The usefulness of the genetic panel in the classification and refinement of diagnostic accuracy of Mexican patients with Marfan syndrome and other connective tissue disorders.

Marfan syndrome (MFS) is a multisystem genetic disorder with over 3000 mutations described in the fibrillin 1 (FBN1) gene. Like MFS, other connective tissue disorders also require a deeper understanding of the phenotype-genotype relationship due to the complexity of the clinical presentation, where diagnostic criteria often overlap. Our objective was to identify mutations in patients with connective tissue disorders using a genetic multipanel and to analyze the genotype-phenotype associations in a cohort of Mexican patients. We recruited 136 patients with MFS and related syndromes from the National Institute of Cardiology. Mutations were identified using next-generation sequencing (NGS). To examine the correlation between mutation severity and severe cardiovascular conditions, we focused on patients who had undergone Bentall-de Bono surgery or aortic valve repair. The genetic data obtained allowed us to reclassify the initial clinical diagnosis across various types of connective tissue disorders. The transforming growth factor beta receptor 2 (TGFBR2) rs79375991 mutation was found in 10 out of 16 (63%) Loeys-Dietz patients. We observed a high prevalence (65%) of more severe mutations, such as frameshift indels and stop codons, among patients requiring invasive treatments like aortic valve-sparing surgery, Bentall and de Bono procedures, or aortic valve replacement due to severe cardiovascular injury. Although our study did not achieve precise phenotype-genotype correlations, it underscores the importance of a multigenetic panel evaluation. This could pave the way for a more comprehensive diagnostic approach and inform medical and surgical treatment decision-making.

Evaluation of the participation of ABCA1 transporter in epicardial and mediastinal adipose tissue from patients with coronary artery disease

ABSTRACT Objective: Recent studies have shown a relationship between adipose tissue and coronary artery disease (CAD). The ABCA1 transporter regulates cellular cholesterol content and reverses cholesterol transport. The aim of this study was to determine the relationship between single nucleotide polymorphisms (SNPs) R230C, C-17G, and C-69T and their expression in epicardial and mediastinal adipose tissue in Mexican patients with CAD. Subjects and methods: The study included 71 patients with CAD and a control group consisting of 64 patients who underwent heart valve replacement. SNPs were determined using TaqMan probes. mRNA was extracted using TriPure Isolation from epicardial and mediastinal adipose tissue. Quantification and expression analyses were done using RT-qPCR. Results: R230C showed a higher frequency of the GG genotype in the CAD group (70.4%) than the control group (57.8%) [OR 0.34, 95% CI (0.14-0.82) p = 0.014]. Similarly, C-17G (rs2740483) showed a statistically significant difference in the CC genotype in the CAD group (63.3%) in comparison to the controls (28.1%) [OR 4.42, 95% CI (2.13-9.16), p = 0.001]. mRNA expression in SNP R230C showed statistically significant overexpression in the AA genotype compared to the GG genotype in CAD patients [11.01 (4.31-15.24) vs. 3.86 (2.47-12.50), p = 0.015]. Conclusion: The results suggest that the GG genotype of R230C and CC genotype of C-17G are strongly associated with the development of CAD in Mexican patients. In addition, under-expression of mRNA in the GG genotype in R230C is associated with patients undergoing revascularization.

Expressions of mRNA and encoded proteins of mitochondrial uncoupling protein genes (UCP1, UCP2, and UCP3) in epicardial and mediastinal adipose tissue and associations with coronary artery disease.

