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Funder: Rosetrees Trust, Aim & methods: To investigate peripheral blood methylation episignatures in KMT2B-related dystonia (DYT-KMT2B), the authors undertook genome-wide methylation profiling of ∼2 M CpGs using a next-generation sequencing-based assay and compared the findings with those in controls and patients with KMT2D-related Kabuki syndrome type 1 (KS1). Results: A total of 1812 significantly differentially methylated CpG positions (false discovery rate < 0.05) were detected in DYT-KMT2B samples compared with controls. Multi-dimensional scaling analysis showed that the 10 DYT-KMT2B samples clustered together and separately from 29 controls and 10 with pathogenic variants in KMT2D. The authors found that most differentially methylated CpG positions were specific to one disorder and that all (DYT-KMT2B) and most (Kabuki syndrome type 1) methylation alterations in CpG islands were gain of methylation events. Conclusion: Using sensitive methylation profiling methodology, the authors replicated recent reports of a methylation episignature for DYT-KMT2B. These findings will facilitate the development of episignature-based assays to improve diagnostic accuracy.

Around 95% of patients with clinical features that meet the diagnostic criteria for von Hippel–Lindau disease (VHL) have a detectable inactivating germline variant in VHL. The VHL protein (pVHL) functions as part of the E3 ubiquitin ligase complex comprising pVHL, elongin C, elongin B, cullin 2 and ring box 1 (VCB-CR complex), which plays a key role in oxygen sensing and degradation of hypoxia-inducible factors. To date, only variants in VHL have been shown to cause VHL disease. We undertook trio analysis by whole-exome sequencing in a proband with VHL disease but without a detectable VHL mutation. Molecular studies were also performed on paired DNA extracted from the proband’s kidney tumour and blood and bioinformatics analysis of sporadic renal cell carcinoma (RCC) dataset was undertaken. A de novo pathogenic variant in ELOC NM_005648.4(ELOC):c.236A>G (p.Tyr79Cys) gene was identified in the proband. ELOC encodes elongin C, a key component [C] of the VCB-CR complex. The p.Tyr79Cys substitution is a mutational hotspot in sporadic VHL-competent RCC and has previously been shown to mimic the effects of pVHL deficiency on hypoxic signalling. Analysis of an RCC from the proband showed similar findings to that in somatically ELOC-mutated RCC (expression of hypoxia-responsive proteins, no somatic VHL variants and chromosome 8 loss). These findings are consistent with pathogenic ELOC variants being a novel cause for VHL disease and suggest that genetic testing for ELOC variants should be performed in individuals with suspected VHL disease with no detectable VHL variant.

Funder: DH | National Institute for Health Research (NIHR); doi: https://doi.org/10.13039/501100000272, Funder: National Health Service, Funder: European Research Council (Advanced Researcher Award) Cancer Research UK Cambridge Cancer Centre Medical Research Council (Infrastructure Award) National Health Service, Gastrointestinal stromal tumour (GIST) is a mesenchymal neoplasm arising in the gastrointestinal tract. A rare subset of GISTs are classified as wild-type GIST (wtGIST) and these are frequently associated with germline variants that affect the function of cancer predisposition genes such as the succinate dehydrogenase subunit genes (SDHA, SDHB, SDHC, SDHD) or NF1. However, despite this high heritability, familial clustering of wtGIST is extremely rare. Here, we report a mother-son diad who developed wtGIST at age 66 and 34 years, respectively. Comprehensive genetic testing revealed germline truncating variants in both SDHA (c.1534C>T (p.Arg512*)) and PALB2 (c.3113G>A (p.Trp1038*)) in both affected individuals. The mother also developed breast ductal carcinoma in-situ at age 70 years. Immunohistochemistry and molecular analysis of the wtGISTs revealed loss of SDHB expression and loss of the wild-type SDHA allele in tumour material. No allele loss was detected at PALB2 suggesting that wtGIST tumourigenesis was principally driven by succinate dehydrogenase deficiency. However, we speculate that the presence of multilocus inherited neoplasia alleles syndrome (MINAS) in this family might have contributed to the highly unusual occurrence of familial wtGIST. Systematic reporting of tumour risks and phenotypes in individuals with MINAS will facilitate the clinical interpretation of the significance of this diagnosis, which is becoming more frequent as strategies for genetic testing for hereditary cancer becomes more comprehensive.

