Elevated Levels of the Cytokine LIGHT in Pediatric Crohn's Disease.

LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes), encoded by the TNFSF14 gene, is a cytokine belonging to the TNF superfamily. On binding to its receptors, herpes virus entry mediator and lymphotoxin ß receptor, it activates inflammatory responses. We conducted this study to determine whether plasma LIGHT levels are elevated in Crohn's disease (CD) in a pediatric population with the aim of nominating this cytokine as a therapeutic target. We used a single-molecule immunoassay to determine the circulating levels of free LIGHT in plasma from pediatric patients with CD in our biobank (n = 183), a panel of healthy pediatric (n = 9) or adult (n = 22) reference samples, and pediatric biobank controls (n = 19). We performed correlational analyses between LIGHT levels and the clinical characteristics of the CD cohort, including age, Montreal classification, family history, medical/surgical therapy, and routine blood test parameters. LIGHT levels were greatly elevated in CD, with an average of 305 versus 32.4 pg/ml for controls from the biobank (p < 0.0001). The outside reference samples showed levels of 57 pg/ml in pediatric controls and 55 pg/ml in adults (p < 0.0001). We found a statistically significant correlation between white blood cell count and free LIGHT (p < 0.046). We conclude that free, soluble LIGHT is increased 5- to 10-fold in pediatric CD across an array of disease subtypes and characteristics.

Novel insights into the phenotypic spectrum and pathogenesis of Hardikar syndrome.

PURPOSE: Hardikar syndrome (HS, MIM #301068) is a female-specific multiple congenital anomaly syndrome characterized by retinopathy, orofacial clefting, aortic coarctation, biliary dysgenesis, genitourinary malformations, and intestinal malrotation. We previously showed that heterozygous nonsense and frameshift variants in MED12 cause HS. The phenotypic spectrum of disease and the mechanism by which MED12 variants cause disease is unknown. We aim to expand the phenotypic and molecular landscape of HS and elucidate the mechanism by which MED12 variants cause disease. METHODS: We clinically assembled and molecularly characterized a cohort of 11 previously unreported individuals with HS. Additionally, we studied the effect of MED12 deficiency on ciliary biology, hedgehog, and yes-associated protein (YAP) signaling; pathways implicated in diseases with phenotypic overlap with HS. RESULTS: We report novel phenotypes associated with HS, including cardiomyopathy, arrhythmia, and vascular anomalies, and expand the molecular landscape of HS to include splice site variants. We additionally demonstrate that MED12 deficiency causes decreased cell ciliation, and impairs hedgehog and YAP signaling. CONCLUSION: Our data support updating HS standard-of-care to include regular cardiac imaging, arrhythmia screening, and vascular imaging. We further propose that dysregulation of ciliogenesis and YAP and hedgehog signaling contributes to the pathogenesis of HS.

A novel MBTPS2 variant associated with BRESHECK syndrome impairs sterol-regulated transcription and the endoplasmic reticulum stress response.

Ichthyosis follicularis, atrichia, and photophobia syndrome (IFAP syndrome) is a rare, X-linked disorder caused by pathogenic variants in membrane-bound transcription factor protease, site 2 (MBTPS2). Pathogenic MBTPS2 variants also cause BRESHECK syndrome, characterized by the IFAP triad plus intellectual disability and multiple congenital anomalies. Here we present a patient with ichthyosis, sparse hair, pulmonic stenosis, kidney dysplasia, hypospadias, growth failure, thrombocytopenia, anemia, bone marrow fibrosis, and chronic diarrhea found by research-based exome sequencing to harbor a novel, maternally inherited MBTPS2 missense variant (c.766 G>A; (p.Val256Leu)). In vitro modeling supports variant pathogenicity, with impaired cell growth in cholesterol-depleted media, attenuated activation of the sterol regulatory element-binding protein pathway, and failure to activate the endoplasmic reticulum stress response pathway. Our case expands both the genetic and phenotypic spectrum of BRESHECK syndrome to include a novel MBTPS2 variant and cytopenias, bone marrow fibrosis, and chronic diarrhea.

Association of a rare NOTCH4 coding variant with systemic sclerosis: a family-based whole exome sequencing study.

