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1.  An integrative functional genomics approach for discovering biomarkers in schizophrenia 
Briefings in Functional Genomics  2011;10(6):387-399.
Schizophrenia (SZ) is a complex disorder resulting from both genetic and environmental causes with a lifetime prevalence world-wide of 1%; however, there are no specific, sensitive and validated biomarkers for SZ. A general unifying hypothesis has been put forward that disease-associated single nucleotide polymorphisms (SNPs) from genome-wide association study (GWAS) are more likely to be associated with gene expression quantitative trait loci (eQTL). We will describe this hypothesis and review primary methodology with refinements for testing this paradigmatic approach in SZ. We will describe biomarker studies of SZ and testing enrichment of SNPs that are associated both with eQTLs and existing GWAS of SZ. SZ-associated SNPs that overlap with eQTLs can be placed into gene–gene expression, protein–protein and protein–DNA interaction networks. Further, those networks can be tested by reducing/silencing the gene expression levels of critical nodes. We present pilot data to support these methods of investigation such as the use of eQTLs to annotate GWASs of SZ, which could be applied to the field of biomarker discovery. Those networks that have association with SNP markers, especially cis-regulated expression, might lead to a more clear understanding of important candidate genes that predispose to disease and alter expression. This method has general application to many complex disorders.
PMCID: PMC3277082  PMID: 22155586
expression quantitative trait loci; cis-regulatory SNPs; GWAS; gene expression; lymphoblastoid cell lines
2.  Evidence of allelic imbalance in the schizophrenia susceptibility gene ZNF804A in human dorsolateral prefrontal cortex 
Schizophrenia research  2013;152(1):111-116.
The rs1344706, an intronic SNP within the zinc-finger protein 804A gene (ZNF804A), was identified as one of the most compelling risk SNPs for schizophrenia (SZ) and bipolar disorder (BD). It is however not clear by which molecular mechanisms ZNF804A increases disease risk. We evaluated the role of ZNF804A in SZ and BD by genotyping the originally associated rs1344706 SNP and an exonic SNP (rs12476147) located in exon four of ZNF804A in a sample of 428 SZ, 385 BD, and 578 controls from the isolated population of the Costa Rica Central Valley. We also investigated the rs1344706 SNP for allelic specific expression (ASE) imbalance in the dorsolateral prefrontal cortex (DLPFC) of 46 heterozygous postmortem brains.
While no significant association between rs1344706 and SZ or BD was observed in the Costa Rica sample, we observed an increased risk of SZ for the minor allele (A) of the exonic rs12476147 SNP (p =0.026). Our ASE assay detected a significant over-expression of the rs12476147 A allele in DLPFC of rs1344706 heterozygous subjects. Interestingly, cDNA allele ratios were significantly different according to the intronic rs1344706 genotypes (p-value = 0.03), with the rs1344706 A allele associated with increased ZNF804A rs12476147 A allele expression (average 1.06, p-value = 0.02, for heterozygous subjects vs. genomic DNA).
In conclusion, we have demonstrated a significant association of rs12476147 with SZ, and using a powerful within-subjects design, an allelic expression imbalance of ZNF804A exonic SNP rs12476147 in the DLPFC. Although this data does not preclude the possibility of other functional variants in ZNF804A, it provides evidence that the rs1344706 SZ risk allele is the cis-regulatory variant directly responsible for this allelic expression imbalance in adult cortex.
PMCID: PMC3947280  PMID: 24315717
schizophrenia; bipolar disorder; ZNF804A; association study; rs1344706; allelic-specific expression
3.  Analysis of miR-137 Expression and rs1625579 in Dorsolateral Prefrontal Cortex 
Journal of psychiatric research  2013;47(9):1215-1221.
MicroRNAs (miRNAs) are small non-coding RNAs that act as potent regulators of gene expression. A recent GWAS reported the rs1625579 SNP, located downstream of miR-137, as the strongest new association with schizophrenia (Ripke et al., 2011). Prior to this GWAS finding, a schizophrenia imaging-genetic study found miR-137 target genes significantly enriched for association with activation in the dorsolateral prefrontal cortex (DLPFC) (Potkin et al., 2010).
We investigated the expression levels of miR-137 and three candidate target genes (ZNF804A, CACNA1C, TCF4) in the DLPFC of postmortem brain tissue from 2 independent cohorts: 1) 26 subjects (10 control (CTR), 7 schizophrenia (SZ), 9 bipolar disorder (BD)) collected at the UCI brain bank; and 2) 99 subjects (33 CTR, 35 SZ, 31 BD) obtained from the Stanley Medical Research Institute (SMRI). MiR-137 expression in the DLPFC did not differ between diagnoses. We also explored the relationship between rs1625579 genotypes and miR-137 expression. Significantly lower miR-137 expression levels were observed in the homozygous TT subjects compared to TG and GG subjects in the control group (30% decrease, p-value=0.03). Moreover, reduced miR-137 levels in TT subjects corresponded to increased levels of the miR-137 target gene TCF4. The miR-137 expression pattern in 9 brain regions was significant for regional effect (ANOVA p-value=1.83E-12), with amygdala and hippocampus having the highest miR-137 expression level. In conclusion, decreased miR-137 expression is associated with the SZ risk allele of rs1625579, and potential regulation of TCF4, another SZ candidate gene. This study offers additional support for involvement of miR-137 and downstream targets as mechanisms of risk for psychiatric disorders.
