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Abstract Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators ESRP1 and ESRP2 regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using esrp1/2 mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human ESRP1/2 gene variants. We found that many variants predicted by in silico methods to be pathogenic were functionally benign. Esrp1 also regulates the alternative splicing of Ctnnd1 and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of ctnnd1 is sufficient to rescue morphogenesis of epithelial-derived structures in esrp1/2 zebrafish mutants. Additionally, we identified 13 CTNND1 variants from genome sequencing of OFC cohorts, confirming CTNND1 as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of Esrp-Ctnnd1 acting in the embryonic epithelium to regulate palatogenesis.

Summary: Background: Phenotypic variability within families with epilepsy is often observed, even when relatives share the same monogenic cause. We aimed to investigate whether common polygenic risk for epilepsy could explain the penetrance and phenotypic expression of rare pathogenic variants in familial epilepsies. Methods: We studied 58 clinically heterogeneous families with genetic epilepsy with febrile seizures plus (GEFS+). Relatives were coded as either unaffected or affected with epilepsy, and graded according to phenotype severity: no seizures, febrile seizures (FS) only, febrile seizures plus (FS+), generalised/focal epilepsy, or developmental and epileptic encephalopathy (DEE). Epilepsy polygenic risk scores (PRSs) were tested for association with epilepsy phenotype. Within families, the mean PRS difference was compared between pairs concordant versus discordant for phenotype severity. Statistical analyses were performed using mixed-effect regression models. Findings: 304 individuals segregating a known, or presumed, rare variant of large effect, were studied. Within families, higher epilepsy polygenic risk was associated with an epilepsy diagnosis (OR = 1.39, 95% CI 1.08, 1.80, padj = 0.040). Relatives with a more severe phenotype had a mean pairwise PRS difference of +0.19 higher than relatives with a milder phenotype (padj = 0.010). The difference increased with greater phenotype discordance between relatives. As the cohort included two rare variants with >30 relatives each, variant-specific genotype–phenotype associations could also be analysed. Whilst the epilepsy PRS effect was strong for relatives segregating the GABRG2 p.Arg82Gln pathogenic variant (padj = 0.0010), the effect was not significant for SCN1B p.Cys121Trp. Interpretation: We provide support for genetic background modifying the penetrance and phenotypic expression of rare variants associated with ‘monogenic’ epilepsies. In GEFS+ families, relatives with higher epilepsy PRSs were more likely to show penetrance (epilepsy diagnosis) and a more severe phenotype. Variant-specific analyses suggest that some rare variants may be more susceptible to PRS modification, carrying important genetic counselling and disease prognostication implications for patients. Funding: National Health and Medical Research Council of Australia, Medical Research Future Fund of Australia.

Summary: Orofacial clefts (OFCs) are common congenital birth defects with various etiologies, including genetic variants. Online Mendelian Inheritance in Man (OMIM) annotated several hundred genes involving OFCs. Furthermore, several hundreds of de novo variants (DNVs) have been identified from individuals with OFCs. Some DNVs are related to known OFC genes or pathways, but there are still many DNVs whose relevance to OFC development is unknown. To explore novel gene functions and their cellular expression profiles, we focused on DNVs in genes that were not listed in OMIM. We collected 960 DNVs in 853 genes from published studies and curated these genes, based on the DNVs’ deleteriousness, into 230 and 23 genes related to cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO), respectively. For comparison, we curated 178 CL/P and 277 CPO genes from OMIM. In CL/P, the pathways enriched in DNV and OMIM genes were significantly overlapped (p = 0.002). Single-cell RNA sequencing (scRNA-seq) analysis of mouse lip development revealed that both gene sets had abundant expression in the ectoderm (DNV genes: adjusted p = 0.032, OMIM genes: adjusted p