The authors compared the DNA methylation patterns in blood from individuals with two rare neurodevelopmental disorders (childhood-onset dystonia [DYT

PURPOSE: Disruptions of genomic imprinting are associated with congenital imprinting disorders (CIDs) and other disease states, including cancer. CIDs are most often associated with altered methylation at imprinted differentially methylated regions (iDMRs). In some cases, multiple iDMRs are affected causing multilocus imprinting disturbances (MLIDs). The availability of accurate, quantitative, and scalable high-throughput methods to interrogate multiple iDMRs simultaneously would enhance clinical diagnostics and research. METHODS: We report the development of a custom targeted methylation sequencing panel that covered most relevant 63 iDMRs for CIDs and the detection of MLIDs. We tested it in 70 healthy controls and 147 individuals with CIDs. We distinguished loss and gain of methylation per differentially methylated region and classified high and moderate methylation alterations. RESULTS: Across a range of CIDs with a variety of molecular mechanisms, ImprintSeq performed at 98.4% sensitivity, 99.9% specificity, and 99.9% accuracy (when compared with previous diagnostic testing). ImprintSeq was highly sensitive for detecting MLIDs and enabled diagnostic criteria for MLID to be proposed. In a child with extreme MLID profile a probable genetic cause was identified. CONCLUSION: ImprintSeq provides a novel assay for clinical diagnostic and research studies of CIDs, MLIDs, and the role of disordered imprinting in human disease states.

The enzyme succinate dehydrogenase (SDH) functions in the citric acid cycle and loss of function predisposes to the development of phaeochromocytoma/paraganglioma (PPGL), wild type gastrointestinal stromal tumour (wtGIST) and renal cell carcinoma. SDH-deficient tumours are most commonly associated with a germline SDH subunit gene (SDHA/B/C/D) mutation but can also be associated with epigenetic silencing of the SDHC gene. However, clinical diagnostic testing for an SDHC epimutation is not widely available. The objective of this study was to investigate the indications for and the optimum diagnostic pathways for the detection of SDHC epimutations in clinical practice. SDHC promoter methylation analysis of 32 paraffin embedded tumours (including 15 GIST and 17 PPGL) was performed using a pyrosequencing technique and correlated with SDHC gene expression. SDHC promoter methylation was identified in 6 (18.7%) tumours. All 6 SDHC epimutation cases presented with SDH deficient wtGIST and 3/6 cases had multiple primary tumours. No case of constitutional SDHC promoter hypermethylation was detected. Whole genome sequencing of germline DNA from three wtGIST cases with an SDHC epimutation, did not reveal any causative sequence anomalies. Herein, we recommend a diagnostic workflow for the detection of an SDHC epimutation in a service setting.

Systemic sclerosis (SSc) is a severe connective tissue disorder characterized by extensive fibrosis, vascular damage, and autoimmune events. During the last years, the number of genetic markers convincingly associated with SSc has exponentially increased. In this report, we aim to offer an updated review of the classical and novel genetic associations with SSc, analyzing the firmest and replicated signals within HLA and non-HLA genes, identified by both candidate gene and genome-wide association (GWA) studies. We will also provide an insight into the future perspectives and approaches that might shed more light into the complex genetic background underlying SSc. In spite of the remarkable advance in the field of SSc genetics during the last decade, the use of the new genetic technologies such as next generation sequencing (NGS), as well as the deep phenotyping of the study cohorts, to fully characterize the genetic component of this disease is imperative.

