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.

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.

Increased levels of plasma IL-1b and BDNF can predict resistant depression patients.

BACKGROUND: There is no strong evidence on the link between inflammatory profile and pattern of drug treatment response in depressive patients that could result in Coronary Artery Disease occurrence. OBJECTIVE: This study aimed to compare the subclinical atherosclerosis markers, inflammatory profile, and BDNF production in Resistant Depression (RD) or Bipolar Affective Disorder (BAD) patients under conventional treatment. METHODS: The population evaluated was comprised of 34 RD, 43 BAD, and 41 controls. Subclinical atherosclerosis markers were evaluated using ultrasonography, tomography, and exercise stress test. Plasma concentrations of TNFα, IL-1ß, IL-6, and BDNF were measured using Luminex100™. The usCRP concentration was measured using turbidimetric immunoassay. IL1B, IL6, and TNFA expression were determined using TaqMan®. For the statistical analysis, the significance level was established at p<0.05. RESULTS: Concerning subclinical atherosclerosis markers, only O2 consumption was reduced in the BAD group (p = 0.001). Although no differences were found in gene expression, BDNF and IL-1ß plasma concentration was increased in the RD group (p = 0.002 and p = 0.005, respectively) even with an antidepressant treatment, which suggests that these drugs have no effect in IL-1ß secretion and that the inflammasome may play a role in therapy response. CONCLUSION: Taken together, both BDNF and IL-1ß plasma concentrations could be used to the early identification of RD patients.

Increased levels of plasma IL-1b and BDNF can predict resistant depression patients

SUMMARY BACKGROUND: There is no strong evidence on the link between inflammatory profile and pattern of drug treatment response in depressive patients that could result in Coronary Artery Disease occurrence. OBJECTIVE: This study aimed to compare the subclinical atherosclerosis markers, inflammatory profile, and BDNF production in Resistant Depression (RD) or Bipolar Affective Disorder (BAD) patients under conventional treatment. METHODS: The population evaluated was comprised of 34 RD, 43 BAD, and 41 controls. Subclinical atherosclerosis markers were evaluated using ultrasonography, tomography, and exercise stress test. Plasma concentrations of TNFα, IL-1β, IL-6, and BDNF were measured using Luminex100™. The usCRP concentration was measured using turbidimetric immunoassay. IL1B, IL6, and TNFA expression were determined using TaqMan®. For the statistical analysis, the significance level was established at p<0.05. RESULTS: Concerning subclinical atherosclerosis markers, only O2 consumption was reduced in the BAD group (p = 0.001). Although no differences were found in gene expression, BDNF and IL-1β plasma concentration was increased in the RD group (p = 0.002 and p = 0.005, respectively) even with an antidepressant treatment, which suggests that these drugs have no effect in IL-1β secretion and that the inflammasome may play a role in therapy response. CONCLUSION: Taken together, both BDNF and IL-1β plasma concentrations could be used to the early identification of RD patients.

RESUMO FUNDAMENTAÇÃO: Não há fortes evidências sobre a associação entre o perfil inflamatório e o padrão de resposta ao tratamento medicamentoso em pacientes depressivos que podem resultar em ocorrência de doença coronariana. OBJETIVO: O objetivo deste estudo foi comparar os marcadores de aterosclerose subclínica, o perfil inflamatório e a produção de BDNF em pacientes com Depressão Resistente (DR) ou Transtorno Afetivo Bipolar (BAD) sob tratamento convencional. MÉTODOS: A população avaliada incluiu 34 RD, 43 BAD e 41 controles. Os marcadores de aterosclerose subclínica foram avaliados por ultrassonografia, tomografia e teste de esforço. As concentrações plasmáticas de TNFα, IL-1β, IL-6 e BDNF foram medidas utilizando Luminex100TM. A concentração de usCRP foi medida por imunoensaio turbidimétrico. A expressão de IL1B, IL6 e TNFA foi determinada usando TaqMan®. Para as análises estatísticas, foi estabelecido o nível de significância de p < 0,05. RESULTADOS: Quanto aos marcadores de aterosclerose subclínica, apenas o consumo de O2 foi reduzido no grupo BAD (p = 0,001). Embora não tenham sido encontradas diferenças na expressão gênica, a concentração plasmática de BDNF e IL-1β foi aumentada no grupo RD (p = 0,002 e p = 0,005, respectivamente) mesmo sob tratamento antidepressivo, o que sugere que esses medicamentos não têm efeito na secreção de IL-1β e que o inflamassomo pode desempenhar um papel na resposta terapêutica. CONCLUSÃO: Juntas, as concentrações BDNF e IL-1β poderiam ser usadas para a identificação precoce de pacientes com DR.