Summary: 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

Summary: Background: The epilepsies are highly heritable conditions that commonly follow complex inheritance. While monogenic causes have been identified in rare familial epilepsies, most familial epilepsies remain unsolved. We aimed to determine (1) whether common genetic variation contributes to familial epilepsy risk, and (2) whether that genetic risk is enriched in familial compared with non-familial (sporadic) epilepsies. Methods: Using common variants derived from the largest epilepsy genome-wide association study, we calculated polygenic risk scores (PRS) for patients with familial epilepsy (n = 1,818 from 1,181 families), their unaffected relatives (n = 771), sporadic patients (n = 1,182), and population controls (n = 15,929). We also calculated separate PRS for genetic generalised epilepsy (GGE) and focal epilepsy. Statistical analyses used mixed-effects regression models to account for familial relatedness, sex, and ancestry. Findings: Patients with familial epilepsies had higher epilepsy PRS compared to population controls (OR 1·20, padj = 5×10−9), sporadic patients (OR 1·11, padj = 0.008), and their own unaffected relatives (OR 1·12, padj = 0.01). The top 1% of the PRS distribution was enriched 3.8-fold for individuals with familial epilepsy when compared to the lowest decile (padj = 5×10−11). Familial PRS enrichment was consistent across epilepsy type; overall, polygenic risk was greatest for the GGE clinical group. There was no significant PRS difference in familial cases with established rare variant genetic etiologies compared to unsolved familial cases. Interpretation: The aggregate effects of common genetic variants, measured as polygenic risk scores, play an important role in explaining why some families develop epilepsy, why specific family members are affected while their relatives are not, and why families manifest specific epilepsy types. Polygenic risk contributes to the complex inheritance of the epilepsies, including in individuals with a known genetic etiology. Funding: National Health and Medical Research Council of Australia, National Institutes of Health, American Academy of Neurology, Thomas B and Jeannette E Laws McCabe Fund, Mirowski Family Foundation.

Exposure to polychlorinated biphenyls (PCBs), an endocrine-disrupting compound, is ubiquitous despite decades-old bans on the manufacture and use of PCBs. Increased exposure to PCBs is associated with adverse health consequences throughout life, including type 2 diabetes and cancer. PCB exposure is also associated with alterations in epigenetic marks and gene transcription, which could lead to adverse health outcomes, but many of these are population-specific. To further investigate the association between PCB and epigenetic marks, DNA methylation was measured at 787,684 CpG sites in 641 peripheral blood samples from the Michigan Polybrominated Biphenyl (PBB) Registry. 1345 CpGs were associated with increased total PCB level after controlling for age, sex, and 24 surrogate variables (FDR < 0.05). These CpGs were enriched in active promoter and transcription associated regions (p

Abstract In 1973, accidental contamination of Michigan livestock with polybrominated biphenyls (PBBs) led to the establishment of a registry of exposed individuals that have been followed for > 40 years. Besides being exposed to PBBs, this cohort has also been exposed to polychlorinated biphenyls (PCBs), a structurally similar class of environmental pollutants, at levels similar to average US exposure. In this study, we examined the association between current serum PCB and PBB levels and various female reproductive health outcomes to build upon previous work and inconsistencies. Participation in this cross-sectional study required a blood draw and completion of a detailed health questionnaire. Analysis included only female participants who had participated between 2012 and 2015 (N = 254). Multivariate linear and logistic regression models were used to identify associations between serum PCB and PBB levels with each gynecological and infertility outcome. Additionally, a generalized estimating equation (GEE) model was used to evaluate each pregnancy and birth outcome in order to account for multiple pregnancies per woman. We controlled for age, body mass index, and total lipid levels in all analyses. A p-value of

Endocrine-disrupting compounds are associated with altered epigenetic regulation and adverse health outcomes, although inconsistent results suggest that people have varied responses to the same exposure. Interpersonal variation in response to environmental exposures is not identified using standard, population-based methods. However, methods that capture an individual’s response, such as analyzing stochastic epigenetic mutations (SEMs), may capture currently missed effects of environmental exposure. To test whether polybrominated biphenyl (PBB) was associated with SEMs, DNA methylation was measured using Illumina’s MethylationEPIC array in PBB-exposed individuals, and SEMs were identified. Association was tested using a linear regression with robust sandwich variance estimators, controlling for age, sex, lipids, and cell types. The number of SEMs was variable (range: 119–18,309), and positively associated with age (p = 1.23e-17), but not with sex (p = 0.97). PBBs and SEMs were only positively associated in people who were older when they were exposed (p = 0.02 vs. p = 0.91). Many subjects had SEMs enriched in biological pathways, particularly in pathways involved with xenobiotic metabolism and endocrine function. Higher number of SEMs was also associated with higher age acceleration (intrinsic: p = 1.70e-3; extrinsic: p = 3.59e-11), indicating that SEMs may be associated with age-related health problems. Finding an association between environmental contaminants and higher SEMs may provide insight into individual differences in response to environmental contaminants, as well as into the biological mechanism behind SEM formation. Furthermore, these results suggest that people may be particularly vulnerable to epigenetic dysregulation from environmental exposures as they age.

