Strong Evidence of a Combination Polymorphism of the Tyrosine Kinase 2 Gene and the Signal Transducer and Activator of Transcription 3 Gene as a DNA-Based Biomarker for Susceptibility to Crohn’s Disease in the Japanese Population

The study subjects were Japanese who were unrelated to one another. The subjects included 112 patients with UC, 83 patients with CD, and 200 gender-matched, healthy volunteers as control subjects. IBD patients were enrolled from eight general hospitals in Nagasaki, Japan from October 2003 to October 2008. The clinical characteristics of the subjects at the end point of this study are shown in Table I. The study protocol was approved by the Committee for Ethical Issue dealing with the Human Genome and Gene Analysis at Nagasaki University, and written informed consent was obtained from each subject.

The diagnosis of IBD was made based on the endoscopic, radiological, histological, and clinical criteria established by both the World Health Organization Council for International Organizations of Medical Sciences and the International Organization for the Study of Inflammatory Bowel Disease [23‐25]. Patients with indeterminate colitis, multiple sclerosis, systemic lupus erythematosus, or any other diagnosed autoimmune diseases were excluded from this study.

Patients with UC were classified into subgroups according to age at onset (≤40 or ≥40 years), extension of disease (proctitis, left-sided colitis, or pancolitis), disease severity (mild, moderate, or severe), and disease activity (active or inactive) (Table I). Likewise, patients with CD were classified into subgroups according to age at onset (≤40 or ≥40 years), the location of lesions (ileal, ileocolonic, colonic, or isolated upper), disease severity (mild, moderate, or severe), disease activity (active or inactive), and the behavior of disease (stricturing, penetrating, or perianal; Table I). The location and extension of UC and CD, disease severity of UC and CD, and behavior of CD were stratified in accordance with the Montreal classification [26] with slight modification. A high clinical activity index (CAI ≥ 5) for UC [27] and a high Crohn’s disease activity index (CDAI ≥ 150) [28] were regarded as active-phase patients.

Genomic DNA was extracted from a whole blood sample from each subject using a DNA Extractor WB-Rapid Kit (Wako, Osaka, Japan) according to the manufacturer’s protocol.

All of SNPs in STAT3 (GenBank accession number, AY572796; MIM 102582) located on chromosome 17q21 [29] and TYK2 (GenBank accession number, AY549314; MIM 176941) located on chromosome 19p13.2 [30] were obtained using data available on the International HapMap Web site [31]. Candidate tag SNPs were selected with priority in a minor allele frequency of more than 5%. Subsequently, linkage disequilibrium blocks and genotyped tag SNPs among the candidate tag SNPs were determined using the iHAP software program [32]. The gene structure and positions of the genotyped tag SNP sites in STAT3 and TYK2 are shown in Figs. 1 and 2, respectively.

Three SNPs, rs8074524 in intron 3, rs2293152 in intron 11, and rs957970 in intron 23, were selected as genotyped tag SNPs (Fig. 1) and were subsequently analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). The polymorphic region was amplified by PCR with a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) using 25 ng of genomic DNA in a 25-µl reaction mixture containing 0.8× GoTaq Green master mix (Promega, Madison, WI, USA) and 15 pmol each of the following primers: forward primer 5′-GTCTGGAAAGCTCCATCTGC-3′ and reverse primer 5′-AGAGGCCAGATTAGTGCTGG-3′ for rs8074524; forward primer 5′-TCCCCTGTGATTCAGATCCC-3′ and reverse primer 5′-CATTCCCACATCTCTGCTCC-3′ for rs2293152; and forward primer 5′-CTGGGCTCAAGTGATCTTCC-3′ and reverse primer 5′-GTACTCATCGCCCTCCATTG-3′ for rs957970. The amplification protocol comprised initial denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 30 s and final extension at 72°C for 5 min. The PCR products were digested with restriction enzyme, Hpa II (Takara Bio Inc., Kyoto, Japan) for rs8074524 and rs2293152; Xsp I (Takara Bio Inc.) for rs957970. The digests were separated by electrophoresis on a 2% agarose gel (Nacalai Tesque, Kyoto, Japan) and visualized with an ultraviolet transilluminator (Alpha Innotech Co., San Leandro, CA, USA) after ethidium bromide (Nacalai Tesque) staining.

Four genotyped tag SNPs in TYK2, rs280496 in intron 3, rs280519 in intron 14, rs2304256 in intron 18, and rs280523 in intron 20 (Fig. 2), were analyzed by PCR-RFLP using 15 pmol each of the following primers: forward primer 5′-CGGGGTGATATGCTCATTGG-3′ and reverse primer 5′-CAACGTGCTGCTGGACAACG-3′ for rs280496; forward primer 5′-CCGCCATGGTGAAAGTTAGC-3′ and reverse primer 5′-ATTTGTGCAGGCCAAGCTGC-3′ for rs280519; forward primer 5′-TCACCAGGCACTTGTTGTCC-3′ and reverse primer 5′-CGGCTTCCAGCATGTGTATG-3′ for rs2304256; and forward primer 5′-ACATTTCCCCCTGCCTACAC-3′ and reverse primer 5′-TTACAGACATGCGCCACCAC-3′ for rs280523. The other constituents of the PCR mixture were the same as described above. The amplification protocol comprised initial denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 64°C (rs280496, rs280519, and rs280523) or 62°C (rs2304256) for 30 s, and extension at 72°C for 30 s and final extension at 72°C for 5 min. The PCR products were digested with Bsl I (New England BioLabs Inc., Beverly, MA, USA) for rs280496, Hpy99 I (New England BioLabs Inc.) for rs280519, Bsm I (New England BioLabs Inc.) for rs2304256, and BsiE I (New England BioLabs Inc.) for rs280523. The digests were then separated on a 2% agarose gel as described above.

Two tag SNPs in TYK2, which showed a close association of susceptibility to CD and were located within the same linkage disequilibrium block (Fig. 2), were utilized to infer the haplotype structure as well as to analyze the haplotype frequency using the SNP Alyze 7.0 standard software package (Dynacom Inc., Yokohama, Japan) to emphasize the variability and to enhance the power of detecting allelic association of rare variants [33, 34].

Differences in age and gender between UC or CD patients and control subjects were evaluated by an unpaired Student’s t test and a chi-square test, respectively, using the SPSS 17 (SPSS Japan Inc., Tokyo, Japan) and Prism 5 (GraphPad Software Inc., San Diego, CA, USA) statistical software packages. The frequencies of the expected alleles were calculated from those of the observed genotypes according to the Hardy–Weinberg equilibrium. The frequencies of the observed and expected alleles were compared by the chi-square test with Yates’ correction using the SNP Alyze 7.0 standard software package. The frequencies and distributions of alleles, genotypes, haplotypes, and diplotypes were statistically compared between UC or CD patients and control subjects by the chi-square test and logistic regression analysis using Prism 5 and SPSS 17. Subsequently, a comparison of the genetic risk factors between the statistically significant genotype of STAT3 and diplotype of TYK2 for susceptibility to CD was carried out by a multivariate logistic regression analysis using SPSS 17. The odds ratio (OR) with 95% confidence interval (CI) was calculated using SPSS 17. A P value of less than 0.05 was considered to be statistically significant.