Genome-wide study of gene-by-sex interactions identifies risks for cleft palate.

Structural birth defects affect 3-4% of all live births and, depending on the type, tend to manifest in a sex-biased manner. Orofacial clefts (OFCs) are the most common craniofacial structural birth defects and are often divided into cleft lip with or without cleft palate (CL/P) and cleft palate only (CP). Previous studies have found sex-specific risks for CL/P, but these risks have yet to be evaluated in CP. CL/P is more common in males and CP is more frequently observed in females, so we hypothesized there would also be sex-specific differences for CP. Using a trio-based cohort, we performed sex-stratified genome-wide association studies (GWAS) based on proband sex followed by a genome-wide gene-by-sex (GxS) interaction testing. There were 13 loci significant for GxS interactions, with the top finding in LTBP1 (RR=3.37 [2.04 - 5.56], p=1.93x10 -6 ). LTBP1 plays a role in regulating TGF-B bioavailability, and knockdown in both mice and zebrafish lead to craniofacial anomalies. Further, there is evidence for differential expression of LTBP1 between males and females in both mice and humans. Therefore, we tested the association between the imputed genetically regulated gene expression of genes with significant GxS interactions and the CP phenotype. We found significant association for LTBP1 in cell cultured fibroblasts in female probands (p=0.0013) but not in males. Taken altogether, we show there are sex-specific risks for CP that are otherwise undetectable in a combined sex cohort, and LTBP1 is a candidate risk gene, particularly in females.

DeepFace: Deep learning-based framework to contextualize orofacial cleft-related variants during human embryonic craniofacial development.

Orofacial clefts (OFC) are among the most common human congenital birth defects. Previous multiethnic studies have identified dozens of associated loci for both cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Although several nearby genes have been highlighted, the 'casual' variants are largely unknown. Here, we developed DeepFace, a convolutional neural network model, to assess the functional impact of variants by SNP activity difference (SAD) scores. The DeepFace model is trained with 204 epigenomic assays from crucial human embryonic craniofacial developmental stages of post-conception week (pcw) 4 to pcw 10. The Pearson Correlation Coefficient between the predicted and actual values for 12 epigenetic features achieved a median range of 0.50 to 0.83. Specifically, our model revealed that SNPs significantly associated with OFC tended to exhibit higher SAD scores across various variant categories compared to less-related groups, indicating a context-specific impact of OFC-related SNPs. Notably, we identified six SNPs with a significant linear relationship to SAD scores throughout developmental progression, suggesting that these SNPs could play a temporal regulatory role. Furthermore, our cell-type specificity analysis pinpointed the trophoblast cell as having the highest enrichment of risk signals associated with OFC. Overall, DeepFace can harness distal regulatory signals from extensive epigenomic assays, offering new perspectives for prioritizing OFC variants using contextualized functional genomic features. We expect DeepFace to be instrumental in accessing and predicting the regulatory roles of variants associated with OFC, and the model that can be extended to study other complex diseases or traits.

Maternal perceptions of assisting preschool-aged children with toothbrushing.

BACKGROUND: Parent-led toothbrushing with fluoride toothpaste is part of an evidence-based strategy to prevent caries in children. There is a gap in the literature regarding perceptions of how and when to assist a child with toothbrushing from the maternal perspective. METHODS: A qualitative cross-sectional study was conducted with participants in North and North Central Appalachia to examine maternal perceptions of when and how to assist with toothbrushing. From 2018 through 2022, 301 mothers of children aged 3 through 5 years volunteered to participate in semistructured interviews from a more extensive parent study (Center for Oral Health Research in Appalachia cohort). The qualitative data were transcribed, coded, and analyzed using Nvivo software, Version 12 (QSR International). The data were analyzed using grounded theory, constant comparative method, and template analysis. RESULTS: A total of 301 mothers were interviewed for this study; 156 (52%) lived in West Virginia and 145 (48%) lived in Pittsburgh, Pennsylvania. Four main themes emerged: (1) assisting with child toothbrushing, (2) ceasing to provide assistance with child toothbrushing, (3) lacking recommendations from dental care professionals on child toothbrushing, and (4) adhering to recommendations from dental care professionals on child toothbrushing assistance. CONCLUSIONS: Understanding the factors that influence how parents brush their children's teeth and the information they receive to guide daily dental hygiene behavior for children is essential in developing effective interventions for preventing caries in children. PRACTICAL IMPLICATIONS: These insights can improve child toothbrushing quality through improved oral hygiene education, recommendations, terminology, and policies from the dental community.