Objective.The PTPN22 rs2476601 genetic variant has been associated with rheumatoid arthritis (RA) and other autoimmune diseases. Some reports suggest that this single-nucleotide polymorphism (SNP) may not be the only causal variant in the region of PTPN22. Our aim was to identify new independent RA-associated common gene variants in the PTPN22 region.Methods.We analyzed Wellcome Trust Case-Control Consortium genome-wide association study data for associations in the 397.2 kb PTPN22 region and selected 9 associated SNP (with p < 5 × 10−3) for replication and dependence analysis. The replication cohorts comprised 2857 patients with RA and 2994 controls from Spain, Netherlands, and Norway.Results.We found that 6 of the 9 selected SNP were associated in the Spanish cohort. Of these, 4 were also associated in the Dutch and Norwegian cohorts, and all 6 were associated with RA in the combined analysis. Conditional analyses showed that none of these associations was independent of rs2476601.Conclusion.The SNP rs2476601 located in the PTPN22 gene is the sole common genetic variant associated with RA in the 1p13.2 region, suggesting that neighbor genes of PTPN22 do not have a major influence in RA.

Objective The risk of rheumatoid arthritis (RA) is increased in the offspring of individuals affected with various autoimmune disorders, including psoriasis. Recently, the deletion of 2 genes from the late cornified envelope (LCE) gene cluster, LCE3C and LCE3B, has been associated with psoriasis in several populations. The purpose of this study was to assess whether this polymorphic gene deletion could also be involved in susceptibility to RA. Methods We tested for association between the LCE3C_LCE3B copy number variant and a single-nucleotide polymorphism in strong linkage disequilibrium with this variant (rs4112788) and RA in 2 independent case–control data sets (197 and 400 samples from patients with RA, respectively, and 411 and 567 samples from control subjects, respectively), collected at 4 Spanish hospitals. All samples were directly typed for presence of the LCE3C_LCE3B deletion (LCE3C_LCE3B-del) by polymerase chain reaction, and association analysis was performed using the SNPassoc R package. Results An association of homozygosity for the LCE3C_LCE3B-del and rs4112788 C allele with the risk of RA was observed in the first data set and was replicated in an independent case–control set. A combined analysis showed an overall P value of 0.0012 (odds ratio [OR] 1.45, 95% confidence interval [95% CI] 1.16–1.81) for association of the LCE3C_LCE3B-del. When the analysis was stratified for serologic data, we observed association in anti–cyclic citrullinated peptide (anti-CCP)–positive patients (P = 0.012, OR 1.51 [95% CI 1.09–2.13]) but not in anti-CCP–negative patients. Conclusion We have identified an association between the LCE3C_LCE3B-del and RA, and we have verified a pleiotropic effect of a common genetic risk factor (LCE3C_LCE3B-del) for autoimmune diseases that is involved in both psoriasis and RA.

In this study, 1,833 systemic sclerosis (SSc) cases and 3,466 controls were genotyped with the Immunochip array. Classical alleles, amino acid residues, and SNPs across the human leukocyte antigen (HLA) region were imputed and tested. These analyses resulted in a model composed of six polymorphic amino acid positions and seven SNPs that explained the observed significant associations in the region. In addition, a replication step comprising 4,017 SSc cases and 5,935 controls was carried out for several selected non-HLA variants, reaching a total of 5,850 cases and 9,401 controls of European ancestry. Following this strategy, we identified and validated three SSc risk loci, including DNASE1L3 at 3p14, the SCHIP1-IL12A locus at 3q25, and ATG5 at 6q21, as well as a suggested association of the TREH-DDX6 locus at 11q23. The associations of several previously reported SSc risk loci were validated and further refined, and the observed peak of association in PXK was related to DNASE1L3. Our study has increased the number of known genetic associations with SSc, provided further insight into the pleiotropic effects of shared autoimmune risk factors, and highlighted the power of dense mapping for detecting previously overlooked susceptibility loci.

