Disruption of DNA methylation-mediated cranial neural crest proliferation and differentiation causes orofacial clefts in mice.

Orofacial clefts of the lip and palate are widely recognized to result from complex gene-environment interactions, but inadequate understanding of environmental risk factors has stymied development of prevention strategies. We interrogated the role of DNA methylation, an environmentally malleable epigenetic mechanism, in orofacial development. Expression of the key DNA methyltransferase enzyme DNMT1 was detected throughout palate morphogenesis in the epithelium and underlying cranial neural crest cell (cNCC) mesenchyme, a highly proliferative multipotent stem cell population that forms orofacial connective tissue. Genetic and pharmacologic manipulations of DNMT activity were then applied to define the tissue- and timing-dependent requirement of DNA methylation in orofacial development. cNCC-specific Dnmt1 inactivation targeting initial palate outgrowth resulted in OFCs, while later targeting during palatal shelf elevation and elongation did not. Conditional Dnmt1 deletion reduced cNCC proliferation and subsequent differentiation trajectory, resulting in attenuated outgrowth of the palatal shelves and altered development of cNCC-derived skeletal elements. Finally, we found that the cellular mechanisms of cleft pathogenesis observed in vivo can be recapitulated by pharmacologically reducing DNA methylation in multipotent cNCCs cultured in vitro. These findings demonstrate that DNA methylation is a crucial epigenetic regulator of cNCC biology, define a critical period of development in which its disruption directly causes OFCs, and provide opportunities to identify environmental influences that contribute to OFC risk.

Frem1 activity is regulated by Sonic hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis.

BACKGROUND: Frem1 has been linked to human face shape variation, dysmorphology, and malformation, but little is known about its regulation and biological role in facial development. RESULTS: During midfacial morphogenesis in mice, we observed Frem1 expression in the embryonic growth centers that form the median upper lip, nose, and palate. Expansive spatial gradients of Frem1 expression in the cranial neural crest cell (cNCC) mesenchyme of these tissues suggested transcriptional regulation by a secreted morphogen. Accordingly, Frem1 expression paralleled that of the conserved Sonic Hedgehog (Shh) target gene Gli1 in the cNCC mesenchyme. Suggesting direct transcriptional regulation by Shh signaling, we found that Frem1 expression is induced by SHH ligand stimulation or downstream pathway activation in cNCCs and observed GLI transcription factor binding at the Frem1 transcriptional start site during midfacial morphogenesis. Finally, we found that FREM1 is sufficient to induce cNCC proliferation in a concentration-dependent manner and that Shh pathway antagonism reduces Frem1 expression during pathogenesis of midfacial hypoplasia. CONCLUSIONS: By demonstrating that the Shh signaling pathway regulates Frem1 expression in cNCCs, these findings provide novel insight into the mechanisms underlying variation in midfacial morphogenesis.

Genome-wide Enrichment of De Novo Coding Mutations in Orofacial Cleft Trios.

Although de novo mutations (DNMs) are known to increase an individual's risk of congenital defects, DNMs have not been fully explored regarding orofacial clefts (OFCs), one of the most common human birth defects. Therefore, whole-genome sequencing of 756 child-parent trios of European, Colombian, and Taiwanese ancestry was performed to determine the contributions of coding DNMs to an individual's OFC risk. Overall, we identified a significant excess of loss-of-function DNMs in genes highly expressed in craniofacial tissues, as well as genes associated with known autosomal dominant OFC syndromes. This analysis also revealed roles for zinc-finger homeobox domain and SOX2-interacting genes in OFC etiology.

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations.

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

Gene-environment interactions: aligning birth defects research with complex etiology.

