Study of the genetic basis of syndromic microcephaly associated with neurodevelopmental disorders

O desenvolvimento embrionário do cérebro humano é intrincado e altamente coordenado espacial e temporalmente, o que é primordial para seu correto funcionamento, assim como foi no passado ao longo da evolução dos primatas. Distúrbios neste processo podem comprometer função e tamanho do órgão, como na microcefalia, uma condição clínica caracterizada pela redução da circunferência occipitofrontal da cabeça em pelo menos 2 desvios-padrão (SD) da média para aquela idade e sexo. A microcefalia pode ser classificada em primária (congênita) ou secundária (pós-natal), sendo considerada leve (-2 a -3 SD) ou grave (≤ -3 SD); também pode ser classificada como sindrômica, se alterações externas ao sistema nervoso central estiverem presentes. Sua incidência é de 2-3% na população mundial e tem etiologias diversas, tanto ambiental quanto genética, com a maior parte dos casos permanecendo idiopática. Por se tratar de uma condição complexa que muitas vezes se apresenta associada a outros fenótipos, ainda há muito a ser explorado em relação às bases genéticas da microcefalia, previamente não investigadas na população brasileira. Este trabalho se propôs a avaliar uma coorte de indivíduos brasileiros afetados por microcefalia associada a distúrbios de neurodesenvolvimento, visando caracterizar as bases genéticas do fenótipo e quadros clínicos associados. Para tanto, realizamos análise cromossômica por microarranjo genômico (CMA) para investigação de alterações de número de cópias (CNVs), bem como sequenciamento de exoma completo (WES) para prospecção de variantes genômicos pontuais em um subgrupo de pacientes. No estudo por CMA, analisamos 185 pacientes microcefálicos, dos quais 39 tinham CNVs clinicamente relevantes. Compilamos uma lista inédita de genes reconhecidos como causa de microcefalia e realizamos o levantamento das síndromes de CNVs associadas. Neste estudo de CNVs raras causais, sugerimos novos genes candidatos para o fenótipo com base na caracterização das regiões afetadas no grupo avaliado de pacientes, assim como em pacientes microcefálicos descritos no banco de dados DECIPHER: genes reconhecidamente associados à microcefalia por SNVs e indels, mas não ainda por CNVs; aqueles com evidência ainda insuficiente para corroborar tal associação; genes conhecidos de macrocefalia; e novos candidatos, propostos no presente estudo, como OTUD7A, BBC3, CNTN6 e NAA15. No estudo por WES, investigamos 45 indivíduos (42 famílias) que tiveram resultado negativo no CMA (100 exomas analisados no total), resultando em uma taxa de diagnóstico molecular (variantes patogênicas ou provavelmente patogênicas) de 47,6%, similar à reportada na literatura. Em relação à apresentação clínica, deficiência intelectual e alterações cerebrais estruturais foram altamente prevalentes, com taxas similares a outras coortes de microcefalia, mas encontramos frequências superiores de dismorfismos faciais, alterações comportamentais, cardiovasculares e musculoesqueléticas, além de perda auditiva. Pela análise dos casos negativos no CMA e exoma, identificamos um gene novo causativo para o fenótipo (CCDC17), que requer estudos funcionais futuros para validação. Em conclusão, contribuímos para o diagnóstico molecular e aconselhamento genético de metade de um total de 48 famílias (no mínimo um indivíduo com microcefalia dentre seus sinais clínicos). Em seis casos, foram identificadas variantes patogênicas ou provavelmente patogênicas do tipo CNV, e em 20 casos foram detectadas variantes do tipo SNV/indel (taxa de diagnóstico molecular global de 54,2%). Estes achados corroboram a microcefalia como um importante indicador de alterações genômicas e evidenciam que o exoma como primeira linha de investigação seria a melhor estratégia para diagnóstico desta condição. Adicionalmente, trouxemos uma visão inédita das causas genéticas da condição na população brasileira e das comorbidades mais frequentes, bem como pudemos sugerir novos genes candidatos ao fenótipo que poderão ser explorados em pesquisas futuras. Ainda assim, aproximadamente metade dos casos avaliados permanece idiopático, apontando para a necessidade de estratégias complementares de investigação de alterações estruturais e