Cis-acting Effects Research Articles

Abstract 619: Regulatory Mechanism of Lmod1 Mediated Predisposition to Coronary Artery Disease

Atherosclerotic coronary artery disease (CAD) continues to be the leading cause of mortality and morbidity worldwide, with an estimated 40% of one’s lifetime risk attributed to genetic factors. Meta-analysis of genome-wide association studies implemented to identify this risk in human populations, has now identified rs2820315 (P=7.7E-10; OR=1.05), in the smooth muscle cell-restricted gene, Leiomodin 1 (LMOD1), as the leading genetic polymorphism associated with CAD. However, the causal mechanism by which this polymorphism is responsible for predisposition to CAD remains to be identified. Expression quantitative trait loci (eQTL) mapping in GTEx and STARNET databases revealed that carriers of the risk allele, rs34091558, which is in tight linkage disequilibrium with rs2820315, display significantly attenuated LMOD1 expression in vascular tissues than carriers of the ancestral allele. Allelic expression imbalance analyses in heterozygous HCASMC donors further demonstrated cis-acting effects of the polymorphism on LMOD1 gene expression. To determine the mechanism responsible for this reduction in LMOD1 expression, we performed position weight matrix (PWM) motif analyses and found that rs34091558 disrupts the binding site of a transcription factor called forkhead box O3 (FOXO3). Subsequent chromatin immunoprecipitation and reporter assays demonstrated reduced FOXO3 binding and transcriptional activity by the risk allele in cultured HCASMCs. Platelet-derived growth factor BB (PDGF-BB) stimulation also significantly reduced LMOD1 expression coincident with FOXO3 knockdown. Finally, both gain and loss-of-function for FOXO3 and LMOD1 in HCASMC delineated a regulatory circuit by which LMOD1 regulates SMC proliferation, migration and contraction, characteristic features of atherosclerotic lesion progression. Taken together, these results provide compelling functional evidence that: 1) rs3091558 is associated with reduced LMOD1 expression, 2) this reduction appears to be mediated through the inhibition of FOXO3 binding and 3) changes in vessel wall processes through LMOD1 dysregulation may partially explain the heritable risk for CAD.

Deletion of a Long-Range Dlx5 Enhancer Disrupts Inner Ear Development in Mice.

Distal enhancers are thought to play important roles in the spatiotemporal regulation of gene expression during embryonic development, but few predicted enhancer elements have been shown to affect transcription of their endogenous genes or to alter phenotypes when disrupted. Here, we demonstrate that a 123.6-kb deletion within the mouse Slc25a13 gene is associated with reduced transcription of Dlx5, a gene located 660 kb away. Mice homozygous for the Slc25a13 deletion mutation [named hyperspin (hspn)] have malformed inner ears and are deaf with balance defects, whereas previously reported Slc25a13 knockout mice showed no phenotypic abnormalities. Inner ears of Slc25a13hspn/hspn mice have malformations similar to those of Dlx5-/- embryos, and Dlx5 expression is severely reduced in the otocyst but not the branchial arches of Slc25a13hspn/hspn embryos, indicating that the Slc25a13hspn deletion affects otic-specific enhancers of Dlx5 In addition, transheterozygous Slc25a13+/hspn Dlx5+/- mice exhibit noncomplementation with inner ear dysmorphologies similar to those of Slc25a13hspn/hspn and Dlx5-/-embryos, verifying a cis-acting effect of the Slc25a13hspn deletion on Dlx5 expression. CRISPR/Cas9-mediated deletions of putative enhancer elements located within the Slc25a13hspn deleted region failed to phenocopy the defects of Slc25a13hspn/hspn mice, suggesting the possibility of multiple enhancers with redundant functions. Our findings in mice suggest that analogous enhancer elements in the human SLC25A13 gene may regulate DLX5 expression and underlie the hearing loss that is associated with split-hand/-foot malformation 1 syndrome. Slc25a13hspn/hspn mice provide a new animal model for studying long-range enhancer effects on Dlx5 expression in the developing inner ear.