Revitalizing the AZT Through of the Selenium: An Approach in Human Triple Negative Breast Cancer Cell Line.

Triple-negative breast cancer represents about 15% of all cases of breast cancer, and still represents a therapeutic challenge. 3'-Azido-3'-deoxythymidine (AZT) is a nucleoside reverse transcriptase inhibitor with antitumor activity. Chalcogenides compounds, such as selenium, are very important intermediates applied in organic synthesis. Our objective was to investigate the effect and the underlying cell death mechanisms of AZT and its derivatives, in human breast cancer cell lines. The inhibitory effect of AZT and derivatives (1072, 1073, and 1079) was determined by MTT assay (0.1, 1, 10, 50, and 100 µM for concentrations and times 4, 24, 48, and 72 h) and Live/Dead in tumor cell lines MCF-7, MDA-MB 231 and also in non-tumor cell line CHO. Gene expression profiles related to apoptosis were investigated by qRT-PCR and induction of apoptosis was investigated by flow cytometry. MTT and Live/Dead assays showed that AZT derivatives decreased the rate of cell proliferation at concentrations of 50 and 100 µM in tumor cell lines MCF-7 and MDA-MB 231 while the commercial AZT presented a low antitumoral potential in all strains tested. In flow cytometry analysis we demonstrated that derivatives of AZT induced apoptosis, with an increase in both initial and late stages in both tumor cell lines evaluated, especially in MDA-MB 231. Our data show that the AZT derivative 1072 increased the expression of transcripts of the genes caspase 3 and 8 in MDA-MB 231 cell line when compared to control, suggesting that the extrinsic pathway of apoptosis was activated. In conclusion, derivatives of AZT, especially 1072, induce cytotoxicity in vitro in the triple negative breast cancer cell line through activation of the extrinsic pathway of apoptosis. These compounds containing selenium in its formulation are potential therapeutic agents for breast cancer.

Brazilian red propolis induces apoptosis-like cell death and decreases migration potential in bladder cancer cells.

Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP) on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 µg/mL) on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay) assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 µg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 µg/mL). Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment.

Methotrexate diethyl ester-loaded lipid-core nanocapsules in aqueous solution increased antineoplastic effects in resistant breast cancer cell line.

Breast cancer is the most frequent cancer affecting women. Methotrexate (MTX) is an antimetabolic drug that remains important in the treatment of breast cancer. Its efficacy is compromised by resistance in cancer cells that occurs through a variety of mechanisms. This study evaluated apoptotic cell death and cell cycle arrest induced by an MTX derivative (MTX diethyl ester [MTX(OEt)2]) and MTX(OEt)2-loaded lipid-core nanocapsules in two MTX-resistant breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231. The formulations prepared presented adequate granulometric profile. The treatment responses were evaluated through flow cytometry. Relying on the mechanism of resistance, we observed different responses between cell lines. For MCF-7 cells, MTX(OEt)2 solution and MTX(OEt)2-loaded lipid-core nanocapsules presented significantly higher apoptotic rates than untreated cells and cells incubated with unloaded lipid-core nanocapsules. For MDA-MB-231 cells, MTX(OEt)2-loaded lipid-core nanocapsules were significantly more efficient in inducing apoptosis than the solution of the free drug. S-phase cell cycle arrest was induced only by MTX(OEt)2 solution. The drug nanoencapsulation improved apoptosis induction for the cell line that presents MTX resistance by lack of transport receptors.

