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A study of 269 children enrolled into a National Registry for children with persistent glomerular hematuria identified 131 individuals with genetically confirmed X-linked Alport Syndrome. A single variant c.1871G>A p.Gly624Asp (G624D) in COL4A5 was predominant and accounted for 39% of X-linked Alport Syndrome in unrelated Polish families (44 of 113). To evaluate its origins, the genetic variation in a 2.79 Mb segment encompassing the COL4A5 locus on chromosome X was assessed. All G624D alleles were found on the same rare haplotype background, indicating a founder effect dating back to the 12-13th century. The phenotypic data of 131 children with X-linked Alport Syndrome and their 195 affected adult relatives revealed that the G624D variant was associated with a significantly milder clinical course in comparison to other pathogenic COL4A5 variants. Furthermore the clinical course of this genetically uniform cohort was milder than that observed in individuals with other COL4A5 missense mutations. In spite of the benign clinical manifestation throughout childhood and early adulthood, the G624D variant confers significant risk for both kidney failure and deafness in males, albeit 20-30 years later than that observed in individuals with other COL4A5 pathogenic variants (50% cumulative risk of starting dialysis at 54 years (95% confidence interval: 50-62) v. 26 years (95% confidence interval: 22-30)). Thus, males with G624D are candidates for existing and emerging therapies for Alport Syndrome.
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Colágeno Tipo IV , Nefrite Hereditária , Insuficiência Renal , Adulto , Criança , Colágeno Tipo IV/genética , Análise Mutacional de DNA , Europa (Continente) , Efeito Fundador , Humanos , Masculino , Pessoa de Meia-Idade , Nefrite Hereditária/genéticaRESUMO
Amino acids (AAs) play a crucial role in cancer cell metabolism. Levels of 22 plasma AAs at the time of diagnosis and after treatment were established among 39 pediatric cancer patients and 33 healthy children. Glutamic acid levels decreased and tryptophan levels increased during treatment. Cancer patients presented significantly lower levels of glutamine and leucine post-treatment while levels of 12 other AAs were higher comparing to controls. Results suggest that plasma free AA profile may serve as a prognostic biomarker.
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Aminoácidos/sangue , Neoplasias/sangue , Adolescente , Biomarcadores Tumorais/sangue , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Masculino , Metabolômica , Neoplasias/diagnóstico , Neoplasias/terapia , PrognósticoRESUMO
BACKGROUND: Genotype-phenotype studies in type 1 diabetes (T1DM) patients are needed for further development of therapy strategies. OBJECTIVE: Our aims were to investigate the distribution of selected PTPN22 and FCRL3 gene polymorphisms and their associations with clinical course of disease in children with newly diagnosed T1DM from the Pomeranian region of Poland. SUBJECTS/METHODS: The prospective, longitudinal study of 147 children with newly diagnosed T1DM-autoimmune subtype was conducted. The PTPN22 c.1858T>C (rs2476601) and FCRL3 -169C>T (rs7528684) polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and DNA sequencing. The frequencies of genotypes were compared between the study and population-matched control group (327 random anonymous samples from the Pomeranian region). Selected patients underwent a 24-monthly follow up [periodic re-evaluation of fasting C-peptide concentration (FCP) and hemoglobin A1c (HbA1c ) level]. RESULTS: A significantly lower coincidence of the PTPN22 c.1858CC and FCRL3 -169CC genotypes was found in the study group compared with controls (P = 0.04). The PTPN22 c.1858CC and FCRL3 -169CC genotype combination, restricted to female patients only, was associated with well-preserved residual ß-cell function throughout the entire follow up (prolonged FCP level increase up to the sixth month of disease, with further very stable dynamics-FCP median level ≥0.67 ng/mL without significant decrease up to the 24th month). HbA1c levels in this subgroup also remained the lowest during the observation period. CONCLUSIONS/INTERPRETATION: Ascertained phenomenon could be explained by an interacting mechanism of the two polymorphisms through estrogen-regulated nuclear factor kappa B signaling in regulatory T (Treg ) lymphocytes. This hypothesis, if confirmed, may lead to further development of Treg administration-based therapies.
