Survival of autoreactive T lymphocytes by microRNA-mediated regulation of apoptosis through TRAIL and Fas in type 1 diabetes.

Autoreactive CD8(+) T cells recognizing autoantigens expressed by pancreatic islets lead to the destruction of insulin-producing beta cells in type 1 diabetes (T1D), but these T cells also occur in healthy subjects. We tested the hypothesis that uncontrolled expansion of diabetogenic T cells in patients occurs, resulting from failure to activate apoptosis. We compared function, transcriptome and epigenetic regulation thereof in relation with fate upon repeated exposure to islet-autoantigen of islet autoreactive T cells from healthy and type 1 diabetic donors with identical islet epitope specificity and HLA-A2 restriction. Patient's T cells proliferated exponentially, whereas those of non-diabetic origin succumbed to cell death. Transcriptome analysis revealed reduced expression of TRAIL, TRAIL-R2, FAS and FASLG (members of the extrinsic apoptosis pathway) in patient-derived compared with healthy donor-derived T cells. This was mirrored by increased expression of microRNAs predicted to regulate these particular genes, namely miR-98, miR-23b and miR-590-5p. Gene-specific targeting by these microRNAs was confirmed using dual-luciferase reporter assays. Finally, transfection of these microRNAs into primary T cells reduced FAS and TRAIL mRNA underscoring their functional relevance. We propose that repression of pro-apoptotic pathways by microRNAs contributes to unrestricted expansion of diabetogenic cytotoxic T cells, implicating microRNA-mediated gene silencing in islet autoimmunity in T1D.

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.

Gain of FAM123B and ARHGEF9 in an Obese Man with Intellectual Disability, Congenital Heart Defects and Multiple Supernumerary Ring Chromosomes.

In a 24-year-old man with mild intellectual disability, congenital heart defects and obesity, we identified up to 4 small supernumerary marker chromosomes (sSMCs) in blood metaphases. The ring-shaped sSMCs were derived from chromosomes 11, 12 and X as well as a fourth, unidentified chromosome. In interphase nuclei of epithelial cells from the urinary tract and buccal mucosa, the presence of the r(11), r(12) and r(X) was confirmed by FISH. Using Illumina Infinium 317K SNP-arrays, we detected 3 copies of the pericentromeric regions of chromosomes 11, 12 and X. The r(X) was present in 84-89% of cells in the various tissues examined, lacks the XIST gene, but contains FAM123B, a potential dosage-sensitive candidate gene for congenital cardiac abnormalities, and ARHGEF9, a candidate gene for intellectual disability. ARHGEF9 encodes collybistin (CB), which is required for localization of the inhibitory receptor-anchoring protein gephyrin and for formation and maintenance of postsynaptic GABAA and glycine receptors. We propose that the 2-fold increase in dosage of ARHGEF9 disturbs the stoichiometry of CB with its interacting proteins at inhibitory postsynapses. SNP alleles and short tandem repeat markers on the r(11) and r(X) were compatible with a maternal origin of both sSMCs through a meiosis II error. The sSMCs may have resulted from predivision chromatid nondisjunction, leading to anaphase lagging, followed by incomplete degradation of the supernumerary chromosomes.

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

Abdominal aortic aneurysm (AAA) is a multifactorial condition. The transforming growth factor beta (TGF-beta) pathway regulates vascular remodeling and mutations in its receptor genes, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysm (TAA). The TGF-beta 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.

Copy number changes of the microcephalin 1 gene (MCPH1) in patients with autism spectrum disorders.

Autism spectrum disorder (ASD) represents a set of neurodevelopmental disorders with a strong genetic aetiology. Chromosomal rearrangements have been detected in 5-10% of the patients with ASD, and recent applications of array comparative genomic hybridisation (aCGH) are identifying further candidate regions and genes. In this study, we present four patients who implicate microcephalin 1 (MCPH1) in band 8p23.1 as an ASD susceptibility gene. Patient 1 was a girl with a syndromic form of autistic disorder satisfying the Autism Diagnostic Interview-Revised (ADI-R), Autism Diagnostic Observation Schedule (ADOS) and Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria. Oligonucleotide aCGH (oaCGH) showed that she had a classic inv dup del(8)(qter-> p23.1::p23.1-> p21.2) containing at least three candidate genes; MCPH1 and DLGAP2 within the 6.9-Mb terminal deletion and NEF3 within the concomitant 14.1-Mb duplication. Three further patients with MCPH1 copy number changes were found using single-nucleotide polymorphism (SNP) array analysis in a cohort of 54 families with ASD patients. Our results show that ASD can be a component of the classical inv dup del(8) phenotype and identify changes in copy number of MCPH1 as a susceptibility factor for ASD in the distal short arm of chromosome 8.

Genetic variations in regulatory pathways of fatty acid and glucose metabolism are associated with obesity phenotypes: a population-based cohort study.

BACKGROUND: As nuclear receptors and transcription factors have an important regulatory function in adipocyte differentiation and fat storage, genetic variation in these key regulators and downstream pathways may be involved in the onset of obesity. OBJECTIVE: To explore associations between single nucleotide polymorphisms (SNPs) in candidate genes from regulatory pathways that control fatty acid and glucose metabolism, and repeated measurements of body mass index (BMI) and waist circumference in a large Dutch study population. METHODS: Data of 327 SNPs across 239 genes were analyzed for 3575 participants of the Doetinchem cohort, who were examined three times during 11 years, using the Illumina Golden Gate assay. Adjusted random coefficient models were used to analyze the relationship between SNPS and obesity phenotypes. False discovery rate q-values were calculated to account for multiple testing. Significance of the associations was defined as a q-value < or = 0.20. RESULTS: Two SNPs (in NR1H4 and SMARCA2 in women only) were significantly associated with both BMI and waist circumference. In addition, two SNPs (in SIRT1 and SCAP in women only) were associated with BMI alone. A functional SNP, in IL6, was strongly associated with waist. CONCLUSION: In this explorative study among participants of a large population-based cohort, five SNPs, mainly located in transcription mediator genes, were strongly associated with obesity phenotypes. The results from whole genome and candidate gene studies support the potential role of NR1H4, SIRT1, SMARCA2 and IL6 in obesity. Although replication of our findings and further research on the functionality of these SNPs and underlying mechanism is necessary, our data indirectly suggest a role of GATA transcription factors in weight control.

