Whole Exome Sequencing Uncovers Genetic Syndromes Associated with Orofacial Clefts presenting with Limb abnormalities in a Sub-Saharan African cohort

Whole Exome Sequencing Uncovers Genetic Syndromes Associated with Orofacial Clefts presenting with Limb abnormalities in a Sub-Saharan African cohort | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Whole Exome Sequencing Uncovers Genetic Syndromes Associated with Orofacial Clefts presenting with Limb abnormalities in a Sub-Saharan African cohort Edna Tackie, Solomon Obiri-Yeboah, Gideon Okyere Mensah, Tamara D. Busch, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8340088/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 18 You are reading this latest preprint version Abstract Background: Orofacial clefts (OFCs) are the most frequent congenital craniofacial anomalies that occur during embryonic development. The incidence is ~1 in 700 live births; it may occur in isolation or with other abnormalities, such as limb deformities. Congenital limb malformations are the second most prevalent birth defect, affecting 1 per 500 to 1000 live births. It can also occur in isolation or as part of a syndrome. This study investigated the genetic aetiology of OFCs co-occurring with limb abnormalities in a Sub-Saharan African cohort. Methods: Nine unrelated probands with concurrent OFC and limb anomalies were recruited, including one multiplex family involving an affected mother and proband. Whole exome sequencing (WES) was performed at 100X on the DNA samples obtained from affected families, utilising paired-end configuration on the Illumina HiSeq platform. Variant calling utilized the Sentieon workflow. Rare, deleterious variants were identified in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines on variant classification. De novo and other variants predicted as pathogenic were prioritized based on all possible Mendelian inheritance patterns, including variable penetrance and expressivity. Pathway enrichment analysis, protein-protein interactions, and gene expression analysis were undertaken to decipher the biological functions of implicated genes. Results: All cases were syndromic, presenting with preaxial and postaxial limb anomalies along with other craniofacial features. WES revealed plausible pathogenic variants in pleiotropic genes ( TP63 , NIPBL , MYH3 , FGFR2 ) in four simplex cases. In four other simplex probands, multiple rare variants were identified in developmentally relevant genes (e.g., RGPD5, FAM90A26, FOXD4L1, FAM170A, DLG1, ANKRD1, TRIM74, TRIM73, PRDM9 ) necessary for normal craniofacial and limb development. The multiplex family had two affected individuals (the mother and the proband), both carrying a TP63 variant, consistent with autosomal dominant inheritance with variable expressivity. Most of the observed variants were de novo , with some being novel. Conclusion: While some cases can be attributed to single-gene syndromes (e.g., NIPBL-associated Cornelia de Lange Syndrome), others may result from multiple co-occurring syndromes. These findings will inform recurrence risk estimates, genetic counselling, and clinical management. Orofacial clefts limb abnormalities genetic syndromes whole exome sequencing pleiotropy polygenic Sub-Saharan Africa Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Orofacial clefts (OFCs) are the most frequent congenital craniofacial anomalies that occur during embryonic development and are mainly characterized by incomplete fusion of the palate, lip, or both. OFCs may manifest in various forms, including bilateral or unilateral, cleft palate (CP) or cleft lip with or without cleft palate, CL/P 1 . It has a global incidence of approximately 1 in 700 live births 2 . However, the incidence varies significantly across populations, with African populations reporting the lowest incidence (~ 1 in 2000 live births), Europeans reporting an intermediate incidence (~ 1 in 1,000 live births), and Asian populations showing the highest rates, ~ 1 in 500 live births 2 , 3 . In Ghana, a prevalence of 6.3 per 1000 individuals and a live birth incidence of 1.31 per 1000 have been reported in different communities 4 , 5 . A child with CL/P may encounter feeding difficulties, in addition to conductive hearing loss, speech impairments, malocclusion, and aesthetic problems. These physical anomalies can have a significant impact on the psychological and social well-being of the affected individual. Furthermore, the families of these individuals often struggle with societal stigmatization and substantial financial burdens, as the estimated mean lifetime treatment cost is approximately $ 92,000 6–8 . OFCs encompass a spectrum, ranging from nonsyndromic, constituting 70% of CL/P and 30% of CP cases, to syndromic clefts, which are associated with other structural anomalies. CP co-occurs in approximately 70% of infants diagnosed with unilateral CL and 85% of those diagnosed with bilateral CL 1,9 . Syndromic clefts are categorized according to their aetiology into monogenic syndromes, chromosomal syndromes, known teratogen exposures, and uncategorized syndromes that might not fit clearly into any well-defined category due to complex genetic interactions or unidentified aetiological factors 1 , 10 . The genetic analysis of OFCs poses a significant challenge, as most cases do not strictly conform to the Mendelian inheritance pattern and often occur sporadically as non-familial cases 9 . While the effect size and functionality of pathogenic variants identified through WES are larger compared to loci discovered via GWAS studies, the sporadic nature of most OFC cases continues to complicate genetic variant detection and characterization 11 . Limb abnormalities encompass a broad range of congenital and postnatal acquired conditions that affect the structure, functionality, or development of the lower and upper extremities. They can be broadly classified into failure of formation of anatomical parts (e.g., amelia, phocomelia, intercalary defects, etc.), failure of differentiation of anatomical parts (e.g., syndactyly, clinodactyly), duplications (e.g., polydactyly), overgrowth and undergrowth (e.g., macrodactyly and brachydactyly), congenital constriction band syndrome, and generalized or systemic skeletal defects 12 . These anomalies may be sporadic and isolated, exhibiting a particular genetic inheritance pattern, or may be associated with specific syndromes. Disruptions during embryonic development, genetic mutations, and environmental factors (e.g., infection and dietary habits) can lead to the development of these anomalies and may impact one or multiple limbs 12 . Limb defects are the second most common congenital abnormalities in neonates, following congenital heart disease, affecting roughly 4.48 per 10,000 live births globally 13 , 14 . Syndactyly occurs in approximately 1 in 2,000 to 3,000 live births, whereas polydactyly has a higher incidence, ranging from 1 in 500 to 1,000 live births 15 . The pooled prevalence of congenital anomalies, including limb defects, is approximately 23.5 per 1000 live births in Africa 16 . Clubfoot (talipes equinovarus) is relatively common, with an estimated incidence of 1 per 1000 live births globally and a prevalence of ~ 1.31 per 1,000 live births in the African population 17 – 19 . Within Ghana, the incidence of clubfoot has been documented at 25.4 per 100,000 live births in the northern population 20 . Syndromic limb malformations carry a substantial mortality risk, with reported survival rates as low as 4% when associated with other organ abnormalities 21 . In Northern Ghana, congenital malformations contribute to an overall neonatal mortality rate of 13.5%, with musculoskeletal system defects ranking as the third most prevalent 22 . Approximately 10 to 30% of congenital limb defects are syndromic, and ~ 13% are associated with multiple congenital anomalies (MCA), depending on the population 23 . Various exome sequencing and targeted gene studies have implicated several genes in congenital limb defects, including polydactyly (e.g., GLI3 , TWIST1, HOXD13 , etc.), syndactyly ( FGFR2 , GLI3 , HOXD13, GJA1 , etc.), and ectrodactyly, e.g., TP63 15,24 . Several syndromes exemplify the dual presentation of OFCs and limb defects with or without other anomalies. These include the TP63 -spectrum of disorders, Apert syndrome, Cornelia de Lange Syndrome (CdLS), and VACTERL/ VATER Association 24 – 27 . To investigate the genetic architecture of the co-occurrence of OFCs and limb defects, we conducted whole exome sequencing (WES) on nine Ghanaian families, comprising twenty-five individuals in total. This included ten affected individuals, comprising nine probands and one affected mother. Sanger sequencing was subsequently performed to validate the implicated variants. We examined whether the observed co-occurrence arises from monogenic pleiotropy, polygenic burden, or a combination thereof. Our goal was to enhance current understanding of the molecular architecture of syndromic OFCs presenting with limb defects and to elucidate the shared developmental pathways that link the face and limbs. Methods Study population, participant recruitment, and ethical considerations The study population consisted of 7 case-parent trios and 2 case-mother dyads, all recruited from the National Cleft Care Centre (NCCC) at Komfo Anokye Teaching Hospital (KATH), Kumasi, Ghana. Each family included a proband diagnosed with OFCs and associated limb malformations, along with one or both of their biological parents. Ethical approval was obtained from the Institutional Review Board (IRB) at KATH (KATH-IRB/AP/032/20), Kumasi, Ghana. Written informed consent was obtained from participating families prior to the collection of data and samples. All participating families were of Ghanaian descent. Recruited probands presented with CL, CP, or CLP, along with digital anomalies and sometimes other anomalies. Sample collection and DNA extraction Saliva samples were collected using Oragene•DISCOVER saliva tool kits ( https://www.dnagenotek.com ). Participants who were old enough to spit provided their saliva samples directly, while for those who were not old enough to spit, cheek swab samples were collected. Genomic DNA was isolated from the buccal swabs and saliva samples using the Oragene protocol at the Human Genetics and Genome (HuGENE) Laboratory at KNUST 19 . The quantity of DNA obtained for each sample was measured using the Qubit Assay (ThermoFisher Scientific, Hampton, USA). XY genotyping using real-time Polymerase Chain Reaction (PCR) was conducted as a quality control measure to verify the genetic sex of study participants 19 . The detailed protocol for DNA processing has been published by Gowans et al. (2016). Whole exome sequencing Whole exome sequencing (WES) was employed to identify genetic variants associated with syndromic OFCs in the recruited Ghanaian trios and dyads. Genomic DNA samples were shipped to commercial sequencing service providers (Azenta Life Sciences, LLC; South Plainfield, NJ, USA), where exome sequencing was conducted. The genomic DNA underwent fragmentation through acoustic shearing (Covaris S220 instrument). Exonic sequences, flanking intronic regions and UTRs were captured using the Twist Human Comprehensive Exome library preparation procedure (Twist Biosciences, South San Francisco, CA, USA). The fragmented DNAs were purified, end-repaired, adenylated at the 3’ ends, and ligated to adapters. Limited-cycle PCR was used to amplify the adapter-ligated DNA fragments. Validation of the adapter-ligated DNA fragments was performed using Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantification was carried out using Qubit 4 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA). The adapter-ligated DNA fragments were subjected to hybridization with biotinylated baits. Streptavidin-coated binding beads were used to capture the resulting hybrid DNAs, after which they were thoroughly washed. The washed captured DNAs were subsequently amplified and indexed using the Illumina indexing primers. Post-captured DNA libraries underwent validation through Agilent TapeStation (Agilent, Santa Clara, CA, USA) and quantification via Qubit 4 Fluorometer and Real-Time PCR (KAPA Biosystems, Wilmington, MA, USA). Generated sequencing libraries were combined and clustered into multiple sections of a flow cell. Following this, the flow cell was inserted into the Illumina HiSeq instrument, where the samples underwent sequencing in a 2 x 150 bp paired-end (PE) configuration at 100X read depth according to the manufacturer’s instructions. Image analysis and base calling procedures were carried out using the HiSeq Control Software (HCS). The initial raw sequence data in binary base call (.bcl) format produced by the Illumina HiSeq system were transformed into fastq files and de-multiplexed using the Illumina bcl2fastq software. A single mismatch was permitted for the identification of index sequences. Bioinformatics analysis for variant calling Following WES, raw FASTQ files (Fig. 1 ) were processed using a standardized bioinformatics pipeline to align reads, call variants, and prioritize candidates. The raw reads generated from WES were quality checked utilising FastQC 0.11.9 and trimmed using Trimmomatic 0.39 29 to remove sequencing adapters and low-quality bases. The processed reads were aligned to the human reference genome (GRCh38), using the Sentieon 202112.01 workflow 30 . Subsequently, PCR/Optical duplicates were identified and marked, generating BAM files. Single-nucleotide variants (SNVs) and small insertions and deletions (INDELs) were called by employing Sentieon DNAscope algorithm. VCF files generated underwent normalization, including left alignment of INDELs and splitting multiallelic sites into distinct sites, using bcftools v1.13 31 . Subsequently, overlapping transcripts were identified for individual variants, and the potential effects of these variants on the transcripts were annotated utilising the Ensembl Variant Effect Predictor (VEP) v104 32 . For downstream cohort analysis, the most severe impact for each variant was chosen. Variant Filtering and Prioritisation Process Likely pathogenic variants were prioritized (Fig. 1 ) using the American College of Medical Genetics and Genomics (ACMG) guidelines on variant classification 33 . The variant filtering process was designed to prioritize high-confidence, functionally relevant genetic variants associated with the syndromic OFCs under study. Protein-altering variants, including missense, frameshift insertions/deletions, stop-gained, stop-loss, start-gained, start-loss, and splice region variants with MAF < 0.01 were prioritized. MAF was ascertained using 1000 Genomes Project ( https://www.internationalgenome.org/ ), Exome Sequencing Project (esp.gs.washington.edu/drupal/), and Gnome Aggregation database consortium, gnomAD ( https://gnomad.broadinstitute.org/ ) v4.1. The pathogenicity of all missense variants was determined using both single and meta-prediction tools embedded in dbNSFP v4.9 34 . The tools included ClinPred, MetaRNN, BayesDel_addAF, REVEL, CADD, AlphaMissense, MutPred2, Polyphen-2, MutationAssessor, Mutation Taster, and SIFT (Supplementary Table S1 ). Missense variants predicted to be pathogenic by at least six out of eleven (i.e., the majority) of the prediction tools were filtered for. The pathogenicity of other variants, such as stop and start gain or loss, was predicted using CADD. Pathogenicity of splice region variants was also determined using SpliceAI embedded in Ensembl VEP. Pathogenic variants in genes associated with craniofacial and limb phenotypes, as well as their associated disorders in humans, were curated using GeneCards ( https://www.genecards.org/ ), Malacards ( https://www.malacards.org/ ), OMIM ( https://omim.org/ ), Genome alliances ( https://www.alliancegenome.org/ ), Mouse Genome Informatics ( https://www.informatics.jax.org ), and Facebase ( https://www.facebase.org/ ). Newly identified genes involved in biological processes relevant to the phenotypes under study, such as primary ciliary function, bone formation and development, cell adhesion, transcription regulation, cell migration, and proliferation, were curated using Genome Alliance, Mouse Genome Informatics, and Facebase. The co-segregation of candidate variants was assessed to confirm whether identified variants followed Mendelian inheritance patterns (e.g., autosomal dominant or recessive). This was done by verifying the presence of the variant in affected individuals and its absence in unaffected family members, where possible. Given the absence of family history in affected individuals in 8 out of the 9 families, candidate variants were prioritized based on those fitting a model of de novo variants or autosomal dominant inheritance with incomplete penetrance. Variants that met the filtering criteria were considered the most likely to cause the observed phenotypes in affected individuals (Fig. 1 ). Sanger sequencing As a quality control step, two novel candidate variants in MYH3 (c.2015G > A, p.Arg672His) and NIPBL (c.7617_7618del, p.Ser2540ProfsTer21) identified via WES were validated using Sanger sequencing. The exact procedure has been previously published 28 . In summary, primers (Supplementary Table S2 ) were designed to flank each variant region using Primer3 ( https://primer3.ut.ee/ ). The genomic sequences flanking the variant site were obtained from the UCSC Genome Browser (GRCh38/hg38; https://genome.ucsc.edu/ ). Each primer pair with closely matched Tm values (within 2°C) was prioritized and validated using BLAT function of UCSC Genome Browser ( https://genome.ucsc.edu/cgi-bin/hgBlat ) to confirm sequence specificity. I n-silico PCR analysis was also performed using the UCSC browser ( https://genome.ucsc.edu/cgi-bin/hgPcr ) to validate the specificity and production of a single amplicon (300 to 700bp) from the primers. PCR was used to amplify genomic DNA (4 ng/µL) in 10 µL reaction volumes. Amplification success was verified using 2% agarose gel electrophoresis. Validated amplicons were subjected to sequencing at Functional Biosciences (Madison, WI, USA) using an ABI 3730XL DNA Sequencer. Generated chromatogram data were analysed using PHRED for base calling, PHRAP for assembly, POLYPHRED for variant detection, and CONSED for visualisation, as previously described 19 . Structural and evolutionary analysis For structural analysis of mutant proteins, the potential impact of missense variants on protein structure was evaluated using in-silico modelling tools. The protein structures of candidate genes were downloaded from AlphaFold ( https://alphafold.ebi.ac.uk/ ) and visualized using UCSF Chimera v1.14 ( https://www.cgl.ucsf.edu/chimera/ ). The effect of the variant on the protein structures was evaluated using PyMOL v2.3.2 ( https://www.pymol.org/ ). Evolutionary conservation of identified variants (Table 1 ) was assessed through multiple sequence alignment of vertebrate orthologs using MAFFT (Multiple Alignment using Fast Fourier Transform) accessed from EMBL-EBI with default parameters 35 . Species included in alignments are indicated in the respective figures (Fig. 2 ; Supplementary Figure S3 ). Protein sequences were obtained from the NCBI RefSeq ( https://www.ncbi.nlm.nih.gov/refseq/ ) database and UniProt ( https://www.uniprot.org/ ). Table 1 Pathogenic variants identified through whole exome sequencing in nine affected probands. Family ID (Sex) Gene (Inheritance) Genomic Co-ordinates Genotype ( Zygosity ) HGVSc HGVSp No. of Tools Predicting Pathogenicity 1 M(XY) RGPD5 (De Novo) chr2:109837829 (rs1553471918) Het c.4708G > A p.Gly1570Arg 7 FAM90A26 (De Novo) chr4:9173181 (rs1450748908) Homo c.10del p.Cys4ValfsTer12 N/A 2 M(XY) FOXD4L1 (De Novo) chr2:113499585 (rs201655302) Het c.329A > C p.Tyr110Ser 9 FAM170A (De Novo) chr5:119634539 (rs754719389) Het c.791T > C p.Met264Thr 7 DLG1 (De Novo) chr3:197090942 (rs769502806) Het c.1730G > A p.Arg577Gln 6 ANKRD1 (De Novo) chr10:90917812 (rs773773073) Het c.472C > T p.His158Tyr 11 3 F(XX) TP63 (De novo) chr3:189868614 (rs886041251) Het c.1027C > T p.Arg343Trp 11 4 F(XX) NIPBL (Unknown)* chr5:37059093 ( Novel ) Het c.7617_7618del p.Ser2540ProfsTer21 N/A 5 F(XX) MYH3 (De novo) chr17:10641317 (rs121913617) Het c.2015G > A p.Arg672His 9 Family ID (Sex) Gene (Inheritance) Genomic Co-ordinates Genotype ( Zygosity ) HGVSc HGVSp No. of Tools Predicting Pathogenicity 6 F(XX) FGFR2 (Unknown)* chr10:121520163 (rs79184941) Het c.755C > G p.Ser252Trp 10 7 M(XY) TRIM74 (De Novo) chr7:72961358 (rs199887265) Het c.487C > T p.Arg163Ter CADD score of 35 TRIM73 (De Novo) chr7:75403732 (rs199982097) Het c.487C > T p.Arg163Ter CADD score of 35 8 M(XY) PRDM9 (De Novo) chr5:23527360 ( Novel ) Homo c.2272_2273insTG p.Arg758LeufsTer182 N/A 9 F(XX) TP63 ( Maternal inheritance ) chr3:189867902 (rs1205536026) Het c.952C > T p.Arg318Cys 11 *Paternal samples of Families 4 and 6 were unavailable; however, the identified variants for these two families were absent in the mothers. Het : heterozygous; Homo : Homozygous. Pathway enrichment and interaction analysis Candidate genes obtained after prioritisation were analysed for biological process annotations. The g:Profiler tool ( https://biit.cs.ut.ee/gprofiler/gost ), a web-based platform, was used to conduct an enrichment analysis of the identified genes against several databases, including Gene Ontology (GO) Biological Process (BP), Molecular Function (MF), and Cellular Component (CC), Reactome, WikiPathways, and Human Phenotype (HP) ontology. g:Profiler performs functional enrichment analysis of gene lists using Fisher’s exact test for over-representation 36 . The analysis was restricted to Homo sapiens (human) to ensure species-specific relevance with a significance threshold of p < 0.05. All other parameters were defaulted. The output generated included enriched pathways, their adjusted P -value, and the number of overlapping genes from the candidate list. The EnrichmentMap plugin in Cytoscape (v3.10.4) 36 , an open-source platform for visualising complex networks, was employed to cluster and visualize pathway enrichment results as an interactive network. This approach clusters functionally related pathways based on gene set overlap. By doing so, it reduces redundancy, reveals higher-order biological themes, and enables interpretation of enrichment results compared to isolated pathway lists. Protein-protein interaction network and hub genes identification A protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database v12.0 with an interacting confidence score of > 0.4 ( http://string-db.org ). The resulting PPI network was exported to Cytoscape for visualisation and further analysis. The CytoHubba plugin, embedded in Cytoscape, was then applied with the maximal clique centrality (MCC) algorithm to identify hub genes 36 , as these highly interconnected nodes are often critical drivers of biological processes. Gene expression analysis using mouse models The gene expression patterns of implicated candidate genes were investigated using the Mouse Genome Informatics (MGI) Gene Expression Database (GXD) v6.24 37 . To refine the search and ensure relevance to the syndromic phenotypes, the curated list of candidate genes was submitted to GXD via the Batch Search interface. A structured filtering approach was implemented to focus on developmentally relevant anatomical regions under the ‘Conceptus’ category. The selected filters include the following terms: 1st branchial arch (mandibular and maxillary components), face mesenchyme, oral region mesenchyme, head surface ectoderm, latero-nasal process, nasal pit, mesenchyme derived from neural crest, and the limb. The ‘heart’ filter was also applied, as two of the probands (Family 2 and Family 4) presented with congenital heart defects. Expression data were retrieved across developmental stages from the earliest post-implantation period through adult stages, with particular emphasis on critical periods of craniofacial (E8.5 to E14.5) and limb (E9.5 to E14.5) morphogenesis. Expression annotations were quantified and visualized via the tissue × gene matrix, with colour intensity corresponding to the number of expression results per anatomical structure. A heatmap was generated using Morpheus 37 , a web-based platform for matrix visualisation and analysis, with qualitative expression levels colour-coded to visualize spatiotemporal gene activity. Results Clinical presentation of affected individuals Eight simplex and one multiplex families (Supplementary Figure S1 ), comprising two dyads and seven trios, with a history of syndromic OFCs presenting with limb abnormalities, were recruited for this study. WES datasets were generated from DNA obtained from case parent trios, except for Families 4 and 6, where the datasets were generated for case mother dyads due to the unavailability of the fathers. Family 1 The proband was a 6-year-old male who presented with a complete cleft palate, ulnar (postaxial) hexadactyly of both hands, mild intellectual disability, speech delay until age 4, mild microcephaly, characteristic facial features (almost V-shaped head with undulating surface), and cephalhematoma (Supplementary Figure S1 A). Family 2 The proband was a 5-month-old male who presented with left complete CL, left unilateral talipes equinovarus, microcephaly, a low heart murmur that was suggestive of a heart defect, and large, low-set ears. A follow-up on this family revealed that the child had no neck control at 8 months, suggestive of global developmental delay (Supplementary Figure S1 B). Family 3 The proband was a 7-day-old female who presented with bilateral incomplete CLP (right complete CLP plus left complete CP), bifid uvula, ectrodactyly of the right hand (missing/hypoplasia of the third digit with normal development of all other digits), and the fifth digit of the right hand was stiff and could not be bent. The thumb, index, and ring fingers of the right hand were also folded on each other. Clinical evaluation established a diagnosis of ectrodactyly ectodermal dysplasia cleft lip/palate (EEC) based on the presented phenotypes (Supplementary Figure S1 C). Family 4 The proband was a 4-month-old female who presented with complete CP, syndactyly of the 2nd and 3rd toes of both feet, severe micrognathia, glossoptosis and breathing difficulty. The proband was re-examined after 8 months, at which point additional phenotypes were observed. She had a short stature, a small and upturned nose, developmental delay, a long philtrum, a thin and downturned upper lip, low-set ears, thick eyebrows, microcephaly, a short fifth finger, arched eyebrows that almost met in the middle, hirsutism, a feeding problem/failure to thrive, long eyelashes, and pulmonary stenosis. The proband was born prematurely during the 8th month of pregnancy with a low birth weight of 1.5 kg. Clinical evaluation established a diagnosis of Cornelia de Lange syndrome based on the presented phenotypes (Supplementary Figure S1 D). Family 5 The proband was a 2-weeks-old female who presented with complete CP, bilateral clubfoot, camptodactyly, a characteristic face that causes her to appear to be whistling, microstomia (small puckered mouth), pursed lip, H-shaped scar-like mark extending from the lower lip towards the bottom of the chin, widely-spaced deep eyes, prominent cheeks, struggles to open the mouth, diminished ability to suck, flat philtrum, as well as high-arched and V-shaped palate. Clinical evaluation established a diagnosis of Distal arthrogryposis type 2A (DA2A), also known as Freeman-Sheldon Syndrome (FSS), Supplementary Figure S1 E. Family 6 The proband was a female who was 1 month 9 days old at the time of recruitment. She presented with incomplete CP, bulging eyes, and finger and toe syndactyly. These clinical presentations were suggestive of Apert syndrome. The proband was born to consanguineous parents (Supplementary Figure S1 F). Family 7 The proband was a 1-week-old male who presented with right complete CLP, unilateral right clubfoot, syndactyly of the 3 middle left toes, left-hand digital hypoplasia with anonychia affecting all fingers except the thumb and right radial clubhand (Supplementary Figure S1 G). Family 8 The proband was a 3-day-old male who presented with right complete CLP and hexadactyly, an extra thumb on the right hand (Supplementary Figure S1 H). Family 9 The proband was a 2-week-old female. She presented with complete bilateral CLP, ectrodactyly, and ectodermal dysplasia that presented as anhidrosis with impaired thermoregulation and nail dystrophy characterized by thickened nails. These phenotypes were clinically suggestive of EEC syndrome. The proband was born to consanguineous parents. The mother presented with bilateral symbrachydactyly (4th finger), right-hand syndactyly (3–4 fingers) and right foot ectrodactyly (Supplementary Figure S1 I). Probable pathogenic variants identified by whole exome sequencing WES analysis of probands with co-occurring OFCs and limb abnormalities unveiled several candidate variants in genes associated with certain developmental pathways and syndromes (Table 1 ; Supplementary Table S3 ). These include variants in genes such as TP63, FGFR2, PRDM9, DLG1, NIPBL and MYH3 . Importantly, many of these variants were de novo , some of which were novel, suggesting that these variants may be population-specific. As a quality control measure, the variants observed in NIPBL and MYH3 were confirmed with Sanger sequencing (Supplementary Figure S2 ). Structural and evolutionary analysis of implicated variants The structural and evolutionary analyses of identified missense variants (Table 1 ) revealed molecular alterations that may underlie the observed phenotypes. The wild-type amino acids are located in highly conserved regions of proteins, with many being perfectly conserved from humans to distant vertebrates such as zebrafish. The structural analyses also revealed a consistent pattern of disruptive alterations across all variants (Fig. 2 ; Supplementary Figure S3 ). As a representative example, the R343W substitution in TP63 occurred in the 8th exon within the DNA-binding domain of the protein, replacing a positively charged arginine with nonpolar tryptophan containing a bulky indole side chain. This substitution eliminates electrostatic interactions between the TP63 protein and the negatively charged DNA phosphate backbone and can destabilize the protein due to unfavourable torsion angles. The de novo R672H variant in MYH3 replaces the positive charge of arginine with a smaller, less charged histidine, potentially weakening ATP binding by reducing electrostatic interactions and decreasing hydrogen bonds critical for the stability of the binding pocket. Pathway enrichment analysis, protein-protein interaction network and hub genes identification Functional enrichment analysis of genes harbouring candidate variants revealed the involvement in several key biological pathways in normal craniofacial and limb development (Fig. 3 ; Supplementary Figure S4 ). A highly significant and coherent enrichment related to coordinated developmental processes, specifically those derived from epithelial-mesenchymal interactions, was observed in both Gene Ontology (GO) categories and Human Phenotype Ontology (HP) terms (Fig. 3 A and B). A profound statistically significant enrichment for developmental pathways directly related to processes governing limb formation and OFC pathogenesis were observed, including appendage morphogenesis (GO: 0035107, Padj = 1.722×10 − 3 ), limb morphogenesis (GO:0035108, Padj = 1.722×10 − 3 ), limb development (GO:0060173, Padj = 4.208×10 − 3 ), cranial skeletal system development (GO: 1904888, Padj = 1.107×10 − 2 ), morphogenesis of an epithelial bud (GO:0060572, Padj = 4.573×10 − 2 ) and embryonic morphogenesis (GO: 0048598, Padj = 1.875×10 − 2 ). These pathways were complemented by human phenotype ontology (HP) terms, highlighting specific defects such as deviation of the thumb (HP:0009603, Padj = 2.177×10 − 2 ) and cutaneous finger syndactyly (HP:0010554, Padj = 2.302×10 − 2 ). A significant enrichment was observed for abnormalities in sensory organs that share developmental origins and pathways with the face and limbs, particularly those derived from cranial placodes and neural crest cells 38 . These included lacrimal duct stenosis (HP:0007678, Padj = 1.016×10 − 3 ), conductive hearing impairment (HP:0000405, Padj = 1.135x10 − 2 ) and nasolacrimal duct obstruction (HP:0000579, Padj = 1.168×10 − 2 ). This pattern of phenotypic enrichment corresponds to established clinical features observed in patients who present with both OFCs and limb malformations 39 . Identification of central regulators (Fig. 3 C and D) within the implicated gene set (Table 1 ) resulted in a PPI network comprising 13 nodes (proteins) and 5 edges (interactions), with an average node degree of 0.769 and a p-value of 0.26 (Supplementary Figure S5 ). The PPI enrichment p-value indicates that the observed number of connections is not statistically significantly greater than what would be expected by chance for a random set of proteins of the same size and degree distribution from the genome. Given the exploratory nature of our study and to capture potential novel interactions within this specific genetic context, a confidence score threshold of 0.150 was employed. This threshold was selected after the default confidence filter (~ 0.400) yielded only a single interaction, which was deemed insufficient for meaningful network analysis. Despite the lack of statistical enrichment, the analysis identified RGPD5 , MYH3 , ANKRD1 , DLG1 , FGFR2 , and TP63 as the top hub genes (Fig. 3 D). Spatiotemporal Expression Profiles of Implicated Genes Based on analysis of the Morpheus heat map, nine of the thirteen candidate genes demonstrated distinctive expression patterns associated with specific morphogenetic processes across various mouse strains, developmental stages, and tissue structures (Fig. 4 ). The other four genes were not represented in the Mouse Genome Informatics (MGI) database utilized for the gene expression analysis. Some genes, including Dlg1 , Nipbl , and Fgfr2 , exhibited peak expression during early embryonic development, while Dlg1 , Nipbl . Prmd9 and Trp63 maintained sustained expression throughout multiple developmental windows (Fig. 4 A). Notable Dlg1 , Fgfr2 , and Trp63 expression was observed from Theiler stages (TS) 1 through to 23 in the branchial arches, including ectoderm and mesenchymal components, facial prominence mesenchyme, and palatal shelf, which are regions essential for lip and palate formation. Concurrently, Dlg1 , Fgfr2 , Myh3 , and Trp63 displayed an overlapping moderate to high expression from TS 15 to 28 in limb mesenchyme, limb ectoderm, and developing skeletal and muscle elements (Fig. 4 B). Discussion The study sought to decipher the genetic aetiology of OFCs co-occurring with limb abnormalities, and in some cases, additional phenotypes (Table 1 ). The co-occurrence of OFCs and limb deformities, though less frequent than isolated OFCs, has significant implications for genetic counselling and clinical management. This co-occurrence is often associated with specific genetic syndromes or different syndromes manifesting concurrently 19 . WES analysis identified multiple pathogenic variants (Table 1 ) in affected individuals, some of which have been previously reported and associated with known genetic syndromes. The proband in Family 1 carried two de novo variants, one in RGPD5 and another in FAM90A26 . RGPD5 interacts with nuclear proteins, including Ras-related nuclear protein (RAN) and transportin-1 40 . A previous study reported RGPD5 deletion in an infant with multiple craniofacial and limb malformations, including unilateral complete cleft lip, microcephaly, craniosynostosis, syndactyly, dysmorphic ears, bilateral congenital talipes equinovarus, bilateral radial club hand, and ectrodactyly 41 . The proband in the current study presented with similar phenotypes. FAM90A26 is expressed in gonad primordial and testicular germline stem cells, with an overexpression of its protein product in the heart ( https://www.bgee.org/ ; https://www.genecards.org/ ). Four de novo variants were identified in the proband from Family 2 (Table 1 ), including variants in FOXD4L1 , FAM170A , DLG1 , and ANKRD1 . FOXD4L1 is a forkhead/winged-helix (FOX) transcriptional factor that is crucial for embryogenesis and regulates neural ectoderm by preserving neural precursor cell pluripotency and repressing transcription factors and other genes that drive neural differentiation. These are crucial for FGF and BMP signalling, regulating neural plate patterning and maintaining neural fate 42 . In the expression analysis (Fig. 4 ), Foxd4 exhibited an early-onset expression pattern, with craniofacial-specific expression beginning at E7.5 and limited expression in some skeletal elements (femur diaphysis, femur metaphysis, and tibia) between postnatal week 5 to 70. FAM170A has been linked with VATER/VACTERL Association 43 , which is defined by the systematic concurrence of vertebral defects (V), anal atresia (A), cardiac malformations (C), tracheoesophageal fistula with or without oesophageal atresia (TE), renal abnormalities (R), and limb anomalies (L) 26 . The proband in the current study presented with CL, talipes equinovarus, global developmental delay, a heart defect, and other abnormalities, which are characteristic of this syndrome. DLG1 , expressed in both mesenchymal and epithelial cells, plays critical roles in palatal morphogenesis, limb elongation, and spatial patterning. Pathogenic variants in DLG1 are associated with CLP and