PMCID: PMC3753093  PMID: 23786914
schizophrenia; bipolar disorder; rs1625579; miR-137; TCF4; gene expression
4.  G protein-linked signaling pathways in bipolar and major depressive disorders 
Frontiers in Genetics  2013;4:297.
The G-protein linked signaling system (GPLS) comprises a large number of G-proteins, G protein-coupled receptors (GPCRs), GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP), phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD) and bipolar disorder (BPD). This study evaluated whether GPLS is altered at the transcript level. The gene expression in the dorsolateral prefrontal (DLPFC) and anterior cingulate (ACC) were compared from MDD, BPD, and control subjects using Affymetrix Gene Chips and real time quantitative PCR. High quality brain tissue was used in the study to control for confounding effects of agonal events, tissue pH, RNA integrity, gender, and age. GPLS signaling transcripts were altered especially in the ACC of BPD and MDD subjects. Transcript levels of molecules which repress cAMP activity were increased in BPD and decreased in MDD. Two orphan GPCRs, GPRC5B and GPR37, showed significantly decreased expression levels in MDD, and significantly increased expression levels in BPD. Our results suggest opposite changes in BPD and MDD in the GPLS, “activated” cAMP signaling activity in BPD and “blunted” cAMP signaling activity in MDD. GPRC5B and GPR37 both appear to have behavioral effects, and are also candidate genes for neurodegenerative disorders. In the context of the opposite changes observed in BPD and MDD, these GPCRs warrant further study of their brain effects.
PMCID: PMC3870297  PMID: 24391664
G-protein coupled receptor (GPCR); transcriptome; bipolar disorder; major depressive disorder; GPR37; GPRC5B; cyclic AMP; phosphatidylinositol
5.  Coding and Noncoding Gene Expression Biomarkers in Mood Disorders and Schizophrenia 
Disease markers  2013;35(1):11-21.
Mood disorders and schizophrenia are common and complex disorders with consistent evidence of genetic and environmental influences on predisposition. It is generally believed that the consequences of disease, gene expression, and allelic heterogeneity may be partly the explanation for the variability observed in treatment response. Correspondingly, while effective treatments are available for some patients, approximately half of the patients fail to respond to current neuropsychiatric treatments. A number of peripheral gene expression studies have been conducted to understand these brain-based disorders and mechanisms of treatment response with the aim of identifying suitable biomarkers and perhaps subgroups of patients based upon molecular fingerprint. In this review, we summarize the results from blood-derived gene expression studies implemented with the aim of discovering biomarkers for treatment response and classification of disorders. We include data from a biomarker study conducted in first-episode subjects with schizophrenia, where the results provide insight into possible individual biological differences that predict antipsychotic response. It is concluded that, while peripheral studies of expression are generating valuable results in pathways involving immune regulation and response, larger studies are required which hopefully will lead to robust biomarkers for treatment response and perhaps underlying variations relevant to these complex disorders.
PMCID: PMC3774957  PMID: 24167345
6.  Mitochondrial Mutations and Polymorphisms in Psychiatric Disorders 
Frontiers in Genetics  2012;3:103.
Mitochondrial deficiencies with unknown causes have been observed in schizophrenia (SZ) and bipolar disorder (BD) in imaging and postmortem studies. Polymorphisms and somatic mutations in mitochondrial DNA (mtDNA) were investigated as potential causes with next generation sequencing of mtDNA (mtDNA-Seq) and genotyping arrays in subjects with SZ, BD, major depressive disorder (MDD), and controls. The common deletion of 4,977 bp in mtDNA was compared between SZ and controls in 11 different vulnerable brain regions and in blood samples, and in dorsolateral prefrontal cortex (DLPFC) of BD, SZ, and controls. In a separate analysis, association of mitochondria SNPs (mtSNPs) with SZ and BD in European ancestry individuals (n = 6,040) was tested using Genetic Association Information Network (GAIN) and Wellcome Trust Case Control Consortium 2 (WTCCC2) datasets. The common deletion levels were highly variable across brain regions, with a 40-fold increase in some regions (nucleus accumbens, caudate nucleus and amygdala), increased with age, and showed little change in blood samples from the same subjects. The common deletion levels were increased in the DLPFC for BD compared to controls, but not in SZ. Full mtDNA genome resequencing of 23 subjects, showed seven novel homoplasmic mutations, five were novel synonymous coding mutations. By logistic regression analysis there were no significant mtSNPs associated with BD or SZ after genome wide correction. However, nominal association of mtSNPs (p < 0.05) to SZ and BD were found in the hypervariable region of mtDNA to T195C and T16519C. The results confirm prior reports that certain brain regions accumulate somatic mutations at higher levels than blood. The study in mtDNA of common polymorphisms, somatic mutations, and rare mutations in larger populations may lead to a better understanding of the pathophysiology of psychiatric disorders.
