An Integrative Genomic Study of Dupuytren's Disease

Dupuytren's Disease (DD) is a fibromatosis in the palmar connective tissue that leads to flexion contractures of fingers. DD has a strong genetic component with an estimated heritability of about 80%. A previous collaborative genome-wide association study (GWAS) has mapped 9 susceptibility loci that were shown to contribute to the increased risk of DD. However, these loci together can only explain a small fraction of heritability. Moreover, the respective genes and functional variants underlying DD remained unclear. Therefore, the present study aims to systematically investigate the genetic architecture of DD at different levels by: i) identifying functional variants contributing to a strong GWAS association signal at a DD risk locus, ii) prioritizing DD phenotype-related genes with rare variant burdens, and iii) characterizing the transcriptional deregulation in DD. An intervall on chromosome 7, 7p14.1, tagged by rs16879765 (G>A), which was most significantly associated with DD in the previous GWAS, has been identified as a DD susceptibility locus. Therefore, we first used a target-enrichment strategy coupled with next generation sequencing (targeted NGS) to assess a 500kb region at 7p14.1. A rare non-synonymous variant, rs149095633 (p.P121L, on haplotypes tagged by the rs16879765*A risk allele), and a common eQTL candidate, rs2044831 (in moderate linkage disequilibrium with rs16879765), were identified in EPDR1, a functional candidate gene contributing to the contractile phenotype of DD primary cells. Second, we performed a pilot whole exome sequencing (WES) study in 50 DD patients with suspected high genetic predisposition and prioritized candidate genes with rare variant burden. 3919 rare coding variants were predicted to be deleterious. 1774 genes with gene burden greater than 2 were filtered for suitable phenotype classes and palmar expression according to Human Phenotype Ontology and the gene intolerance score EvolTol. As a result, 12 genes were prioritized as DD candidate genes with potentially pathogenic rare variants. In particular, 6 of these genes were suggested as functionally important for DD development. Third, we carried out an elaborate transcriptome study in 50 DD/control biopsy samples by RNAseq. By pathway perturbation analysis using gene expression profiles, the Hippo signaling pathway was suggested as a key mechanotransdution pathway to prepare the profibrotic microenvironment in DD. The TGFβ pathway and ECM-receptor interactions were predicted as pathways essential for tissue fibrosis. Moreover, by alternative splicing (AS) analysis, DD tissue was suggested to harbor distinctive isoform profiles and isoform usage, which might provide a mechanism for cells in disease tissue to adapt to fibrosis and further promote fibrosis progression. In summary, this study characterized for the first time a GWAS risk locus for DD and provided an approach for identifying functional variants for DD in the post-GWAS era. The exploratory prioritization of DD-related candidate genes supported the assumption that rare variants can contribute to the development of the disease and nominated candidate genes for follow-up studies. Furthermore, this study proposed key physiological pathways involved in transcriptional regulation of DD and gave a first insight into disease tissue-specific AS and possible AS regulation mechanisms. Overall, our study represents a first step in integrating various genomic approaches to elucidate the mechanistic links between the genetic predisposition and the development of DD

Meta-Analysis of Genome-Wide Association Studies and Network Analysis-Based Integration with Gene Expression Data Identify New Suggestive Loci and Unravel a Wnt-Centric Network Associated with Dupuytren’s Disease

Dupuytren´s disease, a fibromatosis of the connective tissue in the palm, is a common complex disease with a strong genetic component. Up to date nine genetic loci have been found to be associated with the disease. Six of these loci contain genes that code for Wnt signalling proteins. In spite of this striking first insight into the genetic factors in Dupuytren´s disease, much of the inherited risk in Dupuytren´s disease still needs to be discovered. The already identified loci jointly explain ~1% of the heritability in this disease. To further elucidate the genetic basis of Dupuytren´s disease, we performed a genome-wide meta-analysis combining three genome-wide association study (GWAS) data sets, comprising 1,580 cases and 4,480 controls. We corroborated all nine previously identified loci, six of these with genome-wide significance (p-value < 5x10-8). In addition, we identified 14 new suggestive loci (p-value < 10−5). Intriguingly, several of these new loci contain genes associated with Wnt signalling and therefore represent excellent candidates for replication. Next, we compared whole-transcriptome data between patient- and control-derived tissue samples and found the Wnt/β-catenin pathway to be the top deregulated pathway in patient samples. We then conducted network and pathway analyses in order to identify protein networks that are enriched for genes highlighted in the GWAS meta-analysis and expression data sets. We found further evidence that the Wnt signalling pathways in conjunction with other pathways may play a critical role in Dupuytren´s disease

Identification of pathogenic variant enriched regions across genes and gene families

Missense variant interpretation is challenging. Essential regions for protein function are conserved among gene-family members, and genetic variants within these regions are potentially more likely to confer risk to disease. Here, we generated 2871 gene-family protein sequence alignments involving 9990 genes and performed missense variant burden analyses to identify novel essential protein regions. We mapped 2,219,811 variants from the general population into these alignments and compared their distribution with 76,153 missense variants from patients. With this gene-family approach, we identified 465 regions enriched for patient variants spanning 41,463 amino acids in 1252 genes. As a comparison, by testing the same genes individually, we identified fewer patient variant enriched regions, involving only 2639 amino acids and 215 genes. Next, we selected de novo variants from 6753 patients with neurodevelopmental disorders and 1911 unaffected siblings and observed an 8.33-fold enrichment of patient variants in our identified regions (95% C.I. = 3.90-Inf, P-value= 2.72 x 10(-11)). By using the complete ClinVar variant set, we found that missense variants inside the identified regions are 106-fold more likely to be classified as pathogenic in comparison to benign classification (OR = 106.15, 95% C.I = 70.66-Inf, P-value <2.2x10(-16)). All pathogenic variant enriched regions (PERs) identified are available online through "PER viewer," a user-friendly online platform for interactive data mining, visualization, and download. In summary, our gene-family burden analysis approach identified novel PERs in protein sequences. This annotation can empower variant interpretation.Peer reviewe

