Whole genome and exome sequencing of monozygotic twins discordant for Crohn's disease

Background Crohn's disease (CD) is an inflammatory bowel disease caused by genetic and environmental factors. More than 160 susceptibility loci have been identified for IBD, yet a large part of the genetic variance remains unexplained. Recent studies have demonstrated genetic differences between monozygotic twins, who were long thought to be genetically completely identical. Results We aimed to test if somatic mutations play a role in CD etiology by sequencing the genomes and exomes of directly affected tissue from the bowel and blood samples of one and the blood-derived exomes of two further monozygotic discordant twin pairs. Our goal was the identification of mutations present only in the affected twins, pointing to novel candidates for CD susceptibility loci. We present a thorough genetic characterization of the sequenced individuals but detected no consistent differences within the twin pairs. An estimate of the CD susceptibility based on known CD loci however hinted at a higher mutational load in all three twin pairs compared to 1,920 healthy individuals. Conclusion Somatic mosaicism does not seem to play a role in the discordance of monozygotic CD twins. Our study constitutes the first to perform whole genome sequencing for CD twins and therefore provides a valuable reference dataset for future studies. We present an example framework for mosaicism detection and point to the challenges in these types of analyses

Exome analysis of rare and common variants within the NOD signaling pathway

Pediatric inflammatory bowel disease (pIBD) is a chronic heterogeneous disorder. This study looks at the burden of common and rare coding mutations within 41 genes comprising the NOD signaling pathway in pIBD patients. 136 pIBD and 106 control samples underwent whole-exome sequencing. We compared the burden of common, rare and private mutation between these two groups using the SKAT-O test. An independent replication cohort of 33 cases and 111 controls was used to validate significant findings. We observed variation in 40 of 41 genes comprising the NOD signaling pathway. Four genes were significantly associated with disease in the discovery cohort (BIRC2 p = 0.004, NFKB1 p =  0.005, NOD2 p = 0.029 and SUGT1 p = 0.047). Statistical significance was replicated for BIRC2 (p = 0.041) and NOD2 (p = 0.045) in an independent validation cohort. A gene based test on the combined discovery and replication cohort confirmed association for BIRC2 (p = 0.030). We successfully applied burden of mutation testing that jointly assesses common and rare variants, identifying two previously implicated genes (NFKB1 and NOD2) and confirmed a possible role in disease risk in a previously unreported gene (BIRC2). The identification of this novel gene provides a wider role for the inhibitor of apoptosis gene family in IBD pathogenesis

From Next-Generation Sequencing Alignments to Accurate Comparison and Validation of Single-Nucleotide Variants: The Pibase Software

Scientists working with single-nucleotide variants (SNVs), inferred by next-generation sequencing software, often need further information regarding true variants, artifacts and sequence coverage gaps. In clinical diagnostics, e.g. SNVs must usually be validated by visual inspection or several independent SNV-callers. We here demonstrate that 0.5–60% of relevant SNVs might not be detected due to coverage gaps, or might be misidentified. Even low error rates can overwhelm the true biological signal, especially in clinical diagnostics, in research comparing healthy with affected cells, in archaeogenetic dating or in forensics. For these reasons, we have developed a package called pibase, which is applicable to diploid and haploid genome, exome or targeted enrichment data. pibase extracts details on nucleotides from alignment files at user-specified coordinates and identifies reproducible genotypes, if present. In test cases pibase identifies genotypes at 99.98% specificity, 10-fold better than other tools. pibase also provides pair-wise comparisons between healthy and affected cells using nucleotide signals (10-fold more accurately than a genotype-based approach, as we show in our case study of monozygotic twins). This comparison tool also solves the problem of detecting allelic imbalance within heterozygous SNVs in copy number variation loci, or in heterogeneous tumor sequences

CEACAM1 regulates TIM-3-mediated tolerance and exhaustion

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers[superscript 1, 2, 3, 4, 5]. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition[superscript 6, 7, 8, 9, 10]. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.American Association for Cancer Research. Pancreatic Cancer Action Networ

Missense variants in NOX1 and p22phox in a case of very-early-onset inflammatory bowel disease are functionally linked to NOD2

Contains fulltext : 201167.pdf (publisher's version ) (Open Access

Whole genome and exome sequencing of monozygotic twins discordant for Crohn's disease

