Luciano Abreu Brito

Region 8q24 is a susceptibility locus for nonsyndromic oral clefting in Brazil

BACKGROUND Nonsyndromic cleft lip with or without cleft palate is a relatively common craniofacial defect with multifactorial inheritance. The association of the rs987525 single nucleotide variant, located in a gene desert at 8q24.21 region, has been consistently replicated in European populations. We performed a structured association approach combined with transcriptional analysis of the MYC gene to dissect the role of rs987525 in oral clefting susceptibility in the ethnically admixed Brazilian population. METHODS We performed the association study conditioned on the individual ancestry proportions in a sample of 563 patients and 336 controls, and in an independent sample of 221 patients and 261 controls. The correlation between rs987525 genotypes and MYC transcriptional levels in orbicularis oris muscle mesenchymal stem cells was also investigated in 42 patients and 4 controls. RESULTS We found a significant association in the larger sample (p = 0.0016; OR = 1.80 [95% confidence interval {CI}, 1.21-2.69], for heterozygous genotype, and 2.71 [95% CI, 1.47-4.96] for homozygous genotype). We did not find a significant correlation between rs987525 genotypes and MYC transcriptional levels (p = 0.14; r = -0.22, Spearman Correlation). CONCLUSIONS We present a positive association of rs987525 in the Brazilian population for the first time, and it is likely that the European contribution to our population is driving this association. We also cannot discard a role of rs987515 in MYC regulation, because this locus behaves as an expression quantitative locus of MYC in another tissue.

Exomic variants of an elderly cohort of Brazilians in the ABraOM database

Brazilians are highly admixed with ancestry from Europe, Africa, America, and Asia and yet still underrepresented in genomic databanks. We hereby present a collection of exomic variants from 609 elderly Brazilians in a census‐based cohort (SABE609) with comprehensive phenotyping. Variants were deposited in ABraOM (Online Archive of Brazilian Mutations), a Web‐based public database. Population representative phenotype and genotype repositories are essential for variant interpretation through allele frequency filtering; since elderly individuals are less likely to harbor pathogenic mutations for early‐ and adult‐onset diseases, such variant databases are of great interest. Among the over 2.3 million variants from the present cohort, 1,282,008 were high‐confidence calls. Importantly, 207,621 variants were absent from major public databases. We found 9,791 potential loss‐of‐function variants with about 300 mutations per individual. Pathogenic variants on clinically relevant genes (ACMG) were observed in 1.15% of the individuals and were correlated with clinical phenotype. We conducted incidence estimation for prevalent recessive disorders based upon heterozygous frequency and concluded that it relies on appropriate pathogenicity assertion. These observations illustrate the relevance of collecting demographic data from diverse, poorly characterized populations. Census‐based datasets of aged individuals with comprehensive phenotyping are an invaluable resource toward the improved understanding of variant pathogenicity.

IRF6 is a risk factor for nonsyndromic cleft lip in the Brazilian population

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex disorder with a worldwide incidence estimated at 1:700. Among the putative susceptibility loci, the IRF6 gene and a region at 8q24.21 have been corroborated in different populations. To test the role of IRF6 in NSCL/P predisposition in the Brazilian population, we conducted a structured association study with the SNPs rs642961 and rs590223, respectively, located at 5′ and 3′ of the IRF6 gene and not in strong linkage disequilibrium (LD), in patients from five different Brazilian locations. We also evaluated the effect of these SNPs in IRF6 expression in mesenchymal stem cells (MSC). We observed association between rs642961 and cleft lip only (CLO) (P = 0.009; odds ratio (OR) for AA genotype = 1.83 [95% Confidence interval (CI), 0.64–5.31]; OR for AG genotype = 1.72 [95% CI, 1.03–2.84]). This association seems to be driven by the affected patients from Barbalha, a location which presents the highest heritability estimate (H2 = 0.85), and the A allele at rs642961 is acting through a dominant model. No association was detected for the SNP rs590223. We did not find any correlation between expression levels and genotypes of the two loci, and it is possible that these SNPs have a functional role in some specific period of embryogenesis.

