Fatores ambientais associados a ocorrência de fissuras labiais ou palatinas não sindrômicas : Associeted enviromental factors with the occurrence of non-sydromic cleft lip or palate

Rodrigo Soares de Andrade

FATORES AMBIENTAIS ASSOCIADOS À OCORRÊNCIA DE

FISSURAS LABIAIS OU PALATINAS NÃO SINDRÔMICAS

ASSOCIATED ENVIRONMENTAL FACTORS WITH THE

OCCURRENCE OF NON-SYNDROMIC CLEFT LIP OR PALATE

Piracicaba/SP 2019

FATORES AMBIENTAIS ASSOCIADOS À OCORRÊNCIA DE

FISSURAS LABIAIS OU PALATINAS NÃO SINDRÔMICAS

ASSOCIATED ENVIRONMENTAL FACTORS WITH THE

OCCURRENCE OF NON-SYNDROMIC CLEFT LIP OR PALATE

Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutor em Estomatopatologia na Área de Patologia.

Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Pathology in Oral Pathology area.

Orientador: Prof. Dr. Hercílio Martelli Júnior

Este exemplar corresponde à versão final da tese defendida pelo aluno Rodrigo Soares de Andrade, e orientado pelo Prof. Dr. Hercílio Martelli Júnior.

Piracicaba/SP 2019

Universidade Estadual de Campinas

Biblioteca da Faculdade de Odontologia de Piracicaba Marilene Girello - CRB 8/6159

Andrade, Rodrigo Soares de, 1992-

An24f AndFatores ambientais associados a ocorrência de fissuras labiais ou palatinas não sindrômicas / Rodrigo Soares de Andrade. – Piracicaba, SP : [s.n.], 2019. AndOrientador: Hercílio Martelli Júnior.

AndTese (doutorado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.

And1. Fenda palatina. 2. Cafeína. 3. Poluição por fumaça de tabaco. 4. Ácido fólico. 5. Anormalidades dentárias. I. Martelli Júnior, Hercílio. II. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título.

Informações para Biblioteca Digital

Título em outro idioma: Associeted enviromental factors with the occurrence of non-sydromic cleft lip or palate

Palavras-chave em inglês: Cleft palate

Caffeine

Tobacco smoke pollution Folic acid

Tooth abnormalities

Área de concentração:Patologia Titulação: Doutor em Estomatopatologia Banca examinadora:

Hercílio Martelli Júnior [Orientador] Marcelo Sivieri de Araújo

Ana Camila Pereira Messetti Renato Assis Machado Alan Roger dos Santos Silva Data de defesa: 06-12-2019

Programa de Pós-Graduação:Estomatopatologia

Identificação e informações acadêmicas do(a) aluno(a)

- ORCID do autor: http://orcid.org/0000-0001-6114-0929 - Currículo Lattes do autor: http://lattes.cnpq.br/4936205843300438

Faculdade de Odontologia de Piracicaba

A Comissão Julgadora dos trabalhos de Defesa de Tese de Doutorado, em sessão pública realizada em 06 de dezembro de 2019, considerou o candidato RODRIGO SOARES DE ANDRADE aprovado.

PROF. DR. HERCÍLIO MARTELLI JÚNIOR

PROF. DR. MARCELO SIVIERI DE ARAÚJO

PROFª. DRª. ANA CAMILA PEREIRA MESSETTI

PROF. DR. RENATO ASSIS MACHADO

PROF. DR. ALAN ROGER DOS SANTOS SILVA

A Ata da defesa, assinada pelos membros da Comissão Examinadora, consta no SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa da

Dedico este trabalho e essa vitória a minha família, meu pai Moacir Jose de

Andrade, minha mãe Maria Lucia Soares Andrade, minha irmã Nayara Soares de Andrade e meu sobrinho Heitor Soares Caixeta, que sem sombra de tudo, são meu maior

Agradeço ao meu orientador, Prof. Dr. Hercílio Martelli Júnior, por todos esses anos de trabalho árduo, por todos os ensinamentos e oportunidades, e por toda paciência por me formar o profissional que sou hoje. Minha admiração e carinho ultrapassam a relação orientador/orientado e trilhamos um caminho de uma grande parceria e amizade. O vejo como um exemplo de ser humano e um como um profissional admirável! Tenho orgulho de poder ter sido orientado por um profissional tão competente, que no final dessa jornada, podê-lo-ei chamar de amigo.

O presente trabalho foi realizado com apoio das agências Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Código de

Financiamento 001, 88881.188930/2018-01 PDSE; e ao Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq), processo nº 141063/2016-5.

À Universidade Estadual de Campinas (UNICAMP), na pessoa do Magnífico Reitor, Prof. Dr. Marcelo Knobel e à Faculdade de Odontologia de Piracicaba –

UNICAMP, na pessoa do seu Diretor, Prof. Dr. Francisco Haiter Neto e Diretor Associado, Prof. Dr. Flávio Henrique Baggio Aguiar.

À Profa. Dra. Karina Gonzales Silvério Ruiz, coordenadora geral dos programas de Pós-graduação da Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas.

Ao Prof. Dr. Márcio Ajudarte Lopes, coordenador do programa de Pós-graduação em Estomatopatologia da Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas.

Aos docentes das áreas de Semiologia e Patologia da Faculdade de Odontologia de Piracicaba – UNICAMP, Prof. Dr. Márcio Ajudarte Lopes, Prof. Dr. Alan Roger dos

Santos Silva, Prof. Dr. Pablo Agustín Vargas, Prof. Dr. Oslei Paes de Almeida, Prof. Dr. Edgard Graner, Prof. Dr. Ricardo D. Coletta e Prof. Dr. Jacks Jorge Júnior, por todos os

ensinamentos.

A Prof. Dra. Daniella Barbosa Reis Martelli, por todo auxílio e ajuda para elaboração desse trabalho, por sua disponibilidade e por prontidão em me ensinar.

A Universidade José do Rosário Vellano (UNIFENAS) e seus profissionais, por terem me recebido e me acolhido tão carinhosamente.

Aos profissionais e pacientes do Centro de anomalias craniofaciais - Pró Sorriso, onde realizei vários trabalhos, pude evoluir como profissional e como ser humano.O aprendizado e as oportunidades que tive, são impagáveis.

processo.

A Profa. Dra. Adriana Boeri Freiri Tamburini, por todo conhecimento compartilhado, por toda ajuda, e principalmente por toda a amizade e amparo, serei eternamente grato!

A Dra. Flavia Alves, minha psicóloga e terapeuta, que foi de extrema importância para me dar luz e conforto em tempos difíceis durante o meu doutorado.

Aos meus amigos Adriano e Matheus, por toda amizade e carinho, me propiciando os melhores momentos.

