Mikhil Bamne

Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimers disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case–control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer’s disease in seven independent case–control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer’s disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer’s disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer’s disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

Serotonin Gene Polymorphisms and Bipolar I Disorder: Focus on the Serotonin Transporter

The pathogenesis of bipolar disorder may involve, at least in part, aberrations in serotonergic neurotransmission. Hence, serotonergic genes are attractive targets for association studies of bipolar disorder. We have reviewed the literature in this field. It is difficult to synthesize results as only one polymorphism per gene was typically investigated in relatively small samples. Nevertheless, suggestive associations are available for the 5HT2A receptor and the serotonin transporter genes. With the availability of extensive polymorphism data and high throughput genotyping techniques, comprehensive evaluation of these genes using adequately powered samples is warranted. We also report on our investigations of the serotonin transporter, SLC6A4 (17q11.1‐q12). An insertion/deletion polymorphism (5HTTLPR) in the promoter region of this gene has been investigated intensively. However, the results have been inconsistent. We reasoned that other polymorphism/s may contribute to the associations and the inconsistencies may be due to variations in linkage disequilibrium (LD) patterns between samples. Therefore, we conducted LD analyses, as well as association and linkage using 12 polymorphisms, including 5HTTLPR. We evaluated two samples. The first sample consisted of 135 US Caucasian nuclear families having a proband with bipolar I disorder (BDI, DSM IV criteria) and available parents. For case‐control analyses, the patients from these families were compared with cord blood samples from local Caucasian live births (n = 182). Our second, independent sample was recruited through the Systematic Treatment Enhancement Program for Bipolar Disorder (STEP‐BD, 545 cases, 548 controls). No significant associations were detected at the individual polymorphism or haplotype level using the case‐control or family‐based analyses. Our analyses do not support association between SLC6A4 and BDI families. Further studies using sub‐groups of BDI are worthwhile.

Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.

Genetic Association Studies of Antioxidant Pathway Genes and Schizophrenia

The endogenous production of highly reactive oxidation species is an inherent by-product of cellular energy metabolism. Cellular antioxidant defense systems (AODS) comprising various antioxidants counter these damaging effects. Several lines of evidence, including postmortem studies, suggest increased oxidative stress in patients with schizophrenia. Some genetic association studies and gene-expression studies suggest that patients also may have altered ability to mount antioxidative mechanisms. As the genetic associations may provide etiologic evidence in support of the oxidative-stress hypothesis of schizophrenia, a focused review has been conducted. We also suggest avenues for further research.

A comprehensive genetic association and functional study of TNF in schizophrenia risk.

In view of prior reports suggesting associations between polymorphisms of the tumor necrosis factor gene (TNF) and schizophrenia, we sequenced all exons, introns and 7 kb flanking sequence at TNF in DNA pooled from 125 Caucasian schizophrenia cases and 200 controls. We identified 18 SNPs of which we selected and genotyped 8 among 244 cases and 276 controls. We detected no significant genotype or haplotype associations in the entire sample or in subgroups defined by gender or exposure to HSV1, HSV2, CMV, or Toxoplasma gondii. We used a dual-luciferase expression assay to quantify TNF expression driven by each common promoter haplotype in a neuroblastoma cell line. Three haplotypes drove significantly lower levels of TNF expression than the most common haplotype, including a haplotype with -308A, the allele reported to increase risk for schizophrenia (in contrast to earlier reports). We find no evidence to implicate TNF gene polymorphisms for schizophrenia risk in our sample.

Investigation of an amyloid precursor protein protective mutation (A673T) in a North American case-control sample of late-onset Alzheimer's disease

A rare amyloid precursor protein gene variant, A673T (rs63750847) was recently reported to protect against Alzheimers disease and age-related cognitive decline among Icelanders and the same rare variant was observed also in Finnish, Norwegian, and Swedish populations. We investigated this variant in 1674 late-onset Alzheimers disease cases and 2644 elderly control subjects, all North American Whites (US Whites). We did not observe any example of the A673T variant in our large sample. Our findings suggest that this rare variant could be specific to the individuals of the origin from the Nordic countries.

