Identification of pathogenic variants in the brazilian cohort with familial hypercholesterolemia using exon-targeted gene sequencing

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH-related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3'UTR and 5'UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

Identification of pathogenic variants in the Brazilian cohort with Familial hypercholesterolemia using exon-targeted gene sequencing.

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH-related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3'UTR and 5'UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

Perfil de citocinas em biópsia miocárdica de paciente transplantado com COVID-19 / Cytokine profile in myocardial biopsy of a transplant patient with COVID-19

FUNDAMENTO: A inflamação pode estar envolvida na ativação imune do transplante, resultando em disfunção do enxerto. A COVID-19 é caracterizada pela liberação descontrolada e elevada de citocinas pró-inflamatórias e imunidade suprimida. Descrevemos um caso de paciente com transplante de coração e COVID-19 que desenvolveu disfunção do enxerto e os achados do perfil de citocinas na biopsia endomiocárdica. RELATO DE CASO: Paciente com transplante cardíaco há 13 anos apresentou-se no PS com queixa de diarreia com duração de 7 dias e teste rápido para COVID-19 positivo, evoluiu com disfunção ventricular (FE=56% anterior de 66%) com ressonância magnética compatível com miocardite aguda e elevação de troponina. Após cinco dias de pulsoterapia com corticoide em altas doses e melhora da função ventricular e redução da troponina, realizou biópsia endomicárdica, com resultado 0R. A tomografia computadorizada de coronárias mostrou ausência de calcificação coronária ou redução luminal coronária significativa. A última vacina para COVID foi há 6 meses antes da internação e ela não tomou a terceira dose porque estava gripada na época. Recebeu alta hospitalar após 11 dias com recuperação da função do enxerto. No dia da biópsia, o teste rápido para COVID-19 foi negativo e o RT-PCR no tecido miocárdico não detectou o vírus SARS-CoV-2. Usando a mesma amostra de biópsia, a expressão de 84 genes relacionados à regulação da inflamação também foi analisada usando o método RT2 Profiler PCR Array (Qiagen Cat. No. 330213 PAHS-077Z). A interpretação dos resultados foi comparada com uma amostra controle, que teoricamente não tinha COVID-19. Encontramos 15 genes mais expressos em tecidos que tinham COVID-19 (tabela). Por exemplo, o gene CCL16 foi 8,5 vezes mais expresso em tecido com COVID-19 do que o controle. DISCUSSÃO: A literatura recente descreve que os genes CXCL5 e NFKB1 têm envolvimento na biologia do coronavírus e estão envolvidos na resposta imune ou atividade antiviral, IL17A tem envolvimento na biologia do coronavírus e está envolvido na resposta inflamatória da tempestade de citocinas, e KNG1 tem envolvimento na biologia do coronavírus e é relevante para o processo da doença. CONCLUSÃO: Este é um dos primeiros relatos sobre o uso de um perfil de citocinas analisado em uma biópsia miocárdica de um paciente transplantado com COVID-19. Além da detecção de genes que regulam a inflamação, foram identificados alguns genes relacionados ao envolvimento da biologia e resposta imune ou atividade viral, o que pode ser o primeiro passo para o desenvolvimento de futuros estudos clínicos.

Effect of qualitative and quantitative nutritional plan on gene expression in obese patients in secondary prevention for cardiovascular disease

Summary Background & aims: Diet is a modifiable risk factor, which may influence the gene expression and the concentration of inflammatory biomarkers related to obesity and atherosclerosis. In this sub study from Brazilian Cardioprotective Nutritional (BALANCE) Program, we hypothesized that a nutritional intervention based on the usual Brazilian diet modulates the expression of genes involved with atherosclerosis and inflammatory biomarkers in male patients, in the secondary prevention for cardiovascular disease. Methods: Six male patients, aged 45 years or older, obese, were selected to follow a qualitative-quantitative food plan for 6 months. Glycemia, insulinemia, lipid profile, plasma concentration of inflammatory biomarkers (interleukin (IL) -1b), IL-6, IL-8, IL-10, IL-12, tumor necrosis factor alpha, C-reactive protein and adiponectin, and expression of 84 atherosclerosis-related genes in total peripheral blood cells, were measured. Results: After nutritional intervention, the participants reduced weight (p<0.04), waist circumference(p<0.04), Homeostasis Model Assessment index for insulin resistance (p»0.046) and overall leukocyte count (p»0.046) and neutrophils (p»0.028). There was no significant modification in the plasma concentration of the inflammatory biomarkers, however, there was a significant increase in the expression of Apo A1 (p»0.011), ELN (p»0.017) and IL4 (p»0.037) genes. Conclusions: The BALANCE Program, the qualitative-quantitative food plan composed of Brazilian usual foods, did not reduce the concentration of inflammatory biomarkers, but increased in total peripheral blood cells the expression of genes involved in reducing the risk of cardiometabolic in obese patients, in secondary prevention for cardiovascular disease. The clinical trial is registered athttps://clinicaltrials.gov/and the unique identifier is NCT01620398.