Esta tese contemplou dois estudos “distintos” envolvendo pacientes com fissuras orais não sindrômicas. As fissuras de lábio e/ou palato são malformações congênitas que, como consequência, trazem uma série de alterações como comprometimento da estética, da fala e da posição dos dentes. De modo geral, são justificadas pela herança multifatorial, coexistindo fatores genéticos e ambientais, e podem ser associação ou não a síndromes. O primeiro estudo teve como objetivo avaliar fatores de risco ambientais que tenham influência sobre o desenvolvimento de fissuras orais não sindrômicas. A suplementação de ácido fólico, fumo passivo, consumo excessivo de cafeína e de álcool e exposição a produtos químicos como agrotóxicos foram as variáveis estudadas através da aplicação de um questionário. Com isso, observou-se que o consumo de cafeína acima de 100 mg diárias se mostrou maior em mães de filhos com fissuras orais. Esse consumo excessivo acima de 500mg semanais mostrou uma probabilidade 34 vezes maior do desenvolvimento de fissuras nos filhos dessas mães. O fumo passivo acima de 15 minutos diários também se mostrou como fator de risco importante e mais frequentemente encontrado para mães de filhos com fissuras orais não sindrômicas. A suplementação com ácido fólico e sulfato ferroso, juntamente com seus complexos multivitamínicos mostraram fator protetor para evitar o desenvolvimento de fissuras orais não sindrômicas, sendo mais consumido pelas mães de filhos não fissurados. Já o segundo estudo teve como objetivo analisar anomalias dentárias em pacientes com fissuras labiais isoladas. Realizou-se uma comparação entre a quantidade e tipo de anomalia dentária em pacientes com fissuras labiais que envolviam ou não para o rebordo alveolar. Observou-se que a agenesia dentária foi à anomalia mais encontrada, seguida de dentes supranumerários e microdontia. Pacientes com a fissura labial com o envolvimento de rebordo apresentaram mais anomalias dentárias quando comparados a pacientes com fissura labial sem o envolvimento do rebordo alveolar. Em síntese, concluímos que o consumo de cafeína acima de 100 mg diários e o fumo passivo além de 15 minutos ao dia aumentaram o risco do desenvolvimento de fissuras orais não sindrômicas não sindrômicas. A suplementação com ácido fólico, complexos multivitamínicos e alimentos ricos em ferro, utilizados durante a realização do pré-natal, atuam como fatores protetores para o não desenvolvimento de fissuras orais não sindrômicas não sindrômicas. Pacientes com fissuras labiais com o envolvimento de rebordo tem mais anomalias dentárias do que os pacientes com fissuras labiais sem o

Palavras–chave: fissuras orais, cafeína, fumo passivo, ácido fólico, anomalias dentarias,

Abstract

This thesis contemplated two "distinct" studies involving patients with nonsyndromic oral clefts. Clefts of the lip and/or palate are congenital malformations that, as a consequence, bring about a series of alterations such as impairment of aesthetics, speech and tooth position. In general, they are justified by multifactorial inheritance, coexisting genetic and environmental factors, and may or may not be associated with syndromes. The first study aimed to evaluate environmental risk factors that influence the development of nonsyndromic oral clefts. Folic acid supplementation, secondhand smoking, excessive consumption of caffeine and alcohol, and exposure to chemicals such as pesticides were the variables studied by applying a questionnaire. Thus, it was observed that the consumption of caffeine above 100 mg daily was higher in mothers of children with oral clefts. This excessive consumption above 500 mg per week showed a 34 times greater probability of the development of cracks in the children of these mothers. Secondhand smoke above 15 minutes daily was also shown to be an important risk factor and more frequently found for mothers of children with nonsyndromic oral clefts. Supplementation with folic acid and ferrous sulfate, along with its multivitamin complexes showed a protective factor to avoid the development of nonsyndromic oral clefs, being more consumed by mothers of non-cleft children. The second study aimed to analyze dental anomalies in patients with isolated cleft lips. A comparison was made between the amount and type of dental anomaly in patients with cleft lip involving or not the alveolar ridge. It was observed that tooth agenesis was the most commonly found anomaly, followed by supernumerary teeth and microdontia. Patients with cleft lip involving the ridge presented more dental anomalies when compared to patients with cleft lip without the involvement of the alveolar ridge. In summary, we concluded that the consumption of caffeine above 100 mg daily and secondhand smoking beyond 15 minutes a day increased the risk of developing nonsyndromic oral clefts. Folic acid supplementation, multivitamin complexes and iron-rich foods used during prenatal care act as protective factors for the development of nonsyndromic oral clefts. Patients with cleft lip with lip involvement have more dental anomalies than patients with cleft lip without lip involvement and tooth agenesis and supernumerary are the most common dental anomalies in patients with cleft lip alone.

Keywords: oral clefts, caffeine, secondhand smoking, folic acid, dental anomalies, dental

Sumário

1 Introdução ... 14

2 Artigos

2.1 Artigo:

... 17

2.2

Artigo:

... 37

3 Discussão ... 51

4 Conclusão ... 55

Referências ... 56

Apêndice 1

... 62

Anexos

Anexo 1

... 65

Anexo 2

... 80

1 Introdução

1.1 Capítulo 1

As fissuras orais não sindrômicas são as anomalias craniofaciais mais prevalentes na população. A prevalência destas condições em todo o mundo é de aproximadamente 1 em cada 500 nativivos e varia entre diferentes países, etnias e raças (Rahimov et al., 2012). As fissuras orais não sindrômicas podem afetar a vida de uma pessoa e de sua família, tendo uma influência importante na qualidade de vida dos indivíduos.

A etiologia das fissuras orais não sindrômicas envolve fatores ambientais, genéticos e epigenéticos (Lima et al., 2015, Kumari et al., 2018; Machado et al., 2018). Consanguinidade, deficiência nutricional materna durante a gravidez, tabagismo e alcoolismo são considerados como alguns desses fatores de risco (Martelli et al., 2012).O tabagismo, incluindo a forma passiva (Ayu & Samsudin, 2003; McKinney et al., 2016), aumenta o risco de fissuras orais não sindrômicas , devido ao efeito teratogênico dos agentes ativos e radicais livres presentes na fumaça do cigarro (Martelli et al., 2015; Machado et al., 2016; Machado et al., 2018). Little et al. (2004) observaram uma probabilidade de2,1 vezes maior de desenvolvimento de fissuras orais não sindrômicas para mães expostas ao tabagismo passivo.