Objective: To evaluate the expression of UCP1, UCP2, and UCP3 mRNA and encoded proteins in epicardial and mediastinal adipose tissues in patients with coronary artery disease (CAD). Subjects and methods: We studied 60 patients with CAD and 106 patients undergoing valve replacement surgery (controls). Expression levels of UCP1, UCP2, and UCP3 mRNA and encoded proteins were measured by quantitative real-time PCR and Western blot analysis, respectively. Results: : We found increased UCP1, UCP2, and UCP3 mRNA levels in the epicardial adipose tissue in the CAD versus the control group, and higher UCP1 and UCP3 mRNA expression in the epicardial compared with the mediastinal tissue in the CAD group. There was also increased expression of UCP1 protein in the epicardial tissue and UCP2 protein in the mediastinum tissue in patients with CAD. Finally, UCP1 expression was associated with levels of fasting plasma glucose, and UCP3 expression was associated with levels of high-density lipoprotein cholesterol and low-density cholesterol in the epicardial tissue. Conclusion: Our study supports the hypothesis that higher mRNA expression by UCP genes in the epicardial adipose tissue could be a protective mechanism against the production of reactive oxygen species and may guard the myocardium against damage. Thus, UCP levels are essential to maintain the adaptive phase of cardiac injury in the presence of metabolic disorders.

Expressions of mRNA and encoded proteins of mitochondrial uncoupling protein genes (UCP1, UCP2, and UCP3) in epicardial and mediastinal adipose tissue and associations with coronary artery disease

ABSTRACT Objective: To evaluate the expression of UCP1, UCP2, and UCP3 mRNA and encoded proteins in epicardial and mediastinal adipose tissues in patients with coronary artery disease (CAD). Subjects and methods: We studied 60 patients with CAD and 106 patients undergoing valve replacement surgery (controls). Expression levels of UCP1, UCP2, and UCP3 mRNA and encoded proteins were measured by quantitative real-time PCR and Western blot analysis, respectively. Results: We found increased UCP1, UCP2, and UCP3 mRNA levels in the epicardial adipose tissue in the CAD versus the control group, and higher UCP1 and UCP3 mRNA expression in the epicardial compared with the mediastinal tissue in the CAD group. There was also increased expression of UCP1 protein in the epicardial tissue and UCP2 protein in the mediastinum tissue in patients with CAD. Finally, UCP1 expression was associated with levels of fasting plasma glucose, and UCP3 expression was associated with levels of high-density lipoprotein cholesterol and low-density cholesterol in the epicardial tissue. Conclusions: Our study supports the hypothesis that higher mRNA expression by UCP genes in the epicardial adipose tissue could be a protective mechanism against the production of reactive oxygen species and may guard the myocardium against damage. Thus, UCP levels are essential to maintain the adaptive phase of cardiac injury in the presence of metabolic disorders.

Polymorphisms in TNFAIP3, but not in STAT4, BANK1, BLK, and TNFSF4, are associated with susceptibility to Takayasu arteritis.

BACKGROUND: Takayasu arteritis (TAK) is considered a rare disease characterized by nonspecific inflammation of the large arteries, especially the aorta and its major branches. Because TAK is an autoimmune disease (AD), it could share susceptibility loci with other pathologies such as systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), among others. Widely explored polymorphisms in non-HLA genes, including TNFAIP3, STAT4, TNFSF4, BANK1, and BLK have been consistently associated with both SLE and RA, but they have not been evaluated in TAK. OBJECTIVE: The aim of our study was to investigate whether TNFAIP3, STAT4, BANK1, BLK, and TNFSF4 polymorphisms are associated with susceptibility to TAK. METHODS: The TNFAIP3 rs2230926T/G and rs5029924C/T, STAT4 rs7574865G/T, BANK1 10516487G/A, BLK rs2736340T/C, rs13277113A/G, and TNFS4 rs2205960G/T polymorphisms were genotyped in 101 cases and 276 controls by using a TaqMan SNP genotyping assay. An association analysis was performed. RESULTS: The TNFAIP3 rs2230926T/G and rs5029924C/T polymorphisms were in complete linkage disequilibrium and turned out to be risk factors for TAK (OR = 4.88, p = 0.0001). The STAT4, BANK1, BLK, and TNFSF4 polymorphisms were not associated with the disease. CONCLUSIONS: This is the first study documenting an association of TNFAIP3 rs2230926T/G and rs5029924C/T with TAK. Our results provide new information on the genetic bases of TAK.

