Polygenic risk impacts PDGFRA mutation penetrance in non-syndromic cleft lip and palate.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common, severe craniofacial malformation that imposes significant medical, psychosocial and financial burdens. NSCL/P is a multifactorial disorder with genetic and environmental factors playing etiologic roles. Currently, only 25% of the genetic variation underlying NSCL/P has been identified by linkage, candidate gene and genome-wide association studies. In this study, whole-genome sequencing and genome-wide genotyping followed by polygenic risk score (PRS) and linkage analyses were used to identify the genetic etiology of NSCL/P in a large three-generation family. We identified a rare missense variant in PDGFRA (c.C2740T; p.R914W) as potentially etiologic in a gene-based association test using pVAAST (P = 1.78 × 10-4) and showed decreased penetrance. PRS analysis suggested that variant penetrance was likely modified by common NSCL/P risk variants, with lower scores found among unaffected carriers. Linkage analysis provided additional support for PRS-modified penetrance, with a 7.4-fold increase in likelihood after conditioning on PRS. Functional characterization experiments showed that the putatively causal variant was null for signaling activity in vitro; further, perturbation of pdgfra in zebrafish embryos resulted in unilateral orofacial clefting. Our findings show that a rare PDGFRA variant, modified by additional common NSCL/P risk variants, have a profound effect on NSCL/P risk. These data provide compelling evidence for multifactorial inheritance long postulated to underlie NSCL/P and may explain some unusual familial patterns.

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 association and characterization of FSTL5 in isolated clubfoot.

Talipes equinovarus (clubfoot, TEV) is a congenital rotational foot deformity occurring in 1 per 1000 births with increased prevalence in males compared with females. The genetic etiology of isolated clubfoot (iTEV) remains unclear. Using a genome-wide association study, we identified a locus within FSTL5, encoding follistatin-like 5, significantly associated with iTEV. FSTL5 is an uncharacterized gene whose potential role in embryonic and postnatal development was previously unstudied. Utilizing multiple model systems, we found that Fstl5 was expressed during later stages of embryonic hindlimb development, and, in mice, expression was restricted to the condensing cartilage anlage destined to form the limb skeleton. In the postnatal growth plate, Fstl5 was specifically expressed in prehypertrophic chondrocytes. As Fstl5 knockout rats displayed no gross malformations, we engineered a conditional transgenic mouse line (Fstl5LSL) to overexpress Fstl5 in skeletal osteochondroprogenitors. We observed that hindlimbs were slightly shorter and that bone mineral density was reduced in adult male, but not female, Prrx1-cre;Fstl5LSL mice compared with control. No overt clubfoot-like deformity was observed in Prrx1-cre;Fstl5LSL mice, suggesting FSTL5 may function in other cell types to contribute to iTEV pathogenesis. Interrogating published mouse embryonic single-cell expression data showed that Fstl5 was expressed in cell lineage subclusters whose transcriptomes were associated with neural system development. Moreover, our results suggest that lineage-specific expression of the Fstl genes correlates with their divergent roles as modulators of transforming growth factor beta and bone morphogenetic protein signaling. Results from this study associate FSTL5 with iTEV and suggest a potential sexually dimorphic role for Fstl5 in vivo.

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.

Route of delivery does not impact postnatal surgical morbidity in pregnancies affected by fetal achondroplasia.

PURPOSE: Pregnancies affected by maternal or fetal achondroplasia present unique challenges. The optimal route of delivery in fetuses with achondroplasia has not been established. Our objective was to determine whether the route of delivery affects postnatal achondroplasia-related surgical burden. METHODS: We conducted a secondary analysis of Achondroplasia Natural History Study (CLARITY), which is a multicenter natural history cohort study of patients with achondroplasia. Achondroplasia-related surgical morbidity, which we defined as the need for one or more postnatal achondroplasia-related surgeries, was assessed in relation to the route of delivery and whether the mother also had achondroplasia. Rate of each individual surgery type (otolaryngology, brain, foramen magnum, spine, and extremity) was also assessed in relation to the route of delivery. RESULTS: Eight hundred fifty-seven patients with achondroplasia with known route of delivery and known maternal stature were included. Three hundred sixty (42%) patients were delivered vaginally, and 497 (58%) patients were delivered by a cesarean delivery. There was no difference in the odds of requiring any postnatal achondroplasia-related surgery in those with achondroplasia who were delivered vaginally compared with those delivered by cesarean birth (odds ratio 0.95, 95% CI = 0.68-1.34, P = .80). No difference was present in the odds of requiring any postnatal achondroplasia-related surgery when route of delivery was compared for fetuses born to 761 average stature mothers (odds ratio 1.05, 95% CI = 0.74-1.51, P = .78). There was also no difference in the odds of requiring each of the individual achondroplasia-related surgeries by route of delivery, including cervicomedullary decompression. CONCLUSION: Our study suggests that it is reasonable for average stature patients carrying a fetus with achondroplasia to undergo a trial of labor in the absence of routine obstetric contraindications.

