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1.
Epigenomics ; 11(2): 133-145, 2019 02.
Article En | MEDLINE | ID: mdl-30638414

AIM: To determine if nonsyndromic cleft lip with or without cleft palate (nsCL/P) genetic risk variants influence liability to nsCL/P through gene regulation pathways, such as those involving DNA methylation. MATERIALS & METHODS: nsCL/P genetic summary data and methylation data from four studies were used in conjunction with Mendelian randomization and joint likelihood mapping to investigate potential mediation of nsCL/P genetic variants. RESULTS & CONCLUSION: Evidence was found at 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, suggesting that genetic variation at these loci may increase liability to nsCL/P by influencing DNA methylation. Follow-up analyses using different tissues and gene expression data provided further insight into possible biological mechanisms.


Cleft Lip/genetics , Cleft Palate/genetics , DNA Methylation , Genetic Variation , Child , Cleft Lip/pathology , Cleft Palate/pathology , Epigenesis, Genetic , Female , Homeodomain Proteins/genetics , Humans , Male , Mendelian Randomization Analysis , Netrin-1/genetics , Transcription Factors/genetics
2.
Front Genet ; 9: 149, 2018.
Article En | MEDLINE | ID: mdl-29922329

Repeats in coding and non-coding regions have increasingly been associated with many human genetic disorders, such as Richieri-Costa-Pereira syndrome (RCPS). RCPS, mostly characterized by midline cleft mandible, Robin sequence and limb defects, is an autosomal-recessive acrofacial dysostosis mainly reported in Brazilian patients. This disorder is caused by decreased levels of EIF4A3, mostly due to an increased number of repeats at the EIF4A3 5'UTR. EIF4A3 5'UTR alleles are CG-rich and vary in size and organization of three types of motifs. An exclusive allelic pattern was identified among affected individuals, in which the CGCA-motif is the most prevalent, herein referred as "disease-associated CGCA-20nt motif." The origin of the pathogenic alleles containing the disease-associated motif, as well as the functional effects of the 5'UTR motifs on EIF4A3 expression, to date, are entirely unknown. Here, we characterized 43 different EIF4A3 5'UTR alleles in a cohort of 380 unaffected individuals. We identified eight heterozygous unaffected individuals harboring the disease-associated CGCA-20nt motif and our haplotype analyses indicate that there are more than one haplotype associated with RCPS. The combined analysis of number, motif organization and haplotypic diversity, as well as the observation of two apparently distinct haplotypes associated with the disease-associated CGCA-20nt motif, suggest that the RCPS alleles might have arisen from independent unequal crossing-over events between ancient alleles at least twice. Moreover, we have shown that the number and sequence of motifs in the 5'UTR region is associated with EIF4A3 repression, which is not mediated by CpG methylation. In conclusion, this study has shown that the large number of repeats in EIF4A3 does not represent a dynamic mutation and RCPS can arise in any population harboring alleles with the CGCA-20nt motif. We also provided further evidence that EIF4A3 5'UTR is a regulatory region and the size and sequence type of the repeats at 5'UTR may contribute to clinical variability in RCPS.

3.
Nat Commun ; 9(1): 1114, 2018 03 13.
Article En | MEDLINE | ID: mdl-29535311

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.

4.
Nat Commun ; 9(1): 475, 2018 02 02.
Article En | MEDLINE | ID: mdl-29396410

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.


Brain/embryology , Gene Expression , Neural Stem Cells/metabolism , Twins, Dizygotic , Zika Virus Infection/congenital , Brain/metabolism , Brain/virology , Brazil , Case-Control Studies , Female , Genetic Predisposition to Disease , High-Throughput Nucleotide Sequencing , Humans , Induced Pluripotent Stem Cells , Infant , Infant, Newborn , Male , Neural Stem Cells/virology , Sequence Analysis, RNA , TOR Serine-Threonine Kinases/genetics , Wnt Signaling Pathway/genetics , Zika Virus Infection/genetics , Zika Virus Infection/virology
5.
J Clin Endocrinol Metab ; 103(3): 917-925, 2018 03 01.
Article En | MEDLINE | ID: mdl-29342293

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.


Birth Weight/genetics , Fetal Growth Retardation/genetics , Infant, Small for Gestational Age , Comparative Genomic Hybridization , DNA Copy Number Variations , DNA Methylation , Female , Genetic Predisposition to Disease , Genome-Wide Association Study , Gestational Age , Humans , Infant, Newborn , Male , Prospective Studies , Whole Exome Sequencing/methods
6.
Nat. Commun. ; 9: 475, 2018.
Article En | SES-SP, SESSP-IBPROD, SES-SP | ID: but-ib14938

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.

7.
Nat Commun, v. 9, 475, fev. 2018
Article En | SES-SP, SESSP-IBPROD, SES-SP | ID: bud-2422

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.

8.
Sci Rep ; 7(1): 2441, 2017 05 26.
Article En | MEDLINE | ID: mdl-28550290

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.


Cleft Lip/genetics , Cleft Palate/genetics , DNA Methylation , Penetrance , Antigens, CD/genetics , Brazil , Cadherins/genetics , Child , Child, Preschool , Cleft Lip/pathology , Cleft Palate/pathology , Cohort Studies , CpG Islands/genetics , Female , Genetic Predisposition to Disease/genetics , Humans , Male , Promoter Regions, Genetic/genetics
9.
Hum Mutat ; 36(11): 1029-33, 2015 Nov.
Article En | MEDLINE | ID: mdl-26123647

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.