Existem evidências para associações entre alguns hábitos alimentares da mãe durante o primeiro trimestre de gravidez, como o consumo de alimentos e bebidas ricas em cafeína e o desenvolvimento de fissuras orais não sindrômicas (McDonald et al., 1992; Browne, 2006; Bille et al., 2007; Johansen et al., 2009).A ingestão alimentar materna e o estado nutricional desempenham um papel significativo no desenvolvimento das fissuras orais não sindrômicas (Krapels et al., 2006). O baixo consumo de alimentos ricos em ferro pode estar associado à ocorrência de fissuras orais não sindrômicas, mas os mecanismos envolvidos nesta associação ainda não são totalmente compreendidos (McKinney et al., 2016). O uso de multivitamínicos reduz o risco de fissura na maioria dos estudos (Wilcox et al., 2007; Jia et al., 2011). A suplementação com ácido fólico por si só mostra diminuir o risco de defeitos do tubo neural e das fissuras orais não sindrômicas (Rozendaal et al., 2013; De-Regil et al., 2015; Machado et al., 2016).

Como os fatores de risco podem variar de acordo com diversas variáveis, faz-se necessário investigar a etiologia das fissuras orais não sindrômicas em diferentes

localidades para identificar os fatores de risco que são mais frequentes nos diferentes países. No Brasil, a prevalência das fissuras orais não sindrômicas varia de 0,36 a 1,54 por 1.000 nativivos (Martelli-Júnior et al., 2007; Rodrigues et al., 2009), sendo expressiva a prevalência destas anomalias.Por isso, faz-se immportante conhecer e buscar correlacionar a ocorrência de fissuras orais não sindrômicas com os diversos fatores de risco ambientais, genéticos e epigenéticos.

1.2 Capítulo 2

O desenvolvimento facial humano ocorre entre a 4ª e 8ª semanas de vida intrauterina e a união dos processos faciais (maxilares, mandibulares e frontonasais) vão permitir a correta formação e desenvolvimento da face. A ausência na coalescência desses processos anatômicos resulta no aparecimento dos vários tipos de fissuras devido à ruptura da integridade do lábio e/ou do palato (Paranaíba et al., 2013). Este processo é complexo e ocorre sob o controle de mecanismos genéticos (Koukskoura et al., 2011).

As fissuras orais não sindrômicas são caracterizadas por áreas de descontinuidades no lábio e/ou palato, representando as anomalias congênitas mais frequentes na região craniofacial dos seres humanos (Dixon et al., 2011; Marazita et al.,2012; Haet al., 2015). Para a formação do lábio superior e maxila, as proeminências maxilares passam por crescimento medial, produzindo fusão de proeminência nasal com proeminências maxilares (Sarmiento et al., 2017).

Para identificar as fissuras labiopalatinas não sindrômicas (FL/PNS), em função das suas diferentes apresentações clínicas, as mesmas são tradicionalmente divididas em fissuras de lábio (FLNS), fissuras de lábio e palato (FLPNS) e fissuras palatinas (FPNS) (Spina et al.,1972). As FL/PNS afetam aproximadamente 1 em cada 500 a 2.500 nativivos, em todo o mundo (Dixon et al., 2011). Martelli et al. (2012) mostraram a prevalência para cada tipo de fissura, sendo a de maior ocorrência as FLPNS (53,4%), seguida, respectivamente, das FLNS (26,2%) e das FPNS (20,49%). Também mostraram que as FPNS ocorrem com maior frequência no sexo feminino, enquanto as FLPNS e FLNS predominam no masculino. Essas malformações podem levar a prejuízos na audição, fonação e aparência afetando psicologicamente o indivíduo e sua família (Fan et al., 2018). Eslami et al. (2013) mostraram que ocorre um aumento na frequência de ansiedade, baixa autoestima e depressão nos pacientes com fissuras orais não sindrômicas.

Silva et al. (2018) observaram que clinicamente as FLPNS comprometem a saúde bucal, levando a dentes ausentes ou malformados e podem comprometer a higiene oral. Küchler et al. (2011) mostraram que a ocorrência de fissuras orais não sindrômicas e o desenvolvimento dos germes dentários tem uma estreita relação embrionária com relação à cronologia e posição anatômica, pois os eventos críticos dos dentes, lábio e palato ocorrem quase que simultaneamente, sugerindo que fissuras orais não sindrômicas e alterações dentárias tem como determinante, o fator genético. Stahl (2006) relata que a formação da FLPNS interfere no desenvolvimento dentário, o que adiciona risco de ocorrência de anomalias dentárias nos indivíduos com esta malformação congênita. Esses achados podem representar um marcador clínico para definição de subfenótipos das fissuras (Sá et al., 2016).

Sabe-se que a hipodontia é mais frequente no lado da fissura comparado ao contralateral, e também à prevalência de hipodontia aumenta com a gravidade da fissura (Suzuki et al., 2017). As anomalias dentárias podem ser atribuídas à própria fissura ou ao início da correção cirúrgica dos defeitos, além de estarem relacionadas com a extensão da fissura (Jamal et al., 2010). Melo Filho et al. (2015) e Cakan et al. (2018) mostraram que as anomalias dentárias possuem maior frequência em indivíduos fissurados, podendo ser de número, tamanho e forma. Em pacientes com fissuras orais não sindrômicas, a anomalia dentária mais frequente é agenesia, seguida por supranumerário e microdontia (Paranaíba et al., 2013; Sá et al., 2016). Letra et al. (2007) relataram que o desenvolvimento do germe dentário e a incidência de FLPNS tem relação embrionária. As anomalias dentárias podem representar marcadores clínicos adicionais para fissuras orais não sindrômicas , mostrando um fator genético comum entre as condições (Chu et al., 2016).

A partir da constatação de que anomalias dentárias são mais prevalentes em pacientes com fissuras orais não sindrômicas , é importante observar se existe ou não diferença nas anomalias dentárias, seja de número ou forma, e na sua prevalência quando se compara as fissuras labiais completas (envolvendo lábio e rebordo alveolar) e fissuras incompletas (que afetem somente o lábio, sem envolvimento do rebordo alveolar).

Artigo 1

*

Maternal consumption of caffeine and second-hand tobacco smokers risk factor for the development of oral clefts

Rodrigo Soares de Andrade1, Daniella Reis Barbosa Martelli2, Alison José Martelli1, Adriana Boeri Freire Tamburini3_{, Mário Sérgio Oliveira Swerts}3_{, Verônica Oliveira}

Dias2, Hercílio Martelli Júnior1,2

1_{Department of Oral Diagnosis, School of Dentistry, State University of Campinas,}

FOP-UNICAMP, Piracicaba, São Paulo, Brazil.

2_{School of Dentistry, State University of Montes Claros, Unimontes, Minas Gerais}

State, Brazil.

