Obesity is a major health problem worldwide and results from an interplay of social, environmental and genetic factors 1. Genome-wide association (GWA) studies have contributed to the identification of new polygenic variants contributing to inter-individual body mass index (BMI) differences 2345. Recently, Liu et al. 6 reported that variants in the beta catenin-like 1 gene (CTNNBL1) were associated with increased fat mass and obesity in a GWA conducted with 1,000 adult U.S. Caucasians. In the same report, this observation was validated in a French case-control sample (896 class III obese adults; BMI ≥ 40 kg/m² and 2,916 normal weight controls; BMI < 25 kg/m²).

Our study had two objectives. First, we aimed to replicate the association of the obesity risk alleles (rs6013029 T-allele, rs16986921 T-allele, rs6020712 A-allele, rs6020846 G-allele, rs6020395 C-allele, rs16986890 G-allele, rs6096781 C-allele, and rs6020339 C-allele) of CTNNBL1 in two GWA data sets. Second, we explored three other genes SH3PXD2B (rs13356223, rs10077897 and rs13436547), SLIT3 (rs17734503 and rs12654448) and FLJ42133 (rs7363432 and rs6095722), also mentioned by Liu et al. 6 in our GWAs. We analysed three samples: (1) GWA data from 487 cases with early onset extreme obesity and 442 controls; (2) GWA data of 1,644 individuals from a population-based adult cohort and (3) genotyping data of the best CTNNBL1 SNP rs6013029 previously reported in 6 in a sample of 775 independent nuclear families each comprising one or more extremely obese offspring and both parents.

We analysed the data of both GWA studies on the SNPs previously reported in 6 of the CTNNBL1 gene. There was no indication of a deviation from Hardy-Weinberg equilibrium at any of these markers in either GWA sample or among the founders in the families based on the exact test described above (all p-values > 0.05). Furthermore, there was no evidence for an association of any of the SNPs in the CTNNBL1 gene with obesity in our data (Table 1). The strongest signal in the original report (rs6013029) achieved a two-sided p-value of 0.53 in our case-control GWA with an estimated odds ratio (OR) of 0.88 (95% confidence interval (CI) 0.60 – 1.30) for the risk-allele T. Even though there is some overlap in the confidence intervals when comparing our result to the results of the original report's French case-control validation sample with 1.42 (95% CI 1.14 – 1.77) the point estimators indicate different directions of the T-allele effect. Combined with the absence of an observed association of this marker with BMI in the KORA cohort a false positive initial observation is the most likely explanation (Table 1).

As rs6013029 was the main initial finding 6, we nevertheless decided to genotype this variant in 775 independent families ascertained for at least one obese offspring. We detected no evidence for an overtransmission of the T-allele – risk allele in the original study – to the obese offspring (two-sided p = 0.50), and an effect size estimate based on this sample of genotype relative risk (GRR) = 0.933 (95% CI 0.717 – 1.214) failed to exclude unity as well. The other CTNNBL1 SNPs previously described in 6 are displayed in Table 1. In addition, we also tried to explore the unvalidated results of SNPs from three additional genes (FLJ42133: rs7363432 and rs6095722; SH3PXD2B: rs13356223, rs10077897 and rs13436547; SLIT3: rs17734503 and rs12654448) which were also reported to be associated with increased fat mass (FLJ42133) or increased BMI (SH3PXD2B and SLIT3), respectively (Table 2). Once again, no evidence for an association in either of our GWAs was detected. In view of the requirement that replication studies need to be adequately powered, we assessed the power of each or our three samples based on the parameters listed above. For the given samples sizes our family-based replication study had a power > 80%, the cohort had a very limited power of about 10% while the case-control GWA had a slightly larger power of about 54%. If the initially reported values overestimate the true genetic effect, which is presumably quite often the case 11, our data nevertheless contribute to a more precise idea of the impact of CTNNBL1 variants on obesity.

In sum, our results underline the importance of replication of GWA results in independent samples even though independent validations may have been reported within the same initial study. While replication of association with obesity of intron 1 variants in FTO has been demonstrated robustly in almost all subsequent studies comprising obese adults and children 712131415, the study by Liu et al. 6 was an exception as none of the intron 1 FTO SNPs showed evidence for a body weight-related association. Interestingly, however, the study did find some evidence for an association of variants in INSIG2 with obesity 516171819. Both examples underline the difficulties that arise when trying to validate, confirm and replicate associations with such complex traits as obesity. Our failure to replicate the initial findings 6 also does not appear to be a result of population stratification. All recruitment was done in Germany for which population stratification effects have shown to be of minor importance 20.

Another possible explanation for a lack of replication is that our results are mainly based on data for children and adolescents which are different from 6 where only adults were investigated. Again the example of FTO 6 highlights how validated associations found in adults with obesity may also be present in children with extreme obesity 321. Recently, two independent studies comprising more than 32,000 22 and 14,000 23 individuals also did not find significant association of the CTNNBL1 variant rs6013029 and obesity. Our study is a replication and validation attempt with sufficient combined power to independently replicate an initial finding 6, while also providing some evidence to support the decision not to follow-up variants that did not "survive" a validation within the same initial report 6. Although we were not able to replicate the original findings, our data may be useful for a meta-analytical assessment of the association of CTNNBL1 variants and obesity. A retrospective look at the conflicting reports on INSIG2 and the recent reports on CTNNBL1 suggests that research on mediating and moderating variables to more comprehensively assess phenotype-genotype relationships is urgently needed.

We did not detect confirmation of association of variants in CTNNBL1 with obesity in a population of Central European ancestry. Further studies have to be performed to validate or not the initial findings about the association of CTNNBL1 variants and obesity.

The authors declare that they have no competing interests.

CIGV participated of the study design, perform the genotyping and drafted the manuscript; BG performed statistical analysis and drafted the manuscript; AS participated of the study design, performed secondary statistical analyses and drafted the manuscript; TM, SF, HG, IMH, TI, HEW and HS participated of the study design; JH and AH conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.