Item does not contain fulltext INTRODUCTION: A recent genome-wide association study in European systemic sclerosis (SSc) patients identified three loci (PSORS1C1, TNIP1 and RHOB) as novel genetic risk factors for the disease. The aim of this study was to replicate the previously mentioned findings in a large multicentre independent SSc cohort of Caucasian ancestry. METHODS: 4389 SSc patients and 7611 healthy controls from different European countries and the USA were included in the study. Six single nucleotide polymorphisms (SNP): rs342070, rs13021401 (RHOB), rs2233287, rs4958881, rs3792783 (TNIP1) and rs3130573 (PSORS1C1) were analysed. Overall significance was calculated by pooled analysis of all the cohorts. Haplotype analyses and conditional logistic regression analyses were carried out to explore further the genetic structure of the tested loci. RESULTS: Pooled analyses of all the analysed SNPs in TNIP1 revealed significant association with the whole disease (rs2233287 p(MH)=1.94x10(-4), OR 1.19; rs4958881 p(MH)=3.26x10(-5), OR 1.19; rs3792783 p(MH)=2.16x10(-4), OR 1.19). These associations were maintained in all the subgroups considered. PSORS1C1 comparison showed association with the complete set of patients and all the subsets except for the anti-centromere-positive patients. However, the association was dependent on different HLA class II alleles. The variants in the RHOB gene were not associated with SSc or any of its subsets. CONCLUSIONS: These data confirmed the influence of TNIP1 on an increased susceptibility to SSc and reinforced this locus as a common autoimmunity risk factor.

Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P = 1.34×10-8, OR = 1.22, CI 95% = 1.14-1.30; rs2004640: P = 4.60×10-7, OR = 0.84, CI 95% = 0.78-0.90; rs10488631: P = 7.53×10-20, OR = 1.63, CI 95% = 1.47-1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P = 9.04×10-22, OR = 1.75, CI 95% = 1.56-1.97) better explained the observed association (likelihood P-value = 1.48×10-4), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific. © 2013 Carmona et al.

The red cell acid phosphatase (ACP1) gene, which encodes a low-molecular-weight phosphotyrosine phosphatase, has been suggested as a common genetic factor of autoimmunity. In the present study, we aim to investigate the possible association of ACP1 with the susceptibility of systemic lupus erythematosus (SLE). A total of 1,546 SLE patients and 1,947 healthy individuals from 4 Caucasians populations were included in the present study. Four single-nucleotide polymorphisms (SNPs) were genotyped in this study: rs10167992, rs11553742, rs7576247, and rs3828329. ACP1*A, ACP1*B, and ACP1*C codominant ACP1 alleles were determined using 2 of the SNPs and analyzed. After the meta-analysis test was performed, a significant association of rs11553742*T was observed (ppooled = 0.005, odds ratios = 1.37 [1.10-1.70]), retaining significance after multiple testing was applied (pFDR = 0.019). Our data indicate for first time the association of rs11553742*T with increased susceptibility in SLE patients. © 2012 American Society for Histocompatibility and Immunogenetics.

Contains fulltext : 88210.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Genome-wide association studies carried out in rheumatoid arthritis (RA) have led to the discovery of several genetic associations with this disease. Still, the current associated genetic variations can explain only part of the genetic risk involved in RA, and it is well recognized that these genome-wide association studies are likely underpowered to detect all common disease variants. This study was undertaken to explore the genomic regions showing low-significance associations in previous genome-wide association studies of RA. METHODS: To reduce the false-positive signal fraction, we exploited pathway analysis to prioritize regions containing genes most likely to be implicated in RA. We hypothesized that true disease genes would be in a similar pathway. Therefore, genes from similar pathways but located in different regions were prioritized for replication using Prioritizer software. A total of 384 genetic variants selected from previous RA genome-wide association studies were tested in a Spanish case-control discovery cohort comprising 376 RA patients and 478 healthy controls for replication. Statistically significant associations were further validated in replication cohorts from Spain and The Netherlands. The study consisted of a total of 1,818 RA patients and 2,498 controls. RESULTS: We detected a novel genetic association between RA and the MSRA gene (rs10903323) in the Spanish combined population (P = 2.91 x 10(-5) , odds ratio [OR] 1.51). This association was further tested in our independent Dutch replication cohort. Combined analysis showed an overall association of MSRA with RA (P = 3.19 x 10(-4) , OR 1.28). CONCLUSION: Our findings indicate that a novel association in the MSRA gene is related to oxidative stress and support the notion of a major role for this process in RA. 01 november 2010

