Genetic Risk Assessment of Nonsyndromic Cleft Lip with or without Cleft Palate by Linking Genetic Networks and Deep Learning Models.

Recent deep learning algorithms have further improved risk classification capabilities. However, an appropriate feature selection method is required to overcome dimensionality issues in population-based genetic studies. In this Korean case-control study of nonsyndromic cleft lip with or without cleft palate (NSCL/P), we compared the predictive performance of models that were developed by using the genetic-algorithm-optimized neural networks ensemble (GANNE) technique with those models that were generated by eight conventional risk classification methods, including polygenic risk score (PRS), random forest (RF), support vector machine (SVM), extreme gradient boosting (XGBoost), and deep-learning-based artificial neural network (ANN). GANNE, which is capable of automatic input SNP selection, exhibited the highest predictive power, especially in the 10-SNP model (AUC of 88.2%), thus improving the AUC by 23% and 17% compared to PRS and ANN, respectively. Genes mapped with input SNPs that were selected by using a genetic algorithm (GA) were functionally validated for risks of developing NSCL/P in gene ontology and protein-protein interaction (PPI) network analyses. The IRF6 gene, which is most frequently selected via GA, was also a major hub gene in the PPI network. Genes such as RUNX2, MTHFR, PVRL1, TGFB3, and TBX22 significantly contributed to predicting NSCL/P risk. GANNE is an efficient disease risk classification method using a minimum optimal set of SNPs; however, further validation studies are needed to ensure the clinical utility of the model for predicting NSCL/P risk.

Syndromic Oral Clefts: Challenges of Genetic Assessment in Brazil and Suggestions to Improve Health Policies.

Addressing the unmet health needs of persons living with congenital anomalies in low- and middle-income countries (LMIC) is a major challenge. Registries and databases are exemplary tools capable to link research data with health programs. Since 2009, Brazil's Craniofacial Project, a multicenter and voluntary research initiative, collects socioeconomic, medical, and genetic information on individuals with craniofacial anomalies through the Brazilian Database on Craniofacial Anomalies (BDCA). This article discusses challenges to the provision of genetic assessment and counselling for individuals with syndromic oral clefts (SOC) through public health services in LMIC, such as Brazil. Subjects were selected using methods of the BDCA as described elsewhere. Among 800 records, 66 assigned as SOC with no etiologic diagnosis were preselected for genomic imbalance screening. Only 28 have timely completed basic protocol using public health services, and 22 were able to perform chromosomal microarray analysis. Pathogenic genomic imbalances were identified in 4 (18.18%) and a copy number variation of uncertain clinical significance was detected in one. Results exemplify barriers faced by the majority of the population of Brazil to reach whole genetic assessment either through public genetic services or in research settings. In this unfavorable scenario, BDCA has allowed the recognition of individuals with similar needs, optimizing the scarce genetic laboratory facilities in Brazil. Ultimately, BDCA has facilitated the translation of research into care. This experience may be successfully extended to other congenital anomalies and to LMIC with similar characteristics. A set of suggestions focusing on oral clefts is provided.

Machine Learning Models for Genetic Risk Assessment of Infants with Non-syndromic Orofacial Cleft.

The isolated type of orofacial cleft, termed non-syndromic cleft lip with or without cleft palate (NSCL/P), is the second most common birth defect in China, with Asians having the highest incidence in the world. NSCL/P involves multiple genes and complex interactions between genetic and environmental factors, imposing difficulty for the genetic assessment of the unborn fetus carrying multiple NSCL/P-susceptible variants. Although genome-wide association studies (GWAS) have uncovered dozens of single nucleotide polymorphism (SNP) loci in different ethnic populations, the genetic diagnostic effectiveness of these SNPs requires further experimental validation in Chinese populations before a diagnostic panel or a predictive model covering multiple SNPs can be built. In this study, we collected blood samples from control and NSCL/P infants in Han and Uyghur Chinese populations to validate the diagnostic effectiveness of 43 candidate SNPs previously detected using GWAS. We then built predictive models with the validated SNPs using different machine learning algorithms and evaluated their prediction performance. Our results showed that logistic regression had the best performance for risk assessment according to the area under curve. Notably, defective variants in MTHFR and RBP4, two genes involved in folic acid and vitamin A biosynthesis, were found to have high contributions to NSCL/P incidence based on feature importance evaluation with logistic regression. This is consistent with the notion that folic acid and vitamin A are both essential nutritional supplements for pregnant women to reduce the risk of conceiving an NSCL/P baby. Moreover, we observed a lower predictive power in Uyghur than in Han cases, likely due to differences in genetic background between these two ethnic populations. Thus, our study highlights the urgency to generate the HapMap for Uyghur population and perform resequencing-based screening of Uyghur-specific NSCL/P markers.

