Clinically actionable secondary findings in 130 triads from sub-Saharan African families with non-syndromic orofacial clefts.

INTRODUCTION: The frequency and implications of secondary findings (SFs) from genomic testing data have been extensively researched. However, little is known about the frequency or reporting of SFs in Africans, who are underrepresented in large-scale population genomic studies. The availability of data from the first whole-genome sequencing for orofacial clefts in an African population motivated this investigation. METHODS: In total, 130 case-parent trios were analyzed for SFs within the ACMG SFv.3.0 list genes. Additionally, we filtered for four more genes (HBB, HSD32B, G6PD and ACADM). RESULTS: We identified 246 unique variants in 55 genes; five variants in four genes were classified as pathogenic or likely pathogenic (P/LP). The P/LP variants were seen in 2.3% (9/390) of the subjects, a frequency higher than ~1% reported for diverse ethnicities. On the ACMG list, pathogenic variants were observed in PRKAG (p. Glu183Lys). Variants in the PALB2 (p. Glu159Ter), RYR1 (p. Arg2163Leu) and LDLR (p. Asn564Ser) genes were predicted to be LP. CONCLUSION: This study provides information on the frequency and pathogenicity of SFs in an African cohort. Early risk detection will help reduce disease burden and contribute to efforts to increase knowledge of the distribution and impact of actionable genomic variants in diverse populations.

Damaging Mutations in AFDN Contribute to Risk of Nonsyndromic Cleft Lip With or Without Cleft Palate.

Novel or rare damaging mutations have been implicated in the developmental pathogenesis of nonsyndromic cleft lip with or without cleft palate (nsCL ± P). Thus, we investigated the human genome for high-impact mutations that could explain the risk of nsCL ± P in our cohorts.We conducted next-generation sequencing (NGS) analysis of 130 nsCL ± P case-parent African trios to identify pathogenic variants that contribute to the risk of clefting. We replicated this analysis using whole-exome sequence data from a Brazilian nsCL ± P cohort. Computational analyses were then used to predict the mechanism by which these variants could result in increased risks for nsCL ± P.We discovered damaging mutations within the AFDN gene, a cell adhesion molecule (CAMs) that was previously shown to contribute to cleft palate in mice. These mutations include p.Met1164Ile, p.Thr453Asn, p.Pro1638Ala, p.Arg669Gln, p.Ala1717Val, and p.Arg1596His. We also discovered a novel splicing p.Leu1588Leu mutation in this protein. Computational analysis suggests that these amino acid changes affect the interactions with other cleft-associated genes including nectins (PVRL1, PVRL2, PVRL3, and PVRL4) CDH1, CTNNA1, and CTNND1.This is the first report on the contribution of AFDN to the risk for nsCL ± P in humans. AFDN encodes AFADIN, an important CAM that forms calcium-independent complexes with nectins 1 and 4 (encoded by the genes PVRL1 and PVRL4). This discovery shows the power of NGS analysis of multiethnic cleft samples in combination with a computational approach in the understanding of the pathogenesis of nsCL ± P.

Whole-genome sequencing reveals de-novo mutations associated with nonsyndromic cleft lip/palate.

The majority (85%) of nonsyndromic cleft lip with or without cleft palate (nsCL/P) cases occur sporadically, suggesting a role for de novo mutations (DNMs) in the etiology of nsCL/P. To identify high impact protein-altering DNMs that contribute to the risk of nsCL/P, we conducted whole-genome sequencing (WGS) analyses in 130 African case-parent trios (affected probands and unaffected parents). We identified 162 high confidence protein-altering DNMs some of which are based on available evidence, contribute to the risk of nsCL/P. These include novel protein-truncating DNMs in the ACTL6A, ARHGAP10, MINK1, TMEM5 and TTN genes; as well as missense variants in ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TULP4. Many of these protein-altering DNMs were predicted to be pathogenic. Analysis using mouse transcriptomics data showed that some of these genes are expressed during the development of primary and secondary palate. Gene-set enrichment analysis of the protein-altering DNMs identified palatal development and neural crest migration among the few processes that were significantly enriched. These processes are directly involved in the etiopathogenesis of clefting. The analysis of the coding sequence in the WGS data provides more evidence of the opportunity for novel findings in the African genome.

Genome-Wide Scan for Parent-of-Origin Effects in a sub-Saharan African Cohort With Nonsyndromic Cleft Lip and/or Cleft Palate (CL/P).