Analysis of Circulating miR-1, miR-23a, and miR-26a in Atrial Fibrillation Patients Undergoing Coronary Bypass Artery Grafting Surgery.

Atrial fibrillation (AF) is the most common arrhythmia after cardiac surgery. From a pathophysiological point of view, a myriad of factors such as trauma, atrial dilation, ischemia, mechanical myopericarditis, autonomic imbalance, loss of connexins, AF nest remodeling, inflammation, sutures, and dysfunction caused by postextracorporeal circulation can contribute to postoperative atrial fibrillation (POAF) resulting in a longer hospital stay and consequently higher cost. Recent studies showed that short fragments of RNA, called microRNA (miRNA), can contribute to the development of several cardiovascular diseases, including AF. The aim of this study was to evaluate the levels of circulating miRNAs (miR-1, -23a, and -26a) that can be involved in POAF. Patients submitted to coronary artery bypass graft surgery were grouped in POAF (24 patients) and without POAF (24 patients). Results showed older age, longer clamp-time, and more days in the intensive care unit as well as a longer total hospital stay in the POAF group. Preoperative levels of circulating miRNAs were similar. Analysis of miRNAs revealed significantly lower circulating levels of miRNA-23a (P = 0.02) and -26a (P = 0.01) in the POAF group during the postoperative period. Receiver operating characteristic (ROC) analysis showed the area under the ROC curve of miR-23a and miR-26a for predicting FA was 0.63 (95% confidence interval [CI]: 0.51-0.74; P = 0.02) and 0.66 (95% CI: 0.55-0.77; P = 0.01), respectively. Our data suggests that circulating miRNA-23a and -26a may be involved in the underlying biology of postoperative AF development.

Reference genes for studies in infectious parasitic diseases in five types of human tissues

Gene expression analyses based on messenger RNA (mRNA) expression require accurate data normalization. When using endogenous reference genes, these should be carefully validated. Validated reference genes vary greatly depending on tissue, cell subsets and experimental context. This study was aimed to identify reference genes that have more stable mRNA levels among individuals in peripheral blood mononuclear cells (PBMC); fresh skin biopsies; lung and brain autopsies as well as, skin biopsies formalin-fixed paraffin-embedded (FFPE). Therefore, 6 endogenous reference genes were evaluated by quantitative real-time polymerase chain reaction: 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP), beta-2-microbolin (B2M), ubiquitin C (UBC) and mitochondrially encoded ATP synthase 6 (MT-ATP6). Furthermore, validation of their stabilities and performance as reference genes was determined by geNorm and NormFinder programs. The results show that the most stable genes for PBMC and fresh skin biopsies were TBP and UBC; in FFPE lung autopsies and skin biopsies were GAPDH and B2M; and in FFPE brain autopsies were GAPDH and UBC. In addition, 18S rRNA was the least stable of all genes analyzed. These data concluded that even genes constitutively expressed have transcript level variations in different tissues as well as storage and experimental conditions. These observations suggest that suitable reference genes should be selected for normalization of gene expression data analysis.

As análises de expressão gênica baseadas na expressão do RNA mensageiro (mRNA) requerem normalização precisa dos dados. Ao usar genes de referência endógenos, estes devem ser cuidadosamente validados. Os genes de referência validados variam muito, dependendo do tecido, subconjuntos de células e contexto experimental. Este estudo teve como objetivo identificar genes de referência que apresentam níveis de mRNA mais estáveis ​​entre indivíduos em células mononucleares do sangue periférico (PBMC); biópsias de pele fresca; autópsias pulmonares e cerebrais, bem como biópsias de pele fixadas em formalina e embebidas em parafina (FFPE). Portanto, 6 genes de referência endógenos foram avaliados por reação quantitativa em cadeia da polimerase em tempo real: rRNA 18S, gliceraldeído-3-fosfato desidrogenase (GAPDH), proteína de ligação à caixa TATA (TBP), beta-2-microbolina (B2M), ubiquitina C (UBC) e ATP sintase 6 mitocondrialmente codificada (MT-ATP6). Além disso, a validação de suas estabilidades e desempenho como genes de referência foi determinada pelos programas geNorm e NormFinder. Os resultados mostram que os genes mais estáveis ​​para PBMC e biópsias de pele fresca foram TBP e UBC; nas autópsias pulmonares de FFPE e biópsias de pele foram GAPDH e B2M; e nas autópsias cerebrais de FFPE foram GAPDH e UBC. Além disso, o 18S rRNA foi o menos estável de todos os genes analisados. Esses dados concluíram que mesmo os genes expressos constitutivamente apresentam variações no nível de transcrição em diferentes tecidos, bem como condições experimentais e de armazenamento. Essas observações sugerem que genes de referência adequados devem ser selecionados para normalização da análise dos dados de expressão gênica.