Whole genome sequencing identifies associations for nonsyndromic sagittal craniosynostosis with the intergenic region of BMP2 and noncoding RNA gene LINC01428.

Craniosynostosis (CS) is a major birth defect resulting from premature fusion of cranial sutures. Nonsyndromic CS occurs more frequently than syndromic CS, with sagittal nonsyndromic craniosynostosis (sNCS) presenting as the most common CS phenotype. Previous genome-wide association and targeted sequencing analyses of sNCS have identified multiple associated loci, with the strongest association on chromosome 20. Herein, we report the first whole-genome sequencing study of sNCS using 63 proband-parent trios. Sequencing data for these trios were analyzed using the transmission disequilibrium test (TDT) and rare variant TDT (rvTDT) to identify high-risk rare gene variants. Sequencing data were also examined for copy number variants (CNVs) and de novo variants. TDT analysis identified a highly significant locus at 20p12.3, localized to the intergenic region between BMP2 and the noncoding RNA gene LINC01428. Three variants (rs6054763, rs6054764, rs932517) were identified as potential causal variants due to their probability of being transcription factor binding sites, deleterious combined annotation dependent depletion scores, and high minor allele enrichment in probands. Morphometric analysis of cranial vault shape in an unaffected cohort validated the effect of these three single nucleotide variants (SNVs) on dolichocephaly. No genome-wide significant rare variants, de novo loci, or CNVs were identified. Future efforts to identify risk variants for sNCS should include sequencing of larger and more diverse population samples and increased omics analyses, such as RNA-seq and ATAC-seq.

Risk of Postpartum Dental Caries: Survival Analysis of Black/African American and White Women in Appalachia.

Background: Pregnancy is associated with increased risk of caries, but the extent this increase extends into the postpartum period is poorly understood. Study Objective: Describe the epidemiology of dental decay in the postpartum period among Black/African American and White American women and explore associations with potentially modifiable risk factors. Materials and Methods: We analyzed data from 1,131 Black/African American and White women participating in Center for Oral Health Research in Appalachia cohorts. Women were enrolled during the first two trimesters of pregnancy. Calibrated dental professionals completed dental examinations at the prenatal enrollment visit, and 2-month, 1-year, 2-year, and 3-year postpartum visits. Results: Between the prenatal visit and 2-month visit, the incidence of decayed, missing, and filled teeth (DMFT) increase was 6.92/100 person-months, compared to 3.6/100 person-months between the 2-month and 1-year visit. In a multivariate Cox proportional hazard regression predicting incidence of caries up to 3-years postpartum, being younger, having less than college education, a household income <$50,000, smoking cigarettes, a DMFT >0, a very poor or poor Oral hygiene Rating Index, lower salivary pH at enrollment, or frequently drinking 100% juice increased the hazard of new dental caries. Adjusting for race/ethnic group did not affect the direction or magnitude of observed associations. Conclusions: The strong associations of prior DMFT and Oral Rating Index with occurrence of new dental caries postpartum suggests that targeting young women for interventions to improve oral health may be more valuable for reducing caries incidence during pregnancy and in the postpartum period than targeting women only during pregnancy.

Syndrome-informed phenotyping identifies a polygenic background for achondroplasia-like facial variation in the general population.

Human craniofacial shape is highly variable yet highly heritable with genetic variants interacting through multiple layers of development. Here, we hypothesize that Mendelian phenotypes represent the extremes of a phenotypic spectrum and, using achondroplasia as an example, we introduce a syndrome-informed phenotyping approach to identify genomic loci associated with achondroplasia-like facial variation in the normal population. We compared three-dimensional facial scans from 43 individuals with achondroplasia and 8246 controls to calculate achondroplasia-like facial scores. Multivariate GWAS of the control scores revealed a polygenic basis for normal facial variation along an achondroplasia-specific shape axis, identifying genes primarily involved in skeletal development. Jointly modeling these genes in two independent control samples showed craniofacial effects approximating the characteristic achondroplasia phenotype. These findings suggest that both complex and Mendelian genetic variation act on the same developmentally determined axes of facial variation, providing new insights into the genetic intersection of complex traits and Mendelian disorders.

netMUG: a novel network-guided multi-view clustering workflow for dissecting genetic and facial heterogeneity.