Infrared meibography and molecular assessment of p63 gene mutations in a Mexican patient with EEC syndrome. / Meibografía infrarroja y evaluación molecular de la mutación del gen p63 en un paciente mexicano con síndrome EEC.

OBJECTIVE: To report the finding of infrared meibography in a Mexican patient with EEC syndrome (Ectrodactyly-ectodermal dysplasia-cleft syndrome) confirmed by molecular analysis of the p63 gene. CLINICAL CASE: A 31 year-old male patient was seen due to a history of progressive visual loss in both eyes associated with long-term photophobia. The patient was born with cleft lip and palate, ectrodactyly of right hand, and afterwards, displayed nail dysplasia, anodontia and alopecia, with which ectodermal dysplasia was diagnosed. The ophthalmological findings were limited to the adnexa and the ocular surface. In vivo infrared meibography showed total absence of Meibomian glands in the lower eyelids and severe deficiency in the upper eyelids. In addition, it was shown that the patient was a heterozygous carrier of a missense mutation R304W (C → T) in exon 8 of the p63 gene. DISCUSSION: The R304W mutation in the p63 gene region is definitely related to characteristics such as the absence of Meibomian glands.

Integrated assessment of differentially expressed plasma microRNAs in subtypes of nonsyndromic orofacial clefts.

BACKGROUND: Orofacial clefts include cleft lip only (CLO), cleft palate only (CPO), and cleft lip with palate (CLP). Previously, we reported the expression profile of plasma microRNAs in CLO, CPO, and CLP, respectively. However, the interaction of each subtype remains poorly investigated. METHODS: In this study, we integrated the expression profiles of plasma miRNAs in these 3 subtypes, and assessed the distinct and overlapping dysregulated miRNAs using Venn diagrams. Their respective target genes reported in the literature were further analyzed using pathway analysis. RESULTS AND CONCLUSION: The results showed that distinct or overlapping signaling pathways were involved in CLO, CPO, and CLP. The common key gene targets reflected functional relationships to the Wnt, Notch, TGF-beta, and Hedgehog signaling pathways. Further studies should examine the mechanism of the potential target genes, which may provide new avenues for future clinical prevention and therapy.

Dermatoglyphics and Cheiloscopy as Key Tools in Resolving the Genetic Correlation of Inheritance Patterns in Cleft Lip and Palate Patients: An Assessment of 160 Patients.

OBJECTIVE: The objective of this study was to correlate dermatoglyphics and cheiloscopy with genetic inheritance in cleft lip and cleft palate patients. DESIGN AND SETTING: This was a case-control study to look for asymmetry in finger and lip print patterns. All of the participants were divided into two equal groups (40 mothers and 40 fathers in each group). The data were analyzed by three evaluators who were blind to the study to avoid any chances of error. PATIENTS/PARTICIPANTS: A sample of 160 sporadic participants were identified and evaluated. Group A was composed of 80 healthy parents not affected by cleft lip and cleft palate but had at least one child born with nonsyndromic cleft. Group B consisted of 80 healthy parents not affected by cleft lip and cleft palate and had healthy children without cleft lip and cleft palate. MAIN OUTCOME MEASURES: Main outcome measures were marked dermatoglyphic asymmetry and specific lip print pattern in the study group. RESULTS: We found marked asymmetry in various fingerprints and specific type II and type III lip print in the study group when compared with the control group. It was observed that groove count on the lip was significantly more frequent in study group parents. CONCLUSION: Our study determined that there is a significant correlation between increased dermatoglyphic asymmetry and specific type II and type III lip print pattern in parents of children born with cleft. This could act as an important screening marker for the prediction of cleft lip and cleft palate inheritance.

Assessment of differentially expressed plasma microRNAs in nonsyndromic cleft palate and nonsyndromic cleft lip with cleft palate.