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Diabetes Mellitus Tipo 1/genética , Células Secretoras de Insulina/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Receptores Imunológicos/genética , Adolescente , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Lactente , Estudos Longitudinais , MasculinoRESUMO
Huntington disease (HD) is an inherited neurodegenerative disorder caused by mutations in the huntingtin gene. Involvement of mitochondrial dysfunctions in, and especially influence of the level of mitochondrial DNA (mtDNA) on, development of this disease is unclear. Here, samples of blood from 84 HD patients and 79 controls, and dermal fibroblasts from 10 HD patients and 9 controls were analysed for mtDNA levels. Although the type of mitochondrial haplogroup had no influence on the mtDNA level, and there was no correlation between mtDNA level in leukocytes in HD patients and various parameters of HD severity, some considerable differences between HD patients and controls were identified. The average mtDNA/nDNA relative copy number was significantly higher in leukocytes, but lower in fibroblasts, of symptomatic HD patients relative to the control group. Moreover, HD women displayed higher mtDNA levels in leukocytes than HD men. Because this is the largest population analysed to date, these results might contribute to explanation of discrepancies between previously published studies concerning levels of mtDNA in cells of HD patients. We suggest that the size of the investigated population and type of cells from which DNA is isolated could significantly affect results of mtDNA copy number estimation in HD. Hence, these parameters should be taken into consideration in studies on mtDNA in HD, and perhaps also in other diseases where mitochondrial dysfunction occurs.
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DNA Mitocondrial/metabolismo , Fibroblastos/metabolismo , Doença de Huntington/metabolismo , Leucócitos/metabolismo , Adulto , Idoso , DNA Mitocondrial/genética , Feminino , Humanos , Doença de Huntington/genética , Masculino , Pessoa de Meia-Idade , Mutação , Pele/metabolismo , Adulto JovemRESUMO
DNA digestion with endonucleases sensitive to CpG methylation such as HpaII followed by polymerase chain reaction (PCR) quantitation is commonly used in molecular studies as a simple and inexpensive solution for assessment of region-specific DNA methylation. We observed that the results of such analyses were highly overestimated if mock-digested samples were applied as the reference. We determined DNA methylation levels in several promoter regions in two setups implementing different references: mock-digested and treated with a restriction enzyme that has no recognition sites within examined amplicons. Fragmentation of reference templates allowed removing the overestimation effect, thereby improving measurement accuracy.
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Metilação de DNA , Enzimas de Restrição do DNA/metabolismo , Reação em Cadeia da Polimerase/métodosRESUMO
Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by the extension of the CAG repeats in exon 1 of the HTT gene and is transmitted in a dominant manner. The present study aimed to assess whether patients' sex, in the context of mutated and normal allele length, contributes to age on onset (AO) of HD. The study population comprised a large cohort of 3723 HD patients from the European Huntington's Disease Network's REGISTRY database collected at 160 sites across 17 European countries and in one location outside Europe. The data were analyzed using regression models and factorial analysis of variance (ANOVA) considering both mutated allele length and sex as predictors of patients' AO. AO, as described by the rater's estimate, was found to be later in affected women than in men across the whole population. This difference was most pronounced in a subgroup of 1273 patients with relatively short variants of the mutated allele (40-45 CAG repeats) and normal alleles in a higher half of length distribution-namely, more than 17 CAG repeats; however, it was also observed in each group. Our results presented in this observational study point to sex-related differences in AO, most pronounced in the presence of the short mutated and long normal allele, which may add to understanding the dynamics of AO in Huntington's Disease.Trial registration: ClinicalTrials.gov identifier NCT01590589.