Polymorphisms at Regions 1p22.1 (rs560426) and 8q24 (rs1530300) Are Risk Markers for Nonsyndromic Cleft Lip and/or Palate in the Brazilian Population

Polymorphisms at Regions 1p22.1 (rs560426) and 8q24 (rs1530300) Are Risk Markers for Nonsyndromic Cleft Lip and/or Palate in the Brazilian Population Elizabete Bagordakis, L ivia M aris Ribeiro Paranaiba, Luciano Abreu Brito, Sibele Nascimento de Aquino, Ana Camila Messetti, Herc ilio Martelli-Junior, Mario Sergio Oliveira Swerts, Edgard Graner, Maria Rita Passos-Bueno, and Ricardo D. Coletta* Department of Oral Diagnosis, School of Dentistry, State University of Campinas, Piracicaba, S~ao Paulo, Brazil Stomatology Clinic, Dental School, State University of Montes Claros, Montes Claros, Minas Gerais, Brazil Human Genome Research Center, Institute of Biosciences, University of S~ao Paulo, S~ao Paulo, Brazil Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Jos e Ros ario Vellano, Minas Gerais, Brazil

Novel Mutations in IRF6 in Nonsyndromic Cleft Lip With or Without Cleft Palate: When Should IRF6 Mutational Screening be Done?

Novel Mutations in IRF6 in Nonsyndromic Cleft Lip With or Without Cleft Palate: When Should IRF6 Mutational Screening be Done? Fernanda Sarquis Jehee, Beatriz A. Burin, K atia M. Rocha, Roseli Zechi-Ceide, Daniela F. Bueno, Luciano Brito, Josiane Souza, Gabriela Ferraz Leal, Antonio Richieri-Costa, Nivaldo Alonso, Paulo A. Otto, and Maria Rita Passos-Bueno* Centro de Estudos do Genoma Humano, Departamento de Gen etica e Biologia Evolutiva, Instituto de Biociencias, Universidade de S~ao Paulo, S~ao Paulo, SP, Brazil Departamento de Gen etica Cl inica, Hospital de Rehabilitac~ao de Anomalias Craniofaciais (HRAC), Universidade de S~ao Paulo, Bauru, Brazil Sobrapar, Instituto de Cirurgia Pl astica, Campinas, S~ao Paulo, Brazil CAIF, Centro de Atendimento Integral ao Fissurado L abio Palatal, Curitiba, Paran a, Brazil Centro de Atenc~ao aos Defeitos da Face do Instituto Materno-Infantil Prof. Fernando Figueira (CADEFI), Recife, Brazil Departamento de Gen etica e Biologia Evolutiva, Instituto de Biociencias, USP, S~ao Paulo, Brazil

Genetic contribution for non-syndromic cleft lip with or without cleft palate (NS CL/P) in different regions of Brazil and implications for association studies†

Non‐syndromic cleft lip with or without cleft palate (NS CL/P) is a complex disease in which heritability estimates vary widely depending on the population studied. To evaluate the importance of genetic contribution to NS CL/P in the Brazilian population, we conducted a study with 1,042 families from five different locations (Santarém, Fortaleza, Barbalha, Maceió, and Rio de Janeiro). We also evaluated the role of consanguinity and ethnic background. The proportion of familial cases varied significantly across locations, with the highest values found in Santarém (44%) and the lowest in Maceió (23%). Heritability estimates showed a higher genetic contribution to NS CL/P in Barbalha (85%), followed by Santarém (71%), Rio de Janeiro (70%), Fortaleza (64%), and Maceió (45%). Ancestry was not correlated with the occurrence of NS CL/P or with the variability in heritability. Only in Rio de Janeiro was the coefficient of inbreeding significantly larger in NS CL/P families than in the local population. Recurrence risk for the total sample was approximately 1.5–1.6%, varying according to the location studied (0.6–0.7% in Maceió to 2.2–2.8% in Barbalha). Our findings show that the degree of genetic contribution to NS CL/P varies according to the geographic region studied, and this difference cannot be attributed to consanguinity or ancestry. These findings suggest that Barbalha is a promising region for genetic studies. The data presented here will be useful in interpreting results from molecular analyses and show that care must be taken when pooling samples from different populations for association studies.

