Novas aplicações do diagnóstico pré-natal não invasivo pela análise do plasma materno

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2018.A descoberta do DNA fetal livre de células na circulação materna viabilizou o desenvolvimento de tecnologias para o diagnóstico pré-natal não invasivo. Neste trabalho foram desenvolvidos três estudos independentes relacionados a este tema. O objetivo do primeiro estudo foi determinar o haplótipo Y-STR do feto no plasma materno durante a gravidez e estimar, de forma não invasiva, se o suposto pai e feto pertencem à mesma linhagem masculina. O estudo incluiu casais com gravidez única e paternidade supostamente conhecida. O sangue periférico foi coletado em tubos EDTA (mãe) e em papel FTA (pai). O DNA do plasma materno foi extraído usando NucliSens EasyMAG (Biomerieux). O sexo fetal foi determinado por qPCR pesquisando o alvo DYS-14 no plasma materno e também foi confirmado após o parto. Dos voluntários incluídos, 20 mães com fetos masculinos e 10 mães com fetos femininos foram selecionadas para a análise de Y-STR. A moda da idade gestacional foi de 12 semanas. Todas as amostras de DNA foram submetidas a amplificação por PCR com kits PowerPlex Y23, ampFLSTR Yfiler e dois multiplexes próprios, que em conjunto representam 27 diferentes Y-STR. Os produtos de PCR foram detectados no analisador genético 3500 e foram analisados usando o software GeneMapper-IDX (Life Technologies). Os haplótipos fetais (formato Yfiler) foram comparados com outros 5328 haplótipos brasileiros disponíveis no banco de dados de referência de haplótipos cromossômicos Y (YHRD). Como resultado, entre 22 e 27 loci foram amplificados com sucesso a partir do plasma materno em todos os 20 casos de fetos masculinos. Nenhuma das mulheres com fetos femininos apresentou um haplótipo Y-STR falsamente amplificado. O haplótipo detectado no plasma materno correspondeu ao haplótipo do pai alegado em 16 dos 20 casos. Quatro casos apresentaram inconsistências simples e que não configuravam exclusões; 1 caso mostrou uma mutação no locus DYS458 devido à contração de uma unidade repetição e 3 casos apresentaram “dropout” em um dos locus DYS385 I/II. Todas as inconsistências foram 6 confirmadas após o nascimento do bebê. Dezesseis haplótipos fetais não foram encontrados em YHRD e um deles apresentou mutação, o que corresponde à probabilidade de paternidade de 99,9812% e 95,7028%, respectivamente. Três haplótipos fetais ocorreram duas vezes no YHRD, o que corresponde à probabilidade de paternidade de 99,9437%. Em conclusão, um haplótipo Y-STR fetal discriminatório pode ser obtido da análise do plasma materno durante a gravidez a partir da décima segunda semanas de gestação. Todos os fetos masculinos puderam ser atribuídos à linhagem masculina do pai alegado no início da gravidez. A alta probabilidade de paternidade associada a cada caso sugere que a relação não é aleatória e essa estratégia pode ser usada como alternativa para análise de parentesco fetal masculino. Posteriormente foi questionado se o DNA fetal estaria presente na microcirculação materna e se a determinação não invasiva do sexo fetal utilizando o sangue capilar coletado por punção digital é comparável aos resultados obtidos do plasma isolado do sangue venoso. Desta forma, o segundo estudo envolveu 101 voluntárias grávidas. A mediana da idade gestacional (min–max) foi 11 (8–20) semanas. Inicialmente o desenho experimental do estudo foi prospectivo. No entanto, após iniciar os experimentos resultados falsos positivos para o DNA masculino foram identificados nas amostras de sangue capilar usando um protocolo de assepsia com álcool isopropílico. Suspeitou-se da contaminação da pele dos dedos maternos com DNA exógeno masculino. Subsequentemente novos protocolos de assepsia do local de coleta do sangue capilar (ponta dos dedos) foram implementados (hipoclorito de sódio 0,5% tamponado aplicado duas vezes e hipoclorito de sódio 1% tamponado aplicado uma vez). Os resultados obtidos do plasma isolado do sangue venoso e capilar foram comparados com o sexo do recém-nascido ao nascimento. As análises revelaram que a sensibilidade e especificidade da determinação do sexo fetal no sangue venoso comparado ao sexo no nascimento foi 100% (95%IC 93- 100%) e 100% (95%IC 93-100%). No sangue capilar, os resultados foram 100% (95%IC 78-100%) e 58% (95%IC 27-84%) para o grupo do álcool isopropílico, 100% (95%IC 83-100%) e 100% (95%IC 82-100%) para o grupo do hipoclorito de sódio tamponado à 0,5%, e 100% (95%IC 80-100%) e 100% (95%IC 81-100%) para o grupo do hipoclorito de sódio tamponado à 1%, respectivamente. Em conclusão, o DNA fetal está presente no sangue capilar materno permitindo a determinação não invasiva do sexo fetal. O DNA masculino exógeno pode estar presente nas pontas dos dedos das mulheres grávidas e ser detectável gerando resultados falsos positivos. Por isto, a eliminação do DNA masculino exógeno é crítica para uma confiável determinação do sexo fetal usando o sangue capilar materno. As soluções de hipoclorito de sódio diluído e tamponado, entre 0,5% e 1% pode ser usado para eliminar o DNA masculino exógeno das pontas dos dedos das mulheres grávidas. Por fim, o uso do cromossomo Y no diagnóstico pré-natal não invasivo limita sua aplicação aos fetos masculinos. Portanto, seria importante o desenvolvimento de tecnologias para análise do DNA fetal independentemente do sexo fetal e que contemple os cromossomos autossômicos. Assim, hipotetizou-se usar os polimorfismos de inserção e deleção (Indels) presentes no pai e ausentes na mãe como “pequenas ilhas” de diferenças genéticas entre mãe e feto para o estudo pré-natal não invasivo das características genéticas do feto. Entretanto, antes de desenvolver esta metodologia seria necessário conhecer as frequências alélicas e os parâmetros forenses destes Indels usando um conjunto de marcadores que tenha sido previamente validado e de preferência na população local. O tema de pesquisa do terceiro estudo foi avaliar as frequências alélicas de 38 indels na população do Distrito Federal. Os resultados indicam que este conjunto é um sistema genético altamente informativo, pois a probabilidade de discriminar dois indivíduos selecionados ao acaso é de 99,9999999999993% e a probabilidade de excluir um indivíduo falsamente acusado em um caso de paternidade é 99,81%. Com base nas frequências alélicas foram calculadas as distâncias genéticas da população estudada (Distrito Federal) em relação as populações descritas em outros estudos (Portugal, África, Ásia, tribo Terena e do estado do Rio de Janeiro). A análise das distâncias genéticas entre as diferentes populações revelou que o Distrito Federal é mais semelhante ao Rio de Janeiro, assumindo uma posição intermediária em relação à Europa e Rio de Janeiro - Afrodescendente e foi distante dos nativos americanos, africanos e do leste asiático. Esta conclusão está de acordo com a composição da população relatada no Censo 2010, em que a maioria dos imigrantes era do Nordeste e do Sudeste. Estes três estudos foram aprovados por comitês de ética locais e todos os participantes assinaram o termo de consentimento livre esclarecido.The discovery of cell-free fetal DNA in the maternal circulation enabled the development of technologies for noninvasive prenatal diagnosis. In this work, three independent studies related to this topic were developed. The aim of the first study is to determine the fetus YSTR haplotype in maternal plasma during pregnancy and estimate, non-invasively, if the alleged father and fetus belong to the same male lineage. The study enrolled couples with singleton pregnancies and supposedly known paternity. Peripheral blood was collected in EDTA tubes (mother) and in FTA paper (father). Maternal plasma DNA was extracted by using NucliSens EasyMAG. Fetal gender was determined by qPCR targeting DYS-14 in maternal plasma and it was also confirmed after the delivery. From all included volunteers, 20 mothers bearing male fetuses and 10 mothers bearing female fetuses were selected for the Y-STR analysis. The mode gestational age was 12 weeks. All DNA samples were subjected to PCR amplification by PowerPlex Y23, ampFLSTR Yfiler, and two in-house multiplexes, which together accounts for 27 different Y-STR. The PCR products were detected with 3500 Genetic Analyzer and they were analyzed using GeneMapper-IDX. Fetuses’ haplotypes (Yfiler format) were compared to other 5328 Brazilian haplotypes available on Y-chromosome haplotypes reference database (YHRD). As a result, between 22 and 27 loci were successfully amplified from maternal plasma in all 20 cases of male fetuses. None of the women bearing female fetuses had a falsely amplified Y-STR haplotype. The haplotype detected in maternal plasma completely matched the alleged father haplotype in 16 out of the 20 cases. Four cases showed single mismatches and they did not configure exclusions; 1 case showed a mutation in the DYS458 locus due to the loss of one repeat unit and 3 cases showed one DYS385I/II locus dropout. All mismatches were confirmed after the delivery. Seventeen fetuses’ haplotypes were not found in YHRD and one of them had a mutation, which corresponded to the paternity probability of 99.9812% and 95.7028%, respectively. Three fetuses’ haplotypes occurred twice in YHRD, which corresponded to paternity probability of 99.9437%. In conclusion, high discriminatory fetal Y-STR haplotype could be determined from maternal plasma during pregnancy starting at 12 weeks of gestation. All male fetuses could be attributed to the alleged father male lineage early in pregnancy. The high probability of paternity associated with each case suggests that the relationship is not random and this strategy can be use as an alternative for male fetal kinship analysis. Posteriorly, questioned whether the presence of fetal DNA on maternal microcirculation and investigate whether the fetal sex determination performed on plasma isolated from capillary blood is comparable to results obtained using plasma isolated from venous blood. Thus, the second study involved 101 pregnant volunteers. The median (min–max) gestational age was 11 (8–20) weeks. The initial study design was prospective. However, after commencement of the study the appearance of false positive results for male DNA among the capillary samples lead us to suspect maternal skin contamination with exogenous male DNA. We subsequently instituted new skin-cleansing protocols for the capillary samples (isopropyl alcohol, 0.5% buffered sodium hypochlorite applied twice and 1% buffered sodium hypochlorite applied once), and compared with these results with our plasma samples. The analysis reveled that, compared to the clinical examination at birth, sensitivity and specificity of the fetal sex determination in venous blood was 100% (95%CI 93-100%) and 100% (95%CI 93-100%). In capillary blood, results were 100% (95%CI 78- 100%) and 58% (95%CI 27-84%) for the isopropyl alcohol group, 100% (95%CI 83-100%) and 100% (95%CI 82-100%) for the 0.5% buffered sodium hypochlorite group and 100% (95%CI 80-100%) and 100% (95%CI 81- 100%) for 1% buffered sodium hypochlorite group, respectively. In conclusion, fetal DNA is present in maternal capillary blood allowing for non-invasive fetal sex determination. Exogenous male DNA may be present, and detectable, on the fingertips of pregnant women. So, the elimination of the exogenous male DNA is critical for a reliable fetal sex determination using maternal capillary blood. A diluted buffered sodium hypochlorite (0.5% - 1%) can be used to eliminate exogenous male DNA from the fingertips of pregnant women. Exploration of the Y chromosome in non-invasive prenatal diagnosis limits its application to male fetuses. Thus, it is important to develop technologies for fetal DNA analysis regardless of the fetal sex and that contemplate the autosomal Chromosomes. Thus, it was hypothesized to use insertion and deletion polymorphisms (Indels) present in the father and absent in the mother as "small islands" of genetic differences between mother and fetus for the non- invasive prenatal study of genetic characteristics of the fetus. However, before developing this methodology it is necessary to know the allelic frequencies and the forensic parameters of these Indels using a set of markers that has been previously validated and preferably in the local population. Thus, the third study was to evaluate the allelic frequencies of 38 indels in the population of the Federal District. The results indicate that this set is a highly informative genetic system. Therefore, the probability of discriminating two individuals selected at random is 99.9999999999993% and the probability of excluding a falsely accused individuals in a paternity case is 99.81%. Based on the allelic frequencies, the genetic distances of the study population (Federal District) were calculated in relation to the populations described by different studies (Portugal, Africa, Asia, Terena and Rio de Janeiro). Furthermore, analysis of genetic distances between diferents populations revealed that Federal District was plotted closer of Rio de Janeiro. Moreover, it assumed an intermediate position relative to Europe and RJ-AFD and was quite distant from Native Americans, Africans and East Asians. This conclusion agrees with the population composition reported in the 2010 National Survey Inquiries, in which most of the immigrants were from the northeast and the southeast. The studies developed were approved by a local ethics committee and all participants signed the informed consent form