disorganized chondrocytes in the sternum, particularly in the proliferative zone 44 , 45 . Among the implicated genes for this family, Dlg1 exhibited the second most comprehensive temporal and spatial expression profile, starting at E0.5 and persisting postnatally, with prominent signals in craniofacial tissues (first branchial arch, facial prominences, and palatal shelves) from E8.5 and in limb structures from E10.5. We hypothesize that DLG1 , a well-established gene associated with clefting and limb defects, contributes to the severe limb and craniofacial phenotypes exhibited by the proband. The ANKRD1 gene product, cardiac ankyrin repeat protein (CARP), functions as a transcriptional repressor and a sarcomeric component of the titin-binding complex 46 . Pathogenic variants in ANKRD1 have been implicated in cardiomyopathies and septal defects 46 , 47 . The proband in the current study also presented with a heart defect characterized by low murmurs, which may result from the pathogenic variant identified in ANKRD1 . Ankrd1 is expressed in the heart, starting at E7.5 (TS 11), which coincides with cardiogenic plate formation, as well as craniofacial and limb tissues (Fig. 4 ). The phenotypic spectrum in the proband suggests a polygenic aetiology, where these four de novo variants likely work synergistically to produce the observed craniofacial, limb, and cardiac defects. Two heterozygous missense variants in TP63 were identified in individuals from two unrelated families, Family 3 and Family 9, who presented with bilateral cleft lip and palate (CLP), ectrodactyly, and other orofacial and limb malformations. TP63 functions as a key regulator of ectodermal, limb and craniofacial morphogenesis 48 . Heterozygous variants in TP63 account for multiple autosomal dominant disorders defined by three key phenotypes: limb defects, ectodermal dysplasia, and facial clefting. At least eight different TP63 -related syndromes with overlapping phenotypes have been reported 24 , 48 . Trp63 exhibits a broad epithelial-enriched expression profile from early embryogenesis to adulthood, with high expression evident in the limb ectoderm and facial prominence ectoderm during their respective windows (E9.5 to E14.5) of outgrowth and fusion (Fig. 4 ). The first variant (c.1027C > T, p.Arg343Trp) was identified as a de novo mutation. A de novo TP63 variant may represent spontaneous mutagenesis or suggest the presence of undetected germline mosaicism in a phenotypically normal parent 48 . The second variant (c.952C > T, p.Arg318Cys) was inherited from an affected mother. The mother from this consanguineous family (Family 9) presented with bilateral symbrachydactyly, right 3–4 fingers syndactyly, and right foot ectrodactyly, but no orofacial cleft. Both variants observed in TP63 , occurring within the DNA-binding domain (DBD), are clinically associated with ectrodactyly, ectodermal dysplasia, and cleft lip-palate syndrome 3, EEC3 24 . A novel heterozygous frameshift variant in NIPBL was identified in the proband of Family 4, who was clinically diagnosed with CdLs. Pathogenic variants in NIPBL cause CdLS, a complex developmental disorder and the most frequently occurring cohesinopathy. Affected individuals present with impaired growth, abnormal limb development, and developmental impairments 27 . Our spatiotemporal analysis revealed broad early-onset expression of Nipbl , with craniofacial and limb expression observed from E8.5 and E10.5, respectively. Cardiac expression began from E9.5, with presence in the primitive heart tube, atria, and ventricles (Fig. 4 ). This aligns with CdLS clinical phenotypes, where congenital heart defects frequently co-occur with craniofacial and limb malformations 49 . The observed frameshift variant in the current proband may result in a truncated protein, potentially leading to haploinsufficiency of NIPBL 49 , 50 . A de novo heterozygous missense variant in MYH3 was identified in the proband of Family 5, who presented features descriptive of FSS. Heterozygous variants in MYH3 cause Sheldon-Hall syndrome (SHS) or FSS, characterized by distinctive facial features, hand and foot contractures, camptodactyly, oropharyngeal defects, and scoliosis 51 , 52 . The identified variant (c.2015G > A, p.Arg672His) has been previously reported as a recurrent mutational hotspot 52 . Expression analysis revealed early Myh3 activity localized predominantly to muscle-related structures, including craniofacial muscle compartments and limb muscles. Beyond muscular tissues, Myh3 was also detected in skeletal elements (Fig. 4 ). A heterozygous missense variant in FGFR2 was identified in the proband from Family 6. The identified variant (c.755C > G, p.Ser252Trp) has been associated with Apert syndrome (AS) 25 , 53 and it is detected in ~ 59% of all AS cases, with a strong association with CP 54 . Fgfr2 was detected in the developing branchial arch mesenchyme and limb bud from their earliest formation stages (E8.5 and E10.5, respectively), with expression persistence throughout major morphogenetic periods and limited expression in the left ventricle of the heart (Fig. 4 ). De novo heterozygous stop-gained variants in TRIM73 and TRIM74 were observed in the proband from Family 7. These genes have been associated with Williams-Beuren syndrome (WBS), which results from abnormal homologous recombination and unequal crossing-over between tandem segments containing TRIM50 , TRIM73 , and TRIM74 55 . The TRIM proteins function as E3 ubiquitin ligases, conferring substrate specificity to ubiquitin-proteasome complexes and thereby affecting nearly all cellular processes 56 . TRIM73 and TRIM74 share a high degree of similarity (~ 99.8%), indicating redundant paralogs with overlapping functions in developmental processes, potentially providing functional compensation through genetic redundancy. WBS is defined by unique dysmorphic craniofacial features, supravalvular aortic stenosis (SVAS), hypertension, depressed nasal bridge, intellectual disability, premature ageing of the skin, broad forehead, infantile hypercalcemia, and tooth defects 57 , 58 . Interestingly, the phenotypes of this syndrome were palpable in the current proband, who presented with right complete CLP, right clubfoot, left toes 2–4 syndactyly, left-hand digital hypoplasia with anonychia affecting all fingers except the thumb, and right radial clubhand. We hypothesize that the involvement of TRIM proteins in cellular processes, such as apoptosis and cell proliferation 59 , may underlie the observed phenotypes. However, further functional genomics experiments are warranted to confirm these observations. We also identified a novel de novo PRDM9 frameshift variant (c.2272_2273insTG, p.Arg758LeufsTer182) in the proband from Family 8. PRDM9 is specifically expressed in germ cells during meiosis, where its methyltransferase activity increases H3K4me3 at recombination hotspots 60 , thereby acting as a crucial determinant of meiotic recombination in both murine and human models 61 . PRDM9 has been implicated in Smith-Magenis Syndrome (SMS), an autosomal disorder characterized by OFCs, depressed nasal bridge, hand polydactyly, toe syndactyly, clinodactyly of the 5th finger, micrognathia, etc 43 . The current proband presented with complete CLP and hand hexadactyly, characteristic of SMS. Population-specific recombination patterns associated with PRDM9 alleles may contribute to disease-causing genomic rearrangements, particularly in individuals of West African ancestry 62 . In our spatiotemporal analysis, Prdm9 showed expression in craniofacial tissues (branchial arch, facial prominence, head mesenchyme, head surface ectoderm) from E8.5 and limb structures from E10.5 through to adulthood (Fig. 4 ), suggesting a role for this gene in craniofacial development. A consistent pattern of disruptive molecular alteration and loss of conserved amino acid residue was observed for all studied variants in the structural and evolutionary analyses (Fig. 2 ; Supplementary Figure S3 ). Multiple sequence alignment showed that all the wild-type amino acids subject to mutation were highly conserved across vertebrate species. The identified substitutions consistently resulted in detrimental structural changes, including loss of critical electrostatic interactions, introduction of steric clashes, disruption of hydrogen bonding networks, and alteration of binding pocket geometries. Pathway enrichment analysis (Fig. 3 A) revealed that the implicated genes are significantly involved in appendage morphogenesis, cranial skeletal system development, and embryonic morphogenesis pathways in craniofacial and limb development. Human phenotype ontology (HP) terms also revealed enrichment for phenotypes such as nasolacrimal duct obstruction, cutaneous finger syndactyly, and deviation of the thumb, corresponding to the established clinical features observed in patients who present with both OFCs and limb malformations 39 . The EnrichmentMap analysis generated a network of 46 nodes and 140 edges, organized into clusters and individual nodes (Fig. 3 B). The first cluster centred on epithelial and glandular development, indicating that disrupted epithelial-mesenchymal interactions underlie palatal, limb, and glandular malformations (Doshi and Patil, 2012). The second cluster focused on craniofacial development and urogenital morphogenesis. The third cluster was dominated by appendage and limb morphogenesis, co-enriched with abnormalities of the external genitalia, which indicate shared developmental pathways. The protein-protein interaction network (Fig. 3 C) revealed limited connectivity among the candidate genes, with only 5 interactions identified among the 13 proteins (Supplementary Figure S5 ). Despite the sparse network structure (PPI enrichment p-value = 0.26), RGPD5 , MYH3 , ANKRD1 , DLG1 , NIPBL , FGFR2 , and TP63 emerged as key hub genes (Fig. 3 D) with established roles in pathways fundamental to embryogenesis, specifically craniofacial and limb morphogenesis. The extrapolability of the findings of the current study is limited by its small sample size. Again, our inability to obtain paternal samples in two families hindered our ability to determine whether certain variants are de novo . Lastly, the study focused on the analysis of WES datasets to decipher single nucleotide variants (SNVs) and indels in the aetiology of the conditions under study. As a result, potentially pathogenic non-coding and structural variants could have been missed. Conclusion In conclusion, WES analysis of nine Ghanaian families presenting with OFCs and limb abnormalities, and in some instances other abnormalities, identified probable pathogenic genetic variants, including novel and population-specific mutations in various genes, confirming the genetic heterogeneity of OFCs co-occurring with limb malformations. These variants support a model in which both pleiotropic, monogenic, and polygenic mechanisms contribute to phenotypic variations. Future studies incorporating larger, more diverse cohorts and functional validation approaches will help expand variant discovery, clarify biological mechanisms, and improve genetic counselling and clinical management of syndromic OFCs. Abbreviations ANKRD Ankyrin Repeat Domain BMP Bone morphogenic protein CdLS Cornelia de Lange syndrome CL Cleft lip CL/P Cleft lip with or without cleft palate CLP Cleft lip and palate CP Cleft palate DA2A Distal arthrogryposis type 2A DLG1 Discs Large MAGUK Scaffold Protein 1 DNA Deoxyribonucleic acid EEC Ectrodactyly ectodermal dysplasia cleft lip/palate FAM170A Family with Sequence Similarity 170 Member A FAM90A26 Family with Sequence Similarity 90 Member A26 FGF Family with Sequence Similarity 90 Member A26 FOXD4L1 Forkhead Box D4 Like 1 FSS Freeman-Sheldon syndrome GLI3 GLI Family Zinc Finger 3 GWAS Genome-wide association studies HuGENE Human Genetics and Genome KATH Komfo Anokye Teaching Hospital KNUST Kwame Nkrumah University of Science and Technology MAGUKs Membrane-associated guanylate kinase homologs MYH3 Myosin Heavy Chain 3 NIPBL Nipped-B-like protein OFCs Orofacial clefts PRDM9 PR domain-containing 9 RGPD5 RANBP2-Like and GRIP Domain Containing 5 SHS Sheldon-Hall Syndrome TP63 Tumor protein p63 TRIM Tripartite motif WES Whole exome sequencing WGS Whole genome sequencing Declarations Ethics approval and consent to participate This study was approved by the Institutional Review Board (IRB) at KATH (KATH-IRB/AP/032/20), Kumasi, Ghana. Written informed consent was obtained from all participating families. Consent for publication Not applicable Availability of data and materials The whole exome sequencing (WES) dataset reported in this article can be accessed through FaceBase Consortium (https://www.facebase.org) under controlled access, with an accession number 94-D420 (Gowans LJJ, 2025). The informed consent obtained from participants only permits sharing the WES dataset under controlled access. The data and materials that support the findings of this study are available from the corresponding author upon reasonable request. The data and materials that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests Funding This project was supported by the IADR/Smile Train Cleft Research Award, 2023 (LJJG), K43DE029427 by NIDCR/FIC/NIH, USA (LJJG). Authors' contributions L. J. J. Gowans, contributed to conception, design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript; E. Tackie, contributed to design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript; A. Butali, P. Donkor, G. O. Mensah, B. Tsri, S. Obiri-Yeboah, T. D. Busch, A. A. Adeyemo, contributed to design, data acquisition, analysis and interpretation, and critically revised the manuscript; C. O. Asamoah, D. K. Sabbah, G. Plange-Rhule, A. A. Oti, contributed to data acquisition, analysis and interpretation, and critically revised the manuscript. All authors gave their final approval and agreed to be accountable for all aspects of the work. Acknowledgement We sincerely thank the study participants for their participation. 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Doshi RR, Patil AS. A role of genes in craniofacial growth. IIOAB J. 2012;3(2):19–36. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Additional file 1 - Table S1: Prediction tools, scoring ranges, and thresholds used for filtering out pathogenic variants. AdditionalFile2.docx Additional file 2 - Table S2: Primer sequences used for variant validation. AdditionalFile3.docx Additional file 3 - Table S3: In-text Pathogenic Variants. This table provides supplementary details on the top candidate pathogenic variants identified during the analysis, including predictions from various tools not presented in the main results section. AdditionalFile4.docx Additional file 4 - Figure S1: Family Pedigrees. (A-I) Pedigrees showing the inheritance patterns observed in the nine families. AdditionalFile5.docx Additional file 5 - Figure S2:Sanger sequencing chromatograms confirming NIPBL and MYH3 variants. Chromatograms showing heterozygous variants identified by whole-exome sequencing and validated by Sanger sequencing. (A) NIPBL variant (c.7617_7618del, p.Ser2540ProfsTer21). (B) MYH3 variant (c.2015G>A, p.Arg672His). Arrows indicate the positions of the variants. AdditionalFile6.docx Additional file 6 - Figure S3: Evolutionary and structural in silico analysis of variants. (A-D) Multiple sequence alignment (MSA) of regions surrounding missense variant positions in RGPD5 , FOXD4L , FAM170A , DLG1 , ANKRD1 , FGFR2 , and TP63 shows conservation across available species. For most genes, alignments included Homo sapiens (wild-type), Pan troglodytes , Mus musculus , Rattus norvegicus , Canis lupus familiaris , Bos taurus , Danio rerio , and Macaca fascicularis . (E-H) Corresponding molecular docking poses showing predicted structural alterations caused by amino acid substitutions. Note: The alignment for RGPD5 includes only Homo sapiens and Pan troglodytes (Chimpanzee) as conservation beyond primates is limited. FOXD4L includes only Pan troglodytes , Macaca nemestrina , Xenopus tropicalis , and Xenopus laevis due to the absence of clear orthologs in other model organisms AdditionalFile7.docx Additional file 7 - Figure S4: Heatmap view showing the contribution of individual genes to enriched terms, with darker colours indicating stronger associations. AdditionalFile8.docx Additional file 8 - Figure S5: Network statistics for protein-protein interaction analysis of candidate genes. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Feb, 2026 Reviews received at journal 22 Jan, 2026 Reviews received at journal 19 Jan, 2026 Reviews received at journal 18 Jan, 2026 Reviews received at journal 17 Jan, 2026 Reviewers agreed at journal 15 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers agreed at journal 11 Jan, 2026 Reviews received at journal 10 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers invited by journal 07 Jan, 2026 Editor invited by journal 12 Dec, 2025 Editor assigned by journal 11 Dec, 2025 Submission checks completed at journal 11 Dec, 2025 First submitted to journal 11 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8340088","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":560060221,"identity":"34bf221d-7151-4efa-8196-c100690addde","order_by":0,"name":"Edna Tackie","email":"","orcid":"","institution":"Kwame Nkrumah University of Science and Technology (KNUST)","correspondingAuthor":false,"prefix":"","firstName":"Edna","middleName":"","lastName":"Tackie","suffix":""},{"id":560060222,"identity":"535a9ef9-ef3b-4126-a17f-4e4ed4e8c950","order_by":1,"name":"Solomon Obiri-Yeboah","email":"","orcid":"","institution":"Kwame Nkrumah University of Science and Technology (KNUST)","correspondingAuthor":false,"prefix":"","firstName":"Solomon","middleName":"","lastName":"Obiri-Yeboah","suffix":""},{"id":560060223,"identity":"987236c5-091d-40bf-866d-d5e17d1d16ee","order_by":2,"name":"Gideon Okyere Mensah","email":"","orcid":"","institution":"Kwame Nkrumah University of Science and Technology (KNUST)","correspondingAuthor":false,"prefix":"","firstName":"Gideon","middleName":"Okyere","lastName":"Mensah","suffix":""},{"id":560060227,"identity":"0ea83c30-4967-404a-8799-f79f3273b328","order_by":3,"name":"Tamara D. 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1","display":"","copyAsset":false,"role":"figure","size":49729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioinformatics pipeline for the identification and prioritisation of rare genetic variants potentially associated with craniofacial and limb abnormalities. \u003c/strong\u003eThe variants were prioritized based on pathogenicity predictions, craniofacial and limb phenotypes, gene expression profile, and functional roles in biological processes, based on the ACMG guidelines.