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

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

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

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

Investigating the relationship between cancer and orofacial clefts using GWAS significant loci for cancers: A case-control and case-triad study.

Background: Several population-based case-control studies have reported concurrent presentation of cancer and congenital malformations. Many associations have been made between oral clefting and cancers, though some of these results are conflicting. Some studies have reported an increased risk of cancer among 1st-degree relatives of cleft cases and vice versa, and also an excess risk of cancers of the breast, lung, and brain among those with oral clefts. This study aimed to determine if the genetic polymorphisms found in some cancers are also associated with orofacial cleft in an African cohort. Methods: The study was a case-control and case-triad study in which cases were 400 individuals clinically diagnosed with non-syndromic cleft lip and/or palate (CL/P), while controls were 450 individuals without CL/P. Samples were obtained from three African countries while DNA extraction, PCR, and genotyping were carried out at the University of Iowa, US. Eleven SNPs in genes coding for SWI/SNF subunits and 13 GWAS significant SNPs for cancers associated with orofacial cleft were selected. Case-control analysis, transmission disequilibrium test (TDT), and DFAM to combine the parent-offspring trio data and unrelated case/control data in a single analysis were carried out using PLINK. Results: For the case-control analyses that included all the clefts and for the CLP subtype, none of the SNPs were statistically significant. Statistically increased risk for the following SNPs rs34775372 (p = 0.02; OR = 1.54, CI:1.07-2.22), rs55658222 (p = 0.009; OR = 2.64, CI:1.28-5.45) and rs72728755 (p = 0.02; OR=2.27, CI:1.17-4.45) was observed with the CL only sub-group. None of these were significant after Bonferoni correction. In the TDT analyses, a significantly reduced risk with rs10941679 (p = 0.003; OR = 0.43, CI:0.24-0.75) was observed and this was significant after Bonferroni correction. The rs10941679 was also significant (p = 0.003) in the DFAM analyses as well even after Bonferroni correction. Conclusion: The results from this study represent an important starting point for understanding the concurrent presentation of some cancers in orofacial clefts, and cancer risks in cleft patients. The associations observed warrant further investigation in a larger cohort and will set the stage for a more mechanistic approach toward understanding the risk for cancers in families with clefts.

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

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

Comparing the Treatment Outcomes of Absorbable Sutures, Nonabsorbable Sutures, and Tissue Adhesives in Cleft Lip Repair: A Systematic Review.

OBJECTIVES: To examine the literature and synthesize the available reports for the best possible option between absorbable, nonabsorbable, and tissue adhesives in cleft lip skin closure. DESIGN: We conducted systematic searches for randomized controlled trials and controlled clinical trials in PubMed, Cochrane, Ovid Medline, and OpenGrey databases. Identified studies were retrieved and assessed for eligibility. All statistical analyses were done with Revman, version 5.4. INTERVENTIONS: The intervention considered in this systematic review were techniques of cleft lip repair using resorbable sutures, nonabsorbable sutures, medical adhesives, or any combination of these. OUTCOME MEASURES: The primary outcomes assessed in the trials had to include any combination of the following: wound healing cosmesis and wound healing complications. While secondary outcomes considered were quality of life, direct and indirect costs to patients and health services, and participant satisfaction. RESULTS: Only 6 studies met all inclusion criteria and were selected for qualitative analysis. A more favorable wound healing cosmesis was seen when nonabsorbable suture was used in cleft lip repair compared to absorbable sutures and tissue adhesives (CI, 0.65-4.35). This advantage was overshadowed by the significantly higher prevalence of postoperative complications when nonabsorbable sutures are used. CONCLUSION: Although the results point to more favorable cosmesis with nonabsorbable sutures and an overall more favorable outcome with either absorbable sutures or tissue adhesives, the 6 selected studies were assessed at an unclear risk of bias; therefore, the results of this study should be interpreted with caution and regarded as low-certainty evidence.

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

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

Facial Anthropometry Measurements Using Three-Dimensional Stereophotogrammetry Analysis Among Nigerians.

ABSTRACT: This study aimed to determine the normative facial anthropometry measurement among Nigerians using three-dimensional stereophotogrammetry analysis.This study was carried out in Lagos, Nigeria over a period of 3 years. The sample population was Nigerians of diverse ethnic groups, age 16 and above with no history of congenital or acquired craniofacial deformities.A total of 452 subjects participated in the study with 56.2% males and 43.8% females. Most of the participants were between the ages of 25 to 49 (54.4%), 40.7% were less than 25 years of age and only 4.4% were more than 50 years old. The mean body mass index (BMI) for males was 22.7 and 23.4 for females. Mean values of upper facial height, midfacial height, lower facial height, intercanthal distance, interpupillary distance, upper facial width, and lower facial width are 69.13 ±â€Š5.91, 49.89 ±â€Š3.56, 67.85 ±â€Š6.12, 35.19 ±â€Š3.20, 67.04 ±â€Š3.67, 139.43 ±â€Š7.11, and 124.29 ±â€Š9.72 mm, respectively. The upper facial height, commissure width, upper lip length, and lower jaw width were significantly affected by age, while the BMI of an individual was a determinant of the interpupillary distance, facial width, and lower jaw width.This study demonstrated that there was a statistically significant difference in the facial dimensions of males when compared to females across all ages among the study population. The authors also observed that age and BMI are significant predictors of variations in some of the measurements.

