The International Family Study of Nonsyndromic Orofacial Clefts: Design and Methods.

BACKGROUND: The majority of research to understand the risk factors of nonsyndromic orofacial clefts (NSOFCs) has been conducted in high-income populations. Although patients with NSOFCs in low- and middle-income countries (LMICs) are at the highest risk of not receiving care, global health infrastructure allows innovative partnerships to explore the etiologic mechanisms of cleft and targets for prevention unique to these populations. METHODS: The International Family Study (IFS) is an ongoing case-control study with supplemental parental trio data designed to examine genetic, environmental, lifestyle, and sociodemographic risk factors for NSOFCs in 8 LMICs (through August 2020). Interview and biological samples are collected for each family. The interview includes demographics, family history of cleft, diet and water sources, maternal pregnancy history, and other lifestyle and environmental factors. RESULTS: Seven of 8 countries are currently summarized (2012-2017) for a total of 2955 case and 2774 control families with 11 946 unique biological samples from Vietnam, Philippines, Honduras, Madagascar, Morocco, Democratic Republic of the Congo, and Nicaragua. The phenotype distribution was 1641 (55.5%) cases with cleft lip and palate, 782 (26.5%) with cleft lip (CL), and 432 (14.6%) with cleft palate (CP). DISCUSSION: The International Family Study is the largest case set of NSOFCs with an associated biobank in LMICs currently assembled. The biobank, family, and case-control study now include samples from 8 LMICs where local health care infrastructure cannot address the surgical burden of cleft or investigate causal mechanisms. The International Family Study can be a source of information and may collaborate with local public health institutions regarding education and interventions to potentially prevent NSOFCs.

The role of smoke from cooking indoors over an open flame and parental smoking on the risk of cleft lip and palate: A case- control study in 7 low-resource countries.

BACKGROUND: Cleft is one of the most common birth defects globally and the lack of access to surgery means millions are living untreated. Smoke exposure from cooking occurs infrequently in developed countries but represents a high-proportion of smoke exposure in less-developed regions. We aimed to study if smoke exposure from cooking is associated with an increased risk in cleft, while accounting for other smoke sources. METHODS: We conducted a population-sampled case-control study of children with cleft lip and/or palate and healthy newborns from Vietnam, Philippines, Honduras, Nicaragua, Morocco, Congo, and Madagascar. Multivariable regression models were used to assess associations between maternal cooking during pregnancy, parental smoking, and household tobacco smoke with cleft. RESULTS: 2137 cases and 2014 controls recruited between 2012-2017 were included. While maternal smoking was uncommon (<1%), 58.3% case and 36.1% control mothers cooked over an open fire inside. Children whose mothers reported cook smoke exposure were 49% (95% confidence interval (CI) = 1.2-1.8) more likely to have a child with a cleft. This was consistent in five of seven countries. No significant associations were found for any other smoke exposure. CONCLUSIONS: Our finding of maternal cook smoke and cleft in low-resource countries, similar to maternal tobacco smoke in high-resource countries, may reflect a common etiology. This relationship was present across geographically diverse countries with variable socioeconomic statuses and access to care. Exposures specific to low-resource settings must be considered to develop public health strategies that address the populations at increased risk of living with cleft and inform the mechanisms leading to cleft development.

A Synonymous Exonic Splice Silencer Variant in IRF6 as a Novel and Cryptic Cause of Non-Syndromic Cleft Lip and Palate.

Missense, nonsense, splice site and regulatory region variants in interferon regulatory factor 6 (IRF6) have been shown to contribute to both syndromic and non-syndromic forms of cleft lip and/or palate (CL/P). We report the diagnostic evaluation of a complex multigeneration family of Honduran ancestry with a pedigree structure consistent with autosomal-dominant inheritance with both incomplete penetrance and variable expressivity. The proband's grandmother bore children with two partners and CL/P segregates on both sides of each lineage. Through whole-exome sequencing of five members of the family, we identified a single shared synonymous variant, located in the middle of exon 7 of IRF6 (p.Ser307Ser; g.209963979 G>A; c.921C>T). The variant was shown to segregate in the seven affected individuals and through three unaffected obligate carriers, spanning both sides of this pedigree. This variant is very rare, only being found in three (all of Latino ancestry) of 251,352 alleles in the gnomAD database. While the variant did not create a splice acceptor/donor site, in silico analysis predicted it to impact an exonic splice silencer element and the binding of major splice regulatory factors. In vitro splice assays supported this by revealing multiple abnormal splicing events, estimated to impact >60% of allelic transcripts. Sequencing of the alternate splice products demonstrated the unmasking of a cryptic splice site six nucleotides 5' of the variant, as well as variable utilization of cryptic splice sites in intron 6. The ectopic expression of different splice regulatory proteins altered the proportion of abnormal splicing events seen in the splice assay, although the alteration was dependent on the splice factor. Importantly, each alternatively spliced mRNA is predicted to result in a frame shift and prematurely truncated IRF6 protein. This is the first study to identify a synonymous variant as a likely cause of NS-CL/P and highlights the care that should be taken by laboratories when considering and interpreting variants.

Genomic analyses in African populations identify novel risk loci for cleft palate.

