Association of MSX1 and TGFB3 with nonsyndromic clefting in humans.

Nonsyndromic cleft lip with or without cleft palate (CL/P) and nonsyndromic cleft palate only (CPO) are common congenital anomalies with significant medical, psychological, social, and economic ramifications. Both CL/P and CPO are examples of complex genetic traits. There exists sufficient evidence to hypothesize that disease loci for CL/P and CPO can be identified by a candidate-gene linkage-disequilibrium (LD) strategy. Candidate genes for clefting, including TGFA, BCL3, DLX2, MSX1, and TGFB3, were screened for LD with either CL/P or CPO in a predominantly Caucasian population, with both case-control- and nuclear-family-based approaches. Previously reported LD for TGFA with both CL/P and CPO could not be confirmed, except in CL/P patients with a positive family history. Also, in contrast to previous studies, no LD was found between BCL3 and either CL/P or CPO. Significant LD was found between CL/P and both MSX1 and TGFB3 and between CPO and MSX1, suggesting that these genes are involved in the pathogenesis of clefting. In addition, a mutation search in the genes DLX2, MSX1, and TGFB3 was performed in 69 CPO patients and in a subset of the CL/P patients. No common mutations were found in the coding regions of these genes; however, several rare variants of MSX1 and TGFB3 were found that may alter the latters' normal function. These results form the basis for future research, including (a) mutation searches in the MSX1 and TGFB3 genes in Caucasian CL/P patients and (b) extension of the search for MSX1 mutations in CPO patients to the noncoding regions.     

Linkage disequilibrium between GABRB3 gene and nonsyndromic familial cleft lip with or without cleft palate

The malformation of nonsyndromic cleft lip with or without cleft palate (CL/P) is a common congenital disease that affects approximately 1/1000 newborns in Caucasian populations. Genetic studies indicate that CL/P has the characteristics of a complex genetic trait. Linkage analysis and mouse-model knockout studies have suggested several candidate genes mapping in different chromosome regions for CL/P malformation. On these grounds, we have investigated, by linkage disequilibrium (LD) and parametric and nonparametric linkage analyses, five different candidate genes, including those for the beta3 subunit of the gamma-aminobutyric acid receptor (GABRB3), glutamic acid decarboxylase 1 (GAD1), retinoic acid receptor alpha (RARA), transforming growth factor beta3 (TGFB3), and msh ( Drosophila) homeobox homolog 1 (MSX1). Interestingly, a significant LD between GABRB3 and CL/P was obtained ( P-value=0.008 in the allele-wise analysis for multiallelic markers), suggesting that the GABRB3 gene is involved in this congenital disease. This new finding in humans is in agreement with previously reported data obtained with the murine model. Indeed, mouse studies indicate a role for gamma-aminobutyric acid (GABA) and its receptor in normal palate development. Exclusion of the GAD1 gene, which encodes the GABA-producing enzyme, in CL/P pathogenesis was obtained in our study. Moreover, we were unable to confirm the involvement of the MSX1 gene in nonsyndromic CL/P. Modest evidence of LD between marker alleles and CL/P was found at the RARA and TGFB3 loci suggesting a minor role for these genes in our family set of nonsyndromic CL/P.     

Analysis of candidate genes on chromosome 2 in oral cleft case-parent trios from three populations