Congenital heart defects in orofacial cleft: A prospective cohort study.

BACKGROUND: Congenital heart defects (CHDs) are one of the most common associated anomalies in patients with an orofacial cleft (OFC). However, few studies have shown the association between cleft type and CHDs in our population. This study aimed to assess the prevalence of CHDs in a cohort of OFC patients at a tertiary health facility in Nigeria, as well as assess the risk of CHD by OFC type. MATERIALS AND METHODS: This was a prospective study design. Patients with an OFC were consecutively enrolled at a single OFC treatment facility. All subjects were assessed by a paediatric cardiologist and had echocardiography done. They were categorised based on the presence of CHDs, as well as the OFC phenotypic type (cleft lip and/or alveolus, cleft lip and palate and cleft palate only). Statistical analysis was done using STATA version 14 (College Station, Texas), and significance was set at P < 0.05. RESULTS: A total of 150 subjects enrolled in the study over a period of 2 years (2018-2020). The median age of subjects was 6 months (interquartile range: 2-24), and 54.7% were female. The prevalence of CHDs in the subjects reviewed was 30.7%. Based on the severity of CHDs, the majority presented with simple defects (95.6%). Overall, the most common presentation was patent foramen ovale (12.7%), followed by septal defects (8.0%). There was no significant association between cleft type and the odds of a CHD. CONCLUSION: The study reports a relatively high prevalence of CHDs in patients with OFC; however, there was no association between the risk of CHD by cleft type. Although a majority of CHDs may pose a low operative risk, cardiac evaluation is recommended for all cases of OFC to aid the identification of potentially high-risk cases.

Missense Variants Within GJB2 Gene Locus and the Risk of Hearing Defects in Nonsyndromic Cleft Lip and Palate.

AIM: The aim of the study was to investigate the role of variants in GJB2 gene in the etiology of hearing defects in nonsyndromic cleft lip/palate. METHOD: Saliva samples were obtained from cases (subjects with orofacial clefts) and control (subjects without orofacial clefts) who consented to the study. Deoxyribonucleic acid (DNA) was extracted using standardized protocol at Butali Lab (Iowa, IA). Primers for the coding region of GJB2 was designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) and optimized in the Butali lab using a gradient polymerase chain reaction to determine the annealing temperature for each primer set (forward and reverse). We measured the DNA concentration using Qubit and XY genotyping done for quality control. A concentration of 5 ng/µL of DNA was used for Sanger sequencing. RESULTS: A total of 150 subjects were sequenced (66 cases; 84 controls). Mutations in GJB2 gene were detected in 2 individuals with cleft palate. We found p.Arg165Trp variant in 1 case and p.Leu81Val variant in the second case. Although p.Arg165Trp was predicted to be either benign or tolerated by SIFT/POLYPHEN, the single nucleotide change from C>T, that is, CGG>TGG leads to a premature stop codon preventing the protein formation. The p.Leu81Val variant was predicted to be probably damaging/ deleterious. CONCLUSIONS: The present study implicates variants in the GJB2 gene in the etiology of hearing defects in nonsyndromic cleft lip and palate in the Nigerian population. Screening for variations in GJB2 gene is important for genetic counseling especially in high-risk families.

Correlation Between Height and Impacted Third Molars and Genetics Role in Third Molar Impaction.

AIM: This study sought to evaluate the relationship between height of an individual and the presence of impaction of maxillary and mandibular third molars, and to determine the role of genetics in third molar impaction. MATERIALS AND METHODS: This was a case-control study, with cases consisted of 200 subjects with third molar impactions; and 200 controls without third molar impactions. Height of subjects was measured, and saliva samples were collected from all the subjects. DNA was extracted from saliva samples. To investigate the role of selected genes in the etiology of third molar impactions, Taqman Genotyping using SNPs identified for jaw growth, height and tooth agenesis was employed. Five candidate genes were investigated using 11 markers (SNPs). RESULTS: The mean height of cases was significantly lower than that of the control subjects (p = 0.04). No difference was found in allele frequency between cases and controls for 10 of the 11 SNPs. However, for rs6504591 the p value was near significance (p = 0.07) with odd ratio of 2.131. Subjects with lower third molar impactions were significantly shorter than those who have fully erupted third molars. CONCLUSIONS: Subjects with lower third molar impactions were significantly shorter than those who have fully erupted third molars. We observed that individuals with third molar impaction tend to have T allele at the locus, suggesting that the T allele at the locus may increase the risk for having an impacted third molar. The rs6504591 G/T variation on human chromosome 17 (WNT9B gene) appears to increase risk by twofolds for impaction albeit with inability to detect significance due to small sample size.