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Idade de Início , Proteína Huntingtina , Doença de Huntington , Humanos , Doença de Huntington/genética , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Proteína Huntingtina/genética , Alelos , Repetições de Trinucleotídeos/genética , Expansão das Repetições de Trinucleotídeos/genética , Fatores Sexuais , Idoso , Mutação , Europa (Continente)/epidemiologiaRESUMO
Background: Hemorrhagic transformation (HT) in acute ischemic stroke is likely to occur in patients treated with intravenous thrombolysis (IVT) and may lead to neurological deterioration and symptomatic intracranial hemorrhage (sICH). Despite the complex inclusion and exclusion criteria for IVT and some useful tools to stratify HT risk, sICH still occurs in approximately 6% of patients because some of the risk factors for this complication remain unknown. Objective: This study aimed to explore whether there are any differences in circulating microRNA (miRNA) profiles between patients who develop HT after thrombolysis and those who do not. Methods: Using qPCR, we quantified the expression of 84 miRNAs in plasma samples collected prior to thrombolytic treatment from 10 individuals who eventually developed HT and 10 patients who did not. For miRNAs that were downregulated (fold change (FC) <0.67) or upregulated (FC >1.5) with p < 0.10, we investigated the tissue specificity and performed KEGG pathway annotation using bioinformatics tools. Owing to the small patient sample size, instead of multivariate analysis with all major known HT risk factors, we matched the results with the admission NIHSS scores only. Results: We observed trends towards downregulation of miR-1-3p, miR-133a-3p, miR-133b and miR-376c-3p, and upregulation of miR-7-5p, miR-17-3p, and miR-296-5p. Previously, the upregulated miR-7-5p was found to be highly expressed in the brain, whereas miR-1, miR-133a-3p and miR-133b appeared to be specific to the muscles and myocardium. Conclusion: miRNA profiles tend to differ between patients who develop HT and those who do not, suggesting that miRNA profiling, likely in association with other omics approaches, may increase the current power of tools predicting thrombolysis-associated sICH in acute ischemic stroke patients. This study represents a free hypothesis-approach pilot study as a continuation from our previous work. Herein, we showed that applying mathematical analyses to extract information from raw big data may result in the identification of new pathophysiological pathways and may complete standard design works.
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Despite the animal models' complexity, researchers tend to reduce the number of animals in experiments for expenses and ethical concerns. This tendency makes the risk of false-positive results, as statistical significance, the primary criterion to validate findings, often fails if testing small samples. This study aims to highlight such risks using an example from experimental regenerative therapy and propose a machine-learning solution to validate treatment effects. The example analysed was the pharmacological treatment of ear pinna punch wound healing in mice. Wound closure data analysed included eight groups treated with an epigenetic inhibitor, zebularine, and eight control groups receiving vehicle alone, of six mice each. We confirmed the zebularine healing effect for all 64 pairwise comparisons between treatment and control groups but also determined minor yet statistically significant differences between control groups in five of 28 possible comparisons. The occurrences of significant differences between the control groups, regardless of standardised experimental conditions, indicate a risk of statistically significant effects in the case a compound lacking the desired biological activity is tested. Since the criterion of statistical significance itself can be confusing, we demonstrate a machine-learning algorithm trained on datasets representing treatment and control experiments as a helpful tool for validating treatment outcomes. We tested two machine-learning approaches, Naïve Bayes and Support Vector Machine classifiers. In contrast to the Mann-Whitney U-test, indicating enhanced healing effects for some control groups receiving saline alone, both machine-learning algorithms faultlessly assigned all animal groups receiving saline to the controls.
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Experimentação Animal , Animais , Camundongos , Teorema de Bayes , Algoritmos , Aprendizado de Máquina , Resultado do TratamentoRESUMO
Cytokines are responsible for maintaining homeostasis as cell growth, differentiation, migration and apoptosis mediators. They play a pivotal role in immune responses to inflammatory reactions. In oncological diseases, the cross-talk between cells of the immunological system and cells of the tumour microenvironment is led by cytokines. Also, the overproduction of cytokines may change the tumour microenvironment and stimulate tumour development and growth. To test whether pro-inflammatory cytokines or associated with them transcription factor levels are changed in a group of 53 paediatric cancer patients, serum levels of IL-1ß, IL-6, TNF-α and NF-κB were assessed and compared to measures in 25 healthy controls. Increased levels of IL-6 were found among patients in active oncological treatment (P=0.002) but not among patients whose treatment was completed. Our data suggest that IL6, but not IL-1ß, TNF-α and NF-κB, is elevated as a result of the immune response in the microenvironment around the tumour and in blood cancers, among patients who were not infected at the time of blood collection. Thus, IL6 levels might serve as a potential biomarker of oncohematological diseases.