3_{Center for Rehabilitation of Craniofacial Anomalies, Dentária School, University of}

José Rosario Vellano, Alfenas, Minas Gerais, Brazil.

Correspondence: Rodrigo Soares de Andrade,

Oral Pathology, FOP-UNICAMP, Av. Limeira, 901 - Areião, Piracicaba – São Paulo,

Brazil.

CEP: 13414-018 - Phone: +55-19-999665488 E-mail: rodrigosoares002@hotmail.com

ABSTRACT

Objective: The aim of the present study was to evaluate the possibility of

environmental factors such as caffeine, folic acid, multivitamin complexes, alcohol and tobacco (secondhand smoking) to act as oral clefts development risk factors.

Methods: This case-control study included 409 mothers, 132 with children presenting

oral clefts (case) and 277 not presenting oral clefts (control). In both groups, children were 0-2 years-old. A questionnaire was applied to each mother to evaluate their food consumption and habits during pregnancy’s first trimester.

Results: Eighty-four (63.63%) mothers in the case group reported secondhand smoking

and 5 (3.78%) reported alcohol consumption (p=0.797). Mothers more exposed to passive smoking were twice as likely to have a child with an oral cleft. Caffeine consumption was observed in 126 (95.45%) of the mothers in the case group and in 246 (88.80%) in the control group (OR=3.20; 95% CI=1.21-8.43) (p=0.013). Amongst case group mothers, 107 (84.25%) consumed more than 500 mg of caffeine weekly, leading to an observed in 34% increase in the frequency of oral cleft in their children. Folic acid supplementation was observed in 116 (87.87%) cases and 271 (97.83%) were control (p<0.001). As for ferrous sulfate administration, 114 (86.36%) in the case group and 271 (97.83%) in the control group (p=0.617).

Conclusions: The results suggest a direct relationship between both secondhand

smoking and caffeine consumption to nonsyndromic oral clefts, while folic acid, ferrous sulfate and multivitamins consumption could be considered protective factors against nonsyndromic oral clefts occurrence.

Objetivo: Investigar fatores ambientais como cafeína, ácido fólico, complexos

multivitamínicos, álcool e tabaco (tabagismo passivo), que têm sido descritos como fatores de risco para o desenvolvimento das fissuras orais.

Métodos: Este estudo caso-controle incluiu 409 mães, sendo 132 com crianças com

fissura oral (caso) e 277 com crianças sem fissura oral (controle). Em ambos os grupos, as crianças tinham idade entre 0 e 2 anos. Um questionário foi aplicado a cada uma das mães para investigar seus hábitos e consumo de alimentos durante o primeiro trimestre da gestação.

Resultados: Oitenta e quatro (63,63%) mães do grupo caso relataram tabagismo

passivo e 5 (3,78%) relataram uso de álcool (p=0,797). As mães expostas ao tabagismo passivo apresentaram duas vezes maior probabilidade de ter um filho com fissura oral. No grupo caso, 126 mães (95,45%) consumiram cafeína, e no grupo controle 246 (88,80%) (OR=3,20; 95% CI=1,21-8,43) (p=0,013). Entre as mães do grupo caso 107 (84,25%) consumiam semanalmente mais de 500 mg de cafeína, aumentando em 34% a frequência de fissuras orais nos filhos. A suplementação com ácido fólico foi observada em 116 (87,87%) casos e 271 (97,83%) do controle (p<0,001). Em relação ao consumo de sulfato ferroso, 114 (86,36%) no grupo caso aderiram e 271 (97,83%) no controle (p=0.617).

Conclusões: Os resultados sugerem uma relação direta entre tabagismo passivo,

consumo de cafeína e fissuras orais não sindrômicas. O uso de ácido fólico, sulfato ferroso e multivitaminas são fatores protetores para a ocorrência de fissuras orais não sindrômicas.

INTRODUTION

Nonsyndromic oral clefts are amongst the most prevalent congenital abnormalities in humans (1) and is a substantial morbidity and mortality source worldwide with significant social impact (2). In 70% of cases, the cleft lip and/or palate present as non-syndromic (NSCL/P), that is, without structural malformations in other organs and without behavioral and cognitive alterations (3,4). Based on epidemiological features and embryologic timing, NSCL/P is traditionally divided in cleft lip (NSCL), cleft lip and palate (NSCLP) and cleft palate (NSCP) (5).

The incidence of NSCL/P is approximately 1 in 500-2,500 live births, varying according to location, ethnicity and socioeconomic status of the population studied (1). In Brazil, studies on the incidence of NSCL/P are scarce and vary considerably. According to the Brazilian surveys, the incidence of NSCLP ranges from 0.19 to 1.54 for every 1,000 live births (6,7).

The etiology of NSCL/P is attributed to genetic, epigenetic and environmental factors interplay but the exact interactions are poorly known (1,8). This lack of knowledge is probably a reflection of the wide diversity of molecular mechanisms related to facial embryogenesis involving multiple genes and the influence of environmental factors (9-12). Amongst the major environmental risk factors for the development of NSCL/P are medication use, smoking, maternal diet and vitamin supplementation, particularly during pregnancy’s first trimester (13-15).

The aim of the present study was to evaluate the possibility of environmental factors such as caffeine, folic acid, multivitamin complexes, alcohol and tobacco (secondhand smoking) to act as oral clefts development risk factors

MATERIALS AND METHODS

This case-control study was conducted in the Center for Rehabilitation of Craniofacial Anomalies, State of Minas Gerais, Brazil, where 132 mothers were recruited. They were being followed as well as their children to be included in the case group, with children up to 24 months of age with NSCL/P and 277 mothers to the control group of children without craniofacial randomly obtained from the Pediatric Dentistry Clinic at the University. Patients were recruited from February 2018 to February 2019. The sample was chosen for convenience, that is, all patients during the chronological period of the study were included.

Case group included mothers with up to 24 months-old children diagnosed with NSCL/P without gender distinction, while control group included children without any skull alterations or syndrome. Each control will be paired with a case of gender and age equal within 3 months. All children with NSCL/P were evaluated by a multidisciplinary team of specialists to exclude the involvement of other alterations or syndromes.

The sample of the studied population was selected during one year of follow-up, based on our primary exposure of interest, which is secondhand smoking, supplementation with folic acid, ferrous sulfate and multivitamin complexes, in addition to caffeine and alcohol consumption during the first trimester of pregnancy. As reference values, the consumption of the studied variables was taken into consideration. The mothers answered a demographic questionnaire with characteristics, habits and exposures during pregnancy. The questionnaire included questions about consumption of caffeine-containing beverages (coffee, soft drinks with caffeine, energetic beverages with caffeine, and dietary supplements of physical performance) during the first 3 months of pregnancy. For each drink, there was 1 question with 5 response categories: none, number of 100ml cups per week on average (without specifying cup size),

consumption up to 500 mg weekly, from 500 mg to 1000 mg weekly and above 1400 mg weekly (13-15). Questionnaire application method was standardized for mothers from both groups about their food and nutritional supplementation, as well as their habits during pregnancy, focusing on the first trimester of the gestational period.