PMCID: PMC3379031  PMID: 22723804
mitochondria; homoplasmy; common deletion; novel mutations; schizophrenia; bipolar disorder
7.  Analysis Of Whole Genome Biomarker Expression In Blood And Brain 
The consistency of peripheral gene expression data and the overlap with brain expression has not been evaluated in biomarker discovery, nor has it been reported in multiple tissues from the same subjects on a genome wide transcript level. The effects of processing whole blood, transformation, and passaged cell lines on gene expression profiling was studied in healthy subjects using Affymetrix arrays. Ficoll extracted peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus (EBV) transformed lymphocytes, passaged lymphoblastic cell lines (LCLs), and whole blood from Tempus tubes were compared. There were 6,813 transcripts differentially expressed between different methods of blood preparation. Principal component analysis resolved two partitions involving pre- and post-transformation EBV effects.
Combining results from Affymetrix arrays, postmortem subjects' brain and PBMC profiles showed co-expression levels of summarized transcripts for 4,103 of 17,859 (22.9%) RefSeq transcripts. In a control experiment, rat hemi-brain and blood showed similar expression levels for 19% of RefSeq transcripts. After filtering transcripts that were not significantly different in abundance between human cerebellum and PBMCs from the Affymetrix exon array the correlation in mean transcript abundance was high as expected (r = 0.98). Differences in the alternative splicing index in brain and blood were found for about 90% of all transcripts examined. This study demonstrates over 4,100 brain transcripts co-expressed in blood samples can be further examined by in vitro and in vivo experimental studies of blood and cell lines from patients with psychiatric disorders.
PMCID: PMC3098564  PMID: 20127885
biomarker; gene expression; whole genome
8.  Mitochondrial involvement in psychiatric disorders 
Annals of medicine  2008;40(4):281-295.
Recent findings of mitochondrial abnormalities in brains from subjects with neurological disorders have led to a renewed search for mitochondrial abnormalities in psychiatric disorders. A growing body of evidence suggests that there is mitochondrial dysfunction in schizophrenia, bipolar disorder, and major depressive disorder, including evidence from electron microscopy, imaging, gene expression, genotyping, and sequencing studies. Specific evidence of dysfunction such as increased common deletion and decreased gene expression in mitochondria in psychiatric illnesses suggests that direct examination of mitochondrial DNA from postmortem brain cells may provide further details of mitochondrial alterations in psychiatric disorders.
PMCID: PMC3098560  PMID: 18428021
Bipolar disorder; major depressive disorder; mitochondria; mtDNA; schizophrenia
9.  Shared Gene Expression Alterations in Schizophrenia and Bipolar Disorder 
Biological psychiatry  2008;64(2):89-97.
Schizophrenia and bipolar disorder together affect approximately 2.5% of the world population, and their etiologies are thought to involve multiple genetic variants and environmental influences. The analysis of gene expression patterns in brain may provide a characteristic signature for each disorder.
RNA samples from the dorsolateral prefrontal cortex (Brodmann area 46) consisting of individuals with schizophrenia (SZ), bipolar disorder (BPD), and control subjects were tested on the Codelink Human 20K Bioarray platform. Selected transcripts were validated by quantitative real-time polymerase chain reaction (PCR). The strong effects of age, gender, and pH in the analysis of differential gene expression were controlled by analysis of covariance (ANCOVA). Criteria for differential gene expression were 1) a gene was significantly dysregulated in both BPD and SZ compared with control subjects and 2) significant in ANCOVA analysis with samples that have a pH above the median of the sample.
A list of 78 candidate genes passed these two criteria in BPD and SZ and was overrepresented for functional categories of nervous system development, immune system development and response, and cell death. Five dysregulated genes were confirmed with quantitative Q-PCR in both BPD and SZ. Three genes were highly enriched in brain expression (AGXT2L1, SLC1A2, and TU3A). The distribution of AGXT2L1 expression in control subjects versus BPD and SZ was highly significant (Fisher’s Exact Test, p < 10−06).
These results suggest a partially shared molecular profile for both disorders and offer a window into discovery of common pathophysiology that might lead to core treatments.