\u003c/p\u003e","description":"","filename":"Binder21.png","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/08eb0dc71dc21210dda32f21.png"},{"id":98200736,"identity":"6d67dc53-38a4-46d1-9bec-3140c69cd607","added_by":"auto","created_at":"2025-12-15 07:49:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1725772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural and evolutionary analysis of variants in implicated genes.\u003c/strong\u003e \u003cstrong\u003e(A-E) \u003c/strong\u003eMultiple sequence alignment of \u003cem\u003eDLG1\u003c/em\u003e, \u003cem\u003eTP63\u003c/em\u003e,\u003cem\u003eMYH3\u003c/em\u003e, and \u003cem\u003eFGFR2\u003c/em\u003eacross diverse species demonstrated high evolutionary conservation of the wild-type amino acid residue at each position (Highlighted in red boxes). \u0026nbsp;\u003cstrong\u003e(F-J) \u003c/strong\u003eStructural modelling shows the tertiary protein structure and magnified views of amino acid substitutions, which include replacement of positively charged arginine (pink/yellow/green/white) with polar uncharged glutamine (brown) in \u003cem\u003eDLG1\u003c/em\u003e, bulky tryptophan (red) in \u003cem\u003eTP63\u003c/em\u003e, weakly basic histidine (red) in \u003cem\u003eMYH3\u003c/em\u003eand small polar cysteine (rose) in \u003cem\u003eTP63\u003c/em\u003e. In \u003cem\u003eFGFR2\u003c/em\u003e, a small, polar serine (green) is replaced with a bulky aromatic tryptophan (brown).\u003c/p\u003e","description":"","filename":"Binder22.png","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/7d97083e801ed2230d2fb2fe.png"},{"id":98200737,"identity":"12cb539d-14e2-4501-9aea-eaa44ad35f9b","added_by":"auto","created_at":"2025-12-15 07:49:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":364450,"visible":true,"origin":"","legend":"\u003cp\u003ePathway enrichment and Protein-Protein Interaction Network analysis. (\u003cstrong\u003eA\u003c/strong\u003e) Significantly enriched biological pathways, processes, and terms obtained from g:Profiler, ranked by –log10 adjusted p-values. (\u003cstrong\u003eB\u003c/strong\u003e) EnrichmentMap visualisation of the interconnected network of implicated biological pathways, processes and phenotypes. (\u003cstrong\u003eC\u003c/strong\u003e) PPI network constructed from prioritized genes using the STRING database (interaction score: 0.150). (\u003cstrong\u003eD\u003c/strong\u003e) Visual representation of hub genes within the PPI network.\u003c/p\u003e","description":"","filename":"Binder23.png","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/585c5f4f56fe640bfe9fdaef.png"},{"id":98200739,"identity":"2e344ef2-81eb-42d4-9a95-c3e9d5c27981","added_by":"auto","created_at":"2025-12-15 07:49:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":292749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatiotemporal gene expression analysis during mouse development.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eHeatmap displaying expression profiles of 9 genes (\u003cem\u003eAnkrd1\u003c/em\u003e, \u003cem\u003eDlg1\u003c/em\u003e, \u003cem\u003eFam170a\u003c/em\u003e, \u003cem\u003eFgfr2\u003c/em\u003e, \u003cem\u003eFoxd4\u003c/em\u003e, \u003cem\u003eMyh3\u003c/em\u003e, \u003cem\u003eNipbl\u003c/em\u003e, \u003cem\u003ePrdm9\u003c/em\u003e, \u003cem\u003eTrp63\u003c/em\u003e*) across multiple variables. The top panel shows metadata bars indicating structure types, developmental ages, developmental stages, strain information, and sex designation. Expression levels are gradient colour-coded based on transcripts per million (TPM) from white (0 TPM) through light blue (0.5 TPM) to progressively darker blues, with the darkest blue-black representing values \u0026gt;5000 TPM. Genes are displayed along the y-axis, while embryonic tissues, developmental stages and other variables are shown along the x-axis. Craniofacial tissues (e.g., frontonasal process, palatal shelf mesenchyme, facial prominence) and limb-associated tissues (e.g., limb bud, apical ectodermal ridge, limb mesenchyme, ectoderm, and bone) exhibit overlapping expression profiles across multiple genes. \u003cstrong\u003e(B)\u003c/strong\u003e Matrix visualisation of gene expression across anatomical structures for nine implicated genes (\u003cem\u003eAnkrd1\u003c/em\u003e, \u003cem\u003eDlg1\u003c/em\u003e, \u003cem\u003eFam170a\u003c/em\u003e, \u003cem\u003eFgfr2\u003c/em\u003e, \u003cem\u003eFoxd4\u003c/em\u003e, \u003cem\u003eMyh3\u003c/em\u003e, \u003cem\u003eNipbl\u003c/em\u003e, \u003cem\u003ePrdm9\u003c/em\u003e, \u003cem\u003eTrp63\u003c/em\u003e*). The y-axis lists anatomical structures, including branchial arch components, mesenchyme derivatives, limb structures, and neural crest-derived tissues. The x-axis represents individual genes. Expression data is colour-coded according to the number of expression results annotated: dark blue (present in \u0026gt;50 assays), blue (5–50 assays), and light blue (1–4 assays). Red triangles indicate structures where expression was reported as both present and absent across datasets, yellow triangles represent only absent or ambiguous results in substructures, and empty squares signify the absence of any expression annotations for the respective gene-tissue combination. It should be noted that the visualisation of expression data includes nine of the fourteen genes with variants listed in Table 1, corresponding to the genes for which expression annotations were available in the Mouse Genome Informatics (MGI) database. *\u003cem\u003eTP63\u003c/em\u003e is shown as \u003cem\u003e\u003cstrong\u003eTrp63\u003c/strong\u003e\u003c/em\u003e in expression analyses according to mouse ortholog nomenclature\u003c/p\u003e","description":"","filename":"Binder24.png","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/e983325f8e6c8aed67f1bec1.png"},{"id":100356238,"identity":"10737c42-73d8-4011-ac7a-02ba109f90dd","added_by":"auto","created_at":"2026-01-16 06:58:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3947462,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/3ede128d-d9fc-46ce-9c21-af87e65928e0.pdf"},{"id":98431022,"identity":"4ede27e1-75b2-4b48-957b-ebc625540401","added_by":"auto","created_at":"2025-12-17 16:46:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1 - Table S1:\u003c/strong\u003e Prediction tools, scoring ranges, and thresholds used for filtering out pathogenic variants.\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/4ba32cb077e3b028c5f49942.docx"},{"id":98431705,"identity":"0376021a-7be6-4729-9208-a215deb6d3c4","added_by":"auto","created_at":"2025-12-17 16:48:11","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2 - Table S2: \u003c/strong\u003ePrimer sequences used for variant validation.\u003c/p\u003e","description":"","filename":"AdditionalFile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/bef3ed16ea8d856138854fd0.docx"},{"id":98200740,"identity":"8b17027f-6262-4819-9408-151f59fd943e","added_by":"auto","created_at":"2025-12-15 07:49:39","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3 - Table S3: \u003c/strong\u003eIn-text Pathogenic Variants. This table provides supplementary details on the top candidate pathogenic variants identified during the analysis, including predictions from various tools not presented in the main results section.\u003c/p\u003e","description":"","filename":"AdditionalFile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/5bdfb6d642f9600cca506a85.docx"},{"id":98432910,"identity":"93c01dc1-1048-455e-9f20-6607e8cd83c4","added_by":"auto","created_at":"2025-12-17 16:50:06","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":157817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4 - Figure S1:\u003c/strong\u003e Family Pedigrees. (\u003cstrong\u003eA-I\u003c/strong\u003e) Pedigrees showing the inheritance patterns observed in the nine families.\u003c/p\u003e","description":"","filename":"AdditionalFile4.docx","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/1e39c85b75e65e67404d964d.docx"},{"id":98431778,"identity":"64c32a91-52e6-4420-85dd-85f724a97458","added_by":"auto","created_at":"2025-12-17 16:48:20","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":551845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 5 - Figure S2:\u003c/strong\u003eSanger sequencing chromatograms confirming \u003cem\u003eNIPBL\u003c/em\u003e and \u003cem\u003eMYH3\u003c/em\u003evariants. Chromatograms showing heterozygous variants identified by whole-exome sequencing and validated by Sanger sequencing. \u003cstrong\u003e(A)\u003c/strong\u003e NIPBL variant (c.7617_7618del, p.Ser2540ProfsTer21). \u003cstrong\u003e(B)\u003c/strong\u003e MYH3 variant (c.2015G\u0026gt;A, p.Arg672His). Arrows indicate the positions of the variants.\u003c/p\u003e","description":"","filename":"AdditionalFile5.docx","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/8cbde9d6d3b2f89dd42f91b9.docx"},{"id":98433131,"identity":"5049f905-4714-44c4-85ad-d899513a06bf","added_by":"auto","created_at":"2025-12-17 16:50:19","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1294197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 6 - Figure S3: \u003c/strong\u003eEvolutionary and structural\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003ein silico\u003c/em\u003e analysis of variants\u003cstrong\u003e. (A-D) \u003c/strong\u003eMultiple sequence alignment (MSA) of regions surrounding missense variant positions in \u003cem\u003eRGPD5\u003c/em\u003e, \u003cem\u003eFOXD4L\u003c/em\u003e, \u003cem\u003eFAM170A\u003c/em\u003e, \u003cem\u003eDLG1\u003c/em\u003e, \u003cem\u003eANKRD1\u003c/em\u003e, \u003cem\u003eFGFR2\u003c/em\u003e, and \u003cem\u003eTP63\u003c/em\u003eshows conservation across available species. For most genes, alignments included \u003cem\u003eHomo sapiens\u003c/em\u003e (wild-type), \u003cem\u003ePan troglodytes\u003c/em\u003e, \u003cem\u003eMus musculus\u003c/em\u003e, \u003cem\u003eRattus norvegicus\u003c/em\u003e, \u003cem\u003eCanis lupus familiaris\u003c/em\u003e, \u003cem\u003eBos taurus\u003c/em\u003e, \u003cem\u003eDanio rerio\u003c/em\u003e, and \u003cem\u003eMacaca fascicularis\u003c/em\u003e. \u003cstrong\u003e(E-H)\u003c/strong\u003e Corresponding molecular docking poses showing predicted structural alterations caused by amino acid substitutions.\u003cstrong\u003e Note:\u003c/strong\u003e The alignment for \u003cem\u003eRGPD5\u003c/em\u003e includes only \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003ePan troglodytes\u003c/em\u003e (Chimpanzee) as conservation beyond primates is limited. \u003cem\u003eFOXD4L\u003c/em\u003e includes only \u003cem\u003ePan troglodytes\u003c/em\u003e, \u003cem\u003eMacaca nemestrina\u003c/em\u003e, \u003cem\u003eXenopus tropicalis\u003c/em\u003e, and \u003cem\u003eXenopus laevis\u003c/em\u003e due to the absence of clear orthologs in other model organisms\u003c/p\u003e","description":"","filename":"AdditionalFile6.docx","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/c846151fe2d9533ab4456ac8.docx"},{"id":98200747,"identity":"81523aca-7831-4532-8427-93ae779ca326","added_by":"auto","created_at":"2025-12-15 07:49:40","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":284119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 7 - Figure S4: \u003c/strong\u003eHeatmap view showing the contribution of individual genes to enriched terms, with darker colours indicating stronger associations.\u003c/p\u003e","description":"","filename":"AdditionalFile7.docx","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/9cdc8242828d3f051e97bfca.docx"},{"id":98430904,"identity":"5b38dcb5-ef9f-483e-90f7-7f47710e979b","added_by":"auto","created_at":"2025-12-17 16:46:25","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":96469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 8 - Figure S5: \u003c/strong\u003eNetwork statistics for protein-protein interaction analysis of candidate genes.\u003c/p\u003e","description":"","filename":"AdditionalFile8.docx","url":"https://assets-eu.researchsquare.com/files/rs-8340088/v1/818a94f2d1ed48538b5a5563.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole Exome Sequencing Uncovers Genetic Syndromes Associated with Orofacial Clefts presenting with Limb abnormalities in a Sub-Saharan African cohort","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOrofacial clefts (OFCs) are the most frequent congenital craniofacial anomalies that occur during embryonic development and are mainly characterized by incomplete fusion of the palate, lip, or both. OFCs may manifest in various forms, including bilateral or unilateral, cleft palate (CP) or cleft lip with or without cleft palate, CL/P\u003csup\u003e1\u003c/sup\u003e. It has a global incidence of approximately 1 in 700 live births \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, the incidence varies significantly across populations, with African populations reporting the lowest incidence (~\u0026thinsp;1 in 2000 live births), Europeans reporting an intermediate incidence (~\u0026thinsp;1 in 1,000 live births), and Asian populations showing the highest rates, ~\u0026thinsp;1 in 500 live births\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In Ghana, a prevalence of 6.3 per 1000 individuals and a live birth incidence of 1.31 per 1000 have been reported in different communities\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA child with CL/P may encounter feeding difficulties, in addition to conductive hearing loss, speech impairments, malocclusion, and aesthetic problems. These physical anomalies can have a significant impact on the psychological and social well-being of the affected individual. Furthermore, the families of these individuals often struggle with societal stigmatization and substantial financial burdens, as the estimated mean lifetime treatment cost is approximately \u003cspan\u003e$\u003c/span\u003e92,000\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOFCs encompass a spectrum, ranging from nonsyndromic, constituting 70% of CL/P and 30% of CP cases, to syndromic clefts, which are associated with other structural anomalies. CP co-occurs in approximately 70% of infants diagnosed with unilateral CL and 85% of those diagnosed with bilateral CL\u003csup\u003e1,9\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSyndromic clefts are categorized according to their aetiology into monogenic syndromes, chromosomal syndromes, known teratogen exposures, and uncategorized syndromes that might not fit clearly into any well-defined category due to complex genetic interactions or unidentified aetiological factors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The genetic analysis of OFCs poses a significant challenge, as most cases do not strictly conform to the Mendelian inheritance pattern and often occur sporadically as non-familial cases\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. While the effect size and functionality of pathogenic variants identified through WES are larger compared to loci discovered via GWAS studies, the sporadic nature of most OFC cases continues to complicate genetic variant detection and characterization\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eLimb abnormalities encompass a broad range of congenital and postnatal acquired conditions that affect the structure, functionality, or development of the lower and upper extremities. They can be broadly classified into failure of formation of anatomical parts (e.g., amelia, phocomelia, intercalary defects, etc.), failure of differentiation of anatomical parts (e.g., syndactyly, clinodactyly), duplications (e.g., polydactyly), overgrowth and undergrowth (e.g., macrodactyly and brachydactyly), congenital constriction band syndrome, and generalized or systemic skeletal defects\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These anomalies may be sporadic and isolated, exhibiting a particular genetic inheritance pattern, or may be associated with specific syndromes. Disruptions during embryonic development, genetic mutations, and environmental factors (e.g., infection and dietary habits) can lead to the development of these anomalies and may impact one or multiple limbs\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLimb defects are the second most common congenital abnormalities in neonates, following congenital heart disease, affecting roughly 4.48 per 10,000 live births globally\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Syndactyly occurs in approximately 1 in 2,000 to 3,000 live births, whereas polydactyly has a higher incidence, ranging from 1 in 500 to 1,000 live births\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The pooled prevalence of congenital anomalies, including limb defects, is approximately 23.5 per 1000 live births in Africa\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Clubfoot (talipes equinovarus) is relatively common, with an estimated incidence of 1 per 1000 live births globally and a prevalence of ~\u0026thinsp;1.31 per 1,000 live births in the African population\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Within Ghana, the incidence of clubfoot has been documented at 25.4 per 100,000 live births in the northern population\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Syndromic limb malformations carry a substantial mortality risk, with reported survival rates as low as 4% when associated with other organ abnormalities\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In Northern Ghana, congenital malformations contribute to an overall neonatal mortality rate of 13.5%, with musculoskeletal system defects ranking as the third most prevalent\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eApproximately 10 to 30% of congenital limb defects are syndromic, and ~\u0026thinsp;13% are associated with multiple congenital anomalies (MCA), depending on the population\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Various exome sequencing and targeted gene studies have implicated several genes in congenital limb defects, including polydactyly (e.g., \u003cem\u003eGLI3\u003c/em\u003e, \u003cem\u003eTWIST1, HOXD13\u003c/em\u003e, etc.), syndactyly (\u003cem\u003eFGFR2\u003c/em\u003e, \u003cem\u003eGLI3\u003c/em\u003e, \u003cem\u003eHOXD13, GJA1\u003c/em\u003e, etc.), and ectrodactyly, e.g., \u003cem\u003eTP63\u003c/em\u003e\u003csup\u003e15,24\u003c/sup\u003e. Several syndromes exemplify the dual presentation of OFCs and limb defects with or without other anomalies. These include the \u003cem\u003eTP63\u003c/em\u003e-spectrum of disorders, Apert syndrome, Cornelia de Lange Syndrome (CdLS), and VACTERL/ VATER Association\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the genetic architecture of the co-occurrence of OFCs and limb defects, we conducted whole exome sequencing (WES) on nine Ghanaian families, comprising twenty-five individuals in total. This included ten affected individuals, comprising nine probands and one affected mother. Sanger sequencing was subsequently performed to validate the implicated variants. We examined whether the observed co-occurrence arises from monogenic pleiotropy, polygenic burden, or a combination thereof. Our goal was to enhance current understanding of the molecular architecture of syndromic OFCs presenting with limb defects and to elucidate the shared developmental pathways that link the face and limbs.