The effect of WNT5B IVS3C>G on the susceptibility to type 2 diabetes in UK Caucasian subjects

Methods and results Two thousand seven hundred and one Caucasian middle-aged men from the prospective Northwick Park Heart Study II (NPHSII) of whom 153 developed T2D over 15 years and 1268 Caucasian middle-aged patients with T2D (60% male) were genotyped using a TaqMan assay for the IVS3C > G variant (rs2270031) in the WNT5B gene. The frequency of the G allele was 0.026 (0.022–0.031) in controls and 0.031 (0.025–0.039) in patients with diabetes, p = 0.24. In the prospective analysis, G allele carriers with BMI below 26 kg/m 2 had significantly higher T2D hazard risk [3.46 (1.34–8.96), p = 0.01]. Comparing T2D cases with NPHSII controls, the G allele was associated with a significantly higher T2D odds ratio (OR) of 1.50 (1.06–2.12), p = 0.02 in subjects with BMI lower than 30 kg/m 2 . Increasing BMI had a smaller effect on risk in G allele carriers. The effect on risk was not explained by genotype being associated with any classical T2D risk factor. When the combined effect of this SNP and the TCF7L2 IVS3C > T SNP (rs7903146) was evaluated, a 2.07 (1.40–3.07), p < 0.0001 fold higher OR was observed in carriers of both the rare alleles. Conclusion Variation in WNT5B predisposes to T2D in the absence of obesity. The increase in risk conferred by the presence of both WNT5B and TCF7L2 variants strengthens the role of wnt signaling in T2D. Keywords WNT5B gene Type 2 diabetes Obesity Caucasians TCF7L2 gene wnt signaling Introduction Over 150 million people worldwide are currently affected by diabetes, a number which is expected to double by the year 2030 [1] . This increasing prevalence may be due to the lifestyle adopted by western societies but there is also compelling evidence to support a major genetic component to type 2 diabetes (T2D) susceptibility [2] . Genes related to the wingless-type MMTV integration site family (wnt) signaling pathway are implicated in the pathogenesis of T2D due to the pathway's important role in the normal development [3] and function of pancreas. Wnts are cysteine-rich secreted ligands involved in the glucose-induced insulin secretion in pancreatic islets through activation of the lipoprotein-related protein 5 co-receptor [4] , and the secretion of glucagon-like peptide-1 (GLP-1) by the intestinal endocrine L-cells through the transcription factor 7-like 2 (TCF7L2). GLP-1 is essential for both the postprandial insulin secretion and the generation of new beta cells from the ductal precursor cells [5] . Moreover, the wnt canonical pathway has been shown to inhibit adipogenesis [6] which is associated with the developing of insulin resistance and subsequently T2D [7] . The WNT5B gene has recently been reported to confer susceptibility to T2D [8] . In an investigation of 40 single nucleotide polymorphisms (SNPs) in 11 WNT genes, Kanazawa et al. [8] found that the IVS3C > G polymorphism (rs2270031) in WNT5B (referred in their report as intron 4 + 438 C/G IMS-JST024404) was strongly associated with T2D in the Japanese population. In the present study we have evaluated the IVS3C > G variant in the WNT5B gene in Caucasian subjects with respect to obesity status and other traditional T2D risk factor, including interaction with variation in the gene encoding the TCF7L2 transcription factor. Methods Study groups Second Northwick Park Heart Study (NPHSII) The study consists of 3012 unrelated Caucasian men aged 50–64 years, recruited from nine general practices in the UK and followed prospectively for 15 years. Subjects were excluded at baseline if they had unstable angina, a history of myocardial infarction, coronary surgery, cerebrovascular disease, a life-threatening malignancy, or reported using aspirin or anticoagulant therapy, insulin or oral hypoglycaemics. Self-report by questionnaire was used to identify 67 subjects requiring insulin or oral hypoglycaemics as cases at baseline. New cases up to the end of 2005 were identified by searching for physicians' practice notes on diagnosis and treatment of T2D according to the current national guidelines. All men gave written informed consent. Full details have been published elsewhere [9] . University College London Diabetes and Cardiovascular Study (UDACS), Ealing Diabetes Study of Coagulation (EDSC) and PREDICT study UDACS and EDSC are cross-sectional samples of individuals with T2D according to the World