The risk of delivering offspring with an NSCL/P was estimated by odds ratio with a 95% confidence interval in non-conditional logistic regression models. All beverages were summarized and analyzed in the same way as follows. "Cups per week" was calculated from the reported number of cups consumed per day, per week (divided or multiplied by 7). Women whom reported consuming less than 1 cup per week were categorized as zero consumption. The variable “concentration in milligram per week” was analyzed as a continuously variable. Finally, categories were created: 0-100mg per week (reference category), 100mg at 100mg to the level above 1400mg per week (13-15). Trends between categories were evaluated, with zero as the reference. An estimate of caffeine from all sources was computed from data on coffee, soft drinks with caffeine, energetic beverages with caffeine, and dietary supplements of physical performance.

The caffeine content was estimated as 100mg per cup of coffee, 40mg per cup of soft drinks with caffeine, 80 mg per cup of dietary supplements of physical performance, 70mg per thermogenic capsule, drink based on the values of the international health authorities (http://www.matportalen.no/Emner/Gravide). The risk of NSCL/P was assessed by increasing the daily caffeine intake by 100mg (continuous variable) and categories >100mg, increasing every 100 mg up to a proportion greater than 1000 mg of caffeine (13).

Adjustments were made to potential confounders (factors associated with NSCL/P in other studies, most of which were also associated in our study), namely, A

(quartiles), dietary folate (factors associated with NSCL/P, most of which were also associated in our study), folic acid supplementation (yes or no), use of vitamin supplements (yes or no), alcohol consumption in early pregnancy (yes or no), smoking (passive only, yes or no> 15 min per day). Evaluations of possible interactions with coffee intake were performed comparing folic acid supplementation and smoking. When evaluating the effects of coffee or tea separately, we adjusted to the categorized as cups per week

A logistic regression test was performed to calculate gross and adjusted OR, corresponding 95% CI and p values between exposures of interest and NSCL/P. Because of the number of patients, all variations of NSCL/P were combined for our analysis. We also performed subgroup analyzes replicating our conditional logistic regression analysis among those with NSCL/P.

All analyzes were performed using the IBM SPSS Statistics (SPSS®, version 20.0; SPSS Inc., Chicago, Illinois, USA) with a significance level of 5%.To address the impact of uncontrolled bias, p-value, a new measure, was calculated that assesses how far the actual risk estimate can be given without conflicts and bias in a study in a sensitivity analysis.

RESULTS

As for deleterious habits such as secondhand tobacco smoke and alcohol consumption in the first trimester of pregnancy, 84 (63.63%) mothers in the case group reported being secondhand tobacco smokers during the first trimester of pregnancy (p<0.001; OR=6.46 CI=4.09-10.20) and 5 (3.78%) mothers stated the frequent use of alcohol, and we found a significant difference between groups (OR=0.86; 95% CI=0.30-2.52)

(p=0.797). In the control, 59 (21.29%) mothers were secondhand tobacco smokers and 12 (4.33%) consumed alcohol during the first trimester of pregnancy (Table 1).

In the case group, 126 mothers (95.45%) frequently ingested caffeine-containing products during pregnancy, and 277 mothers in the control group (88.80%) (OR=3.20; 95% CI=1.21-8.43) (p=0.013). Of the 127 mothers in the group who consumed caffeine, 83 (65.3%) consumed more than 1,000 mg per week, and of the 246 mothers in the case group, 124 (50.40%) consumed more than 1,000 mg per week (Table 2). Of the 132 mothers of children with NSCL/P interviewed, 8 (6.06%) reported consanguinity of their son because they were married to first-degree relatives (p≥0.065). Of those, 18 (13.63%) reported a history of NSCL/P in the family. When we observed folic acid supplementation in the first trimester of pregnancy, 116 (87.87%) mothers of NSCL/P were used correctly and 271 (97.83%) were case group (OR=6.23; 95% CI=2.37-16.32) (p<0.001).

When questioned about consumption of pre-gestational vitamins such as ferrous sulfate, 114 (86.36%) mothers of the case group had supplementation and 271 (97.83%) of the mothers in the control group (OR=7.13; 95% CI=2.75-18.43. About changing eating habits, 109 (82.57%) mothers in the case group reported having changed their diet in the first trimester of pregnancy, eating healthier foods, rich in vitamins and iron while 223 (80.50%) of the control mothers also reported having improved their eating pattern (OR=0.87; 95% CI=0.50-1.49) (p≥0.61) (Table 1).

DISCUSSION

Some studies have raised the possibility that habits of mothers during the gestational period are directly related to the increased possibility of developing NSCL/P (15,16). In this study we analyzed the mothers in prenatal care comparing the consumption of

dietary supplements related to the good development of the fetus, such as folic acid and ferrous sulfate (17). These are mainly related to fusion of maxillary processes and vision. The results of the present study corroborate Taghavi et al. (2012), who also observed that mothers of children without NSCL/P do prenatal care correctly and follow better supplementation diet than mothers of children with NSCL/P (18).

Secondhand tobacco smoke is considered deleterious when the mother inhaled some nicotine-based agent over 15 minutes daily, a parameter established by the WHO in 2003 (https://www.who.int/tobacco/research/secondhand_smoke/en/). Although mechanisms through which tobacco increases craniofacial abnormalities in infants are not precisely understood, they most likely involve the interaction between smoking and polymorphism of susceptible genes (19).

Inhaled CO2 suffers dissociations within the mother's body releasing free

radicals that have teratogenic effects, wherein the vasoconstrictive action of nicotine can cause reduction of utero-placental blood flow. Carbon monoxide binds to hemoglobin so that less oxygen is available to the placenta (20). In addition, endothelial injury caused by tobacco increases the rupture of placental neovessels, leading to reduced fetal blood supply and hypoxia, which probably results in abnormal facial morphogenesis. Therefore, the sum of exposure to toxins, hypoxia and cellular ischemia results in craniofacial abnormalities (19). Little et al., (2004) analyzed 24 mothers on the smoking habits of pregnant women and the occurrence of NSCL/P in their children (20). There was a statistically significant association between relative maternal smoking and NSCL/P (relative risk=1.34). This form corroborates the evidence of this study, in which 84 of the 132 mothers with cleft children reported being exposed to smoking (63.6%), while 59 of the 277 mothers in the control group were exposed (21.3%), showing an increased risk of children with cleft palate from smoking mothers (21).