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy population, participant recruitment, and ethical considerations\u003c/h2\u003e\n \u003cp\u003eThe study population consisted of 7 case-parent trios and 2 case-mother dyads, all recruited from the National Cleft Care Centre (NCCC) at Komfo Anokye Teaching Hospital (KATH), Kumasi, Ghana. Each family included a proband diagnosed with OFCs and associated limb malformations, along with one or both of their biological parents.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ewas obtained from the Institutional Review Board (IRB) at KATH (KATH-IRB/AP/032/20), Kumasi, Ghana. Written informed consent was obtained from participating families prior to the collection of data and samples. All participating families were of Ghanaian descent. Recruited probands presented with CL, CP, or CLP, along with digital anomalies and sometimes other anomalies.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSample collection and DNA extraction\u003c/h3\u003e\n\u003cp\u003eSaliva samples were collected using Oragene\u0026bull;DISCOVER saliva tool kits (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.dnagenotek.com\u003c/span\u003e\u003c/span\u003e). Participants who were old enough to spit provided their saliva samples directly, while for those who were not old enough to spit, cheek swab samples were collected.\u003c/p\u003e\n\u003cp\u003eGenomic DNA was isolated from the buccal swabs and saliva samples using the Oragene protocol at the Human Genetics and Genome (HuGENE) Laboratory at KNUST \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The quantity of DNA obtained for each sample was measured using the Qubit Assay (ThermoFisher Scientific, Hampton, USA). XY genotyping using real-time Polymerase Chain Reaction (PCR) was conducted as a quality control measure to verify the genetic sex of study participants \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The detailed protocol for DNA processing has been published by Gowans et al. (2016).\u003c/p\u003e\n\u003ch3\u003eWhole exome sequencing\u003c/h3\u003e\n\u003cp\u003eWhole exome sequencing (WES) was employed to identify genetic variants associated with syndromic OFCs in the recruited Ghanaian trios and dyads. Genomic DNA samples were shipped to commercial sequencing service providers (Azenta Life Sciences, LLC; South Plainfield, NJ, USA), where exome sequencing was conducted.\u003c/p\u003e\n\u003cp\u003eThe genomic DNA underwent fragmentation through acoustic shearing (Covaris S220 instrument). Exonic sequences, flanking intronic regions and UTRs were captured using the Twist Human Comprehensive Exome library preparation procedure (Twist Biosciences, South San Francisco, CA, USA). The fragmented DNAs were purified, end-repaired, adenylated at the 3\u0026rsquo; ends, and ligated to adapters. Limited-cycle PCR was used to amplify the adapter-ligated DNA fragments. Validation of the adapter-ligated DNA fragments was performed using Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantification was carried out using Qubit 4 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA). The adapter-ligated DNA fragments were subjected to hybridization with biotinylated baits. Streptavidin-coated binding beads were used to capture the resulting hybrid DNAs, after which they were thoroughly washed. The washed captured DNAs were subsequently amplified and indexed using the Illumina indexing primers. Post-captured DNA libraries underwent validation through Agilent TapeStation (Agilent, Santa Clara, CA, USA) and quantification via Qubit 4 Fluorometer and Real-Time PCR (KAPA Biosystems, Wilmington, MA, USA).\u003c/p\u003e\n\u003cp\u003eGenerated sequencing libraries were combined and clustered into multiple sections of a flow cell. Following this, the flow cell was inserted into the Illumina HiSeq instrument, where the samples underwent sequencing in a 2 x 150 bp paired-end (PE) configuration at 100X read depth according to the manufacturer\u0026rsquo;s instructions. Image analysis and base calling procedures were carried out using the HiSeq Control Software (HCS). The initial raw sequence data in binary base call (.bcl) format produced by the Illumina HiSeq system were transformed into fastq files and de-multiplexed using the Illumina bcl2fastq software. A single mismatch was permitted for the identification of index sequences.\u003c/p\u003e\n\u003ch3\u003eBioinformatics analysis for variant calling\u003c/h3\u003e\n\u003cp\u003eFollowing WES, raw FASTQ files (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) were processed using a standardized bioinformatics pipeline to align reads, call variants, and prioritize candidates. The raw reads generated from WES were quality checked utilising FastQC 0.11.9 and trimmed using Trimmomatic 0.39\u003csup\u003e29\u003c/sup\u003e to remove sequencing adapters and low-quality bases. The processed reads were aligned to the human reference genome (GRCh38), using the Sentieon 202112.01 workflow\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Subsequently, PCR/Optical duplicates were identified and marked, generating BAM files. Single-nucleotide variants (SNVs) and small insertions and deletions (INDELs) were called by employing Sentieon DNAscope algorithm. VCF files generated underwent normalization, including left alignment of INDELs and splitting multiallelic sites into distinct sites, using bcftools v1.13\u003csup\u003e31\u003c/sup\u003e. Subsequently, overlapping transcripts were identified for individual variants, and the potential effects of these variants on the transcripts were annotated utilising the Ensembl Variant Effect Predictor (VEP) v104 \u003csup\u003e32\u003c/sup\u003e. For downstream cohort analysis, the most severe impact for each variant was chosen.\u003c/p\u003e\n\u003ch3\u003eVariant Filtering and Prioritisation Process\u003c/h3\u003e\n\u003cp\u003eLikely pathogenic variants were prioritized (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) using the American College of Medical Genetics and Genomics (ACMG) guidelines on variant classification\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The variant filtering process was designed to prioritize high-confidence, functionally relevant genetic variants associated with the syndromic OFCs under study. Protein-altering variants, including missense, frameshift insertions/deletions, stop-gained, stop-loss, start-gained, start-loss, and splice region variants with MAF\u0026thinsp;\u0026lt;\u0026thinsp;0.01 were prioritized. MAF was ascertained using 1000 Genomes Project (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.internationalgenome.org/\u003c/span\u003e\u003c/span\u003e), Exome Sequencing Project (esp.gs.washington.edu/drupal/), and Gnome Aggregation database consortium, gnomAD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gnomad.broadinstitute.org/\u003c/span\u003e\u003c/span\u003e) v4.1. The pathogenicity of all missense variants was determined using both single and meta-prediction tools embedded in dbNSFP v4.9\u003csup\u003e34\u003c/sup\u003e. The tools included ClinPred, MetaRNN, BayesDel_addAF, REVEL, CADD, AlphaMissense, MutPred2, Polyphen-2, MutationAssessor, Mutation Taster, and SIFT (Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Missense variants predicted to be pathogenic by at least six out of eleven (i.e., the majority) of the prediction tools were filtered for. The pathogenicity of other variants, such as stop and start gain or loss, was predicted using CADD. Pathogenicity of splice region variants was also determined using SpliceAI embedded in Ensembl VEP. Pathogenic variants in genes associated with craniofacial and limb phenotypes, as well as their associated disorders in humans, were curated using GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003c/span\u003e), Malacards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.malacards.org/\u003c/span\u003e\u003c/span\u003e), OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003c/span\u003e), Genome alliances (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.alliancegenome.org/\u003c/span\u003e\u003c/span\u003e), Mouse Genome Informatics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.informatics.jax.org\u003c/span\u003e\u003c/span\u003e), and Facebase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.facebase.org/\u003c/span\u003e\u003c/span\u003e). Newly identified genes involved in biological processes relevant to the phenotypes under study, such as primary ciliary function, bone formation and development, cell adhesion, transcription regulation, cell migration, and proliferation, were curated using Genome Alliance, Mouse Genome Informatics, and Facebase. The co-segregation of candidate variants was assessed to confirm whether identified variants followed Mendelian inheritance patterns (e.g., autosomal dominant or recessive). This was done by verifying the presence of the variant in affected individuals and its absence in unaffected family members, where possible. Given the absence of family history in affected individuals in 8 out of the 9 families, candidate variants were prioritized based on those fitting a model of \u003cem\u003ede novo\u003c/em\u003e variants or autosomal dominant inheritance with incomplete penetrance. Variants that met the filtering criteria were considered the most likely to cause the observed phenotypes in affected individuals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eSanger sequencing\u003c/h2\u003e\n \u003cp\u003eAs a quality control step, two novel candidate variants in \u003cem\u003eMYH3\u003c/em\u003e (c.2015G\u0026thinsp;\u0026gt;\u0026thinsp;A, p.Arg672His) and \u003cem\u003eNIPBL\u003c/em\u003e (c.7617_7618del, p.Ser2540ProfsTer21) identified via WES were validated using Sanger sequencing. The exact procedure has been previously published\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In summary, primers (Supplementary Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e) were designed to flank each variant region using Primer3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://primer3.ut.ee/\u003c/span\u003e\u003c/span\u003e). The genomic sequences flanking the variant site were obtained from the UCSC Genome Browser (GRCh38/hg38; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu/\u003c/span\u003e\u003c/span\u003e). Each primer pair with closely matched Tm values (within 2\u0026deg;C) was prioritized and validated using BLAT function of UCSC Genome Browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu/cgi-bin/hgBlat\u003c/span\u003e\u003c/span\u003e) to confirm sequence specificity. I\u003cem\u003en-silico\u003c/em\u003e PCR analysis was also performed using the UCSC browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu/cgi-bin/hgPcr\u003c/span\u003e\u003c/span\u003e) to validate the specificity and production of a single amplicon (300 to 700bp) from the primers.\u003c/p\u003e\n \u003cp\u003ePCR was used to amplify genomic DNA (4 ng/\u0026micro;L) in 10 \u0026micro;L reaction volumes. Amplification success was verified using 2% agarose gel electrophoresis. Validated amplicons were subjected to sequencing at Functional Biosciences (Madison, WI, USA) using an ABI 3730XL DNA Sequencer. Generated chromatogram data were analysed using PHRED for base calling, PHRAP for assembly, POLYPHRED for variant detection, and CONSED for visualisation, as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eStructural and evolutionary analysis\u003c/h3\u003e\n\u003cp\u003eFor structural analysis of mutant proteins, the potential impact of missense variants on protein structure was evaluated using \u003cem\u003ein-silico\u003c/em\u003e modelling tools. The protein structures of candidate genes were downloaded from AlphaFold (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003c/span\u003e) and visualized using UCSF Chimera v1.14 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cgl.ucsf.edu/chimera/\u003c/span\u003e\u003c/span\u003e). The effect of the variant on the protein structures was evaluated using PyMOL v2.3.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pymol.org/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eEvolutionary conservation of identified variants (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) was assessed through multiple sequence alignment of vertebrate orthologs using MAFFT (Multiple Alignment using Fast Fourier Transform) accessed from EMBL-EBI with default parameters\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Species included in alignments are indicated in the respective figures (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e; Supplementary Figure \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e). Protein sequences were obtained from the NCBI RefSeq (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/refseq/\u003c/span\u003e\u003c/span\u003e) database and UniProt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePathogenic variants identified through whole exome sequencing in nine affected probands.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFamily ID\u003c/p\u003e\n \u003cp\u003e(Sex)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003cp\u003e(Inheritance)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenomic\u003c/p\u003e\n \u003cp\u003eCo-ordinates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenotype\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eZygosity\u003c/em\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHGVSc\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHGVSp\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of Tools Predicting Pathogenicity\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003eM(XY)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRGPD5\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De Novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr2:109837829 (rs1553471918)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.4708G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Gly1570Arg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eFAM90A26 (De Novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr4:9173181 (rs1450748908)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHomo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.10del\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Cys4ValfsTer12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003eM(XY)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eFOXD4L1\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De Novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr2:113499585 (rs201655302)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.329A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Tyr110Ser\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eFAM170A\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De Novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr5:119634539 (rs754719389)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.791T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Met264Thr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDLG1\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De Novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr3:197090942 (rs769502806)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.1730G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Arg577Gln\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eANKRD1\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De Novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr10:90917812 (rs773773073)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.472C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.His158Tyr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003eF(XX)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTP63\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr3:189868614 (rs886041251)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.1027C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Arg343Trp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003eF(XX)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNIPBL\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(Unknown)*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr5:37059093 (\u003cem\u003eNovel\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.7617_7618del\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Ser2540ProfsTer21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003eF(XX)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMYH3\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr17:10641317 (rs121913617)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.2015G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Arg672His\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFamily ID\u003c/p\u003e\n \u003cp\u003e(Sex)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Inheritance)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenomic\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCo-ordinates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenotype\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eZygosity\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHGVSc\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHGVSp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of Tools Predicting Pathogenicity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003eF(XX)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eFGFR2\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(Unknown)*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr10:121520163 (rs79184941)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.755C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Ser252Trp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003cp\u003eM(XY)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTRIM74\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De Novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr7:72961358 (rs199887265)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.487C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Arg163Ter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCADD score of 35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTRIM73\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De Novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr7:75403732 (rs199982097)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.487C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Arg163Ter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCADD score of 35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003eM(XY)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePRDM9\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(De Novo)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr5:23527360\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eNovel\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHomo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.2272_2273insTG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Arg758LeufsTer182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003eF(XX)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTP63\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eMaternal inheritance\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echr3:189867902 (rs1205536026)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHet\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ec.952C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep.Arg318Cys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e*Paternal samples of Families 4 and 6 were unavailable; however, the identified variants for these two families were absent in the mothers. \u003cem\u003eHet\u003c/em\u003e: heterozygous; \u003cem\u003eHomo\u003c/em\u003e: Homozygous.