Renato Assis Machado

Genetic risk factors for nonsyndromic cleft lip with or without cleft palate in a Brazilian population with high African ancestry

Nonsyndromic cleft lip with or without cleft palate (NSCL ± P) is the most common orofacial birth defect, exhibiting variable prevalence around the world, often attributed to ethnic and environmental differences. Linkage analyses and genome‐wide association studies have identified several genomic susceptibility regions for NSCL ± P, mostly in European‐derived or Asian populations. Genetic predisposition to NSCL ± P is ethnicity‐dependent, and the genetic basis of susceptibility to NSCL ± P likely varies among populations. The population of Brazil is highly admixed, with highly variable ancestry; thus, the genetic determinants of NSCL ± P susceptibility may be quite different. This study tested association of 8 single‐nucleotide polymorphisms (SNPs), previously identified by genome‐wide studies in other populations, with NSCL ± P in a Brazilian population with high African ancestry. SNPs rs560426, rs642961, rs1530300, rs987525, rs3758249, rs7078160, rs17085106, and rs13041247 were genotyped in 293 Brazilian patients with NSCL ± P and 352 unaffected Brazilian controls. Each sample was also genotyped for 40 biallelic short insertion/deletion polymorphic markers to characterize genetic ancestry. The average African ancestry background was 31.1% for the NSCL ± P group and 36.7% for the control group. After adjustment for ancestry and multiple testing, the minor alleles of rs3758249 (OR: 1.58, 95% CI: 1.25–2.01, P = 0.0001) and rs7078160 (OR: 1.59, 95% CI: 1.21–2.07, P = 0.0002) were significantly associated with risk of NSCL ± P. Polymorphisms located in IRF6 (rs642961) and 8q24 (rs1530300 and rs987525) showed marginal associations in this Brazilian population with high African ancestry. These results indicate that rs3758249 at 9q22 and rs7078160 at 10q25.3 represent risk loci for NSCL ± P in the Brazilian population with high African ancestry.

Interactions Between Rad51 Rs1801321 And Maternal Cigarette Smoking As Risk Factor For Nonsyndromic Cleft Lip With Or Without Cleft Palate

Interactions Between RAD51 rs1801321 and Maternal Cigarette Smoking as Risk Factor for Nonsyndromic Cleft Lip with or without Cleft Palate Renato Assis Machado, Helenara Salvati Bertolossi Moreira, Sibele Nascimento de Aquino, Hercilio Martelli-Junior, Silvia Regina de Almeida Reis, Darlene Camati Persuhn, Tao Wu, Yuan Yuan, and Ricardo D. Coletta* Department of Oral Diagnosis, School of Dentistry, State University of Campinas, Piracicaba, S~ao Paulo, Brazil Department of Physiotherapy, State University of Western Paran a, Paran a, Brazil Stomatology Clinic, Dental School, State University of Montes Claros, Montes Claros, Minas Gerais, Brazil Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Jos e Ros ario Vellano, Minas Gerais, Brazil Department of Basic Science, Bahiana School of Medicine and Public Health, Salvador, Bahia, Brazil Molecular Biology Department, Federal University of Paraı́ba, Jo~ao Pessoa, Paraı́ba, Brazil Peking University School of Public Health, Beijing, China

Brazilian multicenter study of association between polymorphisms in CRISPLD2 and JARID2 and non-syndromic oral clefts

BACKGROUND Variants in the cysteine-rich secretory protein LCCL domain containing 2 gene (CRISPLD2) and in the jumonji, AT-rich interaction domain 2 gene (JARID2) were previously shown to influence non-syndromic oral cleft susceptibility. Herein, we performed a case-control study to examine the potential association of single-nucleotide polymorphisms (SNPs) in CRISPLD2 and JARID2 with non-syndromic cleft lip and/or palate (NSCL/P) in the Brazilian population. Given the ethnicity-dependent genetic predisposition to NSCL/P, we performed a structured analysis taking into account the genomic ancestry variation of each individual. METHODS Four SNPs in CRISPLD2 (rs1546124, rs8061351, rs2326398, and rs4783099) and four in JARID2 (rs915344, rs2299043, rs2237138, and rs2076056), that were previously reported to be associated with NSCL/P, were genotyped in 785 Brazilian patients with NSCL/P (549 with cleft lip with or without cleft palate-NSCL ± P, and 236 with cleft palate only-NSCPO) and 693 unaffected Brazilian controls. Genomic ancestry was assessed with a set of 40 biallelic short insertion/deletion variants previously validated as ancestry informative markers of the Brazilian population. RESULTS After adjustment of ancestry variations, allelic analysis revealed marginal associations between the CRISPLD2 rs4783099 T allele and increased risk for NSCPO (OR: 1.31, 95% CI: 1.05-1.62, P = 0.01) and between JARID2 rs2237138 and decreased NSCL ± P risk (OR: 0.80, 95% CI: 0.67-0.97, P = 0.02). Haplotype analysis indicated a lack of association between JARID2 haplotypes and non-syndromic oral cleft risk. CONCLUSIONS Our results suggest that CRISPLD2 rs4783099 may represent a risk factor for NSCPO while JARID2 rs2237138 shows a protective effect against NSCL ± P in the Brazilian population.

