Alexandre R. Vieira

A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4

Case-parent trios were used in a genome-wide association study of cleft lip with and without cleft palate. SNPs near two genes not previously associated with cleft lip with and without cleft palate (MAFB, most significant SNP rs13041247, with odds ratio (OR) per minor allele = 0.704, 95% CI 0.635–0.778, P = 1.44 × 10−11; and ABCA4, most significant SNP rs560426, with OR = 1.432, 95% CI 1.292–1.587, P = 5.01 × 10−12) and two previously identified regions (at chromosome 8q24 and IRF6) attained genome-wide significance. Stratifying trios into European and Asian ancestry groups revealed differences in statistical significance, although estimated effect sizes remained similar. Replication studies from several populations showed confirming evidence, with families of European ancestry giving stronger evidence for markers in 8q24, whereas Asian families showed stronger evidence for association with MAFB and ABCA4. Expression studies support a role for MAFB in palatal development.

Mutations in BMP4 Are Associated with Subepithelial, Microform, and Overt Cleft Lip

Cleft lip with or without cleft palate (CL/P) is a complex trait with evidence that the clinical spectrum includes both microform and subepithelial lip defects. We identified missense and nonsense mutations in the BMP4 gene in 1 of 30 cases of microform clefts, 2 of 87 cases with subepithelial defects in the orbicularis oris muscle (OOM), 5 of 968 cases of overt CL/P, and 0 of 529 controls. These results provide confirmation that microforms and subepithelial OOM defects are part of the spectrum of CL/P and should be considered during clinical evaluation of families with clefts. Furthermore, we suggest a role for BMP4 in wound healing.

Defining Subphenotypes for Oral Clefts Based on Dental Development

Individuals with clefts present considerably more dental anomalies than do individuals without clefts. We used dental development to subphenotype clefts with the goal of identifying cleft subgroups that could have specific genetic contributions. We examined 1000 individuals, 500 with clefts and 500 without. We used several clinical features, such as cleft completeness or incompleteness, laterality, and the presence of dental anomalies to assess each individual’s cleft status. We performed chi-square and Fisher’s exact tests to compare the frequencies of observed anomalies between individuals with and individuals without clefts, and among individuals with different cleft subphenotypes. Agenesis of the lateral incisor on the non-cleft side was the most remarkable observation, and may suggest that such cases could be considered incomplete forms of bilateral clefts of the lip.

Enamel Formation Genes Are Associated with High Caries Experience in Turkish Children

There is evidence for a genetic component in caries susceptibility, and studies in humans have suggested that variation in enamel formation genes and their interaction with Streptococcus mutans levels may contribute to caries. For the present study, we used DNA samples collected from 173 unrelated children from Istanbul: 91 children with 4 or more affected tooth surfaces and 82 caries-free children. Six single-nucleotide polymorphism markers were genotyped in selected candidate genes (ameloblastin, amelogenin, enamelin, tuftelin 1 and tuftelin interacting protein 11) that influence enamel formation. Allele and genotype frequencies were compared between groups with distinct caries experience. Regression analysis was used for the evaluation of individual gene effects, environmental effects and gene-environment interactions. Overrepresentation of the C allele of the amelogenin marker was seen in cases with dmft scores higher than 8 (p = 0.01) when compared to controls. Also, overrepresentation of the T allele of the ameloblastin marker was seen in cases with dmfs scores higher than 10 (p = 0.05) when compared to controls. In addition, the CT genotype of the tuftelin rs3790506 marker was overrepresented in cases with dmft scores higher than 5 (p = 0.05) and dmfs scores higher than 6 (p = 0.05) compared to controls. The best-fitting model showed that dmfs is increased when the following factors are present: (1) females and both the anterior and posterior teeth are affected simultaneously, (2) when the T allele of the tuftelin rs3790506 is involved, and (3) the C allele of the amelogenin rs17878486 is involved. Our study provides support that genes involved in enamel formation modify caries susceptibility in humans.

Possible Association of Amelogenin to High Caries Experience in a Guatemalan-Mayan Population

There is evidence for a genetic component in caries susceptibility, but the disease is greatly influenced by environmental factors, which are extremely difficult to control in humans. For the present study, we used DNA samples collected from 110 unrelated, non-cleft individuals older than 12 years of age from Tiquisate, Guatemala: a population with similar cultural, dietary and hygiene habits, similar access to the dentist and fluoride exposure. Forty-four individuals were designated ‘very low caries experience’ (DMFT ≤2), and 66 were designated ‘higher caries experience’ (DMFT ≧3). Single-nucleotide polymorphism markers were genotyped in selected candidate genes (ameloblastin, amelogenin, enamelin, tuftelin-1, and tuftelin interacting protein 11) that influence enamel formation. Having at least one copy of the rare amelogenin marker allele was associated with increased age-adjusted caries experience. This association was stronger in individuals with higher DMFT (DMFT ≧20; p = 0.0000001). Our results suggest that variation in amelogenin may contribute to caries susceptibility in the population studied. The approach of comparing individuals with extremely distinct caries experiences could be valuable for decreasing the potential influence of environmental factors on genetic studies of caries.

