Specific Aims
Orofacial cleft (OFC) is the second most common birth defect, following congenital heart disease, affecting about 1 in 700 live births worldwide. It is characterized by clefting of the lip and/or palate thus, interfering with ingestion and speech, as well as inflicting a social and financial burden on the individual and family. Owing to this psychosocial impact on the family, overall productivity can be impeded and thus their contribution to the global economy is affected. Management of this condition is a life-long process of multiple surgical interventions to repair the cleft, speech therapy and management of the dental malocclusion because of the condition. It is estimated that the cost per individual treatment of this defect is over 100,000 USD. This profound cost of management coupled with the impact of the defect on the productivity of the family makes this congenital defect a public health concern globally.
Primary intervention (such as genetic counseling of couples at risk) will go a long way in reducing the impact of this condition on the family and potential healthcare. This intervention can only be successful when the cause and the mechanisms involved in this defect are well-understood. It has been shown that OFCs are associated with genetic and environmental risk factors. However, there remains much to be discovered regarding the genetic risk associated with this defect. The heterogeneity of the African population presents an opportunity to identify novel candidate genes and how they result in OFCs. The Sonic Hedgehog (SHH) signaling pathway has been proven to be essential for craniofacial development. This pathway acts by regulating the expression of genes downstream. However, these genes are yet to be fully identified.
Consequently, interventions in the form of prevention of new cases are yet to yield the much-needed success. The purpose of this study is to fill this gap by identifying genes that would succinctly explain the genetic etiology of OFC in the African population, the variants associated with OFCs and the mechanism by which they cause the defects. This will help in adequate intervention by early prediction of cases (through genetic counseling of at risk couples) and eliminate the consequent impact on the family and huge financial burden on the healthcare system.
The central hypothesis of this study is that genes downstream of the SHH signaling pathway are involved in the development of the lip and palate. Mutation in these genes will affect the signaling mechanism of the pathway thus, resulting in OFCs. It is believed that identification of these genes and the causative variants will help predict embryonic cleft and this knowledge can be used in prevention and neonatal therapy.
We plan to test this central hypothesis and thereby achieve the objective of this study by pursuing the following specific aims:
1. Identifications of genes involved in craniofacial development that are regulated by the SHH signaling pathway: We plan on searching through databases for the genes that overlap SHH signaling pathway and a reported involvement in craniofacial development (associated with OFCs), in pursuit of identifying candidate genes (known and unknown. We will also use neural crest cell lines to identify new genes that are downstream SHH pathway and confirm those obtained from our searches.
2. Identification of variants that explains genetic factors associated with OFCs: Targeted sequencing of the above candidate genes will be done in DNA samples obtained from African cases (with OFCs) and controls. This is aimed at identifying novel and rare variants that can explain the roles of the genes in the etiology of OFCs.
3. Characterization of the biological functions of the variants identified: We will carry out functional studies in cell lines and animal models
The expected outcome of this study is identification of variants of genes (downstream the SHH pathway) and how they result in OFCs in African population.
SIGNIFICANCE
Importance of the Problem: Orofacial Cleft (OFC) is the second most common congenital defect with a prevalence of about 1/700 livebirths globally [1]. It is a congenital condition that results from disruption in the embryogenesis of the orofacial region [2] [3]. These defects affect feeding of newborn, speech, quality of life and psychosocial well-being of the family [4]. The stigma associated with this defect, especially in African population makes it a huge burden to the well-being of the family. It is estimated that the health care cost per case is over 100,000 USD [4]. Thus, this defect has a huge socio-economic impact and poses a burden financially to the family of the affected [4]. The failure in the fusion of the maxillary processes and their derivatives result in the cleft of the lip and/or palate [5]. This disruption is caused by environmental and genetic factors [5] [6].
Scientific Premise of the Proposed Study: Interaction between the epithelium- that develops from the embryonic ectoderm and mesenchyme-that develops from the ectomessenchyme is essential for the proliferation, migration and outgrowth of cells required for the closure of the clefts during embryogenesis [7]. This interaction is achieved by the Sonic Hedgehog molecules in the Sonic Hedgehog (SHH) signaling pathway [7] [8]. The Sonic Hedgehog signaling pathway is involved in the embryonic development of the brain, limbs and orofacial structures [7]. The SHH signaling molecule is secreted by the ectoderm and binds to the Ptch1 receptor of the ectomessenchyme. This association removes the inhibitory effect of Ptch1 on the Smoothened (Smo). Downstream of this pathway, transcription of target genes is activated by Gli [8]. These target genes regulate the proliferation, outgrowth and migration of the cells to close the cleft [7]. However, little is known about these target genes: their identities and mechanism of biological functions. Thus, our proposed study will identify these target genes and their variants, how they act in the development of the orofacial structures and the effect of the variants identified.