Although here is no exact known mechanism through which coffee ingestion can increase the risk of NSCL/P (14), it seems to involve effects on homocysteine. Evidences that hyperhomocysteinemia of the mother may be linked to the increased risk of clefts are also corroborated (16). Coffee ingestion increases homocysteine plasma concentration (21) as well as secondhand tobacco smoke (22) raising the risk of spontaneous abortions, chromosomal anomalies, multiple congenital aberrations (23) and mainly low birth weight (24). Supplements of folic acid and ferrous sulfate are usually associated with lower risk of NSCL/P (25), and such supplements also reduce plasma homocysteine (21), which justifies the supplementation of these nutrients during the pre-gestational period. In the present study, the association between high coffee intake and NSCL/P was higher in mothers of the case group and also on the use of folic acid and ferrous sulfate supplements that were lower than in the control (87.00%) and case (97.00%) groups respectively.

Similarly, mothers that consumed coffee in large quantities (>500mg/week) are more pre-exposed to chemicals per session than those who do not have NSCL/P. However, alcohol consumption was not statistically significant between groups. Even if adjusted for possible confounding factors, the possibility of residual bias by known factors cannot be ruled out. Although we have no control over the diet, there was no evidence of a worse diet with iron-rich foods.

Considering that the fusion of the maxillary processes occurs relatively late in the first trimester (26), it is possible that the reported coffee intake is higher than the actual consumption in the crucial phase of embryonic development, as over time coffee intake decreases due to nausea and vomiting. A meta-analysis of three studies on maternal coffee consumption and NSCL/P found a slight increase in the risk of oral clefts (27), corroborating our study, which showed that caffeine consumption increases

the likelihood of oral clefts development. All three studies used 0 cups of 100ml as a reference category, as recommended by our methodology. In the study by Kurppa et al. (1983), drinking more than 4 cups of coffee by day was not associated with higher risk of NSCL/P (OR=1.0; 95% CI= 0.6-1.6) (28), while in the study by McDonald et al. (1992), more than 3 cups of coffee per day resulted in an adjusted odds ratio of 1.4 (95%; CI=0.7-2.7) (29), in this study it was observed that caffeine consumption by mothers of the case group was higher, and mothers of children with NSCL/P consumed more caffeine. In the study by Billie et al (2007), with 134 mothers, caffeine consumption was associated with an increased risk of NSCL/P (30), which was corroborated by the results of this study.

Concentration parameters of caffeine in different sources, portion sizes and methods of preparation for coffee should be considered to estimate caffeine ingestion. However, the methodology (13-15) has been extensively validated on the basis of a subpopulation, using various biomarkers as reference measures. The correspondence between food diaries was particularly high for coffee and cola-based soda, which are the main caffeine sources in our population (31,32).

High consumption of caffeine in the first trimester of pregnancy by the mother increased the risk of nonsyndromic oral cleft in the child, as a unsatisfactory prenatal leads to ineffective nutritional supplementation, thus becoming an important risk factor. Harmful habits such as alcohol consumption and especially the inhalation of active tobacco agents, even if passively, may harm the fetus.

We can observe limitations in this study, some of them may not have been standardized, or mistakenly answered by mothers of both groups, but to minimize these biases the questionnaire was applied in a reserved place, giving more privacy and

confounding factors were applied to statistical tests. Due to the rarity of finding children with oral cleft of up to 24 months, the number of research participants was also limited.

In summary, our results suggest a relationship between both secondhand smoking and caffeine consumption to nonsyndromic oral clefts, while folic acid, ferrous sulfate and multivitamins consumption could be considered protective factors against nonsyndromic oral clefts occurrence.

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Radiol. 2015; (120): 744-750.

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Surg. 2018;29(6):e534-e54

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Gynecol. 2016; 215(5): 11-13.

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28. Bille C, Olsen J, Vach W, et al. Oral clefts and life style factors—a case-cohort study based on prospective Danish data. Eur J Epidemiol. 2007; 22(3): 173–181.

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32. Frary, C.D.; Johnson, R.K.; Wang, M.Q. Food sources and intakes of ca_eine in the diets of persons in the United States. J. Am. Diet. Assoc. 2005, 105, 110– 113.

Table 1 - Values referring to the variables evaluated in this study, which may have a direct relationship with the development of nonsyndromic oral clefts. Case n = 132 (%) Control n= 277 (%) p value OD ratio (95%) Gender Female Male 57 (43.18) 75 (56.82) 129(46.57) 148(53.42) 0.520 Reference 1.147 (0.755-1.741) Consanguinity No Yes 127 (96.22) 5 (3.78) 274 (98.92) 3 (1.08) 0.065 R e f e r e n c e 3.596(0.846-15.279) NSCL/P history No Yes 114 (86.36) 18 (13.64) 270 (97.47) 7(2,52) <0.001 Reference 1.188(1.097-1.286)

Folic acid Yes

No 116 (87.88) 16 (12.12) 271 (97.83) 6 (2.16) <0.001 R e f e r e n c e 6.230 (2.378-16.322) Nutritional supplementation (iron) Yes No 109 (82.58) 23 (17.42) 223 (80.50) 54 (19.50) 0.617 Reference 0.871 (0.508-1.494) Vitamin supplementation Yes No 114 (86.37) 18 (13.63) 271 (97.83) 6 (2.17) <0.001 R e f e r e n c e 7.132 (2.759-18.431) Second-hand tobacco smoke > 15 min No Yes 48 (36.37) 84 (63.63) 218 (78.70) 59 (21.30) <0.001 Reference 6.466 (4.096-10.208) Alcohol consumption No Yes 127 (96.22) 5 (3.78) 265 (95.66) 12 (4.34) 0.797 Reference 0.869 (0.300-2.521)

Exposure to chemicals No Yes 127 (96.22) 5 (3.78) 270 (97.47) 7 (2.53) 0.480 Reference 1.419 (0.743-4.877) Caffeine consumption No Yes 5 (3.78) 127 (96.22) 31 (11.19) 246 (88.81) 0.013 Reference 3.201 (1215-8.431) Medicine Consumption No Yes 47 (35.61) 85 (64.39) 187 (67.50) 90 (32.50) <0.001 Reference 3.758 (2.430-5.812)

Table 2 - Reference values of caffeine consumption of the case and control groups Case n= 127 (96.2%) Control n= 246 (88.8%) p value OD ratio (95%) >100<= 500mg 20 (15.74%) 44 (17.88%) 0.070 2.727(0.922-8.066) >500 mg 107 (84.25%) 203 (82.52%) 0.021 3.163 (1.193-8.387) Reference: (0 <=100mg)

Artigo 2

*

Dental anomalies in Brazilian patients with non-syndromic isolated cleft lip with or without alveolar ridge involvement

Rodrigo Soares de Andrade1 _{, Alison José Martelli}1 _{,Adriana Freire Boeri Tamburini}2 _,

Daniella Reis B. Martelli3 _{, Letízia Monteiro de Barros}2 _{, Hercílio Martelli Júnior}2, 3

1_{Department of Oral Diagnosis, School of Dentistry, State University of Campinas,}

FOP-UNICAMP, Piracicaba, São Paulo, Brazil.

