Anna E. Vine

Study of Regions of Extended Homozygosity Provides a Powerful Method to Explore Haplotype Structure of Human Populations

Previous investigations have reported linkage disequilibrium occurring between nearby polymorphisms, a block‐like structure for such relationships, some instances where surprisingly few haplotypes are found and regions of extended homozygosity which are especially marked around centromeres and which are especially common on the X chromosome. We investigated the distribution and nature of regions of extended homozygosity in a sample of 1411 subjects included in a genome wide association study. Regions of extended homozygosity over 1Mb are common, with an average of 35.9 occurring per subject, and containing on average 73 homozygous markers. They have a markedly non‐random distribution. They are relatively common on the X chromosome and are seen at centromeres but are also concentrated at other chromosomal regions where presumably recombination is rare. They seem to be a consequence of some haplotypes being very common in the population and although sometimes this reflects the effect of a very common haplotype we also note that there are examples of two or three common haplotypes, each very different from each other, underlying this effect. Regions of extended homozygosity are commoner than previously appreciated. They result from the presence of extended haplotypes with high population frequency. Such regions concentrate in particular locations. The haplotypes involved are sometimes markedly disparate from each other. These regions offer a valuable opportunity for further investigation, in particular with regard to their ancestral history.

Case-case genome-wide association analysis shows markers differentially associated with schizophrenia and bipolar disorder and implicates calcium channel genes

Objective There are theoretical reasons why comparing marker allele frequencies between cases of different diseases, rather than with controls, may offer benefits. The samples may be better matched, especially for background risk factors common to both diseases. Genetic loci may also be detected which influence which of the two diseases occurs if common risk factors are present. Method We used samples of UK bipolar and schizophrenic cases that had earlier been subject to genome-wide association studies and compared marker allele frequencies between the two samples. When these differed for a marker, we compared the case sample allele frequencies with those of a control sample. Results Eight markers were significant at P value of less than 10−5. Of these, the most interesting finding was for rs17645023, which was significant at P value of less than 10−6 and which lies 36 kb from CACNG5. Control allele frequencies for this marker were intermediate between those for bipolar and schizophrenic cases. Conclusion The application of this approach suggests that it does have some merits. The finding for CACNG5, taken together with the earlier implication of CACNA1C and CACNA1B, strongly suggests a key role for voltage-dependent calcium channel genes in the susceptibility to bipolar disorder and/or schizophrenia.

Confirmation of prior evidence of genetic susceptibility to alcoholism in a genome-wide association study of comorbid alcoholism and bipolar disorder.

Objectives Alcoholism and affective disorders are both strongly comorbid and heritable. We have investigated the genetic comorbidity between bipolar affective disorder and alcoholism. Methods A genome-wide allelic association study of 506 patients from the University College London bipolar disorder case–control sample and 510 ancestrally matched supernormal controls. One hundred forty-three of the bipolar patients fulfilled the Research Diagnostic Criteria diagnosis of alcoholism. A total of 372 193 single nucleotide polymorphisms (SNPs) were genotyped. Genes previously shown to be associated with alcoholism and addiction phenotypes were then tested for association in the bipolar alcoholic sample using gene-wise permutation tests of all SNPs genotyped within a 50-kb region flanking each gene. Results Several central nervous system genes showed significant (P<0.05) gene-wise evidence of association with bipolar alcoholism. The genes implicated, which replicated genes previously shown to be associated with alcoholism were: cadherin 11, collagen type 11 &agr;2, neuromedin U receptor 2, exportin7, and semaphorin-associated protein 5A. The SNPs most strongly implicated in bipolar alcoholism, but, which did not meet conventional genome-wide significance criteria were the insulin-like growth factor-binding protein 7, carboxypeptidase O, cerebellin 2, and the cadherin 12 genes. Conclusion We have confirmed the role of some genes previously shown to be associated with alcoholism in the comorbid bipolar alcoholism subgroup. In this subgroup, bipolar disorder may lower the threshold for the phenotypic expression of these alcoholism susceptibility genes. We also show that some genes may independently increase susceptibility to affective disorder and alcoholism.

No evidence for excess runs of homozygosity in bipolar disorder.

Background Recent studies have reported large common regions of homozygosity (ROHs) that are the result of autozygosity, that is, the cooccurrence within individuals of long haplotypes that have a high frequency in the population. A recent study reports that such regions are found more commonly in individuals with schizophrenia compared with controls, and identified nine ‘risk ROHs’ that were individually more common in cases. Of these, four contained or neighboured genes associated with schizophrenia (NOS1AP/UHMK1, ATF2, NSF and PIK3C3). Methods We have applied the same methodology to a UK sample of 506 cases with bipolar disorder and 510 controls. Results There was no overall excess of common ROHs among bipolar individuals. With one exception, the haplotypes accounting for the ROHs appeared to be distributed according to the Hardy–Weinberg equilibrium. One ROH was individually more common among cases (uncorrected P = 0.0003). This ROH spanned the chromosome 2p23.3 gene ITSN2 (the gene for intersectin 2 isoform 2). However, inspection of the homozygous haplotypes and haplotype-based tests for association failed to provide a clearer understanding of why this ROH was occurring more commonly. Conclusion Overall, we conclude that, in contrast with schizophrenia, common ROHs are rarely associated with susceptibility to bipolar disorder. This supports the idea that predominantly different genes are increasing susceptibility to schizophrenia and bipolar affective disorders.

