case_images: PMC4730728_01
case_images: PMC4730728_02
case_images: PMC4730728_03
case_images: PMC4730728_04
case_images: Identification of the IL10RA mutation associated with infantile-onset inflammatory bowel disease (IOIBD). a A c.537G > A mutation at the 3' end of exon 4 of the IL10RA gene in the family of our patient with IOIBD. Arrows indicate site of mutation. Parts of sequences of exon 4 and intron 4 are shown in upper case and lower case, respectively. b RT-PCR analysis for the IL10RA gene in the patient with c.537G > A mutation. Schematic representation of positions of the primers for RT-PCR and the c.537G > A mutation is given in the upper panel. RT-PCR products from the c.537G > A mutation of the IL10RA gene are shown in the lower panel. The expected 216-bp and 495-bp products of the IL10RA exons 4-5 and exons 2-5, respectively, were detected in a healthy control and the patient, while additional shorter products were observed in the patient (bands 3 and 4). c Sequence analysis of RT-PCR products indicated that the c.537G > A mutation caused 2 kinds of splicing variants; an 18 bp deletion of the 3' end of exon 4 and skipping of exon 4 (170 bp)
case_images: 12876_2016_424_Fig1_HTML.jpg
case_images: PMC4730728_04_12876_2016_424_Fig1_HTML.jpg
case_images: Fig1
case_images: We identified a novel homozygous and heterozygous mutation (c.537G > A, p.T179T) at the 3' end of exon 4 of IL10RA in the patient and his parents, respectively (Fig. 1a). Because the nucleotides at the end of the exon are involved in the splicing process, we examined whether the c.537G > A mutation generated splicing variants by reverse-transcription polymerase chain reaction (RT-PCR) analysis using 3 primer sets: set A, to amplify exons 2 and 3 as a control; set B, to detect deletion or insertion near the boundary between exons 4 and 5; and set C, to amplify exons 3 and 4 (upper panel in Fig. 1b). We detected an additional small PCR product in the patient, as well as the product of expected size using set B (compare band #3 with #2, lower panel in Fig. 1b). Moreover, we found a much smaller product in the patient using set C (band #4, lower panel in Fig. 1b). As shown in Fig. 1c, product #2, which showed expected size, possessed a c.537G > A mutation but indicated intact splicing between exon 4 and 5.
case_images: Loss of function of the IL-10 receptor. Functional analysis of IL-10 receptor complex was performed by determining signal transducer and activator of transcription 3 (STAT3) phosphorylation using flow cytometry. Peripheral blood mononuclear cells from the patient (right panel) and his father (left panel) were stimulated for 15 minutes with IL-10 (20 ng/mL) or IL-6 (20 ng/mL) or kept unstimulated, and later fixed, permeabilized and stained for phosphorylated STAT3 (pSTAT3), which is downstream of the IL-10 and IL-6 receptor complexes
case_images: 12876_2016_424_Fig2_HTML.jpg
case_images: PMC4730728_05_12876_2016_424_Fig2_HTML.jpg
case_images: Fig2
case_images: IL-10R. IL-6-induced pSTAT3, which served as an internal positive control, was intact (right panel in Fig. 2). In contrast, both IL-10 stimulation and IL-6 stimulation of the control cells led to pSTAT3 formation (left panel in Fig. 2).
case_images: PMC4730728_05
case_images: Loss of function of the IL-10 receptor. Functional analysis of IL-10 receptor complex was performed by determining signal transducer and activator of transcription 3 (STAT3) phosphorylation using flow cytometry. Peripheral blood mononuclear cells from the patient (right panel) and his father (left panel) were stimulated for 15 minutes with IL-10 (20 ng/mL) or IL-6 (20 ng/mL) or kept unstimulated, and later fixed, permeabilized and stained for phosphorylated STAT3 (pSTAT3), which is downstream of the IL-10 and IL-6 receptor complexes
case_images: 12876_2016_424_Fig2_HTML.jpg
case_images: PMC4730728_05_12876_2016_424_Fig2_HTML.jpg
case_images: Fig2
case_images: IL-10R. IL-6-induced pSTAT3, which served as an internal positive control, was intact (right panel in Fig. 2). In contrast, both IL-10 stimulation and IL-6 stimulation of the control cells led to pSTAT3 formation (left panel in Fig. 2).