Expressão de Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade (LRP1) em Monócito em Correlação com EIMC em Pacientes Mexicanos Hipertensos / Monocyte Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Expression Correlates with cIMT in Mexican Hypertensive Patients

Resumo Fundamento A hipertensão arterial (HTA) representa um grande fator de risco de morbidade e mortalidade cardiovascular. Ainda não se sabe que mecanismos moleculares específicos estão associados ao desenvolvimento de hipertensão essencial. Objetivo Neste trabalho, analisamos a associação entre expressão mRNA de monócito LRP1, expressão de proteína LRP1, e espessura íntima-média de carótida (EIMC) de pacientes com hipertensão essencial. Métodos A expressão mRNA de monócito LRP1 e os níveis de proteína e EIMC foram quantificados em 200 indivíduos mexicanos, sendo 91 normotensos (NT) e 109 hipertensos (HT) A significância estatística foi definida em p < 0,05. Resultados O grupo de pacientes HT tinha EIMC maior altamente significativa em comparação com os pacientes NT (p = 0,002), e isso está relacionado ao aumento na expressão mRNA de LRP1 (6,54 versus. 2,87) (p = 0,002) e expressão de proteína LRP1 (17,83 versus 6,25), respectivamente (p = 0,001). Essas diferenças foram mantidas mesmo quando dividimos nossos grupos de estudo, levando em consideração apenas aqueles que apresentavam dislipidemia na expressão de mRNA (p = 0,041) e de proteínas (p < 0,001). Também se identificou que a indução de LRP1 mediada por LRP1 em monócitos em de maneira dependente de dose e tempo, com diferença significativa em NT versus HT (0,195 ± 0,09 versus 0,226 ± 0,12, p = 0,046). Conclusão Foi encontrado um aumento em EIMC em indivíduos com hipertensão, associada a expressões de proteína LRP1 e mRNA mais altas em monócitos, independente da presença de dislipidemia em pacientes HT. Esses resultados que a upregulation de LRP1 em monócitos de pacientes hipertensos mexicanos poderia estar envolvida na diminuição da EIMC. (Arq Bras Cardiol. 2021; 116(1):56-65)

Abstract Background Arterial hypertension (HTA) represents a major risk factor for cardiovascular morbidity and mortality. It is not yet known which specific molecular mechanisms are associated with the development of essential hypertension. Objective In this study, we analyzed the association between LRP1 monocyte mRNA expression, LRP1 protein expression, and carotid intima media thickness (cIMT) of patients with essential hypertension. Methods The LRP1 monocyte mRNA expression and protein levels and cIMT were quantified in 200 Mexican subjects, 91 normotensive (NT) and 109 hypertensive (HT). Statistical significance was defined as p < 0.05. Results HT patients group had highly significant greater cIMT as compared to NT patients (p=0.002) and this correlated with an increase in the expression of LRP1 mRNA expression (6.54 vs. 2.87) (p = 0.002) and LRP1 protein expression (17.83 vs. 6.25), respectively (p = 0.001). These differences were maintained even when we divided our study groups, taking into account only those who presented dyslipidemia in both, mRNA (p = 0.041) and proteins expression (p < 0.001). It was also found that Ang II mediated LRP1 induction on monocytes in a dose and time dependent manner with significant difference in NT vs. HT (0.195 ± 0.09 vs. 0.226 ± 0.12, p = 0.046). Conclusion An increase in cIMT was found in subjects with hypertension, associated with higher mRNA and LRP1 protein expressions in monocytes, irrespective of the presence of dyslipidemias in HT patients. These results suggest that LRP1 upregulation in monocytes from Mexican hypertensive patients could be involved in the increased cIMT. (Arq Bras Cardiol. 2021; 116(1):56-65)

Lack of Association between Cytokine Genetic Polymorphisms in Takayasu's Arteritis in Mexican Patients.

