Trio-based GWAS identifies novel associations and subtype-specific risk factors for cleft palate

Orofacial clefts (OFCs) are the most common craniofacial birth defects and are often categorized into two etiologically distinct groups: cleft lip with or without cleft palate (CL/P) and isolated cleft palate (CP). CP is highly heritable, but there are still relatively few established genetic risk factors associated with its occurrence compared to CL/P. Historically, CP has been studied as a single phenotype despite manifesting across a spectrum of defects involving the hard and/or soft palate. We performed GWAS using transmission disequilibrium tests using 435 case-parent trios to evaluate broad risks for any cleft palate (ACP, n=435), as well as subtype-specific risks for any cleft soft palate (CSP, n=259) and any cleft hard palate (CHP, n=125). We identified a single genome-wide significant locus at 9q33.3 (lead SNP rs7035976, p=4.24×10−8) associated with CHP. One gene at this locus, angiopoietin-like 2 ( ANGPTL2 ), plays a role in osteoblast differentiation. It is expressed in craniofacial tissue of human embryos, as well as in the developing mouse palatal shelves. We found 19 additional loci reaching suggestive significance (p<5×10−6), of which only one overlapped between groups (chromosome 17q24.2, ACP and CSP). Odds ratios (ORs) for each of the 20 loci were most similar across all three groups for SNPs associated with the ACP group, but more distinct when comparing SNPs associated with either the CSP or CHP groups. We also found nominal evidence of replication (p<0.05) for 22 SNPs previously associated with cleft palate (including CL/P). Interestingly, most SNPs associated with CL/P cases were found to convey the opposite effect in those replicated in our dataset for CP only. Ours is the first study to evaluate CP risks in the context of its subtypes and we provide newly reported associations affecting the broad risk for CP as well as evidence of subtype-specific risks. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Sequencing services were provided by the Center for Inherited Disease Research (CIDR). CIDR is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University, contract number HHSN268201700006I. Patient recruitment, assembly of phenotypic information, sequencing services, and data analysis were supported by National Institutes of Health (NIH) grants: X01-HG010835 (EL), R01- DE016148 (MM, SW), R01-DE030342 (EL), R01-DE011931 (JH), R01-DE028300 (AB), R01-DE014581 (TB), R37-DE008559 (JM), R00-DE024571 (CB), S21MD001830 (CB) U54GM133807 (CB), T32GM008490 (KR), F31DE032588 (KR). Some of this work was supported through cooperative agreements under PA 96043 from the Centers for Disease Control and Prevention to the Centers for Birth Defects Research and Prevention participating in the NBDPS. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the California Department of Public Health. This article was prepared while Trenell J. Mosley was employed at Emory University. The opinions expressed in this article are the author's own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study makes use of data generated by the DECIPHER community. A full list of centres who contributed to the generation of the data is available from and via email from contact{at}deciphergenomics.org. Funding for the DECIPHER project was provided by Wellcome (grant number WT223718/Z/21/Z). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional Review Boards at the University of Pittsburgh, the University of Iowa, and Emory University gave ethical approval for this work in cooperation with IRBs at local recruitment sites. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Sequence and phenotype data is available from the Database of Genotypes and Phenotypes (dbGaP) under study accession phs002220.v1.p1.