Brandi Rollins

Mitochondrial Variants in Schizophrenia, Bipolar Disorder, and Major Depressive Disorder

Background Mitochondria provide most of the energy for brain cells by the process of oxidative phosphorylation. Mitochondrial abnormalities and deficiencies in oxidative phosphorylation have been reported in individuals with schizophrenia (SZ), bipolar disorder (BD), and major depressive disorder (MDD) in transcriptomic, proteomic, and metabolomic studies. Several mutations in mitochondrial DNA (mtDNA) sequence have been reported in SZ and BD patients. Methodology/Principal Findings Dorsolateral prefrontal cortex (DLPFC) from a cohort of 77 SZ, BD, and MDD subjects and age-matched controls (C) was studied for mtDNA sequence variations and heteroplasmy levels using Affymetrix mtDNA resequencing arrays. Heteroplasmy levels by microarray were compared to levels obtained with SNaPshot and allele specific real-time PCR. This study examined the association between brain pH and mtDNA alleles. The microarray resequencing of mtDNA was 100% concordant with conventional sequencing results for 103 mtDNA variants. The rate of synonymous base pair substitutions in the coding regions of the mtDNA genome was 22% higher (p = 0.0017) in DLPFC of individuals with SZ compared to controls. The association of brain pH and super haplogroup (U, K, UK) was significant (p = 0.004) and independent of postmortem interval time. Conclusions Focusing on haplogroup and individual susceptibility factors in psychiatric disorders by considering mtDNA variants may lead to innovative treatments to improve mitochondrial health and brain function.

NCAM1 association study of bipolar disorder and schizophrenia: polymorphisms and alternatively spliced isoforms lead to similarities and differences

Objective 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. Methods 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. Results 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). Conclusions 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.

Mitochondrial Mutations and Polymorphisms in Psychiatric Disorders

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.

Evidence of allelic imbalance in the schizophrenia susceptibility gene ZNF804A in human dorsolateral prefrontal cortex.

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 422 SZ, 382 BD, and 507 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-subject 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.

Mitochondrial Mutations in Subjects with Psychiatric Disorders

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.

Coding and Noncoding Gene Expression Biomarkers in Mood Disorders and Schizophrenia

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.

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

Genetic evidence has supported the hypothesis that schizophrenia (SZ) is a polygenic disorder caused by the disruption in function of several or many genes. The most common and reproducible cellular phenotype associated with SZ is a reduction in dendritic spines within the neocortex, suggesting alterations in dendritic architecture may cause aberrant cortical circuitry and SZ symptoms. Here, we review evidence supporting a multifactorial model of mitochondrial dysfunction in SZ etiology and discuss how these multiple paths to mitochondrial dysfunction may contribute to dendritic spine loss and/or underdevelopment in some SZ subjects. The pathophysiological role of mitochondrial dysfunction in SZ is based upon genomic analyses of both the mitochondrial genome and nuclear genes involved in mitochondrial function. Previous studies and preliminary data suggest SZ is associated with specific alleles and haplogroups of the mitochondrial genome, and also correlates with a reduction in mitochondrial copy number and an increase in synonymous and nonsynonymous substitutions of mitochondrial DNA. Mitochondrial dysfunction has also been widely implicated in SZ by genome-wide association, exome sequencing, altered gene expression, proteomics, microscopy analyses, and induced pluripotent stem cell studies. Together, these data support the hypothesis that SZ is a polygenic disorder with an enrichment of mitochondrial targets.

The somatic common deletion in mitochondrial DNA is decreased in schizophrenia

Large deletions in mitochondrial DNA (mtDNA) can occur during or result from oxidative stress leading to a vicious cycle that increases reactive oxygen species (ROS) damage and decreases mitochondrial function, thereby causing further oxidative stress. The objective of this study was to determine if disease specific brain differences of the somatic mtDNA common deletion (4977 bp) could be observed in major depressive disorder (MDD), bipolar disorder (BD), and schizophrenia (SZ) compared to a control group. The accumulation of the mtDNA common deletion was measured using a quantitative assay across 10 brain regions (anterior cingulate cortex, amygdala, caudate nucleus, dorsolateral prefrontal cortex, hippocampus, nucleus accumbens, orbitofrontal cortex, putamen, substantia nigra, and thalamus). The correlation with age of the mtDNA deletion was highly significant across brain regions as previously shown. A significant decrease in the global accumulation of common deletion in subjects with SZ compared to MDD, BD, and controls was observed after correcting for age, pH, PMI, and gender. The decreases in SZ were largest in dopaminergic regions. One potential side effect of antipsychotic drugs on mitochondria is the impairment of mitochondria function, which might explain these findings. The decreased global brain mtDNA common deletion levels suggests that mitochondrial function is impaired and might be part of an overall mitochondria dysfunction signature in subjects with schizophrenia.

Lack of association to a NRG1 missense polymorphism in schizophrenia or bipolar disorder in a Costa Rican population.

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.

Coding SNPs included in exon arrays for the study of psychiatric disorders.