2_{Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of José}

Rosario Vellano, Alfenas, Minas Gerais, Brazil.

3_{Dental School, State University of Montes Claros, Unimontes, Minas Gerais State, Brazil.}

Correspondence: Rodrigo Soares de Andrade, Oral Pathology, FOP-UNICAMP.

Avenue Limeira, 901 - Areião, Piracicaba – São Paulo, Zip code: 13414-018

Piracicaba – São Paulo – Brazil.

Abstract

Background: Some studies have demonstrated a high prevalence of dental anomalies in

patients with nonsyndromic cleft lip and/or palate (NSCL/P). Few studies have evaluated these anomalies in patients’ cleft lip with or without alveolus (NSCL±A).

Objective: The aim of the current study was to investigate the prevalence of dental anomalies

in a group of Brazilian patients with NSCL±A isolated.

Methods: Of the 1,528 patients with oral clefts treated at the Centre for Rehabilitation of

Craniofacial Anomalies, 154 (10.07%) subjects with NSCL±A were included in this study. Panoramic and periapical radiographs were performed and dental anomalies of size, number and shape were considered.

Results: Of the 154 patients, 87 (56.50%) were male and 67 (43.50%) female. Ninety

(58.40%) patients presented NSCL+A and 64 (41.60%) without NSCL-A. Of these 70 subjects with dental anomalies, 44 (62.85%) were male and 26 (37.14%) female (p=0.146) and, 98 anomalies were observed, being 66 (67.34%) in the NSCL+A group and 32 (32.65%) in the NSCL-A. Dental agenesis was the most prevalent anomaly, representing 56.12% (n=55). Supernumerary teeth representing 29.59% (n=29) of all anomalies and microdontia representing 12.24% (n=12).

Conclusion: In both groups of NSCL, complete and incomplete, the most common dental

anomalies were the agenesia followed by supranumerary teeth, followed for microdontia. NSCL+A was the group with the highest number of dental anomalies.

Keywords: Cleft lip, dental anomalies, oral clefts, taurodontism, oligodontia. Introduction

Nonsyndromic oral clefts are common congenital birth defects in humans characterized by defective fusions of the facial processes, occurring in 1 in 500-2,500 live births worldwide.1 In Brazil, considering the geographic variation, the prevalence varies between 1:650 to 1:2,700 live birth.2,3 About 70% of cases occur as a nonsyndromic form [nonsyndromic cleft lip and/or cleft palate (NSCL/P)], and the remaining 30% are associated with Mendelian disorders or chromosomal, teratogenic and sporadic conditions.4

According to the anatomical site, NSCL/P are commonly classified in cleft lip with or without alveolus (NSCL±A), cleft lip and palate (NSCLP) or cleft palate only (NSCP).5,6 A complex and heterogeneous etiology with interplay among environmental, genetic and epigenetics factors has been reported, but the factors associated remain partially defined.7,8

The human face and the upper jaw begin their development in the fourth week of gestation by mesenchymal migration and fusion of the primitive facial elements: the paired medial nasal processes and maxillary processes. As the medial nasal processes fuse with each other, they form the anterior portion of the upper jaw – the pre-maxilla – from which the dental incisors originate, and also the medial portion of the upper lip (philtrum) and the primary palate. An isolated soft tissue cleft lip is caused by a disturbance in the approximation, fusion or mesenchyme penetration through the epithelial membrane surrounding the medial, and lateral nasal processes and the maxillary process.9,10

The clinical term cleft lip is often used in the literature to describe both isolated soft tissue cleft lip and cleft of the lip and the alveolar process; both originate in the embryonic formation of the primary palate. However, the two are significantly different from one another from a clinical point of view.10

Studies involving different populations show that patients with NSCL/P have a higher prevalence of dental anomalies in relation to the general population.6,11-14 Although dental

anomalies are more prevalent in the NSCL/P, few studies have investigated these alterations in the isolated NSCL±A.6,10 _{Thus, the aim of the current study was to investigate the}

prevalence of dental anomalies in a group of Brazilian patients with NSCL±A isolated.

Material and methods

This cross-sectional, epidemiological and observational study. All participants were recruited from the same institution (Centre for Rehabilitation of Craniofacial Anomalies). Records from 2013 to 2018 to patients with NSCL/P were reviewed; those without complete dental history, panoramic and periapical radiographs were not included in the final analysis. Similarly, patients with a history of dental extraction, who had previous orthodontic treatment or were at a young age (12 years old), due to the inability to accurately identify all anomalies, with associations or syndromes, with NSCP or NSCLP were excluded as well. Were included only patients with NSCL±A. Dental anomalies in third molar were also not considered in this study. Of the 2.484 patients, only 154 with NSCL±A remained in the study, after the application of the exclusion criteria.

All patients were carefully evaluated by experts of Center for Rehabilitation of Craniofacial Anomalies. In order to eliminate inter examiner differences, dental anomalies were classified by a single calibrated examiner. The determination of cleft phenotype was based on the description in the NSCL-A files and confirmed by examination of the dental casts and radiographs.11,14

All of the subjects were from Minas Gerais, Brazil, where there is an admixed population of Europeans (mostly from Portugal and Italy) and Africans, with a small percentage of native Brazilian Indians.15 All patients presented similar ethnicities and social culture and were assisted by the Brazilian Public Health System.The information collected

was stored in a database and analysed using the statistical program SPSS® version 24.0 (Statistical Package for Social Sciences for Windows, Inc., USA). Comparisons were made by cross-tabulation and standard chi-square test, with statistical significance set at p≤.05. This study was approved by the Research Ethics Committee (#2.746.400).

Results

Of the 1,528 patients treated at the Centre for Rehabilitation of Craniofacial Anomalies, 154 (10.07%) subjects with NSCL±A were included in this study. Of the 154 patients, 87 (56.50%) were male and 67 (43.50%) female. Ninety (58.40%) patients presented NSCL+A and 64 (41.60%) without NSCL-A. Of the 154 patients with NSCL±A, 84 (54.55%) had no dental anomalies and 70 (45.45%) (p=0.353) patients presented 98 dental anomalies in total. Of the 70 patients with dental anomalies, 44 (62.85%) were male and 26 (37.14%) female (p=0.146).