Introduction: Multi-view data offer advantages over single-view data for characterizing individuals, which is crucial in precision medicine toward personalized prevention, diagnosis, or treatment follow-up. Methods: Here, we develop a network-guided multi-view clustering framework named netMUG to identify actionable subgroups of individuals. This pipeline first adopts sparse multiple canonical correlation analysis to select multi-view features possibly informed by extraneous data, which are then used to construct individual-specific networks (ISNs). Finally, the individual subtypes are automatically derived by hierarchical clustering on these network representations. Results: We applied netMUG to a dataset containing genomic data and facial images to obtain BMI-informed multi-view strata and showed how it could be used for a refined obesity characterization. Benchmark analysis of netMUG on synthetic data with known strata of individuals indicated its superior performance compared with both baseline and benchmark methods for multi-view clustering. The clustering derived from netMUG achieved an adjusted Rand index of 1 with respect to the synthesized true labels. In addition, the real-data analysis revealed subgroups strongly linked to BMI and genetic and facial determinants of these subgroups. Discussion: netMUG provides a powerful strategy, exploiting individual-specific networks to identify meaningful and actionable strata. Moreover, the implementation is easy to generalize to accommodate heterogeneous data sources or highlight data structures.

Joint multi-ancestry and admixed GWAS reveals the complex genetics behind human cranial vault shape.

The cranial vault in humans is highly variable, clinically relevant, and heritable, yet its genetic architecture remains poorly understood. Here, we conduct a joint multi-ancestry and admixed multivariate genome-wide association study on 3D cranial vault shape extracted from magnetic resonance images of 6772 children from the ABCD study cohort yielding 30 genome-wide significant loci. Follow-up analyses indicate that these loci overlap with genomic risk loci for sagittal craniosynostosis, show elevated activity cranial neural crest cells, are enriched for processes related to skeletal development, and are shared with the face and brain. We present supporting evidence of regional localization for several of the identified genes based on expression patterns in the cranial vault bones of E15.5 mice. Overall, our study provides a comprehensive overview of the genetics underlying normal-range cranial vault shape and its relevance for understanding modern human craniofacial diversity and the etiology of congenital malformations.

Potential risk factors and genetic variants associated with dental caries incidence in Appalachia using genome-wide survival analysis.

OBJECTIVE: The aim of this study was to identify the potential risk factors and genetic variants associated with dental caries incidence using survival analysis. METHODS: The Center for Oral Health Research in Appalachia recruited and prospectively followed pregnant women and their children. A total of 909 children followed from birth for up to 7 years were included in this study. Annual intra-oral examinations were performed to assess dental caries experience including the approximate time to first caries incidence in the primary dentition. Cox proportional hazards models were used to assess the associations of time to first caries incidence with self-reported risk factors and 4.9 million genetic variants ascertained using a genome-wide genotyping array. RESULTS: A total of 196 of 909 children (21.56%) had their first primary tooth caries event during follow-up. Household income, home water source, and mother's educational attainment were significantly associated with time to first caries incidence in the stepwise Cox model. The heritability (i.e., proportion of variance explained by genetics) of time to first caries was 0.54. Though no specific genetic variants were associated at the genome-wide significance level (P < 5E-8), we identified 14 loci at the suggestive significance level (5E-8 < P < 1E-5), some of which were located within or near genes with plausible biological functions in dental caries. CONCLUSION: Our findings indicate that household income, home water source, and mother's educational attainment are independent risk factors for dental caries incidence. We nominate several suggestive loci for further investigation.

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

Cleft palate (CP) is one of the most common craniofacial birth defects; however, there are relatively few established genetic risk factors associated with its occurrence despite high heritability. Historically, CP has been studied as a single phenotype, although it manifests across a spectrum of defects involving the hard and/or soft palate. We performed a genome-wide association study using transmission disequilibrium tests of 435 case-parent trios to evaluate broad risks for any cleft palate (ACP) (n = 435), and 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 both in craniofacial tissue of human embryos and 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 for the 20 loci were most similar across all 3 groups for SNPs associated with the ACP group, but more distinct when comparing SNPs associated with either subtype. We also found nominal evidence of replication (p < 0.05) for 22 SNPs previously associated with orofacial clefts. Our 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.

Rare variant modifier analysis identifies variants in SEC24D associated with orofacial cleft subtypes.