4 páginas, 2 figuras, 2 tablas.-- Spanish Scleroderma Group., Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs that leads to profound disability and premature death. To identify new SSc susceptibility loci, we conducted the first genome-wide association study in a population of European ancestry including a total of 2,296 individuals with SSc and 5,171 controls. Analysis of 279,621 autosomal SNPs followed by replication testing in an independent case-control set of European ancestry (2,753 individuals with SSc (cases) and 4,569 controls) identified a new susceptibility locus for systemic sclerosis at CD247 (1q22–23, rs2056626, P = 2.09 × 10−7 in the discovery samples, P = 3.39 × 10−9 in the combined analysis). Additionally, we confirm and firmly establish the role of the MHC (P = 2.31 × 10−18), IRF5 (P = 1.86 × 10−13) and STAT4 (P = 3.37 × 10−9) gene regions as SSc genetic risk factors., This work was supported by the following grants: T.R.D.J.R. was funded by the VIDI laureate from the Dutch Association of Research (NWO) and Dutch Arthritis Foundation (National Reumafonds). J.M. was funded by GEN-FER from the Spanish Society of Rheumatology, SAF2009-11110 from the Spanish Ministry of Science, CTS-4977 from Junta de Andalucía, Spain and in part by Redes Temáticas de Investigación Cooperativa Sanitaria Program, RD08/0075 (RIER) from Instituto de Salud Carlos III (ISCIII), Spain (J.M.). R.B. is supported by the I3P Consejo Superior de Investigaciones Científicas program funded by the 'Fondo Social Europeo'. B.Z.A. is supported by the Netherlands Organization for Health Research and Development (ZonMW grant 016.096.121). B.K. is supported by the Dutch Diabetes Research Foundation (grant 2008.40.001) and the Dutch Arthritis Foundation (Reumafonds, grant NR 09-1-408). Genotyping of the Dutch control samples was sponsored by US National Insitutes of Mental Health funding, R01 MH078075 (R.O.A.). The German controls were from the PopGen biobank (to B.K.).

Introduction Acid phosphatase locus 1 (ACP1) encodes a low molecular weight phosphotyrosine phosphatase implicated in a number of different biological functions in the cell. The aim of this study was to determine the contribution of ACP1 polymorphisms to susceptibility to rheumatoid arthritis (RA), as well as the potential contribution of these polymorphisms to the increased risk of cardiovascular disease (CV) observed in RA patients. Methods A set of 1,603 Spanish RA patients and 1,877 healthy controls were included in the study. Information related to the presence/absence of CV events was obtained from 1,284 of these participants. All individuals were genotyped for four ACP1 single-nucleotide polymorphisms (SNPs), rs10167992, rs11553742, rs7576247, and rs3828329, using a predesigned TaqMan SNP genotyping assay. Classical ACP1 alleles (*A, *B and *C) were imputed with SNP data. Results No association between ACP1 gene polymorphisms and susceptibility to RA was observed. However, when RA patients were stratified according to the presence or absence of CV events, an association between rs11553742*T and CV events was found (P = 0.012, odds ratio (OR) = 2.62 (1.24 to 5.53)). Likewise, the ACP1*C allele showed evidence of association with CV events in patients with RA (P = 0.024, OR = 2.43). Conclusions Our data show that the ACP1*C allele influences the risk of CV events in patients with RA., This work was supported by two grants from Fondo de Investigaciones Sanitarias PI06-0024 and PS09/00748 (Spain) and by the RETICS Program, RD08/0075 (RIER) from the Instituto de Salud Carlos III (ISCIII), within the VI PN de I+D+i 2008-2011 (FEDER). MT was supported by the Spanish Ministry of Science through the program Juan de la Cierva (JCI-2010-08227).