Published online Nov 28, 2021. doi: 10.3748/wjg.v27.i44.7705
Peer-review started: May 31, 2021
First decision: July 1, 2021
Revised: July 9, 2021
Accepted: November 9, 2021
Article in press: November 9, 2021
Published online: November 28, 2021
Interleukin 10 receptor alpha (IL10RA) gene mutations constitute the most common monogenic disease in East Asia, affecting the health of children. However, identifying disease-causing mutant sites or copy number variants remains challenging in the clinic.
According to the results of our previous study, severe clinical symptoms as well as significantly increased serum IL-10 indicate IL10RA dysfunction, a monogenic phenotype of very early-onset inflammatory bowel disease (VEO-IBD). In addition, such very early-onset IBD showed a heterozygous IL10RA gene mutation by whole exon sequencing, leading to the employment of WGS and subsequent identification of 333-bp deletions in IL10RA.
We investigated the potential disease-causing gene mutations missed by WES and target gene panel sequencing (TGPS). Our results may contribute to monogenic disease diagnosis.
Four patients clinically diagnosed with VEO-IBD during the past 5 years were recruited for this study. Based on their severe clinical phenotypes and the fact that before hospitalization, three patients harbored an IL10RA mutation (c.301C>T, p.R101W in one patient; c.537G>A, p.T179T in two patients), as detected by TGPS and trio-WES, and because WES did not show conclusive results in the fourth patient, we performed whole-genome sequencing (WGS) on patients A and B and reanalyzed the trio-WES data from patients C and D. To verify the functional change caused by the novel mutation, peripheral blood mononuclear cells (PBMCs) from patient D were isolated and stimulated in vitro with lipopolysaccharide (LPS), IL-10, and LPS + IL-10. Serum IL-10 levels in four patients and tumor necrosis factor-α (TNF-α) in the cell supernatant were determined by ELISA. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705 and Ser727 in PBMCs was determined by western blot analysis.
Results of WGS revealed a novel 333-bp deletion encompassing exon 1 of IL10RA in patients A and B, which was also found in patients C and D after reanalyzing their WES data. Patient D was homozygous for the 333-bp deletion. All four patients showed elevated serum IL-10 levels. In vitro, IL-10-stimulated PBMCs from patient D failed to induce STAT3 phosphorylation at Tyr705 and minimally suppressed TNF-α production induced by LPS. Phosphorylation at Ser727 in PBMCs was not affected by LPS or LPS + IL-10 in both healthy subjects and patient D.
Genome-wide uniformity of coverage of WGS identified a novel 333-bp deletion in IL10RA in four patients with VEO-IBD, whereas the results of initially performed WES were inconclusive. WGS, which was more informative than WES, is the most important comprehensive second-tier genomic test for monogenic diseases in the clinic.
We will customize a multiplex ligation-dependent amplification probe of the 333-bp deletion in IL10RA to help diagnose IL10RA mutation-related monogenic diseases.