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 Alzheimer's 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-ß precursor protein (APP) and extracellular Aß42 and Aß40 (the 42- and 40-residue isoforms of the amyloid-ß peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aß42 and Aß40. 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.

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

OBJECTIVES: 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. METHODS: Fibroblasts from patients with bipolar I disorder (BD-I) (n = 13) and controls (n = 12) were infected ex vivo with a lentiviral reporter incorporating the promoter sequences for Bmal1, a circadian gene to drive expression of the firefly luciferase gene. Following synchronization, the bioluminescence was used to estimate period length. Phase response curves (PRCs) were also generated following forskolin challenge and the phase response patterns were characterized. RESULTS: Period length and PRCs could be estimated reliably from the constructs. There were no significant case-control differences in period length, with a nonsignificant trend for differences in PRCs following the phase-setting experiments. CONCLUSIONS: An ex vivo cellular fibroblast-based model can be used to investigate circadian function in BD-I. It can be generated from specific individuals and this could usefully complement ongoing circadian clinical research.

Systematic association studies of mitochondrial DNA variations in schizophrenia: focus on the ND5 gene.

Postmortem studies, as well as genetic association studies, have implicated mitochondrial dysfunction in schizophrenia (SZ). We conducted multistaged analysis to assess the involvement of mitochondrial DNA (mtDNA) variations in SZ. Initially, the entire mtDNA genome was sequenced in pools of DNA from SZ cases and controls (n = 180 in each group, set 1). Two polymorphisms localized to the NADH dehydrogenase subunit 5 (ND5) gene demonstrated suggestive case control allele frequency differences (mtDNA 13368 G/A, p = .019 and mtDNA 13708G/A, p = .043). Hence, the ND5 gene was sequenced in individual samples from the initial panel of cases and controls. Additional subjects from another independent set of cases and controls (set 2, cases, n = 244, controls n = 508) were also sequenced individually. No significant differences in allele frequencies for mtDNA 13368 G/A, and mtDNA 13708G/A were observed. However, we identified 216 other rare variants, 53 of which were reported earlier in association studies of other mitochondrial disorders. We compared the distribution of polymorphisms in both sets of cases and controls. No significant case-control differences were observed in the smaller, first set. In the second set, cases had more variants overall (p = 0.014), as well as synonymous variants (p = 0.02), but the difference for nonsynonymous variants was not significant (p = 0.19). Screening available first-degree relatives (n = 10) revealed 10 maternally inherited variations, suggesting that not all the variants are somatic mutations. Further investigations are warranted.

Engineered Saccharomyces cerevisiae strain BioS-OS1/2, for the detection of oxidative stress.

One of the major stress factors during space and high-altitude flight is the oxidative damage caused by the release of reactive oxygen intermediates (ROIs) in human tissues. ROIs are released in response to several stress factors including radiation in space. Since ROIs contribute to several pathological conditions, there has been a great interest in developing a biosensor that can monitor the impact of ROIs on biological systems. Toward this goal, we sought to engineer a yeast stain that can monitor oxidative stress and be easily integrated into a biosensor platform. Saccharomyces cerevisiae respond to hyperoxidative stress by activating the expression of many proteins including the transcription factor, Yap1. Activated Yap1 primarily binds to the Yap-1 response elements in the promoters of genes that combat oxidative stress. Based on these observations, we genetically altered the Yap-1 pathway in the YCR094W BY4742 strain of S. cerevisiae by fusing the YREs in the promoter region of TRX2 gene to a cDNA-insert encoding green fluorescent protein (GFP). Exposure of this engineered yeast strain BioS-OS1 to varying levels of oxidative stress, as generated by different concentrations of H(2)O(2) or diamide, elicits robust expression of GFP that can be monitored by the fluorescence of GFP by as early as 1 h. BioS-OS1 can detect a H(2)O(2) concentration from 300 microM onward. We also show that the signaling strength of the strain can be increased by engineering multiple YREs in the upstream of the cDNA-insert encoding GFP. Thus, the results presented here demonstrate that the engineered BioS-OS yeast strain can detect ROI-generating oxidative stress and validate the use of this prototypic strain for the development of a biosensor to detect and monitor oxidative stress factors during space and high altitude flights.

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 Alzheimer's 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.

APOE gene polymorphism and risk of coronary stenosis in Pakistani population.

Genetic variation in lipid regulatory genes, particularly APOE, significantly influences the risk of coronary artery disease (CAD). This study aimed to assess the association between APOE polymorphism and angiographically assessed coronary stenosis in Pakistani population. A total of 695 subjects (22.3% female, mean age = 54 ± 11 years) presenting with chest pain were enrolled after obtaining written informed consent. CAD stenosis/extent was assessed by angiography. Patients were classified as having severe stenosis (≥ 70%), moderate stenosis (30-69%), and mild stenosis (<30%). CAD patients with ≥ 70% stenosis (n = 491) were further categorized based on possessing one, two, or three vessel diseases to assess the disease extent. Genomic DNA from leukocytes was isolated with DNA purification kit (Qiagen) and APOE polymorphisms (E2/E3/E4) were determined using TaqMan assays. Six hundred and seventy-two of 695 subjects were successfully genotyped. The frequency of APOE∗4 carriers (3/4 and 4/4 genotypes) was significantly higher in severe stenosis group (≥ 70%) as compared to mild group (<30%) (22.8% versus 13.01%; P = 0.01). In multiple regression, the odds ratio for APOE∗4 carriers to develop ≥ 70% stenosis was 2.16 (95% CI: 1.29-3.79; P < 0.005). In conclusion, the presence of APOE∗4 allele is a significant risk factor to develop severe coronary stenosis (>70%) among Pakistanis.

