C. de Kovel

Screening for subtelomeric rearrangements in 210 patients with unexplained mental retardation using multiplex ligation dependent probe amplification (MLPA)

Background: Subtelomeric rearrangements contribute to idiopathic mental retardation and human malformations, sometimes as distinct mental retardation syndromes. However, for most subtelomeric defects a characteristic clinical phenotype remains to be elucidated. Objective: To screen for submicroscopic subtelomeric aberrations using multiplex ligation dependent probe amplification (MLPA). Methods: 210 individuals with unexplained mental retardation were studied. A new set of subtelomeric probes, the SALSA P036 human telomere test kit, was used. Results: A subtelomeric aberration was identified in 14 patients (6.7%) (10 deletions and four duplications). Five deletions were de novo; four were inherited from phenotypically normal parents, suggesting that these were polymorphisms. For one deletion, DNA samples of the parents were not available. Two de novo submicroscopic duplications were detected (dup 5qter, dup 12pter), while the other duplications (dup 18qter and dup 22qter) were inherited from phenotypically similarly affected parents. All clinically relevant aberrations (de novo or inherited from similarly affected parents) occurred in patients with a clinical score of ⩾3 using an established checklist for subtelomeric rearrangements. Testing of patients with a clinical score of ⩾3 increased the diagnostic yield twofold to 12.4%. Abnormalities with clinical relevance occurred in 6.3%, 5.1%, and 1.7% of mildly, moderately, and severely retarded patients, respectively, indicating that testing for subtelomeric aberrations among mildly retarded individuals is necessary. Conclusions: The value of MLPA is confirmed. Subtelomeric screening can be offered to all mentally retarded patients, although clinical preselection increases the percentage of chromosomal aberrations detected. Duplications may be a more common cause of mental retardation than has been appreciated.

Coeliac disease-associated risk variants in TNFAIP3 and REL implicate altered NF-κB signalling

Objective: Our previous coeliac disease genome-wide association study (GWAS) implicated risk variants in the human leucocyte antigen (HLA) region and eight novel risk regions. To identify more coeliac disease loci, we selected 458 single nucleotide polymorphisms (SNPs) that showed more modest association in the GWAS for genotyping and analysis in four independent cohorts. Design: 458 SNPs were assayed in 1682 cases and 3258 controls from three populations (UK, Irish and Dutch). We combined the results with the original GWAS cohort (767 UK cases and 1422 controls); six SNPs showed association with p<1×10−04 and were then genotyped in an independent Italian coeliac cohort (538 cases and 593 controls). Results: We identified two novel coeliac disease risk regions: 6q23.3 (OLIG3-TNFAIP3) and 2p16.1 (REL), both of which reached genome-wide significance in the combined analysis of all 2987 cases and 5273 controls (rs2327832 p = 1.3×10−08, and rs842647 p = 5.2×10−07). We investigated the expression of these genes in the RNA isolated from biopsies and from whole blood RNA. We did not observe any changes in gene expression, nor in the correlation of genotype with gene expression. Conclusions: Both TNFAIP3 (A20, at the protein level) and REL are key mediators in the nuclear factor kappa B (NF-κB) inflammatory signalling pathway. For the first time, a role for primary heritable variation in this important biological pathway predisposing to coeliac disease has been identified. Currently, the HLA risk factors and the 10 established non-HLA risk factors explain ∼40% of the heritability of coeliac disease.

Genomewide scan identifies susceptibility locus for dyslexia on Xq27 in an extended Dutch family

Context: Dyslexia is a common disorder with a strong genetic component, but despite significant research effort, the aetiology is still largely unknown. Objective: To identify loci contributing to dyslexia risk. Methods: This was a genomewide linkage analysis in a single large family. Dutch families with at least two first degree relatives suffering from dyslexia participated in the study. Participants were recruited through an advertisement campaign in papers and magazines. The main outcome measure was linkage between genetic markers and dyslexia phenotype. Results: Using parametric linkage analysis, we found strong evidence for a locus influencing dyslexia on Xq27.3 (multipoint lod = 3.68). Recombinations in two family members flanked an 8 cM region, comprising 11 currently confirmed genes. All four males carrying the risk haplotype had very low scores on the reading tests. The presentation in females was more variable, but 8/9 females carrying the risk haplotype were diagnosed dyslexic by our composite score, so we considered the putative risk allele to be dominant with reduced penetrance. Linkage was not found in an additional collection of affected sibling pairs. Conclusions: A locus influencing dyslexia risk is probably located between markers DXS1227 and DXS8091 on the X chromosome, closely situated to a locus indicated by a published genome scan of English sibling pairs. Although the locus may not be a common cause for dyslexia, the relatively small and gene poor region offers hope to identify the responsible gene.

