NIDCR Workshop: Gene-Environment Interactions in Orofacial Clefting Meeting Summary

Executive Summary

The National Institute of Dental and Craniofacial Research (NIDCR), in collaboration with the Centers for Disease Control (CDC) and NIH’s Eunice Kennedy Shriver National Institute of Child Health and Development (NICHD), National Institute of Alcohol Abuse and Alcoholism (NIAAA), National Institute of Drug Abuse (NIDA), and National Institute of Environmental Health Sciences (NIEHS), convened a workshop on September 6th and 7th, 2016 that brought together developmental biologists, epidemiologists, pediatricians, surgeons, representatives from professional associations, and other experts from institutions across the United States. The aim of the meeting was to assess current knowledge on gene-environment (GxE) interactions in the etiology of orofacial clefting, to identify needed resources, and to map out paths for future research.

Mounting evidence suggests that genetic and environmental factors influence the risk of orofacial clefting. However, little is known about the combined effects of gene-environment (GxE) interactions due to difficulties such as small sample sizes, heterogeneity of the birth defect across populations, and the challenge of accurately measuring exposure levels.

Two primary approaches have been used to study orofacial clefting: epidemiological analyses of human populations, and basic science approaches using model organisms such as mice, zebrafish and frogs. By examining both external and internal influences, research on GxE interactions has the potential to identify ways to reduce the risk of the birth defect, which can be associated with significant health complications that require long term follow-up.

Workshop attendees made the following suggestions for speeding the pace of discovery of GxE interactions in orofacial clefting:

Resources and research infrastructure:

  • Formation of a network of institutions focused on investigating the causes of birth defects, including clefting.
  • Strengthening ties between basic science and clinical researchers, perhaps using funding mechanisms that require these types of collaborations.
  • Establishment of a virtual repository for research results and information on tools such as questionnaires and banked tissue samples.
  • Broader utilization of existing tissue banks for research.

Research directions:

  • Examination of the role of environmental factors that have not yet been fully studied, such as the microbiome, opiates, e-cigarettes, and serotonin antagonists.
  • Placing a greater focus on understanding the molecular pathways and developmental processes that lead to the formation of a cleft.

Methods and tools:

  • Development of techniques that could be used to measure the anatomy of the orofacial region to provide quantitative assessments of the range of clefting phenotypes.
  • Identification of better methods to assess an individual’s environmental exposures, such as finding and validating relevant biomarkers.
  • Isolation of new experimental cell lines, such as mouse neural crest cell lines or cells derived from human induced pluripotent stem (iPS) cells that can be used in controlled experiments on the effects of environmental exposures.

Agenda Summary

The workshop began the evening of September 6th with overviews of the clinical care of children with orofacial clefting and the current state of research. The following day, September 7th, was divided into two parts. The day began with presentations focused on the effects of four classes of environmental factors and in the afternoon participants broke out into groups to talk about resource gaps, ways to stimulate translational research and interdisciplinary collaboration, and broad questions in orofacial clefting. The attendees then reconvened to report upon and discuss their ideas in the context of the overall GxE concept.


As an embryo develops during pregnancy, cells from both sides of the developing neural tube move ventrally and join to form the midface structures, including the mouth. If the tissue fails to connect completely, a cleft lip or palate (roof of the mouth), or both, can occur. Each year in the United States, about 4,440 babies are born with cleft lip (with or without cleft palate) and about 2,650 are born with cleft palate alone.

Children with cleft lip and/or palate (CL/P) often have trouble feeding, speaking, and hearing and can have problems with their teeth. Physicians recommend that surgery to repair the cleft occur within the first year or two of life, and additional surgeries may be needed as the child ages. Children with CL/P typically require a variety of services and ideally are treated by multidisciplinary clinical teams that include physicians, speech therapists, dentists, orthodontists, psychologists and social workers.