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.

Genetics and Management of the Patient with Orofacial Cleft

Cleft lip or palate (CL/P) is a common facial defect present in 1 : 700 live births and results in substantial burden to patients. There are more than 500 CL/P syndromes described, the causes of which may be single-gene mutations, chromosomopathies, and exposure to teratogens. Part of the most prevalent syndromic CL/P has known etiology. Nonsyndromic CL/P, on the other hand, is a complex disorder, whose etiology is still poorly understood. Recent genome-wide association studies have contributed to the elucidation of the genetic causes, by raising reproducible susceptibility genetic variants; their etiopathogenic roles, however, are difficult to predict, as in the case of the chromosomal region 8q24, the most corroborated locus predisposing to nonsyndromic CL/P. Knowing the genetic causes of CL/P will directly impact the genetic counseling, by estimating precise recurrence risks, and the patient management, since the patient, followup may be partially influenced by their genetic background. This paper focuses on the genetic causes of important syndromic CL/P forms (van der Woude syndrome, 22q11 deletion syndrome, and Robin sequence-associated syndromes) and depicts the recent findings in nonsyndromic CL/P research, addressing issues in the conduct of the geneticist.

Genetics and genomics in Brazil: a promising future.

Medical Genetics has a recent history in Brazil and started out as a research program in Human Genetics during the 1950s, mainly by a shift in work of Brazilian scientists from genetic studies conducted on drosophila melanogaster to this new emerging field of research, following a worldwide tendency (Otto and Frota-Pessoa 1975; Beiguelman 2000). The Department of Biology, currently Department of Genetics and Evolutionary Biology, at the Institute of Biosciences, University of Sao Paulo, was one of the main pioneers in this field, followed by other initiatives at the Federal Universities at Rio Grande do Sul and Parana (Otto and Frota-Pessoa 1975; Beiguelman 2000). The importance of genetics was recognized by the medical community only as of the 60 and onward, after the advent of human cytogenetics, and with the introduction of Genetics as a subject in a few medical schools (Beiguelman 2000). Nevertheless, Medical Genetics still is regarded as an optional discipline in many medical schools in Brazil. The Brazilian population is highly heterogeneous and admixed, as a result of five centuries of crossbreeding among native Amerindians, Europeans settlers and immigrants, and sub-Saharan Africans, who were mostly brought to this country during the slavery period. The countrys diverse regions underwent slightly different colonization processes: the North region had a larger preservation of indigenous people, the Northeast region had a stronger history of African influence, while Europeans colonized most of the South region (Alves-Silva et al. 2000; Pena et al. 2011). More recently, waves of Asian immigration reached mainly the Southeastern region. This trihybrid admixture process was strongly influenced by a directional mating between European males and African and Amerindian females during the colonization period, as demonstrated by the use of sexual and mitochondrial chromosome markers (Pena et al. 2009; Resque et al. 2010). In the last decade, the availability of genomic ancestry informative markers (AIMs) has encouraged a precise characterization of the contribution of each parental population. It has been shown that European ancestry is evenly preponderant across the country; the African contribution has reached the highest proportion in the Northeast (∼30.3%), followed in decreasing order by the Southeast (∼18.9%), South (∼12.7%), and North (∼10.9%) regions, while the Amerindian contribution is the highest in the North (∼19.4%) region, and relatively evenly spread across the other regions (Santos et al. 2010; Pena et al. 2011). An unexpected high Amerindian contribution is also found in semi-isolated communities founded by African-slaves refugees, the “quilombos” (Lopes Maciel et al. 2011; Kimura et al. 2013; Gontijo et al. 2014). Our colonization history also accounts for the high incidence of some diseases. For example, the autosomal recessive (AR) sickle cell disease (SCD), one of the most common monogenic disorder in Brazil (Cancado and Jesus 2007; Lobo et al. 2014, Table ​Table1),1), has been attributed to the intense African slave trade that occurred between the 16th and 19th centuries (Aygun and Odame 2012). Table 1 Genetic disorders under federal clinical protocols and guidelines for treatment including those from the newborn screening The Brazilian admixture, as briefly explained, constitutes a good example of a population characteristic boosting the research on Genetics in Brazil. Inbreeding in Brazil played a similar role within this field. The cultural diversity, socio-economic status, migration, population density, urbanization, and permissive laws have historically influenced the heterogeneous degree of consanguineous marriages across the country (Machado et al. 2013; Freire-Maia 1957, Fig. ​Fig.1A).1A). Even though inbreeding has been decreasing in all Brazilian regions in the past decades, large differences between Southern and Northeastern populations still remain, with the coefficient of inbreeding (f) 13 times higher in some Northeastern (f = 0.00395; proportion of consanguineous marriage