Isolated oral clefts, including cleft lip with/without cleft palate (CL/P) and cleft palate (CP), have a complex and heterogeneous etiology. Case-parent trios from three populations were used to study genes spanning chromosome 2, where single nucleotide polymorphic (SNP) markers were analyzed individually and as haplotypes. Case-parent trios from three populations (74 from Maryland, 64 from Singapore and 95 from Taiwan) were genotyped for 962 SNPs in 104 genes on chromosome 2, including two well-recognized candidate genes: TGFA and SATB2. Individual SNPs and haplotypes (in sliding windows of 2–5 SNPs) were used to test for linkage and disequilibrium separately in CL/P and CP trios. A novel candidate gene (ZNF533) showed consistent evidence of linkage and disequilibrium in all three populations for both CL/P and CP. SNPs in key regions of ZNF533 showed considerable variability in estimated genotypic odds ratios and their significance, suggesting allelic heterogeneity. Haplotype frequencies for regions of ZNF533 were estimated and used to partition genetic variance into among-and within-population components. Wright’s fixation index, a measure of genetic diversity, showed little difference between Singapore and Taiwan compared with Maryland. The tensin-1 gene (TNS1) also showed evidence of linkage and disequilibrium among both CL/P and CP trios in all three populations, albeit at a lower level of significance. Additional genes (VAX2, GLI2, ZHFX1B on 2p; WNT6–WNT10A and COL4A3–COL4A4 on 2q) showed consistent evidence of linkage and disequilibrium only among CL/P trios in all three populations, and TGFA showed significant evidence in two of three populations.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Mutations of GADD45G in rabbits cause cleft lip by the disorder of proliferation,apoptosis and epithelial-mesenchymal transition (EMT)

The cleft lip with or without cleft palate (CL/P) is one of the most common congenital defects in humans. Genome-wide association studies (GWAS) have been widely used for identifying candidate genes, and different genes or chromosomal regions have shown strong evidence for the presence of causal genes in CL/P. To date, two independent GWAS have identified GADD45G as influencing risk for CL/P. However, there is no animal model evidence about GADD45G related to CL/P. Here, we reported the generation of a novel GADD45G mutated rabbit model by CRISPR/Cas9 and CRISPR-based BE4-Gam systems. The homozygous (GADD45G^−/−) while not heterozygous (GADD45G^+/−) pups died after birth due to severe craniofacial defects of unilateral or bilateral cleft lip (CL). Moreover, the disorder of proliferation, apoptosis and epithelial-mesenchymal transition (EMT) were also determined in the medial and lateral nasal processes (MNP and LNP) of the embryonic day 13 (E13) GADD45G^−/− rabbits, which compared with the normal wild type (WT) rabbits. Thus, our study confirmed for the first time that loss of GADD45G lead to CL at the animal level and provided new insights into the crucial role of GADD45G for upper lip formation and fusion.     

AXIN2 and CDH1 polymorphisms, tooth agenesis, and oral clefts

BACKGROUND: AXIN2 and CDH1 genes play important roles during craniofacial morphogenesis. Mutations in these genes have been described in families presenting colorectal cancer and tooth agenesis, and gastric cancer and cleft lip/palate (CL/P). Oral clefts have been associated with tooth agenesis. We investigated if AXIN2 and CDH1 polymorphisms were associated with clefts or with any associated dental subphenotypes. METHODS: Markers in AXIN2 and CDH1 were genotyped using Taqman chemistry in a sample cohort comprised of 500 cleft individuals and 500 unrelated controls. RESULTS: Comparison between cleft and control groups showed a trend for association for AXIN2 with incomplete cleft palate (p = .006) and CDH1 with unilateral CL/P (p = .03 for left CL/P and p = .04 for right CL/P). Comparison of cleft subphenotypes with tooth agenesis and controls revealed borderline associations for CDH1 (p = .008) and AXIN2 (p = .01) with unilateral right CL/P with tooth agenesis. CONCLUSIONS: We observed only borderline results for the association of AXIN2 and CDH1 with CL/P with and without tooth agenesis. Nevertheless, implication of these genes in the simultaneous occurrence of CL/P and cancer, and in tooth agenesis and cancer, is rather intriguing and warrants further investigations with other geographic and ethnic populations. Birth Defects Research (Part A), 2009. © 2008 Wiley‐Liss, Inc.     

Epidemiological and genetic study of 200 cases of oral cleft in the Emilia Romagna region of northern Italy

Epidemiological and genetic variables in clefts were analyzed during the years 1978-1986 in a case-control study of congenital malformations in the Emilia Romagna region of northern Italy. Among 150,168 newborns, 200 cases of cleft were detected, yielding a prevalence of 1.33 per 1,000. These clefts consisted of 112 (0.075%) cases of cleft lip with or without cleft palate (CL +/- P) and 88 cases (0.058%) of cleft palate (CP). Coexisting abnormalities were found in 32% of cases. The heritability coefficient of CL +/- P was 0.84. No cluster in time or space could be demonstrated. Epilepsy was the only maternal risk factor found to be correlated with clefts. A predominance of males was found among CL +/- P cases.     