A simple method for assessing the strength of evidence for association at the level of the whole gene

Introduction It is expected that different markers may show different patterns of association with different pathogenic variants within a given gene. It would be helpful to combine the evidence implicating association at the level of the whole gene rather than just for individual markers or haplotypes. Doing this is complicated by the fact that different markers do not represent independent sources of information. Method We propose combining the p values from all single locus and/or multilocus analyses of different markers according to the formula of Fisher, X = ∑(−2ln(pi)), and then assessing the empirical significance of this statistic using permutation testing. We present an example application to 19 markers around the HTRA2 gene in a case-control study of Parkinson’s disease. Results Applying our approach shows that, although some individual tests produce low p values, overall association at the level of the gene is not supported. Discussion Approaches such as this should be more widely used in assimilating the overall evidence supporting involvement of a gene in a particular disease. Information can be combined from biallelic and multiallelic markers and from single markers along with multimarker analyses. Single genes can be tested or results from groups of genes involved in the same pathway could be combined in order to test biologically relevant hypotheses. The approach has been implemented in a computer program called COMBASSOC which is made available for downloading.

A pragmatic suggestion for dealing with results for candidate genes obtained from genome wide association studies

BackgroundResearchers may embark on a genome-wide association study before fully investigating candidate regions which have been reported to produce evidence to suggest that they harbour susceptibility loci. If the genome wide study had not been carried out then results which demonstrated only modest statistical significance from candidate regions would be judged to be of interest and would stimulate further investigation. However if hundreds of thousands of markers are typed then inevitably very large numbers of such results will occur by chance and those from candidate regions may attract no special attention.ResultsAn approach is proposed in which differential treatment is afforded to markers from candidate regions and from those that are routinely typed in the context of a genome wide scan. Different prior probabilities are assigned to the two types of marker. A likelihood ratio is derived from the reported p value for each marker, calculated as LR = echiinv(1,p)/2, and the posterior odds in favour of a true positive association are obtained. These odds can be used to rank the markers with a view to suggesting the regions in which further genotyping is indicated. We suggest that prior probabilities be specified such that a candidate marker significant at p = 0.01 and a routine marker significant at p = 0.00001 will yield similar values for the posterior odds. We show that this can be achieved by setting a value for prior probability of association to 0.1 for candidate markers and to 0.00018 for routine markers.ConclusionIt is essential that formal procedures be adopted in order to avoid modestly positively results from candidate regions being swamped by the huge number of nominally significant results which will be obtained when very many markers are genotyped. Software to carry out the conversion from p values to posterior odds is available from http://www.mds.qmul.ac.uk/statgen/grpsoft.html.

Yin yang haplotypes revisited - long, disparate haplotypes observed in European populations in regions of increased homozygosity.

Objectives: Yin yang haplotypes differ at every SNP. A previous study provided striking examples of these haplotypes, but claimed that their distribution across the genome could be attributed to chance. When we studied regions of homozygosity (ROHs) we found haplotypes that tended to differ at several SNPs simultaneously but did not subject this to formal testing. Here, we formally assess whether haplotypes from these regions provide evidence for the yin yang effect. Method: We identified 20 regions where ROHs are common in a sample of European subjects and studied the most frequent haplotypes of SNPs with high minor allele frequency. We calculated the average ‘disparity’ between all haplotype pairs using a ‘disparity score’ calculated as 2d, where d is the number of SNPs at which a pair of haplotypes differ. We also studied these SNPs in HapMap samples. Results: Overall, there was highly significant evidence for excess haplotype disparity. Three of the HapMap populations with European ancestry also had statistically significant haplotype disparity scores. Conclusion: Locations where ROHs are frequent harbour long common haplotypes showing surprising disparity. We believe these haplotypes must represent ancient founder effects and could be valuable for elucidating features of a population’s history.

Investigation into the ability of SNP chipsets and microsatellites to detect association with a disease locus.

We wished to investigate the ability of different SNP chipsets to detect association with a disease and to investigate the linkage disequilibrium (LD) relationships between microsatellites and nearby SNPs in order to assess their potential usefulness to detect association.

PW01-232 - Connectivity genes in comorbid alcoholism and bipolar disorder

Introduction Bipolar disorder (BPD) and alcoholism are strongly comorbid and both have significant genetic influences but no consistent genetic vulnerability has been found. We aimed to find bipolar-alcoholism vulnerability genes. Method A genome-wide association study (GWAS) of 510 patients with bipolar disorder (BPD), of whom 143 met Research Diagnostic Criteria (RDC) alcoholism diagnoses, and 506 ancestrally matched supernormal controls. We genotyped 372K genetic markers on an Affymetrix 500K-array. Chi-square analysis of allelic association using PLINK, and permutation testing for gene-wise association of genes previously associated with alcoholism-related phenotypes using COMBASSOC, were performed. Results No marker met genomewide significance. Gene-wise analyses of markers clustering near genes already implicated in alcoholism, but which were not associated in non-alcoholic BPD, were: Cadherin-11 (CDH11, p = 6 × 10-4), Exportin 7 (XPO7), neuromedin-U receptor 2 (NMUR2), collagen type XI-alpha 2 (COL11A2) and Semaphorin-5A (SEMA5A). Discussion These genes replicated prior genetic reports implicating “connectivity” (adhesion, migration and neuronal signalling) genes in addictions and comorbid BPD. Connectivity genes regulate neuronal connections during development and play roles in later neuroadaptive and mnemonic processes. These processes may influence addiction vulnerability, as seen clinically in denial, cognitive impairment, and repetitive substance misuse and relapse behaviour. We propose that we have identified genes i) increasing susceptibility to alcoholism that could be unmasked or released by the presence of bipolar affective disorder; ii) and genes increasing susceptibility to affective disorder that also predispose to secondary alcoholism. We were limited by small sample size. Larger future studies are needed.