Carol Guy

Confirmation of association between expanded CAG/CTG repeats and both schizophrenia and bipolar disorder

Recent studies have suggested that expanded CAG/CTG repeats contribute to the genetic aetiology of schizophrenia and bipolar disorder. However, the nature of this contribution is uncertain and difficult to predict from other known trinucleotide repeat diseases that display much simpler patterns of inheritance. We have sought to replicate and extend earlier findings using Repeat Expansion Detection in an enlarged sample of 152 patients with schizophrenia, 143 patients with bipolar disorder, and 160 controls. We have also examined DNA from the parents of 62 probands with schizophrenia or bipolar disorder. Our results confirm our earlier, preliminary findings of an association between expanded trinucleotide repeats and both schizophrenia and bipolar disorder. However, our data do not support the hypothesis that trinucleotide repeat expansion can alone explain the complex patterns of inheritance of the functional psychoses neither can this mechanism fully explain apparent anticipation.

The impact on outcome of the addition of all-trans retinoic acid to intensive chemotherapy in younger patients with nonacute promyelocytic acute myeloid leukemia: overall results and results in genotypic subgroups defined by mutations in NPM1,FLT3, and CEBPA

We investigated the benefit of adding all-trans retinoic acid (ATRA) to chemotherapy for younger patients with nonacute promyelocytic acute myeloid leukemia and high-risk myelodysplastic syndrome, and considered interactions between treatment and molecular markers. Overall, 1075 patients less than 60 years of age were randomized to receive or not receive ATRA in addition to daunorubicin/Ara-C/thioguanine chemotherapy with Ara-C at standard or double standard dose. There were data on FLT3 internal tandem duplications and NPM1 mutations (n = 592), CEBPA mutations (n = 423), and MN1 expression (n = 195). The complete remission rate was 68% with complete remission with incomplete count recovery in an additional 16%; 8-year overall survival was 32%. There was no significant treatment effect for any outcome, with no significant interactions between treatment and demographics, or cytarabine randomization. Importantly, there were no interactions by FLT3/internal tandem duplications, NPM1, or CEBPA mutation. There was a suggestion that ATRA reduced relapse in patients with lower MN1 levels, but no significant effect on overall survival. Results were consistent when restricted to patients with normal karyotype. ATRA has no overall effect on treatment outcomes in this group of patients. The study did not identify any subgroup of patients likely to derive a significant survival benefit from the addition of ATRA to chemotherapy.

Further support for an association between a polymorphic CAG repeat in the hKCa3 gene and schizophrenia.

A recent study has suggested that a polymorphism in the hKCa3 potassium channel may be associated with raised susceptibility to schizophrenia.1 Despite its modest statistical significance, the study1 is intriguing for two reasons. First, hKCa3 contains a polymorphic CAG repeat in its coding sequence, with large repeats more common in schizophrenics compared with controls.1 This is interesting in view of several repeat expansion detection (RED) studies2 that have reported an excess of large CAG repeats in psychotic probands.3–7 Second, the hKCa3 gene is a functional candidate gene because studies of antipsychotic and psychotogenic compounds suggest that glutamatergic systems modulated by SKCa channels may be important in schizophrenia pathogenesis.1 In the light of the above, we have tested the hypothesis of an association between schizophrenia and the hKCa3 CAG repeat polymorphism using a case control study design. Under the same model of analysis as the earlier study, schizophrenic probands had a higher frequency of alleles with greater than 19 repeats than controls (χ2 = 2.820, P = 0.047, 1-tail). Our data therefore provide modest support for the hypothesis that polymorphism in the hKCa3 gene may contribute to susceptibility to schizophrenia.

Long Repeat Tracts at SCA8 in Major Psychosis

Expansion at a recently identified unstable trinucleotide repeat on chromosome 13q21 has been reported as the molecular cause for spinocerebellar ataxia type 8 (SCA8). The trinucleotide repeat, which consists of a [CTA]n repeat and adjacent [CTG]n repeat, was reported to have a pathogenic range of 107-127 CTG repeats (or 110-130 combined CTA and CTG repeats) in a large ataxia kindred. This repeat region was also cloned by our group from a bipolar affective disorder (BPAD) patient, who has approximately 600 combined repeats, and large alleles (>100 repeats) were reported to be present in 0.7% of controls and 1.5% of major psychosis patients (n = 710 and n = 1,120, respectively). We have followed up these findings by screening three new samples of BPAD and schizophrenia (SCZ) patients and controls, including 272 individuals from 14 BPAD families from Sweden, 130 individuals from 32 SCZ and BPAD families/trios from the Azores Islands, and 206 SCZ individuals from the United Kingdom and Ireland, and 219 matched controls. We found large repeat alleles above the SCA8 pathogenic range in individuals from 3 of 32 Azorean pedigrees and in 1 of 206 SCZ individuals from the United Kingdom, and repeat alleles within the SCA8 pathogenic range in 1 of 14 Swedish families. Although the rarity of major psychosis patients carrying the SCA8 expansion mutation would require a much larger sample size to reach statistical significance, these results support the previously reported observation of increased occurrence of large repeats at SCA8 in major psychosis. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:873-876, 2000.

CAG repeat length in the hKCa3 gene and symptom dimensions in schizophrenia

BACKGROUND Long CAG repeats in the hKCa3 potassium channel gene have been associated with schizophrenia. We sought evidence for associations between this polymorphism and aspects of the schizophrenia phenotype. METHODS Associations were investigated between CAG repeat length and gender, age of illness onset, and psychotic symptom dimensions in 203 unrelated individuals with DSM-IIIR schizophrenia. RESULTS No association was found between CAG repeat length and gender or age of onset. Long CAG repeats were associated with higher negative symptom dimension scores. CONCLUSIONS This study provides preliminary evidence that genetic liability to negative symptoms in schizophrenia may be partly mediated through the hKCa3 gene.

