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Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

ABSTRACT: 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.

SUBMITTER: Glocker EO

PROVIDER: S-EPMC2787406 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

Glocker Erik-Oliver EO Kotlarz Daniel D Boztug Kaan K Gertz E Michael EM Schäffer Alejandro A AA Noyan Fatih F Perro Mario M Diestelhorst Jana J Allroth Anna A Murugan Dhaarini D Hätscher Nadine N Pfeifer Dietmar D Sykora Karl-Walter KW Sauer Martin M Kreipe Hans H Lacher Martin M Nustede Rainer R Woellner Cristina C Baumann Ulrich U Salzer Ulrich U Koletzko Sibylle S Shah Neil N Segal Anthony W AW Sauerbrey Axel A Buderus Stephan S Snapper Scott B SB Grimbacher Bodo B Klein Christoph C

The New England journal of medicine 20091104 21

<h4>Background</h4>The molecular cause of inflammatory bowel disease is largely unknown.<h4>Methods</h4>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 al ...[more]

PMID: 19890111

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Project description:Abstract: Interleukin-10-deficient (Il10-/-) mice serve as a model for inflammatory bowel disease (IBD). The severity of colitis strongly depends on the inbred strain carrying the disrupted Il10 gene: C3H/HeJBir (C3) confers disease susceptibility, whereas C57BL/6J (B6) confers resistance. Genome-wide scans with microsatellite markers on segregrating backcross and F2 populations resulted in the detection of ten colitogenic quantitative trait loci (QTL). The aim of this study was to reduce the large number of candidate genes within the QTL intervals by identifying those genes which are located within the candidate gene intervals and which are differentially expressed in the colon of IBD-susceptible and -resistant strains. Using this combination of QTL mapping and microarray analysis, we identified 16 genes which were differentially expressed between B6- and C3-Il10-/- mice and were located within the candidate gene intervals. Three of these genes (Pla2g2a, Gbp1, Cd14) showed prominent differences in expression levels between B6- and C3-Il10-/- as well as between B6 and C3 wildtype mice and were considered to be major candidate genes. Pla2g2a and Gbp1 are known to be polymorphic between C3 and B6 mice. Expression data for Cd14 were confirmed by real-time RT PCR using specified pathogen free and germfree Il10-/- mice. In conclusion, the large number of candidate genes was reduced to three major candidates by using a combination of QTL mapping and microarray analysis. All three genes play an important role in inflammatory processes and immune response. Material & Methods:; Two independent microarray experiments were performed using SPF mice: 19 for the first experiment (4 C3 WT [GSM39296], 5 C3-Il10-/- [GSM39297], 5 B6 WT [GSM39290], 5 B6-Il10-/- [GSM39291]) and 18 for the second (4 C3 WT [GSM39295], 4 C3-Il10-/- [GSM39294], 5 B6 WT [GSM39288], 5 B6-Il10-/- [GSM39289]). The mice were euthanized by cervical dislocation. Following a ventral midline incision, the colon (including the rectum, but not the anus) was removed and flushed with PBS. Each colon was opened longitudinally with a pair of scissors, further dissected and homogenized using a micropistill in 1.5 ml peqGOLD Trifast FL. RNA was isolated according to the manufacturerÂ´s protocol, and quality was checked using RNA Nano LabChipÂ® technology (Agilent, BÃ¶blingen, Germany). For each of the two array experiments, equal amounts (7 micro gram) of RNA were pooled from the four or five animals of each mouse strain, resulting in a total of eight samples. For biotin-labeled target synthesis starting from 3-5 Âµg of total RNA, reactions were performed using standard protocols supplied by the manufacturer (Affymetrix; Santa Clara, CA). Briefly, 3-5 Âµg total RNA were converted to dsDNA using 100 pmol of a T7T23V primer (Eurogentec; Seraing, Belgium) containing a T7 promoter. The cDNA was then used directly in an in-vitro transcription reaction in the presence of biotinylated nucleotides. The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 12.5 Âµg of each biotinylated cRNA preparation were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix MG U74Av2 for 16 hours. Data selection and transformation procedures. For normalization, all array experiments were scaled to a target intensity of 150, otherwise using the default values of the Microarray suite 5.0 (MAS 5.0). Filtering of the results was done as follows: Genes were considered as strong regulated if their fold change was greater than or equal 2 or less than or equal -2, the statistical parameter for a significant change was less than 0.01 (change p-Value for changes called Increased) or greater than 0.99 (change p-Value for changes called Decreased). Additionally, the signal difference of a certain gene was greater than 200. Genes were considered as weak regulated if their fold change was greater than or equal 1.5 or less than or equal -1.5, the statistical parameter for a significant change was less than 0.001 or greater than 0.999 and the signal difference of a certain gene was greater than 40.

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Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

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Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

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