PMCID: PMC3098561  PMID: 18191109
AGXT2L1; antipsychotic medication; apoptosis; bipolar disorder; BUB1B; dorsolateral prefrontal cortex; EMX2; ERBB2; FGF2; FTH1; IL2RA; LGALS3; MAFG; microarray; neurogenesis; NFATC1; PVR; quantitative PCR; RERG; schizophrenia; SLC1A2; SMCY; SMO; SOX9; TU3A; TXNIP
10.  Dysregulation of X-Linked Gene Expression in Klinefelter’s Syndrome and Association With Verbal Cognition 
Klinefelter’s Syndrome (KS) is a chromosomal karyotype with one or more extra X chromosomes. KS individuals often show language impairment and the phenotype might be due to overexpression of genes on the extra X chromosome(s). We profiled mRNA derived from lymphoblastoid cell lines from males with documented KS and control males using the Affymetrix U133P microarray platform. There were 129 differentially expressed genes (DEGs) in KS group compared with controls after Benjamini–Hochberg false discovery adjustment. The DEGs included 14 X chromosome genes which were significantly over-represented. The Y chromosome had zero DEGs. In exploratory analysis of gene expression–cognition relationships, 12 DEGs showed significant correlation of expression with measures of verbal cognition in KS. Overexpression of one pseudoautosomal gene, GTPBP6 (GTP binding protein 6, putative) was inversely correlated with verbal IQ (r = −0.86, P < 0.001) and four other measures of verbal ability. Overexpression of XIST was found in KS compared to XY controls suggesting that silencing of many genes on the X chromosome might occur in KS similar to XX females. The microarray findings for eight DEGs were validated by quantitative PCR. The 14 X chromosome DEGs were not differentially expressed in prior studies comparing female and male brains suggesting a dysregulation profile unique to KS. Examination of X-linked DEGs, such as GTPBP6, TAF9L, and CXORF21, that show verbal cognition–gene expression correlations may establish a causal link between these genes, neurodevelopment, and language function. A screen of candidate genes may serve as biomarkers of KS for early diagnosis.
PMCID: PMC2094046  PMID: 17347996
XXY; XY; brain; lymphocytes; microarray; XIST; GTPBP6; TAF9L; CXORF21
11.  NCAM1 association study of bipolar disorder and schizophrenia: polymorphisms and alternatively spliced isoforms lead to similarities and differences 
Psychiatric genetics  2007;17(2):55-67.
The neural cell adhesion molecule (NCAM1) is a multifunction transmembrane protein involved in synaptic plasticity, neurodevelopment, and neurogenesis. Multiple NCAM1 proteins were differentially altered in bipolar disorder and schizophrenia. Single nucleotide polymorphisms (SNPs) in the NCAM1 gene were significantly associated with bipolar disorder in the Japanese population. Bipolar disorder and schizophrenia may share common vulnerability or susceptibility risk factors for shared features in each disorder.
Both SNPs and splice variants in the NCAM1 gene were analysed in bipolar disorder and schizophrenia. A case-control study design for association of SNPs and differential exon expression in the NCAM1 gene was used.
A genotypic association between bipolar disorder and SNP b (rs2303377 near mini-exon b) and a suggestive association between schizophrenia and SNP 9 (rs646558) were found. Three of the two marker haplotypes for SNP 9 and SNP b showed varying frequencies between bipolar and controls (P < 0.0001) as well as between schizophrenia and controls (P < 0.0001). There were nine NCAM1 transcripts present in postmortem brain samples that involve alternative splicing of NCAM1 mini-exons (a, b, c) and the secreted (SEC) exon. Significant differences in the amounts of four alternatively spliced isoforms were found between NCAM1 SNP genotypes. In exploratory analysis, the c—SEC alternative spliced isoform was significantly decreased in bipolar disorder compared to controls for NCAM1 SNP b heterozygotes (P = 0.013).
Diverse NCAM1 transcripts were found with possibly different functions. The results suggest that SNPs within NCAM1 contribute differential risk for both bipolar disorder and schizophrenia possibly by alternative splicing of the gene. Psychiatr Genet 17:55−67 © 2007 Lippincott Williams & Wilkins.
PMCID: PMC2077086  PMID: 17413444
alternative splicing; association; bipolar disorder; genetics; neural cell adhesion molecule 1; schizophrenia; secreted-neural cell adhesion molecule 1; single nucleotide polymorphism; variable alternative spliced exon–secreted-neural cell adhesion molecule 1
12.  Identification of Pathways for Bipolar Disorder A Meta-analysis 
JAMA psychiatry  2014;71(6):657-664.
Genome-wide investigations provide systematic information regarding the neurobiology of psychiatric disorders.
To identify biological pathways that contribute to risk for bipolar disorder (BP) using genes with consistent evidence for association in multiple genome-wide association studies (GWAS).
Four independent data sets with individual genome-wide data available in July 2011 along with all data sets contributed to the Psychiatric Genomics Consortium Bipolar Group by May 2012. A prior meta-analysis was used as a source for brain gene expression data.
The 4 published GWAS were included in the initial sample. All independent BP data sets providing genome-wide data in the Psychiatric Genomics Consortium were included as a replication sample.
We identified 966 genes that contained 2 or more variants associated with BP at P < .05 in 3 of 4 GWAS data sets (n = 12 127 [5253 cases, 6874 controls]). Simulations using 10 000 replicates of these data sets corrected for gene size and allowed the calculation of an empirical P value for each gene; empirically significant genes were entered into a pathway analysis. Each of these pathways was then tested in the replication sample (n = 8396 [3507 cases, 4889 controls]) using gene set enrichment analysis for single-nucleotide polymorphisms. The 226 genes were also compared with results from a meta-analysis of gene expression in the dorsolateral prefrontal cortex.
