Peter Alan Holmans

De novo CNV analysis implicates specific abnormalities of postsynaptic signalling complexes in the pathogenesis of schizophrenia.

A small number of rare, recurrent genomic copy number variants (CNVs) are known to substantially increase susceptibility to schizophrenia. As a consequence of the low fecundity in people with schizophrenia and other neurodevelopmental phenotypes to which these CNVs contribute, CNVs with large effects on risk are likely to be rapidly removed from the population by natural selection. Accordingly, such CNVs must frequently occur as recurrent de novo mutations. In a sample of 662 schizophrenia proband–parent trios, we found that rare de novo CNV mutations were significantly more frequent in cases (5.1% all cases, 5.5% family history negative) compared with 2.2% among 2623 controls, confirming the involvement of de novo CNVs in the pathogenesis of schizophrenia. Eight de novo CNVs occurred at four known schizophrenia loci (3q29, 15q11.2, 15q13.3 and 16p11.2). De novo CNVs of known pathogenic significance in other genomic disorders were also observed, including deletion at the TAR (thrombocytopenia absent radius) region on 1q21.1 and duplication at the WBS (Williams–Beuren syndrome) region at 7q11.23. Multiple de novos spanned genes encoding members of the DLG (discs large) family of membrane-associated guanylate kinases (MAGUKs) that are components of the postsynaptic density (PSD). Two de novos also affected EHMT1, a histone methyl transferase known to directly regulate DLG family members. Using a systems biology approach and merging novel CNV and proteomics data sets, systematic analysis of synaptic protein complexes showed that, compared with control CNVs, case de novos were significantly enriched for the PSD proteome (P=1.72 × 10−6). This was largely explained by enrichment for members of the N-methyl-D-aspartate receptor (NMDAR) (P=4.24 × 10−6) and neuronal activity-regulated cytoskeleton-associated protein (ARC) (P=3.78 × 10−8) postsynaptic signalling complexes. In an analysis of 18u2009492 subjects (7907 cases and 10u2009585 controls), case CNVs were enriched for members of the NMDAR complex (P=0.0015) but not ARC (P=0.14). Our data indicate that defects in NMDAR postsynaptic signalling and, possibly, ARC complexes, which are known to be important in synaptic plasticity and cognition, play a significant role in the pathogenesis of schizophrenia.

Gene-wide analyses of genome-wide association data sets: evidence for multiple common risk alleles for schizophrenia and bipolar disorder and for overlap in genetic risk

Genome-wide association (GWAS) analyses have identified susceptibility loci for many diseases, but most risk for any complex disorder remains unattributed. There is therefore scope for complementary approaches to these data sets. Gene-wide approaches potentially offer additional insights. They might identify association to genes through multiple signals. Also, by providing support for genes rather than single nucleotide polymorphisms (SNPs), they offer an additional opportunity to compare the results across data sets. We have undertaken gene-wide analysis of two GWAS data sets: schizophrenia and bipolar disorder. We performed two forms of analysis, one based on the smallest P-value per gene, the other on a truncated product of P method. For each data set and at a range of statistical thresholds, we observed significantly more SNPs within genes (Pmin for excess<0.001) showing evidence for association than expected whereas this was not true for extragenic SNPs (Pmin for excess>0.1). At a range of thresholds of significance, we also observed substantially more associated genes than expected (Pmin for excess in schizophrenia=1.8 × 10−8, in bipolar=2.4 × 10−6). Moreover, an excess of genes showed evidence for association across disorders. Among those genes surpassing thresholds highly enriched for true association, we observed evidence for association to genes reported in other GWAS data sets (CACNA1C) or to closely related family members of those genes including CSF2RB, CACNA1B and DGKI. Our analyses show that association signals are enriched in and around genes, large numbers of genes contribute to both disorders and gene-wide analyses offer useful complementary approaches to more standard methods.

A family based association study of T102C polymorphism in 5HT2A and schizophrenia plus identification of new polymorphisms in the promoter.

Several studies have shown an association between schizophrenia and the C allele of a T-C polymorphism at nucleotide 102 and the 5HT2A receptor gene. In the present study we observed this association in a sample of 63 parent/offspring trios where the proband received a diagnosis of DSM-III-R schizophrenia using TDT analysis (χ2u2009=u20096.26, Pu2009=u20090.006,χ2u2009=u20099.00, Pu2009=u20090.001 when one affected offspring was selected at random from each family, suggesting that the results are due to association rather than linkage). There was no significant difference between the transmission of C102 from heterozygous fathers and mothers, which fails to support a role for genomic imprinting in this effect. T102C does not result in an alteration of the amino acid sequence of the protein. We therefore screened the promoter of 5HT2A for polymorphisms using single-strand confirmation polymorphism analysis. An A-G polymorphism at −1438 that creates an HpaII restriction site was identified. This was found to be in complete linkage disequilibrium with T102C and is hence a candidate for the pathogenic variant in schizophrenia. Functional analysis of A-1438G using luciferase assay demonstrated significant basal promoter activity in 5HT2A expressing HeLa cells by both the A and G variants. However, comparison of the A and G variants showed no significant differences in basal activity nor when promoter activity was induced by cAMP and protein kinase C-dependent mechanisms.