Characterization of cross-tissue genetic-epigenetic effects and their patterns in schizophrenia

BackgroundOne of the major challenges in current psychiatric epigenetic studies is the tissue specificity of epigenetic changes since access to brain samples is limited. Peripheral tissues have been studied as surrogates but the knowledge of cross-tissue genetic-epigenetic characteristics remains largely unknown. In this work, we conducted a comprehensive investigation of genetic influence on DNA methylation across brain and peripheral tissues with the aim to characterize cross-tissue genetic-epigenetic effects and their roles in the pathophysiology of psychiatric disorders.MethodsGenome-wide methylation quantitative trait loci (meQTLs) from brain prefrontal cortex, whole blood, and saliva were identified separately and compared. Focusing on cis-acting effects, we tested the enrichment of cross-tissue meQTLs among cross-tissue expression QTLs and genetic risk loci of various diseases, including major psychiatric disorders. CpGs targeted by cross-tissue meQTLs were also tested for genomic distribution and functional enrichment as well as their contribution to methylation correlation across tissues. Finally, a consensus co-methylation network analysis on the cross-tissue meQTL targeted CpGs was performed on data of the three tissues collected from schizophrenia patients and controls.ResultsWe found a significant overlap of cis meQTLs (45–73 %) and targeted CpG sites (31–68 %) among tissues. The majority of cross-tissue meQTLs showed consistent signs of cis-acting effects across tissues. They were significantly enriched in genetic risk loci of various diseases, especially schizophrenia, and also enriched in cross-tissue expression QTLs. Compared to CpG sites not targeted by any meQTLs, cross-tissue targeted CpGs were more distributed in CpG island shores and enhancer regions, and more likely had strong correlation with methylation levels across tissues. The targeted CpGs were also annotated to genes enriched in multiple psychiatric disorders and neurodevelopment-related pathways. Finally, we identified one co-methylation network shared between brain and blood showing significant schizophrenia association (p = 5.5 × 10−6).ConclusionsOur results demonstrate prevalent cross-tissue meQTL effects and their contribution to the correlation of CpG methylation across tissues, while at the same time a large portion of meQTLs show tissue-specific characteristics, especially in brain. Significant enrichment of cross-tissue meQTLs in expression QTLs and genetic risk loci of schizophrenia suggests the potential of these cross-tissue meQTLs for studying the genetic effect on schizophrenia. The study provides compelling motivation for a well-designed experiment to further validate the use of surrogate tissues in the study of psychiatric disorders.

Abstract 21021: Functional Regulatory Mechanism of Smooth Muscle Cell-Restricted LMOD1 Coronary Artery Disease Locus

Coronary artery disease (CAD) remains the leading cause of mortality and morbidity worldwide and has an estimated heritability of 40%. Meta-analyses of genome-wide association studies have now identified 73 genome-wide significant loci and the most recent multi-ethnic meta-analysis identified a number of arterial wall-specific loci. One of these loci include the smooth muscle cell-restricted factor, LMOD1 (Leiomodin 1), a member of the actin filament nucleator family that is highly enriched in smooth muscle tissues including main arteries and visceral organs of the gastrointestinal system. We hypothesized that LMOD1 may serve as a potent marker of the phenotypic modulation of smooth muscle cells during atherosclerosis. We recently identified a non-coding regulatory variant, rs34091558, which is in linkage disequilibrium with the lead CAD GWAS variant, rs2820315 (P=7.7E-10; OR=1.05). However, the causal mechanism for how these variants alter LMOD1 expression/function and CAD risk remains unclear. Expression quantitative trait loci (eQTL) mapping in GTEx and STARNET databases confirmed that rs34091558 is among the top eQTLs for LMOD1 in vascular tissues. Allelic expression imbalance analyses in heterozygous HCASMC donors further demonstrated cis-acting effects on LMOD1 gene expression. Position weight matrix (PWM) motif analyses identified the protective allele at rs34091558 to form a consensus forkhead box O3 (FOXO3) binding motif, which is predicted to be disrupted by the risk allele. FOXO3 chromatin immunoprecipitation and reporter assays demonstrated reduced FOXO3 binding and transcriptional activity by the risk allele in HCASMC. Platelet-derived growth factor BB (PDGF-BB) stimulation also significantly reduced LMOD1 expression coincident with FOXO3 knockdown. Finally, both gain and loss-of-function for FOXO3 and LMOD1 in HCASMC delineated a regulatory circuit by which LMOD1 decreased HCASMC proliferation and migration and increased cell contraction. Taken together, these results provide compelling functional evidence that: 1) common genetic variation is associated with dysregulation of LMOD1 expression, and 2) changes in vessel wall processes through LMOD1 dysregulation may partially explain the heritable risk for CAD.

Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1

Although lysyl oxidase-like 1 (LOXL1) is known as the principal genetic risk factor for pseudoexfoliation (PEX) syndrome, a major cause of glaucoma and cardiovascular complications, no functional variants have been identified to date. Here, we conduct a genome-wide association scan on 771 German PEX patients and 1,350 controls, followed by independent testing of associated variants in Italian and Japanese data sets. We focus on a 3.5-kb four-component polymorphic locus positioned spanning introns 1 and 2 of LOXL1 with enhancer-like chromatin features. We find that the rs11638944:C>G transversion exerts a cis-acting effect on the expression levels of LOXL1, mediated by differential binding of the transcription factor RXRα (retinoid X receptor alpha) and by modulating alternative splicing of LOXL1, eventually leading to reduced levels of LOXL1 mRNA in cells and tissues of risk allele carriers. These findings uncover a functional mechanism by which common noncoding variants influence LOXL1 expression.

Mendelian inheritance of trimodal CpG methylation sites suggests distal cis-acting genetic effects.

BackgroundEnvironmentally influenced phenotypes, such as obesity and insulin resistance, can be transmitted over multiple generations. Epigenetic modifications, such as methylation of DNA cytosine-guanine (CpG) pairs, may be carriers of inherited information. At the population level, the methylation state of such “heritable” CpG sites is expected to follow a trimodal distribution, and their mode of inheritance should be Mendelian.MethodsUsing the Illumina Infinium 450 K DNA methylation array, we determined DNA CpG-methylation in blood cells from a family cohort 123 individuals of Arab ethnicity, including 18 elementary father-mother-child trios, we asked whether Mendelian inheritance of CpG methylation is observed, and most importantly, whether it is independent of any genetic signals. Using 40× whole genome sequencing, we therefore excluded all CpG sites with possibly confounding genetic variants (SNP) within the binding regions of the Illumina probes.ResultsWe identified a total of 955 CpG sites that displayed a trimodal distribution and confirmed trimodality in a study of 1805 unrelated Caucasians. Of 955 CpG sites, 99.9% observed a strict Mendelian pattern of inheritance and had no SNP within +/−110 nucleotides of the CpG site by design. However, in 97% of these cases a distal cis-acting SNP within a +/−1 Mbp window was found that explained the observed CpG distribution, excluding the hypothesis of epigenetic inheritance for these clear-cut trimodal sites. Using power analysis, we showed that in 46% of all cases, the closest CpG-associated SNP was located more than 1000 bp from the CpG site.ConclusionsOur findings suggest that CpG methylation is maintained over larger genomic distances. Furthermore, nearly half of the SNPs associated with these trimodal sites were also associated with the expression of nearby genes (P = 4.08 × 10−6), implying a regulatory effect of these trimodal CpG sites.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0295-1) contains supplementary material, which is available to authorized users.

The influence of FKBP5 genotype on expression of FKBP5 and other glucocorticoid-regulated genes, dependent on trauma exposure.

The FK506 binding protein 51 (FKBP5), an intrinsic regulator of the glucocorticoid receptor, has been associated with pathological behaviors particularly in the context of childhood trauma (CT), via a putatively regulatory polymorphism, rs1360780. However, trans- and cis-acting effects of this locus and its interaction with CT are incompletely understood. To study its effects on the expression of glucocorticoid-regulated genes including FKBP5, we used lymphoblastoid cell lines (LCLs) derived from 16 CT-exposed patients with greater than two substance dependence/suicidal behavior diagnoses (casesCT+) and 13 non-CT-exposed controls (controlsCT-). This study in LCLs measures long-term trait-like differences attributable to genotype or lasting epigenetic modification. Through analysis of differential allelic expression (DAE) using an FKBP5 3'-UTR reporter single nucleotide polymorphism (SNP), rs3800373, that is in strong linkage disequilibrium with rs1360780, we confirmed that the rs1360780 risk allele (A) (or conceivably that of a linked SNP) leads to higher FKBP5 expression in controlsCT-. Intriguingly, casesCT+ did not show DAE, perhaps because of a genotype-predicted difference in FKBP5 DNA methylation restricted to casesCT+. Furthermore, through correlation analyses on FKBP5 expression at baseline and after induction by dexamethasone, we observed that casesCT+ had lower induction of FKBP5 expression, indicating that overall they may have strong ultra-short negative-feedback. Only casesCT+ showed an effect of rs1360780 genotype on expression of FKBP5 and other glucocorticoid-regulated genes. Together, these results confirm that the rs1360780 locus alters FKBP5 expression and further that in trans-fashion this locus affects the expression of other glucocorticoid-regulated genes after a glucocorticoid challenge. The CT exposure appears to be essential for trans-effects of rs1360780 on glucocorticoid-regulated genes.