não-codificadoras, baseadas em tecnologias como sequenciamento de genoma completo, sequenciamento de moléculas longas, mapeamento ótico, estudos epigenéticos e investigação de alterações mitocondriais.The embryonic development of the human brain is intricate and spatially and temporally highly coordinated, which is essential for its proper performance, as it was in the past throughout the evolution of primates. Disturbances in this process can compromise the organ function and size, as occurs in microcephaly. It is a clinical condition characterized by reduction of the occipitofrontal circumference by at least 2 standard deviations (SD) from the mean for that age and sex. Microcephaly can be primary (congenital) or secondary (postnatal), considered mild (-2 to -3 SD) or severe (≤ -3 SD); it can also be classified as syndromic if alterations external to the central nervous system are present. Its incidence is 2-3% in the world population and has various etiologies, environmental and genetic, with most cases remaining idiopathic. Because it is a complex condition often associated with other phenotypes, there is still much to be explored about the genetic bases of microcephaly, previously not investigated in the Brazilian population. This work aimed to evaluate a cohort of Brazilian individuals affected by microcephaly associated with neurodevelopmental disorders, aiming to characterize the genetic bases of the phenotype, and associated clinical conditions. To this end, we performed chromosomal microarray analysis (CMA) to investigate copy number variants (CNVs), and, in a subgroup of patients, whole-exome sequencing (WES) to prospect for specific genomic variants. In the CMA study, we analyzed 185 microcephalic patients, of whom 39 had clinically relevant CNVs. We compiled a list of recognized microcephaly genes and surveyed associated CNV syndromes. In this study of rare causal CNVs, we suggested new candidate genes for the phenotype based on the characterization of the affected regions in the evaluated group of patients, as well as evaluating microcephalic patients reported in the DECIPHER database: genes known to be associated with microcephaly by SNVs and indels, but not yet by CNVs; those with insufficient evidence to corroborate such an association; known macrocephaly genes; and new candidates, such as OTUD7A, BBC3, CNTN6, and NAA15. In the WES study, we investigated 45 individuals (42 families) who tested negative for CMA (100 exomes analyzed in total), resulting in a molecular diagnosis rate (pathogenic or likely pathogenic variants) of 47.6%, similar to the yield reported in WES studies from the literature. Regarding the clinical presentation, intellectual disability, and structural brain alterations were highly prevalent, with rates similar to other cohorts of microcephaly, but we found higher frequencies of facial dysmorphisms, behavioral, cardiovascular, and musculoskeletal alterations, in addition to hearing loss. By analyzing the negative cases after CMA and exome analysis, we identified a novel gene for the microcephaly (CCDC17), which requires future functional studies for validation. In conclusion, we contributed to the molecular diagnosis and genetic counseling of half of a total of 48 families with at least one individual with microcephaly among its clinical signs. In six cases, pathogenic or probably pathogenic CNV variants were identified, and in 20 cases SNV/indel variants were detected (overall molecular diagnosis rate 54.2%). These findings have corroborated that microcephaly is an important indicator of genomic alterations and showed that using exome as the first-tier of investigation would be the best strategy for diagnosing this condition. Additionally, we brought an unprecedented view of the genetic causes of microcephaly in a Brazilian population, and the most frequent comorbidities, as well as suggested new candidate genes that could be explored in future research. Even so, approximately half of the evaluated cases remain idiopathic, pointing to the need for complementary strategies for the investigation of structural and non-coding variants, based on technologies such as whole-genome sequencing, sequencing of long reads, optical mapping, epigenetic studies, and investigation of mitochondrial variants