An association screen of myelin-related genes implicates the chromosome 22q11 PIK4CA gene in schizophrenia.

Several lines of evidence, including expression analyses, brain imaging and genetic studies suggest that the integrity of myelin is disturbed in schizophrenia patients. In this study, we first reconstructed a pathway of 138 myelin-related genes, all involved in myelin structure, composition, development or maintenance. Then we performed a two-stage association analysis on these 138 genes using 771 single nucleotide polymorphisms (SNPs). Analysis of our data from 310 cases vs 880 controls demonstrated association of 10 SNPs from six genes. Specifically, we observed highly significant P-values for association in PIK4CA (observed P=6.1 x 10(-6)). These findings remained significant after Bonferroni correction for 771 tests. The PIK4CA gene is located in the chromosome 22q11 deletion syndrome region, which is of particular interest because it has been implicated in schizophrenia. We also report weak association of SNPs in PIK3C2G, FGF1, FGFR1, ARHGEF10 and PSAP (observed P

Chicken single nucleotide polymorphism identification and selection for genetic mapping.

Single nucleotide polymorphisms (SNP) are the ideal markers for high-density genome wide mapping. A total of 327,000 expressed sequence tag (EST) sequences, obtained from the ChickEST project, were examined for the presence of SNP. A total of 32,268 potential chicken SNP were identified and stored in a customized Microsoft Access database and evaluated in silico for their usability for a high-density genetic map. Based on a minimum of 3 for the minor allele occurrence and a minimum of 30% for the minor allele frequency, 5,332 reliable SNP were selected, of which both SNP alleles were present in the database at a high frequency. To test the usefulness of the in silico SNP identification, 24 SNP affecting a BglII site were used for a genotyping study. A functional PCR assay could be designed for 21 of the 24 SNP. It was possible to validate 90% of this marker subset (21 SNP) by BglII restriction analysis. The high percentage of validated markers demonstrates the reliability of the 5,332 chicken SNP markers. Furthermore, the limited number of genomic DNA samples necessary to validate 90% of the SNP markers confirmed the prediction of the high frequency at which both alleles of the selected SNP were present in the tested chicken populations.

A whole-genome scan in 164 Dutch sib pairs with attention-deficit/hyperactivity disorder: suggestive evidence for linkage on chromosomes 7p and 15q.

A genome scan was performed on 164 Dutch affected sib pairs (ASPs) with attention-deficit/hyperactivity disorder (ADHD). All subjects were white and of Dutch descent and were phenotyped according to criteria set out in the Diagnostic and Statistical Manual Of Mental Disorders, 4th edition. Initially, a narrow phenotype was defined, in which all the sib pairs met the full ADHD criteria (117 ASPs). In a broad phenotype, additional sib pairs were included, in which one child had an autistic-spectrum disorder but also met the full ADHD criteria (164 ASPs). A set of 402 polymorphic microsatellite markers with an average intermarker distance of 10 cM was genotyped and analyzed using the Mapmaker/sibs program. Regions with multipoint maximum likelihood scores (MLSs) >1.5 in both phenotypes were fine mapped with additional markers. This genome scan indicated several regions of interest, two of which showed suggestive evidence for linkage. The most promising chromosome region was located at 15q, with an MLS of 3.54 under the broad phenotype definition. This region was previously implicated in reading disability and autism. In addition, MLSs of 3.04 and 2.05 were found for chromosome regions 7p and 9q in the narrow phenotype. Except for a region on chromosome 5, no overlap was found with regions mentioned in the only other independent genome scan in ADHD reported to date.

The cps locus of Streptococcus suis serotype 2: genetic determinant for the synthesis of sialic acid.

The capsule of S. suis serotype 2 is composed of glucose, galatose, N-acetylglucosamine, rhamnose and sialic acid. Recently, we described a major part of the cps2 locus of S. suis serotype 2. Based on sequence homology genes encoding potential glucosyl-, galactosyl-, N-acetylglucosaminyl- and rhamnosyltransferase activities could be identified. However, we did not find genes involved in the synthesis of sialic acid. Here, we describe the cloning and characterization of a remaining part of the cps2 locus. Based on the establish sequence 11 potential genes, designated orf2L, orf2M, orf2N, cps2O to cps2T, orf2U and orf2V were identified. A gene homologous to genes involved in the polymerization of the repeating oligosaccharide unit (cps2O) as well as genes involved in the synthesis of sialic acid (cps2P to cps2T) were identified. Moreover, hybridizing experiments showed that the genes involved in the sialic acid synthesis are present in S. suis serotype 1, 2, 14, 27 and 1/2. The orf2M and orf2N regions showed similarity to proteins involved in the polysaccharide biosynthesis of other Gram-positive bacteria. However, these regions seemed to be truncated or were non-functional as the result of frame-shift or point mutations. At its 3;-end the cps2 locus contained two insertional elements (orf2U and orf2V), both of which seemed to be non-functional.