Lucas Alvizi

Susceptibility to DNA damage as a molecular mechanism for non-syndromic cleft lip and palate

Non-syndromic cleft lip/palate (NSCL/P) is a complex, frequent congenital malformation, determined by the interplay between genetic and environmental factors during embryonic development. Previous findings have appointed an aetiological overlap between NSCL/P and cancer, and alterations in similar biological pathways may underpin both conditions. Here, using a combination of transcriptomic profiling and functional approaches, we report that NSCL/P dental pulp stem cells exhibit dysregulation of a co-expressed gene network mainly associated with DNA double-strand break repair and cell cycle control (p = 2.88×10−2–5.02×10−9). This network included important genes for these cellular processes, such as BRCA1, RAD51, and MSH2, which are predicted to be regulated by transcription factor E2F1. Functional assays support these findings, revealing that NSCL/P cells accumulate DNA double-strand breaks upon exposure to H2O2. Furthermore, we show that E2f1, Brca1 and Rad51 are co-expressed in the developing embryonic orofacial primordia, and may act as a molecular hub playing a role in lip and palate morphogenesis. In conclusion, we show for the first time that cellular defences against DNA damage may take part in determining the susceptibility to NSCL/P. These results are in accordance with the hypothesis of aetiological overlap between this malformation and cancer, and suggest a new pathogenic mechanism for the disease.

Rare Variants in the Epithelial Cadherin Gene Underlying the Genetic Etiology of Nonsyndromic Cleft Lip with or without Cleft Palate

Nonsyndromic orofacial cleft (NSOFC) is a complex disease of still unclear genetic etiology. To investigate the contribution of rare epithelial cadherin (CDH1) gene variants to NSOFC, we target sequenced 221 probands. Candidate variants were evaluated via in vitro, in silico, or segregation analyses. Three probably pathogenic variants (c.760G>A [p.Asp254Asn], c.1023T>G [p.Tyr341*], and c.2351G>A [p.Arg784His]) segregated according to autosomal dominant inheritance in four nonsyndromic cleft lip with or without cleft palate (NSCL/P) families (Lod score: 5.8 at θ = 0; 47% penetrance). A fourth possibly pathogenic variant (c.387+5G>A) was also found, but further functional analyses are needed (overall prevalence of CDH1 candidate variants: 2%; 15.4% among familial cases). CDH1 mutational burden was higher among probands from familial cases when compared to that of controls (P = 0.002). We concluded that CDH1 contributes to NSCL/P with mainly rare, moderately penetrant variants, and CDH1 haploinsufficiency is the likely etiological mechanism.

Stem Cells as a Good Tool to Investigate Dysregulated Biological Systems in Autism Spectrum Disorders

Identification of the causes of autism spectrum disorders (ASDs) is hampered by their genetic heterogeneity; however, the different genetic alterations leading to ASD seem to be implicated in the disturbance of common molecular pathways or biological processes. In this scenario, the search for differentially expressed genes (DEGs) between ASD patients and controls is a good alternative to identify the molecular etiology of such disorders. Here, we employed genome‐wide expression analysis to compare the transcriptome of stem cells of human exfoliated deciduous teeth (SHEDs) of idiopathic autistic patients (n = 7) and control samples (n = 6). Nearly half of the 683 identified DEGs are expressed in the brain (P = 0.003), and a significant number of them are involved in mechanisms previously associated with ASD such as protein synthesis, cytoskeleton regulation, cellular adhesion and alternative splicing, which validate the use of SHEDs to disentangle the causes of autism. Autistic patients also presented overexpression of genes regulated by androgen receptor (AR), and AR itself, which in turn interacts with CHD8 (chromodomain helicase DNA binding protein 8), a gene recently shown to be associated with the cause of autism and found to be upregulated in some patients tested here. These data provide a rationale for the mechanisms through which CHD8 leads to these diseases. In summary, our results suggest that ASD share deregulated pathways and revealed that SHEDs represent an alternative cell source to be used in the understanding of the biological mechanisms involved in the etiology of ASD. Autism Res 2013, ●●: ●●–●●.