Association Between Genes Involved in Craniofacial Development and Nonsyndromic Cleft Lip and/or Palate in the Brazilian Population

Objective To determine the association of single-nucleotide polymorphisms (SNPs) in genes related to craniofacial development, which were previously identified as susceptibility signals for nonsyndromic oral clefts, in Brazilians with nonsyndromic cleft lip and/or palate (NSCL/P). Design The SNPs rs748044 (TNP1), rs1106514 (MSX1), rs28372960, rs15251 and rs2569062 (TCOF1), rs7829058 (FGFR1), rs1793949 (COL2A1), rs11653738 (WNT3), and rs242082 (TIMP3) were assessed in a family-based transmission disequilibrium test (TDT) and a structured case-control analysis based on the individual ancestry proportions. Setting The SNPs were initially analyzed by TDT, and polymorphisms showing a trend toward excess transmission were subsequently studied in an independent case-control sample. Participants The study sample consisted of 189 case-parent trios of nonsyndromic cleft lip with or without cleft palate (NSCL±P), 107 case-parent trios of nonsyndromic cleft palate (NSCP), 318 isolated samples of NSCL±P, 189 isolated samples of NSCP, and 599 healthy controls. Main Outcome Measure Association of alleles with NSCL/P pathogenesis. Results Preferential transmission of SNPs rs28372960 and rs7829058 in NSCL±P trios and rs11653738 in NSCP trios (P = .04) were observed, although the structured case-control analysis did not confirm these associations. The haplotype T-C-C formed by TCOF1 SNPs rs28372960, rs15251, and rs2569062 was more frequently transmitted from healthy parents to NSCL±P offspring, but the P value (P = .01) did not withstand Bonferroni correction for multiple tests. Conclusions With the modest associations, our results do not support the hypothesis that TNP1, MSX1, TCOF1, FGFR1, COL2A1, WNT3, and TIMP3 variants are risk factors for nonsyndromic oral clefts in the Brazilian population.

Clinical relevance of breast and gastric cancer-associated polymorphisms as potential susceptibility markers for oral clefts in the Brazilian population

BackgroundEpidemiological studies have indicated a higher incidence of breast and gastric cancer in patients with nonsyndromic cleft lip with or without cleft palate (NSCL ± P) and their relatives, which can be based on similar genetic triggers segregated within family with NSCL ± P.MethodsThis multicenter study evaluated the association of 9 single nucleotide polymorphisms (SNP) in AXIN2 and CDH1, representing genes consistently altered in breast and gastric tumors, with NSCL ± P in 223 trios (father, mother and patient with NSCL ± P) by transmission disequilibrium test (TDT).ResultsOur results showed that the minor A allele of rs7210356 (p = 0.01) and the T-G-G-A-G haplotype formed by rs7591, rs7210356, rs4791171, rs11079571 and rs3923087 SNPs (p = 0.03) in AXIN2 were significantly under-transmitted to patients with NSCL ± P. In CDH1 gene, the C-G-A-A and A-G-A-G haplotypes composed by rs16260, rs9929218, rs7186053 and rs4783573 polymorphisms were respectively over-transmitted (p = 0.01) and under-transmitted (p = 0.008) from parents to the children with NSCL ± P.ConclusionsThe results suggest that polymorphic variants in AXIN2 and CDH1 may be associated with NSCL ± P susceptibility, and reinforce the putative link between cancer and oral clefts.

Potential genetic markers for nonsyndromic oral clefts in the Brazilian population: A systematic review and meta-analysis

Although various genes and genomic regions were described as of susceptibility for nonsyndromic oral clefts (NOC), recent reports have demonstrated significant interethnic variations in the genetic predisposition, a situation that affects the Brazilian population, one of the most admixed populations in the world. Therefore, the purpose of this review was to describe the available information on genetic risk markers for NOC in the Brazilian population.