Most cases of clefting (between 50 and 80 percent) occur in the absence of other birth defects and are said to be “isolated” or “nonsyndromic.” Non-syndromic clefting tends to cluster in families, providing strong evidence for a genetic component to risk, but the genes involved have been difficult to identify due to a pattern of inheritance that does not follow strict Mendelian genetics. The presentations and discussions at this workshop largely focused on non-syndromic orofacial clefting.

Orofacial Clefting: Clinical Perspectives

This session began with opening remarks by NIDCR Director, Martha J. Somerman, D.D.S., Ph.D., who welcomed attendees and affirmed the Institute’s commitment to advancing CL/P research. In recent years, NIDCR has spent approximately $15-20 million per year on grants related to the birth defect, including clinical, basic and translational research.

The first set of talks presented overviews of the range of phenotypes associated with CL/P and the clinical care of patients. The development of the lip and palate occurs between the fifth and ninth week of gestation, and CL/P can take on different forms, such as unilateral or bilateral, complete or incomplete.

Care of CL/P patients involves a team of specialists and begins at birth with assistance with feeding. Surgery to repair the cleft often occurs within the first few months of life, and clinical intervention continues into adulthood with additional surgical and dental procedures. The goals of care are to maintain the health and function of oral structures, to return to an aesthetic facial form, enhance the patient’s self-esteem. and provide the patient’s family with support and information. The American Cleft Palate-Craniofacial Association (ACPA) has set guidelines for the parameters of care for CL/P patients and has established a process for accrediting the teams that follow these standards. The ACPA maintains a list of accredited teams that is available to families seeking care.

Orofacial Clefting: State-of-the-Science

The next set of presentations focused on CL/P risk factors, and the epidemiological methods and animal models used to study the birth defect.

CL/P is more common in boys than girls, and varies with race, with Native Americans exhibiting the highest prevalence with 3.6 cases/1,000 live births and African Americans being the least often affected with 0.3 cases/1,000 live births. Cleft palate alone occurs less frequently overall, and is more common in girls than boys, this observation and others suggest that CL/P and cleft palate only are developmentally and genetically distinct.

Identifying environmental risk factors has been a challenge, but a number of studies have linked maternal smoking to the risk of clefting, with an odds ratio (OR) of 1.3. Anticonvulsant medications such as topiramate have been found to have a pronounced effect, with ORs as high as 6.3 having been reported. Findings have been mixed with regard to roles for maternal alcohol consumption and use of nutritional supplements.

Scientists have used several technical approaches, such as genome wide association studies (GWAS), to uncover genes and loci associated with orofacial clefting. Whole exome sequencing and whole genome sequencing techniques are increasingly being used to search for additional genetic factors. Some of the genes and loci that have been implicated to date include TGFA, IRF6, BMP4 and chromosomal region 8q24, but our understanding of the biologic and clinical significance of these markers is incomplete.

Epidemiological methods used to identify genetic loci include the case-control model, as well as the case-parent trio. The case-parent trio design is a robust method for estimating relative risk of the fetal, as well as the maternal genotype; the latter may also influence the fetus’ risk of developing CL/P. In addition, the case-parent design can detect multiplicative interactions among genetic factors.

Animal models such as mice have also proven to be useful tools for identifying genes linked to developmental abnormalities. Scientists have identified susceptibility genes using mouse strains that are particularly vulnerable to developmental defects triggered by maternal exposure to a factor such as alcohol. A number of genes, such as SHH and GLI2, whose role in clefting derives from their influence on midline development have been identified in this way. Scientists can also identify candidate genes by performing whole transcriptome sequencing (RNAseq) of relevant tissues in closely related strains that differ in their susceptibility to developmental abnormalities.

Scientists also use forward genetic screens in model organisms to identify genes that influence the risk of CL/P. In this approach, researchers mutate the genome of an animal model such as zebrafish using a chemical mutagen, then screen to recover animals with midline developmental abnormalities. Researchers can then identify the mutated gene by analyzing the organism’s genotype.

During the discussion, participants noted that the window of susceptibility to environmental factors during which CL/P can occur is very small. That window falls within the first few weeks of gestation and may only last three days, which could account for the small and inconsistent effects that have been observed for many environmental factors.

