Low-Frequency IL23R Coding Variant Associated with Crohn's Disease Susceptibility in Japanese Subjects Identified by Personal Genomics Analysis

Abstract

Background: The common disease-common variant hypothesis is insufficient to explain the complexities of Crohn's disease (CD) genetics; therefore, rare variants are expected to be important in the disease. We explored rare variants associated with susceptibility to CD in Japanese individuals by personal genomic analysis.

Methods: Two-step analyses were performed. The first step was a trio analysis with whole-exome sequence (WES) analysis and the second was a follow-up case-control association study. The WES analysis pipeline comprised Burrows-Wheeler Aligner, Picard, Genome Analysis Toolkit, and SAMTOOLS. Single nucleotide variants (SNVs)/indels were annotated and filtered by using programs implemented in ANNOVAR in combination with identity-by-descent (IBD), subsequently were subjected to the linkage based, and de novo based strategies. Finally, we conducted an association study that included 176 unrelated subjects with CD and 358 healthy control subjects.

Results: In family members, 234,067-297,523 SNVs/indels were detected and they were educed to 106-146 by annotation based filtering. Fifty-four CD variants common to both individuals of the affected sib pair were identified. The linkage based strategy detected five candidate variants whereas the de novo based strategy identified no variants. Consequently, five candidates were analyzed in the case-control association study. CD showed a significant association with one variant in exon 4 of IL23R, G149R [rs76418789, P = 3.9E-5, odds ratio (OR) 0.21, 95% confidence interval (CI) 0.09-0.47 for the dominant model (AA + AG versus GG), and P = 7.3E-5, OR 0.21, 95% CI 0.10-0.48 for AG versus GG, and P = 7.2E-5, OR 0.23, 95% CI 0.10-0.50 for the allele model].

Conclusions: The present study, using personal genomics analysis of a small CD pedigree, is the first to show that the low-frequency non-synonymous variant of IL23R, rs76418789, protects against CD development in Japanese subjects.

Fig 1. The study pedigree.

The pedigree comprised healthy parents (father is indicated as ‘1’ and mother as ‘2’), a healthy second son (4), and older (3) and younger sons (5) affected by Crohn’s disease. Age is indicated in years (y) in the right margin.

Fig 2. Representative endoscopic and pathological findings of Crohn’s disease in the eldest son.

(A) An irregular ulcer encompassed by regenerative epithelia on the transverse colon. (B) Hematoxylin and eosin staining of a typical non-caseating granuloma was seen in the mucosa of biopsy specimens.

Fig 3. Workflow of whole-exome sequence.

Orange rounded rectangles indicate unaffected members, while blue round rectangles indicate affected members. The pedigree numbers, 1, 2, 3, 5, 4, above the round rectangles indicate father, mother, the first, second, and third son, respectively. Numbers inside the rectangles indicate the number of variants. Dashed ellipsoids indicate variants from affected members whereas solid line ellipsoids indicate variants from unaffected members. See text for details.

Fig 4. The segregation pattern of rs76418789 in the pedigree.

The results from PCR sequencing of rs76418789 were placed on each member in the pedigree. The arrowheads indicated the SNV portion. Red colored variants were derived from the mother’s risk allele, whereas green colored ones were derived from father’s reference allele. The pedigree comprised healthy parents (father is indicated as ‘1’ and mother as ‘2’), a healthy second son (4), and older (3) and younger sons (5) affected by Crohn’s disease. Age is indicated in years (y) in the right margin.