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003ePathway enrichment and interaction analysis\u003c/h3\u003e\n\u003cp\u003eCandidate genes obtained after prioritisation were analysed for biological process annotations. The g:Profiler tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biit.cs.ut.ee/gprofiler/gost\u003c/span\u003e\u003c/span\u003e), a web-based platform, was used to conduct an enrichment analysis of the identified genes against several databases, including Gene Ontology (GO) Biological Process (BP), Molecular Function (MF), and Cellular Component (CC), Reactome, WikiPathways, and Human Phenotype (HP) ontology. g:Profiler performs functional enrichment analysis of gene lists using Fisher\u0026rsquo;s exact test for over-representation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The analysis was restricted to \u003cem\u003eHomo sapiens\u003c/em\u003e (human) to ensure species-specific relevance with a significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All other parameters were defaulted. The output generated included enriched pathways, their adjusted \u003cem\u003eP\u003c/em\u003e-value, and the number of overlapping genes from the candidate list. The EnrichmentMap plugin in Cytoscape (v3.10.4)\u003csup\u003e36\u003c/sup\u003e, an open-source platform for visualising complex networks, was employed to cluster and visualize pathway enrichment results as an interactive network. This approach clusters functionally related pathways based on gene set overlap. By doing so, it reduces redundancy, reveals higher-order biological themes, and enables interpretation of enrichment results compared to isolated pathway lists.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eProtein-protein interaction network and hub genes identification\u003c/h2\u003e\n \u003cp\u003eA protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database v12.0 with an interacting confidence score of \u0026gt;\u0026thinsp;0.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://string-db.org\u003c/span\u003e\u003c/span\u003e). The resulting PPI network was exported to Cytoscape for visualisation and further analysis. The CytoHubba plugin, embedded in Cytoscape, was then applied with the maximal clique centrality (MCC) algorithm to identify hub genes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, as these highly interconnected nodes are often critical drivers of biological processes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eGene expression analysis using mouse models\u003c/h2\u003e\n \u003cp\u003eThe gene expression patterns of implicated candidate genes were investigated using the Mouse Genome Informatics (MGI) Gene Expression Database (GXD) v6.24\u003csup\u003e37\u003c/sup\u003e. To refine the search and ensure relevance to the syndromic phenotypes, the curated list of candidate genes was submitted to GXD via the Batch Search interface. A structured filtering approach was implemented to focus on developmentally relevant anatomical regions under the \u0026lsquo;Conceptus\u0026rsquo; category. The selected filters include the following terms: 1st branchial arch (mandibular and maxillary components), face mesenchyme, oral region mesenchyme, head surface ectoderm, latero-nasal process, nasal pit, mesenchyme derived from neural crest, and the limb. The \u0026lsquo;heart\u0026rsquo; filter was also applied, as two of the probands (Family 2 and Family 4) presented with congenital heart defects. Expression data were retrieved across developmental stages from the earliest post-implantation period through adult stages, with particular emphasis on critical periods of craniofacial (E8.5 to E14.5) and limb (E9.5 to E14.5) morphogenesis. Expression annotations were quantified and visualized via the tissue \u0026times; gene matrix, with colour intensity corresponding to the number of expression results per anatomical structure. A heatmap was generated using Morpheus\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, a web-based platform for matrix visualisation and analysis, with qualitative expression levels colour-coded to visualize spatiotemporal gene activity.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eClinical presentation of affected individuals\u003c/h2\u003e \u003cp\u003eEight simplex and one multiplex families (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), comprising two dyads and seven trios, with a history of syndromic OFCs presenting with limb abnormalities, were recruited for this study. WES datasets were generated from DNA obtained from case parent trios, except for Families 4 and 6, where the datasets were generated for case mother dyads due to the unavailability of the fathers.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFamily 1\u003c/strong\u003e \u003cp\u003eThe proband was a 6-year-old male who presented with a complete cleft palate, ulnar (postaxial) hexadactyly of both hands, mild intellectual disability, speech delay until age 4, mild microcephaly, characteristic facial features (almost V-shaped head with undulating surface), and cephalhematoma (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFamily 2\u003c/strong\u003e \u003cp\u003eThe proband was a 5-month-old male who presented with left complete CL, left unilateral talipes equinovarus, microcephaly, a low heart murmur that was suggestive of a heart defect, and large, low-set ears. A follow-up on this family revealed that the child had no neck control at 8 months, suggestive of global developmental delay (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFamily 3\u003c/strong\u003e \u003cp\u003eThe proband was a 7-day-old female who presented with bilateral incomplete CLP (right complete CLP plus left complete CP), bifid uvula, ectrodactyly of the right hand (missing/hypoplasia of the third digit with normal development of all other digits), and the fifth digit of the right hand was stiff and could not be bent. The thumb, index, and ring fingers of the right hand were also folded on each other. Clinical evaluation established a diagnosis of ectrodactyly ectodermal dysplasia cleft lip/palate (EEC) based on the presented phenotypes (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFamily 4\u003c/strong\u003e \u003cp\u003eThe proband was a 4-month-old female who presented with complete CP, syndactyly of the 2nd and 3rd toes of both feet, severe micrognathia, glossoptosis and breathing difficulty. The proband was re-examined after 8 months, at which point additional phenotypes were observed. She had a short stature, a small and upturned nose, developmental delay, a long philtrum, a thin and downturned upper lip, low-set ears, thick eyebrows, microcephaly, a short fifth finger, arched eyebrows that almost met in the middle, hirsutism, a feeding problem/failure to thrive, long eyelashes, and pulmonary stenosis. The proband was born prematurely during the 8th month of pregnancy with a low birth weight of 1.5 kg. Clinical evaluation established a diagnosis of Cornelia de Lange syndrome based on the presented phenotypes (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFamily 5\u003c/strong\u003e \u003cp\u003eThe proband was a 2-weeks-old female who presented with complete CP, bilateral clubfoot, camptodactyly, a characteristic face that causes her to appear to be whistling, microstomia (small puckered mouth), pursed lip, H-shaped scar-like mark extending from the lower lip towards the bottom of the chin, widely-spaced deep eyes, prominent cheeks, struggles to open the mouth, diminished ability to suck, flat philtrum, as well as high-arched and V-shaped palate. Clinical evaluation established a diagnosis of Distal arthrogryposis type 2A (DA2A), also known as Freeman-Sheldon Syndrome (FSS), Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFamily 6\u003c/strong\u003e \u003cp\u003eThe proband was a female who was 1 month 9 days old at the time of recruitment. She presented with incomplete CP, bulging eyes, and finger and toe syndactyly. These clinical presentations were suggestive of Apert syndrome. The proband was born to consanguineous parents (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFamily 7\u003c/strong\u003e \u003cp\u003eThe proband was a 1-week-old male who presented with right complete CLP, unilateral right clubfoot, syndactyly of the 3 middle left toes, left-hand digital hypoplasia with anonychia affecting all fingers except the thumb and right radial clubhand (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFamily 8\u003c/strong\u003e \u003cp\u003eThe proband was a 3-day-old male who presented with right complete CLP and hexadactyly, an extra thumb on the right hand (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFamily 9\u003c/strong\u003e \u003cp\u003eThe proband was a 2-week-old female. She presented with complete bilateral CLP, ectrodactyly, and ectodermal dysplasia that presented as anhidrosis with impaired thermoregulation and nail dystrophy characterized by thickened nails. These phenotypes were clinically suggestive of EEC syndrome. The proband was born to consanguineous parents. The mother presented with bilateral symbrachydactyly (4th finger), right-hand syndactyly (3\u0026ndash;4 fingers) and right foot ectrodactyly (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eI).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProbable pathogenic variants identified by whole exome sequencing\u003c/h2\u003e \u003cp\u003eWES analysis of probands with co-occurring OFCs and limb abnormalities unveiled several candidate variants in genes associated with certain developmental pathways and syndromes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). These include variants in genes such as \u003cem\u003eTP63, FGFR2, PRDM9, DLG1, NIPBL\u003c/em\u003e and \u003cem\u003eMYH3\u003c/em\u003e. Importantly, many of these variants were \u003cem\u003ede novo\u003c/em\u003e, some of which were novel, suggesting that these variants may be population-specific. As a quality control measure, the variants observed in \u003cem\u003eNIPBL\u003c/em\u003e and \u003cem\u003eMYH3\u003c/em\u003e were confirmed with Sanger sequencing (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStructural and evolutionary analysis of implicated variants\u003c/h2\u003e \u003cp\u003eThe structural and evolutionary analyses of identified missense variants (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) revealed molecular alterations that may underlie the observed phenotypes. The wild-type amino acids are located in highly conserved regions of proteins, with many being perfectly conserved from humans to distant vertebrates such as zebrafish. The structural analyses also revealed a consistent pattern of disruptive alterations across all variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). As a representative example, the R343W substitution in \u003cem\u003eTP63\u003c/em\u003e occurred in the 8th exon within the DNA-binding domain of the protein, replacing a positively charged arginine with nonpolar tryptophan containing a bulky indole side chain. This substitution eliminates electrostatic interactions between the TP63 protein and the negatively charged DNA phosphate backbone and can destabilize the protein due to unfavourable torsion angles. The \u003cem\u003ede novo\u003c/em\u003e R672H variant in \u003cem\u003eMYH3\u003c/em\u003e replaces the positive charge of arginine with a smaller, less charged histidine, potentially weakening ATP binding by reducing electrostatic interactions and decreasing hydrogen bonds critical for the stability of the binding pocket.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePathway enrichment analysis, protein-protein interaction network and hub genes identification\u003c/h2\u003e \u003cp\u003eFunctional enrichment analysis of genes harbouring candidate variants revealed the involvement in several key biological pathways in normal craniofacial and limb development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Supplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). A highly significant and coherent enrichment related to coordinated developmental processes, specifically those derived from epithelial-mesenchymal interactions, was observed in both Gene Ontology (GO) categories and Human Phenotype Ontology (HP) terms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). A profound statistically significant enrichment for developmental pathways directly related to processes governing limb formation and OFC pathogenesis were observed, including appendage morphogenesis (GO: 0035107, Padj\u0026thinsp;=\u0026thinsp;1.722\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e), limb morphogenesis (GO:0035108, Padj\u0026thinsp;=\u0026thinsp;1.722\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e), limb development (GO:0060173, Padj\u0026thinsp;=\u0026thinsp;4.208\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e), cranial skeletal system development (GO: 1904888, Padj\u0026thinsp;=\u0026thinsp;1.107\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), morphogenesis of an epithelial bud (GO:0060572, Padj\u0026thinsp;=\u0026thinsp;4.573\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) and embryonic morphogenesis (GO: 0048598, Padj\u0026thinsp;=\u0026thinsp;1.875\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). These pathways were complemented by human phenotype ontology (HP) terms, highlighting specific defects such as deviation of the thumb (HP:0009603, Padj\u0026thinsp;=\u0026thinsp;2.177\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) and cutaneous finger syndactyly (HP:0010554, Padj\u0026thinsp;=\u0026thinsp;2.302\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). A significant enrichment was observed for abnormalities in sensory organs that share developmental origins and pathways with the face and limbs, particularly those derived from cranial placodes and neural crest cells\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. These included lacrimal duct stenosis (HP:0007678, Padj\u0026thinsp;=\u0026thinsp;1.016\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e), conductive hearing impairment (HP:0000405, Padj\u0026thinsp;=\u0026thinsp;1.135x10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) and nasolacrimal duct obstruction (HP:0000579, Padj\u0026thinsp;=\u0026thinsp;1.168\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). This pattern of phenotypic enrichment corresponds to established clinical features observed in patients who present with both OFCs and limb malformations\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIdentification of central regulators (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D) within the implicated gene set (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) resulted in a PPI network comprising 13 nodes (proteins) and 5 edges (interactions), with an average node degree of 0.769 and a p-value of 0.26 (Supplementary Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). The PPI enrichment p-value indicates that the observed number of connections is not statistically significantly greater than what would be expected by chance for a random set of proteins of the same size and degree distribution from the genome. Given the exploratory nature of our study and to capture potential novel interactions within this specific genetic context, a confidence score threshold of 0.150 was employed. This threshold was selected after the default confidence filter (~\u0026thinsp;0.400) yielded only a single interaction, which was deemed insufficient for meaningful network analysis. Despite the lack of statistical enrichment, the analysis identified \u003cem\u003eRGPD5\u003c/em\u003e, \u003cem\u003eMYH3\u003c/em\u003e, \u003cem\u003eANKRD1\u003c/em\u003e, \u003cem\u003eDLG1\u003c/em\u003e, \u003cem\u003eFGFR2\u003c/em\u003e, and \u003cem\u003eTP63\u003c/em\u003e as the top hub genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSpatiotemporal Expression Profiles of Implicated Genes\u003c/h2\u003e \u003cp\u003eBased on analysis of the Morpheus heat map, nine of the thirteen candidate genes demonstrated distinctive expression patterns associated with specific morphogenetic processes across various mouse strains, developmental stages, and tissue structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The other four genes were not represented in the Mouse Genome Informatics (MGI) database utilized for the gene expression analysis. Some genes, including \u003cem\u003eDlg1\u003c/em\u003e, \u003cem\u003eNipbl\u003c/em\u003e, and \u003cem\u003eFgfr2\u003c/em\u003e, exhibited peak expression during early embryonic development, while \u003cem\u003eDlg1\u003c/em\u003e, \u003cem\u003eNipbl\u003c/em\u003e. \u003cem\u003ePrmd9\u003c/em\u003e and \u003cem\u003eTrp63\u003c/em\u003e maintained sustained expression throughout multiple developmental windows (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notable \u003cem\u003eDlg1\u003c/em\u003e, \u003cem\u003eFgfr2\u003c/em\u003e, and \u003cem\u003eTrp63\u003c/em\u003e expression was observed from Theiler stages (TS) 1 through to 23 in the branchial arches, including ectoderm and mesenchymal components, facial prominence mesenchyme, and palatal shelf, which are regions essential for lip and palate formation. Concurrently, \u003cem\u003eDlg1\u003c/em\u003e, \u003cem\u003eFgfr2\u003c/em\u003e, \u003cem\u003eMyh3\u003c/em\u003e, and \u003cem\u003eTrp63\u003c/em\u003e displayed an overlapping moderate to high expression from TS 15 to 28 in limb mesenchyme, limb ectoderm, and developing skeletal and muscle elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study sought to decipher the genetic aetiology of OFCs co-occurring with limb abnormalities, and in some cases, additional phenotypes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The co-occurrence of OFCs and limb deformities, though less frequent than isolated OFCs, has significant implications for genetic counselling and clinical management. This co-occurrence is often associated with specific genetic syndromes or different syndromes manifesting concurrently\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWES analysis identified multiple pathogenic variants (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in affected individuals, some of which have been previously reported and associated with known genetic syndromes. The proband in Family 1 carried two \u003cem\u003ede novo\u003c/em\u003e variants, one in \u003cem\u003eRGPD5\u003c/em\u003e and another in \u003cem\u003eFAM90A26\u003c/em\u003e. \u003cem\u003eRGPD5\u003c/em\u003e interacts with nuclear proteins, including Ras-related nuclear protein (RAN) and transportin-1\u003csup\u003e40\u003c/sup\u003e. A previous study reported \u003cem\u003eRGPD5\u003c/em\u003e deletion in an infant with multiple craniofacial and limb malformations, including unilateral complete cleft lip, microcephaly, craniosynostosis, syndactyly, dysmorphic ears, bilateral congenital talipes equinovarus, bilateral radial club hand, and ectrodactyly\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The proband in the current study presented with similar phenotypes. \u003cem\u003eFAM90A26\u003c/em\u003e is expressed in gonad primordial and testicular germline stem cells, with an overexpression of its protein product in the heart (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bgee.org/\u003c/span\u003e\u003cspan address=\"https://www.bgee.