WITHDRAWN: Selection of reference genes in five types of human tissues for normalization of gene expression studies in infectious diseases.

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WITHDRAWN: Selection of reference genes in five types of human tissuesfor normalization of gene expression studies in infectious diseases

Gene expression analyses based on messenger RNA (mRNA) expression require accurate datanormalization. When using endogenous reference genes, these have to be carefully validated. Validated reference genes vary greatly depending on tissue, cell subsets and experimental context.The aim of this study was to identify reference genes that present more stable mRNA levels amongindividuals in peripheral blood mononuclear cells (PBMC); fresh skin biopsies; lung and brainautopsies as well as, skin biopsies formalin-fixed paraffin-embedded (FFPE). Therefore, 6endogenous reference genes were evaluated by quantitative real-time polymerase chain reaction: 18SrRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP),beta-2-microbolin (B2M), ubiquitin C (UBC) and mitochondrially encoded ATP synthase 6 (MTATP6).Furthermore, validation of their stability and suitability as reference genes was determined bythe geNorm program. The results show that in PBMC and fresh skin biopsies, TBP and UBC wereidentified as the most stable, while in FFPE lung autopsies and skin biopsies, GAPDH and B2M, andin FFPE brain autopsies, GAPDH and UBC turned out to be the most stable...

Functional IL18 polymorphism and susceptibility to Chronic Chagas Disease.

BACKGROUND: Chronic Chagas Disease cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi, the rest of the infected subjects remaining asymptomatic (ASY). The Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis. Local expression of IL-18 in CCC myocardial tissue has recently been described. IL-18 could potentially amplify the process by inducing increased expression of IFN-γ which in turn can increase the production of IL-18, thereby creating a positive feedback mechanism. In order to assess the contribution of the IL-18 to susceptibility to Chronic Chagas Disease, we investigated the association between a single nucleotide polymorphism (SNP) located in the IL-18 gene with the risk of developing Chagas cardiomyopathy. METHODS AND RESULTS: We analyzed the rs2043055 marker in the IL18 gene in a cohort of Chagas disease cardiomyopathy patients (n=849) and asymptomatic subjects (n=202). We found a significant difference in genotype frequencies among moderate and severe CCC patients with ventricular dysfunction. CONCLUSIONS: Our analysis suggests that the IL18 rs2043055 polymorphism- or a SNP in tight linkage disequilibrium with it- may contribute to modulating the Chagas cardiomyopathy outcome.

Estudo sequencial do perfil de expressão gênica em biópsias endomiocárdicas parafinadas: associação com rejeição humoral e vasculopatia do aloenxerto cardíaco / Sequential study of gene expression profiles in paraffin embedded endomyocardial biopsies: association with humoral rejection and cardiac allograft vasculopathy

O transplante cardíaco é a última opção terapêutica para pacientes portadores de insuficiência cardíaca grave. Apesar dos avanços na terapia imunossupressora, a rejeição continua sendo o principal obstáculo para o sucesso do transplante. No presente estudo, propõe-se avaliar o perfil de expressão gênica no tecido cardíaco. Com isso, espera-se contribuir para o melhor entendimento do processo de rejeição a nível molecular. Foram analisadas as amostras sequenciais (1, 3 e 6 meses, 1 e 2 anos pós-transplante) de biópsias endomiocárdicas em parafina de 63 indivíduos transplantados cardíacos...

Myocardial chemokine expression and intensity of myocarditis in Chagas cardiomyopathy are controlled by polymorphisms in CXCL9 and CXCL10.

BACKGROUND: Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium. METHODS AND RESULTS: Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2-6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes. CONCLUSIONS: Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.