OBJECTIVE: Nonsyndromic cleft lip and/or cleft palate (NSCL/P) have multifactorial etiology where genetic factors, gene-environment interactions, stochastic factors, gene-gene interactions, and parent-of-origin effects (POEs) play cardinal roles. POEs arise when the parental origin of alleles differentially impacts the phenotype of the offspring. The aim of this study was to identify POEs that can increase risk for NSCL/P in humans using a genome-wide dataset. METHODS: The samples (174 case-parent trios from Ghana, Ethiopia, and Nigeria) included in this study were from the African only genome wide association studies (GWAS) that was published in 2019. Genotyping of individual DNA using over 2 million multiethnic and African ancestry-specific single-nucleotide polymorphisms from the Illumina Multi-Ethnic Genotyping Array v2 15070954 A2 (genome build GRCh37/hg19) was done at the Center for Inherited Diseases Research. After quality control checks, PLINK was employed to carry out POE analysis employing the pooled subphenotypes of NSCL/P. RESULTS: We observed possible hints of POEs at a cluster of genes at a 1 mega base pair window at the major histocompatibility complex class 1 locus on chromosome 6, as well as at other loci encompassing candidate genes such as ASB18, ANKEF1, AGAP1, GABRD, HHAT, CCT7, DNMT3A, EPHA7, FOXO3, lncRNAs, microRNA, antisense RNAs, ZNRD1, ZFAT, and ZBTB16. CONCLUSION: Findings from our study suggest that some loci may increase the risk for NSCL/P through POEs. Additional studies are required to confirm these suggestive loci in NSCL/P etiology.

Variant analyses of candidate genes in orofacial clefts in multi-ethnic populations.

OBJECTIVES: Cleft lip with/without cleft palate and cleft palate only is congenital birth defects where the upper lip and/or palate fail to fuse properly during embryonic facial development. Affecting ~1.2/1000 live births worldwide, these orofacial clefts impose significant social and financial burdens on affected individuals and their families. Orofacial clefts have a complex etiology resulting from genetic variants combined with environmental covariates. Recent genome-wide association studies and whole-exome sequencing for orofacial clefts identified significant genetic associations and variants in several genes. Of these, we investigated the role of common/rare variants in SHH, RORA, MRPL53, ACVR1, and GDF11. MATERIALS AND METHODS: We sequenced these five genes in 1255 multi-ethnic cleft lip with/without palate and cleft palate only samples in order to find variants that may provide potential explanations for the missing heritability of orofacial clefts. Rare and novel variants were further analyzed using in silico predictive tools. RESULTS: Ninteen total variants of interest were found, with variant types including stop-gain, missense, synonymous, intronic, and splice-site variants. Of these, 3 novel missense variants were found, one in SHH, one in RORA, and one in GDF11. CONCLUSION: This study provides evidence that variants in SHH, RORA, MRPL53, ACVR1, and GDF11 may contribute to risk of orofacial clefts in various populations.

Co-occurrence of orofacial clefts and clubfoot phenotypes in a sub-Saharan African cohort: Whole-exome sequencing implicates multiple syndromes and genes.

BACKGROUND: Orofacial clefts (OFCs) are congenital malformations of the face and palate, with an incidence of 1 per 700 live births. Clubfoot or congenital talipes equinovarus (CTEV) is a three-dimensional abnormality of the leg, ankle, and feet that leads to the anomalous positioning of foot and ankle joints and has an incidence of 1 per 1000 live births. OFCs and CTEV may occur together or separately in certain genetic syndromes in addition to other congenital abnormalities. Here, we sought to decipher the genetic etiology of OFC and CTEV that occurred together in six probands. METHODS: At the time of recruitment, the most clinically obvious congenital anomalies in these individuals were the OFC and CTEV. We carried out whole-exome sequencing (WES) on DNA samples from probands and available parents employing the Agilent SureSelect XT kit and Illumina HiSeq2500 platform, followed by bioinformatics analyses. WES variants were validated by clinical Sanger Sequencing. RESULTS: Of the six probands, we observed probable pathogenic genetic variants in four. In three probands with probable pathogenic genetic variants, each individual had variants in three different genes, whereas one proband had probable pathogenic variant in just one gene. In one proband, we observed variants in DIS3L2, a gene associated with Perlman syndrome. A second proband had variants in EPG5 (associated with Vici Syndrome), BARX1 and MKI67, while another proband had potentially etiologic variants in FRAS1 (associated with Fraser Syndrome 1), TCOF1 (associated with Treacher Collins Syndrome 1) and MKI67. The last proband had variants in FRAS1, PRDM16 (associated with Cardiomyopathy, dilated, 1LL/Left ventricular noncompaction 8) and CHD7 (associated with CHARGE syndrome/Hypogonadotropic hypogonadism 5 with or without anosmia). CONCLUSION: Our results suggest that clubfoot and OFCs are two congenital abnormalities that can co-occur in certain individuals with varying genetic causes and expressivity, warranting the need for deep phenotyping.