Computational analysis for GNAQ mutations: New insights on the molecular etiology of Sturge-Weber syndrome

Somatic activating mutations in the GNAQ have been recently associated with several congenital genetic disorders and tumors; however, the molecular mechanism/etiology that leads to GNAQ somatic mosaic mutation are unknown. Here, we reported a case of Sturge-Weber Syndrome (SWS) manifesting cutaneous vascular malformations (hemifacial Port-wine stain), cerebral and ocular vascular abnormalities (including epilepsy and glaucoma) and harboring a c.548G>A (p.R183Q) somatic mosaic mutation in GNAQ. Computational modeling studies were performed to assistant with the comprehension of the functional impact of p.R183Q and p.Q209L mutations in GNAQ, which encodes a G protein subunit alpha q (Gαq). The p.R183Q mutation was predicted to abolish hydrogen bonds between R183 residue and GDP molecule, destabilizing the inactive GDP-bound conformation of the Gαq mutants. Furthermore, replacement of R183 by Q183 residue was predicted to promote conformation changes in protein surface features affecting the switch I region, a key region that undergoes conformational changes triggered by receptor binding during signal transduction. In addition, replacement of Q209 by L209 residue was predicted to affect the molecular interaction between Gαq and Gβ subunit, impairing formation of the inactive heterotrimeric complex. These findings, in association with PPI network analysis, indicate that p.R183Q and p.Q209L mutations result in the over-activation of different downstream effectors, which in turn will determine the distinct cell responses and phenotype. These findings bring new insights on molecular etiology of vascular malformations associated to SWS and on different mechanisms underlying hyperactivation of downstream pathways to Gαq.

EDA mutation by exome sequencing in non-syndromic X-linked oligodontia

To the Editor: Oligodontia is a severe form of selective tooth agenesis (STHAG), a human condition characterized by failure of development of multiple teeth during organogenesis, which can be inherited as an isolated trait or as part of a recognized genetic syndrome. STHAG exhibits high genetic heterogeneity, as result of a number of mutations in genes known to regulate tooth development (1), and many aspects underlying its genetic causes remain elusive. Here, we used a whole-exome sequencing (WES) strategy followed by in silico prediction tools and 3D structure analysis to determine genomic variants associate with non-syndromic oligodontia in an 8-year-old Brazilian Caucasian boy. This study was approved by the Piracicaba Dental School – Institutional Review Board. Detailed clinical and WES information are available at PhenomeCentral (P0003267; https://phenomecentral.org/). Candidate variants were identified after filtering for rare variants (MAF< 1%), protein-coding regions (including non-sense, non-synonymous variants and Indels), splice-site variants absent from public datasets, and putative inheritance pattern (autosomal dominant and X-linked). Next, considering the role of EDA (MIM#300451) and TBX22 (MIM#300307) in craniofacial and tooth development anomalies, we performed mutational analysis by Sanger sequencing to validate segregation patterns for variants in EDA c.1013C>T (p.T338M) and TBX22 c.1033T>C (p.S345P) in 10 related family members. A positive association between STHAG phenotype and segregation patterns for EDA and TBX22 mutations was observed in this family, confirming an X-linked dominant inheritance pattern (Fig. 1a). However, in silico prediction only supported the variant in EDA (c.1013C>T; dbSNP ID rs132630321) as pathogenic (ClinVar ID 11048), deleterious (SIFT score= 0), probably damaging (PolyPhen= 0.974) and probably deleterious (MutationTaster= 0.99997), whereas the variant in TBX22 was found to be tolerated (SIFT score= 1), benign (PolyPhen= 0) and probably harmless (MutationTaster= 0.00005). Phylogenetic analysis supported these findings (Fig. 1b,c). Here, clinical findings are similar to a previous study on a Chinese family, where the affected male, who bearing only one copy of c.1013C>T mutation (hemizygous), showed a more severe phenotype (2). In addition, however, we found that heterozygous female carriers for this EDA mutation (Fig. 1a) may present a highly variable and milder dental phenotype. Against our hypothesis, the novel variant in TBX22 c.1033 T>C (p.S345P), identified in this study, was not critical for the broad phenotypic heterogeneity displayed by heterozygous female carriers, pointing to EDA as the solely causative gene of the congenitally missing teeth in this family. Recent studies have provided evidence that mutation in the TNF homology domain of ectodysplasin A (EDA) is implicated in familial non-syndromic STHAG, and heterozygous female carriers of EDA mutations may present STHAG with a considerable variation in clinical expression as result of differential pattern of X-chromosome inactivation (3), or still as result of modifier effects in other signaling pathways regulated by EDA-1A (4). The EDA gene is located on chromosome Xq13.1 and encodes isoforms of EDA. Among the isoforms, EDA-A1 (Q92838) is a trimeric type II membrane protein, and a collagen domain-containing member of the superfamily of the TNF ligands, involved in the early epithelial–mesenchymal interactions, and playing a critical role in tooth development. Here, analysis of the tertiary structure of EDA-A1 protein showed that the T338 residue is located in coil structure (corresponding to 337–342 amino acid) within the phylogenetically conserved TNF homology domain, on the outer surface of the molecule (Fig. 1c-e). Replacement of T338 native (hydrophilic) by an M338 mutated residue (hydrophobic) was predicted to alter the conformation of the 337–342 loop leading to a potential impact on EDA-A1 receptor binding properties (Fig. 1d-e). Furthermore, residue interaction analysis indicated that p.T338M mutation might lead to an additional hydrogen bond between chain B (T341) and chain C (D316) (Fig. 1g), suggesting that such mutation has the potential to impact on homotrimer stability. Moreover, because our in silico prediction analysis showed that residues within the 337–342 loop form hydrophobic interaction and hydrogen bonds, with other molecule regions (such as I312 and F314) critical for controlling receptor specificity (Fig. 1f-g) (5), our findings bring new evidences about the role of p.T338M mutation in the recognition/specificity of EDA to its receptor, resulting in a high heterogeneous STHAG phenotype.