Expression-based GWAS identifies variants, gene interactions and key regulators affecting intramuscular fatty acid content and composition in porcine meat.

The aim of this work is to better understand the genetic mechanisms determining two complex traits affecting porcine meat quality: intramuscular fat (IMF) content and its fatty acid (FA) composition. With this purpose, expression Genome-Wide Association Study (eGWAS) of 45 lipid-related genes associated with meat quality traits in swine muscle (Longissimus dorsi) of 114 Iberian × Landrace backcross animals was performed. The eGWAS identified 241 SNPs associated with 11 genes: ACSM5, CROT, FABP3, FOS, HIF1AN, IGF2, MGLL, NCOA1, PIK3R1, PLA2G12A and PPARA. Three expression Quantitative Trait Loci (eQTLs) for IGF2, ACSM5 and MGLL were identified, showing cis-acting effects, whereas 16 eQTLs had trans regulatory effects. A polymorphism in the ACSM5 promoter region associated with its expression was identified. In addition, strong candidate genes regulating ACSM5, FOS, PPARA, PIK3R1, PLA2G12A and HIF1AN gene expression were also seen. Notably, the analysis highlighted the NR3C1 transcription factor as a strong candidate gene involved in the regulation of the 45 genes analysed. Finally, the IGF2, MGLL, MC2R, ARHGAP6, and NR3C1 genes were identified as potential regulators co-localizing within QTLs for fatness and growth traits in the IBMAP population. The results obtained increase our knowledge in the functional regulatory mechanisms involved in these complex traits.

Parkinson's disease: Guilt by genetic association.

Certain sequence variants of the α-synuclein gene are linked to the risk of Parkinson's disease. An analysis of these variants using gene-editing technology provides a possible explanation for this increased risk. See Letter p.95 Determining how genetic risk variants associated with complex diseases actually contribute to the pathological features of the disease remains a challenge. Non-coding sequences variants have been proposed to act in cis to modulate the expression of disease-linked genes, but it is difficult to test this hypothesis because of the complexity of the diseases linked to these variants. Rudolf Jaenisch and colleagues have developed a genetically controlled system to dissect out the cis-acting effect of allelic variants on the expression of the α-synuclein gene SCNA, which has been linked to Parkinson's disease development.

Parkinson-associated risk variant in distal enhancer of α-synuclein modulates target gene expression.

Genome-wide association studies (GWAS) have identified numerous genetic variants associated with complex diseases but mechanistic insights are impeded by the lack of understanding of how specific risk variants functionally contribute to the underlying pathogenesis1. It has been proposed that cis-acting effects of non-coding risk variants on gene expression are a major factor for phenotypic variation of complex traits and disease susceptibility. Recent genome-scale chromatin mapping studies have highlighted the enrichment of GWAS variants in regulatory DNA elements of disease-relevant cell types2–6. Furthermore, single nucleotide polymorphism (SNP)-specific changes in transcription factor (TF) binding are correlated with heritable alterations in chromatin state and considered a major mediator of sequence-dependent regulation of gene expression7–10. Here we describe a novel strategy to functionally dissect the cis-acting effect of genetic risk variants in regulatory elements on gene expression by combining genome-wide epigenetic information with clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in human pluripotent stem cells (hPSCs). By generating a genetically precisely controlled experimental system we identify a common Parkinson’s disease (PD)-associated risk variant in a non-coding distal enhancer element that regulates the expression of alpha-synuclein (SNCA), a key gene implicated in the pathogenesis of PD. Our data suggest that the transcriptional deregulation of SNCA is associated with sequence-dependent binding of the brain-specific TFs EMX2 and NKX6-1. This work establishes an experimental paradigm to functionally connect genetic variation with disease relevant phenotypes.