Developmental biologists rely on genetics-based approaches to understand the origins of congenital abnormalities. Recent advancements in genomics have made it easier than ever to investigate the relationship between genes and disease. However, nonsyndromic birth defects often exhibit non-Mendelian inheritance, incomplete penetrance or variable expressivity. The discordance between genotype and phenotype indicates that extrinsic factors frequently impact the severity of genetic disorders and vice versa. Overlooking gene-environment interactions in birth defect etiology limits our ability to identify and eliminate avoidable risks. We present mouse models of sonic hedgehog signaling and craniofacial malformations to illustrate both the importance of and current challenges in resolving gene-environment interactions in birth defects. We then prescribe approaches for overcoming these challenges, including use of genetically tractable and environmentally responsive in vitro systems. Combining emerging technologies with molecular genetics and traditional animal models promises to advance our understanding of birth defect etiology and improve the identification and protection of vulnerable populations.

FAT4 identified as a potential modifier of orofacial cleft laterality.

Orofacial clefts (OFCs) are common (1 in 700 births) congenital malformations that include a cleft lip (CL) and cleft lip and palate (CLP). These OFC subtypes are also heterogeneous themselves, with the CL occurring on the left, right, or both sides of the upper lip. Unilateral CL and CLP have a 2:1 bias towards left-sided clefts, suggesting a nonrandom process. Here, we performed a study of left- and right-sided clefts within the CL and CLP subtypes to better understand the genetic factors controlling cleft laterality. We conducted genome-wide modifier analyses by comparing cases that had right unilateral CL (RCL; N = 130), left unilateral CL (LCL; N = 216), right unilateral CLP (RCLP; N = 416), or left unilateral CLP (LCLP; N = 638), and identified a candidate region on 4q28, 400 kb downstream from FAT4, that approached genome-wide significance for LCL versus RCL (p = 8.4 × 10-8 ). Consistent with its potential involvement as a genetic modifier of CL, we found that Fat4 exhibits a specific domain of expression in the mesenchyme of the medial nasal processes that form the median upper lip. Overall, these results suggest that the epidemiological similarities in left- to right-sided clefts in CL and CLP are not reflected in the genetic association results.

A CCR4-NOT Transcription Complex, Subunit 1, CNOT1, Variant Associated with Holoprosencephaly.

Holoprosencephaly is the incomplete separation of the forebrain during embryogenesis. Both genetic and environmental etiologies have been determined for holoprosencephaly; however, a genetic etiology is not found in most cases. In this report, we present two unrelated individuals with semilobar holoprosencephaly who have the identical de novo missense variant in the gene CCR4-NOT transcription complex, subunit 1 (CNOT1). The variant (c.1603C>T [p.Arg535Cys]) is predicted to be deleterious and is not present in public databases. CNOT1 has not been previously associated with holoprosencephaly or other brain malformations. In situ hybridization analyses of mouse embryos show that Cnot1 is expressed in the prosencephalic neural folds at gestational day 8.25 during the critical period for subsequent forebrain division. Combining human and mouse data, we show that CNOT1 is associated with incomplete forebrain division.

Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis.

Cleft lip is one of the most common human birth defects, yet our understanding of the mechanisms that regulate lip morphogenesis is limited. Here, we show in mice that sonic hedgehog (Shh)-induced proliferation of cranial neural crest cell (cNCC) mesenchyme is required for upper lip closure. Gene expression profiling revealed a subset of Forkhead box (Fox) genes that are regulated by Shh signaling during lip morphogenesis. During cleft pathogenesis, reduced proliferation in the medial nasal process mesenchyme paralleled the domain of reduced Foxf2 and Gli1 expression. SHH ligand induction of Foxf2 expression was dependent upon Shh pathway effectors in cNCCs, while a functional GLI-binding site was identified downstream of Foxf2 Consistent with the cellular mechanism demonstrated for cleft lip pathogenesis, we found that either SHH ligand addition or FOXF2 overexpression is sufficient to induce cNCC proliferation. Finally, analysis of a large multi-ethnic human population with cleft lip identified clusters of single-nucleotide polymorphisms in FOXF2 These data suggest that direct targeting of Foxf2 by Shh signaling drives cNCC mesenchyme proliferation during upper lip morphogenesis, and that disruption of this sequence results in cleft lip.