Encapsulation in lipid-core nanocapsules overcomes lung cancer cell resistance to tretinoin.

Tretinoin is a retinoid derivative that has an antiproliferative effect on several kinds of tumours. Human lung adenocarcinoma epithelial cell lines (A549) exhibit a profound resistance to the effects of tretinoin. Nanocarriers seem to be a good alternative to overcomecellular resistance to drugs. The aim of this study was to test whether tretinoin-loaded lipid-core nanocapsules exert anantitumor effect on A549 cells. A549 cells were incubated with free tretinoin (TTN), blank nanocapsules (LNC) and tretinoin-loaded lipid-core nanocapsules (TTN-LNC). Data from evaluation of DNA content and Annexin V binding assay by flow cytometry showed that TTN-LNC induced apoptosis and cell cycle arrest at the G1-phase while TTN did not. TTN-LNC showed higher cytotoxic effects than TTN on A549 cells evaluated by MTT and LIVE/DEAD cell viability assay. Gene expression profiling identified up-regulated expression of gene p21 by TTN-LNC, supporting the cell cycle arrest effect. These results showed for the first time that TTN-LNC are able to overcome the resistance of adenocarcinoma cell line A549 to treatment with TTN by inducing apoptosis and cell cycle arrest, providing support for their use in applications in lung cancer therapy.

Associação entre atividade física, aptidão cardiorrespiratória e biomarcadores inflamatórios em adultos jovens aparentemente saudáveis / Association between physical activity, cardiorespiratory fitness and inflammatory biomarkers in apparently healthy young adults

A inflamação sistêmica crônica de baixa intensidade está relacionada com maior risco de doenças cardiovasculares. Estudos sugerem que a proteína C-reativa, um dos principais biomarcadores inflamatórios, pode estar inversamente relacionada com a prática de atividade física e com a aptidão física. O objetivo deste estudo transversal foi determinar as associações entre os níveis de atividade física e a aptidão cardiorrespiratória, com biomarcadores inflamatórios em homens adultos jovens (18-30 anos) aparentemente saudáveis (N=85). As amostras foram analisadas pelo método ELISA (Enzyme-Linked Immuno Sorbent Assay), usando kits de alta sensibilidade para proteína C-reativa, interleucina 6, interleucina 1? e TNF-?. A prática de atividade física foi mensurada por questionário e acelerometria. O consumo máximo de oxigênio (VO2max) foi estimado por teste incremental em cicloergômetro. Na análise bruta, a média das concentrações de proteína C-reativa da amostra foi de 1,59±1,16 mg/L, e esteve significativamente correlacionada com o VO2max (r=-0,32; p=0,03), mas não com atividade física medida por acelerometria ou questionário. O índice de massa corporal (IMC) e a circunferência abdominal apresentaram correlação significativa com a proteína C-reativa (r=0,37; p<0,001 e r=0,41; p<0,001, respectivamente). Quando incluímos no modelo o IMC e a circunferência da cintura, a aptidão física perdeu a significância. Não houve relação entre atividade física ou aptidão física com os demais marcadores inflamatórios. Conclui-se que nesse grupo de adultos jovens houve relação entre a proteína C-reativa e o VO2max, e que esta associação é explicada pelas modificações no perfil antropométrico decorrentes de altos níveis de aptidão cardiorrespiratória.

Low-intensity chronic systemic inflammation is related to an increased risk of cardiovascular disease. Studies suggest that C-reactive protein, one of the main inflammatory biomarkers, may be inversely related to physical activity levels and physical fitness. The aim of this cross-sectional study was to evaluate the associations between physical activity, fitness and inflammatory biomarkers in apparently healthy men (N=85) aged 18 to 30 years. C-reactive protein, interleukin 6, interleukin 1? and TNF-? were measured using the ELISA method. Physical activity practice was assessed by questionnaire and accelerometry. Maximum oxygen uptake (VO2max) was estimated based on a cycle ergometer incremental test. In the unadjusted analysis, the mean concentration of C-reactive protein in the sample was 1.59±1.16 mg/L, and was inversely correlated with VO2max (r=-0.32, p=0.03), but not with physical activity estimated by accelerometry or questionnaire. Body mass index (BMI) and waist circumference also significantly correlate with C-reactive protein (r=0.37, p<0.001 and r=0.41, p<0.001, respectively). After adjustment for anthropometric characteristics (BMI and waist circumference), the association with fitness was no longer significant. No association was observed between physical activity or fitness levels and the other inflammatory markers. We conclude that in this group of young adults, there was an inverse association between fitness and C-reactive protein, but this association is explained by the influence of fitness on anthropometry.