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.

CurQ+, a Next-Generation Formulation of Curcumin, Ameliorates Growth Plate Chondrocyte Stress and Increases Limb Growth in a Mouse Model of Pseudoachondroplasia.

Mutations in cartilage oligomeric matrix protein (COMP) causes protein misfolding and accumulation in chondrocytes that compromises skeletal growth and joint health in pseudoachondroplasia (PSACH), a severe dwarfing condition. Using the MT-COMP mice, a murine model of PSACH, we showed that pathological autophagy blockage was key to the intracellular accumulation of mutant-COMP. Autophagy is blocked by elevated mTORC1 signaling, preventing ER clearance and ensuring chondrocyte death. We demonstrated that resveratrol reduces the growth plate pathology by relieving the autophagy blockage allowing the ER clearance of mutant-COMP, which partially rescues limb length. To expand potential PSACH treatment options, CurQ+, a uniquely absorbable formulation of curcumin, was tested in MT-COMP mice at doses of 82.3 (1X) and 164.6 mg/kg (2X). CurQ+ treatment of MT-COMP mice from 1 to 4 weeks postnatally decreased mutant COMP intracellular retention, inflammation, restoring both autophagy and chondrocyte proliferation. CurQ+ reduction of cellular stress in growth plate chondrocytes dramatically reduced chondrocyte death, normalized femur length at 2X 164.6 mg/kg and recovered 60% of lost limb growth at 1X 82.3 mg/kg. These results indicate that CurQ+ is a potential therapy for COMPopathy-associated lost limb growth, joint degeneration, and other conditions involving persistent inflammation, oxidative stress, and a block of autophagy.

Primary Osteoarthritis Early Joint Degeneration Induced by Endoplasmic Reticulum Stress Is Mitigated by Resveratrol.

Increasing numbers of people are living with osteoarthritis (OA) due to aging and obesity, creating an urgent need for effective treatment and preventions. Two top risk factors for OA, age and obesity, are associated with endoplasmic reticulum (ER) stress. The I-ERS mouse, an ER stress-driven model of primary OA, was developed to study the role of ER stress in primary OA susceptibility. The I-ERS mouse has the unique ability to induce ER stress in healthy adult articular chondrocytes and cartilage, driving joint degeneration that mimics early primary OA. In this study, ER stress-induced damage occurred gradually and stimulated joint degeneration with OA characteristics including increased matrix metalloproteinase activity, inflammation, senescence, chondrocyte death, decreased proteoglycans, autophagy block, and gait dysfunction. Consistent with human OA, intense exercise hastened and increased the level of ER stress-induced joint damage. Notably, loss of a critical ER stress response protein (CHOP) largely ameliorated ER stress-stimulated OA outcomes including preserving proteoglycan content, reducing inflammation, and relieving autophagy block. Resveratrol diminished ER stress-induced joint degeneration by decreasing CHOP, TNFα, IL-1ß, MMP-13, pS6, number of TUNEL-positive chondrocytes, and senescence marker p16 INK4a. The finding, that a dietary supplement can prevent ER stressed-induced joint degeneration in mice, provides a preclinical foundation to potentially develop a prevention strategy for those at high risk to develop OA.

Whole Exome Sequencing in Individuals with Idiopathic Clubfoot Reveals a Recurrent Filamin B (FLNB) Deletion.