Aim: To investigate the relation between polymorphisms in the interleukin 10 (IL)-10, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß and interferon (IFN)-γ genes and Takayasu's arteritis in the Mexican population. Methods: A case-control study was performed to investigate the associations of IL-10, TNF-α, TGF-ß and IFN-γ polymorphisms in a sample of 52 Takayasu's arteritis patients, diagnosed according to the criteria of the American College of Rheumatology and EULAR PRINTO criteria when the patients were under 18 years of age; 60 clinically healthy unrelated Mexican individuals by the 5' exonuclease TaqMan polymerase chain reaction. Polymorphic haplotypes were constructed after linkage disequilibrium analysis. Results: Significant differences were not found in the distribution for genotype and allele frequencies of the polymorphisms studied between healthy controls and Takayasu´s arteritis patients. Likewise, significant associations were not detected in the haplotype analysis with the different genes studied. Conclusions: These findings suggest that the polymorphisms in IL-10, TNF-α, TGF-ß and IFN-γ might not contribute to the susceptibility of Takayasu´s arteritis in the Mexican population.

Para-occupational exposure to pesticides, PON1 polymorphisms and hypothyroxinemia during the first half of pregnancy in women living in a Mexican floricultural area.

BACKGROUND: Adequate maternal thyroxine (T4) concentrations during the first half of pregnancy are fundamental to the embryo's or fetus' neural development. Organophosphate pesticides (OP) can act as thyroid disruptors and genetic polymorphisms for paraoxonase 1 (PON1), an enzyme that detoxifies OP, could be involved in individual's susceptibility to them. We assessed the association between para-occupational exposure to pesticides, including OP, during pregnancy and maternal hypothyroxinemia, as well as the potential genetic susceptibility conferred by PON1 polymorphisms. METHODS: We analyzed information from 381 healthy pregnant women (< 17 gestational weeks), who lived in a floricultural region of Mexico where pesticides, including OP, are routinely used. Women who were para-occupationally exposed to pesticides were those whose partner had an occupation involving contact with these products. Thyroid-Stimulating Hormone (TSH) and free T4 concentrations were determined using ELISA, and hypothyroxinemia was defined as free T4 concentrations <0.76 ng/dL. PON1192QR, PON155LM and PON1-108CT polymorphisms were determined through Polymerase Chain Reaction (PCR). The association between para-occupational exposure and genetic polymorphisms and hypothyroxinemia was estimated using logistic regression models. RESULTS: One hundred and sixty two women (42.52%) were classified as para-occupationally exposed to pesticides. Hypothyroxinemia prevalence was 54%, and it was not significantly associated with pesticide para-occupational exposure (OR: 1.21 95% CI 0.75-1.94). Independently of para-occupational exposure, the likelihood of hypothyroxinemia was higher among women who were carriers of PON155MM than in those with PON155LL genotype (OR MM vs LL: 3.03; 95%CI 1.62, 5.70). PON1192 RR (OR RR vs QQ: 1.72; 95%CI 0.93, 3.17) and PON1-108TT (OR TT vs CC: 1.60; 95%CI 0.90, 2.70) genotypes were marginally associated with hypothyroxinemia. No significant interaction was observed between pesticides para-occupational exposure and PON1 polymorphisms. CONCLUSIONS: These results suggest that PON1 polymorphisms could affect thyroid function during pregnancy in women living in areas where pesticides, including OP, are routinely used. Low exposure variability in this population, could be a possible explanation for the lack of association between para-occupational exposure and thyroid function.

DHEA increases epithelial markers and decreases mesenchymal proteins in breast cancer cells and reduces xenograft growth.

Breast cancer is one of the most common neoplasias and the leading cause of cancer death in women worldwide. Its high mortality rate is linked to a great metastatic capacity associated with the epithelial-mesenchymal transition (EMT). During this process, a decrease in epithelial proteins expression and an increase of mesenchymal proteins are observed. On the other hand, it has been shown that dehydroepiandrosterone (DHEA), the most abundant steroid in human plasma, inhibits migration of breast cancer cells; however, the underlying mechanisms have not been elucidated. In this study, the in vitro effect of DHEA on the expression pattern of some EMT-related proteins, such as E-cadherin (epithelial), N-cadherin, vimentin and Snail (mesenchymal) was measured by Western blot and immunofluorescence in MDA-MB-231 breast cancer cells with invasive, metastatic and mesenchymal phenotype. Also, the in vivo effect of DHEA on xenograft tumor growth in nude mice (nu-/nu-) and on expression of the same epithelial and mesenchymal proteins in generated tumors was evaluated. We found that DHEA increased expression of E-cadherin and decreased N-cadherin, vimentin and Snail expression both in MD-MB-231 cells and in the formed tumors, possibly by DHEA-induced reversion of mesenchymal phenotype. These results were correlated with a tumor size reduction in mouse xenografts following DHEA administration either a week earlier or concurrent with breast cancer cells inoculation. In conclusion, DHEA could be useful in the treatment of breast cancer with mesenchymal phenotype.

