ABSTRACT
BACKGROUND AND AIMS: Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS: SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS: These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.
Subject(s)
Anemia, Sickle Cell/complications , Bile/metabolism , Cholestasis/etiology , Hepatic Insufficiency/etiology , Liver/pathology , Adolescent , Adult , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/genetics , Animals , Bile Ducts, Intrahepatic/diagnostic imaging , Bile Ducts, Intrahepatic/pathology , Cholestasis/pathology , Cholestasis/prevention & control , Disease Models, Animal , Female , Gene Knock-In Techniques , Hemoglobin, Sickle/genetics , Hepatic Insufficiency/pathology , Hepatic Insufficiency/prevention & control , Humans , Intravital Microscopy , Liver/diagnostic imaging , Male , Mice , Middle Aged , NF-kappa B/antagonists & inhibitors , NF-kappa B/drug effects , NF-kappa B/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction/drug effects , Young AdultABSTRACT
A variable number tandem repeat polymorphism (VNTR) in the period 3 (PER3) 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 PER3 VNTR polymorphisms to better define their functional consequences. As the PER3 VNTR is located in the exonic (protein coding) region of PER3, 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 PER3 VNTR as well as a representative set of single nucleotide polymorphisms (SNPs) that tag common variants at the PER3 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 (p = 0.003) and between rs10462021 and SRM (p = 0.047); rs11579477 and average delta power (p = 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 PER3 gene. PER3 polymorphisms had a modest impact on selected sleep/circadian variables in our sample, suggesting that PER3 is associated with sleep and circadian function beyond VNTR polymorphisms. Further replicate analyses in larger, independent samples are recommended.
Subject(s)
Gene Expression Regulation/physiology , Period Circadian Proteins/metabolism , Polymorphism, Single Nucleotide , Sleep Initiation and Maintenance Disorders/genetics , Sleep , Aged , Aged, 80 and over , DNA/genetics , Female , Gentian Violet , Humans , Male , Period Circadian Proteins/geneticsABSTRACT
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.
Subject(s)
Alzheimer Disease/genetics , Genetic Association Studies , Membrane Glycoproteins/genetics , Mutation, Missense , Receptors, Immunologic/genetics , Age of Onset , Aged , Alzheimer Disease/epidemiology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Brain/metabolism , Female , Heterozygote , Humans , Male , Phenotype , Risk , White PeopleABSTRACT
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.
Subject(s)
Alleles , Apolipoproteins E/genetics , Coronary Stenosis/genetics , Polymorphism, Genetic , Adult , Aged , Female , Humans , Male , Middle Aged , Pakistan , Risk FactorsABSTRACT
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.
Subject(s)
Alzheimer Disease/genetics , Genetic Predisposition to Disease/genetics , Genomic Imprinting/genetics , Polymorphism, Single Nucleotide/genetics , Aged , Aged, 80 and over , Female , GRB10 Adaptor Protein/genetics , Genetic Association Studies , Haplotypes , Humans , Male , Nerve Tissue Proteins/genetics , Receptors, G-Protein-Coupled/genetics , Ribonucleases/genetics , Transcription Factors/genetics , WT1 Proteins/geneticsSubject(s)
Asian People/genetics , Dementia/ethnology , Dementia/genetics , Genetic Loci/genetics , Genetic Predisposition to Disease/ethnology , Genetic Predisposition to Disease/genetics , Alleles , Cross-Cultural Comparison , Humans , Pakistan , Polymorphism, Genetic/genetics , Proto-Oncogene Proteins c-met/genetics , Risk FactorsABSTRACT
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.
Subject(s)
Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Genetic Association Studies , Mutation/genetics , Aged , Aged, 80 and over , Case-Control Studies , Cognition Disorders/genetics , Cohort Studies , Female , Genotype , Humans , Male , Middle Aged , North America , Risk , White People/geneticsABSTRACT
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.
Subject(s)
Alzheimer Disease/genetics , Genetic Predisposition to Disease/genetics , Genetic Variation/genetics , Phospholipase D/genetics , Black or African American/genetics , Age of Onset , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Brain/metabolism , Case-Control Studies , Europe/ethnology , Exome/genetics , Female , Humans , Male , Peptide Fragments/metabolism , Phospholipase D/deficiency , Phospholipase D/metabolism , Protein Processing, Post-Translational/genetics , ProteolysisABSTRACT
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.