40th Annual David W Smith Workshop on Malformations and Morphogenesis: Abstracts of the 2019 Annual Meeting.

The 40th Annual David W. Smith Workshop on Malformations and Morphogenesis occurred August 23rd -28th, 2019 at the Snowbird Resort in Snowbird, Utah. The Workshop, which honors the legacy of David W. Smith, brought together clinicians and researchers interested in congenital malformations and their underlying mechanisms of morphogenesis from around the world. In addition to mechanisms of morphogenesis and delineation of new syndromes, the Workshop highlighted five themes: deformations, disruptions and fetal dysmorphology; orofacial clefting; central nervous system malformations; ciliopathies; and X-linked intellectual disabilities. This Conference Report includes the abstracts presented at the 2019 Workshop.

Cohesin complex-associated holoprosencephaly.

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.

Prenatal exposure to pesticides and risk for holoprosencephaly: a case-control study.

BACKGROUND: Pesticide exposure during susceptible windows and at certain doses are linked to numerous birth defects. Early experimental evidence suggests an association between active ingredients in pesticides and holoprosencephaly (HPE), the most common malformation of the forebrain in humans (1 in 250 embryos). No human studies to date have examined the association. This study investigated pesticides during multiple windows of exposure and fetal risk for HPE. It is hypothesized that pre-conception and early pregnancy, the time of brain development in utero, are the most critical windows of exposure. METHODS: A questionnaire was developed for this retrospective case-control study to estimate household, occupational, and environmental pesticide exposures. Four windows of exposure were considered: preconception, early, mid and late pregnancy. Cases were identified through the National Human Genome Research Institute's ongoing clinical studies of HPE. Similarly, controls were identified as children with Williams-Beuren syndrome, a genetic syndrome also characterized by congenital malformations, but etiologically unrelated to HPE. We assessed for differences in odds of exposures to pesticides between cases and controls. RESULTS: Findings from 91 cases and 56 controls showed an increased risk for HPE with reports of maternal exposure during pregnancy to select pesticides including personal insect repellants (adjusted odds ratio (aOR) 2.89, confidence interval (CI): 0.96-9.50) and insecticides and acaricides for pets (aOR 3.84, CI:1.04-16.32). Exposure to household pest control products during the preconception period or during pregnancy was associated with increased risk for HPE (aOR 2.60, OR: 0.84-8.68). No associations were found for occupational exposures to pesticides during pregnancy (aOR: 1.15, CI: 0.11-11.42), although exposure rates were low. Higher likelihood for HPE was also observed with residency next to an agricultural field (aOR 3.24, CI: 0.94-12.31). CONCLUSIONS: Observational findings are consistent with experimental evidence and suggest that exposure to personal, household, and agricultural pesticides during pregnancy may increase risk for HPE. Further investigations of gene by environment interactions are warranted.

A forebrain undivided: Unleashing model organisms to solve the mysteries of holoprosencephaly.

Evolutionary conservation and experimental tractability have made animal model systems invaluable tools in our quest to understand human embryogenesis, both normal and abnormal. Standard genetic approaches, particularly useful in understanding monogenic diseases, are no longer sufficient as research attention shifts toward multifactorial outcomes. Here, we examine this progression through the lens of holoprosencephaly (HPE), a common human malformation involving incomplete forebrain division, and a classic example of an etiologically complex outcome. We relate the basic underpinning of HPE pathogenesis to critical cell-cell interactions and signaling molecules discovered through embryological and genetic approaches in multiple model organisms, and discuss the role of the mouse model in functional examination of HPE-linked genes. We then outline the most critical remaining gaps to understanding human HPE, including the conundrum of incomplete penetrance/expressivity and the role of gene-environment interactions. To tackle these challenges, we outline a strategy that leverages new and emerging technologies in multiple model systems to solve the puzzle of HPE.

