Cloning of candidate genes for bipolar disorder starting from expanded trinucleotide repeats

Bipolar (BP) disorder is a severe psychiatric condition affecting about 1% of the population. The clinical signs of BP disorder are alternating episodes of depression and mania. Family, twin and adoption studies provided evidence for a genetic component in the etiology of the disorder, however the exact mode of inheritance remains unclear. Nevertheless, we identified chromosome 18q21.3--q22 as a candidate region by linkage analysis studies in the Belgian pedigree MAD31.;Several studies described anticipation in families transmitting BP disorder. Anticipation is a decrease in age at onset and/or an increase in disease severity in successive generations. In many diseases showing anticipation, this clinical feature has been explained by triplet repeat expansions as the underlying pathogenic mechanism. Therefore the involvement of trinucleotide repeat expansions in the etiology of BP disorder was hypothesized. This hypothesis was further strengthened by experiments with the repeat expansion detection method showing that the distribution of CAG/CTG repeats was significantly shifted to larger sizes in the DNA of BP patients compared with controls. In the work described in this thesis, the hypothesis that a triplet repeat expansion causes BP disorder linked to chromosome 18q21.3--q22 was investigated.;First, a new methodology was developed for the region specific isolation of triplet repeats, based on yeast artificial chromosome (YAC) fragmentation. Novel YAC fragmentation vectors were constructed and tested with different target sequences, both repetitive and unique. To test the triplet repeat hypothesis in BP disorder, vectors were constructed with as target sequence a short triplet repeat. Application of CAG/CTG based YAC fragmentation to the chromosome 18q21.3--q22 BP candidate region resulted in the isolation of 4 CAG/CTG repeats from this region, including the CAG/CTG repeat in the 5'UTR region of the brain-expressed cytoplasmatic antiproteinase 2 (CAP2) gene. Analysis of these CAG/CTG repeats in members of a chromosome 18 linked BP family did not result in any expanded alleles. Moreover, analysis in a BP case/control sample did not show associated alleles nor genotypes. After excluding the involvement of CAG/CTG repeats in BP disorder linked to chromosome 18q21.3--q22, the method was adapted and applied for the isolation of CCG/CGG repeats from the same region. Only one CCG/CGG repeat, the (CCG)6 repeat present in the 5'UTR of the CAP2 gene, was isolated. In addition, 3 potential CpG islands were isolated. One, CCG4, was localised upstream of a novel gene (NCAG1) that was subsequently shown to be expressed in different tissues, including brain. Sequence analysis of the open reading frame of the NCAG1 gene identified 2 polymorphisms, both changing the predicted protein sequence.;Together, these data excluded involvement of CAG/CTG and CCG/CGG repeats in BP disorder, linked to chromosome 18q21.3--q22.