Genomic studies of expanded trinucleotide repeats : focus on neuropsychiatric disorders

Abstract: 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 onginally 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 <0.00001) (paper II). The average size of RED products in affected individuals was found to correlate well with the average repeat size as determined by PCR. Thus, the accuracy as well as the ability of RED to associate novel expanded CAG/CTG repeats with disease was determined. Eight SCA7 families were analyzed for the presence of expanded CAG/CTG repeats (paper III). RED products of 150-240 nt were found in all affected individuals and were associated with disease (p<0.000001). This strongly indicated that a CAG expansion is the cause of SCA7. Based on the previous correlation between RED product size and actual repeat size, we estimated the averagc repcat size in the SCA7 families analyzed to be around 64 repeat copies. The recent cloning of the SCA7 gene has verified these results. Forty-six Parkinson's disease probands and eleven families displaying clear anticipation were studied using the RED technique (paper IV). Despite the marked anticipation, there was no association between CAG/CTG RED products> 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<0.0007) (paper VI). The majority of RED products > = 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<0.03) as well as in the offspring material (p<0.02). Expanded alleles at the CTG 18.1 locus conferred an odds risk ratio of 2.6-2.8 for affective disorder. The majonty of 150-240 nt RED products detected in the affective disorder matenal correlated in size to expanded alleles at the ERDA1 locus on chromosome 17. Together, expanded alleles at the ERDA1 and CTG18.1 loci account for the majority of RED products > 120 nt detected in the Caucasian population (papers VIand 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.