Investigação Citogenômica de Crianças com Doença Cardíaca Congênita: Experiência de um Centro no Brasil / Cytogenomics Investigation of Infants with Congenital Heart Disease: Experience of a Brazilian Center

Resumo Fundamento Algumas síndromes têm características específicas e facilmente reconhecíveis, enquanto outras podem ser mais complexas de se identificar e podem apresentar diferentes manifestações fenotípicas, por exemplo. Um diagnóstico etiológico é importante para entender a natureza da doença, para estabelecer o prognóstico e para começar o tratamento, permitindo a inclusão de pacientes na sociedade e reduzindo o custo financeiro dessas doenças. Objetivo A proposta inicial deste estudo foi a triagem citogenética para detectar a síndrome de deleção 22q11.2 (SD22q11.2) em recém-nascidos e crianças com doença cardíaca congênita utilizando a técnica da amplificação multiplex de sondas dependente de ligação (MLPA). Assim, por meio da pesquisa, outras mudanças genômicas foram identificadas nesses pacientes cardíacos. Nosso objetivo se estendeu a investigar essas outras mudanças citogenéticas. Métodos Investigamos 118 recém-nascidos com doenças cardíacas congênitas nascidos consecutivamente durante um ano, utilizando a técnica da MLPA. Resultados A técnica da MLPA permitiu a detecção da SD22q11.2 em 10/118 pacientes (8,5%). Outras alterações genômicas foram identificadas em 6/118 pacientes (5%): 1p36 del, 8p23 del (2 casos), 7q dup, 12 dup e 8q24 dup. Conclusão Este estudo ressalta a relevância da detecção de alterações genômicas que estão presentes em recém-nascidos e crianças com doenças cardíacas congênitas por meio de ferramentas citogenômicas.

Abstract Background Some syndromes have specific and easily recognizable features, while others may be more complex to identify and may present different phenotypic manifestations, for example. An etiological diagnosis is important to understand the nature of the disease, to establish the prognosis and to start the treatment, allowing the inclusion of patients in society and reducing the financial cost of such diseases. Objective The initial proposal of this study was cytogenetic screening for the detection of the 22q11.2 deletion syndrome in consecutive newborns and infants with congenital heart disease using the multiplex ligation-dependent probe amplification (MLPA) technique. Therefore, throughout our research, other genomic alterations were identified in these cardiac patients. Thus, our objective was extended to investigate these other cytogenetic alterations. Methods We investigated 118 neonates with congenital heart diseases born consecutively during one year using the MLPA technique. Results The MLPA technique allowed the detection of 22q11.2DS in 10/118 patients (8.5%). Other genomic alterations were also identified in 6/118 patients (5%): 1p36 del, 8p23 del (2 cases), 7q dup, 12 dup and 8q24 dup. Conclusion This study highlights the relevance of detecting genomic alterations that are present in newborns and infants with congenital cardiac diseases using cytogenomic tools.

Novel rearrangements between different chromosomes with direct impact on the diagnosis of 5p- syndrome