Empirically significant genes and biological pathways.
Among 966 genes, 226 were empirically significant (P < .05). Seventeen pathways were overrepresented in analyses of the initial data set. Six of the 17 pathways were associated with BP in both the initial and replication samples: corticotropin-releasing hormone signaling, cardiac β-adrenergic signaling, phospholipase C signaling, glutamate receptor signaling, endothelin 1 signaling, and cardiac hypertrophy signaling. Among the 226 genes, 9 differed in expression in the dorsolateral prefrontal cortex in patients with BP: CACNA1C, DTNA, FOXP1, GNG2, ITPR2, LSAMP, NPAS3, NCOA2, and NTRK3.
Pathways involved in the genetic predisposition to BP include hormonal regulation, calcium channels, second messenger systems, and glutamate signaling. Gene expression studies implicate neuronal development pathways as well. These results tend to reinforce specific hypotheses regarding BP neurobiology and may provide clues for new approaches to treatment and prevention.
PMCID: PMC4523227  PMID: 24718920
13.  Mitochondrial Mutations in Subjects with Psychiatric Disorders 
PLoS ONE  2015;10(5):e0127280.
A considerable body of evidence supports the role of mitochondrial dysfunction in psychiatric disorders and mitochondrial DNA (mtDNA) mutations are known to alter brain energy metabolism, neurotransmission, and cause neurodegenerative disorders. Genetic studies focusing on common nuclear genome variants associated with these disorders have produced genome wide significant results but those studies have not directly studied mtDNA variants. The purpose of this study is to investigate, using next generation sequencing, the involvement of mtDNA variation in bipolar disorder, schizophrenia, major depressive disorder, and methamphetamine use. MtDNA extracted from multiple brain regions and blood were sequenced (121 mtDNA samples with an average of 8,800x coverage) and compared to an electronic database containing 26,850 mtDNA genomes. We confirmed novel and rare variants, and confirmed next generation sequencing error hotspots by traditional sequencing and genotyping methods. We observed a significant increase of non-synonymous mutations found in individuals with schizophrenia. Novel and rare non-synonymous mutations were found in psychiatric cases in mtDNA genes: ND6, ATP6, CYTB, and ND2. We also observed mtDNA heteroplasmy in brain at a locus previously associated with schizophrenia (T16519C). Large differences in heteroplasmy levels across brain regions within subjects suggest that somatic mutations accumulate differentially in brain regions. Finally, multiplasmy, a heteroplasmic measure of repeat length, was observed in brain from selective cases at a higher frequency than controls. These results offer support for increased rates of mtDNA substitutions in schizophrenia shown in our prior results. The variable levels of heteroplasmic/multiplasmic somatic mutations that occur in brain may be indicators of genetic instability in mtDNA.
PMCID: PMC4444211  PMID: 26011537
14.  Schizophrenia miR-137 Locus Risk Genotype is Associated with DLPFC Hyperactivation 
Biological psychiatry  2013;75(5):398-405.
MiR-137 dysregulation has been implicated in the etiology of schizophrenia, but its functional role remains to be determined.
Functional magnetic resonance imaging scans were acquired on 48 schizophrenia patients and 63 healthy volunteers (total sample size n=111 subjects), with similar mean age and sex distribution, while subjects performed a Sternberg Item Response Paradigm with memory loads of 1, 3, and 5 numbers. Dorsolateral prefrontal cortex (DLPFC) retrieval activation for the working memory load of 3 numbers, for which hyperactivation had been shown in schizophrenia patients compared with controls, was extracted. The genome-wide association study confirmed schizophrenia risk SNP rs1625579 (miR-137 locus) was genotyped (schizophrenia: GG n=0, GT n=9, TT n=39; healthy volunteers: GG=2, GT n=15, and TT n=46). Fisher's Exact Test examined the effect of diagnosis on rs1625579 allele frequency distribution (p=ns). Mixed model regression analyses examined the effects of diagnosis and genotype on working memory performance measures and DLPFC activation.
Patients showed significantly higher left DLPFC retrieval activation on working memory load 3, lower working memory performance and longer response times compared with controls. There was no effect of genotype on working memory performance or response times in either group. However, individuals with the rs1625579 TT genotype had significantly higher left DLPFC activation than those with the GG/GT genotypes.
Our study suggests that the rs1625579 TT (miR-137 locus) schizophrenia risk genotype is associated with the schizophrenia risk phenotype DLPFC hyperactivation commonly considered a measure of brain inefficiency.
PMCID: PMC4428556  PMID: 23910899
schizophrenia; rs1625579; mir137; imaging; working memory; genes
15.  Molecular and Bioenergetic Differences between Cells with African versus European Inherited Mitochondrial DNA Haplogroups: Implications for Population Susceptibility to Diseases 
Biochimica et biophysica acta  2013;1842(2):208-219.
The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP turnover rates and lower levels of ROS production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.
