Molecular assessment of differences in the duodenal microbiome in subjects with irritable bowel syndrome.

OBJECTIVE: Breath testing and duodenal culture studies suggest that a significant proportion of irritable bowel syndrome (IBS) patients have small intestinal bacterial overgrowth. In this study, we extended these data through 16S rDNA amplicon sequencing and quantitative PCR (qPCR) analyses of duodenal aspirates from a large cohort of IBS, non-IBS and control subjects. MATERIALS AND METHODS: Consecutive subjects presenting for esophagogastroduodenoscopy only and healthy controls were recruited. Exclusion criteria included recent antibiotic or probiotic use. Following extensive medical work-up, patients were evaluated for symptoms of IBS. DNAs were isolated from duodenal aspirates obtained during endoscopy. Microbial populations in a subset of IBS subjects and controls were compared by 16S profiling. Duodenal microbes were then quantitated in the entire cohort by qPCR and the results compared with quantitative live culture data. RESULTS: A total of 258 subjects were recruited (21 healthy, 163 non-healthy non-IBS, and 74 IBS). 16S profiling in five IBS and five control subjects revealed significantly lower microbial diversity in the duodenum in IBS, with significant alterations in 12 genera (false discovery rate < 0.15), including overrepresentation of Escherichia/Shigella (p = 0.005) and Aeromonas (p = 0.051) and underrepresentation of Acinetobacter (p = 0.024), Citrobacter (p = 0.031) and Microvirgula (p = 0.036). qPCR in all 258 subjects confirmed greater levels of Escherichia coli in IBS and also revealed increases in Klebsiella spp, which correlated strongly with quantitative culture data. CONCLUSIONS: 16S rDNA sequencing confirms microbial overgrowth in the small bowel in IBS, with a concomitant reduction in diversity. qPCR supports alterations in specific microbial populations in IBS.

Carrier testing for severe childhood recessive diseases by next-generation sequencing.

Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160x average target coverage, 93% of nucleotides had at least 20x coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.