As one of the most common structural birth defects, orofacial clefts (OFCs) have been studied for decades, and recent studies have demonstrated that there are genetic differences between the different phenotypic presentations of OFCs. However, the contribution of rare genetic variation genome-wide to different subtypes of OFCs has been understudied, with most studies focusing on common genetic variation or rare variation within targeted regions of the genome. Therefore, we used whole-genome sequencing data from the Gabriella Miller Kids First Pediatric Research Program to conduct a gene-based burden analysis to test for genetic modifiers of cleft lip (CL) vs cleft lip and palate (CLP). We found that there was a significantly increased burden of rare variants in SEC24D in CL cases compared to CLP cases (p = 6.86 [Formula: see text] 10-7). Of the 15 variants within SEC24D, 53.3% were synonymous, but overlapped a known craniofacial enhancer. We then tested whether these variants could alter predicted transcription factor binding sites (TFBS), and found that the rare alleles destroyed binding sites for 9 transcription factors (TFs), including Pax1 (p = 0.0009), and created binding sites for 23 TFs, including Pax6 (p = 6.12 [Formula: see text] 10-5) and Pax9 (p = 0.0001), which are known to be involved in normal craniofacial development, suggesting a potential mechanism by which these synonymous variants could have a functional impact. Overall, this study indicates that rare genetic variation may contribute to the phenotypic heterogeneity of OFCs and suggests that regulatory variation may also contribute and warrant further investigation in future studies of genetic variants controlling risk to OFC.

Data-driven trait heritability-based extraction of human facial phenotypes.

A genome-wide association study (GWAS) of a complex, multi-dimensional morphological trait, such as the human face, typically relies on predefined and simplified phenotypic measurements, such as inter-landmark distances and angles. These measures are predominantly designed by human experts based on perceived biological or clinical knowledge. To avoid use handcrafted phenotypes (i.e., a priori expert-identified phenotypes), alternative automatically extracted phenotypic descriptors, such as features derived from dimension reduction techniques (e.g., principal component analysis), are employed. While the features generated by such computational algorithms capture the geometric variations of the biological shape, they are not necessarily genetically relevant. Therefore, genetically informed data-driven phenotyping is desirable. Here, we propose an approach where phenotyping is done through a data-driven optimization of trait heritability, defined as the degree of variation in a phenotypic trait in a population that is due to genetic variation. The resulting phenotyping process consists of two steps: 1) constructing a feature space that models shape variations using dimension reduction techniques, and 2) searching for directions in the feature space exhibiting high trait heritability using a genetic search algorithm (i.e., heuristic inspired by natural selection). We show that the phenotypes resulting from the proposed trait heritability-optimized training differ from those of principal components in the following aspects: 1) higher trait heritability, 2) higher SNP heritability, and 3) identification of the same number of independent genetic loci with a smaller number of effective traits. Our results demonstrate that data-driven trait heritability-based optimization enables the automatic extraction of genetically relevant phenotypes, as shown by their increased power in genome-wide association scans.

A Retrospective and Prospective Cohort Study Comparing Pediatric Patients With Cleft Lip and Palate From the United States and Guatemala.

Orofacial clefts (OFC) remain among the most prevalent congenital abnormalities worldwide. In the United States in 2010 to 2014, 16.2 of 10,000 live births are born with OFC compared with 23.6 of 10,000 in Alta Verapaz, Guatemala in 2012. Demographics and cleft severity scores were retrospectively gathered from 514 patients with isolated OFC at the Children's Hospital of Philadelphia scheduled for surgery from 2012 to 2019 and from 115 patients seen during surgical mission trips to Guatemala City from 2017 to 2020. Risk factors were also gathered prospectively from Guatemalan families. The Guatemalan cohort had a significantly lower prevalence of cleft palate only compared with the US cohort, which may be a result of greater cleft severity in the population or poor screening and subsequent increased mortality of untreated cleft palate. Of those with lip involvement, Guatemalan patients were significantly more likely to have complete cleft lip, associated cleft palate, and right-sided and bilateral clefts, demonstrating an increased severity of Guatemalan cleft phenotype. Primary palate and lip repair for the Guatemalan cohort occurred at a significantly older age than that of the US cohort, placing Guatemalan patients at increased risk for long-term complications such as communication difficulties. Potential OFC risk factors identified in the Guatemalan cohort included maternal cooking-fire and agricultural chemical exposure, poor prenatal vitamin intake, poverty, and risk factors related to primarily corn-based diets. OFC patients who primarily rely on surgical missions for cleft care would likely benefit from more comprehensive screening and investigation into risk factors for more severe OFC phenotypes.