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFour \u003cem\u003ede novo\u003c/em\u003e variants were identified in the proband from Family 2 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), including variants in \u003cem\u003eFOXD4L1\u003c/em\u003e, \u003cem\u003eFAM170A\u003c/em\u003e, \u003cem\u003eDLG1\u003c/em\u003e, and \u003cem\u003eANKRD1\u003c/em\u003e. \u003cem\u003eFOXD4L1\u003c/em\u003e is a forkhead/winged-helix (FOX) transcriptional factor that is crucial for embryogenesis and regulates neural ectoderm by preserving neural precursor cell pluripotency and repressing transcription factors and other genes that drive neural differentiation. These are crucial for FGF and BMP signalling, regulating neural plate patterning and maintaining neural fate\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In the expression analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), \u003cem\u003eFoxd4\u003c/em\u003e exhibited an early-onset expression pattern, with craniofacial-specific expression beginning at E7.5 and limited expression in some skeletal elements (femur diaphysis, femur metaphysis, and tibia) between postnatal week 5 to 70. \u003cem\u003eFAM170A\u003c/em\u003e has been linked with VATER/VACTERL Association\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, which is defined by the systematic concurrence of vertebral defects (V), anal atresia (A), cardiac malformations (C), tracheoesophageal fistula with or without oesophageal atresia (TE), renal abnormalities (R), and limb anomalies (L)\u003csup\u003e26\u003c/sup\u003e. The proband in the current study presented with CL, talipes equinovarus, global developmental delay, a heart defect, and other abnormalities, which are characteristic of this syndrome. \u003cem\u003eDLG1\u003c/em\u003e, expressed in both mesenchymal and epithelial cells, plays critical roles in palatal morphogenesis, limb elongation, and spatial patterning. Pathogenic variants in \u003cem\u003eDLG1\u003c/em\u003e are associated with CLP and disorganized chondrocytes in the sternum, particularly in the proliferative zone \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Among the implicated genes for this family, \u003cem\u003eDlg1\u003c/em\u003e exhibited the second most comprehensive temporal and spatial expression profile, starting at E0.5 and persisting postnatally, with prominent signals in craniofacial tissues (first branchial arch, facial prominences, and palatal shelves) from E8.5 and in limb structures from E10.5. We hypothesize that \u003cem\u003eDLG1\u003c/em\u003e, a well-established gene associated with clefting and limb defects, contributes to the severe limb and craniofacial phenotypes exhibited by the proband. The \u003cem\u003eANKRD1\u003c/em\u003e gene product, cardiac ankyrin repeat protein (CARP), functions as a transcriptional repressor and a sarcomeric component of the titin-binding complex \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Pathogenic variants in \u003cem\u003eANKRD1\u003c/em\u003e have been implicated in cardiomyopathies and septal defects \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The proband in the current study also presented with a heart defect characterized by low murmurs, which may result from the pathogenic variant identified in \u003cem\u003eANKRD1\u003c/em\u003e. \u003cem\u003eAnkrd1\u003c/em\u003e is expressed in the heart, starting at E7.5 (TS 11), which coincides with cardiogenic plate formation, as well as craniofacial and limb tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The phenotypic spectrum in the proband suggests a polygenic aetiology, where these four \u003cem\u003ede novo\u003c/em\u003e variants likely work synergistically to produce the observed craniofacial, limb, and cardiac defects.\u003c/p\u003e \u003cp\u003eTwo heterozygous missense variants in \u003cem\u003eTP63\u003c/em\u003e were identified in individuals from two unrelated families, Family 3 and Family 9, who presented with bilateral cleft lip and palate (CLP), ectrodactyly, and other orofacial and limb malformations. \u003cem\u003eTP63\u003c/em\u003e functions as a key regulator of ectodermal, limb and craniofacial morphogenesis \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Heterozygous variants in \u003cem\u003eTP63\u003c/em\u003e account for multiple autosomal dominant disorders defined by three key phenotypes: limb defects, ectodermal dysplasia, and facial clefting. At least eight different \u003cem\u003eTP63\u003c/em\u003e-related syndromes with overlapping phenotypes have been reported \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eTrp63\u003c/em\u003e exhibits a broad epithelial-enriched expression profile from early embryogenesis to adulthood, with high expression evident in the limb ectoderm and facial prominence ectoderm during their respective windows (E9.5 to E14.5) of outgrowth and fusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The first variant (c.1027C\u0026thinsp;\u0026gt;\u0026thinsp;T, p.Arg343Trp) was identified as a \u003cem\u003ede novo\u003c/em\u003e mutation. A \u003cem\u003ede novo TP63\u003c/em\u003e variant may represent spontaneous mutagenesis or suggest the presence of undetected germline mosaicism in a phenotypically normal parent \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The second variant (c.952C\u0026thinsp;\u0026gt;\u0026thinsp;T, p.Arg318Cys) was inherited from an affected mother. The mother from this consanguineous family (Family 9) presented with bilateral symbrachydactyly, right 3\u0026ndash;4 fingers syndactyly, and right foot ectrodactyly, but no orofacial cleft. Both variants observed in \u003cem\u003eTP63\u003c/em\u003e, occurring within the DNA-binding domain (DBD), are clinically associated with ectrodactyly, ectodermal dysplasia, and cleft lip-palate syndrome 3, EEC3 \u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA novel heterozygous frameshift variant in \u003cem\u003eNIPBL\u003c/em\u003e was identified in the proband of Family 4, who was clinically diagnosed with CdLs. Pathogenic variants in \u003cem\u003eNIPBL\u003c/em\u003e cause CdLS, a complex developmental disorder and the most frequently occurring cohesinopathy. Affected individuals present with impaired growth, abnormal limb development, and developmental impairments \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Our spatiotemporal analysis revealed broad early-onset expression of \u003cem\u003eNipbl\u003c/em\u003e, with craniofacial and limb expression observed from E8.5 and E10.5, respectively. Cardiac expression began from E9.5, with presence in the primitive heart tube, atria, and ventricles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This aligns with CdLS clinical phenotypes, where congenital heart defects frequently co-occur with craniofacial and limb malformations \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The observed frameshift variant in the current proband may result in a truncated protein, potentially leading to haploinsufficiency of \u003cem\u003eNIPBL\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA \u003cem\u003ede novo\u003c/em\u003e heterozygous missense variant in \u003cem\u003eMYH3\u003c/em\u003e was identified in the proband of Family 5, who presented features descriptive of FSS. Heterozygous variants in \u003cem\u003eMYH3\u003c/em\u003e cause Sheldon-Hall syndrome (SHS) or FSS, characterized by distinctive facial features, hand and foot contractures, camptodactyly, oropharyngeal defects, and scoliosis \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The identified variant (c.2015G\u0026thinsp;\u0026gt;\u0026thinsp;A, p.Arg672His) has been previously reported as a recurrent mutational hotspot \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Expression analysis revealed early \u003cem\u003eMyh3\u003c/em\u003e activity localized predominantly to muscle-related structures, including craniofacial muscle compartments and limb muscles. Beyond muscular tissues, \u003cem\u003eMyh3\u003c/em\u003e was also detected in skeletal elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA heterozygous missense variant in \u003cem\u003eFGFR2\u003c/em\u003e was identified in the proband from Family 6. The identified variant (c.755C\u0026thinsp;\u0026gt;\u0026thinsp;G, p.Ser252Trp) has been associated with Apert syndrome (AS) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e and it is detected in ~\u0026thinsp;59% of all AS cases, with a strong association with CP \u003csup\u003e54\u003c/sup\u003e. \u003cem\u003eFgfr2\u003c/em\u003e was detected in the developing branchial arch mesenchyme and limb bud from their earliest formation stages (E8.5 and E10.5, respectively), with expression persistence throughout major morphogenetic periods and limited expression in the left ventricle of the heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDe novo\u003c/em\u003e heterozygous stop-gained variants in \u003cem\u003eTRIM73\u003c/em\u003e and \u003cem\u003eTRIM74\u003c/em\u003e were observed in the proband from Family 7. These genes have been associated with Williams-Beuren syndrome (WBS), which results from abnormal homologous recombination and unequal crossing-over between tandem segments containing \u003cem\u003eTRIM50\u003c/em\u003e, \u003cem\u003eTRIM73\u003c/em\u003e, and \u003cem\u003eTRIM74\u003c/em\u003e \u003csup\u003e55\u003c/sup\u003e. The TRIM proteins function as E3 ubiquitin ligases, conferring substrate specificity to ubiquitin-proteasome complexes and thereby affecting nearly all cellular processes \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eTRIM73\u003c/em\u003e and \u003cem\u003eTRIM74\u003c/em\u003e share a high degree of similarity (~\u0026thinsp;99.8%), indicating redundant paralogs with overlapping functions in developmental processes, potentially providing functional compensation through genetic redundancy. WBS is defined by unique dysmorphic craniofacial features, supravalvular aortic stenosis (SVAS), hypertension, depressed nasal bridge, intellectual disability, premature ageing of the skin, broad forehead, infantile hypercalcemia, and tooth defects \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Interestingly, the phenotypes of this syndrome were palpable in the current proband, who presented with right complete CLP, right clubfoot, left toes 2\u0026ndash;4 syndactyly, left-hand digital hypoplasia with anonychia affecting all fingers except the thumb, and right radial clubhand. We hypothesize that the involvement of TRIM proteins in cellular processes, such as apoptosis and cell proliferation \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, may underlie the observed phenotypes. However, further functional genomics experiments are warranted to confirm these observations.\u003c/p\u003e \u003cp\u003eWe also identified a novel \u003cem\u003ede novo PRDM9\u003c/em\u003e frameshift variant (c.2272_2273insTG, p.Arg758LeufsTer182) in the proband from Family 8. \u003cem\u003ePRDM9\u003c/em\u003e is specifically expressed in germ cells during meiosis, where its methyltransferase activity increases H3K4me3 at recombination hotspots \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, thereby acting as a crucial determinant of meiotic recombination in both murine and human models \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePRDM9\u003c/em\u003e has been implicated in Smith-Magenis Syndrome (SMS), an autosomal disorder characterized by OFCs, depressed nasal bridge, hand polydactyly, toe syndactyly, clinodactyly of the 5th finger, micrognathia, etc \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The current proband presented with complete CLP and hand hexadactyly, characteristic of SMS. Population-specific recombination patterns associated with \u003cem\u003ePRDM9\u003c/em\u003e alleles may contribute to disease-causing genomic rearrangements, particularly in individuals of West African ancestry \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In our spatiotemporal analysis, \u003cem\u003ePrdm9\u003c/em\u003e showed expression in craniofacial tissues (branchial arch, facial prominence, head mesenchyme, head surface ectoderm) from E8.5 and limb structures from E10.5 through to adulthood (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting a role for this gene in craniofacial development.\u003c/p\u003e \u003cp\u003eA consistent pattern of disruptive molecular alteration and loss of conserved amino acid residue was observed for all studied variants in the structural and evolutionary analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Multiple sequence alignment showed that all the wild-type amino acids subject to mutation were highly conserved across vertebrate species. The identified substitutions consistently resulted in detrimental structural changes, including loss of critical electrostatic interactions, introduction of steric clashes, disruption of hydrogen bonding networks, and alteration of binding pocket geometries.\u003c/p\u003e \u003cp\u003ePathway enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) revealed that the implicated genes are significantly involved in appendage morphogenesis, cranial skeletal system development, and embryonic morphogenesis pathways in craniofacial and limb development. Human phenotype ontology (HP) terms also revealed enrichment for phenotypes such as nasolacrimal duct obstruction, cutaneous finger syndactyly, and deviation of the thumb, corresponding to the established clinical features observed in patients who present with both OFCs and limb malformations \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe EnrichmentMap analysis generated a network of 46 nodes and 140 edges, organized into clusters and individual nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The first cluster centred on epithelial and glandular development, indicating that disrupted epithelial-mesenchymal interactions underlie palatal, limb, and glandular malformations (Doshi and Patil, 2012). The second cluster focused on craniofacial development and urogenital morphogenesis. The third cluster was dominated by appendage and limb morphogenesis, co-enriched with abnormalities of the external genitalia, which indicate shared developmental pathways.\u003c/p\u003e \u003cp\u003eThe protein-protein interaction network (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) revealed limited connectivity among the candidate genes, with only 5 interactions identified among the 13 proteins (Supplementary Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Despite the sparse network structure (PPI enrichment p-value\u0026thinsp;=\u0026thinsp;0.26), \u003cem\u003eRGPD5\u003c/em\u003e, \u003cem\u003eMYH3\u003c/em\u003e, \u003cem\u003eANKRD1\u003c/em\u003e, \u003cem\u003eDLG1\u003c/em\u003e, \u003cem\u003eNIPBL\u003c/em\u003e, \u003cem\u003eFGFR2\u003c/em\u003e, and \u003cem\u003eTP63\u003c/em\u003e emerged as key hub genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) with established roles in pathways fundamental to embryogenesis, specifically craniofacial and limb morphogenesis.\u003c/p\u003e \u003cp\u003eThe extrapolability of the findings of the current study is limited by its small sample size. Again, our inability to obtain paternal samples in two families hindered our ability to determine whether certain variants are \u003cem\u003ede novo\u003c/em\u003e. Lastly, the study focused on the analysis of WES datasets to decipher single nucleotide variants (SNVs) and indels in the aetiology of the conditions under study. As a result, potentially pathogenic non-coding and structural variants could have been missed.