Abstract Background Michigan residents were directly exposed to endocrine-disrupting compounds, polybrominated biphenyl (PBB) and polychlorinated biphenyl (PCB). A growing body of evidence suggests that exposure to certain endocrine-disrupting compounds may affect thyroid function, especially in people exposed as children, but there are conflicting observations. In this study, we extend previous work by examining age of exposure’s effect on the relationship between PBB exposure and thyroid function in a large group of individuals exposed to PBB. Methods Linear regression models were used to test the association between serum measures of thyroid function (total thyroxine (T4), total triiodothyronine (T3), free T4, free T3, thyroid stimulating hormone (TSH), and free T3: free T4 ratio) and serum PBB and PCB levels in a cross-sectional analysis of 715 participants in the Michigan PBB Registry. Results Higher PBB levels were associated with many thyroid hormones measures, including higher free T3 (p = 0.002), lower free T4 (p = 0.01), and higher free T3: free T4 ratio (p = 0.0001). Higher PCB levels were associated with higher free T4 (p = 0.0002), and higher free T3: free T4 ratio (p = 0.002). Importantly, the association between PBB and thyroid hormones was dependent on age at exposure. Among people exposed before age 16 (N = 446), higher PBB exposure was associated with higher total T3 (p = 0.01) and free T3 (p = 0.0003), lower free T4 (p = 0.04), and higher free T3: free T4 ratio (p = 0.0001). No significant associations were found among participants who were exposed after age 16. No significant associations were found between TSH and PBB or PCB in any of the analyses conducted. Conclusions This suggests that both PBB and PCB are associated with thyroid function, particularly among those who were exposed as children or prenatally.

In 1973, Michigan residents were exposed to polybrominated biphenyl (PBB) when it was accidentally added to farm animal feed. Highly exposed individuals and their children have experienced endocrine-related health problems, though the underlying mechanism behind these remains unknown. We investigated whether PBB exposure is associated with variation in DNA methylation in peripheral blood samples from 658 participants of the Michigan PBB registry using the MethylationEPIC BeadChip, as well as investigated what the potential function of the affected regions are and whether these epigenetic marks are known to associate with endocrine system pathways. After multiple test correction (FDR

Summary: Nonsyndromic orofacial clefts (OFCs) are a common birth defect and are phenotypically heterogenous in the structure affected by the cleft—cleft lip (CL) and cleft lip and palate (CLP)—as well as other features, such as the severity of the cleft. Here, we focus on bilateral and unilateral clefts as one dimension of OFC severity, because the genetic architecture of these subtypes is not well understood. We tested for subtype-specific genetic associations in 44 bilateral CL (BCL) individuals, 434 unilateral CL (UCL) individuals, 530 bilateral CLP individuals (BCLP), 1,123 unilateral CLP (UCLP) individuals, and unrelated control individuals (N = 1,626), using a mixed-model approach. While no novel loci were found, the genetic architecture of UCL was distinct compared to BCL, with 44.03% of suggestive loci having different effects between the two subtypes. To further understand the subtype-specific genetic risk factors, we performed a genome-wide scan for modifiers and found a significant modifier locus on 20p11 (p = 7.53 × 10−9), 300 kb downstream of PAX1, that associated with higher odds of BCL versus UCL and replicated in an independent cohort (p = 0.0018) with no effect in BCLP (p > 0.05). We further found that this locus was associated with normal human nasal shape. Taken together, these results suggest bilateral and unilateral clefts may have different genetic architectures. Moreover, our results suggest BCL, the rarest form of OFC, may be genetically distinct from the other OFC subtypes. This expands our understanding of modifiers for OFC subtypes and further elucidates the genetic mechanisms behind the phenotypic heterogeneity in OFCs.