Health Organization criteria [10] , designed to study the association between common gene variants and biochemical risk factors implicated in coronary heart disease in patients with diabetes. Ethical approval was obtained from UCL/UCLH and Ealing Hospital ethics committees. The PREDICT study is a prospective study of patients with established T2D, recruited from routine diabetes clinics at London hospitals to assess the predictive value of coronary artery disease classification by electron beam tomography for coronary heart disease events and stroke. This study had obtained approval from all relevant local ethics committees. The Caucasian T2D patients from UDACS ( n = 570), EDSC ( n = 319) and PREDICT ( n = 379) studies were only included in the present analysis (in total n = 1268). The full characteristics of the patients have been reported previously [11] . Genotyping Genotyping of WNT5B IVS3C > G SNP (rs2270031) was carried out using the TaqMan Assay-by-Design platform (Applied Biosciences, ABI, Warrington, UK). Reactions were performed on 384-well microplates and analysed using ABI TaqMan 7900HT software (probe details available on request). Statistical analysis Statistical analysis was performed with Stata (Version 9, StataCorp, TX, USA). Baseline characteristics for all studies were transformed to a normal distribution where appropriate, and differences were tested by unpaired t-test or analysis of covariance. In the prospective analysis, results are presented as hazard ratios (HR) obtained from Cox regression models with their corresponding 95% confidence interval (CI). Ten year risk of diabetes was estimated by combining the baseline hazard (obtained from the Kaplan–Meier failure function) with the hazard ratio estimates. Genotype effects were tested using a dominant model as there were only two GG men in the study and it was not possible to look at the recessive or the additive models. The comparison for the T2D cohorts with diabetes-free controls is presented as odds ratios (OR) obtained from logistic regression models. Frequencies were compared by Chi-squared test or Fisher's exact test where the numbers were small. Statistical significance was taken as p < 0.05. Results Prospective analysis Among the 2782 Caucasian middle-aged men of the NPHSII where DNA was available, the genotype for the WNT5B gene IVS3C > G variant was obtained in 2701 (97%), of whom 67 had T2D at baseline and were excluded from all analyses. Another 153 developed T2D over the 15 year follow-up period. These 153 men had significantly higher systolic blood pressure (SBP), body mass index (BMI), triglycerides, C-reactive protein (CRP) concentration, lower high density lipoprotein cholesterol levels and marginally higher total cholesterol levels at baseline ( Table 1 ). The genotype distribution was as expected from Hardy–Weinberg proportions ( p = 0.47) and the G allele frequency did not differ significantly between those who developed diabetes and those who did not ( Table 1 ). The risk of developing diabetes, adjusted only for age and practice or for age, practice, SBP, BMI, cholesterol, triglycerides and CRP, was not associated with the presence of the G allele [1.09 (0.55–2.14), p = 0.81 or 1.06 (0.46–2.42), p = 0.89 respectively]. Interaction of the WNT5B gene IVS3C > G variant with obesity in the prospective analysis Increasing BMI had a smaller effect on risk in G allele carriers compared to non-carriers [1.71 (1.18–2.48), p = 0.35 vs. 2.57 (1.87–2.56), p < 0.0001, Fig. 1 A]. The interaction of the variant with obesity was statistically significant ( p = 0.002). However, as it is apparent in Fig. 1 A the respective graphs intercepted at a certain BMI (26 kg/m 2 ). The risk was significantly higher [HR: 3.46 (1.34–8.96), p = 0.01] in G allele carriers having a BMI lower than 26 kg/m 2 , while among the men with BMI higher than 26 kg/m 2 the G allele was associated with a non-significantly lower risk [HR: 0.54 (0.20–1.48), p = 0.23]. This effect on risk was not explained by the genotype being associated with differences in BMI, lipid parameters, blood pressure or CRP between the G allele carriers and non-carriers ( Web appendix 1 ). Case-control analysis A total of 1268 Caucasian T2D patients were genotyped for the WNT5B IVS3C > G variant and compared to 2481 NPHSII men without diabetes serving as controls. Although the cases consisted of both men and women the comparison with