Promoter polymorphisms in glutathione-S-transferase genes affect transcription.

The glutathione-S-transferases are a group of enzymes that play a major role in detoxification and defence against toxic, carcinogenic and other compounds. We analysed the proximal promoters of 14 genes encoding glutathione-S-transferase for polymorphism. Ten of the promoters contained sequence variants, nine of which we were able to clone into a reporter gene vector, pGL3. The relative ability of each haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671) using a cotransfected CMV-SEAP plasmid as a control. Four genes (GSTA1, GSTA2, GSTM4 and GSTT2) showed activity differences greater than 1.5-fold between haplotypes, and a fifth gene (MGST1) showed a 1.4-fold difference. The promoter sequence variants in these genes may therefore play a role in human variation, susceptibility to diseases and the effects of drugs.

Characterisation, mutation detection, and association analysis of alternative promoters and 5’UTRs of the human dopamine D3 receptor gene in schizophrenia

The dopamine D3 receptor gene (DRD3) is a candidate for a number of psychiatric conditions including schizophrenia, bipolar disorder and alcohol and drug abuse. Previous studies have reported associations between polymorphisms in DRD3 and these disorders, but these findings may have reflected linkage disequilibrium with pathogenic variants that are further upstream. We have isolated and sequenced approximately 9 kb of genomic sequence upstream of the human DRD3 translational start site. Using 5′ RACE, we have identified within this region three additional exons and two putative promoter regions which show promoter activity in three different cell lines. A 5′ UTR identified only in lymphoblasts is spread over three exons and is 353 bp long. A second 5′ UTR, found in adult and fetal brain, lymphocytes, kidney and placenta is spread over two exons and is 516 bp long. A 260-bp sequence within this 9 kb corresponds to a previously reported EST, but corresponding mRNA could not be found in the tissues above. The EST, 5′ UTRs and putative promoter regions have been analysed for polymorphisms, revealing 10 single nucleotide polymorphisms, seven of which were tested for association in a large sample of unrelated patients with schizophrenia and matched controls. No associations were observed with schizophrenia. In addition we failed to replicate previous findings of association with homozygosity of the Ser9Gly variant. The results from this study imply that neither the coding nor the regulatory region of DRD3 plays a major role in predisposition to schizophrenia.

Linked polymorphisms upstream of exons 1 and 2 of the human cholecystokinin gene are not associated with schizophrenia or bipolar disorder

The evidence for a significant genetic contribution to the functional psychoses (schizophrenia and bipolar disorder) is now well established. However, in both cases, the non-mendelian mode of inheritance has made the identification of susceptibility loci particularly challenging.1–3 The neuropeptide cholecystokinin (CCK) is present both in the gut and the CNS. Studies of CCK-like immunoreactivity and CCK mRNA levels in human brains have revealed high concentrations in numerous loci and shown colocalisation of CCK with, for example, dopamine and tyrosine hydroxylase.4 Furthermore, antagonists of CCK-B receptors, which are found most frequently in the brain, inhibit the activity of brain dopamine neurons.5 Such findings suggest that, with respect to neuropsychiatric disorders, CCK is a suitable candidate for analysis using methods to detect gene variations which have the potential to affect protein structure or expression.6 In the present study, mutation analyses were carried out on the human CCK gene. Linked polymorphisms were found in the promoter region and in intron 1 close to the 3′ mRNA splice acceptor site. However, the allele frequencies of these polymorphisms in samples of individuals affected with either schizophrenia (n = 117) or bipolar disorder (n = 124) did not differ from those of control subjects (n = 234), suggesting that these variations do not confer a predisposition to either of the functional psychoses.

No evidence for expanded polyglutamine sequences in bipolar disorder and schizophrenia

Several recent studies have suggested that expanded CAG repeats may contribute to the genetic transmission of bipolar disorder and schizophrenia. In all known disorders associated with expanded CAG repeats, the repeat sequence is translated into glutamine. Therefore the simplest hypothesis is that one or more proteins with expanded polyglutamine sequences are involved in the pathogenesis of bipolar disorder and schizophrenia. In order to examine this hypothesis, we have used an antibody against expanded polyglutamine sequences to examine Western blots prepared from lymphoblastoid cell lines of patients with schizophrenia and bipolar disorder. We also examined Western blots prepared from left frontal cortex tissue samples obtained from 11 schizophrenics post mortem. With the exception of the TATA- binding protein (TBP), we did not detect any proteins containing expanded polyglutamine sequences. Our data therefore suggest either that the expanded repeats which are associated with these disorders do not encode polyglutamine, or that they are within genes that are not expressed within the tissues investigated here.

A high proportion of chromosome 21 promoter polymorphisms influence transcriptional activity

We have sought to obtain an unbiased estimate of the proportion of polymorphisms in promoters of human genes that have functional effects. We carried out polymorphism discovery on a randomly selected group of 51 gene promoters mapping to human chromosome 21 and successfully analyzed the effect on transcription of 38 of the sequence variants. To achieve this, a total of 53 different haplotypes from 20 promoters were cloned into a modified pGL3 luciferase reporter gene vector and were tested for their abilities to promote transcription in HEK293t and JEG-3 cells. Up to seven (18%) of the 38 tested variants altered transcription by 1.5-fold, confirming that a surprisingly high proportion of promoter region polymorphisms are likely to be functionally important. The functional variants were distributed across the promoters of CRYAA, IFNAR1, KCNJ15, NCAM2, IGSF5, and B3GALT5. Three of the genes (NCAM2, IFNAR1, and CRYAA) have been previously associated with human phenotypes and the polymorphisms we describe here may therefore play a role in those phenotypes.