BACKGROUND: Systemic sclerosis (SSc) is a rheumatologic disease with a multifactorial etiology. Genome-wide association studies imply a polygenic, complex mode of inheritance with contributions from variation at the human leukocyte antigen locus and non-coding variation at a locus on chromosome 6p21, among other modestly impactful loci. Here we describe an 8-year-old female proband presenting with diffuse cutaneous SSc/scleroderma and a family history of SSc in a grandfather and maternal aunt. METHODS: We employed whole exome sequencing (WES) of three members of this family. We examined rare missense, nonsense, splice-altering, and coding indels matching an autosomal dominant inheritance model. We selected one missense variant for Sanger sequencing confirmation based on its predicted impact on gene function and location in a known SSc genetic locus. RESULTS: Bioinformatic analysis found eight candidate variants meeting our criteria. We identified a very rare missense variant in the regulatory NODP domain of NOTCH4 located at the 6p21 locus, c.4245G > A:p.Met1415Ile, segregating with the phenotype. This allele has a frequency of 1.83 × 10-5 by the data of the Exome Aggregation Consortium. CONCLUSION: This family suggests a novel mechanism of SSc pathogenesis in which a rare and penetrant coding variation can substantially elevate disease risk in contrast to the more modest non-coding variation typically found at this locus. These results suggest that modulation of the NOTCH4 gene might be responsible for the association signal at chromosome 6p21 in SSc.

Genome-wide association study of the age of onset of type 1 diabetes reveals HTATIP2 as a novel T cell regulator.

Introduction: Type 1 diabetes, a disorder caused by autoimmune destruction of pancreatic insulin-producing cells, is more difficult to manage when it presents at a younger age. We sought to identify genetic correlates of the age of onset by conducting the first genome-wide association study (GWAS) treating the age of first diagnosis as a quantitative trait. Methods: We performed GWAS with a discovery cohort of 4,014 cases and a replication cohort of 493 independent cases. Genome-wide significant SNPs were mapped to a causal variant by Bayesian conditional analysis and gel shift assay. The causal protein-coding gene was identified and characterized by RNA interference treatment of primary human pan-CD4+ T cells with RNA-seq of the transcriptome. The candidate gene was evaluated functionally in primary cells by CD69 staining and proliferation assays. Results: Our GWAS replicated the known association of the age of diagnosis with the human leukocyte antigen complex (HLA-DQB1). The second signal identified was in an intron of the NELL1 gene on chromosome 11 and fine-mapped to variant rs10833518 (P < 1.54 × 10-9). Homozygosity for the risk allele leads to average age of onset one year earlier. Knock-down of HIV TAT-interacting protein 2 (HTATIP2), but not other genes in the locus, resulted in alterations to gene expression in signal transduction pathways including MAP kinases and PI3-kinase. Higher levels of HTATIP2 expression are associated with increased viability, proliferation, and activation of T cells in the presence of signals from antigen and cytokine receptors. Discussion: This study implicates HTATIP2 as a new type 1 diabetes gene acting via T cell regulation. Larger population sample sizes are expected to reveal additional loci.

Regulation of Janus Kinase 2 by an Inflammatory Bowel Disease Causal Non-coding Single Nucleotide Polymorphism.

BACKGROUND AND AIMS: Among the >240 genetic loci described to date which confer susceptibility to inflammatory bowel disease, a small subset have been fine-mapped to an individual, non-coding single nucleotide polymorphism [SNP]. To illustrate a model mechanism by which a presumed-causal non-coding SNP can function, we analysed rs1887428, located in the promoter region of the Janus kinase 2 [JAK2] gene. METHODS: We utilized comparative affinity purification-mass spectrometry, DNA-protein binding assays, CRISPR/Cas9 genome editing, transcriptome sequencing and methylome quantitative trait locus methods to characterize the role of this SNP. RESULTS: We determined that the risk allele of rs1887428 is bound by the transcription factor [TF] RBPJ, while the protective allele is bound by the homeobox TF CUX1. While rs188748 only has a very modest influence on JAK2 expression, this effect was amplified downstream through the expression of pathway member STAT5B and epigenetic modification of the JAK2 locus. CONCLUSION: Despite the absence of a consensus TF-binding motif or expression quantitative trait locus, we have used improved methods to characterize a putatively causal SNP to yield insight into inflammatory bowel disease mechanisms. PODCAST: This article has an associated podcast which can be accessed at https://academic.oup.com/ecco-jcc/pages/podcast.

Multiple Epistasis Interactions Within MHC Are Associated With Ulcerative Colitis.