BACKGROUND: Clubfoot, a congenital deformity that presents as a rigid, inward turning of the foot, affects approximately 1 in 1000 infants and occurs as an isolated birth defect in 80% of patients. Despite its high level of heritability, few causative genes have been identified, and mutations in known genes are only responsible for a small portion of clubfoot heritability. QUESTIONS/PURPOSES: (1) Are any rare gene variants enriched (that is, shared) in unrelated patients with isolated clubfoot? (2) Are there other rare variants in the identified gene (Filamin B) in these patients with clubfoot? METHODS: Whole-exome sequence data were generated from a discovery cohort of 183 unrelated probands with clubfoot and 2492 controls. Variants were filtered with minor allele frequency < 0.02 to identify rare variants as well as small insertions and deletions (indels) resulting in missense variants, nonsense or premature truncation, or in-frame deletions. A candidate deletion was then genotyped in another cohort of 974 unrelated patients with clubfoot (a replication cohort). Other rare variants in the candidate gene were also investigated. A segregation analysis was performed in multigenerational families of individuals with clubfoot to see if the genotypes segregate with phenotypes. Single-variant association analysis was performed using the Fisher two-tailed exact test (exact p values are presented to give an indication of the magnitude of the association). RESULTS: There were no recurrent variants in the known genes causing clubfoot in this study. A three-base pair in-frame codon deletion of Filamin B (FLNB) (p.E1792del, rs1470699812) was identified in 1.6% (3 of 183) of probands with clubfoot in the discovery cohort compared with 0% of controls (0 of 2492) (odds ratio infinity (inf) [95% CI 5.64 to inf]; p = 3.18 x 10-5) and 0.0016% of gnomAD controls (2 of 125,709) (OR 1.01 x 103 [95% CI 117.42 to 1.64 x 104]; p = 3.13 x 10-8). By screening a replication cohort (n = 974 patients), we found two probands with the identical FLNB deletion. In total, the deletion was identified in 0.43% (5 of 1157) of probands with clubfoot compared with 0% of controls and 0.0016% of gnomAD controls (OR 268.5 [95% CI 43.68 to 2.88 x 103]; p = 1.43 x 10-9). The recurrent FLNB p.E1792del variant segregated with clubfoot, with incomplete penetrance in two families. Affected individuals were more likely to be male and have bilateral clubfoot. Although most patients had isolated clubfoot, features consistent with Larsen syndrome, including upper extremity abnormalities such as elbow and thumb hypermobility and wide, flat thumbs, were noted in affected members of one family. We identified 19 additional rare FLNB missense variants located throughout the gene in patients with clubfoot. One of these missense variants, FLNB p.G2397D, exhibited incomplete penetrance in one family. CONCLUSION: A recurrent FLNB E1792 deletion was identified in 0.43% of 1157 isolated patients with clubfoot. Given the absence of any recurrent variants in our discovery phase (n = 183) for any of the known genes causing clubfoot, our findings support that novel and rare missense variants in FLNB in patients with clubfoot, although rare, may be among the most commonly known genetic causes of clubfoot. Patients with FLNB variants often have isolated clubfoot, but they and their family members may be at an increased risk of having additional clinical features consistent with Larsen syndrome. CLINICAL RELEVANCE: Identification of FLNB variants may be useful for determining clubfoot recurrence risk and comorbidities.

Achondroplasia Natural History Study (CLARITY): a multicenter retrospective cohort study of achondroplasia in the United States.

PURPOSE: Achondroplasia is the most common short stature skeletal dysplasia (1:20,000-30,000), but the risk of adverse health outcomes from cardiovascular diseases, pain, poor function, excess weight, and sleep apnea is unclear. A multicenter retrospective natural history study was conducted to understand medical and surgical practices in achondroplasia. METHODS: Data from patients with achondroplasia evaluated by clinical geneticists at Johns Hopkins University, A.I. duPont Hospital for Children, McGovern Medical School UTHealth, and University of Wisconsin were populated into a REDCap database. All available retrospective medical records of anthropometry (length/height, weight, occipitofrontal circumference), surgery, polysomnography (PSG), and imaging (e.g., X-ray, magnetic resonance imaging) were included. RESULTS: Data from 1,374 patients (48.8% female; mean age 15.4 ± 13.9 years) constitute the primary achondroplasia cohort (PAC) with 496 subjects remaining clinically active and eligible for prospective studies. Within the PAC, 76.0% had a de novo FGFR3 pathologic variant and 1,094 (79.6%) had one or more achondroplasia-related surgeries. There are ≥37,000 anthropometry values, 1,631 PSGs and 10,727 imaging studies. CONCLUSION: This is the largest multicenter achondroplasia natural history study, providing a vast array of medical information for use in caring for these patients. This well-phenotyped cohort is a reference population against which future medical and surgical interventions can be compared.

CLARITY: Co-occurrences in achondroplasia-craniosynostosis, seizures, and decreased risk of diabetes mellitus.