PMCID: PMC4326177  PMID: 24200652
Mitochondrial haplogroups; transmitochondrial cybrids; inflammation; complement; mitochondria; complement activation; innate immunity; haplogroups; cybrids; retina
16.  A splice donor mutation in NAA10 results in the dysregulation of the retinoic acid signaling pathway and causes Lenz microphthalmia syndrome 
Journal of medical genetics  2014;51(3):185-196.
Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition.
Methods and results
Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T→A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells.
We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway.
PMCID: PMC4278941  PMID: 24431331
17.  An anatomically comprehensive atlas of the adult human brain transcriptome 
Nature  2012;489(7416):391-399.
Neuroanatomically precise, genome-wide maps of transcript distributions are critical resources to complement genomic sequence data and to correlate functional and genetic brain architecture. Here we describe the generation and analysis of a transcriptional atlas of the adult human brain, comprising extensive histological analysis and comprehensive microarray profiling of ~900 neuroanatomically precise subdivisions in two individuals. Transcriptional regulation varies enormously by anatomical location, with different regions and their constituent cell types displaying robust molecular signatures that are highly conserved between individuals. Analysis of differential gene expression and gene co-expression relationships demonstrates that brain-wide variation strongly reflects the distributions of major cell classes such as neurons, oligodendrocytes, astrocytes and microglia. Local neighbourhood relationships between fine anatomical subdivisions are associated with discrete neuronal subtypes and genes involved with synaptic transmission. The neocortex displays a relatively homogeneous transcriptional pattern, but with distinct features associated selectively with primary sensorimotor cortices and with enriched frontal lobe expression. Notably, the spatial topography of the neocortex is strongly reflected in its molecular topography— the closer two cortical regions, the more similar their transcriptomes. This freely accessible online data resource forms a high-resolution transcriptional baseline for neurogenetic studies of normal and abnormal human brain function.
PMCID: PMC4243026  PMID: 22996553
Neuroscience; Genetics; Genomics; Databases
18.  Increased CNV-Region deletions in mild cognitive impairment (MCI) and Alzheimer's disease (AD) subjects in the ADNI sample 
Genomics  2013;102(2):112-122.
We investigated the genome-wide distribution of CNVs in the Alzheimer's disease (AD) Neuroimaging Initiative (ADNI) sample (146 with AD, 313 with Mild Cognitive Impairment (MCI), and 181 controls). Comparison of single CNVs between cases (MCI and AD) and controls shows overrepresentation of large heterozygous deletions in cases (p-value < 0.0001). The analysis of CNV-Regions identifies 44 copy number variable loci of heterozygous deletions, with more CNV-Regions among affected than controls (p = 0.005). Seven of the 44 CNV-Regions are nominally significant for association with cognitive impairment. We validated and confirmed our main findings with genome re-sequencing of selected patients and controls. The functional pathway analysis of the genes putatively affected by deletions of CNV-Regions reveals enrichment of genes implicated in axonal guidance, cell–cell adhesion, neuronal morphogenesis and differentiation. Our findings support the role of CNVs in AD, and suggest an association between large deletions and the development of cognitive impairment
PMCID: PMC4012421  PMID: 23583670
Alzheimer's disease; Copy Number Variable Regions (CNV-Regions); Copy Number Variations (CNVs); Genome-wide scan; Next Generation Sequencing (NGS)
19.  Conserved Chromosome 2q31 Conformations Are Associated with Transcriptional Regulation of GAD1 GABA Synthesis Enzyme and Altered in Prefrontal Cortex of Subjects with Schizophrenia 
The Journal of Neuroscience  2013;33(29):11839-11851.
Little is known about chromosomal loopings involving proximal promoter and distal enhancer elements regulating GABAergic gene expression, including changes in schizophrenia and other psychiatric conditions linked to altered inhibition. Here, we map in human chromosome 2q31 the 3D configuration of 200 kb of linear sequence encompassing the GAD1 GABA synthesis enzyme gene locus, and we describe a loop formation involving the GAD1 transcription start site and intergenic noncoding DNA elements facilitating reporter gene expression. The GAD1-TSS-50kbLoop was enriched with nucleosomes epigenetically decorated with the transcriptional mark, histone H3 trimethylated at lysine 4, and was weak or absent in skin fibroblasts and pluripotent stem cells compared with neuronal cultures differentiated from them. In the prefrontal cortex of subjects with schizophrenia, GAD1-TSS-50kbLoop was decreased compared with controls, in conjunction with downregulated GAD1 expression. We generated transgenic mice expressing Gad2 promoter-driven green fluorescent protein-conjugated histone H2B and confirmed that Gad1-TSS-55kbLoop, the murine homolog to GAD1-TSS-50kbLoop, is a chromosomal conformation specific for GABAergic neurons. In primary neuronal culture, Gad1-TSS-55kbLoop and Gad1 expression became upregulated when neuronal activity was increased. We conclude that 3D genome architectures, including chromosomal loopings for promoter-enhancer interactions involved in the regulation of GABAergic gene expression, are conserved between the rodent and primate brain, and subject to developmental and activity-dependent regulation, and disordered in some cases with schizophrenia. More broadly, the findings presented here draw a connection between noncoding DNA, spatial genome architecture, and neuronal plasticity in development and disease.