Successful searching for epistasis is much challenging, which generally requires very large sample sizes and/or very dense marker information. We exploited the largest Crohn's disease (CD) dataset (18,000 cases + 34,000 controls) and ulcerative colitis (UC) dataset (14,000 cases + 34,000 controls) to date. Leveraging its dense marker information and the large sample size of this IBD dataset, we employed a two-step approach to exhaustively search for epistasis. We detected abundant genome-wide significant (p < 1 × 10-13) epistatic signals, all within the MHC region. These signals were reduced substantially when conditional on the additive background, but still nine pairs remained significant at the Immunochip-wide level (P < 1.1 × 10-8) in conditional tests for UC. All these nine epistatic interactions come from the MHC region, and each explains on average 0.15% of the phenotypic variance. Eight of them were replicated in a replication cohort. There are multiple but relatively weak interactions independent of the additive effects within the MHC region for UC. Our promising results warrant the search for epistasis in large data sets with dense markers, exploiting dependencies between markers.

The B cell SH2/PH domain-containing adaptor Bam32/DAPP1 is required for T cell-independent II antigen responses.

BACKGROUND: Bam32/DAPP1 is a B cell adaptor composed of both a PH and an SH2 domain. Previous studies in cell culture and chicken DT40 cells have indicated that Bam32 is critical for normal signaling downstream of the B cell receptor (BCR). RESULTS: We now study the function of Bam32 in mice in which Bam32 has been disrupted by a viral gene trap approach. Although B and T cell development is normal in Bam32(-/-) mice, B cell proliferation is reduced by about 50% after BCR crosslinking when compared with Bam32(+/+) mice. Differences in the activation of Erk, Jnk and p38 Map kinases, PLCgamma, and Ca(2+) flux do not account for the defect in proliferation as activation was similar in Bam32(+/+) and Bam32(-/-) B cells. Interestingly, whereas antibody response to T-dependent (TD) and T-independent (TI)-I antigens was similar between Bam32(+/+) and Bam32(-/-) mice, TI-II responses were defective in Bam32(-/-) mice; Bam32(-/-) mice failed to undergo isotype class switch recombination (CSR) to produce IgG3 antibodies due to a cell-autonomous defect in generation of IgG3 germline transcripts. The defect in TI-II antigen response led to an impaired antibody response to immunization with type 3 Streptococcus pneumoniae capsular polyschaccharide (PS), resulting in a markedly increased susceptibility to infection by Streptococcus pneumoniae. CONCLUSIONS: These findings indicate that Bam32 specifically couples an upstream signal to the IgG3 isotype heavy chain CSR and suggest that defects in Bam32 may account for the increased susceptibility to encapusulated organisms in a subset of immunodeficient patients.

Pathway-based Genome-wide Association Studies Reveal the Association Between Growth Factor Activity and Inflammatory Bowel Disease.

BACKGROUND: The inflammatory bowel diseases known as Crohn's disease (CD) and ulcerative colitis (UC) are related autoimmune conditions with a complex etiology composed of genetic and environmental factors. Genetic studies have revealed 200 susceptibility loci for inflammatory bowel diseases, but these only account for a small fraction of the genetic heritability of the disease. We employed pathway-based approaches to identify genes that cooperatively make contributions to the genetic etiology of CD. METHODS: We exploited the largest CD dataset (20,000 cases + 28,000 controls) and UC dataset (17,000 cases + 33,500 controls) to date. We conducted a meta-analysis of 5 CD cohorts of European ancestry using 3 pathway-based approaches and further performed replication studies in an independent cohort genotyped on the Immunochip and in another pediatric cohort of European ancestry. Similar meta-analysis was performed for UC cohorts. RESULTS: In addition to the multiple immune-related pathways that have been implicated in the genetic etiology of inflammatory bowel diseases before, we found significant associations involving genes in growth factor signaling for CD. This result was replicated in 2 independent cohorts of European ancestry. This association with growth factor activity is not unique to CD. We found a similar significant association with UC cohorts. CONCLUSIONS: Our findings suggest that genes involved in pathways of growth factor signaling may make joint contributions to the etiology of CD and UC, providing novel insight into the genetic mechanisms of these diseases.

CNV Analysis Associates AKNAD1 with Type-2 Diabetes in Jordan Subpopulations.