Genetic variants near MLST8 and DHX57 affect the epigenetic age of the cerebellum

DNA methylation (DNAm) levels lend themselves for defining an epigenetic biomarker of aging known as the ‘epigenetic clock'. Our genome-wide association study (GWAS) of cerebellar epigenetic age acceleration identifies five significant (P<5.0 × 10−8) SNPs in two loci: 2p22.1 (inside gene DHX57) and 16p13.3 near gene MLST8 (a subunit of mTOR complex 1 and 2). We find that the SNP in 16p13.3 has a cis-acting effect on the expression levels of MLST8 (P=6.9 × 10−18) in most brain regions. In cerebellar samples, the SNP in 2p22.1 has a cis-effect on DHX57 (P=4.4 × 10−5). Gene sets found by our GWAS analysis of cerebellar age acceleration exhibit significant overlap with those of Alzheimer's disease (P=4.4 × 10−15), age-related macular degeneration (P=6.4 × 10−6), and Parkinson's disease (P=2.6 × 10−4). Overall, our results demonstrate the utility of a new paradigm for understanding aging and age-related diseases: it will be fruitful to use epigenetic tissue age as endophenotype in GWAS.

Putative effectors for prognosis in lung adenocarcinoma are ethnic and gender specific.

Lung adenocarcinoma possesses distinct patterns of EGFR/KRAS mutations between East Asian and Western, male and female patients. However, beyond the well-known EGFR/KRAS distinction, gender and ethnic specific molecular aberrations and their effects on prognosis remain largely unexplored. Association modules capture the dependency of an effector molecular aberration and target gene expressions. We established association modules from the copy number variation (CNV), DNA methylation and mRNA expression data of a Taiwanese female cohort. The inferred modules were validated in four external datasets of East Asian and Caucasian patients by examining the coherence of the target gene expressions and their associations with prognostic outcomes. Modules 1 (cis-acting effects with chromosome 7 CNV) and 3 (DNA methylations of UBIAD1 and VAV1) possessed significantly negative associations with survival times among two East Asian patient cohorts. Module 2 (cis-acting effects with chromosome 18 CNV) possessed significantly negative associations with survival times among the East Asian female subpopulation alone. By examining the genomic locations and functions of the target genes, we identified several putative effectors of the two cis-acting CNV modules: RAC1, EGFR, CDK5 and RALBP1. Furthermore, module 3 targets were enriched with genes involved in cell proliferation and division and hence were consistent with the negative associations with survival times. We demonstrated that association modules in lung adenocarcinoma with significant links of prognostic outcomes were ethnic and/or gender specific. This discovery has profound implications in diagnosis and treatment of lung adenocarcinoma and echoes the fundamental principles of the personalized medicine paradigm.

Cis regulatory effects on A-to-I RNA editing in related Drosophila species.

Adenosine-to-inosine RNA editing modifies maturing mRNAs through the binding of adenosine deaminase acting on RNA (Adar) proteins to double-stranded RNA structures in a process critical for neuronal function. Editing levels at individual editing sites span a broad range and are mediated by both cis-acting elements (surrounding RNA sequence and secondary structure) and trans-acting factors. Here, we aim to determine the roles that cis-acting elements and trans-acting factors play in regulating editing levels. Using two closely related Drosophila species, D. melanogaster and D. sechellia, and their F1 hybrids, we dissect the effects of cis sequences from trans regulators on editing levels by comparing species-specific editing in parents and their hybrids. We report that cis sequence differences are largely responsible for editing level differences between these two Drosophila species. This study presents evidence for cis sequence and structure changes as the dominant evolutionary force that modulates RNA editing levels between these Drosophila species.