the NPHSII controls which consisted of men only was possible, since the WNT5B IVS3 G allele frequency (95% CI) was very similar in male and female cases [0.030 (0.022–0.039 vs. 0.033 (0.024–0.047)), p = 0.53]. As shown in Table 1 , patients (60% male) had a mean age of 64.9 (10.9) years and a mean age of diabetes onset of 55 (12.2) years. The duration of diabetes was 10.1 (8.7) years and their BMI 29.4 (5.4) kg/m 2 . The G allele frequency was higher in T2D cases compared to NPHSII controls ( Table 1 ) but the difference was not statistically significant ( p = 0.24). The odds ratio for T2D was not significantly higher in G allele carriers compared to non-carriers [1.19 (0.89–1.57), p = 0.24]. When female cases were excluded from the analysis the results were similar [1.14 (0.80–1.62), p = 0.46]. Interaction of the WNT5B gene IVS3C > G variant with obesity in the case-control analysis Increasing BMI was associated with a lower odds ratio increase in those carrying the G allele, compared to CC subjects [OR: 1.66 (1.24–2.22), p < 0.0001 vs. 2.43 (2.23–2.65), p = 0.001, Fig. 1 B]. The interaction of the variant with obesity was statistically significant ( p = 0.01). Nevertheless, there was a point in BMI (30 kg/m 2 ), as is evident in Fig. 1 B, below which the odds ratio for T2D in G allele carriers was higher [OR: 1.50 (1.06–2.12), p = 0.02] compared to non-carriers. On the contrary, among subjects with a BMI higher than 30 kg/m 2 , the G allele was associated with a non-significantly lower odds ratio [OR: 0.67 (0.38–1.20), p = 0.18]. The association of the variant with T2D risk was not explained by direct effects of the genotype upon BMI, lipid parameters, blood pressure, CRP or glucose ( Web appendix 1 ). When cases included only men, the results were similar ( Web appendix 2 ). Combined effect of WNT5B gene IVS3C > G and TCF7L2 gene IVS3C > T variants in the case-control analysis The TCF7L2 IVS3C > T variant (rs7903146) has been previously genotyped in the same cohort [11] . The TCF7L2 IVS3 T allele frequency (95% CI) did not differ significantly in male and female cases; therefore the comparison of combined T2D patients with the NPHSII healthy men was possible in this case as well. In the WNT5B CC subjects, the TCF7L2 CT and TT genotypes were associated with a significant 1.53 (1.32–1.76), p < 0.0001, fold higher OR for T2D compared to the TCF7L2 CC genotype. Among the TCF7L2 CC subjects, carriers of the WNT5B G allele did not have a higher OR of T2D [1.05 (0.67–1.64)]. However, in the carriers of both the rare alleles ( TCF7L2 T and WNT5B G) had a 2.07 (1.40–3.07), p < 0.0001, significantly higher OR ( Fig. 1 C). Thus, the effect of the two SNPs on risk was essentially additive, with the interaction between the two SNPs not being statistically significant ( p = 0.39). Discussion In the prospective NPHSII there was evidence for a significantly higher risk in WNT5B G allele carriers among men with a BMI below 26 kg/m 2 (where the statistical power at the 5% significance level was <50%). Obesity was expected to be associated with higher risk, as it was the strongest predictor for the disease in NPHSII [12] . However, the effect of increasing BMI on risk was smaller in men carrying the WNT5B G allele. The men who developed T2D in NPHSII were few ( n = 153); therefore the analysis was also conducted with 1268 diabetic patients in a case-control design, where the power to detect differences in risk was higher. In the case-control analysis the G allele was associated with a significantly higher risk in subjects with a BMI below 30 kg/m 2 (where the statistical power at the 5% significance level was >50%) and was possibly protective in subjects with a BMI above 30 kg/m 2 . The variant's effect on risk was apparent and followed the same pattern in both the prospective and the case-control analyses. This is particularly interesting when considering that the net contribution of the G allele to diabetes risk was likely to be small given its low frequency in our study groups. Experiments with 3T3-L1 preadipocytes have shown that WNT5B overexpression promotes adipogenesis and adipocytokine expression [8] . The WNT5B activates the wnt/Ca 2+ pathway and it has been suggested to promote adipogenesis by antagonizing the canonical/β-catenin pathway [13] . Given that a higher T2D risk associated with the WNT5B IVS3C > G variant was observed in subjects of a lower BMI, this variant