Plasma microRNAs (miRNAs) have recently emerged as a new class of regulatory molecules that influence many biological functions. However, the expression profile of plasma microRNAs in nonsyndromic cleft palate (NSCP) or nonsyndromic cleft lip with cleft palate (NSCLP) remains poorly investigated. In this study, we used Agilent human miRNA microarray chips to monitor miRNA levels in three NSCP plasma samples (mixed as the CP group), three NSCLP plasma samples (mixed as the CLP group) and three normal plasma samples (mixed as the Control group). Six selected plasma miRNAs were validated in samples from an additional 16 CP, 33 CLP and 8 healthy children using qRT-PCR. Using Venn diagrams, distinct and overlapping dysregulated miRNAs were identified. Their respective target genes were further assessed using gene ontology and pathway analysis. The results show that distinct or overlapping biological processes and signalling pathways were involved in CP and CLP. Our study showed that the common key gene targets reflected functional relationships to the Notch, Wnt, phosphatidylinositol and Hedgehog signalling pathways. Further studies should examine the mechanism of the potential target genes, which may provide new avenues for future clinical prevention and therapy.

A genome-wide study of inherited deletions identified two regions associated with nonsyndromic isolated oral clefts.

BACKGROUND: DNA copy number variants play an important part in the development of common birth defects such as oral clefts. Individual patients with multiple birth defects (including oral clefts) have been shown to carry small and large chromosomal deletions. METHODS: We investigated the role of polymorphic copy number deletions by comparing transmission rates of deletions from parents to offspring in case-parent trios of European ancestry ascertained through a cleft proband with trios ascertained through a normal offspring. DNA copy numbers in trios were called using the joint hidden Markov model in the freely available PennCNV software. All statistical analyses were performed using Bioconductor tools in the open source environment R. RESULTS: We identified a 67 kb region in the gene MGAM on chromosome 7q34, and a 206 kb region overlapping genes ADAM3A and ADAM5 on chromosome 8p11, where deletions are more frequently transmitted to cleft offspring than control offspring. CONCLUSIONS: These genes or nearby regulatory elements may be involved in the etiology of oral clefts.

Local strategies to address health needs of individuals with orofacial clefts in alagoas, Brazil.

Objective : To describe demographic and clinical-genetic characteristics of patients from a poor area of Brazil and to share experience on how the local genetic unit has addressed their major health needs. Design : Descriptive cohort. Setting : A clinical-genetic unit, a cytogenetics unit, and a regional cleft team located in the northeast and southeast of Brazil. Participants : A total of 133 individuals with orofacial clefts who attended the surgical call of a nongovernmental organization. From this group, 125, 77, and 13 patients completed phases 1, 2, and 3, respectively. Methods : Phase 1 comprised a description of demographic characteristics recorded through interviews. Phase 2 included a clinical-genetic evaluation using a pretested form, as well as cytogenetic analyses of selected patients. Phase 3 comprised collaborative action to address major health needs of patients without primary surgery. The Fisher test was used for statistics with p value < .05. Results : A majority of patients were rural residents with isolated cleft lip with cleft palate. Ages ranged between 0 and 30 years. Fifty percent had never undergone surgery; whereas, 100% had never attended a genetic evaluation. Isolated cleft was diagnosed in 77.9%, syndromes in 14.3%, and multiple congenital abnormalities in 7.8%. Positive familial history of clefts occurred in 28%; whereas, parental consanguinity was present in 7.8% cases. A total of 23 individuals without cleft surgery were registered for multidisciplinary treatment. Conclusions : Findings revealed high levels of unmet medical needs and provided an evidence base for health care planning. Collaborative action was crucial and might be applied to other regions in Brazil.

Assessment of the posteroanterior cephalograms of the parents of children with cleft lip and/or cleft palate in Latvia.