A combined analysis of D22S278 marker alleles in affected sib-pairs: Support for a susceptibility locus for schizophrenia at chromosome 22q12

Several groups have reported weak evidence for linkage between schizophrenia and genetic markers located on chromosome 22q using the lod score method of analysis. However these findings involved different genetic markers and methods of analysis, and so were not directly comparable. To resolve this issue we have performed a combined analysis of genotypic data from the marker D22S278 in multiply affected schizophrenic families derived from 11 independent research groups worldwide. This marker was chosen because it showed maximum evidence for linkage in three independent datasets (Vallada et al., Am J Med Genet 60:139-146, 1995; Polymeropoulos et al., Neuropsychiatr Genet 54:93-99, 1994; Lasseter et al., Am J Med Genet, 60:172-173, 1995. Using the affected sib-pair method as implemented by the program ESPA, the combined dataset showed 252 alleles shared compared with 188 alleles not share (chi-square 9.31, 1df, P = 0.001) where parental genotype data was completely known. When sib-pairs for whom parental data was assigned according to probability were included the number of alleles shared was 514.1 compared with 437.8 not shared (chi-square 6.12, 1df, P = 0.006). Similar results were obtained when a likelihood ratio method for sib-pair analysis was used. These results indicate that may be a susceptibility locus for schizophrenia at 22q12.

Multicenter Linkage Study of Schizophrenia Candidate Regions on Chromosomes 5q, 6q, 10p, and 13q: Schizophrenia Linkage Collaborative Group III *

Schizophrenia candidate regions 33-51 cM in length on chromosomes 5q, 6q, 10p, and 13q were investigated for genetic linkage with mapped markers with an average spacing of 5.64 cM. We studied 734 informative multiplex pedigrees (824 independent affected sibling pairs [ASPs], or 1,003 ASPs when all possible pairs are counted), which were collected in eight centers. Cases with diagnoses of schizophrenia or schizoaffective disorder (DSM-IIIR criteria) were considered affected (n=1,937). Data were analyzed with multipoint methods, including nonparametric linkage (NPL), ASP analysis using the possible-triangle method, and logistic-regression analysis of identity-by-descent (IBD) sharing in ASPs with sample as a covariate, in a test for intersample heterogeneity and for linkage with allowance for intersample heterogeneity. The data most supportive for linkage to schizophrenia were from chromosome 6q; logistic-regression analysis of linkage allowing for intersample heterogeneity produced an empirical P value <.0002 with, or P=.0004 without, inclusion of the sample that produced the first positive report in this region; the maximum NPL score in this region was 2.47 (P=.0046), the maximum LOD score (MLS) from ASP analysis was 3.10 (empirical P=.0036), and there was significant evidence for intersample heterogeneity (empirical P=.0038). More-modest support for linkage was observed for chromosome 10p, with logistic-regression analysis of linkage producing an empirical P=. 045 and with significant evidence for intersample heterogeneity (empirical P=.0096).

A meta-analysis and transmission disequilibrium study of association between the dopamine D3 receptor gene and schizophrenia

We performed a meta-analysis of over 30 case-control studies of association between schizophrenia and a bi-allelic, BalI polymorphism in exon 1 of the dopamine D3 receptor gene. We observed a significant excess of both forms of homozygote in patients (P = 0.0009, odds ratio (OR) = 1.21, 95% Confidence Interval (CI) = 1.07–1.35) in the combined sample of 5351 individuals. No significant heterogeneity was detected between samples and the effects did not appear to be the product of publishing bias. In addition we undertook an independent, family-based association study of this polymorphism in 57 parent/proband trios, taken from unrelated European multiplex families segregating schizophrenia. A transmission disequilibrium test (TDT) showed a significant excess of homozygotes in schizophrenic patients (P = 0.004, odds ratio (OR) = 2.7, 95% CI = 1.35–5.86). Although no significant allelic association was observed, a significant association was detected with the 1–1 genotype alone (P = 0.02, OR = 2.32, 95% CI = 1.13–4.99). In addition when the results of the family-based association study were included in the meta-analysis, the homozygosity effect increased in significance (Pu2009=u20090.0002, OR = 1.23, 95% CI = 1.09–1.38). The results of the meta-analysis and family-based association study provide independent support for a relationship between schizophrenia and homozygosity at the BalI polymorphism of the D3 receptor gene, or between a locus in linkage disequilibrium with it.