Subject(s)
Bipolar Disorder/pathology , Bipolar Disorder/physiopathology , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Adult , Antiemetics/pharmacology , Cell Line , Dexamethasone/pharmacology , Female , Fibroblasts/drug effects , Gene Expression Regulation/drug effects , Humans , Male , Middle Aged , Time Factors , Transfection , Young AdultABSTRACT
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.
Subject(s)
Cytomegalovirus/physiology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/virology , Neurons/cytology , Neurons/virology , Animals , Cell Differentiation , Cell Survival , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/virology , Gene Expression Profiling , Humans , Induced Pluripotent Stem Cells/metabolism , Mice , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neural Stem Cells/virology , Neurons/metabolism , Viral TropismABSTRACT
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.
Subject(s)
HLA Antigens/genetics , Schizophrenia/genetics , Adult , Black or African American/genetics , Black or African American/psychology , Butyrophilins , Case-Control Studies , Chromosomes, Human, Pair 6 , Cytomegalovirus , Cytomegalovirus Infections , Female , Genetic Predisposition to Disease , Genotype , Herpes Simplex/complications , Herpesvirus 1, Human , Humans , Male , Membrane Proteins/genetics , Middle Aged , Multivariate Analysis , Polymorphism, Single Nucleotide , Risk Factors , Schizophrenia/parasitology , Schizophrenia/virology , Toxoplasmosis, Cerebral/complicationsABSTRACT
We have recently found that consanguinity is a risk factor for bipolar I disorder (BP1) and schizophrenia (SZ) in Egypt. Inbreeding has been associated with increased cellular stress and impaired physiological function in plants and animals. Previous studies have reported that telomere length (TL), an index of oxidative stress and cellular senescence is significantly reduced among patients with SZ or mood disorders compared with control individuals. Hence we evaluated TL as a possible mediator of the observed association between consanguinity and BP1/SZ risk. Patients with BP1 (n=108), or SZ (n=60) were compared with screened adult controls in separate experiments. TL was estimated using a quantitative PCR (qPCR) based assay. The inbreeding coefficient/consanguinity rate was estimated in two ways: using 64 DNA polymorphisms ('DNA-based' rate); and from family history data ('self report'). Significant correlation between TL and DNA based inbreeding was not observed overall, though suggestive trends were present among the SZ cases. No significant case-control differences in TL were found after controlling for demographic variables. In conclusion, reduced TL may not explain a significant proportion of observed associations between consanguinity and risk for BP1/SZ.
Subject(s)
Bipolar Disorder/genetics , Inbreeding , Schizophrenia/genetics , Telomere/genetics , Adult , Analysis of Variance , Bipolar Disorder/epidemiology , Case-Control Studies , Egypt/epidemiology , Female , Genetic Predisposition to Disease , Humans , Linear Models , Male , Risk Factors , Schizophrenia/epidemiology , Young AdultABSTRACT
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.
Subject(s)
Antioxidants/metabolism , Genetic Association Studies , Schizophrenia/genetics , Aryldialkylphosphatase/genetics , Chromosome Mapping , Genes, Mitochondrial , Glutamate-Cysteine Ligase/genetics , Glutathione/metabolism , Glutathione Peroxidase/genetics , Glutathione Synthase/genetics , Glutathione Transferase/genetics , Humans , Methionine Sulfoxide Reductases/genetics , Nitric Oxide Synthase Type I/genetics , Quinone Reductases/genetics , Receptors, Mineralocorticoid/genetics , Schizophrenia/enzymology , Superoxide Dismutase/genetics , Glutathione Peroxidase GPX1ABSTRACT
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. © 2010 Wiley-Liss, Inc.
Subject(s)
Alternative Splicing , Dopamine Plasma Membrane Transport Proteins/genetics , Schizophrenia/genetics , Alleles , Base Sequence , Exons , Female , Gene Frequency , Genetic Association Studies , Genetic Predisposition to Disease , Genotype , Haplotypes , Humans , Introns , Linkage Disequilibrium , Male , Open Reading Frames/genetics , Polymorphism, Single Nucleotide , Schizophrenia/metabolism , Substantia Nigra/metabolismABSTRACT
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.