MSX1, PAX9, and TGFA Contribute to Tooth Agenesis in Humans

In this study, we sought to determine the association between tooth agenesis and DNA sequence variation in the genes MSX1 and PAX9 in an ethnically diverse human population. Since cleft lip/palate is also associated with both tooth agenesis and the gene TGFA, we included TGFA in the analysis as well. Cheek swab samples were obtained for DNA analysis from 116 case/parent trios. Probands had at least one developmentally missing tooth, excluding third molars. Genotyping was performed by single-strand conformational polymorphism or kinetic polymerase chain-reaction assays. Transmission distortion of the marker alleles and DNA sequence analysis was performed. Results showed that tooth agenesis is associated with markers of the genes MSX1 and TGFA. No mutations were found in MSX1 or PAX9 coding regions. There were statistically significant data suggesting that MSX1 interacts with PAX9. These findings suggest that MSX1, PAX9, and TGFA play a role in isolated dental agenesis.

Genome Scan, Fine-Mapping, and Candidate Gene Analysis of Non-Syndromic Cleft Lip with or without Cleft Palate Reveals Phenotype-Specific Differences in Linkage and Association Results

Objectives: Non-syndromic orofacial clefts, i.e. cleft lip (CL) and cleft palate (CP), are among the most common birth defects. The goal of this study was to identify genomic regions and genes for CL with or without CP (CL/P). Methods: We performed linkage analyses of a 10 cM genome scan in 820 multiplex CL/P families (6,565 individuals). Significant linkage results were followed by association analyses of 1,476 SNPs in candidate genes and regions, utilizing a weighted false discovery rate (wFDR) approach to control for multiple testing and incorporate the genome scan results. Results: Significant (multipoint HLOD ≥3.2) or genome-wide-significant (HLOD ≥4.02) linkage results were found for regions 1q32, 2p13, 3q27-28, 9q21, 12p11, 14q21-24 and 16q24. SNPs in IRF6 (1q32) and in or near FOXE1 (9q21) reached formal genome-wide wFDR-adjusted significance. Further, results were phenotype dependent in that the IRF6 region results were most significant for families in which affected individuals have CL alone, and the FOXE1 region results were most significant in families in which some or all of the affected individuals have CL with CP. Conclusions: These results highlight the importance of careful phenotypic delineation in large samples of families for genetic analyses of complex, heterogeneous traits such as CL/P.

Genome-wide Scan Finds Suggestive Caries Loci

Here we report the first genome-wide scan performed for caries. Evidence from twin studies suggests a genetic component to caries. We aimed to identify genetic factors contributing to caries in a population similarly influenced by confounding factors, such as diet, oral hygiene habits, fluoride exposure, and access to dental care. Forty-six families with similar cultural and behavioral habits, and living in the Philippines, were studied, and genome-wide genotype data and DMFT (Decayed, Missing due to caries, Filled Teeth) scores were evaluated. Suggestive loci logarithmic odds (LOD) scores above 2.0 or non-parametric p-values below 0.0009) were found for low caries susceptibility (5q13.3, 14q11.2, and Xq27.1) and high caries susceptibility (13q31.1 and 14q24.3). Genes that may be related to saliva flow and diet preferences are proposed as possible candidates. A protective locus for caries in the X chromosome may explain the gender differences seen in caries frequency.

Dental Anomalies as Part of the Cleft Spectrum

Objective: The aim of this study was to determine subphenotypes of clefts based on tooth development. Dental phenotypes (tooth agenesis, supernumerary and impacted teeth, transposition, and structural anomalies) outside the cleft area in the permanent dentition of oral cleft individuals were investigated. Design: Evaluation of dental records of cleft patients. Setting: National Museum of Health and Medicine, Washington, D.C. Patients, Participants: A collection of dental casts, radiographic and clinical records of 146 patients with oral clefts was evaluated. Main Outcome Measure: Association of dental anomalies with preferential subtypes of clefts. Results: Forty-seven individuals (32.19%) presented at least one dental anomaly outside the cleft area. Individuals with complete cleft lip and palate (CLP) presented more dental anomalies than individuals with incomplete CLP (p  =  .04). Cleft palate individuals presented more dental anomalies than CLP individuals (p  =  .048). Maxillary lateral incisors and premolars were the most affected teeth. High incidence of maxillary second premolar agenesis was observed in individuals with bilateral CLP (p  =  .04). In cases with unilateral CLP, 12.5% presented dental anomalies of the maxillary lateral incisors on the noncleft side. Cleft palate individuals presented a high incidence of mandibular premolar anomalies (p  =  .004). Conclusion: Future studies should consider the inclusion of a complete dental description to aid in the definition of cleft subphenotypes to be studied.

Interferon regulatory factor 6 (IRF6) and fibroblast growth factor receptor 1 (FGFR1) contribute to human tooth agenesis.

Phenotypic characteristics expressed in syndromes give clues to the factors involved in the cause of isolated forms of the same defects. We investigated two genes responsible for craniofacial syndromes, FGFR1 and IRF6, in a collection of families with isolated tooth agenesis. Cheek swab samples were obtained for DNA analysis from 116 case/parent trios. Probands had at least one developmentally missing tooth, excluding third molars. In addition, we studied 89 cases and 50 controls from Ohio to replicate any positive findings. Genotyping was performed by kinetic polymerase chain‐reaction or TaqMan assays. Linkage disequilibrium analysis and transmission distortion of the marker alleles were performed. The same variants in the IRF6 gene that are associated with isolated orofacial clefts are also associated with human tooth agenesis (rs861019, P = 0.058; rs17015215—V274I, P = 0.0006; rs7802, P = 0.004). Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. The craniofacial phenotypic characteristics of these syndromes include oral clefts and preferential tooth agenesis of incisors and premolars, besides pits on the lower lips. Also it appears that preferential premolar agenesis is associated with FGFR1 (P = 0.014) and IRF6 (P = 0.002) markers. There were statistically significant data suggesting that IRF6 interacts not only with MSX1 (P = 0.001), but also with TGFA (P = 0.03).