A genome-wide association study of rheumatoid arthritis without antibodies against citrullinated peptides

Introduction Rheumatoid arthritis (RA) patients can be classified based on presence or absence of anticitrullinated peptide antibodies (ACPA) in their serum. This heterogeneity among patients may reflect important biological differences underlying the disease process. To date, the majority of genetic studies have focused on the ACPA-positive group. Therefore, our goal was to analyse the genetic risk factors that contribute to ACPA-negative RA. Methods We performed a large-scale genome-wide association study (GWAS) in three Caucasian European cohorts comprising 1148 ACPA-negative RA patients and 6008 controls. All patients were screened using the Illumina Human Cyto-12 chip, and controls were genotyped using different genome-wide platforms. Population-independent analyses were carried out by means of logistic regression. Meta-analysis with previously published data was performed as follow-up for selected signals (reaching a total of 1922 ACPA-negative RA patients and 7087 controls). Imputation of classical HLA alleles, amino acid residues and single nucleotide polymorphisms was undertaken. Results The combined analysis of the studied cohorts resulted in identification of a peak of association in the HLA-region and several suggestive non-HLA associations. Meta-analysis with previous reports confirmed the association of the HLA region with this subset and an observed association in the CLYBL locus remained suggestive. The imputation and deep interrogation of the HLA region led to identification of a two amino acid model (HLA-B at position 9 and HLA-DRB1 at position 11) that accounted for the observed genome-wide associations in this region. Conclusions Our study shed light on the influence of the HLA region in ACPA-negative RA and identified a suggestive risk locus for this condition.

Clinical and genetic aspects of PCDH19-related epilepsy syndromes and the possible role of PCDH19 mutations in males with autism spectrum disorders

Epilepsy and mental retardation limited to females (EFMR), caused by PCDH19 mutations, has a variable clinical expression that needs further exploration. Onset of epilepsy may be provoked by fever and can resemble Dravet syndrome. Furthermore, transmitting males have no seizures, but are reported to have rigid personalities suggesting possible autism spectrum disorders (ASD). Therefore, this study aimed to determine the phenotypic spectrum associated with PCDH19 mutations in Dravet-like and EFMR female patients and in males with ASD. We screened 120 females suffering from Dravet-like epilepsy, 136 females with EFMR features and 20 males with ASD. Phenotypes and genotypes of the PCDH19 mutation carriers were compared with those of 125 females with EFMR reported in the literature. We report 15 additional patients with a PCDH19 mutation. Review of clinical data of all reported patients showed that the clinical picture of EFMR is heterogeneous, but epilepsy onset in infancy, fever sensitivity and occurrence of seizures in clusters are key features. Seizures remit in the majority of patients during teenage years. Intellectual disability and behavioural disturbances are common. Fifty percent of all mutations are missense mutations, located in the extracellular domains only. Truncating mutations have been identified in all protein domains. One ASD proband carried one missense mutation predicted to have a deleterious effect, suggesting that ASD in males can be associated with PCDH19 mutations.