Abstract Objectives Copy Number Variations (CNVs) in the human genome account for common populational variations but can also be responsible for genetic syndromes depending on the affected region. Although a deletion in 5p is responsible for a syndrome with highly recognizable phenotypical features, other chromosomal abnormalities might overlap phenotypes, especially considering that most studies in 5p use traditional cytogenetic techniques and not molecular techniques. Methods The authors have investigated 29 patients with clinical suspicion of 5p- syndrome using Chromosomal Microarray (CMA), and have gathered information on previous tests, clinical signs, symptoms, and development of the patients. Results The results showed 23 pure terminal deletions, one interstitial deletion, one deletion followed by a 3 Mb duplication in 5p, three cases of 5p deletion concomitant to duplications larger than 20 Mb in chromosomes 2, 9, and 18, and one 5p deletion with a chromosome Y deletion. CMA showed relevant CNVs not typically associated with 5p- that may have contributed to the final phenotype in these patients. Conclusions The authors have identified three novel rearrangements between chromosomes 5 and 2 (Patient 27), 5 and 18 (Patient 11), and 5 and Y (Patient 22), with breakpoints and overlapped phenotypes that were not previously described. The authors also highlight the need for further molecular investigation using CMA, in different chromosomes beyond chromosome 5 (since those cases did not show only the typical deletion expected for the 5p- syndrome) to explain discordant chromosomal features and overlapped phenotypes to unravel the cause of the syndrome in atypical cases. HIGHLIGHTS The authors The authors have described three novel rearrangements between chromosomes 5 and 2, 5 and 18, and 5 and Y with chromosomal breakpoints and overlapped phenotypes that were not previously described. One of the main atypical features for 5p- syndrome that the authors report was the presence of seizures that was found in the three patients with rearrangements between different chromosomes and in a patient with a deletion followed by duplication in 5p. The authors suggest physicians conduct further molecular investigation in the presence of atypical clinical features for patients with 5p- syndrome suspicion.

Cytogenomic assessment of the diagnosis of 93 patients with developmental delay and multiple congenital abnormalities: The Brazilian experience

OBJECTIVE: The human genome contains several types of variations, such as copy number variations, that can generate specific clinical abnormalities. Different techniques are used to detect these changes, and obtaining an unequivocal diagnosis is important to understand the physiopathology of the diseases. The objective of this study was to assess the diagnostic capacity of multiplex ligation-dependent probe amplification and array techniques for etiologic diagnosis of syndromic patients. METHODS: We analyzed 93 patients with developmental delay and multiple congenital abnormalities using multiplex ligation-dependent probe amplifications and arrays. RESULTS: Multiplex ligation-dependent probe amplification using different kits revealed several changes in approximately 33.3% of patients. The use of arrays with different platforms showed an approximately 53.75% detection rate for at least one pathogenic change and a 46.25% detection rate for patients with benign changes. A concomitant assessment of the two techniques showed an approximately 97.8% rate of concordance, although the results were not the same in all cases. In contrast with the array results, the MLPA technique detected ∼70.6% of pathogenic changes. CONCLUSION: The obtained results corroborated data reported in the literature, but the overall detection rate was higher than the rates previously reported, due in part to the criteria used to select patients. Although arrays are the most efficient tool for diagnosis, they are not always suitable as a first-line diagnostic approach because of their high cost for large-scale use in developing countries. Thus, clinical and laboratory interactions with skilled technicians are required to target patients for the most effective and beneficial molecular diagnosis.

Intraoperative analysis of flow dynamics in arteriovenous composite y grafts

Abstract Objective: Composite graft of left internal thoracic artery and great saphenous vein in revascularization of the left coronary system is a technique well described in literature. The aim of this study is to analyze blood flow dynamics in this configuration of composite graft especially in what concerns left internal thoracic artery's adaptability and influence of great saphenous vein segment on left internal thoracic artery's flow. Methods: Revascularization of left coronary system with composite graft, with left internal thoracic artery revascularizing the anterior interventricular artery and a great saphenous vein segment, anastomosed to the left internal thoracic artery, revascularizing another branch of the left coronary system, was performed in 23 patients. Blood flow was evaluated by transit time flowmetry in all segments of the composite graft (left internal thoracic artery proximal segment, left internal thoracic artery distal segment and great saphenous vein segment). Measures were performed in baseline condition and after dobutamine-induced stress, without and with non-traumatic temporary clamping of the distal segments of the composite graft. Results: Pharmacological stress resulted in increase of blood flow values in the analyzed segments (P<0.05). Non-traumatic temporary clamping of great saphenous vein segment did not result in statistically significant changes in the flow of left internal thoracic artery distal segment, both in baseline condition and under pharmacological stress. Similarly, non-traumatic temporary clamping of left internal thoracic artery distal segment did not result in statistically significant changes in great saphenous vein segment flow. Conclusion: Composite grafts with left internal thoracic artery and great saphenous vein for revascularization of left coronary system, resulted in blood flow dynamics with physiological adaptability, both at rest and after pharmacological stress, according to demand. Presence of great saphenous vein segment did not alter physiological blood flow dynamics in distal segment of left internal thoracic artery.

