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.

Perinatal Mental Health Outcomes Following Natural Disasters.

This Viewpoint discusses mental health outcomes associated with natural disaster­related stressors during the perinatal period.

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.

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.

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.

Pre- and peri-natal hurricane exposure alters DNA methylation patterns in children.

Hurricane Maria was the worst recorded natural disaster to affect Puerto Rico. Increased stress in pregnant women during and in the aftermath of the hurricane may have induced epigenetic changes in their infants, which could affect gene expression. Stage of gestation at the time of the event was associated with significant differences in DNA methylation in the infants, especially those who were at around 20-25 weeks of gestation when the hurricane struck. Significant differences in DNA methylation were also associated with maternal mental status assessed after the hurricane, and with property damage. Hurricane Maria could have long lasting consequences to children who were exposed to this disaster during pregnancy.

Dental anomaly detection using intraoral photos via deep learning.

Children with orofacial clefting (OFC) present with a wide range of dental anomalies. Identifying these anomalies is vital to understand their etiology and to discern the complex phenotypic spectrum of OFC. Such anomalies are currently identified using intra-oral exams by dentists, a costly and time-consuming process. We claim that automating the process of anomaly detection using deep neural networks (DNNs) could increase efficiency and provide reliable anomaly detection while potentially increasing the speed of research discovery. This study characterizes the use of` DNNs to identify dental anomalies by training a DNN model using intraoral photographs from the largest international cohort to date of children with nonsyndromic OFC and controls (OFC1). In this project, the intraoral images were submitted to a Convolutional Neural Network model to perform multi-label multi-class classification of 10 dental anomalies. The network predicts whether an individual exhibits any of the 10 anomalies and can do so significantly faster than a human rater can. For all but three anomalies, F1 scores suggest that our model performs competitively at anomaly detection when compared to a dentist with 8 years of clinical experience. In addition, we use saliency maps to provide a post-hoc interpretation for our model's predictions. This enables dentists to examine and verify our model's predictions.

Genome-wide association study of multiethnic nonsyndromic orofacial cleft families identifies novel loci specific to family and phenotypic subtypes.

Nonsyndromic orofacial clefts (OFCs) are among the most common craniofacial birth defects worldwide, and known to exhibit phenotypic and genetic heterogeneity. Cleft lip plus cleft palate (CLP) and cleft lip only (CL) are commonly combined together as one phenotype (CL/P), separately from cleft palate alone. In comparison, our study analyzes CL and CLP separately. A sample of 2218 CL and CLP cases, 4537 unaffected relatives of cases, and 2673 pure controls with no family history of OFC were selected from the Pittsburgh Orofacial Cleft (Pitt-OFC) multiethnic study.genome-wide association studies were run for seven specific phenotypes created based on the cleft type(s) observed within these families, as well as the combined CL/P phenotype. Five novel genome-wide significant associations, 3q29 (rs62284390), 5p13.2 (rs609659), 7q22.1 (rs6465810), 19p13.3 (rs628271), and 20q13.33 (rs2427238), and nine associations (p ≤ 1.0E-05) within previously confirmed OFC loci-PAX7, IRF6, FAM49A, DCAF4L2, 8q24.21, ARID3B, NTN1, TANC2 and the WNT9B:WNT3 gene cluster-were observed. We also found that single nucleotide polymorphisms within a subset of the associated loci, both previously known and novel, differ substantially in terms of their effects across cleft- or family-specific phenotypes, indicating not only etiologic differences between CL and CLP, but also genetic heterogeneity within each of the two OFC subtypes.

Genetic Analyses of Enamel Hypoplasia in Multiethnic Cohorts.

