Disease-associated DNA Variants Research Articles

Sequence-to-expression approach to identify etiological non-coding DNA variations in P53 and cMYC-driven diseases.

Disease risk prediction based on genomic sequence and transcriptional profile can improve disease screening and prevention. Despite identifying many disease-associated DNA variants, distinguishing deleterious non-coding DNA variations remains poor for most common diseases. In this study, we designed in vitro experiments to uncover the significance of occupancy and competitive binding between P53 and cMYC on common target genes. Analyzing publicly available ChIP-seq data for P53 and cMYC in embryonic stem cells showed that ~344-366 regions are co-occupied, and on average, two cis-overlapping motifs (CisOMs) per region were identified, suggesting that co-occupancy is evolutionarily conserved. Using U2OS and Raji cells untreated and treated with doxorubicin to increase P53 protein level while potentially reducing cMYC level, ChIP-seq analysis illustrated that around 16 to 922 genomic regions were co-occupied by P53 and cMYC, and substitutions of cMYC signals by P53 were detected post doxorubicin treatment. Around 187 expressed genes near co-occupied regions were altered at mRNA level according to RNA-seq data analysis. We utilized a computational motif-matching approach to illustrate that changes in predicted P53 binding affinity in CisOMs of co-occupied elements significantly correlate with alterations in reporter gene expression. We performed a similar analysis using SNPs mapped in CisOMs for P53 and cMYC from ChIP-seq data, and expression of target genes from GTEx portal. We found significant correlation between change in cMYC-motif binding affinity in CisOMs and altered expression. Our study brings us closer to developing a generally applicable approach to filter etiological non-coding variations associated with common diseases.

Complexities of Understanding Function from CKD-Associated DNA Variants.

Genome-wide association studies (GWASs) have facilitated the unbiased discovery of hundreds of genomic loci associated with CKD and kidney function. The vast majority of disease-associated DNA variants are noncoding. Those that are causal in CKD pathogenesis likely modulate transcription of target genes in a cell type-specific manner. To gain novel biological insights into mechanisms driving the development of CKD, the causal variants (which are usually not the most significant variant reported in a GWAS), their target genes, and causal cell types need to be identified. This functional validation requires a large number of new data sets, complex bioinformatics analyses, and experimental cellular and in vivo studies. Here, we review the basic principles and some of the current approaches being leveraged to assign functional significance to a genotype-phenotype association.

O11 Lymphocyte DNA methylation mediates genetic risk at RA risk loci that are shared with other immune mediated diseases