Subject(s)
Genetic Predisposition to Disease , Sleep Wake Disorders/classification , Sleep Wake Disorders/genetics , Sleep/genetics , Chromosome Mapping , Circadian Rhythm/genetics , Gene Expression/genetics , Genetic Linkage/genetics , Humans , Point Mutation/genetics , Sleep/physiology , Sleep Disorders, Circadian Rhythm/genetics , Sleep Disorders, Circadian Rhythm/physiopathology , Sleep Wake Disorders/diagnosis , Sleep Wake Disorders/physiopathologyABSTRACT
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.
Subject(s)
Genetic Variation , Herpesvirus 1, Human/immunology , Histocompatibility Antigens Class I/genetics , Prefrontal Cortex/virology , Schizophrenia , Adolescent , Adult , Analysis of Variance , Female , Genotype , Humans , Image Processing, Computer-Assisted/methods , Magnetic Resonance Imaging/methods , Male , Multivariate Analysis , Neuropsychological Tests , Prefrontal Cortex/pathology , Psychiatric Status Rating Scales , Schizophrenia/genetics , Schizophrenia/pathology , Schizophrenia/virology , Young AdultABSTRACT
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.
Subject(s)
Alleles , Dopamine Plasma Membrane Transport Proteins/genetics , Polymorphism, Single Nucleotide/genetics , Cell Line, Tumor , Chromosomes , Electrophoretic Mobility Shift Assay , Exons/genetics , Genetic Variation/genetics , Haplotypes , Humans , Neuroblastoma , Promoter Regions, Genetic/genetics , Regulatory Elements, Transcriptional/genetics , Schizophrenia/genetics , Transcription Factors/genetics , Transcription Initiation SiteABSTRACT
We evaluated the hypothesis that dopaminergic polymorphisms are risk factors for schizophrenia (SZ). In stage I, we screened 18 dopamine-related genes in two independent US Caucasian samples: 150 trios and 328 cases/501 controls. The most promising associations were detected with SLC6A3 (alias DAT), DRD3, COMT and SLC18A2 (alias VMAT2). In stage II, we comprehensively evaluated these four genes by genotyping 68 SNPs in all 478 cases and 501 controls from stage I. Fifteen (23.1%) significant associations were found (p < or = 0.05). We sought epistasis between pairs of SNPs providing evidence of a main effect and observed 17 significant interactions (169 tests); 41.2% of significant interactions involved rs3756450 (5' near promoter) or rs464049 (intron 4) at SLC6A3. In stage III, we confirmed our findings by genotyping 65 SNPs among 659 Bulgarian trios. Both SLC6A3 variants implicated in the US interactions were overtransmitted in this cohort (rs3756450, p = 0.035; rs464049, p = 0.011). Joint analyses from stages II and III identified associations at all four genes (p(joint) < 0.05). We tested 29 putative interactions from stage II and detected replication between seven locus pairs (p < or = 0.05). Simulations suggested our stage II and stage III interaction results were unlikely to have occurred by chance (p = 0.008 and 0.001, respectively). In stage IV we evaluated rs464049 and rs3756450 for functional effects and found significant allele-specific differences at rs3756450 using electrophoretic mobility shift assays and dual-luciferase promoter assays. Our data suggest that a network of dopaminergic polymorphisms increase risk for SZ.
Subject(s)
Catechol O-Methyltransferase/genetics , Dopamine Plasma Membrane Transport Proteins/genetics , Genetic Variation , Receptors, Dopamine D3/genetics , Schizophrenia/genetics , Vesicular Monoamine Transport Proteins/genetics , Alleles , Bulgaria , Case-Control Studies , Cohort Studies , Data Interpretation, Statistical , Female , Gene Frequency , Genes, Reporter , Humans , Linkage Disequilibrium , Logistic Models , Luciferases, Renilla/metabolism , Odds Ratio , Pedigree , Polymorphism, Single Nucleotide , Risk Factors , Sex Factors , United StatesABSTRACT
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 (r(2) > .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.
Subject(s)
Linkage Disequilibrium/genetics , Polymorphism, Single Nucleotide/genetics , RGS Proteins/genetics , Schizophrenia/genetics , Exons/genetics , Genotype , Haplotypes/genetics , Humans , Introns/geneticsABSTRACT
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.