Psychiatric disorders are thought to be multifactorial complex traits with partial predisposition driven by genetic variation altering expression or protein function. While genetic variants have been linked to certain psychiatric conditions and gene expression approaches have identified expression alterations, few studies have combined genetic and expression alterations in neuropsychiatric conditions. Exon arrays have been proposed for the study of gene- and exon-level expression alterations, such as alternative splicing alterations. For this purpose, the Affymetrix Human Exon 1.0 ST array, containing over 5.5 million probes regrouped into 1.4 million probe sets, can be used to interrogate expression of the entire human transcriptome at the individual exon level. Here, we propose a method to use exon arrays also for the study of genetic variation in coding exons taking advantage of the presence of a large number of probes in regions of the transcriptome that contain single nucleotide polymorphisms (SNPs) (1.99 million SNPs in more than 5.5 million total probes). We explored patterns of gene expression in 10 male subjects from Costa Rica, five schizophrenia probands and five gender-matched relatives using lymphoblastoid cell line (LCL) RNA and the Affymetrix Human 1.0 ST Exon Arrays. Although many brain-specific genes are not expressed in LCLs (Vawter MP, unpublished result) and it is not possible to model in LCLs the complex processes and anatomical interactions taking place in a human brain, LCLs have several advantages compared to studying tissue such as brain. Viable lymphocytes readily obtained from large numbers of cases and controls can be experimentally manipulated compared to human brain samples from autopsies or biopsies. Drug effects and other confounds can be controlled in culture to reduce the direct effects on gene expression. Gene- and exon-level changes were observed and confirmed by quantitative-polymerase chain reaction (Q-PCR) between affected and unaffected controls. These expression changes often did not involve probes with coding SNPs. The entire microarray experiment was repeated from independently grown LCLs from the same subjects with similar expression profiles. Interestingly, high exon-level individual variation (110–32 000 fold) was observed on 8151 probe sets, suggestive of alternative splicing differences; however, at least one-half of these probes contained an SNP. Some subjects exhibited background-level expression for one exon within a gene, while other exons showed normal expression levels. Such variation might be due to alternative splicing of exons or perhaps hybridization effects due to coding SNPs. DPM2 (dolichyl-phosphate mannosyltransferase polypeptide 2, regulatory subunit) gene exon level analysis is presented here as an example (Figure 1a). The fourth exon of this gene presented a 100-fold change variation among subjects (subject effect P=10−11). A marked reduction in expression was also observed for the four probes (322624.1–322624.4) composing the probe set (data not shown). Two SNPs (rs7997 and rs6781) were found to be present in the DPM2 exon 4 probe set target sequence and their impact was further explored by allele-specific Q-PCR. Different sets of primers were designed to detect expression from the C (C-primer) and the G (G-primer) alleles of the rs7997 SNP leading to a Ser/Thr missense mutation. A third set of primers were designed to detect expression levels irrespective of the genotype for normalization purposes. Figure 1 The Affymetrix human exon array 1.0 ST contains probes that show differential hybridization pattern for coding SNPs. The DPM2 gene shows a pattern suggestive of alternative splicing across subjects and varies by ~100-fold at exon 4. A similar pattern ... A genotype-specific pattern of expression was observed in the Q-PCR experiment with the allele-specific primers for DPM2 exon 4 (Figure 1b). This indicated that the observed expression pattern was due to the presence of an SNP in the probe. When the proper allele-specific primers were used, normal expression levels were in fact observed for all subjects (Figure 1b). The absence of alternative splicing was also confirmed with another set of primers designed to detect expression in the DPM2 exon 4 irrespective of the genotype. Each subjects DNA was also resequenced to confirm the genotypes detected with allele-specific PCR of cDNA. We also confirmed that the same DPM2 SNP affected microarray hybridization in an independent sample of control subjects of northern European ancestry and validated the same effect by allele-specific Q-PCR. This experiment illustrates the power and specificity of exon arrays to detect not only exon-specific expression but also allele-specific expression within an exon (Figure 1) and underlines the utility and importance to characterize expression in a more comprehensive manner taking into account also genetic information. Although the Affymetrix exon array has been used for tissue-specific alternative splicing (http://www.affymetrix.com/support/technical/technotes/id_altsplicingevents_technote.pdf), we observed that multiple instances of alternative splicing might be more consistent with coding SNPs influencing probe hybridization in one tissue type. Further, since many researchers use algorithms to average individual probes into probe set values; this will inadvertently include probes with hybridization artifacts. There is currently no commercial high throughput platform to capture the effect of both genetics and gene expression conjointly, although results using both cDNA and nuclear DNA on separate platforms have been recently published.1 It has been suggested in the past that probe sets with SNPs can confound expression analysis using Affymetrix expression microarrays.2 We leveraged this phenomenon to study genetic variation of coding exons in psychiatric disorders. This could be particularly useful considering it has been demonstrated that gene expression is under genetic control and therefore, highly dependent on ethnic background.3 So far gene expression studies investigating neuropsychiatric disorders using postmortem tissue have in general not taken into consideration the influence of the ethnic background on gene expression variation. Two sources of gene expression variation could explain ethnic gene expression differences, that is, population specific due to presence of a relatively new SNP or stratification of a common SNP by population. Thus, gene expression studies using subjects with mixed genetic backgrounds can be affected by genetic variation that alters probe hybridization on microarray platforms. We caution that ethnic gene expression differences recently reported by Spielman et al.3 may be accounted for by ethnic specific probes causing differential hybridization to cDNA since ~40% of the Affymetrix probes contain SNPs. In conclusion, we propose using exon array probe-level analysis to obtain coding SNPs genotypic information by comparing probes that contain SNPs to probes that do not contain SNPs. Of the 1.99 million Affymetrix probes on the exon array that contain an SNP, it is estimated that the strongest disruption of hybridization will occur when the SNP is located within the middle 15 bp of probe sequence, which occurs 127 404 times on the exon array with a minor-allele frequency ⩾5%. This means that there are at least 127 404 common exonic SNPs with a maximal hybridization effect on the chip. This analysis can provide in one array genotype-specific expression in cDNA. The combination of expression and genotypic data from the same subjects using the same platform should allow a very efficient study of cis-regulatory variation.