Abstract Background The aetiology of rheumatoid arthritis (RA) is complex. In particular, the vast majority of disease-associated variants implicated by genome-wide association studies are non-coding, leaving genetic mechanisms of adaptive immune dysregulation unresolved. The contribution to this process of epigenetic factors, including the addition of methyl groups to DNA, also remains uncertain. To address these issues and prioritise causal genes for downstream study, genome-wide data incorporating DNA methylation and gene expression measurements from lymphocyte subsets in an early arthritis inception cohort, were available. Methods Whole genome methylation and transcription data from isolated CD4+ T cells and B cells of > 100 well-characterised inflammatory arthritis patients, all of whom were naïve to immunomodulatory treatments and of Northern European ancestry, were obtained (Illumina HumanHT-12 v4 Expression BeadChip and Infinium MethylationEPIC BeadChip arrays, respectively). Genotyping was undertaken using the Illumina Human CoreExome-24 version 1-0 array. After independent pre-processing, normalisation and quality control of paired CD4+ and B lymphocyte data, methylation quantitative trait loci (meQTLs) were first modelled using the MatrixEQTL package in each cell type. Next, at RA risk-associated cis-CpGs, correlations between site-specific methylation and the expression of genes within ±500Kb identified quantitative trait methylations (eQTMs). To infer directionality of SNP-CpG-transcript associations a causal inference test (CIT) was applied. Multiple testing was accounted for, and in vitro assays were used to validate meQTLs at loci of interest and confirm regulatory mechanisms. Further analysis integrated GWAS data from other immune mediated diseases (IMDs) and additional publically available resources. Results We found strong evidence that disease-associated DNA variants regulate cis-CpG methylation of DNA in CD4+ T and/or B cells at 37% RA loci. In general we observed these variants to preferentially modify methylation at sites mapping to lymphocyte enhancers and regions flanking transcription start sites, and at positions bound by the NFκB transcription factor. Using paired, cell-specific transcriptomic data and a statistical approach to infer causality, we then identified examples where site-specific DNA methylation in turn mediates gene expression, including ORMDL3/GSDMB, IL6ST/ANKRD55, FCRL3 and JAZF1 in CD4+ lymphocytes. Leveraging GWAS data we noted that a number of genes regulated in this way highlight mechanisms common to RA, multiple sclerosis and asthma, distinguishing these IMDs from osteoarthritis which is considered a primarily degenerative disease. To validate our findings, cis-meQTL effects at sentinel loci were replicated by pyrosequencing in an independent cohort of genotyped early arthritis patients, and methylation-mediated regulation of FCRL3 expression downstream of the regulatory SNP was confirmed experimentally using a luciferase reporter assay in Jurkat T-cells. Conclusion Our observations highlight important mechanisms of genetic risk in RA and the wider context of autoimmunity. They confirm the utility of DNA methylation profiling as a tool for causal gene prioritisation and, potentially, therapeutic targeting in complex IMD. Disclosures A.D. Clark None. N. Nair None. A.E. Anderson None. N. Thalayasingam None. N. Naamane None. A.J. Skelton None. J. Diboll None. A. Barton None. S. Eyre None. J.D. Isaacs None. L.N. Reynard None. A.G. Pratt Grants/research support; I am a recipient of an unrestricted, investigator initiated research grant from Pfizer, paid to Newcastle University.

Lymphocyte DNA methylation mediates genetic risk at shared immune-mediated disease loci

BackgroundDefining regulatory mechanisms through which noncoding risk variants influence the cell-mediated pathogenesis of immune-mediated disease (IMD) has emerged as a priority in the post–genome-wide association study era.ObjectivesWith a focus on rheumatoid arthritis, we sought new insight into genetic mechanisms of adaptive immune dysregulation to help prioritize molecular pathways for targeting in this and related immune pathologies.MethodsWhole-genome methylation and transcriptional data from isolated CD4+ T cells and B cells of more than 100 genotyped and phenotyped patients with inflammatory arthritis, all of whom were naive to immunomodulatory treatments, were obtained. Analysis integrated these comprehensive data with genome-wide association study findings across IMDs and other publicly available resources.ResultsWe provide strong evidence that disease-associated DNA variants regulate cis-CpG methylation in CD4+ T and/or B cells at 37% RA loci. Using paired, cell-specific transcriptomic data and causal inference testing, we identify examples where site-specific DNA methylation in turn mediates gene expression, including FCRL3 in both cell types and ORMDL3/GSDMB, IL6ST/ANKRD55, and JAZF1 in CD4+ T cells. A number of genes regulated in this way highlight mechanisms common to RA and other IMDs including multiple sclerosis and asthma, in turn distinguishing them from osteoarthritis, a primarily degenerative disease. Finally, we corroborate the observed effects experimentally.ConclusionsOur observations highlight important mechanisms of genetic risk in RA and the wider context of immune dysregulation. They confirm the utility of DNA methylation profiling as a tool for causal gene prioritization and, potentially, therapeutic targeting in complex IMD.

M90 - FUNCTIONAL CHARACTERIZATION OF PSYCHIATRIC DISEASE-ASSOCIATED CACNA1C VARIANTS THROUGH GENETIC, MOLECULAR AND CELLULAR APPROACHES