Association of the TGF-beta receptor genes with abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a multifactorial condition. The transforming growth factor β (TGF-β) pathway regulates vascular remodeling and mutations in its receptor genes, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysm (TAA). The TGF-β pathway may be involved in aneurysm development in general. We performed an association study by analyzing all the common genetic variants in TGFBR1 and TGFBR2 using tag single nucleotide polymorphisms (SNPs) in a Dutch AAA case–control population in a two-stage genotyping approach. In stage 1, analyzing 376 cases and 648 controls, three of the four TGFBR1 SNPs and nine of the 28 TGFBR2 SNPs had a P<0.07. Genotyping of these SNPs in an independent cohort of 360 cases and 376 controls in stage 2 confirmed association (P<0.05) for the same allele of one SNP in TGFBR1 and two SNPs in TGFBR2. Joint analysis of the 736 cases and 1024 controls showed statistically significant associations of these SNPs, which sustained after proper correction for multiple testing (TGFBR1 rs1626340 OR 1.32 95% CI 1.11–1.56 P=0.001 and TGFBR2 rs1036095 OR 1.32 95% CI 1.12–1.54 P=0.001 and rs4522809 OR 1.28 95% CI 1.12–1.46 P=0.0004). We conclude that genetic variations in TGFBR1 and TGFBR2 associate with AAA in the Dutch population. This suggests that AAA may develop partly by similar defects as TAA, which in the future may provide novel therapeutic options.

Erythematous nodes, urticarial rash and arthralgias in a large pedigree with NLRC4‐related autoinflammatory disease, expansion of the phenotype

DEAR EDITOR, Autoinflammatory disorders (AID) are a heterogeneous group of diseases, characterized by an unprovoked innate immune response, resulting in recurrent or ongoing systemic inflammation and fever. Inflammasomes are protein complexes with an essential role in pyroptosis and the caspase1-mediated activation of the proinflammatory cytokines interleukin (IL)-1b, IL-17 and IL-18 (reviewed in Mariathasan and Monack and Lamkanfi and Dixit). Excessive activation of inflammasomes results in systemic autoinflammatory disease. Various inflammasomes have been identified, of which the NLRP3 inflammasome is most widely studied. Gain-of-function mutations in the NLRP3 gene are associated with cryopyrinassociated periodic syndromes (CAPS), with urticarial skin rash as one of the hallmarks. Recently, gain-of-function mutations in NLRC4 (IPAF/CARD12) were found to associate with a clinically heterogeneous AID, characterized by recurrent episodes of fever, periodic urticarial rash, enterocolitis, splenomegaly and macrophage activation syndrome. Five different mutations in NLRC4 have been reported (reviewed in Vance). Gastrointestinal symptoms and haemophagocytosis distinguish the NLRC4-associated phenotype from CAPS. In this study, we describe the phenotype and skin histology of a large pedigree with 13 affected family members due to a novel mutation in NLRC4 (pedigree in Supplementary Fig. S1). Disease onset occurred in infancy in all patients. Symptoms could be triggered by changes in weather conditions, emotional stress and infection. Disease episodes were characterized by skin lesions, conjunctivitis, arthralgias and – in two patients – colitis. Skin lesions, the predominant feature, varied in severity and localization. In contrast to previous reports, we observed different skin phenotypes between the paediatric and adult patients. The adult patients had painful erythematous nodes on their lower legs and feet, either isolated or in combination with urticarial patches on arms, legs, trunk and face. In two patients, the soles were involved, which had not been reported before in AIDs. Remarkably, the paediatric cases in our cohort suffered from urticarial rash only (Fig. 1a). One patient had enterocolitis with histological signs of inflammatory bowel disease, a feature that was previously described. Histological evaluation of colon biopsies revealed active inflammation, with moderate infiltration of neutrophilic granulocytes and a submucosal mononuclear infiltrate. In contrast to the other reports where neonatal-onset enterocolitis spontaneously resolved after the age of 1 year, the onset of enterocolitis in our patient was in adulthood, with a chronic course. The low number of patients with gastrointestinal symptoms in this family is remarkable, as enterocolitis was thought to distinguish the NLRC4-associated AID from CAPS. Haemophagocytosis was not observed. Response to treatment with an IL-1 receptor antagonist (anakinra) varied (Table 1). A detailed description of the methods is given in Supplementary Methods S1. Whole exome sequencing revealed the novel heterozygous c.1333T>C (p.Ser445Pro) variant in NLRC4 in all affected family members. This variant is absent in the dbSNP or ExAC databases. Prediction software programs (Sift score 0 01, Polyphen-2 score 0 997) indicate that this mutation is probably damaging. The mutation is located next to the recently described pathogenic c.1328A>C (p.His443Pro) mutation and segregated with the disease phenotype (LOD score 3 58). Serum analysis of IL-1b, IL-6, IL-10, IL-18, tumour necrosis factor (TNF)-a and interferon-c showed extremely elevated IL-18 concentrations in eight studied patients (median 4324 pg mL , range 3097–13 984 pg mL , normal 0–34 pg mL ). Concentrations of the other cytokines were in the normal range. Skin biopsies were taken from nodes on the shins and/or calf of three adult patients. Histopathological examination in patient V5 showed a deep dermal and subcutaneous lymphocytic–histiocytic infiltrate with (para)septal and lobular panniculitis. In patient VI5, a perivascular infiltrate of lymphocytes and perivascular oedema was observed. Patient VI1 showed a perivascular and interstitial infiltrate of lymphocytes, and in the deep dermis an infiltrate of lymphocytes, histiocytes and a few mast cells (Fig. 1b). Vasculitis was absent in all samples. Skin biopsies of clinically uninvolved skin (V5 and VI1) showed no abnormalities (not shown). Skin biopsies in CAPS and in the related Schnitzler syndrome mostly show a neutrophilic dermal infiltrate without vasculitis. The absence of a neutrophilic infiltrate in the skin biopsies of our patients is remarkable. Immunofluorescence studies did not detect IL-1b in the skin biopsies, whereas it was found in mast cells in a skin biopsy from a patient with Schnitzler syndrome (data not shown). NLRC4 could not be detected in affected or unaffected skin (not shown), whereas it was present in the positive control (spleen).