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2): structure,gene mapping,polymorphisms, and candidate evaluation for human orofacial clefts

BACKGROUND: Nonsyndromic orofacial clefts have an estimated incidence of 1/1000 live births. Population genetic and embryologic studies suggest that cleft palate only (CPO) may be a distinct clinical entity from cleft lip with or without cleft palate (CL/P). Both CPO and CL/P are thought to be multifactorial in etiology, with evidence indicating that genetic, environmental, and developmental determinants may all play a role. The ARNT2 gene localizes to a conserved linkage group on mouse chromosome 7 that is syntenic with human chromosome 15q23-25. This chromosomal region was previously identified as a teratogen-induced clefting susceptibility locus in a genome-wide scan of AXB and BXA recombinant inbred mice. Arnt2 is expressed in the first branchial arch in mice. The teratogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts through the aryl hydrocarbon receptor (Ahr) pathway to produce dose-dependent CPO and thymic wasting in mice exposed in utero. Arnt2 and Ahr proteins dimerize in vitro. TCDD exposure is also associated with orofacial clefting in children of parents involved in agricultural work. METHODS: To determine whether ARNT2 influences human craniofacial development, we identified the human ARNT2 gene and conducted genomic structural analysis. Mutational screening was performed in infants with nonsyndromic CPO or CL/P who were identified by the Iowa Birth Defects Registry. RESULTS: A common amino acid polymorphism was detected but, no obvious disease-causing mutations were detected by SSCP analysis. The microsatellite marker, GATA89D04 (D15S823) was identified within intron 11 of the human ARNT2 gene, and linkage disequilibrium of nonsyndromic CPO and CL/P parent-infant trios was conducted. CONCLUSIONS: No association was demonstrated with CPO (n = 45) and CL/P (n = 37). Teratology 66:85-90, 2002.     

Genetic Determinants of Facial Clefting: Analysis of 357 Candidate Genes Using Two National Cleft Studies from Scandinavia

Background

Facial clefts are common birth defects with a strong genetic component. To identify fetal genetic risk factors for clefting, 1536 SNPs in 357 candidate genes were genotyped in two population-based samples from Scandinavia (Norway: 562 case-parent and 592 control-parent triads; Denmark: 235 case-parent triads).

Methodology/Principal Findings

We used two complementary statistical methods, TRIMM and HAPLIN, to look for associations across these two national samples. TRIMM tests for association in each gene by using multi-SNP genotypes from case-parent triads directly without the need to infer haplotypes. HAPLIN on the other hand estimates the full haplotype distribution over a set of SNPs and estimates relative risks associated with each haplotype. For isolated cleft lip with or without cleft palate (I-CL/P), TRIMM and HAPLIN both identified significant associations with IRF6 and ADH1C in both populations, but only HAPLIN found an association with FGF12. For isolated cleft palate (I-CP), TRIMM found associations with ALX3, MKX, and PDGFC in both populations, but only the association with PDGFC was identified by HAPLIN. In addition, HAPLIN identified an association with ETV5 that was not detected by TRIMM.

Conclusion/Significance

Strong associations with seven genes were replicated in the Scandinavian samples and our approach effectively replicated the strongest previously known association in clefting—with IRF6. Based on two national cleft cohorts of similar ancestry, two robust statistical methods and a large panel of SNPs in the most promising cleft candidate genes to date, this study identified a previously unknown association with clefting for ADH1C and provides additional candidates and analytic approaches to advance the field.     