Identification of sonic hedgehog-regulated genes and biological processes in the cranial neural crest mesenchyme by comparative transcriptomics.

BACKGROUND: The evolutionarily conserved Sonic Hedgehog (Shh) signaling pathway is essential for embryogenesis and orofacial development. SHH ligand secreted from the surface ectoderm activates pathway activity in the underlying cranial neural crest cell (cNCC)-derived mesenchyme of the developing upper lip and palate. Disruption of Shh signaling causes orofacial clefts, but the biological action of Shh signaling and the full set of Shh target genes that mediate normal and abnormal orofacial morphogenesis have not been described. RESULTS: Using comparative transcriptional profiling, we have defined the Shh-regulated genes of the cNCC-derived mesenchyme. Enrichment analysis demonstrated that in cultured cNCCs, Shh-regulated genes are involved in smooth muscle and chondrocyte differentiation, as well as regulation of the Forkhead family of transcription factors, G1/S cell cycle transition, and angiogenesis. Next, this gene set from Shh-activated cNCCs in vitro was compared to the set of genes dysregulated in the facial primordia in vivo during the initial pathogenesis of Shh pathway inhibitor-induced orofacial clefting. Functional gene annotation enrichment analysis of the 112 Shh-regulated genes with concordant expression changes linked Shh signaling to interdependent and unique biological processes including mesenchyme development, cell adhesion, cell proliferation, cell migration, angiogenesis, perivascular cell markers, and orofacial clefting. CONCLUSIONS: We defined the Shh-regulated transcriptome of the cNCC-derived mesenchyme by comparing the expression signatures of Shh-activated cNCCs in vitro to primordial midfacial tissues exposed to the Shh pathway inhibitor in vivo. In addition to improving our understanding of cNCC biology by determining the identity and possible roles of cNCC-specific Shh target genes, this study presents novel candidate genes whose examination in the context of human orofacial clefting etiology is warranted.

Academic Achievement Among Children With Nonsyndromic Orofacial Clefts : A Population-Based Study.

OBJECTIVE: Children with orofacial clefts (OFCs) may experience poor reading proficiency, learning disabilities, and academic underachievement. We examined the association between nonsyndromic (NS) OFCs and end-of-grade (EOG) performance in reading and math from third through eighth grade in a sample subgroup. PARTICIPANTS: We identified a cohort of 559 children with NS-OFCs and 6822 children without birth defects, classifying cleft type by cleft lip alone, with or without cleft alveolar ridge (CL); cleft lip with cleft palate (CL+P); and cleft palate only (CP). MAIN OUTCOME MEASURES: Using logistic regression, we estimated the odds of not meeting grade-level standards among children with NS-OFCs compared to unaffected peers. Using longitudinal analyses, we estimated the odds of not meeting grade-level standards and average change in test scores through eighth grade. RESULTS: Children with NS-OFCs were 1.22 (95% CI: 0.96, 1.83) times as likely not to meet grade-level standards in reading compared to unaffected peers. The effect was similar for math (OR: 1.17; 95% CI: 0.92, 1.48). Children with CL+P were 1.33 (95% CI: 0.86, 1.83) and 1.74 (95% CI: 1.19, 2.56) times as likely not to meet grade-level standard in reading and in both subjects, respectively, compared to unaffected peers. The average rate of change in both scores was similar for children with and without OFCs. CONCLUSIONS: Poor academic performance appears greatest for children with CL+P, a finding compatible with previous observations and hypothesized mechanisms associating orofacial clefts with subtle abnormalities in brain development. Academic performance monitoring and referral for academic assistance is warranted.

Human germline hedgehog pathway mutations predispose to fatty liver.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. METHODS: Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2+/-) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. RESULTS: Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2+/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2+/- mice exposed to a high-fat diet. CONCLUSIONS: Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous buildup (scar tissue) and inflammation of the liver tissue. For the first time patients with holoprosencephaly, a disease caused by SHH signaling mutations, are shown to have increased liver steatosis independent of obesity. This observation was recapitulated in a mouse model of attenuated SHH signaling that also showed increased liver steatosis but with decreased fibrosis and inflammation. While SHH inhibition is associated with a good NAFLD prognosis, this increase in liver fat accumulation in the context of SHH signaling inhibition must be studied prospectively to evaluate its long-term effects, especially in individuals with Western-type dietary habits.

