Pamela Sklar

Characterization of single-nucleotide polymorphisms in coding regions of human genes

Nature Genet. 14, 415– 420 (1996). Due to a cloning error, the sequence reported for ING1 was incorrect. The error appears to have been a result of a compression introducing a frameshift and of the ING1 gene encoding several differentially spliced isoforms that contain a common 3′ exon, one of whichis of a size very similar to that reported in the publication above.

Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse

Single-nucleotide polymorphisms (SNPs) have been the focus of much attention in human genetics because they are extremely abundant and well-suited for automated large-scale genotyping. Human SNPs, however, are less informative than other types of genetic markers (such as simple-sequence length polymorphisms or microsatellites) and thus more loci are required for mapping traits. SNPs offer similar advantages for experimental genetic organisms such as the mouse, but they entail no loss of informativeness because bi-allelic markers are fully informative in analysing crosses between inbred strains. Here we report a large-scale analysis of SNPs in the mouse genome. We characterized the rate of nucleotide polymorphism in eight mouse strains and identified a collection of 2,848 SNPs located in 1,755 sequence-tagged sites (STSs) using high-density oligonucleotide arrays. Three-quarters of these SNPs have been mapped on the mouse genome, providing a first-generation SNP map of the mouse. We have also developed a multiplex genotyping procedure by which a genome scan can be performed with only six genotyping reactions per animal.

Support for involvement of neuregulin 1 in schizophrenia pathophysiology

Schizophrenia is a common, multigenic psychiatric disorder. Linkage studies, including a recent meta-analysis of genome scans, have repeatedly implicated chromosome 8p12-p23.1 in schizophrenia susceptibility. More recently, significant association with a candidate gene on 8p12, neuregulin 1 (NRG1), has been reported in several European and Chinese samples. We investigated NRG1 for association in schizophrenia patients of Portuguese descent to determine whether this gene is a risk factor in this population. We tested NRG1 markers and haplotypes for association in 111 parent-proband trios, 321 unrelated cases, and 242 control individuals. Associations were found with a haplotype that overlaps the risk haplotype originally reported in the Icelandic population (‘HapICE’), and two haplotypes located in the 3′ end of NRG1 (all P<0.05). However, association was not detected with HapICE itself. Comparison of NRG1 transcript expression in peripheral leukocytes from schizophrenia patients and unaffected siblings identified 3.8-fold higher levels of the SMDF variant in patients (P=0.039). Significant positive correlations (P<0.001) were found between SMDF and HRG-beta 2 expression and between HRG-gamma and ndf43 expression, suggesting common transcriptional regulation of NRG1 variants. In summary, our results suggest that haplotypes across NRG1 and multiple NRG1 variants are involved in schizophrenia.

Genome wide significant associations in schizophrenia to ITIH3/4, CACNA1C and SDCCAG8, and extensive replication of associations reported by the Schizophrenia PGC

The Schizophrenia Psychiatric Genome-Wide Association Study Consortium (PGC) highlighted 81 single-nucleotide polymorphisms (SNPs) with moderate evidence for association to schizophrenia. After follow-up in independent samples, seven loci attained genome-wide significance (GWS), but multi-locus tests suggested some SNPs that did not do so represented true associations. We tested 78 of the 81 SNPs in 2640 individuals with a clinical diagnosis of schizophrenia attending a clozapine clinic (CLOZUK), 2504 cases with a research diagnosis of bipolar disorder, and 2878 controls. In CLOZUK, we obtained significant replication to the PGC-associated allele for no fewer than 37 (47%) of the SNPs, including many prior GWS major histocompatibility complex (MHC) SNPs as well as 3/6 non-MHC SNPs for which we had data that were reported as GWS by the PGC. After combining the new schizophrenia data with those of the PGC, variants at three loci (ITIH3/4, CACNA1C and SDCCAG8) that had not previously been GWS in schizophrenia attained that level of support. In bipolar disorder, we also obtained significant evidence for association for 21% of the alleles that had been associated with schizophrenia in the PGC. Our study independently confirms association to three loci previously reported to be GWS in schizophrenia, and identifies the first GWS evidence in schizophrenia for a further three loci. Given the number of independent replications and the power of our sample, we estimate 98% (confidence interval (CI) 78–100%) of the original set of 78 SNPs represent true associations. We also provide strong evidence for overlap in genetic risk between schizophrenia and bipolar disorder.