The proposed study will involve a two-stage database search and targeted sequencing of samples from individuals that have the defect. The first stage database search involves the search for all genes that have been identified to be associated with OFCs. Sources of these genes include genome-wide analyses study in African population that has identified susceptibility loci [9] and candidate genes that have been identified by Exome sequencing [10]. The second stage search include all genes that have been reported to be regulated by the SHH signaling pathway. The two stages are matched and genes that overlaps-found to occur in both stages- are noted. Saliva samples from individuals in the African population that have these defects are processed and targeted sequencing of genes identified from the 2-stage database search is done. The aim of this sequencing is to identify novel and rare variants of these genes that may affect the normal function. We will also perform functional studies using migration and reporter assays in the cranial neural crest derived cell lines to determine the effect of this variants in vitro. Functional studies in vivo will involve the use of mouse models to study how the variants of the genes identified cause OFCs.
Significance of the expected research contribution: This project will help us to understand the genes that are directly involved downstream of the SHH signaling pathway in the development of the craniofacial structures and how their variants result in OFCs in the African populations. The outcome of this study will have obvious implications in African American populations with this defect.
Impact of the project on Scientific Knowledge: African populations have greater genomic diversity than European, American and Asian populations [11], thus this proposal has a major strength in the population to be studied. Samples from this ancestral population will help define the actions of SHH signaling target genes that play a role in these defects and allow us to discover variants that are involved in these defects.
INNOVATION: We will conduct a 2-stage database search. The first database search is the search for all the genes that are involved in craniofacial development and have been associated with OFCs [12]. The second stage search will involve a database search of all the genes in the SHH signaling pathway [7] with focus on those genes downstream the pathway. Genes that overlap in these stages are moved to another level of the study. In vitro cell line study (using cranial neural crest cell line) will be carried out to confirm these genes obtained from our 2-stage search. These cell lines study will involve the inhibition of the SHH molecules thus shutting-off the signaling pathway [7]. The expression of the genes (from our searches) will be assayed using RNA-Seq to ascertain the genes from our searches.
The next level involves targeted sequencing of these genes in DNA samples of cases and controls from African populations to identify novel and rare variants that may explain the genetic factors in the etiology of OFCs.
We will collaborate with a biostatistician and bioinformatics expert to ensure reproducibility and rigor of this study. The genomic diversity of the African population [11] is one of the major strength of this study. This will increase our knowledge of these defects which will be critical for identifying families with the high risk of having babies with these defects, as well as provide effective genetic counselling. This additional understanding of the roles of the genetic risk factors will be one of the many steps that will help in the development of effective therapeutic approach to reduce incidence of these defects.
RIGOR AND REPRODUCIBILITY: The cases that will be recruited in this study will have the OFC phenotype only (non-syndromic). This phenotype will be diagnosed by an experienced Oral and Maxillofacial Surgeon and cases must have satisfied the conditions to be included in the study. Some of these conditions include absence of any other congenital defects or disease condition. Primers for the sequencing will be designed, blasted/blatted, in-silico PCR will be done to assess the quality of the primers and gradient PCR will be done to ascertain the annealing temperatures of the primers. Sequencing of the DNA will be done twice: forward and reverse. The DNA samples will first be sequenced in the forward direction that is using the right (forward) primers, the amplified gene will be sequenced. Variants will be called based on the result of the sequencing in the forward direction. The samples that have variants of interest will be sequenced in the reversed direction. This is a quality control step which helps to ascertain the presence of the variant. Variant of interest in this context means novel variants or known variants with minor allele frequency (MAF) less than 1% (rare variants).
The sample size will be calculated based on a power of 80%. A Chi-square tests of homogeneity will be used to test the fact that our samples are from the same population. Homogeneity of age and gender distributions will also be tested using this Statistical test. Our significance level will be set at 0.05 for all our tests.
Model Organism: Mice will be used for the functional work of the study. Mice are mammals and have very high genetic similarities with humans [13]. The biological function of the candidate genes as well as the variants identified will be studied in mice. Transgenic mice with the genes and variants will be produced. The expression sites of these genes will be assessed using in-situ hybridization and the expression levels of their variants will be assessed by immunostaining.
APPROACH
Strategies: Genes that are associated with OFCs have been published on databases that are accessible. These genes will be searched for and documented. The National Center for Biotechnology Information (NCBI) gene database will be used for the searches. The NCBI is part of the United States National Library of Medicine, a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland. It gives free access to the gene database which contains the genes and the conditions they have been associated with. The searched will include genes that have been associated with OFCs and those in the SHH signaling pathway. Overlapping genes will move to the next phase of the study. We will confirm these genes (from our 2-stage search) by assessing their expression levels in neural crest cell lines. This cell line study will involve the inhibition of the binding of SHH molecules to the Ptch1 receptor on the ectomessenchyme. This inhibition maintains the inhibitory action of Ptch1 on Smo thereby shuts-down the SHH signaling pathway [7]. The genes downstream this pathway will have an alteration in their expression levels as a result of this process. These expression levels of the genes (from our searches) will be assayed using RNA-Seq. This step will help us to confirm that indeed the genes from our searches are downstream the SHH signaling pathway.