PMCID: PMC3713726  PMID: 23864674
20.  Methodological Considerations For Gene Expression Profiling Of Human Brain 
Journal of neuroscience methods  2007;163(2):10.1016/j.jneumeth.2007.03.022.
Gene expression profiles of postmortem brain tissue represent important resources for understanding neuropsychiatric illnesses. The impact(s) of quality covariables on the analysis and results of gene expression studies are important questions. This paper addressed critical variables which might affect gene expression in two brain regions. Four broad groups of quality indicators in gene expression profiling studies (clinical, tissue, RNA, and microarray quality) were identified. These quality control indicators were significantly correlated, however one quality variable did not account for the total variance in microarray gene expression. The data showed that agonal factors and low pH correlated with decreased integrity of extracted RNA in two brain regions. These three parameters also modulated the significance of alterations in mitochondrial-related genes. The average F-ratio summaries across all transcripts showed that RNA degradation from the AffyRNAdeg program accounted for higher variation than all other quality factors. Taken together, these findings confirmed prior studies, which indicated that quality parameters including RNA integrity, agonal factors, and pH are related to differences in gene expression profiles in postmortem brain. Individual candidate genes can be evaluated with these quality parameters in posthoc analysis to help strengthen the relevance to psychiatric disorders. We find that clinical, tissue, RNA, and microarray quality are all useful variables for collection and consideration in study design, analysis, and interpretation of gene expression results in human post-mortem studies.
PMCID: PMC3835340  PMID: 17512057
21.  The First Decade and Beyond of Transcriptional Profiling in Schizophrenia 
Neurobiology of disease  2011;45(1):23-36.
Gene expression changes in brains of individuals with schizophrenia (SZ) have been hypothesized to reflect possible pathways related to pathophysiology and/or medication. Other factors have robust effects on gene expression profiling in brain and possibly influence the schizophrenia transcriptome such as age and pH are examined. Pathways of curated gene expression or gene correlation networks reported in SZ (white matter, apoptosis, neurogenesis, synaptic plasticity, glutamatergic and GABAergic neurotransmission, immune and stress-response, mitochondrial, and neurodevelopment) are not unique to SZ and have been associated with other psychiatric disorders. Suggestions going forward to improve the next decade of profiling: consider multiple brain regions that are carefully dissected, release large datasets from multiple brain regions in controls to better understand neurocircuitry, integrate genetics and gene expression, measure expression variants on genome wide level, peripheral biomarker studies, and analyze the transcriptome across a developmental series of brains. Gene expression, while an important feature of the genomic landscape, requires further systems biology to advance from control brains to a more precise definition of the schizophrenia interactome.
PMCID: PMC3178722  PMID: 21396449
microarray; gene expression; schizophrenia; bipolar disorder; psychiatry; gene-based biomarker discovery; next generation sequencing; allele specific expression; copy number variation
22.  Next Generation Sequence Analysis and Computational Genomics Using Graphical Pipeline Workflows 
Genes  2012;3(3):545-575.
Whole-genome and exome sequencing have already proven to be essential and powerful methods to identify genes responsible for simple Mendelian inherited disorders. These methods can be applied to complex disorders as well, and have been adopted as one of the current mainstream approaches in population genetics. These achievements have been made possible by next generation sequencing (NGS) technologies, which require substantial bioinformatics resources to analyze the dense and complex sequence data. The huge analytical burden of data from genome sequencing might be seen as a bottleneck slowing the publication of NGS papers at this time, especially in psychiatric genetics. We review the existing methods for processing NGS data, to place into context the rationale for the design of a computational resource. We describe our method, the Graphical Pipeline for Computational Genomics (GPCG), to perform the computational steps required to analyze NGS data. The GPCG implements flexible workflows for basic sequence alignment, sequence data quality control, single nucleotide polymorphism analysis, copy number variant identification, annotation, and visualization of results. These workflows cover all the analytical steps required for NGS data, from processing the raw reads to variant calling and annotation. The current version of the pipeline is freely available at These applications of NGS analysis may gain clinical utility in the near future (e.g., identifying miRNA signatures in diseases) when the bioinformatics approach is made feasible. Taken together, the annotation tools and strategies that have been developed to retrieve information and test hypotheses about the functional role of variants present in the human genome will help to pinpoint the genetic risk factors for psychiatric disorders.
PMCID: PMC3490498  PMID: 23139896
Next Generation Sequencing (NGS); LONI pipeline; SNPs; CNVs; workflow; bioinformatics
23.  Lack of association to a NRG1 missense polymorphism in schizophrenia or bipolar disorder in a Costa Rican population 
Schizophrenia research  2011;131(1-3):52-57.