from 6% to 12%) populations as compared to Southern (f = 0.0003) populations (Freire-Maia 1957, 1989; Brito et al. 2011; Weller et al. 2012; Machado et al. 2013), where the frequency of consanguineous marriage (<5%) is comparable to the estimates found for most of the other countries around the world (Hamamy et al. 2011). Figure 1 Inbreeding and Infant mortality rate across Brazil; (A) Inbreeding occurrence in Brazil, adapted from Salzano and Freire-Maia 1967. The diameter of black dots are proportional to the coefficient of inbreeding (f). According to that study, Brazil has a ... It is widely accepted that consanguineous marriage is a risk factor for AR and multifactorial inheritance disorders. Indeed, Brazilian consanguineous families have been useful study subjects for identifying pathogenic mutations underlying AR disorders, some of them originally described in Brazil, such as acheiropodia, Richieri-Costa-Pereira syndrome and Spastic paraplegia, optic atrophy, and neuropathy (SPOAN, Freire-Maia et al. 1975; Ianakiev et al. 2001; Macedo-Souza et al. 2005; Guion-Almeida et al. 2009; Lines et al. 2012; Favaro et al. 2014), in addition to others already described, such as Knobloch syndrome (Sertie et al. 2000). Still, high rates of inbreeding were recently reported to increase prevalence of some rare AR disorders, such as mucopolysaccharidoses and short stature, among others, in small Brazilian villages (Salvatori et al. 1999; Costa-Motta et al. 2011). Recently, National Institute of Populational Genetics (INAGEMP) has launched a census concerning clusters of genetic disorders in Brazil. Preliminary data list more than 100 small villages with a likely higher prevalence of a specific disorder. At a national level, however, the degree of inbreeding in Brazil has never been associated with increased overall prevalence of AR disorder. The impact of inbreeding rate in the etiology of complex disorders in the Brazilian population is still quite unexplored. Nevertheless, it has been shown to confer increased risk for hypertension in quilombos (Kimura et al. 2012), but not for nonsyndromic cleft lip and palate (Brito et al. 2011). The population admixture has always posed a challenge to the gene mapping studies of complex traits in Brazil, including pharmacogenomic studies, particularly when designing case–control studies, which are prone to produce spurious association in the presence of population structure. Accordingly, AIMs have proven to be useful for inferring the putative biogeographic ancestry of individuals, estimating the ancestry proportions of admixed individuals and populations (Shriver et al. 1997; Halder et al. 2008; Pena et al. 2011; Pereira et al. 2012; Suarez-Kurtz et al. 2012; Manta et al. 2013), correcting case–control studies for stratification bias (Brito et al. 2012a,b; Bagordakis et al. 2013), and performing admixture mapping approaches. Many of such studies have benefited from AIM panels built by Brazilian research groups, focused specifically on discriminating Brazilian parental populations (Bastos-Rodrigues et al. 2006; Santos et al. 2010; Brito et al. 2012a; Manta et al. 2013). In addition, in an ethnically admixed population, it is possible to evaluate the impact of certain at-risk alleles on the occurrence of some disorders or to drug response among different ethnicities simultaneously, under similar environmental and socio-economical conditions (Suarez-Kurtz et al. 2014). Brazil, with 26 states (Fig. ​(Fig.1A),1A), is the worlds 5th largest country in area (8,515,767 km2) and population (199,242,462 million individuals; IBGE, 2012). The age profile still remains as that of a young country, with 25% of the population under 15 years old and just 7% over 65 years old. Life expectancy at birth is estimated as 73.8 years old. Despite presenting the 7th largest gross domestic product (GDP), it ranks 61st and 85th in GDP per capita and in human development index (0.73), respectively (IBGE, 2012; reviewed in Acosta et al. 2013; World Bank, 2014, data.worldbank.org), reflecting an outstanding social inequality. As a related example, Brazil still has a high infant mortality rate, but it varies considerably across the country (Fig. ​(Fig.1B).1B). On behalf of policies with impact on sanitation and health, this rate has progressively decreased, and as part of the Millenium Development Goals from the United Nations (UN), Brazil reached the goal of decreasing the mortality rate to 2/3 of the one observed in 1990, 2 years before the deadline, set for 2015 (United Nation Development Programme, 2014). In this scenario, congenital malformations have become the second most common cause of death in children under 1 year of age surpassing infectious diseases, and represents more than 15% of the total number of infant deaths since 2000 (Fig. ​(Fig.2;2; DATASUS, tabnet.datasus.gov.br). This data highlight the increasing importance of Genetics for the health of the Brazilian population. Figure 2 Evolution of infant mortality (under 1 year old) by year, from 1980 to 2013. With the observed overall decrease in deaths caused by different conditions, congenital malformations became the second cause of infant mortality in Brazil (DATASUS). In this manuscript, we focus on describing the genetic services provided in the country, how they are working and some of our laws with direct impact on the quality of life of families with rare genetic disorders, as well as including Federal government support for treatment of some rare genetic disorders.