Clinically actionable secondary findings in 130 triads from sub-Saharan African families with non-syndromic orofacial clefts.

INTRODUCTION: The frequency and implications of secondary findings (SFs) from genomic testing data have been extensively researched. However, little is known about the frequency or reporting of SFs in Africans, who are underrepresented in large-scale population genomic studies. The availability of data from the first whole-genome sequencing for orofacial clefts in an African population motivated this investigation. METHODS: In total, 130 case-parent trios were analyzed for SFs within the ACMG SFv.3.0 list genes. Additionally, we filtered for four more genes (HBB, HSD32B, G6PD and ACADM). RESULTS: We identified 246 unique variants in 55 genes; five variants in four genes were classified as pathogenic or likely pathogenic (P/LP). The P/LP variants were seen in 2.3% (9/390) of the subjects, a frequency higher than ~1% reported for diverse ethnicities. On the ACMG list, pathogenic variants were observed in PRKAG (p. Glu183Lys). Variants in the PALB2 (p. Glu159Ter), RYR1 (p. Arg2163Leu) and LDLR (p. Asn564Ser) genes were predicted to be LP. CONCLUSION: This study provides information on the frequency and pathogenicity of SFs in an African cohort. Early risk detection will help reduce disease burden and contribute to efforts to increase knowledge of the distribution and impact of actionable genomic variants in diverse populations.

Rare variants found in clinical gene panels illuminate the genetic and allelic architecture of orofacial clefting.

PURPOSE: Orofacial clefts (OFCs) are common birth defects including cleft lip, cleft lip and palate, and cleft palate. OFCs have heterogeneous etiologies, complicating clinical diagnostics because it is not always apparent if the cause is Mendelian, environmental, or multifactorial. Sequencing is not currently performed for isolated or sporadic OFCs; therefore, we estimated the diagnostic yield for 418 genes in 841 cases and 294 controls. METHODS: We evaluated 418 genes using genome sequencing and curated variants to assess their pathogenicity using American College of Medical Genetics criteria. RESULTS: 9.04% of cases and 1.02% of controls had "likely pathogenic" variants (P < .0001), which was almost exclusively driven by heterozygous variants in autosomal genes. Cleft palate (17.6%) and cleft lip and palate (9.09%) cases had the highest yield, whereas cleft lip cases had a 2.80% yield. Out of 39 genes with likely pathogenic variants, 9 genes, including CTNND1 and IRF6, accounted for more than half of the yield (4.64% of cases). Most variants (61.8%) were "variants of uncertain significance", occurring more frequently in cases (P = .004), but no individual gene showed a significant excess of variants of uncertain significance. CONCLUSION: These results underscore the etiological heterogeneity of OFCs and suggest sequencing could reduce the diagnostic gap in OFCs.

Rare variants found in multiplex families with orofacial clefts: Does expanding the phenotype make a difference?

Exome sequencing (ES) is now a relatively straightforward process to identify causal variants in Mendelian disorders. However, the same is not true for ES in families where the inheritance patterns are less clear, and a complex etiology is suspected. Orofacial clefts (OFCs) are highly heritable birth defects with both Mendelian and complex etiologies. The phenotypic spectrum of OFCs may include overt clefts and several subclinical phenotypes, such as discontinuities in the orbicularis oris muscle (OOM) in the upper lip, velopharyngeal insufficiency (VPI), microform clefts or bifid uvulas. We hypothesize that expanding the OFC phenotype to include these phenotypes can clarify inheritance patterns in multiplex families, making them appear more Mendelian. We performed exome sequencing to find rare, likely causal genetic variants in 31 multiplex OFC families, which included families with multiple individuals with OFCs and individuals with subclinical phenotypes. We identified likely causal variants in COL11A2, IRF6, SHROOM3, SMC3, TBX3, and TP63 in six families. Although we did not find clear evidence supporting the subclinical phenotype hypothesis, our findings support a role for rare variants in the etiology of OFCs.

netMUG: a novel network-guided multi-view clustering workflow for dissecting genetic and facial heterogeneity.