More evidence for association of a rare TREM2 mutation (R47H) with Alzheimer's disease risk.

Over 20 risk loci have been identified for late-onset Alzheimer's disease (LOAD), most of which display relatively small effect sizes. Recently, a rare missense (R47H) variant, rs75932628 in TREM2, has been shown to mediate LOAD risk substantially in Icelandic and Caucasian populations. Here, we present more evidence for the association of the R47H with LOAD risk in a Caucasian population comprising 4567 LOAD cases and controls. Our results show that carriers of the R47H variant have a significantly increased risk for LOAD (odds ratio = 7.40, p = 3.66E-06). In addition to Alzheimer's disease risk, we also examined the association of R47H with Alzheimer's disease-related phenotypes, including age-at-onset, psychosis, and amyloid deposition but found no significant association. Our results corroborate those of other studies implicating TREM2 as an LOAD risk locus and indicate the need to determine its biological role in the context of neurodegeneration.

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 Alzheimer's 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 Alzheimer's 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.

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.

Functional analysis of upstream common polymorphisms of the dopamine transporter gene.

The human dopamine transporter (DAT, SLC6A3) has been extensively investigated because of its potential involvement in neuropsychiatric disorders. The core elements responsible for its transcription have been identified. A regulatory role for certain genomic variants upstream to the core promoter is known. Recently, other single-nucleotide polymorphisms (SNPs) have been identified in this region and are thought to be associated with schizophrenia and bipolar I disorder. Hence, we have investigated the impact of common SNPs in a 2.8-kilobase region flanking the core promoter region (-2.7 to +63 base pair) in the neuroblastoma cell line SH-SY5Y. Haplotypes generated by site-directed mutagenesis revealed varying impact of individual SNPs on promoter activity using dual luciferase assays. In silico analyses also predicted allele-specific binding of transcription factors for some of these SNPs. Though electrophoretic mobility shift assays indicated several factors that appeared to bind to specific sites within this region, allele-specific binding was not detected for any SNP apart from rs3756450. We have thus identified novel putative regulatory domains flanking the core promoter of DAT that merit further investigation.

Approaches to unravel the genetics of sleep.

Sleep and circadian rhythms are complex and inter-connected physiological processes. Relative to the remarkable progress made in identifying the genetic basis of circadian rhythms and some specific sleep disorders, efforts to identify genetic variants associated with normal variation in sleep have progressed more slowly. Two key issues concerning the design of such studies must be addressed in order to facilitate further progress. The first concerns the sleep related traits to be targeted. The second issue is the choice of the gene-mapping method (linkage, candidate gene association or genome-wide association). This paper discusses these issues, reviews published studies of sleep phenotypes, and recommends cost-effective methods to advance knowledge of the genetic determinants of normal sleep patterns.

Grey matter changes associated with host genetic variation and exposure to Herpes Simplex Virus 1 (HSV1) in first episode schizophrenia.

BACKGROUND: We previously reported reduced prefrontal cortex (PFC) grey matter volume among first episode, antipsychotic-naïve schizophrenia subjects (SZ) exposed to HSV1 but not among healthy subjects (HS) (Prasad et al., 2007). Independently, rs1051788, an exonic polymorphism of the MHC Class I polypeptide-related sequence B (MICB) gene was associated with HSV1 seropositivity, as well as SZ risk. In this study, we examined whether PFC grey matter changes associated with HSV1 exposure varied against the background of MICB genotypes. METHODS: We examined Caucasian individuals from the sample we studied in our previous report (Prasad et al., 2007) (SZ, n=21 and HS, n=19). Whole brain voxelwise analysis of structural MRI scans was conducted using Statistical Parametric Mapping, ver 5 (SPM5). The impact of rs1051788 variation and HSV1 seropositivity on grey matter volumes was examined using regression models on the combined sample of cases and controls, and then within each diagnostic group. RESULTS: In the combined sample of cases and controls, we observed the main effects of HSV1 seropositivity and genotypes, and a significant joint effect of HSV1 seropositivity and genotype mainly in the PFC. The joint effect was more prominent among cases than among controls. DISCUSSION: Our observations suggest that rs1051788 and HSV1 seropositivity are associated individually and jointly with reduced PFC grey matter volume. The patterns of these associations differ by diagnostic status, and these factors explain only a "small" portion of the variance in the grey matter volume reductions.