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NF-kappa B , Neoplasias , Criança , Humanos , Citocinas , Fator de Necrose Tumoral alfa , Interleucina-6 , Microambiente TumoralRESUMO
Cardiovascular (CV) events are the number one cause of lifetime disability and deaths worldwide. It is well known that traditional risk factors do not fully correlate with clinical outcomes; therefore, searching for other markers that would explain CV events' occurrence seems essential. Of importance, one of the main factors at the origin of CV events is oxidative stress, causing inflammation and atherosclerotic plaque instability. Therefore, the present study was conducted to evaluate eight carefully selected genetic polymorphisms related to oxidative stress as risk modifiers for CV events. A cohort of 1020 patients with coronary atherosclerosis was analysed in a 7-year follow-up observational study. The following end points were assessed: CV death, myocardial infarction (MI) and a combined end point of CV death/MI/stroke. Our results show that single polymorphisms are not significant cardiovascular disease risk factors, but genetic risk score (GRS), defined as the accumulation of our eight studied polymorphisms, was significantly associated with the three. Specifically, low GRS was associated with a higher risk of CV death, MI and CV death/MI/stroke. In conclusion, when regarding CV events, GRS investigated here can become clinically meaningful and undoubtedly adds to the knowledge in stratifying the risk of CV events.
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Infarto do Miocárdio , Acidente Vascular Cerebral , Humanos , Seguimentos , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/genética , Fatores de Risco , Polimorfismo Genético , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/genéticaRESUMO
BACKGROUND: Low folate and high homocysteine (Hcy) concentrations are associated with pregnancy-related pathologies such as spina bifida. Polymorphisms in folate/Hcy metabolic enzymes may contribute to this potentially pathogenic biochemical phenotype. METHODS: The study comprised 26 Caucasian and 23 African-American premenopausal women. Subjects gave fasting blood samples for biochemical phenotyping and genotyping. Total Hcy (tHcy) and both plasma and red blood cell (RBC) folate derivatives (i.e. tetrahydrofolate [THF], 5-methylTHF [5-MTHF], and 5,10-methenylTHF [5,10-MTHF]) were measured using stable isotope dilution liquid chromatography, multiple reaction monitoring, and mass spectrometry. Eleven polymorphisms from nine folate/Hcy pathway genes were genotyped. Tests of association between genetic, lifestyle, and biochemical variables were applied. RESULTS: In African American women, tHcy concentrations were associated (p < 0.05) with total RBC folate, RBC 5-MTHF, B(12), and polymorphisms in methionine synthase (MTR) and thymidylate synthase (TYMS). In Caucasian women, tHcy concentrations were not associated with total folate levels, but were associated (p < 0.05) with RBC THF, ratios of RBC 5-MTHF:THF, and polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR) and MTR. In African Americans, folate derivative levels were associated with smoking, B(12), and polymorphisms in MTR, TYMS, methionine synthase reductase (MTRR), and reduced folate carrier1 (RFC1). In Caucasians, folate derivative levels were associated with vitamin use, B(12), and polymorphisms in MTHFR, TYMS, and RFC1. CONCLUSIONS: Polymorphisms in the folate/Hcy pathway are associated with tHcy and folate derivative levels. In African American and Caucasian women, different factors are associated with folate/Hcy phenotypes and may contribute to race-specific differences in the risks of a range of pregnancy-related pathologies.