Frequências alélicas de 15 STRs autossômicos em uma amostra populacional do Distrito Federal do Brasil – um território que surgiu do nada

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2013.O Distrito Federal do Brasil foi criado em 1960 na Região Centro-Oeste em uma área anteriormente despovoada. Em 2010, este território artificial estava povoado por 2.556.121 habitantes. Neste estudo, analisou-se na população do Distrito Federal as variações genéticas dos quinze loci STRs incluídos no kit AmpFlSTR® NGM™ (Life Technologies) (D10S1248, D22S1045, D2S441, D1S1656, D12S391, D2S1338, D3S1358, D8S1179, D16S539, D18S51, D19S433, D21S11, FGA, TH01 e vWA). Os resultados indicam que o NGM™ é um sistema genético altamente informativo. Pois, a probabilidade de discriminar dois indivíduos selecionados ao acaso é de 99,999999999999999995% e a probabilidade de excluir um indivíduo falsamente acusado em um caso de paternidade é 99,99998%. Ademais, a análise das distâncias genéticas entre as populações das cinco macrorregiões brasileiras revelou que o Distrito Federal é mais semelhante ao Sudeste, ao Nordeste e a população brasileira como um todo do que as populações do Sul, Centro-Oeste e Norte. Esta conclusão está de acordo com a composição da população relatada no Censo 2010, em que a maioria dos imigrantes era do Nordeste e do Sudeste. ________________________________________________________________________________ ABSTRACTThe Federal District of Brazil was created in 1960 in the Central-West Region of Brazil in a previously unpopulated area. In 2010, this artificially founded district was populated by 2,562,963 inhabitants. In this study, we analyzed the genetic variations of the fifteen STRs loci (D10S1248, D22S1045, D2S441, D1S1656, D12S391, D2S1338, D3S1358, D8S1179, D16S539, D18S51, D19S433, D21S11, FGA, TH01 and vWA) included in the kit AmpFlSTR® NGM™ (Life Technologies) in this population. The results indicate that the NGM™ is a highly informative genetic system. Therefore, the probability of discriminating two individuals selected at random is 99.999999999999999995% and the probability of excluding a falsely accused individuals in a paternity case is 99.99998%. Furthermore, analysis of genetic distances between populations of the five macroregions revealed that the Brazilian Federal District is more similar to the southeastern, northeastern and overall Brazil population compared to Southern, Central-Western and Northern populations. This conclusion agrees with the population composition reported in the 2010 National Survey Inquiries, in which most of the immigrants were from the northeast and the southeast

EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples

AbstractObjetivesThe extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present study were to compare the DNase activity in these specimens and to investigate its ex-vivo impact over the circulating cell-free DNA yield (ccfDNA), using the circulating cell-free fetal DNA (ccffDNA) as a tool.Design and methodsEDTA-plasma and serum from women bearing male fetus were submitted to an endogenous DNase activity assay based on qPCR hydrolysis probe degradation, they were treated with DNAse I to investigate the action of an exogenous nuclease and also submitted to different temperature conditions to investigate the temperature-dependent degradation of the ccffDNA. In all instances, all male ccffDNA were quantified by qPCR targeting the Y chromosome-specific sequence DYS-14. Moreover, a serial dilution of EDTA was added to nonanticoagulated plasma and serum before the endogenous DNAse activity assay, to investigate the EDTA-mediated inhibition of the blood's DNase.ResultsThe endogenous nuclease activity was 14.9-fold higher in serum compared to EDTA-plasma. The DNAse I treatment did not alter the ccffDNA yields in EDTA-plasma, but completely degraded it in serum. The addition of increasing doses of EDTA to nonanticoagulated plasma and serum resulted in a stepwise inhibition of their nucleases activity. Finally, we observed a much more pronounced temperature-mediated decrease on the ccffDNA amount in serum compared to EDTA-plasma.ConclusionThe exogenous and endogenous DNases are more active in serum, the anticoagulant EDTA indirectly inhibits blood DNases, and consequently ccfDNA is protected from the blood's DNase preanalytical impact in EDTA-plasma

Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method—nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18–23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020

Association between suspected Zika virus disease during pregnancy and giving birth to a newborn with congenital microcephaly: a matched case–control study

Abstract Objective In early 2015, an outbreak of an acute exanthematous illness with dengue-like symptoms occurred in northeastern Brazil. By the end of the same year, an unexpected increase in the number of cases of microcephaly was observed in the region. The microcephaly outbreak cause was unknown and rumors pointing to various potential causes arose. Since we were unaware at the time if this scenario would attract the interest of the broader scientific community, due to the neglected regions associated and as often happens with many others health conditions related to infectious diseases in Latin America. This coupled with the fact that diagnostic testing for Zika virus was not available, prompted us to design a study that could demonstrate the correlation between the development of an exanthematous illness with Zika-like symptoms during pregnancy and the delivery of a newborn with congenital microcephaly. Results Mothers who experienced symptoms associated with the Zika virus during pregnancy had 10 times higher odds of delivering newborns with congenital microcephaly when compared with mothers who did not exhibit Zika-like symptoms. Thus, the acute exanthematous illness outbreak could be associated with the congenital microcephaly outbreak. We could not distinguish which virus caused the acute exanthematous illness in the study subjects (Zika, dengue or chikungunya), but these results could help to reduce the misquided speculation in regards to the cause of the microcephaly and could have expedited public health policies intended for controlling the mosquito vector. In addition to the lower head circumference, microcephalic neonates also had lower thoracic circumference, lower height and lower weight compared to non-microcephalic babies suggesting intrauterine growth restriction. Additionally, we found borderline association between mothers classified as homemakers and, who had past dengue infections with microcephaly. Prior contraction of dengue virus seems to play a role in the risk for the condition reflecting the domestication of the Aedes Aegypti and the enhancement of the Zika virus infection by dengue antibodies, respectively. The limitations of this study are: (a) participants recall bias, (b) absence of laboratory test results for Zika virus and other arboviruses and (c) incomplete test results for other pathogens that could lead to microcephaly. The study protocol was registered at ClinicalTrial.gov under the identifier NCT02741882. Registered on April 13th, 201