Genomewide Significant Linkage to Recurrent, Early-Onset Major Depressive Disorder on Chromosome 15q

A genome scan was performed on the first phase sample of the Genetics of Recurrent Early-Onset Depression (GenRED) project. The sample consisted of 297 informative families containing 415 independent affected sibling pairs (ASPs), or, counting all possible pairs, 685 informative affected relative pairs (555 ASPs and 130 other pair types). Affected cases had recurrent major depressive disorder (MDD) with onset before age 31 years for probands or age 41 years for other affected relatives; the mean age at onset was 18.5 years, and the mean number of depressive episodes was 7.3. The Center for Inherited Disease Research genotyped 389 microsatellite markers (mean spacing of 9.3 cM). The primary linkage analysis considered allele sharing in all possible affected relative pairs with the use of the Z(lr) statistic computed by the ALLEGRO program. A secondary logistic regression analysis considered the effect of the sex of the pair as a covariate. Genomewide significant linkage was observed on chromosome 15q25.3-26.2 (Zlr=4.14, equivalent LOD = 3.73, empirical genomewide P=.023). The linkage was not sex specific. No other suggestive or significant results were observed in the primary analysis. The secondary analysis produced three regions of suggestive linkage, but these results should be interpreted cautiously because they depended primarily on the small subsample of 42 male-male pairs. Chromosome 15q25.3-26.2 deserves further study as a candidate region for susceptibility to MDD.

A systematic genomewide linkage study in 353 sib pairs with schizophrenia

We undertook a genomewide linkage study in a total of 353 affected sib pairs (ASPs) with schizophrenia. Our sample consisted of 179 ASPs from the United Kingdom, 134 from Sweden, and 40 from the United States. We typed 372 microsatellite markers at approximately 10-cM intervals. Our strongest finding was a LOD score of 3.87 on chromosome 10q25.3-q26.3, with positive results being contributed by all three samples and a LOD-1 interval of 15 cM. This finding achieved genomewide significance (P<.05), on the basis of simulation studies. We also found two regions, 17p11.2-q25.1 (maximum LOD score [MLS] = 3.35) and 22q11 (MLS = 2.29), in which the evidence for linkage was highly suggestive. Linkage to all of these regions has been supported by other studies. Moreover, we found strong evidence for linkage (genomewide P<.02) to 17p11.2-q25.1 in a single pedigree with schizophrenia. In our view, the evidence is now sufficiently compelling to undertake detailed mapping studies of these three regions.

Association of slow acetylator genotype for N-acetyltransferase 2 with familial Parkinson's disease

BACKGROUNDnEpidemiological studies have identified positive family history and exposure to environmental toxins as risk factors for Parkinsons disease (PD). An inherited defect of xenobiotic metabolism could result in increased susceptibility to such toxins. We investigated the frequency of functionally relevant polymorphisms in six detoxification enzymes among patients with PD to elucidate the relation between these polymorphisms and the disease.nnnMETHODSnWe obtained brain-tissue samples from 100 patients with apparently sporadic PD and blood samples from 100 living patients with familial PD. For the control group, we extracted DNA from the tissue of 100 pathologically normal brains. The six enzymes analysed in these three groups were: CYP2D6, CYP2E1, NAD(P)H-menadione reductase, glutathione transferases M1 and T1, and N-acetyltransferase 2. We also investigated N-acetyltransferase 2 in 100 blood samples from patients with genetically proven Huntingtons disease. We used PCR-based methods and restriction-enzyme analysis to detect polymorphisms.nnnFINDINGSnThe slow acetylator genotype for N-acetyltransferase 2 was more common in the familial PD group (69%) than in all controls (37%). Even after correction for multiple comparisons, this result remained highly significant (p = 0.002) for familial PD compared with normal controls (odds ratio 3.79 [95% CI 2.08-6.90]) and compared with Huntingtons disease (2.45 [1.37-4.38], p = 0.004). The slow acetylator frequency for N-acetyltransferase 2 for sporadic PD was between that for Huntingtons disease and familial PD. The frequencies of all the other polymorphisms were similar in the two study groups and the normal control group.nnnINTERPRETATIONnWe found an association between the slow acetylator genotype for N-acetyltransferase 2 and familial PD. Further studies are needed to investigate the biological relevance of these findings, but slow acetylation could lead to impaired ability of patients with familial PD to handle neurotoxic substances.

A simple method for analyzing microsatellite allele image patterns generated from DNA pools and its application to allelic association studies

Allelic association studies provide the most powerful method for locating genes of small effect contributing to complex diseases and traits. However, in outbred populations, allelic association is usually maintained only over distances of <=1 cM. Therefore, systematic searches over large regions are costly. Here we present a method involving DNA pooling that can be used as a rapid preliminary screen for allelic association with the most common class of polymorphic markers, single-sequence repeats. Patient and control samples are pooled separately, and markers are typed in the two pools. By use of primers with fluorescent 5 ends, PCR products can be analyzed on an automated sequencing apparatus. Allele image patterns (AIPs) produced for the two groups are overlaid and differences in pattern area between pools computed. From this, a DeltaAIP statistic is calculated from the difference in areas between the two AIPs expressed as a fraction of the total shared and nonshared area. AIPs of a range of different-sized pools were generated by computer simulation for markers with a range of allele sizes and frequencies. DeltaAIPs from pools and chi2 values for individual genotypings were compared, with both simulated and real data from microsatellite markers. The results demonstrated a high correlation between DeltaAIP and chi2 values. DeltaAIP analysis of real microsatellite data indicated the feasibility of using this method in systematic searches for allelic association and generated a small number of false positives but few false negatives. We conclude that DeltaAIP analysis of DNA pools can be used effectively and efficiently as a rapid screen for allelic association in case-control studies.