Clinicopathological significance of SNPs in RAD51 and XRCC3 in oral and oropharyngeal carcinomas

BACKGROUND This study investigated the influence of single nucleotide polymorphisms (SNP) in RAD51 and XRCC3 on susceptibility to oral and oropharyngeal squamous cell carcinomas (SCC) and determined their clinicopathological significance. SUBJECTS AND METHODS SNPs rs1801320 and rs1801321 in RAD51 and rs861539 in XRCC3 were genotyped in 81 patients presenting oral SCC, 45 presenting oropharyngeal SCC, and 130 healthy controls, using TaqMan allelic discrimination assays. Multiple logistic regression models were used to explore the association between SNPs and cancer development, as well as gene-gene (GxG) interaction and gene-environmental factor (GxE) interaction. Clinicopathological associations were verified through the chi-square test, and univariate and multivariate methods were applied for survival analyses. RESULTS Although allelic and genotypic models and the GxG interaction analysis were nonsignificant, the GxE analysis revealed synergistic effects of the risk alleles of rs1801320, rs1801321, and rs861539 with smoking and alcohol consumption on susceptibility to oral and oropharyngeal SCC. Furthermore, oropharyngeal SCC patients carrying the XRCC3 rs861539 GT/TT genotype (T risk allele) presented a shorter overall survival than GG genotype carriers. CONCLUSION Combined effects of RAD51 (rs1801320 and rs1801321) and XRCC3 (rs861539) SNPs with environmental carcinogens (tobacco and alcohol) are associated with oral and oropharyngeal SCC development.

Interactions between superoxide dismutase and paraoxonase polymorphic variants in nonsyndromic cleft lip with or without cleft palate in the Brazilian population: Interactions between SNPs in SODs and PONs in nonsyndromic oral clefts

During development, oxidative stress is hypothesized to mediate embryotoxicity, which may be intensified by exposition to environmental factors and by genetic variations in the enzymes involved in protecting cells from these damaging effects, including superoxide dismutase (SOD) and paraoxonase (PON). The aim of this study was to evaluate the influence of single‐nucleotide polymorphisms (SNP) in genes associated with the neutralization of oxidative stress (SOD and PON family members) in the risk of nonsyndromic oral cleft in the Brazilian population. Initially, we tested for association between 28 SNP in SOD1, SOD2, SOD3, PON1, PON2, and PON3 among 325 nonsyndromic cleft lip with or without cleft palate (NSCL±P) case‐parent trios. Multiple logistic regression analyses were used to explore gene, GxG and GxE, involving factors that induce oxidative stress accumulation during pregnancy. Signals still significant after both Bonferroni correction and in permutation test were subsequently confirmed in an ancestry‐structured case–control analysis with 722 NSCL±P and 866 controls from the same population. In the trio sample, transmission disequilibrium test (TDT) (allele and haplotype) and GxE analysis showed no significant associations, but multiple pairwise GxG interactions involving 10 SNP in PON1, PON2, and PON3 were detected and further examined in the case–control sample. The PON1 rs2237583 and PON2 rs17166879 yielded significant evidence of SNP‐SNP interactions after adjustment for multiple tests (both Bonferroni correction and 10,000 permutation test). The C allele and the CT genotype of PON1 rs2237583 were associated with significant protective effects against NSCL±P, while rs3917490 showed a significant association only in the sample composed of patients displaying high African ancestry. Our results reveal associations between rs2237583 and rs3917490 in PON1 and GxG interactions containing rs2237583 and rs17166879 with the susceptibility of NSCL±P in the Brazilian population. Furthermore, this study underlines the recent tendency of taking into account potential GxG interactions to clarify the underlying mechanisms associated with the etiology of this common malformation. Environ. Mol. Mutagen. 60: 185–196, 2019.