Multi-view data offer advantages over single-view data for characterizing individuals, which is crucial in precision medicine toward personalized prevention, diagnosis, or treatment follow-up. Here, we develop a network-guided multi-view clustering framework named netMUG to identify actionable subgroups of individuals. This pipeline first adopts sparse multiple canonical correlation analysis to select multi-view features possibly informed by extraneous data, which are then used to construct individual-specific networks (ISNs). Finally, the individual subtypes are automatically derived by hierarchical clustering on these network representations. We applied netMUG to a dataset containing genomic data and facial images to obtain BMI-informed multi-view strata and showed how it could be used for a refined obesity characterization. Benchmark analysis of netMUG on synthetic data with known strata of individuals indicated its superior performance compared with both baseline and benchmark methods for multi-view clustering. In addition, the real-data analysis revealed subgroups strongly linked to BMI and genetic and facial determinants of these classes. NetMUG provides a powerful strategy, exploiting individual-specific networks to identify meaningful and actionable strata. Moreover, the implementation is easy to generalize to accommodate heterogeneous data sources or highlight data structures.

Bacterial community modifies host genetics effect on early childhood caries.

Background: By age five approximately one-fifth of children have early childhood caries (ECC). Both the oral microbiome and host genetics are thought to influence susceptibility. Whether the oral microbiome modifies genetic susceptibility to ECC has not been tested. We test whether the salivary bacteriome modifies the association of a polygenic score (PGS, a score derived from genomic data that summarizes genetic susceptibility to disease) for primary tooth decay on ECC in the Center for Oral Health Research in Appalachia 2 longitudinal birth cohort. Methods: Children were genotyped using the Illumina Multi-Ethnic Genotyping Array and underwent annual dental examinations. We constructed a PGS for primary tooth decay using weights from an independent, genome-wide association meta-analysis. Using Poisson regression, we tested for associations between the PGS (high versus low) and ECC incidence, adjusting for demographic characteristics (n=783). An incidence-density sampled subset of the cohort (n=138) had salivary bacteriome data at 24- months of age. We tested for effect modification of the PGS on ECC case status by salivary bacterial community state type (CST). Results: By 60-months, 20.69% of children had ECC. High PGS was not associated with an increased rate of ECC (incidence-rate ratio:1.09 (95% confidence interval (CI): 0.83, 1.42)). However, having a cariogenic salivary bacterial CST at 24-months was associated with ECC (odds ratio (OR): 7.48 (95%CI: 3.06, 18.26)), which was robust to PGS adjustment. An interaction existed between the salivary bacterial CST and the PGS on the multiplicative scale (P= 0.04). The PGS was associated with ECC (OR: 4.83 (95% CI: 1.29, 18.17)) only among individuals with a noncariogenic salivary bacterial CST (n=70). Conclusions: Genetic causes of caries may be harder to detect when not accounting for cariogenic oral microbiomes. As certain salivary bacterial CSTs increased ECC-risk across genetic-risk strata, preventing colonization of cariogenic microbiomes would be universally beneficial.

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.

Rare genetic variants in SEC24D modify orofacial cleft phenotypes.

As one of the most common structural birth defects, orofacial clefts (OFCs) have been studied for decades, and recent studies have demonstrated that there are genetic differences between the different phenotypic presentations of OFCs. However, the contribution of rare genetic variation genome-wide to different subtypes of OFCs has been understudied, with most studies focusing on common genetic variation or rare variation within targeted regions of the genome. Therefore, we used whole-genome sequencing data from the Gabriella Miller Kids First Pediatric Research Program to conduct a gene-based burden analysis to test for genetic modifiers of cleft lip (CL) vs cleft lip and palate (CLP). We found that there was a significantly increased burden of rare variants in SEC24D in CL cases compared to CLP cases (p=6.86×10-7). Of the 15 variants within SEC24D, 53.3% were synonymous, but overlapped a known craniofacial enhancer. We then tested whether these variants could alter predicted transcription factor binding sites (TFBS), and found that the rare alleles destroyed binding sites for 9 transcription factors (TFs), including Pax1 (p=0.0009), and created binding sites for 23 TFs, including Pax6 (p=6.12×10-5) and Pax9 (p= 0.0001), which are known to be involved in normal craniofacial development, suggesting a potential mechanism by which these synonymous variants could have a functional impact. Overall, this study demonstrates that rare genetic variation contributes to the phenotypic heterogeneity of OFCs and suggests that regulatory variation may also contribute and warrant further investigation in future studies of genetic variants controlling risk to OFC.