A Qualitative Analysis of Factors Impacting Comprehensive Cleft Lip and Palate Care in Ghana.

OBJECTIVE: The objective of this study was to examine practices regarding cleft lip and palate (CLP) among medical professionals and caregivers of children with CLP and to identify barriers and facilitators to comprehensive CLP care at a hospital in West Africa. DESIGN: Qualitative methods used consisted of individual semistructured interviews with caregivers of children with CLP and one focus group with CLP team members. SETTING: A majority of the interviews took place in the hospital, with some occurring during home visits. The focus group was conducted in the same hospital. PARTICIPANTS: Forty-five caregivers of children with CLP and 1 adult with CLP completed an interview. Additionally, 2 of the caregivers had CLP and completed an interview from their perspective. The focus group consisted of 13 CLP team members from a comprehensive CLP team in Ghana. INTERVENTIONS: Interviews consisted of semistructured, open-ended questions, and the focus group relied on a discussion guide. Line-by-line coding was used to identify common themes regarding barriers and facilitators to CLP care. RESULTS: Barriers to CLP care that were consistent across caregiver interviews and the focus group were lack of knowledge regarding CLP, stigma and cultural beliefs surrounding CLP, transportation, financial, and feeding/nutrition issues. Barriers to care identified in the interviews and focus group were similar; however, facilitators to care varied greatly between the 2. CONCLUSIONS: Two different qualitative methods provided unique perspectives on barriers and facilitators to CLP care. However, patients and caregivers continue to face substantial barriers to obtaining care.

Non-random distribution of deleterious mutations in the DNA and protein-binding domains of IRF6 are associated with Van Der Woude syndrome.

BACKGROUND: The development of the face occurs during the early days of intrauterine life by the formation of facial processes from the first Pharyngeal arch. Derangement in these well-organized fusion events results in Orofacial clefts (OFC). Van der Woude syndrome (VWS) is one of the most common causes of syndromic cleft lip and/or palate accounting for 2% of all cases. Mutations in the IRF6 gene account for 70% of cases with the majority of these mutations located in the DNA-binding (exon 3, 4) or protein-binding domains (exon 7-9). The current study was designed to update the list of IRF6 variants reported for VWS by compiling all the published mutations from 2013 to date as well as including the previously unreported VWS cases from Africa and Puerto Rico. METHODS: We used PubMed with the search terms; "Van der Woude syndrome," "Popliteal pterygium syndrome," "IRF6," and "Orofacial cleft" to identify eligible studies. We compiled the CADD score for all the mutations to determine the percentage of deleterious variants. RESULTS: Twenty-one new mutations were identified from nine papers. The majority of these mutations were in exon 4. Mutations in exon 3 and 4 had CADD scores between 20 and 30 and mutations in exon 7-9 had CADD scores between 30 and 40. The presence of higher CADD scores in the protein-binding domain (exon 7-9) further confirms the crucial role played by this domain in the function of IRF6. In the new cases, we identified five IRF6 mutations, three novel missense mutations (p.Phe36Tyr, p.Lys109Thr, and p.Gln438Leu), and two previously reported nonsense mutations (p.Ser424*and p.Arg250*). CONCLUSION: Mutations in the protein and DNA-binding domains of IRF6 ranked among the top 0.1% and 1% most deleterious genetic mutations, respectively. Overall, these findings expand the range of VWS mutations and are important for diagnostic and counseling purposes.

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.

Congenital infiltrating lipomatosis of the face with hyperplastic mandibular, maxillary and pterygoid bones: case report and a review of literature.

Congenital infiltrating lipomatosis of the face (CILF) is a rare lipomatous lesion, commonly seen in childhood, and it is characterized by collections of mature, unencapsulated adipose tissues that infiltrate facial soft and hard tissues. The lesion is seen as an overgrowth of bone and soft tissue and is generally present clinically as slow-growing painless masses. In this case report, we described one case of CILF, which is one of the first cases reported in Ghana and Africa as a whole, along with a literature review on the diagnosis and current treatment strategies.

Identification of paternal uniparental disomy on chromosome 22 and a de novo deletion on chromosome 18 in individuals with orofacial clefts.