DNA methylation abnormalities in congenital heart disease

Congenital heart defects represent the most common malformation at birth, occurring also in ∼50% of individuals with Down syndrome. Congenital heart defects are thought to have multifactorial etiology, but the main causes are largely unknown. We have explored the global methylation profile of fetal heart DNA in comparison to blood DNA from control subjects: an absolute correlation with the type of tissue was detected. Pathway analysis revealed a significant enrichment of differential methylation at genes related to muscle contraction and cardiomyopathies in the developing heart DNA. We have also searched for abnormal methylation profiles on developing heart-tissue DNA of syndromic and non-syndromic congenital heart defects. On average, 3 regions with aberrant methylation were detected per sample and 18 regions were found differentially methylated between groups. Several epimutations were detected in candidate genes involved in growth regulation, apoptosis and folate pathway. A likely pathogenic hypermethylation of several intragenic sites at the MSX1 gene, involved in outflow tract morphogenesis, was found in a fetus with isolated heart malformation. In addition, hypermethylation of the GATA4 gene was present in fetuses with Down syndrome with or without congenital heart defects, as well as in fetuses with isolated heart malformations. Expression deregulation of the abnormally methylated genes was detected. Our data indicate that epigenetic alterations of relevant genes are present in developing heart DNA in fetuses with both isolated and syndromic heart malformations. These epimutations likely contribute to the pathogenesis of the malformation by cis-acting effects on gene expression.

Extensive cis-regulatory variation robust to environmental perturbation in Arabidopsis.

cis- and trans-acting factors affect gene expression and responses to environmental conditions. However, for most plant systems, we lack a comprehensive map of these factors and their interaction with environmental variation. Here, we examined allele-specific expression (ASE) in an F1 hybrid to study how alleles from two Arabidopsis thaliana accessions affect gene expression. To investigate the effect of the environment, we used drought stress and developed a variance component model to estimate the combined genetic contributions of cis- and trans-regulatory polymorphisms, environmental factors, and their interactions. We quantified ASE for 11,003 genes, identifying 3318 genes with consistent ASE in control and stress conditions, demonstrating that cis-acting genetic effects are essentially robust to changes in the environment. Moreover, we found 1618 genes with genotype x environment (GxE) interactions, mostly cis x E interactions with magnitude changes in ASE. We found fewer trans x E interactions, but these effects were relatively less robust across conditions, showing more changes in the direction of the effect between environments; this confirms that trans-regulation plays an important role in the response to environmental conditions. Our data provide a detailed map of cis- and trans-regulation and GxE interactions in A. thaliana, laying the ground for mechanistic investigations and studies in other plants and environments.

Genetic influences on translation in yeast.

Heritable differences in gene expression between individuals are an important source of phenotypic variation. The question of how closely the effects of genetic variation on protein levels mirror those on mRNA levels remains open. Here, we addressed this question by using ribosome profiling to examine how genetic differences between two strains of the yeast S. cerevisiae affect translation. Strain differences in translation were observed for hundreds of genes. Allele specific measurements in the diploid hybrid between the two strains revealed roughly half as many cis-acting effects on translation as were observed for mRNA levels. In both the parents and the hybrid, most effects on translation were of small magnitude, such that the direction of an mRNA difference was typically reflected in a concordant footprint difference. The relative importance of cis and trans acting variation on footprint levels was similar to that for mRNA levels. There was a tendency for translation to cause larger footprint differences than expected given the respective mRNA differences. This is in contrast to translational differences between yeast species that have been reported to more often oppose than reinforce mRNA differences. Finally, we catalogued instances of premature translation termination in the two yeast strains and also found several instances where erroneous reference gene annotations lead to apparent nonsense mutations that in fact reside outside of the translated gene body. Overall, genetic influences on translation subtly modulate gene expression differences, and translation does not create strong discrepancies between genetic influences on mRNA and protein levels.

High-resolution transcriptome analysis with long-read RNA sequencing.

RNA sequencing (RNA-seq) enables characterization and quantification of individual transcriptomes as well as detection of patterns of allelic expression and alternative splicing. Current RNA-seq protocols depend on high-throughput short-read sequencing of cDNA. However, as ongoing advances are rapidly yielding increasing read lengths, a technical hurdle remains in identifying the degree to which differences in read length influence various transcriptome analyses. In this study, we generated two paired-end RNA-seq datasets of differing read lengths (2×75 bp and 2×262 bp) for lymphoblastoid cell line GM12878 and compared the effect of read length on transcriptome analyses, including read-mapping performance, gene and transcript quantification, and detection of allele-specific expression (ASE) and allele-specific alternative splicing (ASAS) patterns. Our results indicate that, while the current long-read protocol is considerably more expensive than short-read sequencing, there are important benefits that can only be achieved with longer read length, including lower mapping bias and reduced ambiguity in assigning reads to genomic elements, such as mRNA transcript. We show that these benefits ultimately lead to improved detection of cis-acting regulatory and splicing variation effects within individuals.

