BACKGROUND

The molecular cause of inflammatory bowel disease is largely unknown.

METHODS

We performed genetic-linkage analysis and candidate-gene sequencing on samples from two unrelated consanguineous families with children who were affected by early-onset inflammatory bowel disease. We screened six additional patients with early-onset colitis for mutations in two candidate genes and carried out functional assays in patients' peripheral-blood mononuclear cells. We performed an allogeneic hematopoietic stem-cell transplantation in one patient.

RESULTS

In four of nine patients with early-onset colitis, we identified three distinct homozygous mutations in genes IL10RA and IL10RB, encoding the IL10R1 and IL10R2 proteins, respectively, which form a heterotetramer to make up the interleukin-10 receptor. The mutations abrogate interleukin-10-induced signaling, as shown by deficient STAT3 (signal transducer and activator of transcription 3) phosphorylation on stimulation with interleukin-10. Consistent with this observation was the increased secretion of tumor necrosis factor alpha and other proinflammatory cytokines from peripheral-blood mononuclear cells from patients who were deficient in IL10R subunit proteins, suggesting that interleukin-10-dependent "negative feedback" regulation is disrupted in these cells. The allogeneic stem-cell transplantation performed in one patient was successful.

CONCLUSIONS

Mutations in genes encoding the IL10R subunit proteins were found in patients with early-onset enterocolitis, involving hyperinflammatory immune responses in the intestine. Allogeneic stem-cell transplantation resulted in disease remission in one patient.

MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA.

BACKGROUND

Interleukin-10 (IL-10) signaling genes are attractive inflammatory bowel disease (IBD) candidate genes as IL-10 restricts intestinal inflammation, IL-10 polymorphisms have been associated with IBD in genome-wide association studies, and mutations in IL-10 and IL-10 receptor (IL-10R) genes have been reported in immunodeficient children with severe infantile-onset IBD. Our objective was to determine if IL-10R polymorphisms were associated with early-onset IBD (EO-IBD) and very-early-onset IBD (VEO-IBD).

METHODS

Candidate-gene analysis of IL10RA and IL10RB was performed after initial sequencing of an infantile onset-IBD patient identified a novel homozygous mutation. The discovery cohort included 188 EO-IBD subjects and 188 healthy subjects. Polymorphisms associated with IBD in the discovery cohort were genotyped in an independent validation cohort of 422 EO-IBD subjects and 480 healthy subjects.

RESULTS

We identified a homozygous, splice-site point mutation in IL10RA in an infantile-onset IBD patient causing a premature stop codon (P206X) and IL-10 insensitivity. IL10RA and IL10RB sequencing in the discovery cohort identified five IL10RA polymorphisms associated with ulcerative colitis (UC) and two IL10RB polymorphisms associated with Crohn's disease (CD). Of these polymorphisms, two IL10RA single nucleotide polymorphisms, rs2228054 and rs2228055, were associated with VEO-UC in the discovery cohort and replicated in an independent validation cohort (odds ratio [OR] 3.08, combined P = 2 x 10(-4); and OR 2.93, P = 6 x 10(-4), respectively).

CONCLUSIONS

We identified IL10RA polymorphisms that confer risk for developing VEO-UC. Additionally, we identified the first splice site mutation in IL10RA resulting in infantile-onset IBD. This study expands the phenotype of IL10RA polymorphisms to include both severe arthritis and VEO-UC.

OBJECTIVE

Early onset inflammatory bowel diseases (EO-IBD) developing during the first year of life are likely to reflect inherited defects in key mechanism(s) controlling intestinal homeostasis, as recently suggested for interleukin 10 (IL10). Thus, we aimed to further elaborate the hypothesis of defective anti-inflammatory responses in patients with IBD.

METHODS

The capacities of transforming growth factor β (TGFβ) and IL10 to inhibit proinflammatory cytokine production by monocyte-derived dendritic cells (MoDC) or peripheral blood cells (PBMC) was analyzed in 75 children with IBD, including 13 infants with EO-IBD (in whom autoimmune diseases or classical immunodeficiencies were ruled out). IL10 receptor-A/-B expression, STAT3 activation in response to IL6, IL10, IL21, IL22 were analyzed by FACS and western blotting. IL10RA and B genes were sequenced. The response to IL22 was tested in ileal/colonic tissue cultures. Tissue gene expression was analyzed by Taqman real-time polymerase chain reaction.