Doppler echocardiographic criteria in patency assessment of composite grafts from left internal thoracic artery / Parâmetros Dopplerfluxométricos de perviedade do enxerto composto de artéria torácica interna esquerda

OBJECTIVES: The purpose of our study was to establish, with an entirely noninvasive method, transthoracic Doppler echocardiography, criteria for patency of composite left internal thoracic artery grafts when placed on the left anterior descending artery and other branches of the left coronary system. METHODS: The control group comprised 20 patients with single graft and 20 patients with composite graft; all forty having their patency confirmed by coronary angiogram (CA). In this control group, two Doppler echocardiographic variables, diastolic mean velocity-time and integral diastolic peak velocity to systolic peak velocity ratio were recorded. For each variable, established cut-off points were established, using the ROC (Receiver Operator Characteristic) curves, to identify criteria which could differentiate the composite grafts. Only patients with composite grafts were included in the 159-patients study group. The criteria established by the cut-off points in the control group were then applied to detect patency using a diastolic fraction of > 0.5 as the gold standard. The sensitivity, specificity, and positive and negative predictive values of these two criteria were determined. RESULTS: In the control group, cut-off points of 0.71 and 0.09m were established for the diastolic peak velocity/systolic peak velocity ratio and for diastolic mean velocity-time integral, respectively. In the study group phase, the sensitivity and negative predictive value of the diastolic peak velocity/systolic peak velocity > 0.71 criterion were 36% and 11%, respectively. Diastolic mean velocity-time integral > 0.09m criterion, were 40% and 10.48%. The specificities and positive predictive values of each criterion were 100%. CONCLUSION: Values reaching the criteria established for each variable indicate high probability of composite graft patency. Lower values have a large proportion of false negatives and are not conclusive as patency criteria.

OBJETIVO: O objetivo deste estudo é estabelecer parâmetros preditores de perviedade, avaliados por Dopplerfluxometria, do enxerto composto de artéria torácica interna esquerda, quando revasculariza a artéria interventricular anterior e outro ramo do sistema esquerdo. MÉTODOS: O grupo controle foi formado por 20 pacientes com enxerto simples e 20 pacientes com enxerto, composto cuja perviedade foi confirmada por cineangiocoronariografia. No grupo controle, as variáveis de fluxo relação velocidade pico diastólico/velocidade pico sistólico e integral da velocidade média/tempo na diástole foram registradas. Para cada variável, estabeleceram-se pontos de corte para identificar enxertos compostos, usando-se curvas ROC (receiver operator characteristic). No grupo estudo, foram avaliados 159 pacientes com enxerto composto, determinando-se os dois parâmetros de fluxo. Pontos de cortes estabelecidos no grupo controle foram usados para determinar sensibilidade, especificidade, valores preditivos positivo e negativo de cada variável relacionada à perviedade dos enxertos, tomando-se como referência a fração diastólica > 0,5. RESULTADOS: No grupo controle, os pontos de corte estabelecidos para as variáveis velocidade pico diastólico/velocidade pico sistólico e integral velocidade média/tempo na diástole foram, respectivamente, 0,71 e 0,09m. No grupo estudo, a sensibilidade para a velocidade pico diastólico/velocidade pico sistólico e integral da velocidade média/tempo na diástole, considerando seus pontos de corte, foi de 36,4% e 40%, respectivamente. Os respectivos valores preditivos negativos foram 11% e 10.48%, enquanto especificidade e valor preditivo positivo foram de 100% para os dois parâmetros. CONCLUSÃO: Valores maiores ou iguais aos estabelecidos para cada variável indicam alta probabilidade de perviedade do enxerto composto. Valores inferiores apresentam grande proporção de falsos negativos, não sendo conclusivos quanto à perviedade.