Using human induced pluripotent stem cells to investigate neurodevelopmental effects of human cytomegalovirus

Human cytomegalovirus (HCMV) is one of the leading prenatal causes of mental retardation and congenital deformities, world-wide. Its pathogenesis has generally been investigated using animal models. Human studies in vitro have been limited to neurospheres prepared using forebrain tissues from fetal abortuses. This approach is limited and does not permit analysis of individual specific cells. We generated iPS cells from adult human fibroblasts. iPS cells were differentiated into neurospheres, that were expanded as monolayer culture of neuroprogenitors (NPs). Furthermore, neurospheres were differentiated into neurons that could be stained for Tuj1, tyrosine hydroxylase and NR4A2. Functional competency was confirmed by live imaging of intracellular calcium. NPs and neurons were infected with HCMV (MOI = 3). Cell viability was assessed by FACS analysis. Cytopathic effects of HCMV were observed on the 10th day post infection in neuroprogenitor cells. Earlier, the adherence of these cells to the matrix was reduced. Neurons were much more refractory. Reduced cell density and shortening of neuritic processes was only observed at day 15 after infection. We are presently examining the intracellular effects of HCMV. Human iPS cells can efficiently generate neurospheres, which can be expanded as almost pure cultures of neuroprogenitors or differentiated into neurons. iPS cells-derived NP and neurons offer powerful cellular models to investigate the effect of neurotropic viral agents on neurodevelopment

Fine-mapping reveals novel alternative splicing of the dopamine transporter

Center for Human Genetic Research, Massachusetts General Hospital and Department of Neurology, Harvard Medical School, Harvard University, Boston, Massachusetts.Graduate Program in Biology and Biomedical Science, Yale University, New Haven, Connecticut.The dopamine transporter gene (, ) has been implicated in the pathogenesis of numerous psychiatric and neurodevelopmental disorders, including schizophrenia (SZ). We previously detected association between SZ and intronic 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 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 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. © 2010 Wiley-Liss, Inc

Dopamine Genes and Schizophrenia: Case Closed or Evidence Pending?

The dopamine hypothesis of schizophrenia (SZ) has motivated a large number of genetic association studies but few if any dopaminergic (DA) polymorphisms are accepted as credible risk factors at present. To evaluate whether dopamine-related genes have been investigated adequately, we surveyed public genetic databases and published SZ association studies with regard to 14 conventional DA genes and 7 selected dopamine-interacting proteins. We estimate that 325 polymorphisms would be required to evaluate the impact of common variation on SZ risk among Caucasian samples. To date, 98 polymorphisms have been analyzed in published association studies. We estimate that only 19 of these variations have been evaluated in samples with at least 50% power to detect an association of the effect size commonly found in genetically complex disorders. While it is possible that DA genes do not harbor genetic risk factors for SZ, our review suggests that satisfactory conclusions for most genes cannot be drawn at present. Whole-genome association studies have begun to fill this void, but additional analyses are likely to be needed. Recommendations for future association studies include analysis of adequately powered samples, judiciously selected polymorphisms, multiple ethnic groups, and concurrent evaluation of function at associated single-nucleotide polymorphisms

Linkage Disequilibrium Patterns and Functional Analysis of RGS4 Polymorphisms in Relation to Schizophrenia

The regulator of G-protein signaling 4 (RGS4, chromosome 1q23.3) plays a critical role in G-protein function. Four common single-nucleotide polymorphisms (SNPs) localized between the 5′ upstream sequence and the first intron, as well as 2 haplotypes derived from these SNPs may confer liability to schizophrenia (SZ). However, the pattern of associations varies among samples. To help clarify the putative associations, we report the following analyses: (1) a comprehensive catalog of common polymorphisms, (2) linkage disequilibrium (LD) and association analyses using these SNPs, and (3) functional analysis based on dual-luciferase promoter assays. We identified 62 SNPs from a 20-kb genomic region spanning RGS4, of which 26 are common polymorphisms with a minor allele frequency (MAF) of >5%. LD analysis suggested 5 clusters of SNPs (r2 > .8). Association analyses using the novel SNPs were consistent with the prior reports, but further localization was constrained by significant LD across the region. The 2 haplotypes reported to confer liability to SZ had significant promoter activity compared with promoterless constructs, suggesting a functional role for both haplotypes. Further analyses of promoter sequences are warranted to understand transcriptional regulation at RGS4. This information will be useful for further analysis of samples in which genetic association of RGS4 polymorphisms with SZ has been reported

Associations between period 3 gene polymorphisms and sleep- /chronotype-related variables in patients with late-life insomnia

<p>A variable number tandem repeat polymorphism (VNTR) in the period 3 (<i>PER3</i>) gene has been associated with heritable sleep and circadian variables, including self-rated chronotypes, polysomnographic (PSG) variables, insomnia and circadian sleep-wake disorders. This report describes novel molecular and clinical analyses of <i>PER3</i> VNTR polymorphisms to better define their functional consequences. As the <i>PER3</i> VNTR is located in the exonic (protein coding) region of <i>PER3</i>, we initially investigated whether both alleles (variants) are transcribed into messenger RNA in human fibroblasts. The VNTR showed bi-allelic gene expression. We next investigated genetic associations in relation to clinical variables in 274 older adult Caucasian individuals. Independent variables included genotypes for the <i>PER3</i> VNTR as well as a representative set of single nucleotide polymorphisms (SNPs) that tag common variants at the <i>PER3</i> locus (linkage disequilibrium (LD) between genetic variants < 0.5). In order to comprehensively evaluate variables analyzed individually in prior analyses, dependent measures included PSG total sleep time and sleep latency, self-rated chronotype, estimated with the Composite Scale (CS), and lifestyle regularity, estimated using the social rhythm metric (SRM). Initially, genetic polymorphisms were individually analyzed in relation to each outcome variable using analysis of variance (ANOVA). Nominally significant associations were further tested using regression analyses that incorporated individual ANOVA-associated DNA variants as potential predictors and each of the selected sleep/circadian variables as outcomes. The covariates included age, gender, body mass index and an index of medical co-morbidity. Significant genetic associations with the VNTR were not detected with the sleep or circadian variables. Nominally significant associations were detected between SNP rs1012477 and CS scores (<i>p</i> = 0.003) and between rs10462021 and SRM (<i>p</i> = 0.047); rs11579477 and average delta power (<i>p</i> = 0.043) (analyses uncorrected for multiple comparisons). In conclusion, alleles of the VNTR are expressed at the transcript level and may have a functional effect in cells expressing the <i>PER3</i> gene. <i>PER3</i> polymorphisms had a modest impact on selected sleep/circadian variables in our sample, suggesting that <i>PER3</i> is associated with sleep and circadian function beyond VNTR polymorphisms. Further replicate analyses in larger, independent samples are recommended.</p