Enamel hypoplasia causes reduction in the thickness of affected enamel and is one of the most common dental anomalies. This defect is caused by environmental and/or genetic factors that interfere with tooth formation, emphasizing the importance of investigating enamel hypoplasia on an epidemiological and genetic level. A genome-wide association of enamel hypoplasia was performed in multiple cohorts, overall comprising 7,159 individuals ranging in age from 7-82 years. Mixed-models were used to test for genetic association while simultaneously accounting for relatedness and genetic population structure. Meta-analysis was then performed. More than 5 million single-nucleotide polymorphisms were tested in individual cohorts. Analyses of the individual cohorts and meta-analysis identified association signals close to genome-wide significance (P < 510-8), and many suggestive association signals (510-8 < P < 510-6) near genes with plausible roles in tooth/enamel development. The strongest association signal (P = 1.5710-9) was observed near BMP2K in one of the individual cohorts. Additional suggestive signals were observed near genes with plausible roles in tooth development in the meta-analysis, such as SLC4A4 which can influence enamel hypoplasia. Additional human genetic studies are needed to replicate these results and functional studies in model systems are needed to validate our findings.

The impact of prenatal maternal stress due to potentially traumatic events on child temperament: A systematic review.

The objective of the current study was to complete a systematic review of the relationship between prenatal maternal stress due to potentially traumatic events (PTEs) and child temperament. Eligible studies through June 2020 were identified utilizing a search strategy in PubMed and PsycInfo. Included studies examined associations between prenatal maternal stress due to PTE and child temperament. Two independent coders extracted study characteristics and three coders assessed study quality. Of the 1969 identified studies, 20 met full inclusion criteria. Studies were classified on two dimensions: (1) disaster-related stress and (2) intimate partner violence during pregnancy. For disaster-related prenatal maternal stress, 75% (nine out of 12) of published reports found associations with increased child negative affectivity, 50% (five out of 10) also noted associations with lower effortful control/regulation, and 38% (three out of eight) found associations with lower positive affectivity. When considering prenatal intimate partner violence stress, 80% (four out of five) of published reports found associations with higher child negative affectivity, 67% (four out of six) found associations with lower effortful control/regulation, and 33% (one out of three) found associations with lower positive affectivity. Prenatal maternal stress due to PTEs may impact the offspring's temperament, especially negative affectivity. Mitigating the effects of maternal stress in pregnancy is needed in order to prevent adverse outcomes on the infant's socioemotional development.

The Influence of Sex and Ancestry on Three-Dimensional Palate Shape.

ABSTRACT: Modern human palate shape has been reported to vary by sex and ancestry, but limitations in the methods used to quantify shape and in population coverage have led to inconsistent findings. In the present study, the authors aim to characterize the effects of sex and ancestry on normal-range three-dimensional palate shape through landmark-based morphometrics.Three-dimensional digital dental casts were obtained and landmarked from 794 adults of European (n = 429), African (n = 295), and East Asian (n = 70) ancestry. Principal component analysis was conducted to identify patterns of shape variation present in our cohort, and canonical variates analysis was performed to test for shape differences between sexes and ancestries.Principal component analysis showed that 3 principal components, explaining 76.52% of variance, linked higher palatal vault with either a relative reduction in anteroposterior or mediolateral dimensions. Canonical variates analysis showed that males had wider and shorter palates with more posteriorly located maximum vault depth than females. Individuals of African ancestry, having higher vaults with more posteriorly located maximal depths, also had wider and shorter palates, whereas individuals of European ancestry had narrower and longer palates with more anteriorly located maximum vault depths. Individuals of East Asian ancestry showed the shallowest vaults.It was found that both sex and ancestry influence palate shape, suggesting a possible genetic component underlying this variation. Additionally, our findings indicate that vault height tends to co-vary with anteroposterior or mediolateral dimensions. Further investigation of these morphological patterns may shed light on possible links to common congenital anomalies such as orofacial clefting.

Genome-Wide Association Study (GWAS) of dental caries in diverse populations.

