Post-Covid condition and clinic characteristics associated with SARS-CoV-2 infection: a 2-year follow-up to Brazilian cases.

Until January 2023, Brazil recorded 37 million COVID-19 cases despite the decrease in mortality due to mass vaccination efforts against COVID-19. The infection continues to challenge researchers and health professionals with the persistent symptoms and onset manifestations after the acute phase of the disease, namely Post-Covid Condition (PCC). Being one of the countries with the highest infection rate, Brazil must prepare for a growing number of patients with chronic health consequences of COVID-19. Longitudinal studies that follow patients over extended periods are crucial in understanding the long-term impacts of COVID-19, including potential health consequences and the effects on quality of life. We describe the clinical profile of a cohort of COVID-19 patients infected during the first year of the pandemic in Brazil and a follow-up after two years to investigate the health impacts of SARS-CoV-2 infection. The first wave of SARS-CoV-2 infection in Brazil featured extensive drug misuse, notably the ineffective COVID kit comprised of ivermectin, antimalarials and azithromycin, and elevated in-hospital mortality. In the second phase of the study, Post-Covid Condition was reported by symptomatic COVID-19 subjects across different severity levels two years after infection. Long haulers are more likely to be women, previously hospitalized, and reported a range of symptoms from muscle pain to cognitive deficit. Our longitudinal study is essential to inform public health authorities to develop strategies and policies to control the spread of the virus and mitigate its impacts on society.

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.

In silico analysis of upstream variants in Brazilian patients with familial hypercholesterolemia

ABSTRACT: Familial hypercholesterolemia (FH) is a prevalent autosomal genetic disease associated with increased risk of early cardiovascular events and death due to chronic exposure to very high levels of low-density lipoprotein cholesterol (LDL-c). Pathogenic variants in the coding regions of LDLR, APOB and PCSK9 account for most FH cases, and variants in non-coding regions maybe involved in FH as well. Variants in the upstream region of LDLR, APOB and PCSK9 were screened by targeted next-generation sequencing and their effects were explored using in silico tools. Twenty-five patients without pathogenic variants in FH-related genes were selected. 3 kb upstream regions of LDLR, APOB and PCSK9 were sequenced using the AmpliSeq (Illumina) and Miseq Reagent Nano Kit v2 (Illumina). Sequencing data were analyzed using variant discovery and functional annotation tools. Potentially regulatory variants were selected by integrating data from public databases, published data and context-dependent regulatory prediction score. Thirty-four single nucleotide variants (SNVs) in upstream regions were identified (6 in LDLR, 15 in APOB, and 13 in PCSK9). Five SNVs were prioritized as potentially regulatory variants (rs934197, rs9282606, rs36218923, rs538300761, g.55038486A > G). APOB rs934197 was previously associated with increased rate of transcription, which in silico analysis suggests that could be due to reducing binding affinity of a transcriptional repressor. Our findings highlight the importance of variant screening outside of coding regions of all relevant genes. Further functional studies are necessary to confirm that prioritized variants could impact gene regulation and contribute to the FH phenotype.

In silico analysis of upstream variants in Brazilian patients with Familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a prevalent autosomal genetic disease associated with increased risk of early cardiovascular events and death due to chronic exposure to very high levels of low-density lipoprotein cholesterol (LDL-c). Pathogenic variants in the coding regions of LDLR, APOB and PCSK9 account for most FH cases, and variants in non-coding regions maybe involved in FH as well. Variants in the upstream region of LDLR, APOB and PCSK9 were screened by targeted next-generation sequencing and their effects were explored using in silico tools. Twenty-five patients without pathogenic variants in FH-related genes were selected. 3 kb upstream regions of LDLR, APOB and PCSK9 were sequenced using the AmpliSeq (Illumina) and Miseq Reagent Nano Kit v2 (Illumina). Sequencing data were analyzed using variant discovery and functional annotation tools. Potentially regulatory variants were selected by integrating data from public databases, published data and context-dependent regulatory prediction score. Thirty-four single nucleotide variants (SNVs) in upstream regions were identified (6 in LDLR, 15 in APOB, and 13 in PCSK9). Five SNVs were prioritized as potentially regulatory variants (rs934197, rs9282606, rs36218923, rs538300761, g.55038486A > G). APOB rs934197 was previously associated with increased rate of transcription, which in silico analysis suggests that could be due to reducing binding affinity of a transcriptional repressor. Our findings highlight the importance of variant screening outside of coding regions of all relevant genes. Further functional studies are necessary to confirm that prioritized variants could impact gene regulation and contribute to the FH phenotype.