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Homocisteína/sangue , Estilo de Vida , Pré-Menopausa/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Adolescente , Adulto , Negro ou Afro-Americano/genética , Criança , Pré-Escolar , Suplementos Nutricionais , Eritrócitos/química , Feminino , Estudos de Associação Genética , Homocisteína/genética , Humanos , Lactente , Proteínas de Membrana Transportadoras/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Polimorfismo Genético , Gravidez , Pré-Menopausa/genética , Disrafismo Espinal/epidemiologia , Disrafismo Espinal/etiologia , Disrafismo Espinal/genética , Disrafismo Espinal/metabolismo , Tetra-Hidrofolatos/análise , Timidilato Sintase/genética , Vitaminas/administração & dosagem , População Branca/genética , Adulto JovemRESUMO
The CYBA gene encodes the regulatory subunit of NADPH oxidase, which maintains the redox state within cells and in the blood vessels. That led us to investigate the course of coronary artery disease (CAD) with regards to CYBA polymorphisms. Thus, we recruited 1197 subjects with coronary atherosclerosis and observed them during 7-year follow-up. Three CYBA polymorphisms: c.214C>T (rs4673), c.-932G>A (rs9932581), and c.*24G>A (1049255) were studied for an association with death, major adverse cardiovascular events (MACE) and an elective percutaneous coronary intervention or coronary artery bypass grafting (PCI/CABG). We found an association between the CYBA c.214C>T polymorphism and two end points: death and PCI/CABG. CYBA c.214TT genotype was associated with a lower risk of death than C allele (9.5% vs. 21%, p < 0.05) and a higher risk of PCI/CABG than C allele (69.3% vs. 51.7%, p < 0.01). This suggests that the CYBA c.214TT genotype may be a protective factor against death OR = 0.47 (95%CI 0.28-0.82; p < 0.01), while also being a risk factor for an elective PCI/CABG OR = 2.36 (95%CI 1.15-4.82; p < 0.05). Thus, we hypothesize that among patients with coronary atherosclerosis, the CYBA c.214TT genotype contributes to atherosclerotic plaque stability by altering the course of CAD towards chronic coronary syndrome, thereby lowering the incidence of fatal CAD-related events.
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Oxidative stress is believed to play a critical role in atherosclerosis initiation and progression. In line with this, in a group of 1099 subjects, we determined eight single nucleotide polymorphisms (SNPs) related to oxidative stress (PON1 c.575A>G, MPO c.-463G>A, SOD2 c.47T>C, GCLM c.-590C>T, NOS3 c.894G>T, NOS3 c.-786T>C, CYBA c.214C>T, and CYBA c.-932A>G) and assessed the extent of atherosclerosis in coronary arteries based on Gensini score. An increased risk of having a Gensini score in the higher half of the distribution was observed for the PON1 c.575G allele (odds ratio (OR) = 1.27, 95% confidence interval (CI): 1.004-1.617, p = 0.046). Next, the genetic risk score (GRS) for the additive effect of the total number of pro-oxidative alleles was assessed. We noted an increase in the risk of having a Gensini score above the median with the maximum number of risk alleles (OR = 2.47, 95% CI: 1.19-5.23, p = 0.014). A univariate Spearman's test revealed significant correlation between the total number of pro-oxidant alleles (GRS) and the Gensini score (ρ = 0.068, p = 0.03). In conclusion, the PON1 c.575A>G variant and the high number of risk alleles (GRS) were independent risk factors for a high Gensini score. We suggest, however, that GRS might occur as a more valuable component in adding a predictive value to the genetic background of atherosclerosis.
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Low folate status may be a consequence of suboptimal intake, transport or cellular utilization of folate and, together with elevated homocysteine, is a recognized risk factor or marker for several human pathologies. As folate transport across cell membranes is mediated in part by the reduced folate carrier (RFC1), variants within SLC19A1, the gene that encodes RFC1, may influence disease risk via an effect on folate and/or homocysteine levels. The present study was undertaken to assess the association between the SLC19A1 c.80G>A polymorphism and folate/homocysteine concentrations in healthy young adults from Northern Ireland. The SLC19A1 c.80G>A polymorphism was not strongly associated with either serum folate or homocysteine concentrations in either men or women. However, in women, but not in men, this polymorphism explained 5% of the variation in red blood cell (RBC) folate levels (P= 0.02). Relative to women with the SLC19A1 c.80GG genotype, women with the GA and AA genotypes had higher RBC folate concentrations. Consequently, compared to women with the SLC19A1 c.80GA and AA genotypes, women who are homozygous for the 80G allele may be at increased risk of having a child affected with a neural tube defect and of developing pathologies that have been associated with folate insufficiency, such as cardiovascular disease.