RESULTS

Production of IL10 in response to bacterial motifs was normal in all IBD patients. In contrast to our original hypothesis, no defect of the anti-inflammatory potential of TGFβ and IL10 was observed in children with IBD or EO-IBD except two infants who presented with granuloma-positive colitis at 3 months of life: no response to IL10 was observed secondary to mutations in the α (p.R262C) or β (p.E141X) chain of IL10R, respectively, although a fully functional Jak-STAT3 pathway was present in both patients. When analyzing the regulation of intestinal bacterial clearance, we detected a defect in the patient with absent IL10 RB to upregulate protective transcripts in response to IL22, whereas all other EO-IBD patients, including the patient with an abnormal α chain, responded normally.

CONCLUSIONS

Impaired IL10 signaling characterizes a subgroup of IBD patients, whereas the majority of children with severe IBD including EO forms normally produces and responds to IL10. Defective IL22 signaling may additionally impair intestinal epithelial clearance. Our data point out the complexity of IBD, which represent a group of distinct diseases with several pathogenetic abnormalities.

Interleukin 10 (IL10) is a key anti-inflammatory cytokine that can inhibit proinflammatory responses of both innate and adaptive immune cells. An association between IL10 and intestinal mucosal homeostasis became clear with the discovery that IL10 and IL10 receptor (IL10R)-deficient mice develop spontaneous intestinal inflammation. Similarly, patients with deleterious mutations in IL10, IL10RA, or IL10RB present with severe enterocolitis within the first months of life. Here, we review recent findings on how IL10- and IL10R-dependent signaling modulates innate and adaptive immune responses in the murine gastrointestinal tract, with implications of their role in the prevention of inflammatory bowel disease (IBD). In addition, we discuss the impact of IL10 and IL10R signaling defects in humans and their relationship to very early-onset IBD (VEO-IBD).

Interactions between environment and immune system play an essential role in the aetiology of immunopathologies, including lymphomas. Toll-like receptors (TLR) belong to a group of pattern recognition receptors, with importance for innate immune response and inflammatory processes. Interleukin-10 (IL-10) is a key regulatory cytokine and has been implicated in lymphomagenesis. Functional polymorphisms in these inflammation-associated genes may affect the susceptibility towards lymphoma. To test this hypothesis, we have genotyped DNA of 710 lymphoma cases and 710 controls within the context of a population-based epidemiological study for 11 functionally important single-nucleotide polymorphisms in TLR1, -2, -4, -5, -9, IL10 and IL10 receptor (IL10RA). The IL10RA Ser138Gly variant was underrepresented among lymphoma cases (odds ratio (OR)=0.81, 95 per cent confidence interval (95% CI)=0.65-1.02), mainly owing to an inverse association with Hodgkin's lymphoma (HL). The TLR2 -16933T>A variant was associated with a 2.8-fold increased risk of follicular lymphoma (95% CI=1.43-5.59) and a decreased risk of chronic lymphocytic leukaemia (OR=0.61, 95% CI=0.38-0.95). Furthermore, the TLR4 Asp299Gly variant was positively associated with the risk of mucosa-associated lymphoid tissue lymphoma (OR=2.76, 95% CI=1.12-6.81) and HL (OR=1.80, 95% CI=0.99-3.26). In conclusion, this study suggests an effect of polymorphisms in factors of the innate immune response in the aetiology of some lymphoma subtypes.