Chave de identificação ilustrada dos Phlebotominae (Diptera, Psychodidae) do estado de São Paulo, Brasil

Phlebotomine sand flies are medically important insects, responsible for the transmission of Leishmania parasites between humans and non-human animals, which are found throughout São Paulo state, Brazil. The correct identification of species implicated in the transmission of diseases is paramount to elaborate strategies for vector control. In order to produce an illustrated key to the 67 sand flies male species and 57 female species recorded in São Paulo state we examined and illustrated specimens mounted in microscope slides from entomological collections.Flebotomíneos são insetos de importância médica, responsáveis pela transmissão de parasitas do gênero Leishmania entre humanos e animais não humanos, os quais são encontrados no estado de São Paulo, Brasil. A correta identificação das espécies envolvidas na transmissão de doenças é fundamental para elaboração de estratégias para o controle desses vetores. Para produzir uma chave ilustrada para as 67 espécies de machos e 57 espécies de fêmeas de flebotomíneos registradas no estado de São Paulo, examinamos e ilustramos espécimes montados em lâminas de microscopia obtidos de coleções entomológicas

Geographical distribution of American cutaneous leishmaniasis and its phlebotomine vectors (Diptera: Psychodidae) in the state of São Paulo, Brazil

<p>Abstract</p> <p>Background</p> <p>American cutaneous leishmaniasis (ACL) is a re-emerging disease in the state of São Paulo, Brazil. It is important to understand both the vector and disease distribution to help design control strategies. As an initial step in applying geographic information systems (GIS) and remote sensing (RS) tools to map disease-risk, the objectives of the present work were to: (i) produce a single database of species distributions of the sand fly vectors in the state of São Paulo, (ii) create combined distributional maps of both the incidence of ACL and its sand fly vectors, and (iii) thereby provide individual municipalities with a source of reference material for work carried out in their area.</p> <p>Results</p> <p>A database containing 910 individual records of sand fly occurrence in the state of São Paulo, from 37 different sources, was compiled. These records date from between 1943 to 2009, and describe the presence of at least one of the six incriminated or suspected sand fly vector species in 183/645 (28.4%) municipalities. For the remaining 462 (71.6%) municipalities, we were unable to locate records of any of the six incriminated or suspected sand fly vector species (<it>Nyssomyia intermedia</it>, <it>N. neivai</it>, <it>N. whitmani</it>, <it>Pintomyia fischeri</it>, <it>P. pessoai </it>and <it>Migonemyia migonei</it>). The distribution of each of the six incriminated or suspected vector species of ACL in the state of São Paulo were individually mapped and overlaid on the incidence of ACL for the period 1993 to 1995 and 1998 to 2007. Overall, the maps reveal that the six sand fly vector species analyzed have unique and heterogeneous, although often overlapping, distributions. Several sand fly species - <it>Nyssomyia intermedia </it>and <it>N. neivai </it>- are highly localized, while the other sand fly species - <it>N. whitmani, M. migonei, P. fischeri </it>and <it>P. pessoai </it>- are much more broadly distributed. ACL has been reported in 160/183 (87.4%) of the municipalities with records for at least one of the six incriminated or suspected sand fly vector species, while there are no records of any of these sand fly species in 318/478 (66.5%) municipalities with ACL.</p> <p>Conclusions</p> <p>The maps produced in this work provide basic data on the distribution of the six incriminated or suspected sand fly vectors of ACL in the state of São Paulo, and highlight the complex and geographically heterogeneous pattern of ACL transmission in the region. Further studies are required to clarify the role of each of the six suspected sand fly vector species in different regions of the state of São Paulo, especially in the majority of municipalities where ACL is present but sand fly vectors have not yet been identified.</p

Dipterofauna Associated with Sus scrofa

Cadaverous entomofauna successions vary according to the region, environment, and climate, and such differences may occur within the same country due to seasonal variations. The present study aimed to analyze and compare the dipterofauna that visit or colonize carcasses in the urban and coastal areas of São Paulo, Brazil, during summer and winter seasons. Four swine (Sus scrofa Linné, 1758) carcasses of approximately 12 kg were used. The animals were previously euthanized and then placed in metal cages covered with a flight intercept trap (Shannon, modified). In total, 10,495 flies from 39 families were collected, with 15 species belonging to the Calliphoridae family, 14 species belonging to the Fanniidae family, 43 species belonging to the Muscidae family, and 22 species belonging to the Sarcophagidae family. Flies from these four families visited all carcasses; however, they did not show the highest visitation frequencies in all of the trials. Species variations occurred between the experiments that were performed at different locations and in different seasons. Furthermore, difference in the number of insects attracted to each stage of decomposition was observed. In addition to the four families highlighted above, the families Phoridae, Sepsidae, Otitidae, and Piophilidae were observed in all carcasses

Human visceral leishmaniasis and relationship with vector and canine control measures

