Our results from the candidate gene data suggest that dental anomalies are part of an extended cleft phenotype. In addition, some genes may contribute to clefts in association with dental anomalies. However, there are obvious limitations in our study. Although the Filipino families included in our study tend to have large sibships, it was not always possible to examine all potential subjects in all families. A number of reasons account for that, such as having a job in another city and not being available at the time of data collection, or choosing not to participate in the study. Another limitation is that this family dataset is probably not representative of the Filipino population. Although it is possible that this group of families may be representative of the Cebu province or even the Central Visayas region, the lack of official population-based records of birth defects in the Philippines does not allow us to make any assumptions regarding the Filipino population as a whole.
The association we found between families with clefts and IRF6
confirms our previous work10
with this same population. It is remarkable that the association is still evident with only 42 families, which corroborates that IRF6
is a major contributor to clefts in Filipinos. While concerned about multiple testing, we did not apply the strict Bonferroni correction as it would increase type II errors and a major focus of this study was to identify putative associations with the combined dental anomaly/cleft phenotype for further studies. For example, under the Bonferroni correction, we would have lowered the alpha to 0.00003 (0.05/1489) and the known association with IRF6
(interferon regulatory factor 6; p = 0.001) would have been missed. Therefore we report here all results with p-values below 0.05. However, our data must be carefully interpreted since it is expected that some of the p-values below 0.05 can be due to chance.
Analyses under both the narrow and broad affection statuses resulted in significant evidence of over-transmission for markers in 6q21-q23.2, 9q21, and 17q12. The 6q21-q23.2 and 9q21 regions previously showed linkage to clefts in a meta-analysis of genome wide scan data from seven populations15
. In the current study, markers in 6q21-q23.2 yielded p-values between 0.009 and 0.003, and those in 9q21 yielded p-values between 0.009 and 0.0004. The most significantly over-transmitted marker in 9q21 was rs4742741 in ANKS6
(ankyrin repeat and sterile alpha motif domain containing 6) located at 9q22.33 (p = 0.001 for clefts only, and p = 0.0004 for clefts and dental anomalies). Adrenomedulin, a vasodilator peptide, prevents the suppression of the inhibitory SMAD6 (SMAD, mother against DPP homolog 6) protein by TGFB1
(transforming growth factor beta 1) and restores SMAD2-ANKS6
complex formation in human renal tubular epithelial cell lines45
. TGFB/BMP (bone morphogenetic protein) signals rely on SMAD-dependent pathways in the ectomesenchyme to mediate epithelial-mesenchymal interactions that control the first branchial arch patterning and tooth development46
The rs1810132 marker in ERBB2
(receptor tyrosine-protein kinase erbB-2, precursor), located in 17q12, yielded p-values of 0.0006. Previous work has suggested that RARA
(retinoic acid receptor alpha), located at 17q21.1, is associated with isolated cleft lip and palate47,48
is 642,088 base pairs upstream from RARA
. Since they are relatively near to each other, the previous association suggested for RARA
could actually be due to variation in ERBB2. ERBB2
is an essential component of a neuregulin-receptor complex but it is not activated by EGF
(ectodermal growth factor) or TGFA
(transforming growth factor alpha). Erbb2
-deficient mice die at birth and display defects in pre-synaptic development49
. Ethanol consumption during pregnancy affects the expression of Erbb2
and induces a delay in murine fetal dental morphogenesis50
has not been previously considered as a candidate gene for clefts.
In contrast to the above results, suggestive over-transmission of markers in GART (phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase), DPF3 (D4, zinc and double PHD fingers, family 3), and NRXN3 (neurexin 3) were seen only when the dental anomaly phenotype was included in the analysis. These genes have not been shown to be expressed during tooth development and their function is still largely unknown. According to the Entrez database, GART is required for de novo purine biosynthesis, NRXN3 functions in the vertebrate nervous system as cell adhesion molecules and receptors, and DPF3 is probably involved in RNA transcription.
In summary, our results support the hypothesis that increasing the complexity of the clinical description by adding dental anomalies information will provide new opportunities to map susceptibility loci for clefts. Here we report, for the first time, an extensive candidate gene analysis for cleft susceptibility loci using dental anomalies to subphenotype clefts. This approach appears to be a promising one and may help in the identification of genetic variants that increase cleft susceptibility, which would be a crucial step that may allow better estimates of recurrence risks for individual families.