Many genes implicated in schizophrenia can be related to glutamatergic transmission and neuroplasticity, oligodendrocyte function, and other families clearly related to neurobiology and schizophrenia phenotypes. Others appear rather to be involved in the life cycles of the pathogens implicated in the disease. For example, aspartylglucosaminidase (AGA), PLA2, SIAT8B, GALNT7, or B3GAT1 metabolize chemical ligands to which the influenza virus, herpes simplex, cytomegalovirus (CMV), rubella, or Toxoplasma gondii bind. The epidermal growth factor receptor (EGR/EGFR) is used by the CMV to gain entry to cells, and a CMV gene codes for an interleukin (IL-10) mimic that binds the host cognate receptor, IL10R. The fibroblast growth factor receptor (FGFR1) is used by herpes simplex. KPNA3 and RANBP5 control the nuclear import of the influenza virus. Disrupted in schizophrenia 1 (DISC1) controls the microtubule network that is used by viruses as a route to the nucleus, while DTNBP1, MUTED, and BLOC1S3 regulate endosomal to lysosomal routing that is also important in viral traffic. Neuregulin 1 activates ERBB receptors releasing a factor, EBP1, known to inhibit the influenza virus transcriptase. Other viral or bacterial components bind to genes or proteins encoded by CALR, FEZ1, FYN, HSPA1B, IL2, HTR2A, KPNA3, MED12, MED15, MICB, NQO2, PAX6, PIK3C3, RANBP5, or TP53, while the cerebral infectivity of the herpes simplex virus is modified by Apolipoprotein E (APOE). Genes encoding for proteins related to the innate immune response, including cytokine related (CCR5, CSF2RA, CSF2RB, IL1B, IL1RN, IL2, IL3, IL3RA, IL4, IL10, IL10RA, IL18RAP, lymphotoxin-alpha, tumor necrosis factor alpha [TNF]), human leukocyte antigen (HLA) antigens (HLA-A10, HLA-B, HLA-DRB1), and genes involved in antigen processing (angiotensin-converting enzyme and tripeptidyl peptidase 2) are all concerned with defense against invading pathogens. Human microRNAs (Hsa-mir-198 and Hsa-mir-206) are predicted to bind to influenza, rubella, or poliovirus genes. Certain genes associated with schizophrenia, including those also concerned with neurophysiology, are intimately related to the life cycles of the pathogens implicated in the disease. Several genes may affect pathogen virulence, while the pathogens in turn may affect genes and processes relevant to the neurophysiology of schizophrenia. For such genes, the strength of association in genetic studies is likely to be conditioned by the presence of the pathogen, which varies in different populations at different times, a factor that may explain the heterogeneity that plagues such studies. This scenario also suggests that drugs or vaccines designed to eliminate the pathogens that so clearly interact with schizophrenia susceptibility genes could have a dramatic effect on the incidence of the disease.

BACKGROUND

Vaccination against hepatitis B virus infection (HBV) is safe and effective; however, vaccine-induced antibody level wanes over time. Peak vaccine-induced anti-HBs level is directly related to antibody decay, as well as risk of infection and persistent carriage despite vaccination. We investigated the role of host genetic factors in long-term immunity against HBV infection based on peak anti-HBs level and seroconversion to anti-HBc.

METHODS

We analyzed 715 SNP across 133 candidate genes in 662 infant vaccinees from The Gambia, assessing peak vaccine-induced anti-HBs level and core antibody (anti-HBc) status, whilst adjusting for covariates. A replication study comprised 43 SNPs in a further 393 individuals.

RESULTS

In our initial screen we found variation in IFNG, MAPK8, and IL10RA to affect peak anti-HBs level (GMTratio of < 0.6 or>> 1.5 and P < or = 0.001) and lesser associations in other genes. Odds of core-conversion was associated with variation in CD163. A coding change in ITGAL (R719V) with likely functional relevance showed evidence of association with increased peak anti-HBs level in both screens (1st screen: s595_22 GMTratio 1.71, P = 0.013; 2nd screen: s595_22 GMTratio 2.15, P = 0.011).

CONCLUSIONS

This is to our knowledge the largest study to date assessing genetic determinants of HBV vaccine-induced immunity. We report on associations with anti-HBs level, which is directly related to durability of antibody level and predictive of vaccine efficacy long-term. A coding change in ITGAL, which plays a central role in immune cell interaction, was shown to exert beneficial effects on induction of peak antibody level in response to HBV vaccination. Variation in this gene does not appear to have been studied in relation to immune responses to viral or vaccine challenges previously. Our findings suggest that genetic variation in loci other than the HLA region affect immunity induced by HBV vaccination.

BACKGROUND

Atherosclerosis affects aorta, coronary, carotid, and iliac arteries most frequently than any other body vessel. There may be common molecular pathways sustaining this process. Plaque presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading to plaque rupture and thrombosis.