Brain/abnormalities , Cadherins/genetics , Cleft Lip/genetics , Cleft Palate/genetics , Genetic Variation , Alleles , Amino Acid Substitution , Animals , Antigens, CD , Cadherins/chemistry , Cell Line , Cleft Lip/diagnosis , Cleft Palate/diagnosis , DNA Mutational Analysis , Genotype , Germ-Line Mutation , Humans , Mutation , Open Reading Frames , Penetrance
10.
Stem Cell Rev Rep ; 11(4): 635-44, 2015 Aug.
Article En | MEDLINE | ID: mdl-25931278

Mesenchymal stem cell (MSC) osteogenic differentiation potential varies according to factors such as tissue source and cell population heterogeneity. Pre-selection of cell subpopulations harboring higher osteopotential is a promising strategy to achieve a thorough translation of MSC-based therapies to the clinic. Here, we searched for novel molecular markers predictive of osteopotential by comparing MSC populations from two sources harboring different osteogenic potentials. We show that MSCs from human deciduous teeth (SHED) have an intrinsically higher osteogenic potential when compared with MSCs from human adipose tissue (hASCs) under the same in vitro controlled induction system. Transcriptome profiling revealed IGF2 to be one of the top upregulated transcripts before and during early in vitro osteogenic differentiation. Further, exogenous IGF2 supplementation enhanced alkaline phosphatase activity and matrix mineralization, and inhibition of IGF2 lessened these parameters in SHED and hASCs, validating IGF2 as an osteogenic factor in these MSCs. Further, we found IGF2 to be biallelically expressed in SHED, but not in hASCs. We observed a 4 % methylation increase in the imprinting control region within the IGF2-H19 locus in SHED, and this is mainly due to 2 specific CpG sites. Thus, we suggest that IGF2 upregulation in SHED is due to loss of imprinting. This study unravels osteogenic properties in SHED, implying IGF2 as a potential biomarker of MSCs with higher osteopotential, and unveils IGF2 loss-of-imprinting in SHED.


Adipose Tissue/metabolism , Cell Differentiation/genetics , Dental Pulp/metabolism , Insulin-Like Growth Factor II/genetics , Osteogenesis/genetics , Adipose Tissue/cytology , Adult , Aged , Base Sequence , Benzopyrans/pharmacology , Blotting, Western , Child , Cluster Analysis , Dental Pulp/cytology , Gene Expression Profiling/methods , Humans , Insulin-Like Growth Factor II/antagonists & inhibitors , Insulin-Like Growth Factor II/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Middle Aged , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Transcriptional Activation/drug effects
11.
Am J Hum Genet ; 94(1): 120-8, 2014 Jan 02.
Article En | MEDLINE | ID: mdl-24360810

Richieri-Costa-Pereira syndrome is an autosomal-recessive acrofacial dysostosis characterized by mandibular median cleft associated with other craniofacial anomalies and severe limb defects. Learning and language disabilities are also prevalent. We mapped the mutated gene to a 122 kb region at 17q25.3 through identity-by-descent analysis in 17 genealogies. Sequencing strategies identified an expansion of a region with several repeats of 18- or 20-nucleotide motifs in the 5' untranslated region (5' UTR) of EIF4A3, which contained from 14 to 16 repeats in the affected individuals and from 3 to 12 repeats in 520 healthy individuals. A missense substitution of a highly conserved residue likely to affect the interaction of eIF4AIII with the UPF3B subunit of the exon junction complex in trans with an expanded allele was found in an unrelated individual with an atypical presentation, thus expanding mutational mechanisms and phenotypic diversity of RCPS. EIF4A3 transcript abundance was reduced in both white blood cells and mesenchymal cells of RCPS-affected individuals as compared to controls. Notably, targeting the orthologous eif4a3 in zebrafish led to underdevelopment of several craniofacial cartilage and bone structures, in agreement with the craniofacial alterations seen in RCPS. Our data thus suggest that RCPS is caused by mutations in EIF4A3 and show that EIF4A3, a gene involved in RNA metabolism, plays a role in mandible, laryngeal, and limb morphogenesis.


Clubfoot/genetics , DEAD-box RNA Helicases/genetics , Eukaryotic Initiation Factor-4A/genetics , Hand Deformities, Congenital/genetics , Pierre Robin Syndrome/genetics , Alleles , Amino Acid Sequence , Animals , Bone and Bones/abnormalities , Child , Child, Preschool , Chromosome Mapping , DEAD-box RNA Helicases/metabolism , Eukaryotic Initiation Factor-4A/metabolism , Female , Humans , Male , Molecular Sequence Data , Mutation, Missense , Protein Conformation , Zebrafish/abnormalities
12.
PLoS One ; 8(6): e65677, 2013.
Article En | MEDLINE | ID: mdl-23776525

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.


Cleft Lip/genetics , Cleft Palate/genetics , DNA Breaks, Double-Stranded , Dental Pulp/cytology , Gene Regulatory Networks/genetics , Stem Cells/metabolism , BRCA1 Protein/genetics , Cell Cycle/genetics , Cells, Cultured , Child , DNA Primers/genetics , DNA Repair/genetics , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Flow Cytometry , Gene Expression Profiling , Humans , In Situ Hybridization , MutS Homolog 2 Protein/genetics , Protein Array Analysis , Rad51 Recombinase/genetics , Real-Time Polymerase Chain Reaction
13.
Autism Res ; 6(5): 354-61, 2013 Oct.
Article En | MEDLINE | ID: mdl-23801657

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.


Child Development Disorders, Pervasive/genetics , Dental Pulp/cytology , Gene Expression Profiling , Genetic Association Studies , Genome-Wide Association Study , Stem Cells/physiology , Transcriptome/genetics , Alternative Splicing/genetics , Cell Adhesion/genetics , Child , Cytoskeleton/genetics , DNA-Binding Proteins/genetics , Genetic Predisposition to Disease/genetics , Humans , Oligonucleotide Array Sequence Analysis , Protein Biosynthesis/genetics , Transcription Factors/genetics
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