OBJECTIVE: To compare the craniofacial morphology of parents of children with cleft lip with or without palate (CL±P), children with isolate cleft palate (CP) and individuals without family history of orofacial clefting in Latvia. MATERIALS AND METHODS: Posteroanterior (PA) cephalograms were obtained from all participants: 37 couples of noncleft biological parents of children with nonsyndromic CL±P and 17 couples of noncleft biological parents of children with nonsyndromic CP (the parents groups were made dividing the parents after gender and children cleft type). The control groups consisted of 40 females and 42 males, who had no history of clefts in the family. A conventional cephalometric analysis was used to measure various measurements of facial widths. RESULTS: Statistically significant differences (decreased facial and biorbital width) were found between fathers of children with CP and males from the control group. Results showed asymmetry of zygomatic width (left side dominance) in all parents groups compared with the control groups. The asymmetry was detected in maxillary part (left side dominance) in CP children mothers and females and males control groups. CONCLUSION: Some statistical significant differences in the PA cephalometric measurements among parents groups of children with CL±P and CP, and control groups were found. However the differences among study groups and the control groups were small, often not larger than variations in the population.

Family history and socioeconomic risk factors for non-syndromic cleft lip and palate: a matched case-control study in a less developed country.

INTRODUCTION: From an epidemiological point of view, non-syndromic orofacial clefts are the most common oral congenital deformities worldwide. OBJECTIVE: Family histories were traced and socioeconomic risk factors were identified for non-syndromic cleft lip with or without cleft palate. MATERIAL AND METHODS: A case-control study was carried out with 208 cases of non-syndromic cleft lip with or without cleft palate, and matched by age and sex with 416 controls. Cases were patients attending a referral clinic from 2002 through 2004 in Campeche, Mexico. A questionnaire was administered to collect sociodemographic and socioeconomic variables as well as familial background relevant to non-syndromic cleft lip with or without cleft palate. Conditional logistic regression models were used; adjusted odds ratios and 95% confidence intervals were calculated. RESULTS: In the multivariate model, the following risk factors were identified: 1) low socioeconomic status; 2) birth in the southern region of Campeche state; 3) home delivery or delivery in a publicly funded hospital; 4) occurrence of prior non-syndromic cleft lip with or without cleft palate cases in the father's or mother's family: 5) having a sibling with non-syndromic cleft lip with or without cleft palate; 6) the proband having another malformation, and 7) a history of infections during pregnancy. Prenatal care consisting of vitamin supplementation was a protective factor for non-syndromic cleft lip with or without cleft palate (odds ratio=0.29). CONCLUSIONS: A "social gradient in health" was seen to link oral malformation with diet components, and several socioeconomic and socio-demographic factors broadly encompassed in low socioeconomic status. Further characterization of risk factors will guide the assemblage of a pro-active counseling and prevention program for families at risk for non-syndromic cleft lip and cleft palate.

Differential parental transmission of markers in BCL3 among Korean cleft case-parent trios.

OBJECTIVES: Isolated cleft lip with or without cleft palate (CL/P) is among the most common human birth defects, with a prevalence of approximately 1 in 700 live births. The B-Cell Leukemia/lymphoma 3 (BCL3) gene has been suggested as a candidate gene for CL/P based on association and linkage studies in some populations. This study tests for an association between markers in BCL3 and isolated, non-syndromic CL/P using a case-parent trio design, while considering parent-of-origin effects. METHODS: Forty case-parent trios were genotyped for two single nucleotide polymorphisms (SNPs) in the BCL3 gene. We performed a transmission disequilibrium test (TDT) on individual SNPs, and the FAMHAP package was used to estimate haplotype frequencies and to test for excess transmission of multi-SNP haplotypes. RESULTS: The odds ratio for transmission of the minor allele, OR (transmission), was significant for SNP rs8100239 (OR=3.50, p=0.004) and rs2965169 (OR=2.08, p=0.027) when parent-of-origin was not considered. Parent-specific TDT revealed that SNP rs8100239 showed excess maternal transmission. Analysis of haplotypes of rs2965169 and rs8100239 also suggested excess maternal transmission. CONCLUSIONS: BCL3 appears to influence risk of CL/P through a parent-of-origin effect with excess maternal transmission.

Linkage disequilibrium between MSX1 and non-syndromic cleft lip/palate in the Chilean population.