Histopathological prognosis of papillary thyroid carcinoma associated with nutritional status of vitamins A and E.

BACKGROUND/OBJECTIVES: Experimental and clinical studies have shown that vitamins A and E can inhibit cancer formation and progression. The unfavourable status of these vitamins can represent risk factors for the disease. This study aimed to evaluate the associations between the nutritional status of vitamins A and E (serum levels and dietary intake) and histopathological outcomes in Papillary Thyroid Carcinoma (PTC) patients. SUBJECTS/METHODS: We applied a cross-sectional study (2017-2018) and quantified retinol (ROH) and α-tocopherol (TOH) serum levels and vitamins dietary intake of 46 PTC patients. Serum vitamins were quantified by high efficiency liquid chromatography and vitamins dietary intake was analyzed by 24-hr dietary recalls. RESULTS: Patients with lower ROH serum levels were more likely to present lymph node metastasis and/or angiolymphatic invasion (p = 0.025). In addition, higher vitamin A and vitamin E intake are related to the absence of extrathyroidal extension (p = 0.013) and lymph node metastasis (p = 0.007), respectively. Our findings suggest that a ROH serum level greater than 2.65 µmol/L in PTC patients may be a protective factor against the presence of lymph node metastasis and angiolymphatic invasion. In addition, vitamin A and E intake may protect against extrathyroidal extension and lymph node metastasis. CONCLUSIONS: A favourable nutritional status (higher serum levels and/or intake) of vitamin A and E may be associated with less aggressive tumours in PTC patients.

Genomics, epigenomics and pharmacogenomics of Familial Hypercholesterolemia (FHBGEP): A study protocol

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disease that affects millions of people worldwide. OBJECTIVES: The study protocol FHBGEP was design to investigate the main genomic, epigenomic, and pharmacogenomic factors associated with FH and polygenic hypercholesterolemia (PH). METHODS: FH patients will be enrolled at six research centers in Brazil. An exon-targeted gene strategy will be used to sequence a panel of 84 genes related to FH, PH, pharmacogenomics and coronary artery disease. Variants in coding and regulatory regions will be identified using a proposed variant discovery pipeline and classified according to the American College Medical Genetics guidelines. Functional effects of variants in FH-related genes will be investigated by in vitro studies using lymphocytes and cell lines (HepG2, HUVEC and HEK293FT), CRISPR/Cas9 mutagenesis, luciferase reporter assay and other technologies. Functional studies in silico, such as molecular docking, molecular dynamics, and conformational analysis, will be used to explore the impact of novel variants on protein structure and function. DNA methylation profile and differential expression of circulating non-coding RNAs (miRNAs and lncRNAs) will be analyzed in FH patients and normolipidemic subjects (control group). The influence of genomic and epigenomic factors on metabolic and inflammatory status will be analyzed in FH patients. Pharmacogenomic studies will be conducted to investigate the influence of genomic and epigenomic factors on response to statins in FH patients. SUMMARY: The FHBGEP protocol has the potential to elucidate the genetic basis and molecular mechanisms involved in the pathophysiology of FH and PH, particularly in the Brazilian population. This pioneering approach includes genomic, epigenomic and functional studies, which results will contribute to the improvement of the diagnosis, prognosis and personalized therapy of FH patients.

Genomics, epigenomics and pharmacogenomics of familial hypercholesterolemia (FHBGEP): A study protocol.

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disease that affects millions of people worldwide. OBJECTIVES: The study protocol FHBGEP was design to investigate the main genomic, epigenomic, and pharmacogenomic factors associated with FH and polygenic hypercholesterolemia (PH). METHODS: FH patients will be enrolled at six research centers in Brazil. An exon-targeted gene strategy will be used to sequence a panel of 84 genes related to FH, PH, pharmacogenomics and coronary artery disease. Variants in coding and regulatory regions will be identified using a proposed variant discovery pipeline and classified according to the American College Medical Genetics guidelines. Functional effects of variants in FH-related genes will be investigated by in vitro studies using lymphocytes and cell lines (HepG2, HUVEC and HEK293FT), CRISPR/Cas9 mutagenesis, luciferase reporter assay and other technologies. Functional studies in silico, such as molecular docking, molecular dynamics, and conformational analysis, will be used to explore the impact of novel variants on protein structure and function. DNA methylation profile and differential expression of circulating non-coding RNAs (miRNAs and lncRNAs) will be analyzed in FH patients and normolipidemic subjects (control group). The influence of genomic and epigenomic factors on metabolic and inflammatory status will be analyzed in FH patients. Pharmacogenomic studies will be conducted to investigate the influence of genomic and epigenomic factors on response to statins in FH patients. SUMMARY: The FHBGEP protocol has the potential to elucidate the genetic basis and molecular mechanisms involved in the pathophysiology of FH and PH, particularly in the Brazilian population. This pioneering approach includes genomic, epigenomic and functional studies, which results will contribute to the improvement of the diagnosis, prognosis and personalized therapy of FH patients.