SCN1A variants from bench to bedside-improved clinical prediction from functional characterization

Variants in the SCN1A gene are associated with a wide range of disorders including genetic epilepsy with febrile seizures plus (GEFS+), familial hemiplegic migraine (FHM), and the severe childhood epilepsy Dravet syndrome (DS). Predicting disease outcomes based on variant type remains challenging. Despite thousands of SCN1A variants being reported, only a minority has been functionally assessed. We review the functional SCN1A work performed to date, critically appraise electrophysiological measurements, compare this to in silico predictions, and relate our findings to the clinical phenotype. Our results show, regardless of the underlying phenotype, that conventional in silico software correctly predicted benign from pathogenic variants in nearly 90%, however was unable to differentiate within the disease spectrum (DS vs. GEFS+ vs. FHM). In contrast, patch-clamp data from mammalian expression systems revealed functional differences among missense variants allowing discrimination between disease severities. Those presenting with milder phenotypes retained a degree of channel function measured as residual whole-cell current, whereas those without any whole-cell current were often associated with DS (p = .024). These findings demonstrate that electrophysiological data from mammalian expression systems can serve as useful disease biomarker when evaluating SCN1A variants, particularly in view of new and emerging treatment options in DS

Identification of pathogenic variant enriched regions across genes and gene families

Missense variant interpretation is challenging. Essential regions for protein function are conserved among gene family members, and genetic variants within these regions are potentially more likely to confer risk to disease. Here, we generated 2,871 gene family protein sequence alignments involving 9,990 genes and performed missense variant burden analyses to identify novel essential protein regions. We mapped 2,219,811 variants from the general population into these alignments and compared their distribution with 76,153 missense variants from patients. With this gene family approach, we identified 465 regions enriched for patient variants spanning 41,463 amino acids in 1,252 genes. As a comparison, testing the same genes individually we identified less patient variant enriched regions involving only 2,639 amino acids and 215 genes. Next, we selected de novo variants from 6,753 patients with neurodevelopmental disorders and 1,911 unaffected siblings, and observed an 8.33-fold enrichment of patient variants in our identified regions (95% C.I.=3.90-Inf, p-value = 2.72x10-11). Using the complete ClinVar variant set, we found that missense variants inside the identified regions are 106-fold more likely to be classified as pathogenic in comparison to benign classification (OR = 106.15, 95% C.I = 70.66-Inf, p-value < 2.2 x 10-16). All pathogenic variant enriched regions (PERs) identified are available online through the “PER viewer” a user-friendly online platform for interactive data mining, visualization and download. In summary, our gene family burden analysis approach identified novel pathogenic variant enriched regions in protein sequences. This annotation can empower variant interpretation

Variant Score Ranker-a web application for intuitive missense variant prioritization

Motivation: The correct classification of missense variants as benign or pathogenic remains challenging. Pathogenic variants are expected to have higher deleterious prediction scores than benign variants in the same gene. However, most of the existing variant annotation tools do not reference the score range of benign population variants on gene level. Results: We present a web-application, Variant Score Ranker, which enables users to rapidly annotate variants and perform gene-specific variant score ranking on the population level. We also provide an intuitive example of how gene- and population-calibrated variant ranking scores can improve epilepsy variant prioritization

Variant Score Ranker - a web application for intuitive missense variant prioritization

The correct classification of missense variants as benign or pathogenic remains challenging. Pathogenic variants are expected to have higher deleterious prediction scores than benign variants in the same gene. However, most of the existing variant annotation tools do not reference the score range of benign population variants on gene level. Here, we present a web-application, Variant Score Ranker, which enables users to rapidly annotate variants and perform gene-specific variant score ranking on the population level. We also provide an intuitive example of how gene- and population-calibrated variant ranking scores can improve epilepsy variant prioritization

Differential excitatory vs inhibitory SCN expression at single cell level regulates brain sodium channel function in neurodevelopmental disorders

The four voltage-gated sodium channels SCN1/2/3/8A have been associated with heterogeneous types of developmental disorders, each presenting with disease specific temporal and cell type specific gene expression. Using single-cell RNA sequencing transcriptomic data from humans and mice, we observe that SCNIA is predominantly expressed in inhibitory neurons. In contrast, SCN2/3/8A are profoundly expressed in excitatory neurons with SCN2/3A starting prenatally, followed by SCN1/8A neonatally. In contrast to previous observations from low resolution RNA screens, we observe that all four genes are expressed in both excitatory and inhibitory neurons, however, exhibit differential expression strength. These findings provide molecular evidence, at single-cell resolution, to support the hypothesis that the excitatory/inhibitory (E/I) neuronal expression ratios of sodium channels are important regulatory mechanisms underlying brain homeostasis and neurological diseases. Modulating the Ell expression balance within cell types of sodium channels could serve as a potential strategy to develop targeted treatment for Nay-associated neuronal developmental disorders. (C) 2019 Published by Elsevier Ltd on behalf of European Paediatric Neurology Society

Top Ranking SNPs of the Genome-Wide Meta-Analysis in Dupuytren’s Disease.

<p>Top Ranking SNPs of the Genome-Wide Meta-Analysis in Dupuytren’s Disease.</p

Genes Identified by Pathway Analysis for the Top 23 Meta-Analysis Loci.

<p>Genes Identified by Pathway Analysis for the Top 23 Meta-Analysis Loci.</p