Attendees also pointed out that, to date, studies of interactions between genes and environmental exposures in CL/P have themselves been small and limited in scope. Many studies have not been replicated and finding agreement between data from human and animal studies has been a challenge. In addition, resistance to collaboration and the relative short-term nature of most grant funding opportunities have been found to be obstacles to advancement.

Wednesday, September 7, 2016

Evidence for Gene-Environment Interactions in Orofacial Clefting

The presentations in the second day’s sessions focused on current knowledge of the role of environmental factors in incidence of CL/P. There was general agreement that risk of CL/P was likely to involve the combined small effects of multiple environmental and genetic determinants, as well as stochastic factors.

Maternal Smoking, Alcohol Use and Nutritional Status

Maternal smoking has been one of the most well-studied environmental risk factors, and there have been numerous reports of a small effect on occurrence of CL/P, with an OR of about 1.3. Attendees noted that to better understand the impact of maternal smoking, methods are needed to isolate the ingredients of cigarette smoke and to test their effects individually. Differences among brands or types of cigarettes may partly account for the relatively small effects that have been measured to date. Results on the effects of maternal alcohol consumption have been inconsistent, however some studies have reported a modest increased risk with maternal binge drinking.

In terms of nutritional factors, a healthy diet such as one resembling the DASH (Dietary Approaches to Stop Hypertension) or Mediterranean diets, together with micronutrient supplements, have been shown to modestly reduce the risk of orofacial clefts, but the effects of individual micronutrients (for example, riboflavin, folate, vitamin B12 and others involved in one-carbon metabolism) alone have been inconsistent among differing populations. Being underweight or obese has been linked to a small increased risk of orofacial clefts, with ORs of about 1.2.

A number of research challenges were brought up during the discussion. Despite CL/P being the most common craniofacial birth defect, its frequency still limits the power of epidemiological analyses, and the phenotypic heterogeneity of the birth defect could be a confounding factor. Most analyses group all forms of clefting together. However, different genetic variants or environmental factors may be involved in its different forms. In addition, measurements of micronutrients in blood serum may not detect deficiencies that are tissue-specific in nature; such tissue-specific effects might directly and locally affect the developing lip and palate.

Participants also noted that while genetic factors may seem to outweigh environmental ones, there nevertheless could be environmental solutions.  For example, a nutrient supplement such as folate may compensate for a genetic defect in the one-carbon metabolism pathway.

While the impact of the microbiome on risk of clefting has not yet been studied, attendees viewed it as a promising new area of research. The microbiome is known to have a significant effect on nutrition, suggesting that it may affect maternal nutritional status and clefting.

Participants also noted that the effect of in utero exposure to marijuana and its active compound, tetrahydrocannabinol (THC), on CL/P occurrence has not been fully assessed.  Additional studies pursuing certain preliminary findings should be conducted for understanding effect size and underlying mechanisms.

Medications, Infections and Occupational Exposures

The impact of medications taken and infections that occurred during pregnancy has also been studied. Among medications, maternal use of isotretinoin or anticonvulsants has shown the strongest link with CL/P. Anticonvulsants such as topiramate have an especially pronounced effect, increasing the risk of having a child with CL/P by about 500 percent. While most cases of CL/P are unrelated to use of medications, the marked effects of these drugs may enable us to learn more about orofacial development and how clefts occur.

Infection with rubella, zika, cytomegalovirus, herpes simplex virus and others have been associated with birth defects but there has been no clear link with orofacial clefts.

People are, at times, exposed to chemicals or environmental toxicants through the workplace or through their everyday lives, but few of these substances have shown a strong association with CL/P risk.

There was general agreement during the discussion that the inability to accurately measure maternal and fetal environmental exposures is a major obstacle. Participants suggested that primary teeth could be used to identify exposures that occurred at particular time periods, much as rings in a tree trunk can reveal past environmental conditions. Primary teeth develop in the second trimester, after midface development, however second trimester exposure levels may provide better estimates of exposures early in the pregnancy than current methods. It’s also possible that maternal hair or nails could be used to assess maternal or fetal exposures.

