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Activation of cryptic splice sites in three patients with chronic granulomatous disease.

ABSTRACT: BACKGROUND:Chronic granulomatous disease (CGD) is a primary immune deficiency caused by mutations in the genes encoding the structural components of the phagocyte NADPH oxidase. As a result, the patients cannot generate sufficient amounts of reactive oxygen species required for killing pathogenic microorganisms. METHODS:We analyzed NADPH oxidase activity and component expression in neutrophils, performed genomic DNA and cDNA analysis, and used mRNA splicing prediction tools to evaluate the impact of mutations. RESULTS:In two patients with CGD, we had previously found mutations that cause aberrant pre-mRNA splicing. In one patient an exonic mutation in a cryptic donor splice site caused the deletion of the 3' part of exon 6 from the mRNA of CYBB. This patient suffers from X-linked CGD. The second patient, with autosomal CGD, has a mutation in the donor splice site of intron 1 of CYBA that activates a cryptic donor splice site downstream in intron 1, causing the insertion of intronic sequences in the mRNA. The third patient, recently analyzed, also with autosomal CGD, has a mutation in intron 4 of CYBA, 15 bp from the acceptor splice site. This mutation weakens a branch site and activates a cryptic acceptor splice site, causing the insertion of 14 intronic nucleotides into the mRNA. CONCLUSION:We found three different mutations, one exonic, one in a donor splice site and one intronic, that all caused missplicing of pre-mRNA. We analyzed these mutations with four different splice prediction programs and found that predictions of splice site strength, splice enhancer and splice silencer protein binding and branch site strength are all essential for correct prediction of pre-mRNA splicing.

SUBMITTER: de Boer M

PROVIDER: S-EPMC6732321 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Activation of cryptic splice sites in three patients with chronic granulomatous disease.

de Boer Martin M van Leeuwen Karin K Hauri-Hohl Mathias M Roos Dirk D

Molecular genetics & genomic medicine 20190730 9

<h4>Background</h4>Chronic granulomatous disease (CGD) is a primary immune deficiency caused by mutations in the genes encoding the structural components of the phagocyte NADPH oxidase. As a result, the patients cannot generate sufficient amounts of reactive oxygen species required for killing pathogenic microorganisms.<h4>Methods</h4>We analyzed NADPH oxidase activity and component expression in neutrophils, performed genomic DNA and cDNA analysis, and used mRNA splicing prediction tools to eva ...[more]

PMID: 31364312

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Project description:Background and aims: Gene mutations or variants leading to insufficient reactive oxygen species (ROS) production have been associated with inflammatory bowel disease (IBD). In particular, 40-50% of patients with chronic granulomatous disease have IBD (CGD-IBD). CGD is caused by inherited defects in any one of the 5 subunits forming the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived ROS production. While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection in this immune compromised population. Understanding the impact of NOX2 defects on the composition and function of the intestinal microbiota may lead to the identification of treatments for CGD-IBD. Methods: We evaluated GI symptom and quality of life scores, and clinical biomarkers of local (i.e. fecal occult blood and calprotectin) and systemic (i.e. CBC, CRP, ESR, and albumin) inflammation in a cohort of 79 patients with CGD, 8 mutation carriers and 17 healthy controls followed at the National Institutes of Health (NIH). We profiled the intestinal microbiome by 16S rRNA (V4 region) sequencing and the stool metabolome by mass spectrometry in all fecal samples, and further validated our findings by profiling the stool microbiome in a second cohort of 36 patients with CGD recruited from 11 centers across North-America through the Primary Immune Deficiency Treatment Consortium (PIDTC). Predictive functional profiling of the microbial communities based on 16S rRNA sequencing was also performed. Results: After controlling for significant variables, we show decreased alpha diversity and identified distinct intestinal microbiome and metabolomic profiles in patients with CGD compared to healthy individuals. In particular, we observed enrichment for Erysipelatoclostridium spp., Sellimonas spp. and Lachnoclostridium spp. in stool samples from patients with CGD. Despite differences in alpha and beta diversity in samples from the NIH compared to the PIDTC cohort, there were several bacterial taxa that correlated significantly between both cohorts. We further demonstrate that patients with active IBD and/or a history of IBD have a distinct microbiome and metabolomic profile compared to patients without CGD-IBD and identified bacterial taxa to be evaluated as potential markers of disease severity, as well as targets for future therapeutic interventions. Conclusions: Intestinal microbiome and metabolomic signatures distinguished patients with CGD and CGD-IBD and identified microbial and metabolomic candidates to be further evaluated as potential targets to improve the management of patients with CGD-IBD.

Dataset Information

Activation of cryptic splice sites in three patients with chronic granulomatous disease.

Publications

Activation of cryptic splice sites in three patients with chronic granulomatous disease.

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