Non-syndromic cleft lip/palate (NSCLP) is a complex genetic trait. Linkage and association studies have suggested that a clefting locus could be located on chromosome 4p. Sixty Chilean families were recruited for this study; from these, we used unrelated trios to evaluate the possible linkage disequilibrium between MSX1 and NSCLP. An intragenic marker, MSX1-CA, and an extragenic marker, D4S432 at a distance of 0.8 cM from MSX1, were analyzed by means of polymerase chain-reaction with fluorescent-labeled forward primers, followed by electrophoresis on a laser-fluorescent sequencer. We carried out a transmission/disequilibrium test (TDT) for multiple alleles to evaluate the presence of linkage disequilibrium. Results showed a preferential transmission of the 169-bp allele of MSX1 (p = 0.03). Although there was no preferential transmission for the D4S432 marker, the overall extended TDT (ETDT) showed a significant result (p = 0.01). The authors' findings support the hypothesis of the contribution of MSX1 in the etiology of NSCLP in the Chilean population.

Association between 10 microsatellite markers and nonsyndromic cleft lip palate in the Chilean population.

OBJECTIVES: The objective of this case-control study was to evaluate the possible association between nonsyndromic cleft lip/palate (NSCLP) and 10 genetic markers in four chromosomal regions in the admixed Spanish-Amerindian Chilean population. SETTING: Study participants included 56 patients with NSCLP identified and interviewed for positive family history during the course of clinical examinations at different rehabilitation centers in the cities of Santiago and Talca, Chile. A control group of 59 normal individuals without known familial antecedents of clefting was obtained from blood bank donors of the University Hospital, University of Chile. Cases and controls belonged to low- to low-middle socioeconomic strata. RESULTS: Ten markers from chromosome 4p, 4q, 6p, 17q, and 19q were assessed (MSX1, D4S175, D4S192, F13A1, EDN1, D6S89, D6S105, D6S109, D17S579, BCL3). Four of them showed significant deviations from Hardy-Weinberg expectations in controls, according to the exact test (D4S192, BCL3, F13A1, and D6S89). The case-control comparison by means of the CLUMP program showed significant differences only in BCL3, and D6S109 almost reached statistical significance. CONCLUSIONS: Most of the genetic regions with positive results in Caucasian populations may not be involved in NSCLP in Chile, regardless of the positive evidence for the candidate region on chromosome 19. Similar findings have been reported recently in the Chinese population.

Nonsyndromic cleft lip with or without cleft palate in China: assessment of candidate regions.

OBJECTIVE: Although Asians have the highest birth prevalence of oral-facial clefts, the majority of gene mapping studies of cleft lip with or without cleft palate (CL/P) have been in European or American Caucasians. Therefore, the objective of this study of Chinese families was to evaluate linkage and association between CL/P and 10 genetic markers in five chromosomal regions that have shown positive results in Caucasians. SETTING: Families were ascertained through nonsyndromic CL/P surgical probands from hospitals throughout Shanghai, China. PARTICIPANTS: Study participants included 671 individuals from 60 families with two or more members affected with oral-facial clefts. Of the 671 total individuals, 145 were affected. RESULTS: Ten markers from chromosomes 2, 4, 6, 17, and 19 were assessed (TGFA, MSX1, D4S194, D4S175, F13A1, GATA185H, D17S250, D17S579, D19S49, APOC2). LOD scores were calculated between each of the 10 markers and CL/P as well as model-free statistics of linkage (SimIBD) and association (TDT). None of the markers showed significantly positive LOD scores with CL/P. A significantly positive result (p =.01) was seen using SimIBD for APOC2 on chromosome 19, and a positive TDT result (p =.004) was obtained for D19S49, near APOC2. CONCLUSIONS: This is the first gene mapping study of CL/P in China. These results indicate that most of the genetic regions with positive results in Caucasian families may not be involved in CL/P found in China, although there is some positive evidence for the candidate region on chromosome 19.

A case-control study of nonsyndromic oral clefts in Maryland.