Glutathione system participation in thoracic aneurysms from patients with Marfan syndrome.

BACKGROUND: Aortic dilatation in Marfan syndrome (MFS) is progressive. It is associated with oxidative stress and endothelial dysfunction that contribute to the early acute dissection of the vessel and can result in rupture of the aorta and sudden death. We evaluated the participation of the glutathione (GSH) system, which could be involved in the mechanisms that promote the formation and progression of the aortic aneurysms in MFS patients. PATIENTS AND METHODS: Aortic aneurysm tissue was obtained during chest surgery from eight control subjects and 14 MFS patients. Spectrophotometrical determination of activity of glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), lipid peroxidation (LPO) index, carbonylation, total antioxidant capacity (TAC), and concentration of reduced and oxidized glutathione (GSH and GSSG respectively), was performed in the homogenate from aortic aneurysm tissue. RESULTS: LPO index, carbonylation, TGF-ß1, and GR activity were increased in MFS patients (p < 0.04), while TAC, GSH/GSSG ratio, GPx, and GST activity were significantly decreased (p < 0.04). CONCLUSIONS: The depletion of GSH, in spite of the elevated activity of GR, not only diminished the activity of GSH-depend GST and GPx, but increased LPO, carbonylation and decreased TAC. These changes could promote the structural and functional alterations in the thoracic aorta of MFS patients.

Dehydroepiandrosterone inhibits events related with the metastatic process in breast tumor cell lines.

Dehydroepiandrosterone (DHEA), an adrenal hormone, has a protective role against cancer. We previously shown that DHEA inhibits the proliferation and migration of cell lines derived from breast cancer; however, the role of DHEA in others events related with these effects are unknown. We hypothesized that DHEA inhibits the expression of proteins and some events related with cell migration and metastasis. We determined the migration in Boyden chambers, the invasion in matrigel, anchorage-independent growth and the formation of spheroids in 3 cell lines (MCF-7, MDA-MB-231, ZR-75-30) derived from breast cancer exposed to DHEA. The secretion of metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and several pro-inflammatory molecules in the secretome of these cells was also evaluated. DHEA inhibited the migration in transwells and the invasion in matrigel of MCF-7 and MDA-MB-231 cells. Besides, DHEA inhibited the anchorage-independent growth on agar and decreased the size of spheroids, and also reduced the secretion of IL-1α, IL-6, IL-8, and TNF-α in all cell lines. Metalloproteinase-1 (MMP-1) secretion was slightly decreased by DHEA treatment in MDA-MB-231 cells. Our results also showed that inhibition of migration and invasion induced by DHEA in breast cancer cells is correlated with the decrease of cytokine/chemokine secretion and the diminution of tumor cells growth. MCF-7 cells were the most responsive to the exposure to DHEA, whereas ZR-75-30 cells responded less to this hormone, suggesting that DHEA could be used in the treatment of breast cancer in early stages.

Association between PON1 genetic polymorphisms and miscarriage in Mexican women exposed to pesticides.

