[Interleukin-10 receptor mutations in children with neonatal onset inflammatory bowel disease: genetic diagnosis and pathogenesis].

To explore use of interleukin-10 receptor (IL-10R) gene mutation in diagnosis and pathogenesis of neonatal inflammatory bowel disease (IBD) in 2 suspected cases.Two cases of sibling brothers who had suspected IBD from Guangzhou Women and Children's Medical Center Affiliated to Guangzhou Medical University during the year 2010-2014 were enrolled in the study. The proband, male, 26 days old, weight 3.73 kg, presented with recurrent fever, increased stool frequency since 9 days of age, and was hospitalized at the age of 6 months in 2014. The proband's brother, male, 6 months old, weight 8 kg, had repeated bloody and mucous diarrhea for more than five months, recurrent fever five days, and was hospitalized in 2010. The blood samples were collected from the children and their families for IL-10 receptor genes including IL-10 receptor α subunit (IL-10RA) and β subunit (IL-10RB) PCR amplification. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the proband IL-10RA transcripts. Sequencing was performed on the PCR products forward and reversely. Western blot analysis was used for protein expression of the proband and normal control's IL-10RA and P-STAT3 (Tyr705) expression after IL-10 stimulation, TNF-α level was detected using Human TNF-α ELISA Kit after PBMC was cultured and stimulated.The proband and his brother were IBD patients. Genome sequencing showed mutation in c.537G>A, namely the exon 4 and intron 4 connections changed CA/GT for CG/GT. Sequencing of the RT-PCR products and T-A clone showed that the mutation was (c.519-537del GGTGCCGGGAAACTTCAC, p.LYS173ASNfs*7), as the splice mutation. Two gene mutations were novel mutation. The parents were the mutations carrier. Both of the children were compound heterozygous mutations in IL-10RA. The Western blot analysis showed that the patient and normal children can express IL-10RA protein, however, the function of IL-10RA had obvious defects in the patient, IL-10RA downstream signaling pathways P-STAT3 had no expression. The average level of TNF-α secreted by PBMC after LPS + IL-10 co-stimulation in patient was significantly increased as compared with control group ((2 100±356) vs. (200±50) ng/L, t=9.154, P=0.001), suggesting that interleukin-10-dependent negative feedback regulation is disrupted in the patient.IL-10 receptor mutations can cause neonatal-IBD, for which common treatment effect is poor. Early diagnosis and allogeneic stem-cell transplantation performed may save the children's life.