OBJECTIVE: Estimate the coverage of control measures of visceral leishmaniasis and relate them with the occurrence of human visceral leishmaniasis in endemic urban area. METHODS: Cases of human and canine visceral leishmaniasis were considered as study population and evaluated by a serological survey conducted in Araçatuba, state São Paulo, from 2007 to 2015. The cases of human visceral leishmaniasis were geocoded by the address of the patients and the canine disease by the address of the dogs’ owners. The coverage of serological survey, euthanasia, and insecticide spraying was calculated, as well as the canine seroprevalence and the incidence rates of human visceral leishmaniasis. The relationship between human visceral leishmaniasis and control measures was evaluated, as well as the seroprevalence by comparing maps and by linear regression. The relationship between the canine and the human disease was also evaluated by the Ripley’s K function. RESULTS: The incidence rates of human visceral leishmaniasis showed a period of decline (2007 to 2009) and a period of stability (2010 to 2015), a behavior similar to that of canine seroprevalence. In general, the coverage of control measures was low, and the non-association with the incidence of human visceral leishmaniasis can be a result of the period analyzed and of the small number of analyzed units (sectors of the Superintendence for the Control of Endemic Diseases). The distribution of human cases showed spatial dependence with the distribution of seropositive dogs from 2007 to 2009. CONCLUSIONS: This study reaffirmed the relationship between the occurrence of the disease in humans and dogs, it verified a decrease in the rates of visceral leishmaniasis in Araçatuba over time, even at low coverage of control activities. However, further studies are needed to determine if factors beyond monitoring and control measures are involved in the reduction of incidences.OBJETIVO: Estimar a cobertura das atividades de controle da leishmaniose visceral e relacioná-las com a ocorrência de leishmaniose visceral em humanos em área urbana endêmica. MÉTODOS: Foram considerados como população de estudo os casos de leishmaniose visceral em humanos e em cães avaliados por inquérito sorológico censitário realizado em Araçatuba, SP, de 2007 a 2015. Os casos de leishmaniose visceral em humanos foram geocodificados pelo endereço de residência dos pacientes e, os cães, pelo endereço de residências dos respectivos tutores. Foram calculadas as coberturas do inquérito sorológico, da eutanásia e de borrifação de inseticida, as soroprevalências caninas e as taxas de incidência de leishmaniose visceral em humanos. A relação entre a leishmaniose visceral em humanos e as medidas de controle, bem como a soroprevalência foram avaliadas por comparação de mapas e por meio de regressão linear. A relação entre a doença canina e a humana também foi avaliada por meio da função K de Ripley. RESULTADOS: As taxas de incidência de leishmaniose visceral em humanos apresentaram um período de declínio (2007 a 2009) e um período de estabilidade (2010 a 2015), comportamento semelhante ao das soroprevalências caninas. Em geral, a cobertura das medidas de controle foi baixa e a não associação com a incidência de leishmaniose visceral em humanos pode ser consequência do período analisado e do número pequeno de unidades analisadas (setores da Superintendência de Controle de Endemias). A distribuição dos casos humanos apresentou dependência espacial com a distribuição dos cães soropositivos de 2007 a 2009. CONCLUSÕES: Este trabalho reafirmou a relação entre a ocorrência da doença no homem e no cão, verificou a diminuição das taxas de leishmaniose visceral em humanos e em cães em Araçatuba ao longo do tempo, mesmo em baixa cobertura das atividades de controle. Entretanto, novos estudos são necessários para averiguar se fatores além das atividades de vigilância e controle estariam envolvidos na diminuição das incidências

Spatial epidemiology of American cutaneous leishmaniasis in a municipality of west São Paulo State, Brazil

Background & objectives: In the last decade, in the state of São Paulo, 5898 cases of cutaneous leishmaniasis (CL) were reported. This study was undertaken to analyze the epidemiology of CL in the municipality of Teodoro Sampaio, in São Paulo State, Brazil, based on a geographic approach, as very little is known of the relationship between CL and the spatial transformation process. Methods: This is a population-based quantitative, descriptive and cross-sectional case study. Surveys of the official notifications in the healthcare center and official sources from 1998 to 2011 were analysed. The data were described based on statistics and the Kernel method to detect hotspots of transmission. Results: The age group between 21 and 40 yr was most affected, with 24 cases (57.9%). Of the 41 cases reported between 1998 and 2011, 33 cases were having low education status and 31 cases (75.6%) were males. The spatial and temporal distribution was aggregated in three-year periods which permitted the identification of two microfoci, in periods I (1998–2000) and III (2005–2007). Interpretation & conclusion: The disease has presented, in recent years, a pattern of sporadic transmission or microfoci, and continues to maintain enzootic cycles of Leishmania in a sylvatic environment, ensuring the perpetuation of the pathogen in nature, and the risk of emergence of new cases of CL in domestic animals and humans

Investigação epidemiológica de, um novo caso de leishmaniose visceral ocorrido na Grande São Paulo, Brasil

An epidemiological investigation was made in the urban zone of S. Paulo, Brazil, to elucidate a case of visceral leishmaniasis that occurred in a two-year old child, born and always resident in that place. The possibility of congenital transmission was eliminated. The transmission by blood transfusion and biological vector was discussed on the basis of the past history of the child and the results of a serological survery in human population with indirect imunofluorescence, passive hemagglutination and leishmanin tests, serological survey on dogs and furthermore entomological research in residual forest situated in the neighbourhood of the patient's house.Descreve-se uma investigação epidemiológica realizada em zona urbana do município de São Paulo, Brasil, para esclarecer um caso de leishmaniose visceral ocorrido em criança de 2 anos de idade, nascida e sempre residente no local. Afastou-se a possibilidade de transmissão por via transfusional e por vetor biológico, tendo como base os dados levantados da anamnese do doente, os resultados de inquéritos realizados na área em população humana, utilizando testes de imunofluorescência indireta, hemaglutinação passiva e intradermoreação de Montenegro, em população canina com o teste de imunofluorescência indireta, além de pesquisa entomológica em mata residual