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Eritrócitos/metabolismo , Ácido Fólico/metabolismo , Proteínas de Membrana Transportadoras/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Criança , Feminino , Ácido Fólico/sangue , Predisposição Genética para Doença , Homocisteína/sangue , Humanos , Masculino , Proteína Carregadora de Folato Reduzido , Caracteres Sexuais , Adulto JovemRESUMO
BACKGROUND: Folate and homocysteine are involved in DNA synthesis and methylation processes, which are deregulated during carcinogenesis. OBJECTIVES: The aim of this study was to assess the relationship between folate/homocysteine concentrations, the functional polymorphisms of folate/homocysteine genes and lung cancer risk among cigarette smokers. STUDY DESIGN: The study included 132 lung cancer patients and 396 controls from northern Poland, matched by sex, age and smoking status. The median cigarette pack-years of smoking among both cases and controls was 30.0. Serum, red blood cell (RBC) folates and serum homocysteine concentrations were measured. The genotypes in selected polymorphic sites of the MTHFR, CBS, SHMT1, MTHFD1, MTRR, MTR, TYMS DHFR, TCN2, and SLC19A1 genes were determined. All study participants underwent scanning with low-dose computed tomography. RESULTS: Serum folate concentrations above the median (> 17.5 nmol/l among the healthy controls) were associated with an increased lung cancer risk (odds ratio [OR], 1.54, 95% confidence intervals [CI], 1.04-2.29, P = 0.031). An analogous trend was observed when the population was analysed after subdivision according to RBC folate concentrations, that is, above a value of 506.5 nmol/l (OR, 1.53; 95% CI, 0.95-2.47; P = 0.084). Additionally, in a subset of women, an increased risk of lung cancer development was associated with the SLC19A1 c.80AA genotype (c.80AA versus GG OR, 3.14; 95% CI, 1.32-7.46; P = P = 0.010). CONCLUSION: These results suggest that, in the population consisting of heavy smokers, high folate levels add to the cancerogenic effect of smoking.
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Ácido Fólico/sangue , Homocisteína/sangue , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , Fumar/efeitos adversos , Fatores Etários , Idoso , Feminino , Genótipo , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Razão de Chances , Polônia/epidemiologia , Polimorfismo de Nucleotídeo Único , Proteína Carregadora de Folato Reduzido/genética , Reprodutibilidade dos Testes , Fatores de Risco , Fatores SexuaisRESUMO
A low serum folate and high homocysteine phenotype is associated with an increased risk of neural tube defects (NTDs), cardiovascular diseases and other pathologies. Thus defining both genetic and non-genetic factors that may impact folate/homocysteine metabolism will enhance our understanding of the etiologic mechanisms underlying these conditions and facilitate risk assessment. Dihydrofolate reductase catalyzes the reduction of folic acid to dihydrofolate and thereafter to tetrahydrofolate. The impact of the dihydrofolate reductase (DHFR) c.86 + 60_78 insertion/deletion (ins/del) polymorphism on folate and homocysteine concentrations was analyzed using data from healthy young adults from Northern Ireland, collected as part of visit three of the Young Hearts Project. Among men the DHFR c.86 + 60_78 polymorphism was not significantly associated with serum or red blood cell folate concentrations, or with homocysteine concentrations. Among women the DHFR c.86 + 60_78 polymorphism explained 2% of the variation in RBC folate levels and 5% of the variation in serum folate levels, but did not appear to have an independent effect on homocysteine. Relative to women with the DHFR c.86 + 60_78 ins/ins and ins/del genotypes, del/del homozygotes had increased serum and red blood cell folate concentrations and may therefore be at decreased risk of having offspring affected by NTDs and of other adverse reproductive and health outcomes attributable to low folate.
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Eritrócitos/metabolismo , Ácido Fólico/sangue , Polimorfismo Genético/genética , Tetra-Hidrofolato Desidrogenase/sangue , Tetra-Hidrofolato Desidrogenase/genética , Adolescente , Estudos de Casos e Controles , Criança , Cromatografia Líquida de Alta Pressão , Feminino , Ácido Fólico/genética , Genótipo , Homocisteína/sangue , Humanos , Estudos Longitudinais , Masculino , Irlanda do Norte , Reação em Cadeia da Polimerase , Fatores de Risco , Complexo Vitamínico B/sangueRESUMO
A high homocysteine phenotype, often accompanied by low folate, is associated with several pathologies including cardiovascular disease and birth defects. This phenotype appears to be influenced by both genetic and environmental factors, which may act in a sex-dependent manner. The present analyses were undertaken to identify the determinants of homocysteine concentrations in young men and women, and are based on data from a cohort of young, reproductive age (20-26 years old) individuals in Northern Ireland. Multivariate modeling indicated that homocysteine concentrations are associated with red blood cell (RBC) folate, vitamin B(12), MTHFR 677C>T genotype and smoking status in both males and females. However, the inter-relationships between these variables appear to differ between the sexes. Specifically, homocysteine levels in males were significantly associated with interactions between MTHFR 677C>T genotype and both RBC folate and smoking status. In contrast, homocysteine levels in females were significantly associated with interactions between smoking status and RBC folate. These results suggest that the characteristics of individuals who are at the highest risk for a high homocysteine phenotype differ for males and females. Among males, those with the MTHFR 677TT genotype appear to be at the highest risk and to be the most vulnerable to factors (e.g. smoking, low RBC folate) that are associated with homocysteine raising effects. Among females, smokers (regardless of MTHFR genotype) appear to be at the highest risk, and to be the most vulnerable to a single factor (i.e. RBC folate) that is associated with homocysteine raising effects.
