Abstract MicroRNAs (miRNAs) are a large family of small non-coding RNAs which negatively control gene expression at both the mRNA and protein levels. The number of miRNAs identified is growing ...rapidly and approximately one-third is expressed in the brain where they have been shown to affect neuronal differentiation, synaptosomal complex localization and synapse plasticity, all functions thought to be disrupted in schizophrenia. Here we investigated the expression of 667 miRNAs (miRBase v.13) in the prefrontal cortex of individuals with schizophrenia (SZ, N = 35) and bipolar disorder (BP, N = 35) using a real-time PCR-based Taqman Low Density Array (TLDA). After extensive QC steps, 441 miRNAs were included in the final analyses. At a FDR of 10%, 22 miRNAs were identified as being differentially expressed between cases and controls, 7 dysregulated in SZ and 15 in BP. Using in silico target gene prediction programs, the 22miRNAs were found to target brain specific genes contained within networks overrepresented for neurodevelopment, behavior, and SZ and BP disease development. In an initial attempt to corroborate some of these predictions, we investigated the extent of correlation between the expressions of hsa-mir-34a, -132 and -212 and their predicted gene targets. mRNA expression of tyrosine hydroxylase (TH), phosphogluconate dehydrogenase (PGD) and metabotropic glutamate receptor 3 (GRM3) was measured in the SMRI sample. Hsa-miR-132 and -212 were negatively correlated with TH (p = 0.0001 and 0.0017) and with PGD (p = 0.0054 and 0.017, respectively).
Deep sequencing has become a popular tool for novel miRNA detection but its data must be viewed carefully as the state of the field is still undeveloped. Using three different programs, miRDeep (v1, ...2), miRanalyzer and DSAP, we have analyzed seven data sets (six biological and one simulated) to provide a critical evaluation of the programs performance. We selected these software based on their popularity and overall approach toward the detection of novel and known miRNAs using deep-sequencing data. The program comparisons suggest that, despite differing stringency levels they all identify a similar set of known and novel predictions. Comparisons between the first and second version of miRDeep suggest that the stringency level of each of these programs may, in fact, be a result of the algorithm used to map the reads to the target. Different stringency levels are likely to affect the number of possible novel candidates for functional verification, causing undue strain on resources and time. With that in mind, we propose that an intersection across multiple programs be taken, especially if considering novel candidates that will be targeted for additional analysis. Using this approach, we identify and performed initial validation of 12 novel predictions in our in-house data with real-time PCR, six of which have been previously unreported.
Highlights • We examine the relationship between variation in CRHR1 and post-hurricane PTSD. • We found that rs12938031 and rs4792887 remained associated with PTSD symptoms. • This is the first study ...examining the association between CRHR1 and PTSD in adults. • Results justify additional research to characterize how CRHR1 relates to PTSD. • Identifying genetic variants is valuable for resource allocation post-trauma.
•Endophenotypic measures of panic/anxiety may aid in gene identification efforts.•Carbon dioxide hypersensitivity is a reliable panic/anxiety endophenotype.•This study tested associations between 11 ...genes and CO2 hypersensitivity.•rs1108923 in the ASIC1 gene was associated with baseline respiratory rate.•No other candidate genes were related to physiological or subjective CO2 response.
Few replicable genetic variants have been identified in the etiology of heritable anxiety disorders such as panic disorder. Endophenotypic measures that have reduced heterogeneity may provide more powerful targets for gene identification. We assessed hypersensitivity to carbon dioxide (a reliable endophenotype of panic and anxiety) in 174 Caucasian college students, who were genotyped on 26 polymorphic markers from 11 genes previously associated with panic/anxiety. Individual trajectories of respiratory and subjective anxiety response to carbon dioxide were measured and tested for association with these genetic markers. One marker in the acid-sensing ion channel 1 (ASIC1) gene, rs1108923, had a significant association with respiratory rate. No genes had a significant association with subjective anxiety response. Our findings support previously reported associations between ASIC1 and panic/anxiety, but not other genes previously associated with anxiety disorders. The use of endophenotypic markers is a promising avenue for gene identification in anxiety and other complex disorders.