Modulation of all-trans retinoic acid-induced MiRNA expression in neoplastic cell lines: a systematic review.

BACKGROUND: Cancer is a genetic and epigenetic disease that involves inactivation of tumor suppressor genes and activation of proto-oncogenes. All-trans retinoic acid (ATRA) is an isomer of retinoic acid involved in the onset of differentiation and apoptosis of a number of normal and cancer cells, functioning as an anti-cancer agent in several neoplasms. Ectopic changes in the expression of certain microRNAs (miRNAs) occur in response to ATRA, leading to phenotypic alterations in neoplastic cell lines. Moreover, the modulation of miRNA patterns upon ATRA-treatment may represent an effective chemopreventive and anti-cancer therapy strategy. The present systematic review was performed to provide an overview of the modulation of ATRA-induced miRNA expression in different types of neoplastic cells and identify the efficacy of intervention factors (i.e., concentration and duration of treatment) and how they influence expression profiles of oncogenesis-targeting miRNAs. METHODS: A systematic search was conducted according to the PRISMA statement via the US National Library of Medicine MEDLINE/PubMed bibliographic search engine. RESULTS: The search identified 31 experimental studies involving human cell lines from nine different cancer types (neuroblastoma, acute myeloid leukemia, breast cancer, lung cancer, pancreatic cancer, glioma, glioblastoma, embryonal carcinoma, and colorectal cancer) treated with ATRA at concentrations ranging from 10- 3 µmol/L to 102 µmol mol/L for 24 h to 21 days. CONCLUSION: The concentrations used and the duration of treatment of cancer cells with ATRA varied widely. The presence of ATRA in the culture medium of cancer cells was able to modulate the expression of more than 300 miRNAs, and inhibit invasive behavior and deregulated growth of cancer cells, resulting in total tumor remission in some cases. ATRA may thus be broadly effective for neoplasm treatment and prevention, although these studies may not accurately represent in vivo conditions. Additional studies are required to elucidate ATRA-induced miRNA modulation during neoplasm treatment.

TREML4 mRNA Expression and Polymorphisms in Blood Leukocytes are Associated with Atherosclerotic Lesion Extension in Coronary Artery Disease.

Members of the triggering receptor expressed on myeloid cells (TREM) family are associated with atherosclerosis risk and progression. TREML4 is upregulated in the early phase of acute coronary syndrome. We investigated the relationship between the mRNA expression of 13 genes in blood leukocytes, TREML4 polymorphisms, and coronary artery lesion extension (Friesinger index) in patients with coronary artery disease (CAD) (n = 137). TREML4 rs2803495 (A > G) and rs2803496 (T > C) variants and leukocyte mRNA expression were analysed by qRT-PCR. TREML4 expression was higher in patients with major coronary artery lesions than in subjects without or with low and intermediate lesions (p < 0.05). However, TREML4 polymorphisms were not associated with coronary lesion extent. Presence of the rs2803495 G allele was not associated with increased TREML4 mRNA expression. Patients carrying the rs2803496 C allele (TC/CC genotypes) were more likely to express TREML4 mRNA than non-C allele carriers (allele C: OR 7.3, and 95% CI 1.9-27.5, p = 0.03). In conclusion, increased TREML4 mRNA expression in blood leukocytes is influenced by gene polymorphisms and is associated with more severe coronary artery lesions, suggesting its potential as a biomarker of the extent of coronary lesions in patients with CAD.

Relationship between circulating VCAM-1, ICAM-1, E-selectin and MMP9 and the extent of coronary lesions.

OBJECTIVES: Inflammatory molecules play a role in the development of atherosclerosis, which is the primary origin of cardiovascular disorders. However, to the best of our knowledge, no study has attempted to investigate the relationship between these circulating molecules and the prediction of cardiovascular risk. The present study aimed to investigate the relationships of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 serum concentrations with the extent of coronary lesions. METHODS: Seventy-four individuals who were undergoing coronary angiography for the first time for diagnostic purposes were enrolled in this study. The extent of the coronary lesion was assessed using the Friesinger Index, and subjects were classified into four groups: no lesions, minor lesions, intermediate lesions and major lesions. Serum biochemical parameters and serum concentrations of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 were analyzed. RESULTS: The vascular cell adhesion molecule-1 concentration was higher than 876 ng/mL in individuals with intermediate and major lesions (p<0.001 and p=0.020, respectively). Moreover, logistic regression analysis showed that these patients had an increased risk of having an intermediate lesion (p=0.007). Interestingly, all individuals with major lesions had vascular cell adhesion molecule-1 concentrations higher than 876 ng/mL. No association was found between the concentrations of the other proteins and the Friesinger Index. CONCLUSIONS: Serum vascular cell adhesion molecule-1 may be associated with the extent of coronary lesions. Moreover, it may represent an alternative to improve the cardiovascular risk classification in patients without acute coronary syndrome.