RESULTS

We used DNA microarrays and meta-analysis to study how the presence of calcified plaque modifies human coronary and carotid gene expression. We identified a series of potential human atherogenic genes that are integrated in functional networks involved in atherosclerosis. Caveolae and JAK/STAT pathways, and S100A9/S100A8 interacting proteins are certainly involved in the development of vascular disease. We found that the system of caveolae is directly connected with genes that respond to hormone receptors, and indirectly with the apoptosis pathway. Cytokines, chemokines and growth factors released in the blood flux were investigated in parallel. High levels of RANTES, IL-1ra, MIP-1 alpha, MIP-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-17, PDGF-BB, VEGF and IFN-gamma were found in plasma of atherosclerotic patients and might also be integrated in the molecular networks underlying atherosclerotic modifications of these vessels.

CONCLUSIONS

The pattern of cytokine and S100A9/S100A8 up-regulation characterizes atherosclerosis as a proinflammatory disorder. Activation of the JAK/STAT pathway is confirmed by the up-regulation of IL-6, STAT1, ISGF3G and IL10RA genes in coronary and carotid plaques. The functional network constructed in our research is an evidence of the central role of STAT protein and the caveolae system to contribute to preserve the plaque. Moreover, Cav-1 is involved in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the atherosclerotic phenotype.

B cells not only play a pivotal role in humoral immunity, but also are involved in a broad spectrum of immune responses, including antigen presentation and T-cell function regulation. The identification of cell-surface CD molecules derived from a series of Human Leukocyte Differentiation Antigens (HLDA) Workshops has been instrumental to the discovery and functional characterization of human B-cell populations. Moreover, many events regulating B-cell development, activation, and effector functions are orchestrated by these cell-surface molecules. During the Ninth HLDA Workshop (HLDA9) eighteen new CDs were allocated to cell-surface molecules expressed on B cells: CD210a (IL10RA), CD215 (IL15RA), CD270 (TNFRSF14), CD307a (FCRL1), CD307b (FCRL2), CD307c (FCRL3), CD307d (FCRL4), CD351 (FCAMR), CD352 (SLAMF6), CD353 (SLAMF8), CD354 (TREM1), CD355 (CRTAM), CD357 (TNFRSF18), CD358 (TNFRSF21), CD360 (IL21RA), CD361 (EVI2B), CD362 (SDC2), and CD363 (S1PR1). Here we present their expression patterns on leukocytes, including T lymphocytes, NK cells, granulocytes, monocytes, plasmacytoid and monocyte-derived dendritic cells, and several B-cell subsets. These new CD molecules are expressed on B cells at various stages of differentiation; from bone marrow precursor pro-B cells to plasma cells. Three of them, CD307a, CD307b and CD307d, exhibit a B-cell restricted expression pattern, whereas the rest are also present on other leukocytes. In this paper we also review the structural characteristics, expression, and function of these new CD molecules. The availability of monoclonal antibodies directed against novel B cell-surface molecules will have broad implications not only for B-cell biology, but also for the development of new diagnostic and therapeutic tools.

BACKGROUND

Although the extreme condition of typical profound T-cell dysfunction (TD), severe combined immunodeficiency (SCID), has been carefully defined, we are currently in the process of better defining less typical T-cell deficiencies, which tend to present with autologous circulating T-cell combined immunodeficiency (CID). Because autologous cells might interfere with the outcome of bone marrow transplantation, protocols usually include conditioning regimens. Therefore it is important to define the numbers of autologous cells usually detected in patients with CID versus those with SCID.

OBJECTIVE

We sought to determine the number of circulating T cells in patients with SCID as opposed to those with CID, to study their function, and to evaluate their possible detection during newborn screening using T-cell receptor excision circle (TREC) analysis.

METHODS

Numbers of circulating CD3(+) T cells (as determined by means of flow cytometry), in vitro responses to PHA, and TREC levels, all measured at presentation, were compiled from the research charts of the entire cohort of patients followed prospectively for T-cell immunodeficiency at the Hospital for Sick Children. Clinical data were ascertained retrospectively from the patient's hospital charts.