Few associations of candidate genes with nonsyndromic orofacial clefts in the population of Lithuania

Nonsyndromic orofacial clefting (NS-OFC) is a common complex multifactorial trait with a considerable genetic component and a number of candidate genes suggested by various approaches. Twenty biallelic and microsatellite DNA markers in the strong candidate loci TGFA, TGFB3, GABRB3, RARA, and BCL3 were analysed for allelic association with the NS-OFC phenotype in 112 nuclear families (proband + both parents) from Lithuania by using the transmission disequilibrium test (TDT). Associations were found between the TGFA gene marker rs2166975 and nonsyndromic cleft palate only (CPO) phenotype (p = 0.045, df 1) as well as between the D2S292 marker and the cleft lip with or without cleft palate (CL/CP) phenotype in allele-wise TDT (P = 0.005, df 9) and genotype-wise TDT (P = 0.021, df 24). A weak association (P = 0.085, df 3) of the BCL3 marker (BCL3 gene) with the risk of CPO was also found. Thus our initial results support the contribution of allelic variation in the TGFA locus to the aetiology of CL/CP in the population of Lithuania but they do not point to TGFA as a major causal gene. Different roles of the TGFA and BCL3 genes in the susceptibility to NS-OFC phenotypes are suggested.     

Mitochondrial DNA origins of the Latvian clefting population

Latvia has one of the highest prevalence of isolated cleft lip with or without cleft palate (CL/P) in Europe. To clarify the genetic origins of the Latvian cleft population and establish a method for genetic mapping, mitochondrial DNA variation was studied in a population affected with clefting. One-hundred and seven subjects and 351 samples from unrelated healthy volunteers representing four anthropologically, archaeologically and ethno-linguistically different regions of Latvia were selected. The case group showed a higher frequency of haplogroups U4 (p=0.02) and U5 (p=0.0003) than in non-U haplogroups. We hypothesize that U4 and U5 mtDNA haplotype carriers may also carry susceptibility genes for clefts. Future studies will take into consideration these definitions based on mtDNA haplotypes when analyzing genetic variations and their possible contribution to CL/P susceptibility.     

Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly,Cleft Lip,and Cleft Palate

The Hedgehog (Hh) signaling pathway mediates multiple spatiotemporally-specific aspects of brain and face development. Genetic and chemical disruptions of the pathway are known to result in an array of structural malformations, including holoprosencephaly (HPE), clefts of the lip with or without cleft palate (CL/P), and clefts of the secondary palate only (CPO). Here, we examined patterns of dysmorphology caused by acute, stage-specific Hh signaling inhibition. Timed-pregnant wildtype C57BL/6J mice were administered a single dose of the potent pathway antagonist vismodegib at discrete time points between gestational day (GD) 7.0 and 10.0, an interval approximately corresponding to the 15^th to 24^th days of human gestation. The resultant pattern of facial and brain dysmorphology was dependent upon stage of exposure. Insult between GD7.0 and GD8.25 resulted in HPE, with peak incidence following exposure at GD7.5. Unilateral clefts of the lip extending into the primary palate were also observed, with peak incidence following exposure at GD8.875. Insult between GD9.0 and GD10.0 resulted in CPO and forelimb abnormalities. We have previously demonstrated that Hh antagonist-induced cleft lip results from deficiency of the medial nasal process and show here that CPO is associated with reduced growth of the maxillary-derived palatal shelves. By defining the critical periods for the induction of HPE, CL/P, and CPO with fine temporal resolution, these results provide a mechanism by which Hh pathway disruption can result in “non-syndromic” orofacial clefting, or HPE with or without co-occurring clefts. This study also establishes a novel and tractable mouse model of human craniofacial malformations using a single dose of a commercially available and pathway-specific drug.     

Maternal Transmission Effect of a PDGF-C SNP on Nonsyndromic Cleft Lip with or without Palate from a Chinese Population

Cleft lip with or without palate (CL/P) is a common congenital anomaly with a high birth prevalence in China. Based on a previous linkage signal of nonsyndromic CL/P (NSCL/P) on the chromosomal region 4q31–q32 from the Chinese populations, we screened the 4q31–q32 region for susceptibility genes in 214 trios of Han Chinese. PDGF-C, an important developmental factor, resides in the region and has been implicated in NSCL/P. However, in our family-based association test (transmission disequilibrium test; TDT), we could not conclude an association between PDGF-C and NSCL/P as previously suggested. Instead, we found strong evidence for parent-of-origin effect at a PDGF-C SNP, rs17035464, by a likelihood ratio test (unadjusted p-value = 0.0018; I_m = 2.46). The location of rs17035464 is 13 kb downstream of a previously reported, NSCL/P-associated SNP, rs28999109. Furthermore, a patient from our sample trios was observed with a maternal segmental uniparental isodisomy (UPD) in a region containing rs17035464. Our findings support the involvement of PDGF-C in the development of oral clefts; moreover, the UPD case report contributes to the collective knowledge of rare variants in the human genome.     

