High Incidence and Geographic Distribution of Cleft Palate Cases in Finland Are Associated with a Regulatory Variant in IRF6

In Finland the frequency of isolated cleft palate (CP) is higher than that of isolated cleft lip with or without cleft palate (CL/P). This trend contrasts to that in other European countries but its genetic underpinnings are unknown. We performed a genome-wide association study for orofacial clefts, which include CL/P and CP, in the Finnish population. We identified rs570516915, a single nucleotide polymorphism that is highly enriched in Finns and Estonians, as being strongly associated with CP ( P = 5.25 × 10-34, OR = 8.65, 95% CI 6.11-12.25), but not with CL/P ( P = 7.2 × 10-5), with genome-wide significance. The risk allele frequency of rs570516915 parallels the regional variation of CP prevalence in Finland, and the association was replicated in independent cohorts of CP cases from Finland ( P = 8.82 × 10-28) and Estonia ( P = 1.25 × 10-5). The risk allele of rs570516915 disrupts a conserved binding site for the transcription factor IRF6 within a previously characterized enhancer upstream of the IRF6 gene. Through reporter assay experiments we found that the risk allele of rs570516915 diminishes the enhancer activity. Oral epithelial cells derived from CRISPR-Cas9 edited induced pluripotent stem cells demonstrate that the CP-associated allele of rs570516915 concomitantly decreases the binding of IRF6 and the expression level of IRF6 , suggesting impaired IRF6 autoregulation as a molecular mechanism underlying the risk for CP. ### Competing Interest Statement Fedik Rahimov is current employees of AbbVie, Inc. Karol Bomsztyk is co-founder of Matchstick Technologies, Inc and a co-inventor of PIXUL (US Patents 10809166, 11592366). ### Funding Statement The FinnGen project is funded by two grants from Business Finland (HUS 4685/31/2016 and UH 4386/31/2016) and the following industry partners: AbbVie Inc., AstraZeneca UK Ltd, Biogen MA Inc., Bristol Myers Squibb (and Celgene Corporation & Celgene International II Sarl), Genentech Inc., Merck Sharp & Dohme LCC, Pfizer Inc., GlaxoSmithKline Intellectual Property Development Ltd., Sanofi US Services Inc., Maze Therapeutics Inc., Janssen Biotech Inc, Novartis Pharma AG, and Boehringer Ingelheim International GmbH. This project was also partially funded by grants from the U.S. National Institutes of Health, DE027362 (R.A.C.), DE023575 (R.A.C. and B.C.S.), HG010855 (KB) and CA246503 (KB). We would like to acknowledge support from the Centre of Excellence for Genomics and Translational Medicine (SLTMR16142T/TK142), and the Erasmus+ program of the European Union (2018-1-EL01-KA202-047907; 2018-1-SE01-KA202-039066). W.D.F. acknowledges support from the NIGMS grant R15-GM122030-01. ### 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 Coordinating Ethics Committee of the Hospital District of Helsinki and Uusimaa (HUS) statement number for the FinnGen study is Nr HUS/990/2017. The FinnGen study is approved by the Finnish Institute for Health and Welfare (THL, permit numbers [PN]: THL/2031/6.02.00/2017, THL/1101/5.05.00/2017, THL/341/6.02.00/2018, THL/2222/6.02.00/2018, THL/283/6.02.00/2019, THL/1721/5.05.00/2019 and THL/1524/5.05.00/2020); the Digital and Population Data Services Agency (PN: VRK43431/2017-3, VRK/6909/2018-3, VRK/4415/2019-3); the Social Insurance Institution (PN: KELA 58/522/2017, KELA 131/522/2018, KELA 70/522/2019, KELA 98/522/2019, KELA 134/522/2019, KELA 138/522/2019, KELA 2/522/2020, KELA 16/522/2020); Findata (PN: THL/2364/14.02/2020, THL/4055/14.06.00/2020, THL/3433/14.06.00/2020, THL/4432/14.06/2020, THL/5189/14.06/2020, THL/5894/14.06.00/2020, THL/6619/14.06.00/2020, THL/209/14.06.00/2021, THL/688/14.06.00/2021, THL/1284/14.06.00/2021, THL/1965/14.06.00/2021, THL/5546/14.02.00/2020, THL/2658/14.06.00/2021, THL/4235/14.06.00/2021); Statistics Finland (PN: TK-53-1041-17 and TK/143/07.03.00/2020 [earlier TK-53-90-20], TK/1735/07.03.00/2021, TK/3112/07.03.00/2021) and the Finnish Registry for Kidney Diseases (permission/extract from the meeting minutes on July 4, 2019). 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 All data produced in the present study are available upon reasonable request to the authors