Differential methylation is associated with non-syndromic cleft lip and palate and contributes to penetrance effects

Non-syndromic cleft lip and/or palate (NSCLP) is a common congenital malformation with a multifactorial model of inheritance. Although several at-risk alleles have been identified, they do not completely explain the high heritability. We postulate that epigenetic factors as DNA methylation might contribute to this missing heritability. Using a Methylome-wide association study in a Brazilian cohort (67 NSCLP, 59 controls), we found 578 methylation variable positions (MVPs) that were significantly associated with NSCLP. MVPs were enriched in regulatory and active regions of the genome and in pathways already implicated in craniofacial development. In an independent UK cohort (171 NSCLP, 177 controls), we replicated 4 out of 11 tested MVPs. We demonstrated a significant positive correlation between blood and lip tissue DNA methylation, indicating blood as a suitable tissue for NSCLP methylation studies. Next, we quantified CDH1 promoter methylation levels in CDH1 mutation-positive families, including penetrants, non-penetrants or non-carriers for NSCLP. We found methylation levels to be significantly higher in the penetrant individuals. Taken together, our results demonstrated the association of methylation at specific genomic locations as contributing factors to both non-familial and familial NSCLP and altered DNA methylation may be a second hit contributing to penetrance.

Impact of rare variants in ARHGAP29 to the etiology of oral clefts: role of loss‐of‐function vs missense variants

Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is a prevalent, complex congenital malformation. Genome‐wide association studies (GWAS) on NSCL/P have consistently identified association for the 1p22 region, in which ARHGAP29 has emerged as the main candidate gene. ARHGAP29 re‐sequencing studies in NSCL/P patients have identified rare variants; however, their clinical impact is still unclear. In this study we identified 10 rare variants in ARHGAP29, including five missense, one in‐frame deletion, and four loss‐of‐function (LoF) variants, in a cohort of 188 familial NSCL/P cases. A significant mutational burden was found for LoF (Sequence Kernel Association Test, p = 0.0005) but not for missense variants in ARHGAP29, suggesting that only LoF variants contribute to the etiology of NSCL/P. Penetrance was estimated as 59%, indicating that heterozygous LoF variants in ARHGAP29 confer a moderate risk to NSCL/P. The GWAS hits in IRF6 (rs642961) and 1p22 (rs560426 and rs4147811) do not seem to contribute to the penetrance of the phenotype, based on co‐segregation analysis. Our data show that rare variants leading to haploinsufficiency of ARHGAP29 represent an important etiological clefting mechanism, and genetic testing for this gene might be taken into consideration in genetic counseling of familial cases.

Richieri-Costa-Pereira syndrome: Expanding its phenotypic and genotypic spectrum

Richieri‐Costa‐Pereira syndrome is a rare autosomal recessive acrofacial dysostosis that has been mainly described in Brazilian individuals. The cardinal features include Robin sequence, cleft mandible, laryngeal anomalies and limb defects. A biallelic expansion of a complex repeated motif in the 5′ untranslated region of EIF4A3 has been shown to cause this syndrome, commonly with 15 or 16 repeats. The only patient with mild clinical findings harbored a 14‐repeat expansion in 1 allele and a point mutation in the other allele. This proband is described here in more details, as well as is his affected sister, and 5 new individuals with Richieri‐Costa‐Pereira syndrome, including a patient from England, of African ancestry. This study has expanded the phenotype in this syndrome by the observation of microcephaly, better characterization of skeletal abnormalities, less severe phenotype with only mild facial dysmorphisms and limb anomalies, as well as the absence of cleft mandible, which is a hallmark of the syndrome. Although the most frequent mutation in this study was the recurrent 16‐repeat expansion in EIF4A3, there was an overrepresentation of the 14‐repeat expansion, with mild phenotypic expression, thus suggesting that the number of these motifs could play a role in phenotypic delineation.