RESULTS

One hundred three patients had CD3(+) determinations, and 80 of them had a genetic diagnosis. All patients considered to have typical SCID had CD3(+) T-cell counts of fewer than 500 cells/μL. Some variability was observed among different genotypes. In vitro responses to PHA were recorded in 88 patients, of whom 68 had a genetic diagnosis. All patients with low CD3(+) T-cell numbers (<500 cells/μL) also had markedly decreased responses to PHA (typical SCIDs). However, responses ranged widely in the groups of patients with TD who had more than 500 CD3(+) autologous circulating T cells per microliter. Although patients with Omenn syndrome and ζ chain-associated protein, 70 kDa (ZAP70), and purine nucleoside phosphorylase (PNP) deficiencies had low responses, patients with the p.R222C mutation in the IL-2 receptor γ(IL2RG) gene as well as IL-10 receptor and CD40 ligand deficiencies had normal or near-normal mitogen responses. Finally, 51 patients had TREC levels measured. All patients with typical SCID, Omenn syndrome, and ZAP70 deficiency had low TREC levels. In contrast, patients with mutations in forkhead box protein 3 (FOXP3), CD40 ligand (CD40L), and IL-10 receptor α(IL10RA), as well as patients with the p.R222C mutation in the IL2RG gene, had normal TREC levels.

CONCLUSIONS

Patients with typical SCID can be defined as having fewer than 500 circulating CD3(+) T cells. Most patients with autologous T cells still have profound TD, as defined by reduced in vitro function and thymus output. Some patients with conditions including TD have normal TREC levels and will therefore not be detected in a TREC-based newborn screening program.

Gene expression microarrays are widely used to investigate molecular targets in cancers, including lung cancer. In this study, we analyzed online non-small cell lung cancer (NSCLC) microarray databases, to screen the key genes and pathways related to NSCLC by bioinformatics analyses. And then, the expression levels of two selected genes in the down-regulated co-pathways, myosin light chain kinase (MYLK) and myosin regulatory light chain 9 (MYL9), were determined in tumor, paired paraneoplastic, and normal lung tissues. First, gene set enrichment analysis and meta-analysis were conducted to identify key genes and pathways that contribute to NSCLC carcinogenesis. Second, using the total RNA and protein extracted from lung cancer tissues (n = 240), adjacent non-cancer tissues (n = 240), and normal lung tissues (n = 300), we examined the MYLK and MYL9 expression levels by quantitative real-time PCR and Western blot. Finally, we explored the correlations between mRNA and protein expressions of these two genes and the clinicopathological parameters of NSCLC. Fifteen up-regulated and nine down-regulated co-pathways were observed. A number of differentially expressed genes (CALM1, THBS1, CSF3, BMP2, IL6ST, MYLK, ROCK2, IL3RA, MYL9, PPP2CA, CSF2RB, CNAQ, GRIA2, IL10RA, IL10RB, IL11RA, LIFR, PLCB4, and RAC3) were identified (P < 0.01) in the down-regulated co-pathways. The expression levels of MYLK and MYL9, which act downstream of the vascular smooth muscle contraction signal pathway and focal adhesion pathway, were significantly lower in cancer tissue than those in the paraneoplastic and normal tissues (P < 0.05). Moreover, the expression levels of these two genes in stages III and IV NSCLC were significantly increased, when compared to stages I and II, and expressions levels in NSCLC with lymphatic metastasis were higher than that without lymphatic metastasis (P < 0.05). Additionally, significant lower expression levels of the two genes were found in smokers than in nonsmokers (P < 0.05). In contrast, gender, differentiated degrees, and pathohistological type appeared to have no impact on these gene expressions (P>> 0.05). These findings suggested that low MYLK and MYL9 expressions might be associated with the development of NSCLC. These genes may be also relevant to NSCLC metastasis. Future investigations with large sample sizes needed to verify these findings.

OBJECTIVE

Early-onset inflammatory bowel disease starting within the first months of life could be due to a particular genetic defect. We set up the GENetically determined ImmUne-mediated enteropathieS (GENIUS) network and collected infants with a proven defect of the IL10 axis for accurate phenotyping of disease presentation and evolution.

METHODS

Ten patients with early-onset inflammatory bowel disease with confirmed mutations in IL10, IL10RA, or IL10RB genes were characterized on clinical, endoscopic-histological, immunobiological, and radiological findings. Functional assays to confirm defective responses to IL10 were performed on peripheral blood mononuclear cells.