Circulating miRNAs in acute new-onset atrial fibrillation and their target mRNA network.

BACKGROUND: MicroRNAs (miRNAs) are involved in the pathogenesis of atrial fibrillation (AF), acting on development and progression. Our pilot study investigated the expression of six miRNAs and their miRNA-mRNA interactions in patients with acute new-onset AF, well-controlled AF, and normal sinus rhythm (controls). METHODS AND RESULTS: Plasma of acute new-onset AF patients (n = 5) was collected in the emergency room when patients presented with irregular and fast-atrial fibrillation rhythm. Samples from well-controlled AF (n = 16) and control (n =  15) patients were collected during medical appointments following an ECG. Expression of miR-21, miR-133a, miR-133b, miR-150, miR-328, and miR-499 was analyzed by real-time PCR. Ingenuity Pathway Analysis and the TargetScan database identified the top 30 mRNA targets of these miRNA, seeking the miRNA-mRNA interactions in cardiovascular process. Increased expression of miR-133b (1.4-fold), miR-328 (2.0-fold), and miR-499 (2.3-fold) was observed in patients with acute new-onset AF, compared with well-controlled AF and control patients. Decreased expression of miR-21 was seen in patients with well-controlled AF compared to those with acute new-onset AF and controls (0.6-fold). The miRNA-mRNA interaction demonstrated that SMAD7 and FASLG genes were the targets of miR-21, miR-133b, and miR-499 and were directly related to AF, being involved in apoptosis and fibrosis. CONCLUSION: The miRNAs had different expression profiles dependent on the AF condition, with higher expression in the acute new-onset AF than well-controlled AF. Clinically, this may contribute to an effective assessment for patients, leading to early detection of AF and monitoring to reduce the risk of other serious cardiovascular events.

Association of Serum Alpha-Tocopherol and Retinol with the Extent of Coronary Lesions in Coronary Artery Disease.

Background and aims: Fat-soluble vitamins play an important role in the pathogenesis of cardiovascular disease and progression of atherosclerosis. This study aimed at investigating the relationship of the serum levels of alpha-tocopherol and retinol with the extent of coronary lesions in patients with coronary artery disease. Methods. Patients with coronary artery disease (n=177) aged 30-74 years, who underwent their first coronary angiography, were enrolled. The extent of coronary lesions was assessed using the Friesinger index (FI). Accordingly, patients were grouped as follows: FI = 0-4 (n=90), FI = 5-9 (n=50), and FI = 10-15 (n=37). Serum levels of vitamins were ‬determined via high-performance liquid chromatography and serum biochemical analysis. Results. Assessment of FI-based groups revealed that 50.8% patients had a coronary artery lesion to a low extent (FI 0-4). Individuals in this group were younger and had lower glucose and serum alpha-tocopherol levels than the other groups (p < 0.05). Low levels of alpha-tocopherol were more frequent in the FI 0-4 group than that in the other groups (p=0.03). No difference was observed between the mean serum retinol levels among the FI-based groups (n=0.492), and the low frequency of retinol was consistent among the FI groups (n=0.348). Conclusions. The low level of alpha-tocopherol together with the presence of dyslipidemia is probably associated with the initial events in atherosclerosis. Increased alpha-tocopherol levels in patients with more extensive coronary artery lesions may have resulted from altered vitamin E metabolism with increased oxidative stress.

Relationship between circulating VCAM-1, ICAM-1, E-selectin and MMP9 and the extent of coronary lesions

