ETS1 variants confer susceptibility to ankylosing spondylitis in Han Chinese

We recruited 2,899 unrelated subjects in this case-control study from 2008 to 2011, including 1,015 patients (701 male, 69.1%) with AS from Shandong Provincial Hospital and Qilu Hospital in Jinan, Shandong province. All patients, at a mean age of 38 ± 8.3 years, had no history of inflammatory bowel disease and/or psoriasis and met the modified New York criteria for AS. We also recruited 1,132 unrelated, randomly sampled healthy controls (603 male, 53.3%) from a health check-up center at the hospital during the same period; the mean age was 40 ± 7.2 years old. All subjects were ethnic Han living in Shandong, an eastern coastal province of North China. More detailed information about the samples was reported previously [13]. The replication samples of 352 AS patients and 400 controls from Ningxia (a province in northwest China) were provided by Professor Yang Ze, with detailed information [14]. Flow cytometry was used to immunophenotype HLA-B27 in patients and controls, and only HLA-B27-positive patients and HLA-B27-negative controls were recruited.

Genomic DNA from peripheral blood leukocytes was extracted by a standard phenol-chloroform method. The study was approved by the ethics committee for human studies at the Shandong University School of Medicine, and all subjects provided their informed consent.

All nine coding exons and 1,000 bp of the 5′ upstream sequence of ETS1 were sequenced in DNA samples from 100 AS cases to screen for genetic variants. ETS1 was amplified by PCR before sequencing. The PCR reaction was run in a total volume of 50 μL solution containing 5 × PCR buffer, 10 μL; 5 × dNTP (1 mmol/L each of dATP, dTTP, dGTP and dCTP), 10 μL; 50 ng genomic DNA; 0.4 μM of each primer; and 1 U Taq DNA polymerase. The PCR amplification conditions were optimized with an initial denaturation step at 94°C for 5 minutes followed by 35 cycles of 94°C for 40 sec, optimal annealing temperature for 40 sec, extension at 72°C for 40 sec and a final extension step of 72°C for 10 minutes. All sequencing was performed by the Genomic Analysis Facility (Shenzhen Huada, Shenzhen, China) and analyzed with use of Chromas 2.33 software.

Because we were unable to detect novel variants in the sequencing region, we selected seven SNPs (rs12574073, rs1128334, rs4937333, rs4254089, rs7951925, rs7116578 and rs4937342) in ETS1 with minor allele frequency ≥0.05 and r² >0.8 in data for Han Chinese in Beijing (HCB) in HapMap [15] and genotyped by TaqMan SNP genotyping assay using assay-on-demand probes and primers (C_31697960_10 for rs12574073, C_7539918_10 for rs1128224, C_278298_10 for rs4937333, C_449693_10 for rs4254089 C_220576_10 for rs7951925, C_29363433_10 for rs7116578, C_27979762_20 for rs4937342; Applied Biosystems, Foster City, CA, USA). rs1128334 and rs4937333 were reported to be strongly associated with SLE and were therefore selected and investigated. We also selected rs12574073 as a candidate SNP because it was a proxy SNP for rs6590330, demonstrated to be associated with SLE in a previous GWAS [9]. The locations of these SNPs are shown in Figure 1. All genotyping involved use of the Lightcycler 480 real-time PCR system (Roche, Mannheim, Germany). The PCR reaction was run in a total volume of 5 μL solution containing 2 × Premix EX Taq (Takara, Otsu, Shiga, Japan), 2.5 μL; >10 ng genomic DNA; 2.5 μM of each primer; and 0.05 μL probes. The PCR amplification conditions were 95°C for 30 sec, 45 cycles of 95°C for 5 sec, 60°C for 20 sec and 40°C for 30 sec for cooling. Genotyping accuracy in the samples was confirmed by direct sequencing of PCR products for 5% of randomly chosen samples.