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, WES analysis of nine Ghanaian families presenting with OFCs and limb abnormalities, and in some instances other abnormalities, identified probable pathogenic genetic variants, including novel and population-specific mutations in various genes, confirming the genetic heterogeneity of OFCs co-occurring with limb malformations. These variants support a model in which both pleiotropic, monogenic, and polygenic mechanisms contribute to phenotypic variations. Future studies incorporating larger, more diverse cohorts and functional validation approaches will help expand variant discovery, clarify biological mechanisms, and improve genetic counselling and clinical management of syndromic OFCs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eANKRD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/em\u003eAnkyrin Repeat Domain\u003c/p\u003e\n\u003cp\u003eBMP Bone morphogenic protein\u003c/p\u003e\n\u003cp\u003eCdLS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cornelia de Lange syndrome\u003c/p\u003e\n\u003cp\u003eCL\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cleft lip\u003c/p\u003e\n\u003cp\u003eCL/P\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cleft lip with or without cleft palate\u003c/p\u003e\n\u003cp\u003eCLP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cleft lip and palate\u003c/p\u003e\n\u003cp\u003eCP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cleft palate\u003c/p\u003e\n\u003cp\u003eDA2A\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Distal arthrogryposis type 2A\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDLG1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003eDiscs Large MAGUK Scaffold Protein 1\u003c/p\u003e\n\u003cp\u003eDNA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003eEEC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ectrodactyly ectodermal dysplasia cleft lip/palate\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFAM170A\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003eFamily with Sequence Similarity 170 Member A\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFAM90A26\u003c/em\u003e Family with Sequence Similarity 90 Member A26\u003c/p\u003e\n\u003cp\u003eFGF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Family with Sequence Similarity 90 Member A26\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFOXD4L1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003eForkhead Box D4 Like 1\u003c/p\u003e\n\u003cp\u003eFSS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Freeman-Sheldon syndrome\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGLI3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003eGLI Family Zinc Finger 3\u003c/p\u003e\n\u003cp\u003eGWAS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Genome-wide association studies\u003c/p\u003e\n\u003cp\u003eHuGENE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Human Genetics and Genome\u003c/p\u003e\n\u003cp\u003eKATH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Komfo Anokye Teaching Hospital\u003c/p\u003e\n\u003cp\u003eKNUST\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Kwame Nkrumah University of Science and Technology\u003c/p\u003e\n\u003cp\u003eMAGUKs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Membrane-associated guanylate kinase homologs\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMYH3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003eMyosin Heavy Chain 3\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNIPBL\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/em\u003eNipped-B-like protein\u003c/p\u003e\n\u003cp\u003eOFCs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Orofacial clefts\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePRDM9\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/em\u003ePR domain-containing 9\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRGPD5\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/em\u003eRANBP2-Like and GRIP Domain Containing 5\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSHS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003eSheldon-Hall Syndrome\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTP63\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/em\u003eTumor protein p63\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTRIM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/em\u003eTripartite motif\u003c/p\u003e\n\u003cp\u003eWES\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Whole exome sequencing\u003c/p\u003e\n\u003cp\u003eWGS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Whole genome sequencing\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board (IRB) at KATH (KATH-IRB/AP/032/20), Kumasi, Ghana. Written informed consent was obtained from all participating families.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe whole exome sequencing (WES) dataset reported in this article can be accessed through FaceBase Consortium (https://www.facebase.org) under controlled access, with an accession number 94-D420\u0026nbsp;(Gowans LJJ, 2025). The informed consent obtained from participants only permits sharing the WES dataset under controlled access. The data and materials that support the findings of this study are available from the corresponding author upon reasonable request. The data and materials that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by the IADR/Smile Train Cleft Research Award, 2023 (LJJG), K43DE029427 by NIDCR/FIC/NIH, USA (LJJG).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL. J. J. \u0026nbsp;Gowans, contributed to conception, design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript; E. Tackie, contributed to design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript; A. Butali, P. Donkor, G. O. Mensah, B. Tsri, S. Obiri-Yeboah, T. D. Busch, A. A. Adeyemo, contributed to design, data acquisition, analysis and interpretation, and critically revised the manuscript; C. O. Asamoah, D. K. Sabbah, G. Plange-Rhule, A. A. Oti, contributed to data acquisition, analysis and interpretation, and critically revised the manuscript. All authors gave their final approval and agreed to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank the study participants for their participation. We also thank all members of the Cleft-Craniofacial Team at the National Cleft Care Center (NCCC), Komfo Anokye Teaching Hospital (KATH), Kumasi, Ghana, for supporting the recruitment of study participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMerritt L. Part 1. Understanding the embryology and genetics of cleft lip and palate. 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Nat Rev Genet. 2018;19(10):649\u0026ndash;66.\u003c/li\u003e\n \u003cli\u003eGowans LJJ, Adeyemo WL, Eshete M, Mossey PA, Busch T, Aregbesola B, et al. Association studies and direct DNA sequencing implicate genetic susceptibility loci in the etiology of nonsyndromic orofacial clefts in sub-Saharan African populations. J Dent Res. 2016;95(11):1245\u0026ndash;56.\u003c/li\u003e\n \u003cli\u003eBolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics [Internet]. 2014 Aug 1;30(15):2114\u0026ndash;20.\u003c/li\u003e\n \u003cli\u003eFreed D, Aldana R, Weber JA, Edwards JS. The Sentieon Genomics Tools - A fast and accurate solution to variant calling from next-generation sequence data. bioRxiv. 2017 Jan 1;115717.\u003c/li\u003e\n \u003cli\u003eDanecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. Twelve years of SAMtools and BCFtools. Gigascience [Internet]. 2021 Feb 1;10(2):giab008.\u003c/li\u003e\n \u003cli\u003eMcLaren W, Gil L, Hunt SE, Riat HS, Ritchie GRS, Thormann A, et al. The Ensembl Variant Effect Predictor. Genome Biol [Internet]. 2016;17(1):122.\u003c/li\u003e\n \u003cli\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine. 2015;17(5):405\u0026ndash;24.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLiu X, Li C, Mou C, Dong Y, Tu Y. dbNSFP v4: a comprehensive database of transcript-specific functional predictions \u0026nbsp;and annotations for human nonsynonymous and splice-site SNVs. Genome Med. 2020 Dec;12(1):103.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMadeira F, Madhusoodanan N, Lee J, Eusebi A, Niewielska A, Tivey ARN, et al. The EMBL-EBI Job Dispatcher sequence analysis tools framework in 2024. Nucleic Acids Res. 2024 Jul;52(W1):W521\u0026mdash;W525.\u003c/li\u003e\n \u003cli\u003eReimand J, Isserlin R, Voisin V, Kucera M, Tannus-Lopes C, Rostamianfar A, et al. Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap. Nat Protoc. 2019;14(2):482\u0026ndash;517.\u003c/li\u003e\n \u003cli\u003eRingwald M, Richardson JE, Baldarelli RM, Blake JA, Kadin JA, Smith C, et al. Mouse Genome Informatics (MGI): latest news from MGD and GXD. Mamm Genome. 2022 Mar;33(1):4\u0026ndash;18.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLaMantia AS. Why Does the Face Predict the Brain? Neural Crest Induction, Craniofacial Morphogenesis, and Neural Circuit Development. Front Physiol. 2020; 11:2020.\u003c/li\u003e\n \u003cli\u003eSoğukpınar M, Utine GE, Boduroğlu K, Şimşek-Kiper P\u0026Ouml;. A spectrum of TP63-related disorders with eight affected individuals in five unrelated families. Eur J Med Genet. 2024; 68:104911.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCai Y, Gao Y, Sheng Q, Miao S, Cui X, Wang L, et al. Characterization and potential function of a novel testis-specific nucleoporin \u0026nbsp;BS-63. Mol Reprod Dev. 2002 Jan;61(1):126\u0026ndash;34.\u003c/li\u003e\n \u003cli\u003eJaiswal SK, Kumar A, Ali A, Rai AK. Co-occurrence of mosaic supernumerary isochromosome 18p and intermittent 2q13 deletions in a child with multiple congenital anomalies. Gene. 2015;559(1):94\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eYan B, Neilson KM, Moody SA. Microarray identification of novel downstream targets of FoxD4L1/D5, a critical component of the neural ectodermal transcriptional network. Developmental Dynamics. 2010;239(12):3467\u0026ndash;80.\u003c/li\u003e\n \u003cli\u003eStelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M, Fishilevich S, et al. The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses. Curr Protoc Bioinformatics. 2016 Jun;54:1.30.1-1.30.33.\u003c/li\u003e\n \u003cli\u003eMostowska A, Gaczkowska A, Żukowski K, Ludwig KU, Hozyasz KK, W\u0026oacute;jcicki P, et al. Common variants in DLG1 locus are associated with non-syndromic cleft lip with or without cleft palate. Clin Genet. 2018;93(4):784\u0026ndash;93.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eIizuka-Kogo A, Senda T, Akiyama T, Shimomura A, Nomura R, Hasegawa Y, et al. Requirement of DLG1 for cardiovascular development and tissue elongation during cochlear, enteric, and skeletal development: Possible role in convergent extension. PLoS One. 2015;10(4):1\u0026ndash;19.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDuboscq-Bidot L, Charron P, Ruppert V, Fauchier L, Richter A, Tavazzi L, et al. Mutations in the ANKRD1 gene encoding CARP are responsible for human dilated cardiomyopathy. Eur Heart J. 2009;30(17):2128\u0026ndash;36.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhong L, Chiusa M, Cadar AG, Lin A, Samaras S, Davidson JM, et al. Targeted inhibition of ANKRD1 disrupts sarcomeric ERK-GATA4 signal transduction and abrogates phenylephrine-induced cardiomyocyte hypertrophy. Cardiovasc Res. 2015 May;106(2):261\u0026ndash;71.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRinne T, Brunner HG, Van Bokhoven H. P63-Associated Disorders. Cell Cycle. 2007;6(3):262\u0026ndash;8.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMills JA, Herrera PS, Kaur M, Leo L, McEldrew D, Tintos-Hernandez JA, et al. NIPBL(+/-) haploinsufficiency reveals a constellation of transcriptome \u0026nbsp;disruptions in the pluripotent and cardiac states. Sci Rep. 2018 Jan;8(1):1056.\u003c/li\u003e\n \u003cli\u003eMei L, Liang D, Huang Y, Pan Q, Wu L. Two novel NIPBL gene mutations in Chinese patients with Cornelia de Lange syndrome. Gene [Internet]. 2015;555(2):476\u0026ndash;80.\u003c/li\u003e\n \u003cli\u003eKimber E, Tajsharghi H, Kroksmark A karin, Oldfors A, Tulinius M. Distal arthrogryposis : clinical and genetic findings. 2012;877\u0026ndash;87.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eToydemir RM, Rutherford A, Whitby FG, Jorde LB, Carey JC, Bamshad MJ. Mutations in embryonic myosin heavy chain ( MYH3 ) cause Freeman-Sheldon syndrome and Sheldon-Hall syndrome. 2006;38(5):561\u0026ndash;5.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eIbarra-Arce A, Almaraz-Salinas M, Mart\u0026iacute;nez-Rosas V, Ortiz de Z\u0026aacute;rate-Alarc\u0026oacute;n G, Flores-Pe\u0026ntilde;a L, Romero-Valdovinos M, et al. Clinical study and some molecular features of Mexican patients with syndromic \u0026nbsp;craniosynostosis. Mol Genet Genomic Med. 2020 Aug;8(8):e1266.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eReynolds K, Zhang S, Sun B, Garland MA. Genetics and signaling mechanisms of orofacial clefts. 2020;(March):1588\u0026ndash;634.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHan K, Lou DI, Sawyer SL. Identification of a Genomic Reservoir for New TRIM Genes in Primate Genomes. 2011;7(12).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGushchina L V, Kwiatkowski TA, Bhattacharya S, Weisleder NL. Conserved structural and functional aspects of the tripartite motif gene family point towards therapeutic applications in multiple diseases\u0026nbsp;☆. Pharmacol Ther. 2018;185(October 2017):12\u0026ndash;25.\u003c/li\u003e\n \u003cli\u003eDentici ML, Bergonzini P, Scibelli F, Caciolo C, Rose P De, Cumbo F, et al. brain sciences Clinical and Neurobehavioral Profiling. 2020;(Mim 194050):1\u0026ndash;19.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePober BR. Williams-Beuren syndrome. N Engl J Med. 2010 Jan;362(3):239\u0026ndash;52.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWatanabe M, Hatakeyama S. JB Special Review \u0026mdash; Recent Topics in Ubiquitin-Proteasome System and Autophagy TRIM proteins and diseases. 2017;161(2):135\u0026ndash;44.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGrey C, Barth\u0026egrave;s P, Chauveau-Le Friec G, Langa F, Baudat F, de Massy B. Mouse PRDM9 DNA-Binding Specificity Determines Sites of Histone H3 Lysine 4 Trimethylation for Initiation of Meiotic Recombination. PLoS Biol [Internet]. 2011 Oct 18;9(10):e1001176.\u003c/li\u003e\n \u003cli\u003eBaudat F, Buard J, Grey C, Fledel-Alon A, Ober C, Przeworski M, et al. PRDM9 Is a Major Determinant of Meiotic Recombination Hotspots in Humans and Mice. Science. 2010 Feb 12;327(5967):836\u0026ndash;40.\u003c/li\u003e\n \u003cli\u003eHinch AG, Tandon A, Patterson N, Song Y, Rohland N, Palmer CD, et al. The landscape of recombination in African Americans. Nature. 2011;476(7359):170\u0026ndash;5.\u003c/li\u003e\n \u003cli\u003eDoshi RR, Patil AS. A role of genes in craniofacial growth. IIOAB J. 2012;3(2):19\u0026ndash;36.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Orofacial clefts, limb abnormalities, genetic syndromes, whole exome sequencing, pleiotropy, polygenic, Sub-Saharan Africa","lastPublishedDoi":"10.21203/rs.3.rs-8340088/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8340088/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eOrofacial clefts (OFCs) are the most frequent congenital craniofacial anomalies that occur during embryonic development. The incidence is ~1 in 700 live births; it may occur in isolation or with other abnormalities, such as limb deformities. Congenital limb malformations are the second most prevalent birth defect, affecting 1 per 500 to 1000 live births. It can also occur in isolation or as part of a syndrome. This study investigated the genetic aetiology of OFCs co-occurring with limb abnormalities in a Sub-Saharan African cohort.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eNine unrelated probands with concurrent OFC and limb anomalies were recruited, including one multiplex family involving an affected mother and proband. Whole exome sequencing (WES) was performed at 100X on the DNA samples obtained from affected families, utilising paired-end configuration on the Illumina HiSeq platform. Variant calling utilized the Sentieon workflow. Rare, deleterious variants were identified in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines on variant classification. \u003cem\u003eDe novo \u003c/em\u003eand other variants predicted as pathogenic were prioritized based on all possible Mendelian inheritance patterns, including variable penetrance and expressivity. Pathway enrichment analysis, protein-protein interactions, and gene expression analysis were undertaken to decipher the biological functions of implicated genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAll cases were syndromic, presenting with preaxial and postaxial limb anomalies along with other craniofacial features. WES revealed plausible pathogenic variants in pleiotropic genes (\u003cem\u003eTP63\u003c/em\u003e, \u003cem\u003eNIPBL\u003c/em\u003e, \u003cem\u003eMYH3\u003c/em\u003e, \u003cem\u003eFGFR2\u003c/em\u003e) in four simplex cases. In four other simplex probands, multiple rare variants were identified in developmentally relevant genes (e.g., \u003cem\u003eRGPD5, FAM90A26, FOXD4L1, FAM170A, DLG1, ANKRD1, TRIM74, TRIM73, PRDM9\u003c/em\u003e) necessary for normal craniofacial and limb development. The multiplex family had two affected individuals (the mother and the proband), both carrying a \u003cem\u003eTP63 \u003c/em\u003evariant, consistent with autosomal dominant inheritance with variable expressivity. Most of the observed variants were \u003cem\u003ede novo\u003c/em\u003e, with some being novel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eWhile some cases can be attributed to single-gene syndromes (e.g., NIPBL-associated Cornelia de Lange Syndrome), others may result from multiple co-occurring syndromes. These findings will inform recurrence risk estimates, genetic counselling, and clinical management.\u003c/p\u003e","manuscriptTitle":"Whole Exome Sequencing Uncovers Genetic Syndromes Associated with Orofacial Clefts presenting with Limb abnormalities in a Sub-Saharan African cohort","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 07:49:34","doi":"10.21203/rs.3.rs-8340088/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-05T13:25:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-22T13:21:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-19T06:39:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-18T13:46:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-17T13:22:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331216771155156744219503086584691823428","date":"2026-01-15T20:27:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112034619674997101370926358893028667778","date":"2026-01-12T12:14:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288312317879032710627232705668155611392","date":"2026-01-11T11:48:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-10T17:19:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266061005035317507103727917578166338686","date":"2026-01-09T11:16:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330985108618856929964335127319524767785","date":"2026-01-08T07:24:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137401511071421803017739912290033644416","date":"2026-01-08T03:15:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149401222211831530800475082347210072686","date":"2026-01-07T08:11:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-07T07:27:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-12T12:58:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-12T02:49:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-12T02:48:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Medical Genomics","date":"2025-12-11T21:15:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6574f2a9-5206-4d36-8fc5-b31b0efc2889","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T09:10:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 07:49:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8340088","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8340088","identity":"rs-8340088","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}