Background Despite recent advancements in human genomics and identifying hundreds of risk loci for psychiatric disorders, drug development in psychiatry remains a challenge. While Genome-Wide Association Studies (GWAS) provide us with a great starting point by highlighting risk regions of the genome, translating disease-associated DNA variants to physiological function has been quite sluggish. This is mostly due to polygenic and multifactorial nature of psychiatric diseases which in turn have made development of relevant cellular and in vivo models particularly difficult. One recent GWAS identified over hundred genes that are strongly associated with schizophrenia but contribution of each gene to the disease seems to be small. Each risk gene is identified with tens of SNPs, most of which are common SNPs residing in the intronic regions of DNA. Methods Therefore, establishing cellular models to understand the directionality (loss or gain) and functional defect caused by each risk SNP within each gene can be an enormous undertaking. On the other hand, rare exon variants found in the disease population can serve as a more reliable starting point to understand how the function of a risk gene is affected in the disease. Results Here we examined effects of rare variants reported for CACNA1C, a top schizophrenia GWAS hit, in cellular models. CACNA1C is also linked to bipolar disorder, ADHD, depression and autism where schizophrenia GWAS identified over 160 risk SNPs for this gene. We used linkage disequilibrium information to identify a small number of risk SNPs that have the highest chance of affecting the overall function of CACNA1C gene. We have combined these two approaches, built neuronal functional assays and identified several important disease related phenotypes. Discussion These studies show a proof of concept in the context of the CACNA1C gene but this approach can be used as a platform to characterize other GWAS hits several other disease areas.

Interdisciplinary Models for Research and Clinical Endeavors in Genomic Medicine: A Scientific Statement From the American Heart Association.

The completion of the Human Genome Project has unleashed a wealth of human genomics information, but it remains unclear how best to implement this information for the benefit of patients. The standard approach of biomedical research, with researchers pursuing advances in knowledge in the laboratory and, separately, clinicians translating research findings into the clinic as much as decades later, will need to give way to new interdisciplinary models for research in genomic medicine. These models should include scientists and clinicians actively working as teams to study patients and populations recruited in clinical settings and communities to make genomics discoveries-through the combined efforts of data scientists, clinical researchers, epidemiologists, and basic scientists-and to rapidly apply these discoveries in the clinic for the prediction, prevention, diagnosis, prognosis, and treatment of cardiovascular diseases and stroke. The highly publicized US Precision Medicine Initiative, also known as All of Us, is a large-scale program funded by the US National Institutes of Health that will energize these efforts, but several ongoing studies such as the UK Biobank Initiative; the Million Veteran Program; the Electronic Medical Records and Genomics Network; the Kaiser Permanente Research Program on Genes, Environment and Health; and the DiscovEHR collaboration are already providing exemplary models of this kind of interdisciplinary work. In this statement, we outline the opportunities and challenges in broadly implementing new interdisciplinary models in academic medical centers and community settings and bringing the promise of genomics to fruition.

Impact of autoimmune risk alleles on the immune system.

Genetic analyses of autoimmune diseases have revealed hundreds of disease-associated DNA variants, but the identity and function of the causal variants are understudied and warrant deeper mechanistic studies. Here, we highlight methods for deciphering how alleles that are associated with autoimmune disease alter the human immune system, and suggest strategies for future autoimmune genetic research.

Mutations in USP9X Are Associated with X-Linked Intellectual Disability and Disrupt Neuronal Cell Migration and Growth

With a wealth of disease-associated DNA variants being recently reported, the challenges of providing their functional characterization are mounting. Previously, as part of a large systematic resequencing of the X chromosome in 208 unrelated families with nonsyndromic X-linked intellectual disability, we identified three unique variants (two missense and one protein truncating) in USP9X. To assess the functional significance of these variants, we took advantage of the Usp9x knockout mouse we generated. Loss of Usp9x causes reduction in both axonal growth and neuronal cell migration. Although overexpression of wild-type human USP9X rescued these defects, all three USP9X variants failed to rescue axonal growth, caused reduced USP9X protein localization in axonal growth cones, and (in 2/3 variants) failed to rescue neuronal cell migration. Interestingly, in one of these families, the proband was subsequently identified to have a microdeletion encompassing ARID1B, a known ID gene. Given our findings it is plausible that loss of function of both genes contributes to the individual's phenotype. This case highlights the complexity of the interpretations of genetic findings from genome-wide investigations. We also performed proteomics analysis of neurons from both the wild-type and Usp9x knockout embryos and identified disruption of the cytoskeleton as the main underlying consequence of the loss of Usp9x. Detailed clinical assessment of all three families with USP9X variants identified hypotonia and behavioral and morphological defects as common features in addition to ID. Together our data support involvement of all three USP9X variants in ID in these families and provide likely cellular and molecular mechanisms involved.