may cause downregulation of the gene, or impair splicing, leading to a less effective gene product. It is also possible that the variant itself may not be functional, but rather a marker for a functional variant elsewhere in the gene. In both the prospective and the case-control analyses the variant's effect on risk was not explained by the genotype being associated with any classical T2D risk factor. This suggests that the observed risk effect is not mediated by any of the biochemical traits measured. The wnt pathway is implicated in the pathogenesis of T2D directly through its effect on pancreatic cell development [3] and function [4,5] and indirectly by regulating adipogenesis [6] . Impaired adipogenesis leads to hypertrophic adipocytes that are resistant to insulin and have decreased adipocytokine expression [14] . The interaction of WNT5B IVS3C > G with obesity observed in the present study may reflect the tendency for risk allele carriers to develop T2D at a lower BMI due to early reduction in beta cell function. The wnt signaling pathway results in transcription of target genes through interactions with the TCF7L2 nuclear factor [5] . Variation in TCF7L2 has been shown to be a major risk factor for T2D in many separate studies [2,11] . In previous work carried out in our laboratory the TCF7L2 IVS3C > T SNP was found to predispose to T2D in the same cohorts [11] . Analysis of the combined effect of WNT5B IVS3C > G and TCF7L2 IVS3C > T variants suggested that, for variation in the WNT5B gene to produce a biologically important impact on disease development, the presence of variation in the gene encoding the pathway's downstream effector TCF7L2 is necessary. The strong T2D risk association observed by Kanazawa and colleagues for the WNT5B IVS3C > G variant in the Japanese cohort [ x 2 = 15.6, p = 0.00008; odds ratio = 1.74 (1.32–2.29)] [8] was not replicated in Caucasian subjects. The G allele frequency in the present Caucasian sample was much lower than in the Japanese sample and the statistical power achieved in both study designs was less than 50% at the 5% significance level. This or a different LD structure between the variant genotyped and the functional site in individuals of different ethnic origin may explain the inconsistency of the findings. Limitations to these findings should be taken into consideration. The number of subjects who developed diabetes in the prospective study is relatively small and the results need to be confirmed in other larger studies. For the case-control analysis, the cases recruited from three different studies were combined in order to achieve maximal power; therefore between-study heterogeneity may affect the results. However, this is unlikely to be the case given that the overall characteristics of the patients were similar and the genotype distribution did not differ significantly between the studies (Ref.  [15] and data not shown). The current study shows that variation in WNT5B predisposes to T2D in the absence of obesity while increasing obesity has a smaller effect on risk in carriers of the WNT5B IVS3 G variant. Moreover, it suggests the WNT5B G allele has a significant effect on T2D risk only in carriers of the TCF7L2 risk allele. This observation strengthens the role of wnt signaling in the disease pathogenesis and shows the need for improved understanding of the wnt signaling role in metabolic processes, as well as the possibility for novel therapeutic modalities. Acknowledgments NPHSII was supported by the UK Medical Research Council, the US National Institutes of Health (grant NHLBI 33014); UDACS was supported by Diabetes UK and EDSC by the Coronary Thrombosis Trust. We acknowledge the support of the British Heart Foundation to KDS (FS/06/053), DRG (FS/04/012), JAC and SEH (RG2005/014). Financial support for the PREDICT study was provided by the British Heart Foundation and the Tompkins Foundation. IFG is supported by the Heart Disease and Diabetes Research Trust. Financial contributions were also received from AstraZeneca UK Ltd., Fournier Pharmaceuticals Ltd. and Takeda UK Ltd. The contribution of other members of the PREDICT study group is gratefully acknowledged including A. Dunlop and A. Widdowson. Appendix Supplementary Data Web appendix 2 The risk for type 2 diabetes by BMI in NPHSII controls and T2D male cases stratified by the WNT5B IVS3C > G genotype. 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