Ewing Sarcoma: influence of TP53 Arg72Pro and MDM2 T309G SNPs.

The Ewing Sarcoma is an important tumor of bone and soft tissue. The SNPs Arg72Pro of TP53 and T309G of MDM2 have been associated with many cancer types and have been differently distributed among populations worldwide. Based on a case-control design, this study aimed to assess the role of these SNPs in 24 Ewing Sarcoma patients, compared to 91 control individuals. DNA samples were extracted from blood and genotyped for both SNPs by PCR-RFLP and confirmed by DNA sequencing. The results showed an association between the G allele of the T309G and Ewing Sarcoma (P=0.02). Comparing to the TT carriers, the risk of G allele carriers was 3.35 (95% CI=1.22-9.21) with P=0.02. At the genotypic level, an association of the TT genotype with the control group (P=0.03) was found. Comparing to the TT genotype, the risk of TG and GG was 2.97 (95% CI=1.03-8.58) with P=0.04 and 5.00 (95% CI=1.23-20.34) with P=0.02, respectively. No associations regarding the Arg72Pro SNP were found. Considering that the T309G has been associated with several types of cancer, including sarcomas, our results indicate that this SNP may also be important to Ewing Sarcoma predisposition.

Association between single nucleotide polymorphisms in p53 and abortion in Thoroughbred mares.

Single nucleotide polymorphisms (SNPs) in the p53 gene have been studied extensively in humans. The aims of this study were to determine the frequency of the Arg/Pro SNP in p53 in Thoroughbred mares on one stud in Brazil and to correlate p53 genotypes with reproductive performance. SNPs were detected by PCR-restriction fragment length polymorphism in blood samples from 105 horses and confirmed by sequencing. The allele frequency in Thoroughbred mares at codon 72 in exon 4 was 73.3% Arg/Pro, 17.1% Arg/Arg and 9.6% Pro/Pro. The presence of Arg/Pro was significantly associated with abortion (P=0.02), while Pro/Pro mares had a lower probability of abortion (P<0.05). Using a logistic regression model, the dominance effect was significant (P=0.044; odds ratio 7.94) for abortion and additive effects were not significant (P=0.26). p53 may play a role in equine reproduction.

TP53 gene polymorphism: importance to cancer, ethnicity and birth weight in a Brazilian cohort.

Arg72Pro SNP of p53 has been associated with many types of cancer as well as with survival and longevity. We evaluated the Arg72Pro SNP frequencies of a Brazilian birth cohort and their association with current, demographic and birth epidemiological parameters available. In 1982, all hospital births of Pelotas, southern Brazil, were identified and studied prospectively. In 2004-5, blood samples were collected and DNA extracted. PCR-RFLP was used to genotype the Arg72Pro SNP in 3794 individual samples of the Brazil birth cohort and DNA sequencing was performed to confirm the genotypes. The genotype distribution, which was in Hardy-Weinberg equilibrium, showed a predominance of the arginine amino acid with a frequency of 46.9% Arg/Arg, 42.2% Arg/Pro and 10.9% Pro/Pro. The allele frequency was 0.68 of Arginine and 0.32 of Proline. The Arg72Pro SNP genotype and allelic frequency were related to skin colour where proline amino acid was observed more among black subjects, while arginine amino acid was observed more among white subjects. The individuals without family history of cancer and those with low birth weight were associated with arginine amino acid. The Arg72Pro SNP was strongly associated with important epidemiological variables confirming that genetic profiles on cohort studies can improve our understanding of the susceptibility of diseases and its risk factors.