Multiplex meta-analysis of RNA expression to identify genes with variants associated with immune dysfunction

ObjectiveWe demonstrate a genome-wide method for the integration of many studies of gene expression of phenotypically similar disease processes, a method of multiplex meta-analysis. We use immune dysfunction as an example disease process.DesignWe use a heterogeneous collection of datasets across human and mice samples from a range of tissues and different forms of immunodeficiency. We developed a method integrating Tibshirani's modified t-test (SAM) is used to interrogate differential expression within a study and Fisher's method for omnibus meta-analysis to identify differentially expressed genes across studies. The ability of this overall gene expression profile to prioritize disease associated genes is evaluated by comparing against the results of a recent genome wide association study for common variable immunodeficiency (CVID).ResultsOur approach is able to prioritize genes associated with immunodeficiency in general (area under the ROC curve = 0.713) and CVID in particular (area under the ROC curve = 0.643).ConclusionsThis approach may be used to investigate a larger range of failures of the immune system. Our method may be extended to other disease processes, using RNA levels to prioritize genes likely to contain disease associated DNA variants.

Revealing the human mutome

The number of known mutations in human nuclear genes, underlying or associated with human inherited disease, has now exceeded 100,000 in more than 3700 different genes (Human Gene Mutation Database). However, for a variety of reasons, this figure is likely to represent only a small proportion of the clinically relevant genetic variants that remain to be identified in the human genome (the 'mutome'). With the advent of next-generation sequencing, we are currently witnessing a revolution in medical genetics. In particular, whole-genome sequencing (WGS) has the potential to identify all disease-causing or disease-associated DNA variants in a given individual. Here, we use examples of recent advances in our understanding of mutational/pathogenic mechanisms to guide our thinking about possible locations outwith gene-coding sequences for those disease-causing or disease-associated variants that are likely so often to have been overlooked because of the inadequacy of current mutation screening protocols. Such considerations are important not only for improving mutation-screening strategies but also for enhancing the interpretation of findings derived from genome-wide association studies, whole-exome sequencing and WGS. An improved understanding of the human mutome will not only lead to the development of improved diagnostic testing procedures but should also improve our understanding of human genome biology.

FAT tumour suppressor gene is mutated by both homozygous deletion and point mutation in a variety of different cancer types

With a wealth of disease-associated DNA variants being recently reported, the challenges of providing their functional characterization are mounting. Previously, as part of a large systematic resequencing of the X chromosome in 208 unrelated families with nonsyndromic X-linked intellectual disability, we identified three unique variants (two missense and one protein truncating) in USP9X. To assess the functional significance of these variants, we took advantage of the Usp9x knockout mouse we generated. Loss of Usp9x causes reduction in both axonal growth and neuronal cell migration. Although overexpression of wild-type human USP9X rescued these defects, all three USP9X variants failed to rescue axonal growth, caused reduced USP9X protein localization in axonal growth cones, and (in 2/3 variants) failed to rescue neuronal cell migration. Interestingly, in one of these families, the proband was subsequently identified to have a microdeletion encompassing ARID1B, a known ID gene. Given our findings it is plausible that loss of function of both genes contributes to the individual's phenotype. This case highlights the complexity of the interpretations of genetic findings from genome-wide investigations. We also performed proteomics analysis of neurons from both the wild-type and Usp9x knockout embryos and identified disruption of the cytoskeleton as the main underlying consequence of the loss of Usp9x. Detailed clinical assessment of all three families with USP9X variants identified hypotonia and behavioral and morphological defects as common features in addition to ID. Together our data support involvement of all three USP9X variants in ID in these families and provide likely cellular and molecular mechanisms involved.

FitSNPs: highly differentially expressed genes are more likely to have variants associated with disease

Differential expressed genes are more likely to have variants associated with disease. A new tool, fitSNP, prioritizes candidate SNPs from association studies.