Background Crohn's disease (CD) is an inflammatory bowel disease caused by genetic and environmental factors. More than 160 susceptibility loci have been identified for IBD, yet a large part of the genetic variance remains unexplained. Recent studies have demonstrated genetic differences between monozygotic twins, who were long thought to be genetically completely identical. Results We aimed to test if somatic mutations play a role in CD etiology by sequencing the genomes and exomes of directly affected tissue from the bowel and blood samples of one and the blood-derived exomes of two further monozygotic discordant twin pairs. Our goal was the identification of mutations present only in the affected twins, pointing to novel candidates for CD susceptibility loci. We present a thorough genetic characterization of the sequenced individuals but detected no consistent differences within the twin pairs. An estimate of the CD susceptibility based on known CD loci however hinted at a higher mutational load in all three twin pairs compared to 1,920 healthy individuals. Conclusion Somatic mosaicism does not seem to play a role in the discordance of monozygotic CD twins. Our study constitutes the first to perform whole genome sequencing for CD twins and therefore provides a valuable reference dataset for future studies. We present an example framework for mosaicism detection and point to the challenges in these types of analyses

Improving mapping and SNP-calling performance in multiplexed targeted next-generation sequencing

Abstract Background Compared to classical genotyping, targeted next-generation sequencing (tNGS) can be custom-designed to interrogate entire genomic regions of interest, in order to detect novel as well as known variants. To bring down the per-sample cost, one approach is to pool barcoded NGS libraries before sample enrichment. Still, we lack a complete understanding of how this multiplexed tNGS approach and the varying performance of the ever-evolving analytical tools can affect the quality of variant discovery. Therefore, we evaluated the impact of different software tools and analytical approaches on the discovery of single nucleotide polymorphisms (SNPs) in multiplexed tNGS data. To generate our own test model, we combined a sequence capture method with NGS in three experimental stages of increasing complexity (E. coli genes, multiplexed E. coli, and multiplexed HapMap BRCA1/2 regions). Results We successfully enriched barcoded NGS libraries instead of genomic DNA, achieving reproducible coverage profiles (Pearson correlation coefficients of up to 0.99) across multiplexed samples, with Conclusions We recommend applying our general ‘two-step’ mapping approach for more efficient SNP discovery in tNGS. Our study has also shown the benefit of computing inter-sample SNP-concordances and inspecting read alignments in order to attain more confident results.</p

Exome Sequencing Identifies a Novel MAP3K14 Mutation in Recessive Atypical Combined Immunodeficiency

Primary immunodeficiency disorders (PIDs) render patients vulnerable to infection with a wide range of microorganisms and thus provide good in vivo models for the assessment of immune responses during infectious challenges. Priming of the immune system, especially in infancy, depends on different environmental exposures and medical practices. This may determine the timing and phenotype of clinical appearance of immune deficits as exemplified with early exposure to Bacillus Calmette-Guérin (BCG) vaccination and dissemination in combined immunodeficiencies. Varied phenotype expression poses a challenge to identification of the putative immune deficit. Without the availability of genomic diagnosis and data analysis resources and with limited capacity for functional definition of immune pathways, it is difficult to establish a definitive diagnosis and to decide on appropriate treatment. This study describes the use of exome sequencing to identify a homozygous recessive variant in MAP3K14, NIKVal345Met, in a patient with combined immunodeficiency, disseminated BCG-osis, and paradoxically elevated lymphocytes. Laboratory testing confirmed hypogammaglobulinemia with normal CD19, but failed to confirm a definitive diagnosis for targeted treatment decisions. NIKVal345Met is predicted to be deleterious and pathogenic by two in silico prediction tools and is situated in a gene crucial for effective functioning of the non-canonical nuclear factor-kappa B signaling pathway. Functional analysis of NIKVal345Met- versus NIKWT-transfected human embryonic kidney-293T cells showed that this mutation significantly affects the kinase activity of NIK leading to decreased levels of phosphorylated IkappaB kinase-alpha (IKKα), the target of NIK. BCG-stimulated RAW264.7 cells transfected with NIKVal345Met also presented with reduced levels of phosphorylated IKKα, significantly increased p100 levels and significantly decreased p52 levels compared to cells transfected with NIKWT. Ideally, these experiments would have been conducted in patient-derived immune cells, but we were unable to source these cells from the patient. The functional analysis described in this paper supports previous illustrations of the importance of NIK in human immune responses and demonstrates the involvement of function-altering mutations in MAP3K14 in PIDs. The genomic approach used for this patient demonstrates its value in the diagnosis of an unusual PID and as a tool for detecting rarer mutations to help guide treatment approaches