Maternal Genes and Facial Clefts in Offspring: A Comprehensive Search for Genetic Associations in Two Population-Based Cleft Studies from Scandinavia

Background

Fetal conditions can in principle be affected by the mother''s genotype working through the prenatal environment.

Methodology/Principal Findings

Genotypes for 1536 SNPs in 357 cleft candidate genes were available from a previous analysis in which we focused on fetal gene effects [1]. After data-cleaning, genotypes for 1315 SNPs in 334 autosomal genes were available for the current analysis of maternal gene effects. Two complementary statistical methods, TRIMM and HAPLIN, were used to detect multi-marker effects in population-based samples from Norway (562 case-parent and 592 control-parent triads) and Denmark (235 case-parent triads). We analyzed isolated cleft lip with or without cleft palate (iCL/P) and isolated cleft palate only (iCP) separately and assessed replication by looking for genes detected in both populations by both methods. In iCL/P, neither TRIMM nor HAPLIN detected more genes than expected by chance alone; furthermore, the selected genes were not replicated across the two methods. In iCP, however, FLNB was identified by both methods in both populations. Although HIC1 and ZNF189 did not fully satisfy our stringency criterion for replication, they were strongly associated with iCP in TRIMM analyses of the Norwegian triads.

Conclusion/Significance

Except for FLNB, HIC1 and ZNF189, maternal genes did not appear to influence the risk of clefting in our data. This is consistent with recent epidemiological findings showing no apparent difference between mother-to-offspring and father-to-offspring recurrence of clefts in these two populations. It is likely that fetal genes make the major genetic contribution to clefting risk in these populations, but we cannot rule out the possibility that maternal genes can affect risk through interactions with specific teratogens or fetal genes.     

Linkage analysis in a large Brazilian family with van der Woude syndrome suggests the existence of a susceptibility locus for cleft palate at 17p11.2-11.1.

van der Woude syndrome (VWS), which has been mapped to 1q32-41, is characterized by pits and/or sinuses of the lower lip, cleft lip/palate (CL/P), cleft palate (CP), bifid uvula, and hypodontia (H). The expression of VWS, which has incomplete penetrance, is highly variable. Both the occurrence of CL/P and CP within the same genealogy and a recurrence risk <40% for CP among descendants with VWS have suggested that the development of clefts in this syndrome is influenced by modifying genes at other loci. To test this hypothesis, we have conducted linkage analysis in a large Brazilian kindred with VWS, considering as affected the individuals with CP, regardless of whether it is associated with other clinical signs of VWS. Our results suggest that a gene at 17p11.2-11.1, together with the VWS gene at 1p32-41, enhances the probability of CP in an individual carrying the two at-risk genes. If this hypothesis is confirmed in other VWS pedigrees, it will represent one of the first examples of a gene, mapped through linkage analysis, which modifies the expression of a major gene. It will also have important implications for genetic counseling, particularly for more accurately predicting recurrence risks of clefts among the offspring of patients with VWS.     

Association of genetic variation of the transforming growth factor-alpha gene with cleft lip and palate.

Complex segregation analysis of pedigrees having nonsyndromic cleft lip with or without cleft palate (CL/P) (Chung et al. 1986; Marazita et al. 1986) has shown that a major-locus model best explains the observed recurrence of CL/P in Caucasian families. To identify this major gene, we compared the frequencies of 12 RFLPs at five loci-epidermal growth factor, transforming growth factor-alpha, epidermal growth factor receptor, glucocorticoid receptor, and estrogen receptor-in both a group of 80 subjects with nonsyndromic CL/P and 102 controls. These candidate genes were selected because studies in rodents had suggested their possible involvement in palatogenesis. A significant association was observed between two RFLPs at the transforming-growth-factor-alpha (TGFA) locus and the occurrence of clefting (P = .0047 and P = .0052). This suggests that either the TGFA gene itself or DNA sequences in an adjacent region contribute to the development of a portion of cases of CL/P in humans and provides an opportunity to begin to examine the molecular events underlying lip and palate formation.     