RESULTS

A functional defect in IL10 signaling was confirmed in all IL10R patients tested. Disease started with severe diarrhea within the first 12 weeks in all patients. All infants showed Crohn's disease-like ulcerations limited to the colon with marked perianal inflammation (fissures, abscess, and fistula); disease progression to the small bowel occurred in only 1 patient. Four of the 10 patients had granulomata on histology, and all patients showed Crohn's disease-like mesenteric infiltration on imaging. Disease pattern was indistinguishable between IL10R alpha or beta chain or IL10 defects; autoimmunity was not observed. Mutations in IL10 were more frequently associated with bacterial and viral infections. Patients responded partially to treatment with steroids or anti-tumor necrosis factor drugs, whereas hematopoietic stem cell transplantation proved efficacious.

CONCLUSIONS

The importance of the IL10 pathway within the colonic mucosa is highlighted by the development of severe colitis within a few weeks in infants with mutations in IL10, IL10RA, or IL10RB. Immunosuppression failed to correct the defect in this pathway, which seems to be a key to controlling inflammation in the colon.

The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.

Over the past two decades, investigators have used whole genome linkage and genome-wide association studies, including the "Immunochip" study, to identify a surprising number (over 163) of genetic loci containing susceptibility genes for inflammatory bowel disease (IBD) and its 2 major phenotypes, Crohn's disease (CD) and ulcerative colitis (UC). These loci, although nearly all low-risk, have provided important lessons regarding the nature of IBD etiopathogenesis, including that most loci cause both CD and UC risk; one-third of loci have risk for other common autoimmune diseases; numerous loci contain genes that regulate immunity to microbes; Th17 cells are disproportionately influenced by genes within IBD loci; and the HLA region influences UC far greater than CD. Interleukin-10 receptor subunit (IL10RA and IL10RB) and IL10 cytokine gene mutations cause a rare, severe, infantile-onset, autosomal recessive CD, and this knowledge has allowed curative treatment by bone marrow transplant. Key tasks for broader clinical translation include discovery of risk variants for non-white populations; discovery of the less frequent but higher penetrance and familial risk variants by next-generation sequencing; and determining which of numerous associated variations within loci result in specific gene dysfunction causing IBD risk-as only a small number of genes within IBD loci, including NOD2, IL23R, ATG16L1, IRGM, and PTPN22 have specific functional variations characterized. Studying the effect of IBD susceptiblity gene dysfunctions in tissue cultures and animal models will allow the ultimate translational benefits of developing novel treatments for and potentially preventing IBD in those having specific genetic risk factors.

OBJECTIVE

Very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed at 5 years of age or younger, frequently presents with a different and more severe phenotype than older-onset IBD. We investigated whether patients with VEO-IBD carry rare or novel variants in genes associated with immunodeficiencies that might contribute to disease development.

METHODS

Patients with VEO-IBD and parents (when available) were recruited from the Children's Hospital of Philadelphia from March 2013 through July 2014. We analyzed DNA from 125 patients with VEO-IBD (age, 3 wk to 4 y) and 19 parents, 4 of whom also had IBD. Exome capture was performed by Agilent SureSelect V4, and sequencing was performed using the Illumina HiSeq platform. Alignment to human genome GRCh37 was achieved followed by postprocessing and variant calling. After functional annotation, candidate variants were analyzed for change in protein function, minor allele frequency less than 0.1%, and scaled combined annotation-dependent depletion scores of 10 or less. We focused on genes associated with primary immunodeficiencies and related pathways. An additional 210 exome samples from patients with pediatric IBD (n = 45) or adult-onset Crohn's disease (n = 20) and healthy individuals (controls, n = 145) were obtained from the University of Kiel, Germany, and used as control groups.

RESULTS

Four hundred genes and regions associated with primary immunodeficiency, covering approximately 6500 coding exons totaling more than 1 Mbp of coding sequence, were selected from the whole-exome data. Our analysis showed novel and rare variants within these genes that could contribute to the development of VEO-IBD, including rare heterozygous missense variants in IL10RA and previously unidentified variants in MSH5 and CD19.

CONCLUSIONS

In an exome sequence analysis of patients with VEO-IBD and their parents, we identified variants in genes that regulate B- and T-cell functions and could contribute to pathogenesis. Our analysis could lead to the identification of previously unidentified IBD-associated variants.

OBJECTIVE

Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT).

METHODS

Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry.

RESULTS

We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C>> G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3' splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient's cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients.

CONCLUSIONS

Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.