Orofacial clefts are common developmental disorders that pose significant clinical, economical and psychological problems. We conducted genome-wide association analyses for cleft palate only (CPO) and cleft lip with or without palate (CL/P) with ~17 million markers in sub-Saharan Africans. After replication and combined analyses, we identified novel loci for CPO at or near genome-wide significance on chromosomes 2 (near CTNNA2) and 19 (near SULT2A1). In situ hybridization of Sult2a1 in mice showed expression of SULT2A1 in mesenchymal cells in palate, palatal rugae and palatal epithelium in the fused palate. The previously reported 8q24 was the most significant locus for CL/P in our study, and we replicated several previously reported loci including PAX7 and VAX1.

Paternal Risk Factors for Oral Clefts in Northern Africans, Southeast Asians, and Central Americans.

While several studies have investigated maternal exposures as risk factors for oral clefts, few have examined paternal factors. We conducted an international multi-centered case-control study to better understand paternal risk exposures for oral clefts (cases = 392 and controls = 234). Participants were recruited from local hospitals and oral cleft repair surgical missions in Vietnam, the Philippines, Honduras, and Morocco. Questionnaires were administered to fathers and mothers separately to elicit risk factor and family history data. Associations between paternal exposures and risk of clefts were assessed using logistic regression adjusting for potential confounders. A father's personal/family history of clefts was associated with significantly increased risk (adjusted OR: 4.77; 95% CI: 2.41-9.45). No other significant associations were identified for other suspected risk factors, including education (none/primary school v. university adjusted OR: 1.29; 95% CI: 0.74-2.24), advanced paternal age (5-year adjusted OR: 0.98; 95% CI: 0.84-1.16), or pre-pregnancy tobacco use (adjusted OR: 0.96; 95% CI: 0.67-1.37). Although sample size was limited, significantly decreased risks were observed for fathers with selected occupations. Further research is needed to investigate paternal environmental exposures as cleft risk factors.

Intraflagellar transport 88 (IFT88) is crucial for craniofacial development in mice and is a candidate gene for human cleft lip and palate.

Ciliopathies are pleiotropic human diseases resulting from defects of the primary cilium, and these patients often have cleft lip and palate. IFT88 is required for the assembly and function of the primary cilia, which mediate the activity of key developmental signaling pathways. Through whole exome sequencing of a family of three affected siblings with isolated cleft lip and palate, we discovered that they share a novel missense mutation in IFT88 (c.915G > C, p.E305D), suggesting this gene should be considered a candidate for isolated orofacial clefting. In order to evaluate the function of IFT88 in regulating craniofacial development, we generated Wnt1-Cre;Ift88fl/fl mice to eliminate Ift88 specifically in cranial neural crest (CNC) cells. Wnt1-Cre;Ift88fl/flpups died at birth due to severe craniofacial defects including bilateral cleft lip and palate and tongue agenesis, following the loss of the primary cilia in the CNC-derived palatal mesenchyme. Loss of Ift88 also resulted in a decrease in neural crest cell proliferation during early stages of palatogenesis as well as a downregulation of the Shh signaling pathway in the palatal mesenchyme. Importantly, Osr2KI-Cre;Ift88fl/flmice, in which Ift88 is lost specifically in the palatal mesenchyme, exhibit isolated cleft palate. Taken together, our results demonstrate that IFT88 has a highly conserved function within the primary cilia of the CNC-derived mesenchyme in the lip and palate region in mice and is a strong candidate as an orofacial clefting gene in humans.

Parental risk factors for oral clefts among Central Africans, Southeast Asians, and Central Americans.

BACKGROUND: Several lifestyle and environmental exposures have been suspected as risk factors for oral clefts, although few have been convincingly demonstrated. Studies across global diverse populations could offer additional insight given varying types and levels of exposures. METHODS: We performed an international case-control study in the Democratic Republic of the Congo (133 cases, 301 controls), Vietnam (75 cases, 158 controls), the Philippines (102 cases, 152 controls), and Honduras (120 cases, 143 controls). Mothers were recruited from hospitals and their exposures were collected from interviewer-administered questionnaires. We used logistic regression modeling to estimate odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Family history of clefts was strongly associated with increased risk (maternal: OR = 4.7; 95% CI, 3.0-7.2; paternal: OR = 10.5; 95% CI, 5.9-18.8; siblings: OR = 5.3; 95% CI, 1.4-19.9). Advanced maternal age (5 year OR = 1.2; 95% CI, 1.0-1.3), pregestational hypertension (OR = 2.6; 95% CI, 1.3-5.1), and gestational seizures (OR = 2.9; 95% CI, 1.1-7.4) were statistically significant risk factors. Lower maternal (secondary school OR = 1.6; 95% CI, 1.2-2.2; primary school OR = 2.4, 95% CI, 1.6-2.8) and paternal education (OR = 1.9; 95% CI, 1.4-2.5; and OR = 1.8; 95% CI, 1.1-2.9, respectively) and paternal tobacco smoking (OR = 1.5, 95% CI, 1.1-1.9) were associated with an increased risk. No other significant associations between maternal and paternal factors were found; some environmental factors including rural residency, indoor cooking with wood, chemicals and water source appeared to be associated with an increased risk in adjusted models. CONCLUSION: Our study represents one of the first international studies investigating risk factors for clefts among multiethnic underserved populations. Our findings suggest a multifactorial etiology including both maternal and paternal factors.