Previous studies have identified a number of single nucleotide polymorphisms (SNPs) associated with type-2 diabetes (T2D), but copy number variation (CNV) association has rarely been addressed, especially in populations from Jordan. To investigate CNV associations for T2D in populations in Jordan, we conducted a CNV analysis based on intensity data from genome-wide SNP array, including 34 T2D cases and 110 healthy controls of Chechen ethnicity, as well as 34 T2D cases and 106 healthy controls of Circassian ethnicity. We found a CNV region in protein tyrosine phosphatase receptor type D (PTPRD) with significant association with T2D. PTPRD has been reported to be associated with T2D in genome-wide association studies (GWAS). We additionally identified 16 CNV regions associated with T2D which overlapped with gene exons. Of particular interest, a CNV region in the gene AKNA Domain Containing 1 (AKNAD1) surpassed the experiment-wide significance threshold. Endoplasmic reticulum (ER)-related pathways were significantly enriched among genes which are predicted to be functionally associated with human or mouse homologues of AKNAD1. This is the first CNV analysis of a complex disease in populations of Jordan. We identified and experimentally validated a significant CNVR in gene AKNAD1 associated with T2D.

Meta-analysis of shared genetic architecture across ten pediatric autoimmune diseases.

Genome-wide association studies (GWASs) have identified hundreds of susceptibility genes, including shared associations across clinically distinct autoimmune diseases. We performed an inverse χ(2) meta-analysis across ten pediatric-age-of-onset autoimmune diseases (pAIDs) in a case-control study including more than 6,035 cases and 10,718 shared population-based controls. We identified 27 genome-wide significant loci associated with one or more pAIDs, mapping to in silico-replicated autoimmune-associated genes (including IL2RA) and new candidate loci with established immunoregulatory functions such as ADGRL2, TENM3, ANKRD30A, ADCY7 and CD40LG. The pAID-associated single-nucleotide polymorphisms (SNPs) were functionally enriched for deoxyribonuclease (DNase)-hypersensitivity sites, expression quantitative trait loci (eQTLs), microRNA (miRNA)-binding sites and coding variants. We also identified biologically correlated, pAID-associated candidate gene sets on the basis of immune cell expression profiling and found evidence of genetic sharing. Network and protein-interaction analyses demonstrated converging roles for the signaling pathways of type 1, 2 and 17 helper T cells (TH1, TH2 and TH17), JAK-STAT, interferon and interleukin in multiple autoimmune diseases.

Genetic sharing and heritability of paediatric age of onset autoimmune diseases.

Autoimmune diseases (AIDs) are polygenic diseases affecting 7-10% of the population in the Western Hemisphere with few effective therapies. Here, we quantify the heritability of paediatric AIDs (pAIDs), including JIA, SLE, CEL, T1D, UC, CD, PS, SPA and CVID, attributable to common genomic variations (SNP-h(2)). SNP-h(2) estimates are most significant for T1D (0.863±s.e. 0.07) and JIA (0.727±s.e. 0.037), more modest for UC (0.386±s.e. 0.04) and CD (0.454±0.025), largely consistent with population estimates and are generally greater than that previously reported by adult GWAS. On pairwise analysis, we observed that the diseases UC-CD (0.69±s.e. 0.07) and JIA-CVID (0.343±s.e. 0.13) are the most strongly correlated. Variations across the MHC strongly contribute to SNP-h(2) in T1D and JIA, but does not significantly contribute to the pairwise rG. Together, our results partition contributions of shared versus disease-specific genomic variations to pAID heritability, identifying pAIDs with unexpected risk sharing, while recapitulating known associations between autoimmune diseases previously reported in adult cohorts.

Transcriptome profiling of human ulcerative colitis mucosa reveals altered expression of pathways enriched in genetic susceptibility loci.

Human colonic mucosa altered by inflammation due to ulcerative colitis (UC) displays a drastically altered pattern of gene expression compared with healthy tissue. We aimed to understand the underlying molecular pathways influencing these differences by analyzing three publically-available, independently-generated microarray datasets of gene expression from endoscopic biopsies of the colon. Gene set enrichment analysis (GSEA) revealed that all three datasets share 87 gene sets upregulated in UC lesions and 8 gene sets downregulated (false discovery rate <0.05). The upregulated pathways were dominated by gene sets involved in immune function and signaling, as well as the control of mitosis. We applied pathway analysis to genotype data derived from genome-wide association studies (GWAS) of UC, consisting of 5,584 cases and 11,587 controls assembled from eight European-ancestry cohorts. The upregulated pathways derived from the gene expression data showed a highly significant overlap with pathways derived from the genotype data (33 of 56 gene sets, hypergeometric P = 1.49 × 10(-19)). This study supports the hypothesis that heritable variation in gene expression as measured by GWAS signals can influence key pathways in the development of disease, and that comparison of genetic susceptibility loci with gene expression signatures can differentiate key drivers of inflammation from secondary effects on gene expression of the inflammatory process.