Achondroplasia is the most common disproportionate short statured skeletal dysplasia with a prevalence of approximately 1:20,000-30,000. We created the largest database to date of a historical cohort of 1374 patients with achondroplasia (CLARITY-aChondropLasia nAtuRal hIsTory studY). This cohort was queried for the presence of unrecognized or under-recognized features associated with achondroplasia. Craniosynostosis was found to co-occur with achondroplasia in 9 (0.65%) patients in this cohort, which is much higher than the general population prevalence of 3.1-7.2 per 10,000. In addition, 27 patients had seizures (2.0%), an apparent excess as compared to the general population. Only two people had diabetes despite a high rate of adult obesity. This report documents for the first time an increased prevalence of craniosynostosis in persons with achondroplasia, and adds support to previous observations of an apparently higher than expected prevalence of seizures and lower prevalence of diabetes mellitus.

Joint Degeneration in a Mouse Model of Pseudoachondroplasia: ER Stress, Inflammation, and Block of Autophagy.

Pseudoachondroplasia (PSACH), a short limb skeletal dysplasia associated with premature joint degeneration, is caused by misfolding mutations in cartilage oligomeric matrix protein (COMP). Here, we define mutant-COMP-induced stress mechanisms that occur in articular chondrocytes of MT-COMP mice, a murine model of PSACH. The accumulation of mutant-COMP in the ER occurred early in MT-COMP articular chondrocytes and stimulated inflammation (TNFα) at 4 weeks, and articular chondrocyte death increased at 8 weeks while ER stress through CHOP was elevated by 12 weeks. Importantly, blockage of autophagy (pS6), the major mechanism that clears the ER, sustained cellular stress in MT-COMP articular chondrocytes. Degeneration of MT-COMP articular cartilage was similar to that observed in PSACH and was associated with increased MMPs, a family of degradative enzymes. Moreover, chronic cellular stresses stimulated senescence. Senescence-associated secretory phenotype (SASP) may play a role in generating and propagating a pro-degradative environment in the MT-COMP murine joint. The loss of CHOP or resveratrol treatment from birth preserved joint health in MT-COMP mice. Taken together, these results indicate that ER stress/CHOP signaling and autophagy blockage are central to mutant-COMP joint degeneration, and MT-COMP mice joint health can be preserved by decreasing articular chondrocyte stress. Future joint sparing therapeutics for PSACH may include resveratrol.

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.

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.

Evidence for craniofacial enhancer variation underlying nonsyndromic cleft lip and palate.

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect for which only ~ 20% of the underlying genetic variation has been identified. Variants in noncoding regions have been increasingly suggested to contribute to the missing heritability. In this study, we investigated whether variation in craniofacial enhancers contributes to NSCLP. Candidate enhancers were identified using VISTA Enhancer Browser and previous publications. Prioritization was based on patterning defects in knockout mice, deletion/duplication of craniofacial genes in animal models and results of whole exome/whole genome sequencing studies. This resulted in 20 craniofacial enhancers to be investigated. Custom amplicon-based sequencing probes were designed and used for sequencing 380 NSCLP probands (from multiplex and simplex families of non-Hispanic white (NHW) and Hispanic ethnicities) using Illumina MiSeq. The frequencies of identified variants were compared to ethnically matched European (CEU) and Los Angeles Mexican (MXL) control genomes and used for association analyses. Variants in mm427/MSX1 and hs1582/SPRY1 showed genome-wide significant association with NSCLP (p ≤ 6.4 × 10-11). In silico analysis showed that these enhancer variants may disrupt important transcription factor binding sites. Haplotypes involving these enhancers and also mm435/ABCA4 were significantly associated with NSCLP, especially in NHW (p ≤ 6.3 × 10-7). Importantly, groupwise burden analysis showed several enhancer combinations significantly over-represented in NSCLP individuals, revealing novel NSCLP pathways and supporting a polygenic inheritance model. Our findings support the role of craniofacial enhancer sequence variation in the etiology of NSCLP.

Whole genome sequencing of orofacial cleft trios from the Gabriella Miller Kids First Pediatric Research Consortium identifies a new locus on chromosome 21.

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 the genetic etiology of non-syndromic OFCs is only partially determined. Here, we analyze whole genome sequence (WGS) data for association with risk of OFCs in European and Colombian families selected from a multicenter family-based OFC study. This is the first large-scale WGS study of OFC in parent-offspring trios, and a part of the Gabriella Miller Kids First Pediatric Research Program created for the study of childhood cancers and structural birth defects. WGS provides deeper and more specific genetic data than using imputation on present-day single nucleotide polymorphic (SNP) marker panels. Genotypes of case-parent trios at single nucleotide variants (SNV) and short insertions and deletions (indels) spanning the entire genome were called from their sequences using human GRCh38 genome assembly, and analyzed for association using the transmission disequilibrium test. Among genome-wide significant associations, we identified a new locus on chromosome 21 in Colombian families, not previously observed in other larger OFC samples of Latin American ancestry. This locus is situated within a region known to be expressed during craniofacial development. Based on deeper investigation of this locus, we concluded that it contributed risk for OFCs exclusively in the Colombians. This study reinforces the ancestry differences seen in the genetic etiology of OFCs, and underscores the need for larger samples when studying for OFCs and other birth defects in populations with diverse ancestry.