Genome-wide scan in Portuguese Island families identifies 5q31-5q35 as a susceptibility locus for schizophrenia and psychosis.

Schizophrenia is a common psychiatric disorder with a complex genetic etiology. To understand the genetic basis of this syndrome in Portuguese Island populations, we performed a genome-wide scan of 29 families with schizophrenia, which identified a single region on 5q31–5q35 with strong linkage (NPL=3.09, P=0.0012 at D5S820). Empirical simulations set a genome-wide threshold of NPL=3.10 for significant linkage. Additional support for this locus in schizophrenia comes from higher-density mapping and mapping of 11 additional families. The combined set of 40 families had a peak NPL=3.28 (P=0.00066) at markers D5S2112–D5S820. These data and previous linkage findings from other investigators provide strong and consistent evidence for this genomic region as a susceptibility locus for schizophrenia. Exploratory analyses of a novel phenotype, psychosis, in families with schizophrenia and bipolar disorder detected evidence for linkage to the same markers as found in schizophrenia (peak NPL=3.03, P=0.0012 at D5S820), suggesting that this locus may be responsible for the psychotic symptoms observed in both diseases.

Replication of bipolar disorder susceptibility alleles and identification of two novel genome-wide significant associations in a new bipolar disorder case-control sample.

We have conducted a genotyping study using a custom Illumina Infinium HD genotyping array, the ImmunoChip, in a new UK sample of 1218 bipolar disorder (BD) cases and 2913 controls that have not been used in any studies previously reported independently or in meta-analyses. The ImmunoChip was designed before the publication of the Psychiatric Genome-Wide Association Study Consortium Bipolar Disorder Working Group (PGC-BD) meta-analysis data. As such 3106 single-nucleotide polymorphisms (SNPs) with a P-value <1 × 10−3 from the BD meta-analysis by Ferreira et al. were genotyped. We report support for two of the three most strongly associated chromosomal regions in the Ferreira study, CACNA1C (rs1006737, P=4.09 × 10−4) and 15q14 (rs2172835, P=0.043) but not ANK3 (rs10994336, P=0.912). We have combined our ImmunoChip data (569 quasi-independent SNPs from the 3016 SNPs genotyped) with the recently published PGC-BD meta-analysis data, using either the PGC-BD combined discovery and replication data where available or just the discovery data where the SNP was not typed in a replication sample in PGC-BD. Our data provide support for two regions, at ODZ4 and CACNA1C, with prior evidence for genome-wide significant (GWS) association in PGC-BD meta-analysis. In addition, the combined analysis shows two novel GWS associations. First, rs7296288 (P=8.97 × 10−9, odds ratio (OR)=0.9), an intergenic polymorphism on chromosome 12 located between RHEBL1 and DHH. Second, rs3818253 (P=3.88 × 10−8, OR=1.16), an intronic SNP on chromosome 20q11.2 in the gene TRPC4AP, which lies in a high linkage disequilibrium region along with the genes GSS and MYH7B.

Association analysis of NOTCH4 loci in schizophrenia using family and population-based controls

A genetic association between NOTCH4 and schizophrenia has previously been proposed. Unsing all markers previously shown to be associated, we found no evidence for such in three independent family-based samples (n=519 parent-offspring trios), and a case-control sample derived from the same ethnic background as the original observaton. These data strongly suggest that NOTCH4 is not a significant susceptibility allele for schizophrenia.

