Genetic variant of WIF1 gene is functionally associated with developmental dysplasia of the hip in Han Chinese population

Developmental dysplasia of the hip (DDH) is a common skeletal disorder. Studies have demonstrated a significant role of WIF1 gene in skeletal development. The present study was conducted to reveal the association between DDH and gene WIF1. A two-stage case-control candidate gene association study was conducted in total 1573 samples (586 DDH patients and 987 healthy controls) in this study. Polymorphism rs3782499 was genotyped in all samples. Difference of WIF1 expression in hip joint tissue was compared between the patients and the controls. WIF1 expression was compared among different genotypes in DDH patients. The SNP rs3782499 was found significantly associated with DDH in the two-stage study with 585 patients and 987 controls. There was a significant difference in allele frequency (p = 4.37 * 10−5) and genotype distribution in a recessive model (AG + GG vs. AA). DDH patients were found to have significantly higher WIF1 expression than controls. Moreover, Patients with rs3782499 genotype AA have a significantly increased expression of WIF1 than those with GG. To conclude, polymorphism rs3782499 of WIF1 gene is a functional variant regulating the expression of WIF1 in DDH in Chinese Han population, which might be a potential biomarker for the early diagnosis of DDH.

an important role in skeletal development. Nevertheless, there is no study of WIF1 concerning developmental dysplasia of the hip. In this study, we explore the association between WIF1 gene and DDH in the Han Chinese population.

Materials and Methods
Patients. A two-stage method was applied in this study. In stage 1, we enrolled 386 radiologically confirmed DDH patients and 558 healthy controls. In stage 2, 200 independent X-ray confirmed DDH patients and 429 healthy controls were recruited. In total, 585 DDH patients and 987 healthy controls were recruited for this case -control candidate gene association study. DDH patients were consecutively recruited from the department of orthopedics, the first affiliated hospital of Soochow University. Controls were recruited from physical examination center in the first affiliated hospital of Soochow University. All the subjects were Han Chinese. The study was approved by the Soochow University Ethics Committee and obtained informed consents from all patients and controls. All methods were performed in accordance with the relevant guidelines and regulations.
Sample collection. After  Genotyping of targeted locus. According to the manufacture's protocol, the DNA of all the subjects was extracted either from the buccal swabs using the DNA IQ System (Promega, Madison, WI) or peripheral blood using the NucleoSpin Blood QuickPure Kit (Macherey-Nagel GmbH & Co. KG, Düren, German). All the samples were genotyped with Taqman assay. The sample was genotyped by uninformed laboratory personnel. Genotyping, data input and statistical results were examined by two authors independently. Five percent samples were randomly selected to repeat, and 100% consistency was obtained.
Tissue expression of the WIF1 gene in DDH patients and controls. The tissue expression of WIF1 was measured with real-time PCR using gene-specific primers as follows: forward 5′-TCATGGC AGATCCAACCGTC-3′, reverse 5′-CCACTTCAAATGCTGCCACC-3′ for the WIF1 gene, and forward 5′-GAGTC AACGGATTTGGTCGT-3′, reverse 5′-TTGATTTTGGAGGGATCTCG-3′ for the endogenous control gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For real-time PCR, 1 μL of cDNA was amplified for 40 cycles by SYBRPremix Ex TaqTM II (TaKaRa) in ABI 7900HT mentioned above. Melting curve analysis was done at the end of the reaction to assess the quality of the final PCR products. All samples were analyzed in triplicate using the 2 −ΔΔCt method.

Statistics.
Hardy-Weinberg equilibrium was calculated by chi-squared test in both control and case groups.
The association between the DDH patients and the control subjects in the stages was tested by SAS software (version 9.2 -SAS Institute, Cary, NC, USA). Bilateral chi square tests were conducted to determine the significance of differences in allelic frequencies and P < 0.05 was considered to be statistically significant. The Student t test was used to compare the difference of WIF1 expression between the patients and the controls. DDH patients were classified into three groups according to the genotypes of each SNP, and One-way ANOVA test was used to compare the WIF1 expression among different genotypes.