35(th) Annual David W Smith Workshop on Malformations and Morphogenesis: abstracts of the 2014 annual meeting.

The 35(th) Annual David W Smith Workshop on Malformations and Morphogenesis was held on July 25-30, 2014 at the University of Wisconsin, Madison, Wisconsin. The conference, which honors the legacy of David Smith, brought together over 130 clinicians and researchers interested in congenital malformations and their underlying mechanisms of morphogenesis. As is the tradition of the meeting, the Workshop highlighted five themes besides mechanisms of morphogenesis: Evolution and Development, Minor Malformations, CHARGE syndrome, Craniofacial Development/ Malformations, and Disorders of Chromatin Remodeling. This Conference Report includes the abstracts presented at the Workshop.

Gene-environment interactions in birth defect etiology: Challenges and opportunities.

Birth defects are relatively common congenital outcomes that significantly impact affected individuals, their families, and communities. Effective development and deployment of prevention and therapeutic strategies for these conditions requires sufficient understanding of etiology, including underlying genetic and environmental causes. Tremendous progress has been made in defining the genetic basis of familial and syndromic forms of birth defects. However, the majority of birth defect cases are considered nonsyndromic and thought to result from multifactorial gene-environment interactions. While substantial advances have been made in elucidating the genetic landscape of these etiologically complex conditions, significant biological and technical constraints have stymied progress toward a refined knowledge of environmental risk factors. Defining specific gene-environment interactions in birth defect etiology is even more challenging. However, progress has been made, including demonstration of critical proofs of concept and development of new conceptual and technical approaches for resolving complex gene-environment interactions. In this review, we discuss current views of multifactorial birth defect etiology, comparing them with other diseases that also involve gene-environment interactions, including primary immunodeficiency and cancer. We describe how various model systems have illuminated mechanisms of multifactorial etiology and these models' individual strengths and weaknesses. Finally, suggestions for areas of future emphasis are proposed.

Pharmacokinetic analysis of acute and dietary exposure to piperonyl butoxide in the mouse.

Piperonyl butoxide (PBO) is a popular insecticide synergist present in thousands of commercial, agricultural, and household products. PBO inhibits cytochrome P450 activity, impairing the ability of insects to detoxify insecticides. PBO was recently discovered to also inhibit Sonic hedgehog signaling, a pathway required for embryonic development, and rodent studies have demonstrated the potential for in utero PBO exposure to cause structural malformations of the brain, face, and limbs, or more subtle neurodevelopmental abnormalities. The current understanding of the pharmacokinetics of PBO in mice is limited, particularly with respect to dosing paradigms associated with developmental toxicity. To establish a pharmacokinetic (PK) model for oral exposure, PBO was administered to female C57BL/6J mice acutely by oral gavage (22-1800 mg/kg) or via diet (0.09 % PBO in chow). Serum and adipose samples were collected, and PBO concentrations were determined by HPLC-MS/MS. The serum concentrations of PBO were best fit by a linear one-compartment model. PBO concentrations in visceral adipose tissue greatly exceeded those in serum. PBO concentrations in both serum and adipose tissue decreased quickly after cessation of dietary exposure. The elimination half-life of PBO in the mouse after gavage dosing was 6.5 h (90 % CI 4.7-9.5 h), and systemic oral clearance was 83.3 ± 20.5 mL/h. The bioavailability of PBO in chow was 41 % that of PBO delivered in olive oil by gavage. Establishment of this PK model provides a foundation for relating PBO concentrations that cause developmental toxicity in the rodent models to Sonic hedgehog signaling pathway inhibition.

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.

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.