Multi-locus genome-wide association analysis supports the role of glutamatergic synaptic transmission in the etiology of major depressive disorder

Major depressive disorder (MDD) is a common psychiatric illness characterized by low mood and loss of interest in pleasurable activities. Despite years of effort, recent genome-wide association studies (GWAS) have identified few susceptibility variants or genes that are robustly associated with MDD. Standard single-SNP (single nucleotide polymorphism)-based GWAS analysis typically has limited power to deal with the extensive heterogeneity and substantial polygenic contribution of individually weak genetic effects underlying the pathogenesis of MDD. Here, we report an alternative, gene-set-based association analysis of MDD in an effort to identify groups of biologically related genetic variants that are involved in the same molecular function or cellular processes and exhibit a significant level of aggregated association with MDD. In particular, we used a text-mining-based data analysis to prioritize candidate gene sets implicated in MDD and conducted a multi-locus association analysis to look for enriched signals of nominally associated MDD susceptibility loci within each of the gene sets. Our primary analysis is based on the meta-analysis of three large MDD GWAS data sets (total N=4346 cases and 4430 controls). After correction for multiple testing, we found that genes involved in glutamatergic synaptic neurotransmission were significantly associated with MDD (set-based association P=6.9 × 10−4). This result is consistent with previous studies that support a role of the glutamatergic system in synaptic plasticity and MDD and support the potential utility of targeting glutamatergic neurotransmission in the treatment of MDD.

Cis-Acting Regulation of Brain-Specific ANK3 Gene Expression by a Genetic Variant Associated with Bipolar Disorder

Several genome-wide association studies for bipolar disorder (BD) have found a strong association of the Ankyrin 3 (ANK3) gene. This association spans numerous linked single-nucleotide polymorphisms (SNPs) in an ∼250-kb genomic region overlapping ANK3. The associated region encompasses predicted regulatory elements as well as two of the six validated alternative first exons, which encode distinct protein domains at the N-terminus of the protein also known as Ankyrin-G. Using RNA ligase-mediated rapid amplification of cDNA ends to identify novel transcripts in conjunction with a highly sensitive, exon-specific multiplexed mRNA expression assay, we detected differential regulation of distinct ANK3 transcription start sites and coupling of specific 5′ ends with 3′ mRNA splicing events in postmortem human brain and human stem cell-derived neural progenitors and neurons. Furthermore, allelic variation at the BD-associated SNP rs1938526 correlated with a significant difference in cerebellar expression of a brain-specific ANK3 transcript. These findings suggest a brain-specific cis-regulatory transcriptional effect of ANK3 that may be relevant to BD pathophysiology.

Evidence for association of DNA sequence variants in the phosphatidylinositol-4-phosphate 5-kinase IIα gene (PIP5K2A) with schizophrenia

Linkage studies in schizophrenia have identified a candidate region on chromosome 10p14–11 as reported for several independent samples. We investigated association of DNA sequence variants in a plausible candidate gene located in this region, the gene for phosphatidylinositol-4-phosphate 5-kinase IIα (PIP5K2A), in a sample of 65 sib-pair families for which linkage had been reported. Evidence for association was obtained for 15 polymorphisms spanning 73.6u2009kb in the genomic region of the gene between intron 4 and the 3′ untranslated region, a region with high degree of linkage disequilibrium. Single nucleotide polymorphism (SNP) rs10828317 located in exon 7 and causing a non-synonymous amino-acid exchange (asparagine/serine) produced a P-value of 0.001 (experiment-wide significance level 0.00275) for over-transmission of the major allele coding for serine, analysed by transmission disequilibrium test using FAMHAP. Association of this SNP with schizophrenia has been also described in a sample of 273 Dutch schizophrenic patients and 580 controls (P=0.0004). PIP5K2A is involved in the biosynthesis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), one of the key metabolic crossroads in phosphoinositide signalling. PI(4,5)P2 plays a role in membrane transduction of neurotransmitter signals as well as in intracellular signalling, pathways that may be impaired in schizophrenia.