed for direct detection of expanded repeat sequences was the repeat expansion detection (RED) technique. This method detects expanded repeats with no prior knowledge of the flanking sequence or the genomic location of the repeat. RED was originally used to detect expanded CAG/CTG repeats, and the method was subsequently optimised to include detection of short repeats of other motifs in the genome. Eight out of the ten trinucleotide repeat sequence motifs were optimised successfully to permit screening for expanded repeats in disorders showing anticipation (paper I and summary). Expanded repeats were found in five novel motifs, ATG/CAT, CTT/AAG, TGG/CCA, CCT/AGG and GTT/AAC. Machado-Joseph disease (MJD/SCA3) and spinocerebellar ataxia type 7 (SCA7) belong to a group of autosomal dominant cerebellar ataxias showing progressive degeneration of cerebellar pathways as well as anticipation. CAG/CTG RED products of 180-270 nt were associated with disease in five families with MJD/SCA3 (p 120 nt and disease. Anticipation has also been observed in affective disorder. In two independent large samples of bipolar affective disorder patients, CAG/CTG RED products > = 180 nt were associated with disease (p =180 nt as compared to 29 % of controls. Expanded RED products were associated with disease also in in a sample consisting of affected and unaffected offspring from 12 affective disorder families displaying anticipation (p = 300 nt seen in the Swedish patient material were found to correspond to expanded repeats at the CTG18.1 locus on chromosome 18. Expansions at the CTG18.1 locus were associated with disease in this sample (p 120 nt detected in the Caucasian population (papers VI and VII). A combination of RED analysis and PCR analysis of the ERDA1 and CTG18.1 loci thus markedly increases the sensitivity of the RED method in the search for pathogenic repeats. A novel isolation strategy for sequences flanking expanded repeats has been developed to enable isolation of disease genes. The isolation of expanded repeat sequences is complicated by the instability of large repeats in prokaryotic hosts. The new strategy is based on the ability of RED to identify pools or fractions of DNA containing the expanded repeat in each of a series of physical purification steps. Cloning is facilitated by the enrichment of repeat-containing fragments prior to cloning as well as by careful selection of prokaryotic hosts and procedures