Multiethnic GWAS Reveals Polygenic Architecture of Earlobe Attachment.

The genetic basis of earlobe attachment has been a matter of debate since the early 20th century, such that geneticists argue both for and against polygenic inheritance. Recent genetic studies have identified a few loci associated with the trait, but large-scale analyses are still lacking. Here, we performed a genome-wide association study of lobe attachment in a multiethnic sample of 74,660 individuals from four cohorts (three with the trait scored by an expert rater and one with the trait self-reported). Meta-analysis of the three expert-rater-scored cohorts revealed six associated loci harboring numerous candidate genes, including EDAR, SP5, MRPS22, ADGRG6 (GPR126), KIAA1217, and PAX9. The large self-reported 23andMe cohort recapitulated each of these six loci. Moreover, meta-analysis across all four cohorts revealed a total of 49 significant (p < 5 × 10-8) loci. Annotation and enrichment analyses of these 49 loci showed strong evidence of genes involved in ear development and syndromes with auricular phenotypes. RNA sequencing data from both human fetal ear and mouse second branchial arch tissue confirmed that genes located among associated loci showed evidence of expression. These results provide strong evidence for the polygenic nature of earlobe attachment and offer insights into the biological basis of normal and abnormal ear development.

Novel mTORC1 Mechanism Suggests Therapeutic Targets for COMPopathies.

Cartilage oligomeric matrix protein (COMP) is a large, multifunctional extracellular protein that, when mutated, is retained in the rough endoplasmic reticulum (ER). This retention elicits ER stress, inflammation, and oxidative stress, resulting in dysfunction and death of growth plate chondrocytes. While identifying the cellular pathologic mechanisms underlying the murine mutant (MT)-COMP model of pseudoachondroplasia, increased midline-1 (MID1) expression and mammalian target of rapamycin complex 1 (mTORC1) signaling was found. This novel role for MID1/mTORC1 signaling was investigated since treatments shown to repress the pathology also reduced Mid1/mTORC1. Although ER stress-inducing drugs or tumor necrosis factor α (TNFα) in rat chondrosarcoma cells increased Mid1, oxidative stress did not, establishing that ER stress- or TNFα-driven inflammation alone is sufficient to elevate MID1 expression. Since MID1 ubiquitinates protein phosphatase 2A (PP2A), a negative regulator of mTORC1, PP2A was evaluated in MT-COMP growth plate chondrocytes. PP2A was decreased, indicating de-repression of mTORC1 signaling. Rapamycin treatment in MT-COMP mice reduced mTORC1 signaling and intracellular retention of COMP, and increased proliferation, but did not change inflammatory markers IL-16 and eosinophil peroxidase. Lastly, mRNA from tuberous sclerosis-1/2-null mice brain tissue exhibiting ER stress had increased Mid1 expression, confirming the relationship between ER stress and MID1/mTORC1 signaling. These findings suggest a mechanistic link between ER stress and MID1/mTORC1 signaling that has implications extending to other conditions involving ER stress.

The first reported case of Loeys-Dietz syndrome in a patient with biallelic SMAD3 variants.

Loeys-Dietz syndrome (LDS), a connective tissue disorder characterized by its vascular, skeletal, craniofacial, and cutaneous manifestations is caused by mutations in one of six genes (TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2, and TGFB3). Until recently, all reported cases of LDS have been attributed to heterozygous pathogenic variants in these genes. Here, we report the first case of Loeys-Dietz syndrome due to SMAD3 biallelic likely pathogenic variants in a 15-year-old male with classic Loeys-Dietz features, including dysmorphic facial features, significant scoliosis, and pectus excavatum, arachnodactyly, severe aortic root dilation, and diffuse arterial tortuosity. His parents are each heterozygous for the likely pathogenic variant and are more mildly affected. To our knowledge, this represents the first reported case of biallelic SMAD3-related Loeys-Dietz syndrome and the third case in the literature of biallelic LDS, indicating that there are multiple genetic modes of inheritance underlying this disorder.