The next phase involves targeted sequencing of the genes in the DNA samples of cases and control to identify novel and/ or rare variants of the genes. Saliva samples will be collected from cases (individuals born with OFCs) and their families (unaffected and affected). The variants that will be called include Single nucleotide polymorphisms (SNPs: missense or nonsense), splice site variants(intronic), insertions and deletions(indels). Bioinformatic tools will be used to predict the functional effects of the identified variants. Functional work in cell lines and animal models will help us understand the effect of the variants in living tissues.
Data Collection:
Sample Collection and Processing: This is a collaborative study that will involve 3 institutions. The other 2 institutions will be charged with collection of biological samples. Cases will be diagnosed based on presence of the OFCs. Phenotyping of Cases and Parents will be done by Oral and Maxillofacial Surgeons in the collaborating institutions. Cases and Parents saliva samples will be collected from Africans recruited for this study. These samples will be collected following the Oragene processing protocol for collection of saliva samples for DNA extraction and shipped down to our laboratory. Participants of this study will be well informed of the purpose of this study, procedures involved, and informed consent will be obtained from them or guardians. Local IRB approval will be obtained in the various institutions that will be involved in this study.
Data Analyses:
DNA extracted from the samples will be sequenced. The genes that overlapped in our different searches will be sequenced and the results will be analyzed. Analysis of the sequencing result involves:
Variant Calling: The sequencing results will be aligned with the reference genome (consensus) and variants in the study population will be called. The variants that will be called include Single nucleotide polymorphisms (SNPs: missense or nonsense), splice site variants(intronic), insertions and deletions(indels).
Annotation of Variants: Variants called will be annotated using published algorithms such as University of California, Santa Cruz (UCSC) genome browser, Ensembl’s Variant Effect Predictor (VEP) software, the Combined Annotation-Dependent Depletion (CADD) algorithm and the web-based ANNOtate Variation (wANNOVAR) software. These tools will also help to predict the effect of the variants. Our focus will be on novel and rare variants that are within coding region and splice site.
Functional Analyses: Due to the less reliability of the Bioinformatic tools in the prediction of the effects of variants, we want to take a step further by doing functional work in cell lines and mice. We will collaborate with the lab that specializes in these works. The aim of these analyses is to assess the effect of the variants called in living tissues. Clones of the genes with (mutant-type) and without (wild-type) the annotated variants will be incorporated into a plasmid or virus as vectors. These vectors will be transfected into neural crest cells lines. The transfection of cell lines will be used to assess the expression levels of the mutant-type (mt) compared to the expression levels in wild-type (wt). This will be achieved by doing a qPCR. The stability of the protein as a result of the variant will be assessed by doing Western-Blot of the transfected cell lines.
The mouse work will involve production of transgenic germ lines of mice (with genes containing the variants of interest). We will assess the tissues where these genes are expressed in the wild-type (wt) embryos using in-situ hybridization and the expression levels of their variants will be assessed by immunostaining. These assessments will be done at different time points during development. This will help us identify the period of development when the different genes are expressed in relation to the development of the craniofacial structures paying more attention to the development of the lip and palate. The mt-embryos phenotypes will be assessed looking for any defects as a result of the variant of the individual genes we are studying. The mutant-type (mt) will be compared with the wild-type (wt) at different time points of embryonic development.
POTENTIAL PITFALLS:
During this research, some of the pitfalls that I may encounter include:
Wrong matching of sample details: XY genotyping will help as a quality control and any sample whose gender does not match the XY genotype will not be included in the study. Incompletely labelled samples will not be included in the study as well.
Failure of optimization of Primers: New set of primers will be designed.
Poor Sequencing results: Resequencing will be done with a new set of primers.
UPDATE COURSES:
I plan to take the following courses to improve my expertise while carrying out this study:
Biostatistics: I will update myself with new statistical tools that will enhance the rigor and reproducibility of this study.
Bioinformatics: There are newer tools that will help in understanding the biological function of genetic systems. I plan to take those classes that will contribute to the success of this research.
Conferences and Workshops: I will attend conferences and workshop relevant to this research. This will keep me abreast of the happenings in the field.
TIMELINE: We plan to carry out this research for a period of two (2) years and a follow up study for another 3years. The follow up study will involve extending this research to other populations.
This research will help us understand the downstream (target) genes through which SHH signaling pathway regulates craniofacial development, the variants of these genes associated with OFCs and biological mechanisms involved in this effect.