BACKGROUND: Placental oxidative stress has been involved in the pathogenesis of certain reproductive adverse effects, including miscarriage. Paraxonase 1 (PON1) is a high-density lipoprotein(HDL)-linked enzyme that prevents oxidation of low-density lipoproteins (LDL) and is involved in detoxification from organophosphate pesticides. OBJECTIVE: To assess the association between maternal PON1 polymorphisms (PON1192Q/R, PON155 L/M y PON1-108C/T) and the risk of miscarriage in women chronically exposed to organophosphate pesticides in Mexico. METHODS: In a cross-sectional study, socio-demographic data, reproductive history data, environmental exposures, and other variables of concern were collected by means of a questionnaire from 264 women (floriculturists and wives of floriculturists) who had been pregnant sometime during the 10 years preceding the study. Blood samples were also collected from them. PON1192 and PON155 genotypes were determined by PCR amplification, and PON1-108 genotypes, by a TaqMan real-time polymerase chain reaction assay. Complete information regarding the results of pregnancy and maternal genotype tests was obtained for 514 pregnancies (35 miscarriages and 479 controls). The association between PON1 genotypes and miscarriage was evaluate through GEE models. RESULTS: The risk of miscarriage by mothers with PON1192RR genotype was 2.2 higher than by mothers with PON1192QR/PON1192QQ genotype (95% CI 0.93-5.17). The risk was close to 4 times higher in mothers with PON155MM/PON155LM genotype than in mothers with PON155LL genotype (OR=3.9; 95% CI 1.38-11.0). No significant differences were found in risk of miscarriage based on the maternal PON1-108C/T genotype. No evidence was found of an interaction between the various PON1 genotypes and the mothers' floricultural activity during pregnancy. CONCLUSIONS: This study suggests that there is an effect of genetic maternal PON1 polymorphisms on miscarriage and provides additional evidence that combines with the growing information about the ways in which certain PON1 genotypes can affect the development of the fetus in utero.

Actividades paraoxonasa y arilesterasa bajas en sujetos mexicanos con enfermedad arterial coronaria / Low paraoxonase and arylesterase plasma activities in mexican patients with coronary artery disease

PURPOSE: To determine the Paraoxonase-1 (PON1) activity as well as its pheno- and genotypes at position 192 in Mexican subjects with diagnosis of coronary heart disease (CHD). METHODS: We determined the PON1-192 polymorphism by PCR-RFLP, and serum PON1 activity, using either paraoxon (PONase activity) or phenylacetate (ARE activity) as substrates, in 155 clinically healthy individuals (control group), and 155 patients with at least one myocardial infarction (CHD group). The biochemical A/B phenotype was determined by the ratio of the NaCI 1 M-stimulated PONase activity divided by the ARE activity. RESULTS: We found significantly lower PONase and ARE activities in CHD patients as compared to controls (233.1 +/- 102.1 vs. 295.8 +/- 159.1 nmol/min/mL, and 103.1 +/- 33.7 vs 220.2 +/- 120.7 micromol/min/mL, respectively, p<0.05 for both). Allele and genotype frequencies for PON1-192 were similar in CHD patients and healthy controls. Moreover, in the control group, the PON1-192 Q/R genotype did not matched with the A/B phenotype as has been proposed by other studies. CONCLUSIONS: There were important differences in the ARE and PONase activities between Mexican CHD patients and controls, suggesting that PON1 activity could be a good marker of CHD risk, whereas PON1-192 lacks of value to assess such risk.

Dehydroepiandrosterone delays LDL oxidation in vitro and attenuates several oxLDL-induced inflammatory responses in endothelial cells.

Dehydroepiandrosterone (DHEA) has a protective role against atherosclerosis, most likely mediating an anti-inflammatory action. In order to understand the mechanisms involved in this protection, we evaluated the effects of DHEA on several molecules involved in the inflammatory response. Reactive oxygen species (ROS), expression of adhesion molecules, activation of the NF-kappaB/IkappaB-alpha pathway and of the AP-1 transcription factor were evaluated in human umbilical vein endothelial cells (HUVECs) treated with oxidized low density lipoproteins (oxLDL) and DHEA. We also determined if DHEA affected LDL oxidation in vitro. 100 microM DHEA-treatment inhibited the oxLDL-induced expression of ICAM-1, VCAM-1, PECAM-1, ROS production, and U937 cells adhesion to HUVECs. DHEA also delayed the kinetics of LDL oxidation in vitro. While DHEA did not affect the translocation of NF-kappaB neither the degradation IkappaB-alpha, it led to an increased translocation of AP-1. Our results suggest that DHEA inhibits the expression of molecules involved in the inflammatory process in endothelial cells activated with oxLDL, therefore its potential anti-inflammatory properties should be evaluated for the treatment of chronic inflammatory diseases such as atherosclerosis.