Contribution of polymorphisms in genes associated with craniofacial development to the risk of nonsyndromic cleft lip and/or palate in the Brazilian population.

Background and Objective: Nonsyndromic cleft lip and/or palate (NSCL/P) is a complex disease associated with both genetic and environmental factors. One strategy for identifying of possible NSCL/P genetic causes is to evaluate polymorphic variants in genes involved in the craniofacial development. Design: We carried out a case-control analysis of 13 single nucleotide polymorphisms in 9 genes related to craniofacial development, including TBX1, PVRL1, MID1, RUNX2, TP63, TGF?3, MSX1, MYH9 and JAG2, in 367 patients with NSCL/P and 413 unaffected controls from Brazil to determine their association with NSCL/P. Results: Four out of 13 polymorphisms (rs28649236 and rs4819522 of TBX1, rs7940667 of PVRL1 and rs1057744 of JAG2) were presented in our population. Comparisons of allele and genotype frequencies revealed that the G variant allele and the AG/GG genotypes of TBX1 rs28649236 occurred in a frequency significantly higher in controls than in the NSCL/P group (OR: 0.41; 95% CI: 0.25-0.67; p=0.0002). The frequencies of rs4819522, rs7940667 and rs1057744 minor alleles and genotypes were similar between control and NSCL/P group, without significant differences. No significant associations among cleft types and polymorphisms were observed. Conclusion: The study suggests for the first time evidences to an association of the G allele of TBX1 rs28649236 polymorphism and NSCL/P. Key words:Cleft lip, cleft palate, polymorphism, genetic.