BACKGROUND: Orofacial clefts are the most common malformations of the head and neck region. Genetic and environmental factors have been implicated in the etiology of these traits. METHODS: We recently conducted genotyping of individuals from the African population using the multiethnic genotyping array (MEGA) to identify common genetic variation associated with nonsyndromic orofacial clefts. The data cleaning of this dataset allowed for screening of annotated sex versus genetic sex, confirmation of identify by descent and identification of large chromosomal anomalies. RESULTS: We identified the first reported orofacial cleft case associated with paternal uniparental disomy (patUPD) on chromosome 22. We also identified a de novo deletion on chromosome 18. In addition to chromosomal anomalies, we identified cases with molecular karyotypes suggesting Klinefelter syndrome, Turner syndrome and Triple X syndrome. CONCLUSION: Observations from our study support the need for genetic testing when clinically indicated in order to exclude chromosomal anomalies associated with clefting. The identification of these chromosomal anomalies and sex aneuploidies is important in genetic counseling for families that are at risk. Clinicians should share any identified genetic findings and place them in context for the families during routine clinical visits and evaluations.

Novel GREM1 Variations in Sub-Saharan African Patients With Cleft Lip and/or Cleft Palate.

OBJECTIVE: Cleft lip and/or cleft palate (CL/P) are congenital anomalies of the face and have multifactorial etiology, with both environmental and genetic risk factors playing crucial roles. Though at least 40 loci have attained genomewide significant association with nonsyndromic CL/P, these loci largely reside in noncoding regions of the human genome, and subsequent resequencing studies of neighboring candidate genes have revealed only a limited number of etiologic coding variants. The present study was conducted to identify etiologic coding variants in GREM1, a locus that has been shown to be largely associated with cleft of both lip and soft palate. PATIENTS AND METHOD: We resequenced DNA from 397 sub-Saharan Africans with CL/P and 192 controls using Sanger sequencing. Following analyses of the sequence data, we observed 2 novel coding variants in GREM1. These variants were not found in the 192 African controls and have never been previously reported in any public genetic variant database that includes more than 5000 combined African and African American controls or from the CL/P literature. RESULTS: The novel variants include p.Pro164Ser in an individual with soft palate cleft only and p.Gly61Asp in an individual with bilateral cleft lip and palate. The proband with the p.Gly61Asp GREM1 variant is a van der Woude (VWS) case who also has an etiologic variant in IRF6 gene. CONCLUSION: Our study demonstrated that there is low number of etiologic coding variants in GREM1, confirming earlier suggestions that variants in regulatory elements may largely account for the association between this locus and CL/P.

The prevalence, penetrance, and expressivity of etiologic IRF6 variants in orofacial clefts patients from sub-Saharan Africa.

BACKGROUND: Orofacial clefts are congenital malformations of the orofacial region, with a global incidence of one per 700 live births. Interferon Regulatory Factor 6 (IRF6) (OMIM:607199) gene has been associated with the etiology of both syndromic and nonsyndromic orofacial clefts. The aim of this study was to show evidence of potentially pathogenic variants in IRF6 in orofacial clefts cohorts from Africa. METHODS: We carried out Sanger Sequencing on DNA from 184 patients with nonsyndromic orofacial clefts and 80 individuals with multiple congenital anomalies that presented with orofacial clefts. We sequenced all the nine exons of IRF6 as well as the 5' and 3' untranslated regions. In our analyses pipeline, we used various bioinformatics tools to detect and describe the potentially etiologic variants. RESULTS: We observed that potentially etiologic exonic and splice site variants were nonrandomly distributed among the nine exons of IRF6, with 92% of these variants occurring in exons 4 and 7. Novel variants were also observed in both nonsyndromic orofacial clefts (p.Glu69Lys, p.Asn185Thr, c.175-2A>C and c.1060+26C>T) and multiple congenital anomalies (p.Gly65Val, p.Lys320Asn and c.379+1G>T) patients. Our data also show evidence of compound heterozygotes that may modify phenotypes that emanate from IRF6 variants. CONCLUSIONS: This study demonstrates that exons 4 and 7 of IRF6 are mutational 'hotspots' in our cohort and that IRF6 mutants-induced orofacial clefts may be prevalent in the Africa population, however, with variable penetrance and expressivity. These observations are relevant for detection of high-risk families as well as genetic counseling. In conclusion, we have shown that there may be a need to combine both molecular and clinical evidence in the grouping of orofacial clefts into syndromic and nonsyndromic forms.