Among the 70 patients with dental anomalies, 98 changes were observed, being 66 (67.34%) in the NSCL+A group and 32 (32.65%) in the NSCL-A (Table 1 and 2). Dental agenesis was the most prevalent anomaly, representing 56.12% (n=55) of the 98 observed. In the NSCL+A group, the agenesis accounted for 56.06% (n=37) and in the NSCL-A occurred in 56.25% (n=18) of the cases. It is emphasized that of 55 dental agenesis in both groups studied, 46 (83.63%) affected the lateral incisors.

Supernumerary teeth were the second most common dental anomalies, representing 29.59% (n=29) of all anomalies. In the NSCL+A group, the supernumerary teeth accounted for 30.30% (n=20) and in the NSCL-A occurred in 39.13% (n=9) of the cases. In both groups, the highest occurrence of supernumerary teeth was observed in the lateral incisors (n=14), as well as the dental agenesis.

Subsequently, the third most observed dental anomaly was microdontia, representing 12.24% (n=12) of all dental anomalies. In the NSCL+A group, the microdontia accounted for 12.12% (n=8) and in the NSCL-A occurred in 12.50% (n=4) of the cases. In the NSCL+A group, the occurrence of microdontia was also more observed among the lateral incisors (n=6). In addition to the described dental anomalies, it was observed the occurrence of one case of taurodontism (NSCL+A group) and one case (NSCL-A group) of gemination.

Discussion

The NSCL is an anatomical form of the cleft, implies less mesenchymal tissue, allowing greater incidences of crown malformation of central and lateral incisors, supernumerary reflected in sufficient space for the dichotomous development of the dental root.16 NSCL is caused by a disturbance in the mesenchymal fusion of the medial and lateral nasal processes with the maxillary process.17 The process of tooth formation involves from the dental blade to the mesenchymal tissue around it (also called the dental papilla), and this mesenchymal tissue becomes the dentinofibrous complex, which is the main topografic influence of the dental tissue.18

The isolated soft tissue NSCL without evidence of any type of bone involvement is a relatively rare phenomenon.19,20 Sekhon et al. (2009) reported a prevalence of 2.1%, equally distributed among male and female patients21_{, different from this study, where we found a}

higher prevalence in male patients. These findings corroborate the findings of many studies that have emphasized sexual dimorphism in oral clefts.22-25

The reason for the high prevalence of dental abnormalities is not fully understood, but the assumption is that etiology is a prenatal outcome that interacts with an altered genotype.26 Dental agenesis is largely genetically determined and transmitted by autosomal dominant

inheritance, with incomplete penetration and variable expression.27,28 The agenesis of one or more permanent teeth is the most common congenital anomaly found in humans, affecting about 20% of the world population29, which is still present in NSCL patients, as we observed in this study where agenesis was the most frequent dental anomaly (78.5%).

Oral clefts in humans are usually associated with delayed development of teeth and abnormalities in tooth number, size and shape, both on the cleft side and on the non-cleft side.30 _{Rawashdeh and Bakir (2007) showed a that reduction of tooth size occurred in all types}

of permanent teeth and between early and late teeth in patients with cleft compared to controls31_{, which in this work we observed the presence of microdontia in 8 patients with}

NSCL+A, more frequent in upper lateral incisors, corroborating with others studies29,31,32, who also found a higher frequency of microdontia in upper lateral incisors.

The results of this study support this assumption, that the oral cleft patient has a higher prevalence of almost all types of dental anomalies and was evaluated in this study and found to be more accurate, the group more affected by the NSCL+A compared to the NSCL-A group, with the normal population32. In this study, dental anomalies were analyzed only in the permanent dentition, indicating a high percentage of supernumerary teeth about 41.4%. The reported prevalence of supernumerary teeth in the general population for permanent dentition ranges from 0.1% to 3.8%33, in patients with NSCL+A, twice the frequency (28.5%) when compared to patients with NSCL-A (12.8%).

The knowledge of dental anomalies, such a dental agenesis or supernumerary teeth, is fundamental for oral treatment planning, since such anomalies can lead to edentulous spaces in the maxillary arch that must be closed by orthodontic movements, prostheses or implants.30 Microdontia was observed in 12.12% (NSCL+A) and 12.50% (NSCL-A) in this sample. In this sample of microdontia was observed mainly in the upper lateral incisors. It has

been reported that taurodontism is associated with certain syndromes and disorders of dental development33_{. The results of the present study showed that the general prevalence of}

taurodontism was 1.42% for individuals with NSCL and it was found only in patients with NSCL+A. However, when we compare this prevalence with normal and healthy individuals (0.1%)31 it seems that this result is in agreement with Burzynski & Escobar (2003), Aizenbud et al. (2011) and Mangione et al. (2018) that found that taurodontism occurred more frequently in individuals with oral clefts.9,17,28

Regarding the differences in the prevalence of dental anomalies between NSCL+A and NSCL-A, dental agenesis, supernumerary and microdontia, were significantly higher in individuals with NSCL+A. This is probably due to multiple anatomical, genetic and environmental factors, mesenchymal deficiency, and the direct effect of cleft on the primordial tissues.19,25,28,29,32

It can be concluded from the results of this study that the frequency of certain dental anomalies is strongly related to the phenomena of NSCL is a correlated to have the involvement of the component tissues of the alveolar ridge. Sample size was limited due to the rarity of isolated NSCL and the limited number of patients. We also observed as a limitation of the study that the sample was established geographically in the same region. More evidence to establish the exact nature of this relationship with each dental anomaly is necessary.

Acknowledgments

The Minas Gerais State Research Foundation – Fapemig, Brazil and National Council for Scientific and Technological Development – CNPq, Brazil

Conflict of interest

None declared.

Ethical approval

Approval for this retrospective study was granted by the research ethics committee of the Jose do Rosário Vellano University and the Piracicaba School of Dentistry/University of Campinas (Registration number: 3.574.351). All clinical data were anonymized, and all potential patient identifiers were removed after the return of the database surveys.

Patient consent

Not required.

ORCID

Rodrigo Soares de Andrade http://orcid.org/ 0000-0001-6114-0929

Alison José Martelli http://orcid.org/0000-0002-5361-9240,

Adriana Freire Boeri Tamburini http://orcid.org/0000-0002-0153-3875

Daniella Reis B. Martelli http://orcid.org/0000-0002-3979-7497

Letízia Monteiro de Barros http://orcid.org/0000-0002-5181-6224

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