PURPOSE: Isolated, nonsyndromic oral clefts cases (n = 171) and unaffected controls (n = 182) were used to identify both genetic and environmental risk factors. METHODS: Infants born in Maryland between 1992 to 1998 with an isolated, nonsyndromic oral cleft [cleft lip (CL), cleft lip and palate (CLP), or cleft palate (CP)] were recruited and exposure plus family history data were collected. Controls were unaffected infants. DNA was collected from all cases and their parents, plus controls. RESULTS: No statistically significant association was found between any of the following: maternal smoking, vitamin use, urinary tract infection, or recreational drug use in either univariate analysis or after adjusting for maternal age and education. More control mothers reported alcohol use during the critical time period of pregnancy (one month before conception through the first trimester) as compared to case mothers. There was a 10-fold increase in risk to siblings of cases as compared to siblings of controls. Markers at four candidate genes were examined: transforming growth factor alpha (TGF alpha), transforming growth factor beta 3 (TGF beta 3), MSX1, and BCL3. Only MSX1 showed significant differences in allele frequencies between CP cases and controls. MSX1 also showed significant evidence of linkage disequilibrium with a susceptibility gene controlling risk for CP. CONCLUSION: Most environmental risk factors examined here gave little evidence of association with risk to isolated, nonsyndromic oral clefts, although any alcohol consumption seemed protective. MSX1 showed evidence of linkage disequilibrium in both case-control and case-parent trio analysis.

[Environment and genetics in the etiology of cleft lip and cleft palate with reference to the role of folic acid]. / Ambiente e genetica nell'eziologia delle labioschisi e palatoschisi con particolare riferimento al ruolo dell'acido folico.

The combined birth prevalence of cleft palate [CP] and cleft lip with or without cleft palate [CL(P)] in Europe is approximately one in 700 with characteristic regional variations. Orofacial clefting (OC) is therefore now one of the most frequent congenital anomalies, with a higher birth prevalence that Down's Syndrome or Neural Tube defects, but still lower than cardiovascular malformation. Babies with OC require a multidisciplinary medical approach, surgery and rehabilitative treatments over time. This means an important effort in terms of social organization as well as economical costs for the health care system. In Italy, the health care costs for approximately 800 children born with orofacial clefting per year has been estimated at around 150 billion Lire (80 million Euros). The etiology of OC is complex and heterogeneous both for isolated and associated defects; causes linked to environment, genetics and gene-environment interaction are known, although there is still a lot to do, especially in clarifying the role of genetics in producing susceptibility to the environment. Four categories of genes for which there are results suggestive of a genetic susceptibility to OCs are: 1) genes expressed in a particular area of the embryo or in a particular period of the palatine arch development, such as the transforming growth factors alpha and beta (TGF alpha, TGF beta 2, TGF beta 3); 2) genes having biological activities linked to the OC's pathogenesis without direct involvement (e.g. the retinoic acid receptor (RARA), the methylenetetrahydrofolate reductase receptor (MTHFR) and the folic acid receptor (FOLR1); 3) genes or locus identified in experimental animals as the homeotic genes MSX-1 and MSX-2; 4) genes involved in the interaction with the xenobiotics metabolism as those in P-450 cytochrome system. Several environmental factors have been implicated in the OC etiology; among those, the folic acid supplementation during the periconceptional period that was found effective in the prevention of neural tube defects. In fact, folic acid deficiency may be responsible for different malformations through a common mechanism that interferes with the embryonic development, depending on the maternal or embryo genotype. Further investigation is required to study in depth how the genotype would modify the role of environmental factors like folic acid. Well-designed and conducted epidemiological studies seem to be able to give worthwhile information. Studies carried out in Europe on these issues are a few, particularly those on gene-environment interaction. Recent results obtained in molecular biology and the availability of wealth of data can allow to perform ad hoc investigations, being important not only for the basic research but also for their public health implications. For this objective a specific scientific network at the European level has been set by the European Science Foundation (ESF), whose first step will be to establish consistent case ascertainment and data collection across Europe and to develop standardized protocols and methods of analysis. It is hoped that in the longer term such multicentre collaborative research will enable combined analysis and lead to the identification of genetic susceptibility to certain environmental factors, including nutrition. Such studies would inform the current debate about the efficacy of folic acid and other nutritional factors in prevention of disease in the developing embryo. Subsequent public health measures targeted according to risk might reduce the prevalence of disorders such as orofacial clefting.

Assessment of the patient with cleft lip and palate. A developmental approach.

Children with cleft lip and palate require interdisciplinary team care from infancy through adolescence. An understanding of developmental stages allows the cleft palate team to adapt and integrate its services into the rapidly changing life of the child. This article discusses the maturational, developmental stages of childhood and the services the child with cleft lip and palate and the child's family deserve through each stage. Health care providers in all settings may continue to provide appropriate care for all patients with cleft lip and palate, despite the challenges of a changing health care environment, by emphasizing the needs of the child in all developmental stages.