Genetic Analyses in Small-for-Gestational-Age Newborns

Context Small for gestational age (SGA) can be the result of fetal growth restriction, which is associated with perinatal morbidity and mortality. Mechanisms that control prenatal growth are poorly understood. Objective The aim of the current study was to gain more insight into prenatal growth failure and determine an effective diagnostic approach in SGA newborns. We hypothesized that one or more copy number variations (CNVs) and disturbed methylation and sequence variants may be present in genes associated with fetal growth. Design A prospective cohort study of subjects with a low birth weight for gestational age. Setting The study was conducted at an academic pediatric research institute. Patients A total of 21 SGA newborns with a mean birth weight below the first centile and a control cohort of 24 appropriate-for-gestational-age newborns were studied. Interventions Array comparative genomic hybridization, genome-wide methylation studies, and exome sequencing were performed. Main Outcome Measures The numbers of CNVs, methylation disturbances, and sequence variants. Results The genetic analyses demonstrated three CNVs, one systematically disturbed methylation pattern, and one sequence variant explaining SGA. Additional methylation disturbances and sequence variants were present in 20 patients. In 19 patients, multiple abnormalities were found. Conclusion Our results confirm the influence of a large number of mechanisms explaining dysregulation of fetal growth. We concluded that CNVs, methylation disturbances, and sequence variants all contribute to prenatal growth failure. These genetic workups can be an effective diagnostic approach in SGA newborns.

DNA methylation mediates genetic liability to non-syndromic cleft lip/palate

Background Non-syndromic cleft lip/palate (nsCL/P) is a complex trait with genetic and environmental risk factors. Around 40 distinct genetic risk loci have been identified for nsCL/P, but many reside in non-protein-coding regions with an unclear function. We hypothesised that one possibility is that the genetic risk variants influence susceptibility to nsCL/P through gene regulation pathways, such as those involving DNA methylation. Methods Using nsCL/P Genome-wide association study summary data and methylation data from four studies, we used Mendelian randomization and joint likelihood mapping to identify putative loci where genetic liability to nsCL/P may be mediated by variation in DNA methylation in blood. Results There was evidence at three independent loci, VAX1 (10q25.3), LOC146880 (17q23.3) and NTN1 (17p13.1), that liability to nsCL/P and variation in DNA methylation might be driven by the same genetic variant. Follow up analyses using DNA methylation data, derived from lip and palate tissue, and gene expression catalogues provided further insight into possible biological mechanisms. Conclusions Genetic variation may increase liability to nsCL/P by influencing DNA methylation and gene expression at VAX1, LOC146880 and NTN1.

Genetics of Cleft Lip and Cleft Palate: Perspectives in Surgery Management and Outcome

Clefts of the lip (with or without palate) and of the palate (CLP) comprise a complex, clinically, and etiologically heterogeneous group of craniofacial malformations that affect around 1:700 live births (Dixon et al. 2011). Expressivity varies from mild defects, such as discreet orbicularis oris muscle discontinuity and submucous cleft palate, to more severe phenotypes in which several orofacial tissues are affected. Patients afflicted by CLP often require extensive treatment for their functional rehabilitation and social integration, as they are subject to several surgical interventions and management by a multidisciplinary team for many years (Hamm and Robin 2015). Given the high incidence of CLP and the important psychosocial and health care burden it entails, understanding the etiology of this disorder is of utmost importance, as it may lead to the development or improvement of preventive and therapeutic strategies.

Publisher Correction: Discordant congenital Zika syndrome twins show differential in vitro viral susceptibility of neural progenitor cells

The original PDF version of this Article contained errors in the spelling of Luiz Carlos Caires-Júnior, Uirá Souto Melo, Bruno Henrique Silva Araujo, Alessandra Soares-Schanoski, Murilo Sena Amaral, Kayque Alves Telles-Silva, Vanessa van der Linden, Helio van der Linden, João Ricardo Mendes de Oliveira, Nivia Maria Rodrigues Arrais, Joanna Goes Castro Meira, Ana Jovina Barreto Bispo, Esper Abrão Cavalheiro, and Robert Andreata-Santos, which were incorrectly given as Luiz Carlos de Caires Jr., UiráSouto Melo, Bruno Silva Henrique Araujo, Alessandra Soares Schanoski, MuriloSena Amaral, Kayque Telles Alves Silva, Vanessa Van der Linden, Helio Van der Linden, João Mendes Ricardo de Oliveira, Nivia Rodrigues Maria Arrais, Joanna Castro Goes Meira, Ana JovinaBarreto Bispo, EsperAbrão Cavalheiro, and Robert Andreata Santos. Furthermore, in both the PDF and HTML versions of the Article, the top panel of Fig. 3e was incorrectly labeled ‘10608-1’ and should have been ‘10608-4’, and financial support from CAPES and DECIT-MS was inadvertently omitted from the Acknowledgements section. These errors have now been corrected in both the PDF and HTML versions of the Article.