CCT is an important clinical measurement of human eyes. Recent studies have highlighted CCT as a prognosticator for the development of glaucoma, one of the leading causes of irreversible blindness worldwide, with a thin CCT potentially increasing the risk of developing a subtype known as open-angle glaucoma (OAG) 
. The genetic aetiology of OAG is not well understood, with only one major gene myocilin
. Given that OAG has a complex molecular aetiology, the breakdown of the dichotomous trait (i.e., “affected” or “unaffected” status) into its quantitative measurement will aid in the search for disease-susceptibility genes. It is therefore of highly clinical significance to explore the genetic factors that contribute to CCT variation. Thus, we conducted a multi-stage study on over 5,000 samples with the purpose of detecting the genetic variants for the human CCT. We conducted GWA studies on the discovery sample of the two twin cohorts from Australia and the UK. Another set of GWA studies were performed on the two population-based cohorts in pool genotyping design and the results were further validated by individual genotyping the extended cohort (pooled samples plus additional samples with extreme phenotypes).
We have identified a novel locus near FOXO1
(overall p-value of 4.6×10−10
for SNP rs2721051), which accounts for ~1.2% variation in normal human CCT. FOXO1
, located at 13q14.1 is a 111kb gene belonging to the forkhead family of transcription factors and characterized by a distinct forkhead domain. Whilst the specific functions of this gene are unknown, it may play a role in myogenic growth and differentiation (RefSeq, NCBI). Translocation of this gene with PAX3
has been associated with alveolar rhabdomyosarcoma (RefSeq, NCBI). A recent study by Berry et al. reported that the transcription factor gene FOXC1
(6p25) regulates the expression of FOXO1
and binds to a conserved element in the FOXO1
is a major transcription factor involved in the development of the anterior segment of the eye, which is involved in both anterior segment dysgenesis and congenital glaucoma phenotypes 
In the twin cohorts we obtained genome-wide significant association for the genotyped SNPs rs12447690 (p
) and rs9938149 (p
), ~140kb and ~108kb respectively from the gene ZNF469
(16q24). By individually genotyping the population-based samples with extreme CCT values, we showed that rs12447690 was well replicated with an overall p-value of 8.95×10−11
, accounting for 1.29% of the variation in CCT. ZNF469
was recently implicated in a study of the rare disorder BCS 
. Abu et al. showed that rare sequence variants in ZNF469
segregated with BCS. The SNPs we report near ZNF469
have high MAFs – for example rs12447690 has MAF 0.44 in HapMap CEU samples, with a similar value in the cohorts presented here. Given the recombination hotspot () and the large difference in allele frequency between such variants and the rare variants identified by Abu et al., our findings are unlikely to be explained by linkage disequilibrium (LD) between the rare and common variants (the r2
parameter cannot be high between such polymorphisms). At the 16q24 locus there are four putative genes nearer to rs12447690 than ZNF469
. However, in each case the putative genes are poorly characterized with only a hypothetical protein role.
Interestingly, one of the clinical features of BCS patients is hyperlaxity of the joints 
. A small part of the AU twin cohort overlaps with samples from a pelvic floor study by Hansell et al 
, which included measurements of joint mobility 
). Based on a small sample size of 102 individuals, CCT was inversely correlated (Pearson correlation −0.221, P
0.02583) with thumb bending degree, but was uncorrelated with the other two measurements of joint mobility (Figure S7
). To minimize multiple testing, we only tested for association of the thumb bending measure of joint mobility, and focused on 31 SNPs in the ZNF469
region of interest. Despite the limited power in this study, 2 SNPs rs7198446 and rs7500421 in the underlying region were nominally associated with thumb bending degree, with p-values of 0.0298 and 0.0471 respectively (Figure S8
). These SNPs are halfway (~60kb to both sides) between the top SNPs on chromosome16 found in the CCT study and the gene ZNF469
. We also checked the associations of these variants with CCT in this sample, but none of them was significant.
We have demonstrated a flexible approach to GWA studies using different designs. By taking into account of the thresholds used to determine high and low pools for the quantitative trait (together with population allele frequencies), we mapped the estimates of the differences in pooling allele frequency between high and low pools to the effect sizes on additive scale of the quantitative trait.
In summary, we identified a novel QTL for CCT near gene FOXO1
(13q14.1) with p
. Common variants near ZNF469
(16q24) were found in this study as associated with CCT with p
. Our findings suggest that in addition to rare variants in ZNF469
underlying CCT variation in BCS patients, more common variants near this gene may contribute to CCT variation in the general population.