A missense polymorphism in the NRG1 gene, Val > Leu in exon 11, was reported to increase the risk of schizophrenia in selected families from the Central Valley region of Costa Rica (CVCR). The present study investigated the relationship between three NRG1 genetic variants, rs6994992, rs3924999, and Val > Leu missense polymorphism in exon 11, in cases and selected controls from an isolated population from the CVCR. Isolated populations can have less genetic heterogeneity and increase power to detect risk variants in candidate genes. Subjects with bipolar disorder (BD, n = 358), schizophrenia (SZ, n = 273), or unrelated controls (CO, n = 479) were genotyped for three NRG1 variants. The NRG1 promoter polymorphism (rs6994992) was related to altered expression of NRG1 Type IV in other studies. The expression of NRG1 type IV in the dorsolateral prefrontal cortex (DLPFC) and the effect of the rs6994992 genotype on expression were explored in a postmortem cohort of BD, SZ, major depressive disorder (MDD) cases, and controls. The missense polymorphism Val > Leu in exon 11 was not significantly associated with schizophrenia as previously reported in a family sample from this population, the minor allele frequency is 4%, thus our sample size is not large enough to detect an association. We observed however an association of rs6994992 with NRG1 type IV expression in DLPFC and a significantly decreased expression in MDD compared to controls. The present results while negative do not rule out a genetic association of these SNPs with BD and SZ in CVCR, perhaps due to small risk effects that we were unable to detect and potential intergenic epistasis. The previous genetic relationship between expression of a putative brain specific isoform of NRG1 type IV and SNP variation was replicated in postmortem samples in our preliminary study.
PMCID: PMC3159824  PMID: 21745728
neuregulin 1 isoform expression; schizophrenia; isolated population; Costa Rica; bipolar disorder; major depressive disorder; hippocampus; dorsolateral prefrontal cortex
24.  Next Generation Sequence Analysis and Computational Genomics Using Graphical Pipeline Workflows 
Genes  2012;3(3):545-575.
Whole-genome and exome sequencing have already proven to be essential and powerful methods to identify genes responsible for simple Mendelian inherited disorders. These methods can be applied to complex disorders as well, and have been adopted as one of the current mainstream approaches in population genetics. These achievements have been made possible by next generation sequencing (NGS) technologies, which require substantial bioinformatics resources to analyze the dense and complex sequence data. The huge analytical burden of data from genome sequencing might be seen as a bottleneck slowing the publication of NGS papers at this time, especially in psychiatric genetics. We review the existing methods for processing NGS data, to place into context the rationale for the design of a computational resource. We describe our method, the Graphical Pipeline for Computational Genomics (GPCG), to perform the computational steps required to analyze NGS data. The GPCG implements flexible workflows for basic sequence alignment, sequence data quality control, single nucleotide polymorphism analysis, copy number variant identification, annotation, and visualization of results. These workflows cover all the analytical steps required for NGS data, from processing the raw reads to variant calling and annotation. The current version of the pipeline is freely available at These applications of NGS analysis may gain clinical utility in the near future (e.g., identifying miRNA signatures in diseases) when the bioinformatics approach is made feasible. Taken together, the annotation tools and strategies that have been developed to retrieve information and test hypotheses about the functional role of variants present in the human genome will help to pinpoint the genetic risk factors for psychiatric disorders.
PMCID: PMC3490498  PMID: 23139896
Next Generation Sequencing (NGS); LONI pipeline; SNPs; CNVs; workflow; bioinformatics
25.  P2RX7: Expression Responds to Sleep Deprivation and Associates with Rapid Cycling in Bipolar Disorder Type 1 
PLoS ONE  2012;7(8):e43057.
Rapid cycling is a severe form of bipolar disorder with an increased rate of episodes that is particularly treatment-responsive to chronotherapy and stable sleep-wake cycles. We hypothesized that the P2RX7 gene would be affected by sleep deprivation and be implicated in rapid cycling.
To assess whether P2RX7 expression is affected by total sleep deprivation and if variation in P2RX7 is associated with rapid cycling in bipolar patients.
Gene expression analysis in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and case-case and case-control SNP/haplotype association analyses in patients.
Healthy volunteers at the sleep research center, University of California, Irvine Medical Center (UCIMC), USA (n = 8) and Swedish outpatients recruited from specialized psychiatric clinics for bipolar disorder, diagnosed with bipolar disorder type 1 (n = 569; rapid cycling: n = 121) and anonymous blood donor controls (n = 1,044).
P2RX7 RNA levels were significantly increased during sleep deprivation in PBMCs from healthy volunteers (p = 2.3*10−9). The P2RX7 rs2230912 _A allele was more common (OR = 2.2, p = 0.002) and the ACGTTT haplotype in P2RX7 (rs1718119 to rs1621388) containing the protective rs2230912_G allele (OR = 0.45–0.49, p = 0.003–0.005) was less common, among rapid cycling cases compared to non-rapid cycling bipolar patients and blood donor controls.
Sleep deprivation increased P2RX7 expression in healthy persons and the putatively low-activity P2RX7 rs2230912 allele A variant was associated with rapid cycling in bipolar disorder. This supports earlier findings of P2RX7 associations to affective disorder and is in agreement with that particularly rapid cycling patients have a more vulnerable diurnal system.
PMCID: PMC3429455  PMID: 22952630

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