A recent genome-wide association study reported association between schizophrenia and the ZNF804A gene on chromosome 2q32.1. We attempted to replicate these findings in our Irish Case-Control Study ...of Schizophrenia (ICCSS) sample (N=1021 cases, 626 controls). Following consultation with the original investigators, we genotyped three of the most promising single-nucleotide polymorphisms (SNPs) from the Cardiff study. We replicate association with rs1344706 (trend test one-tailed P=0.0113 with the previously associated A allele) in ZNF804A. We detect no evidence of association with rs6490121 in NOS1 (one-tailed P=0.21), and only a trend with rs9922369 in RGRIP1L (one-tailed P=0.0515). On the basis of these results, we completed genotyping of 11 additional linkage disequilibrium-tagging SNPs in ZNF804A. Of 12 SNPs genotyped, 11 pass quality control criteria and 4 are nominally associated, with our most significant evidence of association at rs7597593 (P=0.0013) followed by rs1344706. We observe no evidence of differential association in ZNF804A on the basis of family history or sex of case. The associated SNP rs1344706 lies in approximately 30 bp of conserved mammalian sequence, and the associated A allele is predicted to maintain binding sites for the brain-expressed transcription factors MYT1l and POU3F1/OCT-6. In controls, expression is significantly increased from the A allele of rs1344706 compared with the C allele. Expression is increased in schizophrenic cases compared with controls, but this difference does not achieve statistical significance. This study replicates the original reported association of ZNF804A with schizophrenia and suggests that there is a consistent link between the A allele of rs1344706, increased expression of ZNF804A and risk for schizophrenia.
Background
Alcohol dependence (AD) shows evidence for genetic liability, but genes influencing risk remain largely unidentified.
Methods
We conducted a genomewide association study in 706 related AD ...cases and 1,748 unscreened population controls from Ireland. We sought replication in 15,496 samples of European descent. We used model organisms (MOs) to assess the role of orthologous genes in ethanol (EtOH)‐response behaviors. We tested 1 primate‐specific gene for expression differences in case/control postmortem brain tissue.
Results
We detected significant association in COL6A3 and suggestive association in 2 previously implicated loci, KLF12 and RYR3. None of these signals are significant in replication. A suggestive signal in the long noncoding RNA LOC339975 is significant in case:control meta‐analysis, but not in a population sample. Knockdown of a COL6A3 ortholog in Caenorhabditis elegans reduced EtOH sensitivity. Col6a3 expression correlated with handling‐induced convulsions in mice. Loss of function of the KLF12 ortholog in C. elegans impaired development of acute functional tolerance (AFT). Klf12 expression correlated with locomotor activation following EtOH injection in mice. Loss of function of the RYR3 ortholog reduced EtOH sensitivity in C. elegans and rapid tolerance in Drosophila. The ryanodine receptor antagonist dantrolene reduced motivation to self‐administer EtOH in rats. Expression of LOC339975 does not differ between cases and controls but is reduced in carriers of the associated rs11726136 allele in nucleus accumbens (NAc).
Conclusions
We detect association between AD and COL6A3, KLF12, RYR3, and LOC339975. Despite nonreplication of COL6A3, KLF12, and RYR3 signals, orthologs of these genes influence behavioral response to EtOH in MOs, suggesting potential involvement in human EtOH response and AD liability. The associated LOC339975 allele may influence gene expression in human NAc. Although the functions of long noncoding RNAs are poorly understood, there is mounting evidence implicating these genes in multiple brain functions and disorders.
A GWAS of alcohol dependence identified signals in the brain‐expressed extracellular matrix protein collagen 6A3 (COL6A3) gene and the previously implicated Krueppel‐like factor 12 (KLF12) and ryanodine receptor 3 (RYR3) genes. Expression of COL6A3 in mouse brain is strongly correlated with handling‐induced convulsions (top inset). Knockout of the C. elegans KLF12 ortholog, Klf‐3, blocks development of acute functional tolerance (middle inset). Transheterozygous or homozygous transposon insertion in the Drosophila RYR3 ortholog, Ryr, significantly reduces the development of rapid tolerance.
Studies of alcohol dependence (AD) have consistently found evidence of linkage on chromosome 4q21–q32. A genome-wide linkage scan in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) ...sample also provided its strongest evidence of linkage on chromosome 4q22–q32 using an index of AD severity based on the count of DSM-IV AD symptoms (ADSX; LOD = 4.59). We conducted a systematic, gene-centric association study using 518 LD-tagging single nucleotide polymorphisms (SNPs) in the 65 known and predicted genes within the 1-LOD interval surrounding the linkage peak. Case-only regression analysis with the quantitative variable of ADSX was performed in the 562 genetically independent cases; nominal support for association was demonstrated by 32 tagging SNPs in 14 genes. We did not observe study-wide significance, but gene-wise correction for multiple testing with the Nyholt procedure yielded empirical evidence of association with two genes, DKK2 (dickkopf homolog 2) (P = 0.007) and EGF (epidermal growth factor) (P = 0.025) in the IASPSAD sample. Three SNPs in DKK2 (rs427983; rs419558; rs399087) demonstrated empirical significance. Assessment of possible replication in 847 cases of European descent from a large independent sample, the Collaborative Study of the Genetics of Alcoholism, yielded replication for DKK2 but not EGF. We observed genotypic and phenotypic replication for DKK2 with the three SNPs yielding significant association with ADSX in the IASPSAD sample. Haplotype-specific expression measurements in post-mortem tissue samples suggested a functional role for DKK2. This evidence notwithstanding, replication is needed before confidence can be placed in these findings.
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