A novel mutation in NLRC4 in a large pedigree with an anakinra responsive autoinflammatory disease

Autoinflammatory disorders (AID) are characterized by chronic or recurrent systemic inflammation associated with various clinical presentations. It is a genetically heterogeneous group of diseases. Recently, gain of function mutations in NLRC4 have been described to be associated with autoinflammatory disease. Here, we report a novel NLRC4 mutation in a large pedigree with an anakinra responsive autoinflammaotry disease.

OP0021 Genetic Factors for the Severity of ACPA-Negative Rheumatoid Arthritis in Two Cohorts of Early Disease: A Genome-Wide Study

Background ACPA-negative and ACPA-positive Rheumatoid Arthritis (RA) are increasingly regarded as separate clinical entities. Although ACPA-negative patients have a less severe disease course at group level, considerable inter-individual differences in the amount of joint destruction occur. Objectives As no studies focusing on genetic risk factors underlying the differences in joint destruction in ACPA-negative patients have been performed thus far, we performed the present study. Methods A Genome-Wide Association Study was performed using Illumina Human CytoSNP-12v2 in relation to radiographic joint destruction in 276 ACPA-negative early RA-patients included in the Leiden Early Arthritis Clinic (EAC). According to the Bonferroni correction on the number of tested SNPs, the threshold for genome wide significance was p<2x10-7. Subsequently, the significant SNPs were evaluated for association with the progression of radiographic joint destruction in 253 ACPA-negative early RA-patients included in the BARFOT-study. As 11 uncorrelated SNPs were tested, the Bonferroni threshold for significance was 0.0045. In all patients, joint destruction was measured by Sharp-van der Heijde Score with good reproducibility. Results 33 SNPs associated significantly to the severity of joint damage (p<2x10-7) in phase-1. In phase-2, two SNPs showed a trend towards a significant association with joint damage, rs2833522 (p=0.0049) and rs17763915 (p=0.047). A combined analysis of both the Leiden and BARFOT datasets of rs2833522 showed a highly significant association with joint destruction (p=3.57x10-9), the presence of the minor allele associated with more severe damage. Conclusions Rs2833522 might be associated with the severity of joint damage in ACPA-negative RA. Larger, longitudinal, studies are needed for confirmation. Acknowledgements This work is supported by the Masterswitch project and BtheCure project. The work of Annette van der Helm-van Mil is supported by the Dutch organization for scientific research (ZonMW). The Dutch Arthritis Foundation (Reumafonds) provided financial support for the genotyping. Disclosure of Interest None Declared