The NIH-supported resources, Children’s Health Exposure Analysis Resource (CHEAR) and the Environmental Influences on Child Health Outcomes (ECHO), may provide data on exposures and biomarkers during pregnancy that could inform CL/P studies.

Small Group Breakout Sessions

During this final session, participants were divided into three groups and asked to consider various aspects of the future of CL/P research and ways to facilitate progress. The entire group then reconvened to review each group’s thoughts.

Group 1–Resources

This group outlined the research resources that are currently available and discussed additional resources that would help advance the pace of discovery. New resources that would benefit the research community include:

  • A virtual repository cataloging research results and tools, such as questionnaires used to assess exposures and banked biological materials.
  • Biomarkers that reflect environmental exposures, for example, validated metabolomic and methylomic panels that can detect previous environmental insults.
  • Additional experimental cell lines, such as mouse neural crest cell lines, or cells derived from human induced pluripotent stem (iPS) cells.
  • Quantitative ways to phenotype people with CL/P. Quantitative ways to measure the range of orofacial phenotypes, perhaps using morphometrics, may enable researchers to distinguish among different forms of the birth defect.

Group 2—Translation and Interdisciplinary Collaboration

This group discussed ways to speed the movement of research results to clinical applications and approaches to foster collaboration among basic scientists and clinical researchers. Factors that could hasten the advent of clinical applications include:

  • Greater interaction between clinical geneticists and basic scientists, perhaps through funding mechanisms fostering such collaborations.
  • Use of model organisms, especially fish and frogs, for high-throughput screens to identify compounds or chemical classes that impact the risk of CL/P.
  • Examination of the impact of a wider range of environmental factors, such as the microbiome, e-cigarettes, marijuana and THC, chewing tobacco, prescription pain medications (opiates), and serotonin antagonists.
  • Use of a broader array of model organisms.
  • A greater focus on the molecular mechanisms that direct orofacial development.
  • More powerful analytic methods for determining maternal genetic effects.

Group 3—Orofacial Clefting: Broad Questions

This group focused on overall challenges to progress in the field of CL/P research and potential solutions to address them. Some of these challenges and solutions include:

  • Lack of communication between basic scientists and clinicians could be addressed using grant mechanisms to leverage these types of relationships.  Partnerships with organizations such as the American Cleft Palate Association, the American Association for Dental Research, and patient advocacy groups could also help.
  • There is a lack of consensus about how much preliminary data from model organisms is sufficient to warrant moving forward with epidemiological studies of human populations. Investigators in the field should seek to establish a standard or guidelines to address this issue.
  • Collaboration and sharing of resources could be promoted through the formation of a network of designated institutions outside NIH focused on studying birth defects, including CL/P. The network could be modeled on NCI’s Cancer Centers Program and Surveillance, Epidemiology, and End Results Program (SEER), or practice-based research networks (PBRNs) like the NIDCR-supported National Dental PBRN.
  • The creation of novel, long-term study cohorts consisting of mothers who have had a child with CL/P (and are more likely to have subsequent children with CL/P than the overall population) would address the challenge of low frequency of the birth defect.
  • Harmonization of data obtained from small cohorts would enable meta-analyses that have greater statistical power than the original individual studies.
  • Research on the combined effects of multiple exposures is lacking, and further examination of common pathways that feed into occurrence of CL/P is needed.



The recommendations emerging from this workshop resulted in a concept for a Funding Opportunity Announcement (FOA) that has been approved by the National Advisory Dental and Craniofacial Research Council. The objectives of this FOA include, but are not limited to, fostering the use of animal or in vitro approaches to:

  • study the mechanisms behind relevant exposures already identified in humans;
  • model gene-gene and gene-environment interactions already identified in humans
  • develop new animal or organoid models for probing the mechanisms behind gene-environment interactions.


Last Reviewed
July 2018