BACKGROUND: Dental caries is one of the most common chronic diseases and is influenced by a complex interplay of genetic and environmental factors. Most previous genetic studies of caries have focused on identifying genes that contribute to dental caries in specific ethnic groups, usually of European descent. METHODS: The aim of this study is to conduct a genome-wide association study (GWAS) to identify associations affecting susceptibility to caries in a large multiethnic population from Argentina, the Philippines, Guatemala, Hungary, and the USA, originally recruited for studies of orofacial clefts (POFC, N = 3686). Ages of the participants ranged from 2 to 12 years for analysis of the primary dentition, and 18-60 years for analysis of the permanent dentition. For each participant, dental caries was assessed by counts of decayed and filled teeth (dft/DFT) and genetic variants (single nucleotide polymorphisms, SNPs) were genotyped or imputed across the entire genome. Caries was analyzed separately for the primary and permanent dentitions, with age, gender, and presence/absence of any type of OFC treated as covariates. Efficient Mixed-Model Association eXpedited (EMMAX) was used to test genetic association, while simultaneously accounting for relatedness and stratification. RESULTS: We identified several suggestive loci (5 × 10-8 < P < 5 × 10-6) within or near genes with plausible biological roles for dental caries, including a cluster of taste receptor genes (TAS2R38, TAS2R3, TAS2R4, TASR25) on chromosome 7 for the permanent dentition analysis, and DLX3 and DLX4 on chromosome 17 for the primary dentition analysis. Genome-wide significant results were seen with SNPs in the primary dentition only; however, none of the identified genes near these variants have known roles in cariogenesis. CONCLUSION: The results of this study warrant further investigation and may lead to a better understanding of cariogenesis in diverse populations, and help to improve dental caries prediction, prevention, and/or treatment in future.

Genome-Wide Association Study of Non-syndromic Orofacial Clefts in a Multiethnic Sample of Families and Controls Identifies Novel Regions.

Orofacial clefts (OFCs) are among the most prevalent craniofacial birth defects worldwide and create a significant public health burden. The majority of OFCs are non-syndromic and vary in prevalence by ethnicity. Africans have the lowest prevalence of OFCs (~ 1/2,500), Asians have the highest prevalence (~1/500), Europeans and Latin Americans lie somewhere in the middle (~1/800 and 1/900, respectively). Thus, ethnicity appears to be a major determinant of the risk of developing OFC. The Pittsburgh Orofacial Clefts Multiethnic study was designed to explore this ethnic variance, comprising a large number of families and individuals (~12,000 individuals) from multiple populations worldwide: US and Europe, Asians, mixed Native American/Caucasians, and Africans. In this current study, we analyzed 2,915 OFC cases, 6,044 unaffected individuals related to the OFC cases, and 2,685 controls with no personal or family history of OFC. Participants were grouped by their ancestry into African, Asian, European, and Central and South American subsets, and genome-wide association run on the combined sample as well as the four ancestry-based groups. We observed 22 associations to cleft lip with or without cleft palate at 18 distinct loci with p-values < 1e-06, including 10 with genome-wide significance (<5e-08), in the combined sample and within ancestry groups. Three loci - 2p12 (rs62164740, p = 6.27e-07), 10q22.2 (rs150952246, p = 3.14e-07), and 10q24.32 (rs118107597, p = 8.21e-07) are novel. Nine were in or near known OFC loci - PAX7, IRF6, FAM49A, DCAF4L2, 8q24.21, NTN1, WNT3-WNT9B, TANC2, and RHPN2. The majority of the associations were observed only in the combined sample, European, and Central and South American groups. We investigated whether the observed differences in association strength were (a) purely due to sample sizes, (b) due to systematic allele frequency difference at the population level, or (c) due to the fact certain OFC-causing variants confer different amounts of risk depending on ancestral origin, by comparing effect sizes to observed allele frequencies of the effect allele in our ancestry-based groups. While some of the associations differ due to systematic differences in allele frequencies between groups, others show variation in effect size despite similar frequencies across ancestry groups.

Multivariate GWAS of Structural Dental Anomalies and Dental Caries in a Multi-Ethnic Cohort.