Evaluation of HLA Polymorphisms in Relation to Schizophrenia Risk and Infectious Exposure

BACKGROUND Genome-wide association studies (GWAS) implicate single nucleotide polymorphisms (SNPs) on chromosome 6p21.3-22.1, the human leukocyte antigen (HLA) region, as common risk factors for schizophrenia (SZ). Other studies implicate viral and protozoan exposure. Our study tests chromosome 6p SNPs for effects on SZ risk with and without exposure. METHOD GWAS-significant SNPs and ancestry-informative marker SNPs were analyzed among African American patients with SZ (n = 604) and controls (n = 404). Exposure to herpes simplex virus, type 1 (HSV-1), cytomegalovirus (CMV), and Toxoplasma gondii (TOX) was assayed using specific antibody assays. RESULTS Five SNPs were nominally associated with SZ, adjusted for population admixture (P < .05, uncorrected for multiple comparisons). These SNPs were next analyzed in relation to infectious exposure. Multivariate analysis indicated significant association between rs3130297 genotype and HSV-1 exposure; the associated allele was different from the SZ risk allele. CONCLUSIONS We propose a model for the genesis of SZ incorporating genomic variation in the HLA region and neurotropic viral exposure for testing in additional, independent African American samples.

Fine-mapping reveals novel alternative splicing of the dopamine transporter

The dopamine transporter gene (SLC6A3, DAT) has been implicated in the pathogenesis of numerous psychiatric and neurodevelopmental disorders, including schizophrenia (SZ). We previously detected association between SZ and intronic SLC6A3 variants that replicated in two independent Caucasian samples, but had no obvious function. In follow‐up analyses, we sequenced the coding and intronic regions of SLC6A3 to identify complete linkage disequilibrium patterns of common variations. We genotyped 78 polymorphisms, narrowing the potentially causal region to two correlated clusters of associated SNPs localized predominantly to introns 3 and 4. Our computational analysis of these intronic regions predicted a novel cassette exon within intron 3, designated E3b, which is conserved among primates. We confirmed alternative splicing of E3b in post‐mortem human substantia nigra (SN). As E3b introduces multiple in‐frame stop codons, the SLC6A3 open reading frame is truncated and the spliced product may undergo nonsense mediated decay. Thus, factors that increase E3b splicing could reduce the amount of unspliced product available for translation. Observations consistent with this prediction were made using cellular assays and in post‐mortem human SN. In mini‐gene constructs, the extent of splicing is also influenced by at least two common haplotypes, so the alternative splicing was evaluated in relation to SZ risk. Meta‐analyses across genome‐wide association studies did not support the initial associations and further post‐mortem studies did not suggest case‐control differences in splicing. These studies do not provide a compelling link to schizophrenia. However, the impact of the alternative splicing on other neuropsychiatric disorders should be investigated.

Genetic Variation in Imprinted Genes is Associated with Risk of Late-Onset Alzheimer's Disease

Epigenetic changes including genomic imprinting may affect risk of late-onset Alzheimers disease (LOAD). There are >100 known imprinted genes and most of them are expressed in human brain. In this study, we examined the association of single nucleotide polymorphisms (SNPs) in 93 imprinted genes with LOAD risk in 1291 LOAD cases and 958 cognitively normal controls. We performed single-site, gene-based, and haplotype analyses. Single-site analysis showed 14 significant associations at p < 0.01. The most significant SNP (rs11770199; p = 0.0003) in single-site analysis was located on chromosome 7 in the GRB10 gene. Gene-based analyses revealed four significant associations in the WT1, ZC3H12C, DLGAP2, and GPR1 genes at p < 0.05. The haplotype analysis also revealed significant associations with three genes (ZC3H12C, DLGAP2, and GPR1). These findings suggest a possible role of imprinted genes in AD pathogenesis that show specific expression in the brain.

Application of an ex vivo cellular model of circadian variation for bipolar disorder research: a proof of concept study.

Disruption of circadian function has been observed in several human disorders, including bipolar disorder (BD). Research into these disorders can be facilitated by human cellular models that evaluate external factors (zeitgebers) that impact circadian pacemaker activity. Incorporating a firefly luciferase reporter system into human fibroblasts provides a facile, bioluminescent readout that estimates circadian phase, while leaving the cells intact. We evaluated whether this system can be adapted to clinical BD research and whether it can incorporate zeitgeber challenge paradigms.