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Homocisteína/sangue , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Adulto , Eritrócitos/química , Feminino , Ácido Fólico/sangue , Genótipo , Humanos , Masculino , Análise de Regressão , Caracteres Sexuais , Fumar/sangue , Vitamina B 12/sangueRESUMO
BACKGROUND: Women with the AA genotype at the (-2518)A>G promoter polymorphism of CCL-2, which encodes the potent pro-inflammatory chemokine monocyte chemoattractant protein 1 (MCP-1), may be at increased risk for having offspring affected by spina bifida. As the A allele at this locus has been associated with decreased transcription of MCP-1 mRNA relative to the G allele, the observed genetic association suggests that the risk of spina bifida may be increased in the offspring of women with low MCP-1 levels. The present study was undertaken to identify potential determinants of MCP-1 levels in women of reproductive age. METHODS: A small cohort of Caucasian and African-American women of reproductive age was recruited to participate in an exploratory investigation of the determinants of several disease-related, biochemical phenotypes, including MCP-1. Subjects completed a brief questionnaire and provided a fasting blood sample for biochemical and genetic studies. Potential biochemical, genetic, and lifestyle factors were assessed for their association with MCP-1 levels using linear regression analyses. RESULTS: In this cohort, MCP-1 levels were significantly higher in Caucasians as compared to African-Americans. Further, among women of both races, there was evidence that MCP-1 levels were associated with smoking status, MTHFR 677C>T genotype, and red blood cell tetrahydrofolate levels. CONCLUSIONS: The results of these analyses indicate that, if maternal CCL-2 genotype is related to the risk of spina bifida, this relationship is likely to be more complex than initially hypothesized, perhaps depending upon folate intake, MTHFR 677C>T genotype, the distribution of folate derivatives, and immune/inflammatory activity.
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Quimiocina CCL2/sangue , Quimiocina CCL2/genética , Defeitos do Tubo Neural/genética , Adulto , Negro ou Afro-Americano , Estudos de Coortes , Feminino , Genótipo , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Defeitos do Tubo Neural/epidemiologia , Polimorfismo Genético , Fatores de Risco , População BrancaRESUMO
A bifunctional protein consisting of MutS, a mismatch binding protein and a beta-galactosidase reporter domain has been constructed. The fusion of beta-galactosidase to the MutS C-terminus was obtained by cloning the Escherichia coli lacZ gene encoding beta-galactosidase into a plasmid vector carrying the Thermus thermophilus mutS gene. Milligram amounts of this huge chimeric protein (217 kDa monomer) were purified from 1l of overexpressing E. coli cells using metal-chelate affinity chromatography. The mismatch binding properties of the fusion protein were confirmed by DNA mobility shift assay in polyacrylamide gels. Binding to biotinylated mismatched DNA immobilized on streptavidin microplates followed by colorimetric reaction with X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), demonstrated both mismatch recognition and beta-galactosidase activity of the chimeric protein. The activity of beta-galactosidase domain of the fusion was similar to that of the native enzyme. A colorimetric assay for beta-galactosidase activity using X-Gal supplemented with NBT (nitro blue tetrazolium) allowed detection of 50 and 500 fmol of the chimeric protein with naked eye in 45 microl volumes after 120 and 15 min incubation, respectively.