OBJECTIVES: Inflammatory molecules play a role in the development of atherosclerosis, which is the primary origin of cardiovascular disorders. However, to the best of our knowledge, no study has attempted to investigate the relationship between these circulating molecules and the prediction of cardiovascular risk. The present study aimed to investigate the relationships of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 serum concentrations with the extent of coronary lesions. METHODS: Seventy-four individuals who were undergoing coronary angiography for the first time for diagnostic purposes were enrolled in this study. The extent of the coronary lesion was assessed using the Friesinger Index, and subjects were classified into four groups: no lesions, minor lesions, intermediate lesions and major lesions. Serum biochemical parameters and serum concentrations of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 were analyzed. RESULTS: The vascular cell adhesion molecule-1 concentration was higher than 876 ng/mL in individuals with intermediate and major lesions (p<0.001 and p=0.020, respectively). Moreover, logistic regression analysis showed that these patients had an increased risk of having an intermediate lesion (p=0.007). Interestingly, all individuals with major lesions had vascular cell adhesion molecule-1 concentrations higher than 876 ng/mL. No association was found between the concentrations of the other proteins and the Friesinger Index. CONCLUSIONS: Serum vascular cell adhesion molecule-1 may be associated with the extent of coronary lesions. Moreover, it may represent an alternative to improve the cardiovascular risk classification in patients without acute coronary syndrome.

Circulating MicroRNAs as Potential Biomarkers of Atrial Fibrillation.

Atrial fibrillation (AF) is the most common supraventricular arrhythmia in the population. MicroRNAs (small endogenous noncoding RNAs) are attractive candidates as biomarkers for AF, especially considering that miRNAs are stable and are detected within easily accessible biofluids such as blood. In this review, we selected twelve studies (2012 to 2016) that were classified according to the sample type. We aimed to provide an overview of the role of circulating miRNAs in AF and to discuss the variability of the results, seeking to improve the perspective of the use of miRNAs as potential noninvasive biomarkers for this heart disease.

The relationship of the oleic acid level and ECHDC3 mRNA expression with the extent of coronary lesion.

BACKGROUND: The fatty acid profile is associated with the risk and progression of several diseases, probably via mechanisms including its influence on gene expression. We previously reported a correlation between ECHDC3 upregulation and the severity of acute coronary syndrome. Here, we assessed the relationship of serum fatty acid profile and ECHDC3 expression with the extent of coronary lesion. METHODS: Fifty-nine individuals aged 30 to 74 years and undergoing elective cinecoronariography for the first time were enrolled in the present study. The extent of coronary lesion was assessed by the Friesinger index and patients were classified as without lesion (n = 18), low lesion (n = 17), intermediate lesion (n = 17) and major lesion (n = 7). Serum biochemistry, fatty acid concentration, and ECHDC3 mRNA expression in blood were evaluated. RESULTS: Elevated serum levels of oleic acid and total monounsaturated fatty acids were observed in patients with low and intermediate lesion, when compared to patients without lesion (p < 0.05). ECHDC3 mRNA expression was 1.2 fold higher in patients with low lesion than in patients without lesion (p = 0.020), and 1.8 fold lower in patients with major lesion patients than in patients with low lesion (p = 0.023). CONCLUSION: Increased levels of monounsaturated fatty acids, especially oleic acid, and ECHDC3 upregulation in patients with coronary artery lesion suggests that these are independent factors associated with the initial progression of cardiovascular disease.

Avaliação d qualidade do DNA obtido de amostras preservadas em parafina / Quality evaluation of DNA from samples preserved in parafin

A obtenção de um material genético íntegro e com concentrações satisfatórias para a amplificação de de uma determinada sequência são os principais desafios para o processamento de amostras de tecido preservados em parafina. O presente estudo teve como objetivo comparar três diferentes protocolos de extração de DNA que têm em comum entre si o princípio de extração por silica-gel. Vinte e sete amostras de tecidos tireoidianos preservados em parafina foram processadas por meio de cortes em triplicata de 20um. O método A utilizou tr~es horas na etapa de digestão do tecido, os métodos B e C utilizaram 16 horas para a digestão, sendo que o último utilizou diferentes solventes para reidratar o tecido e solução de fenol-clorofórmio-álcool isoamílico para purificação. Todos os métodos utilizaram em comum o conjunto de reagentes QIAamp DNA mini kit. Na comparação entre as concentrações obtidas de DNA, o método B spresentou maior média (3,7 ng/iL) já o método A obteve valor médio que 1,2 ng/uL e o método C, de 0,7 ng/uL. Todos os protocolos obtiveram valores da relação 260/280 e 260/230 fora dos valores de refer~encia esperado, evidenciando uma perda na qualidade do DNA extraído. O resultado da integridade do DNA, por meio de eletroforese em gel agarose também demonstrou o alto grau de degradação de todos os métodos analisados. Entretanto, foi possível realizar a genotipagem do polimorfismo 174G>C do gene da interleucina 6 em 100% das amostras testadas para o método C, e 66% par a o método A e B. O método C foi o mais reprodutivo para genotipagem , apesar do maior custo e tempo de processamento, sendo este método mais indicado para a implementação na rotina laboratorial de biologia molecular para amostras preservadas em parafina.