Results
Association of rs3782499 with DDH. The distribution of genotypes in the two groups was consistent with Hardy Weinberg equilibrium (p > 0.01). In stage 1, the distribution frequency of genotype and allele of the locus rs3782499 in the Chinese Han population DDH group and the healthy controls are shown in Table 1. The results showed a significant difference in the allele frequency between the two groups (p = 0.002). There was a significant difference in genotypic distribution in the recessive pattern (AG + GG vs. AA) between the two groups (p = 0.005). A stage 2 study with independent samples was conducted to confirm the findings in stage 1. In stage 2, the significant difference in the allele frequency between the two groups was confirmed and shown in Table 1 (p = 0.008). A significant difference in genotype distribution in a recessive model (AG + GG vs. AA) between two groups (P = 0.018) was demonstrated. In total, with 585 patients and 987 controls, we demonstrated that there was a significant difference in genotype distribution in a recessive model (AG + GG vs. AA) between the two groups (p = 0.0002). The difference in the allele frequency between the two groups was shown in Table 1 (p = 4.37 * 10 −5 ).
Tissue expression of the WIF1 in DDH and the controls. Figure 1

Discussion
In this study, we for the first time explored the association between WIF1 and DDH. A significant association between rs3782499 in gene WIF1 and DDH was identified in our two-stage study. The minor allele frequency of rs3782499 in the control group in our study (0.20) is close to that reported in HapMap for Chinese Han population (0.18). A significant difference of allele frequency between the DDH group and control group was demonstrated. Further analysis also showed significant difference in genotype distribution in a recessive model (AG + GG vs. AA) between the two groups, suggesting association between WIF1 and DDH occurrence in the Han Chinese population. We investigated the expression of WIF1 gene in hip joint capsule and ligament of DDH patients. DDH patients were found to have a significantly increased expression of WIF1 in both the capsule and ligament than the control groups. Besides, patients with genotype AA of rs3782499 were found to have a remarkably increased expression of WIF1 than those with GG. Allele A is indicative of higher expression of WIF1 as well as a higher risk of the development of DDH. Taken together, rs3782499 could be a functional variant regulating the expression of WIF1, which can further play a role in the development of DDH.  Table 1. Association between rs3782499 and DDH in the two-stage population. a OR, Odds Ratio; OR(95% confidence interval(CI)) was shown as effect allele vs reference allele. P values were derived from Bilateral chi square tests to determine the significance of differences.  DDH is a complex multigene hereditary disease, whereby multiple susceptible genes have been reported in literature. The research conducted by Jia et al. showed that rs726252 in pregnancy -associated plasma protein -A2 gene was associated with DDH, suggesting a significantly greater distribution of TT genotype in DDH patients 2 . Shi et al. reported that ASPN was an important regulator in the etiology of DDH, reporting significant association between the D repeat polymorphism of ASPN gene and DDH 3 . In addition, Wang et al. found that a single nucleotide polymorphism in Tbx4 was also associated with DDH 4 while other studies indicated GDF5 and TGFB! as susceptible genes of congenital hip dysplasia 5,6 . Furthermore, The study by Basit et al. indicated that there might be a functional epistatic interaction between HSPG2 and ATP2B4 in familiar DDH 7 . Basit et al. also identified runs of homozygosity on chromosomes 15q13.3 and 19p13.2 in a family segregating DDH with whole genome SNP genotyping 10 . All of these are suggestive of the complex genetic components in DDH.
Wnt signaling cascades are related to many stages of vertebrate limb development. They are involved in proximal-distal outgrowth and dorso-ventral patterning in the early stage and later in the development and maintenance of cartilage, bone, muscles, or joints 11 . Dysregulation of Wnt signaling has been described in several mouse lines exhibiting various limb phenotypes 12 . The classical Wnt signaling pathway is negatively regulated by a variety of soluble factors (WIF1, SFRPs and DKK) in the extracellular environment. WIF1 gene, with a unique and highly conserved WIF domain and five epidermal growth factor-like repeats, encodes a secreted protein, binding to several Wnt proteins and inhibits their activity 13 . As a secreted antagonist of WNT signaling, WIF1 is expressed mainly at the superficial layer of epiphyseal and articular cartilage and promotes chondrogenesis 14 .
In a mouse arthritis model, loss of WIF1 aggravates the destruction of the cartilage 15 . Higher WIF1 expression was detected in DDH patients in the present study. We suggest WIF1 overexpression might be associated with the change of Hip cartilage content and remodeling of the macro-morphology of the hip through the suppression of Wnt signaling. Close relationship between WIF1 gene and Wnt signaling is suggestive of a significant role of WIF1 in hip joint development. This present study was the first report concerning WIF1 in DDH to date. Further experiment to explore the function of WIF1 in DDH is necessary.
In conclusion, we demonstrated the polymorphism rs3782499 of WIF1 gene was associated with DDH in Chinese Han population. Genotype AA of rs3782499 is associated with higher expression of WIF1 in DDH, which further adds to our understanding of its functional role in the development of DDH. Genetic variants of WIF1 could be a potential biomarker for the early diagnosis of DDH. Replication needs to be carried out in other ethnic populations.