Odontogenesis is a complex process, where disruption can result in dental anomalies and/or increase the risk of developing dental caries. Based on previous studies, certain dental anomalies tend to co-occur in patients, suggesting that these traits may share common genetic and etiological components. The main goal of this study was to implement a multivariate genome-wide association study approach to identify genetic variants shared between correlated structural dental anomalies and dental caries. Our cohort (N = 3,579) was derived from the Pittsburgh Orofacial Clefts Study, where multiple dental traits were assessed in both the unaffected relatives of orofacial cleft (OFC) cases (n = 2,187) and unaffected controls (n = 1,392). We identified four multivariate patterns of correlated traits in this data: tooth agenesis, impaction, and rotation (AIR); enamel hypoplasia, displacement, and rotation (HDR); displacement, rotation, and mamelon (DRM); and dental caries, tooth agenesis and enamel hypoplasia (CAH). We analyzed each of these four models using genome-wide multivariate tests of association. No genome-wide statistically significant results were found, but we identified multiple suggestive association signals (P < 10-5) near genes with known biological roles during tooth development, including ADAMTS9 and PRICKLE2 associated with AIR; GLIS3, WDR72, and ROR2 associated with HDR and DRM; ROBO2 associated with DRM; BMP7 associated with HDR; and ROBO1, SMAD2, and MSX2 associated with CAH. This is the first study to investigate genetic associations for multivariate patterns of correlated dental anomalies and dental caries. Further studies are needed to replicate these results in independent cohorts.

Parents of Children With Nonsyndromic Orofacial Clefting Show Altered Palate Shape.

OBJECTIVE: The unaffected relatives of individuals with nonsyndromic orofacial clefts have been shown to exhibit subtle craniofacial differences compared with the general population. Here, we investigate whether these morphological differences extend to the shape of the palate. DESIGN: We conducted a geometric morphometric analysis to compare palate shape in the clinically unaffected parents of children with nonsyndromic cleft lip with or without cleft palate and adult controls of European, Asian, and African ancestry. We conducted pairwise group comparisons using canonical variates analysis, and then confirmed and characterized findings of shape differences using Euclidean distance matrix analysis. RESULTS: Significant differences in palate shape were detected in unaffected mothers (but not fathers) compared to demographically matched controls. The differences in shape were ancestry-specific; mothers of Asian-derived and African-derived ancestry showed wider and shorter palates with higher posterior palatal vaults, while mothers of European-derived ancestry showed narrower palates with higher anterior palatal vaults. CONCLUSIONS: Our findings suggest that altered palate shape is a subclinical phenotypic feature, which may be indicative of elevated orofacial cleft risk. The risk phenotype varied by sex and ancestry, suggesting possible etiologic heterogeneity among demographic groups. Understanding the genetic basis of these informative palate shape traits may reveal new genes and pathways relevant to nonsyndromic orofacial clefting.

A systematic genetic analysis and visualization of phenotypic heterogeneity among orofacial cleft GWAS signals.

Phenotypic heterogeneity is a hallmark of complex traits, and genetic studies of such traits may focus on them as a single diagnostic entity or by analyzing specific components. For example, in orofacial clefting (OFC), three subtypes-cleft lip (CL), cleft lip and palate (CLP), and cleft palate (CP) have been studied separately and in combination. To further dissect the genetic architecture of OFCs and how a given associated locus may be contributing to distinct subtypes of a trait we developed a framework for quantifying and interpreting evidence of subtype-specific or shared genetic effects in complex traits. We applied this technique to create a "cleft map" of the association of 30 genetic loci with three OFC subtypes. In addition to new associations, we found loci with subtype-specific effects (e.g., GRHL3 [CP], WNT5A [CLP]), as well as loci associated with two or all three subtypes. We cross-referenced these results with mouse craniofacial gene expression datasets, which identified additional promising candidate genes. However, we found no strong correlation between OFC subtypes and expression patterns. In aggregate, the cleft map revealed that neither subtype-specific nor shared genetic effects operate in isolation in OFC architecture. Our approach can be easily applied to any complex trait with distinct phenotypic subgroups.