Total peripheral leukocyte RNA was extracted from 50 AS patients and 161 healthy controls by the TRIzol reagent method (Invitrogen, Carlsbad, CA, USA), then underwent reverse transcription by use of a reverse transcript kit (Takara, Otsu, Shiga, Japan). Allelic-specific expression of ETS1 was analyzed only in healthy controls. RNA samples were treated with RNase free DNAase to eliminate genomic DNA contamination before being reverse-transcribed into cDNA. Real-time quantitative RT-PCR to amplify cDNA involved the Roche 480 real-time PCR system with SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Relative expression was analyzed by the comparative threshold cycle (Ct) method and normalized to that of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calculated by the 2^-△△Ct method and log¹⁰ transformed. The primer sequences for ETS1 were sense 5′-TACACAGGCAGTGGACCAATC-3′ and antisense 5′-CCCCGCTGTCTTGTGGATG-3′; and GAPDH, sense 5′-CCAGGTGGTCTCCTCTGACTT-3′ and antisense 5′-GTTGCTGTAGCCAAATTCGTTGT-3′. Melting curve analysis was used to confirm specificity, with four duplicate wells used for each subject [16].

The SNPs were tested for adherence to Hardy-Weinberg equilibrium (HWE) by chi-square test. Comparison of the allele frequency of the seven SNPs for patients and controls involved Fisher’s exact test with use of Plink 1.07 [17]. The Bonferroni correction was used for multiple test correction. Pairwise linkage disequilibrium (LD) was measured with use of Haploview 4.2. Three-locus haplotypes were calculated by use of a full precise-iteration algorithm with the online SHEsis software [18]. Differences in allelic expression were tested by t- test and analysis of variance (ANOVA) by use of GraphPad Prism v5.01 (GraphPad Software, La Jolla, CA, USA). P <0.05 was considered statistically significant.

We obtained allele, genotype and haplotype distributions for seven markers in ETS1 in chromosome 11q23 for 1,132 controls and 1,015 patients with AS in Shandong. The allele frequency for each SNP was analyzed (Table 1), and none deviated significantly from HWE, as determined at the 0.05 significance level. Frequency of the rs1128334 allele A was significantly higher in AS patients (P = 0.005, odds ratio (OR) 1.204, 95% CI 1.06, 1.37) than in healthy controls. Even after the Bonferroni correction, the association remained significant (Pc = 0.035). rs12574073 and rs4937333 were associated with AS but not after the Bonferroni correction. Then, we replicated the associated SNP rs1128334 in the Ningxia cohort of 352 cases and 400 matched controls. The minor allele frequency of rs1128334 was greater for cases than controls (P = 0.015, OR 1.31, 95% CI 1.06, 1.62).

rs1128334 was in strong LD with rs12574073 (r² = 0.95) (Figure 2). We identified eight different haplotypes in the studied population, but only three showed frequency >3% (Table 2). As expected, the haplotype results were similar to the single-site association results, and haplotype TAT formed by rs12574073, rs1128334 and rs4937333 was strongly associated with increased risk of AS (OR 1.24, 95% CI 1.05, 1.47, P = 0.01), whereas haplotype CGC reduced the risk (OR 0.82, 95% CI 0.70, 0.96, P = 0.01).

We examined the mRNA levels of ETS1 in 50 AS patients and 161 healthy controls. Relative ETS1 mRNA expression was lower in cases than controls (P <0.0001, Figure 3).

To determine whether rs1128334 was associated with ETS1 expression, we genotyped 161 healthy control samples for rs1128334 to compare the ETS1 expression in subjects with different genotypes of the SNP. ETS1 mRNA level was lower in AA (n = 28) and AG (n = 50) than GG (n = 83) homozygotes (P <0.01, Figure 4). Thus, carriers with the risk allele A of rs1128334 may exhibit significantly lower ETS1 mRNA expression than those with the allele G. In addition, analysis of healthy controls involved only rs1128334 and rs4937333 because of the high LD between rs12574073 and rs1128334 (r² = 0.95, Figure 2). Subjects carrying haplotype A-T (n = 28) for rs1128334/rs4937333 had a significantly lower level of ETS1 than those carrying haplotype GC (n = 69) (P <0.05, Figure 5).