Report of two cases with Van der Woude syndrome: a child and her mother

Report of two cases with Van der Woude syndrome: a child and her mother: Congenital pits of the lower lip are rare malformations. They are closely associated with cleft lip (CL), cleft lip/palate (CL/CP) or isolated cleft palate (CP) and if so this condition is known as Van der Woude syndrome, which is inherited in an autosomal dominant fashion with high penetrance. Two individuals, one with lower lip pits and cleft palate and the other with isolated lower lip pit from the same family are described. Autosomal dominant pattern of inheritance was observed in this family and treatment consisted of complete removal of sinus tracts in one patient. Pathological features of sinus tracts consisted of stratified nonkeratinized squamous epithelium and a lamina propria of dense connective tissue. Importance of genetic counseling is emphasized as at least half of gene carriers have some kind of clefting.     

Genome-Wide Association Studies in Dogs and Humans Identify ADAMTS20 as a Risk Variant for Cleft Lip and Palate

Cleft lip with or without cleft palate (CL/P) is the most commonly occurring craniofacial birth defect. We provide insight into the genetic etiology of this birth defect by performing genome-wide association studies in two species: dogs and humans. In the dog, a genome-wide association study of 7 CL/P cases and 112 controls from the Nova Scotia Duck Tolling Retriever (NSDTR) breed identified a significantly associated region on canine chromosome 27 (unadjusted p=1.1 x 10^-13; adjusted p= 2.2 x 10^-3). Further analysis in NSDTR families and additional full sibling cases identified a 1.44 Mb homozygous haplotype (chromosome 27: 9.29 – 10.73 Mb) segregating with a more complex phenotype of cleft lip, cleft palate, and syndactyly (CLPS) in 13 cases. Whole-genome sequencing of 3 CLPS cases and 4 controls at 15X coverage led to the discovery of a frameshift mutation within ADAMTS20 (c.1360_1361delAA (p.Lys453Ilefs*3)), which segregated concordant with the phenotype. In a parallel study in humans, a family-based association analysis (DFAM) of 125 CL/P cases, 420 unaffected relatives, and 392 controls from a Guatemalan cohort, identified a suggestive association (rs10785430; p =2.67 x 10^-6) with the same gene, ADAMTS20. Sequencing of cases from the Guatemalan cohort was unable to identify a causative mutation within the coding region of ADAMTS20, but four coding variants were found in additional cases of CL/P. In summary, this study provides genetic evidence for a role of ADAMTS20 in CL/P development in dogs and as a candidate gene for CL/P development in humans.     

Nonsyndromic cleft lip with or without cleft palate: evidence of linkage to BCL3 in 17 multigenerational families.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common craniofacial developmental defect. Recent segregation analyses have suggested that major genes play a role in the etiology of CL/P. Linkage to 22 candidate genes was tested in 11 multigenerational families with CL/P, and 21 of these candidates were excluded. APOC2, 19q13.1, which is linked to the proto-oncogene BCL3, gave suggestive evidence for linkage to CL/P. The study was expanded to include a total of 39 multigenerational CL/P families. Linkage was tested in all families, using an anonymous marker, D19S178, and intragenic markers in BCL3 and APOC2. Linkage was tested under two models, autosomal dominant with reduced penetrance and affecteds only. Homogeneity testing on the two-point data gave evidence of heterogeneity at APOC2 under the affecteds-only model. Both models showed evidence of heterogeneity, with 43% of families linked at zero recombination to BCL3 when marker data from BCL3 and APOC2 were included. A maximum multipoint LOD score of 7.00 at BCL3 was found among the 17 families that had posterior probabilities > = 50% in favor of linkage. The transmission disequilibrium test provided additional evidence for linkage with the 3 allele of BCL3 more often transmitted to affected children. These results suggest that BCL3, or a nearby gene, plays a role in the etiology of CL/P in some families.