A novel BHLHE41 variant is associated with short sleep and resistance to sleep deprivation in humans.

STUDY OBJECTIVES: Earlier work described a mutation in DEC2 also known as BHLHE41 (basic helix-loophelix family member e41) as causal in a family of short sleepers, who needed just 6 h sleep per night. We evaluated whether there were other variants of this gene in two well-phenotyped cohorts. DESIGN: Sequencing of the BHLHE41 gene, electroencephalographic data, and delta power analysis and functional studies using cell-based luciferase. RESULTS: We identified new variants of the BHLHE41 gene in two cohorts who had either acute sleep deprivation (n = 200) or chronic partial sleep deprivation (n = 217). One variant, Y362H, at another location in the same exon occurred in one twin in a dizygotic twin pair and was associated with reduced sleep duration, less recovery sleep following sleep deprivation, and fewer performance lapses during sleep deprivation than the homozygous twin. Both twins had almost identical amounts of non rapid eye movement (NREM) sleep. This variant reduced the ability of BHLHE41 to suppress CLOCK/BMAL1 and NPAS2/BMAL1 transactivation in vitro. Another variant in the same exome had no effect on sleep or response to sleep deprivation and no effect on CLOCK/BMAL1 transactivation. Random mutagenesis identified a number of other variants of BHLHE41 that affect its function. CONCLUSIONS: There are a number of mutations of BHLHE41. Mutations reduce total sleep while maintaining NREM sleep and provide resistance to the effects of sleep loss. Mutations that affect sleep also modify the normal inhibition of BHLHE41 of CLOCK/BMAL1 transactivation. Thus, clock mechanisms are likely involved in setting sleep length and the magnitude of sleep homeostasis. CITATION: Pellegrino R, Kavakli IH, Goel N, Cardinale CJ, Dinges DF, Kuna ST, Maislin G, Van Dongen HP, Tufik S, Hogenesch JB, Hakonarson H, Pack AI. A novel BHLHE41 variant is associated with short sleep and resistance to sleep deprivation in humans. SLEEP 2014;37(8):1327-1336.

Impact of exome sequencing in inflammatory bowel disease.

Approaches to understanding the genetic contribution to inflammatory bowel disease (IBD) have continuously evolved from family- and population-based epidemiology, to linkage analysis, and most recently, to genome-wide association studies (GWAS). The next stage in this evolution seems to be the sequencing of the exome, that is, the regions of the human genome which encode proteins. The GWAS approach has been very fruitful in identifying at least 163 loci as being associated with IBD, and now, exome sequencing promises to take our genetic understanding to the next level. In this review we will discuss the possible contributions that can be made by an exome sequencing approach both at the individual patient level to aid with disease diagnosis and future therapies, as well as in advancing knowledge of the pathogenesis of IBD.

Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli.

Transcription of the bacterial genome by the RNA polymerase must terminate at specific points. Transcription can be terminated by Rho factor, an essential protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits Rho, to assess its role on a genome-wide scale. Rho is revealed as a global regulator of gene expression that matches Escherichia coli transcription to translational needs. We also found that genes in E. coli that are most repressed by Rho are prophages and other horizontally acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E. coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign genes.

An allosteric path to transcription termination.

Transcription termination signals in bacteria occur in RNA as a strong hairpin followed by a stretch of U residues at the 3' terminus. To release the transcript, RNA polymerase (RNAP) is thought to translocate forward without RNA synthesis. Here we provide genetic and biochemical evidence supporting an alternative model in which extensive conformational changes across the enzyme lead to termination without forward translocation. In this model, flexible parts of the RNA exit channel (zipper, flap, and zinc finger) assist the initial step of hairpin folding (nucleation). The hairpin then invades the RNAP main channel, causing RNA:DNA hybrid melting, structural changes of the catalytic site, and DNA-clamp opening induced by interaction with the G(trigger)-loop. Our results envision the elongation complex as a flexible structure, not a rigid body, and establish basic principles of the termination pathway that are likely to be universal in prokaryotic and eukaryotic systems.