Association of low-frequency genetic variants in regulatory regions with nonsyndromic orofacial clefts.

Genome-wide scans have shown that common risk alleles for orofacial clefts (OFC) tend to be located in noncoding regulatory elements and cumulatively explain only part of the heritability of OFCs. Low-frequency variants may account for some of the "missing" heritability. Therefore, we scanned low-frequency variants located within putative craniofacial enhancers to identify novel OFC risk variants and implicate new regulatory elements in OFC pathogenesis. Analyses were performed in a multiethnic sample of 1,995 cases of cleft lip with or without cleft palate (CL/P), 221 cases with cleft palate (CP) only, and 1,576 unaffected controls. One hundred and nineteen putative craniofacial enhancers identified from ChIP-Seq studies in craniofacial tissues or cell lines contained multiple low-frequency (0.01-1%) variants, which we genotyped in participants using a custom Illumina panel. Two complementary statistical approaches, sequence kernel association test and combined multivariate and collapsing, were used to test association of the aggregated low-frequency variants across each enhancer region with CL/P and CP. We discovered a significant association between CP and a branchial arch enhancer near FOXP1 (mm60; p-value = .0002). Additionally, we observed a suggestive association between CL/P and a forebrain enhancer near FOXE1 (hs1717; p-value = .001). These findings suggest that low-frequency variants in craniofacial enhancer regions contribute to the complex etiology of nonsyndromic OFCs.

Genome-wide interaction studies identify sex-specific risk alleles for nonsyndromic orofacial clefts.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect in humans and is notable for its apparent sexual dimorphism where approximately twice as many males are affected as females. The sources of this disparity are largely unknown, but interactions between genetic and sex effects are likely contributors. We examined gene-by-sex (G × S) interactions in a worldwide sample of 2,142 NSCL/P cases and 1,700 controls recruited from 13 countries. First, we performed genome-wide joint tests of the genetic (G) and G × S effects genome-wide using logistic regression assuming an additive genetic model and adjusting for 18 principal components of ancestry. We further interrogated loci with suggestive results from the joint test ( p < 1.00 × 10 -5 ) by examining the G × S effects from the same model. Out of the 133 loci with suggestive results ( p < 1.00 × 10 -5 ) for the joint test, we observed one genome-wide significant G × S effect in the 10q21 locus (rs72804706; p = 6.69 × 10 -9 ; OR = 2.62 CI [1.89, 3.62]) and 16 suggestive G × S effects. At the intergenic 10q21 locus, the risk of NSCL/P is estimated to increase with additional copies of the minor allele for females, but the opposite effect for males. Our observation that the impact of genetic variants on NSCL/P risk differs for males and females may further our understanding of the genetic architecture of NSCL/P and the sex differences underlying clefts and other birth defects.

GWAS reveals loci associated with velopharyngeal dysfunction.

Velopharyngeal dysfunction (VPD) occurs when the muscular soft palate (velum) and lateral pharyngeal walls are physically unable to separate the oral and nasal cavities during speech production leading to hypernasality and abnormal speech reduction. Because VPD is often associated with overt or submucous cleft palate, it could be present as a subclinical phenotype in families with a history of orofacial clefting. A key assumption to this model is that the overt and subclinical manifestations of the orofacial cleft phenotype exist on a continuum and therefore share common etiological factors. We performed a genome-wide association study in 976 unaffected relatives of isolated CP probands, 54 of whom had VPD. Five loci were significantly (p < 5 × 10-8) associated with VPD: 3q29, 9p21.1, 12q21.31, 16p12.3 and 16p13.3. An additional 15 loci showing suggestive evidence of association with VPD were observed. Several genes known to be involved in orofacial clefting and craniofacial development are located in these regions, such as TFRC, PCYT1A, BNC2 and FREM1. Although further research is necessary, this could be an indication for a potential shared genetic architecture between VPD and cleft palate, and supporting the hypothesis that VPD is a subclinical phenotype of orofacial clefting.