Breastfeeding Practices Among Mothers of Children With Orofacial Clefts in an African Cohort.

OBJECTIVE: To carry out a survey of breastfeeding practices and related challenges among mothers with orofacial cleft babies attending the cleft clinic of a tertiary health institution. METHODOLOGY: This was a cross-sectional descriptive study using an interviewer-administered questionnaire. Sample population was all mothers of babies aged between 1 and 18 months with nonsyndromic orofacial clefts attending the cleft clinic of a tertiary health institution in Nigeria. RESULT: A total of 65 mothers participated in the study. Initiation of breastfeeding was reported by the majority (83%, n = 54) of the mothers, and only 18.5% (n = 10) of this proportion continued exclusive breastfeeding. Inability of the babies to suck was reported by 46% (n = 30) of the mothers as being the most important challenge in breastfeeding. There was a significant correlation between the type of cleft and challenge in breastfeeding (Fisher exact P = .001). Sixty-three percent (n = 41) of the mothers reported they received no counseling on overcoming challenges associated with feeding their babies with a cleft at the facility where they delivered. Sixty-nine percent (n = 45) reported they first received nutritional information from the cleft clinic at presentation. The most adopted substitute for breastfeeding was the use of regular feeding bottles (n = 24, 43.6%). CONCLUSION: Rate of initiation of breastfeeding for children with orofacial clefts in this African cohort is higher than reported in other populations despite the low level of nutritional counseling of the mothers after delivery.

Management of Orofacial Cleft in Nigeria - A Retrospective Study.

INTRODUCTION: Orofacial clefts (OFCs) are among the most common craniofacial developmental abnormalities worldwide and a significant cause of childhood morbidity and mortality. This study aimed to identify patterns of patient presentation, treatment approaches, and changes in our overall cleft care service between 2007 and 2019. METHODS AND METHODOLOGY: A retrospective review of patients managed at a tertiary health facility in Nigeria of all OFC cases operated between 2007 and 2019 was done using the postintervention data retrieved from the Smile Train database. Data of all OFC cases operated within the period were analyzed using the Statistical Package for the Social Sciences. Descriptive statistics were performed using the Statistical Package for the Social Sciences version 20.0. RESULTS: A total number of 740 OFC surgeries were performed in 565 patients, consisting of 269 females (48.2%) and 289 males (51.8%). The majority (63%) of the patients presented before the age of 2 years. Thirty-seven percent presented with cleft lip and alveolus, 27.1% with cleft palate only, and 36.7% with cleft lip, alveolus, and palate. Primary cleft lip repair was the most performed surgery (n = 320, 43.2%), the mean age at repair was 2.1 years. Since 2017, additional services such as speech therapy, mixed dentition orthodontics, and nutritional support were added to services provided to our cleft patients. Fifteen patients have undergone speech assessment and three have completed speech treatment. Eight patients have undergone mixed dentition stage orthodontic treatment. DISCUSSION: Our services have evolved from simply providing surgical care to comprehensive care with a multidisciplinary team approach and provision of a wide range of services including nutritional counseling, pediatric care, orthodontic services, and speech therapy. We believe these will improve the overall well-being of our patients while we continue to improve on services based on clinical research outcomes.

Comparison of sensitivity of bacteria isolated in odontogenic infections to ceftriaxone and amoxicillin-clavulanate.

BACKGROUND: Odontogenic infections is a cause of mortality and morbidity in maxillofacial patients. This is largely due to resistance of organisms to antibiotics prescribed. OBJECTIVES: To isolate organisms involved in odontogenic infections and compare the sensitivity of the organisms to Ceftriaxone and Amoxicillin-Clavulanate. METHODS: The causative organisms and antibiotic sensitivity were determined by the following steps: Aspiration of pus done with needle, sample of pus or exudate collected using sterile swab if aspiration was unsuccessful and specimen were placed in transport media (thioglycolatebroth) and sent immediately to microbiology laboratory for culture of organisms and antibiotic sensitivity. RESULTS: Out of a total 55 samples taken for bacteriology, 42 (76.4%) yielded positive culture for bacteria. A total number of 21 bacteria species were identified from the positive cultures. Overall, 52% of isolated organisms were sensitive to amoxicillin-clavulanate, 70% were sensitive to Ceftriaxone while 24% were resistant to both antibiotics (Table 3). Ceftriaxone was statistically significantly more potent in inhibiting bacteria growth than amoxicillin-clavulanate (P =0.009). [Table: see text].