Summary Background Stricturing and penetrating complications account for substantial morbidity and health-care costs in paediatric and adult onset Crohn’s disease. Validated models to predict risk for complications are not available, and the effect of treatment on risk is unknown. Methods We did a prospective inception cohort study of paediatric patients with newly diagnosed Crohn’s disease at 28 sites in the USA and Canada. Genotypes, antimicrobial serologies, ileal gene expression, and ileal, rectal, and faecal microbiota were assessed. A competing-risk model for disease complications was derived and validated in independent groups. Propensity-score matching tested the effect of anti-tumour necrosis factor α (TNFα) therapy exposure within 90 days of diagnosis on complication risk. Findings Between Nov 1, 2008, and June 30, 2012, we enrolled 913 patients, 78 (9%) of whom experienced Crohn’s disease complications. The validated competing-risk model included age, race, disease location, and antimicrobial serologies and provided a sensitivity of 66% (95% CI 51–82) and specificity of 63% (55–71), with a negative predictive value of 95% (94–97). Patients who received early anti-TNFα therapy were less likely to have penetrating complications (hazard ratio [HR] 0·30, 95% CI 0·10–0·89; p=0·0296) but not stricturing complication (1·13, 0·51–2·51; 0·76) than were those who did not receive early anti-TNFα therapy. Ruminococcus was implicated in stricturing complications and Veillonella in penetrating complications. Ileal genes controlling extracellular matrix production were upregulated at diagnosis, and this gene signature was associated with stricturing in the risk model (HR 1·70, 95% CI 1·12–2·57; p=0·0120). When this gene signature was included, the model’s specificity improved to 71%. Interpretation Our findings support the usefulness of risk stratification of paediatric patients with Crohn’s disease at diagnosis, and selection of anti-TNFα therapy. Funding Crohn’s and Colitis Foundation of America, Cincinnati Children’s Hospital Research Foundation Digestive Health Center.

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Temperature-sensitive (Ts) mutants are a powerful tool with which to study gene function in vivo. Ts mutants are typically generated by random mutagenesis followed by laborious screening procedures. By using the Escherichia coli cytotoxin CcdB as a model system, simple procedures for generating Ts mutants at high frequency through site-directed mutagenesis were developed. Putative buried, hydrophobic residues are selected through analysis of the protein sequence. Residue burial is confirmed by ensuring that substitution of the residue by Asp leads to protein inactivation. At such sites, a Ts phenotype can typically be generated either by (i) substitution of two predicted, buried residues with the 18 remaining amino acids or (ii) introduction of Lys, Ser, Ala, and Trp at three to four predicted buried sites. By using these design strategies, 17 tight Ts mutants of CcdB were isolated at four predicted buried sites. The rules were further verified by making several Ts mutants of yeast Gal4 at residues 68, 69, and 70. No Ts mutants of either protein have been previously reported. Such Ts mutants of Gal4 can be used for conditional expression of a variety of genes by using the well characterized upstream-activatingsequence-Gal4 system.

Genetic defects that affect intestinal epithelial barrier function can present with very early onset inflammatory bowel disease (VEOIBD). Using whole genome sequencing, a novel hemizygous defect in NOX1 encoding NAPDH oxidase 1 was identified in a patient with ulcerative colitis-like VEOIBD. Exome screening of 1,878 paediatric patients identified further seven male IBD patients with rare NOX1 mutations. Loss-of-function was validated in p.N122H and p.T497A, and to a lesser degree in p.Y470H, p.R287Q, p.I67M, p.Q293R as well as the previously described p.P330S and the common NOX1 SNP p.D360N (rs34688635) variant. The missense mutation p.N122H abrogated reactive oxygen species (ROS) production in cell lines, ex-vivo colonic explants and patient-derived colonic organoid cultures. Within colonic crypts, NOX1 constitutively generates a high level of ROS in the crypt lumen. Analysis of 9,513 controls and 11,140 IBD patients of non-Jewish European ancestry did not reveal an association between p.D360N and IBD. Our data suggest that loss-of-function variants in NOX1 do not cause a Mendelian disorder of high penetrance but are a context specific modifier. Our results implicate that variants in NOX1 change brush border ROS within colonic crypts at the interface between the epithelium and luminal microbes.

We identified missense mutations in genes that encode NADPH oxidases in children with CD; these were associated with a more aggressive disease course and reduced ROS production by neutrophils from the patients.

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BackgroundThe enormous throughput and low cost of second-generation sequencing platforms now allow research and clinical geneticists to routinely perform single experiments that identify tens of thousands to millions of variant sites. Existing methods to annotate variant sites using information from publicly available databases via web browsers are too slow to be useful for the large sequencing datasets being routinely generated by geneticists. Because sequence annotation of variant sites is required before functional characterization can proceed, the lack of a high-throughput pipeline to efficiently annotate variant sites can act as a significant bottleneck in genetics research.ResultsSeqAnt (Sequence Annotator) is an open source web service and software package that rapidly annotates DNA sequence variants and identifies recessive or compound heterozygous loci in human, mouse, fly, and worm genome sequencing experiments. Variants are characterized with respect to their functional type, frequency, and evolutionary conservation. Annotated variants can be viewed on a web browser, downloaded in a tab-delimited text file, or directly uploaded in a BED format to the UCSC genome browser. To demonstrate the speed of SeqAnt, we annotated a series of publicly available datasets that ranged in size from 37 to 3,439,107 variant sites. The total time to completely annotate these data completely ranged from 0.17 seconds to 28 minutes 49.8 seconds.ConclusionSeqAnt is an open source web service and software package that overcomes a critical bottleneck facing research and clinical geneticists using second-generation sequencing platforms. SeqAnt will prove especially useful for those investigators who lack dedicated bioinformatics personnel or infrastructure in their laboratories.