A functional polymorphism affecting the APOA5 gene expression is causally associated with plasma triglyceride levels conferring coronary atherosclerosis risk in Han Chinese Population

Apolipoprotein A5 (APOA5) gene plays a key role in plasma triglyceride (TG) metabolism, and shows the involvement in coronary artery disease (CAD). A set of single nucleotide polymorphisms around the APOA5 gene was identified to be associated with plasma TG levels. It is of biological and clinical importance to discern the genuine genetic determinants. A polymorphism in 3′ untranslated region of the APOA5 gene, rs2266788, is deserving of investigation for suggestive clues from the association in multiple independent studies. In this study, rs2266788 was genotyped in 3222 unrelated subjects consisting of 2062 CAD cases and 1160 controls. The statistical analyses indicated that the minor C allele of rs2266788 was significantly associated with elevated plasma TG levels and higher CAD risk. In normal human liver tissues, comparison of global APOA5 mRNA levels among genotypes and allelic expression imbalance analysis showed the decreased gene expression for the C allele. Luciferase assays confirmed a concordant result that transcriptional activity was lowered for the C allele compared with the T allele in four cell lines. Multiple lines of evidence in our study supported that rs2266788 was causally associated with plasma TG levels conferring CAD risk in Han Chinese population owing to a cis-acting effect to the APOA5 gene expression.

Common genetic variants on 1p13.2 associate with risk of autism

Autism is a highly heritable neurodevelopmental disorder, and known genetic variants, mostly rare, account only for a small proportion of cases. Here we report a genome-wide association study on autism using two Chinese cohorts as gene discovery (n=2150) and three data sets of European ancestry populations for replication analysis of top association signals. Meta-analysis identified three single-nucleotide polymorphisms, rs936938 (P=4.49 × 10(-8)), non-synonymous rs6537835 (P=3.26 × 10(-8)) and rs1877455 (P=8.70 × 10(-8)), and related haplotypes, AMPD1-NRAS-CSDE1, TRIM33 and TRIM33-BCAS2, associated with autism; all were mapped to a previously reported linkage region (1p13.2) with autism. These genetic associations were further supported by a cis-acting regulatory effect on the gene expressions of CSDE1, NRAS and TRIM33 and by differential expression of CSDE1 and TRIM33 in the human prefrontal cortex of post-mortem brains between subjects with and those without autism. Our study suggests TRIM33 and NRAS-CSDE1 as candidate genes for autism, and may provide a novel insight into the etiology of autism.

DominantPRPF31Mutations Are Hypostatic to a RecessiveCNOT3Polymorphism in Retinitis Pigmentosa: A Novel Phenomenon of “LinkedTrans-Acting Epistasis”

SummaryMutations in PRPF31 are responsible for autosomal dominant retinitis pigmentosa (adRP, RP11 form) and affected families show nonpenetrance. Differential expression of the wildtype PRPF31 allele is responsible for this phenomenon: coinheritance of a mutation and a higher expressing wildtype allele provide protection against development of disease.It has been suggested that a major modulating factor lies in close proximity to the wildtype PRPF31 gene on Chromosome 19, implying that a cis-acting factor directly alters PRPF31 expression. Variable expression of CNOT3 is one determinant of PRPF31 expression. This study explored the relationship between CNOT3 (a trans-acting factor) and its paradoxical cis-acting nature in relation to RP11.Linkage analysis on Chromosome 19 was performed in mutation-carrying families, and the inheritance of the wildtype PRPF31 allele in symptomatic–asymptomatic sibships was assessed—confirming that differential inheritance of wildtype chromosome 19q13 determines the clinical phenotype (P < 2.6 × 10−7).A theoretical model was constructed that explains the apparent conflict between the linkage data and the recent demonstration that a trans-acting factor (CNOT3) is a major nonpenetrance factor: we propose that this apparently cis-acting effect arises due to the intimate linkage of CNOT3 and PRPF31 on Chromosome 19q13—a novel mechanism that we have termed “linked trans-acting epistasis.”