Relationship of autonomic imbalance and circadian disruption with obesity and type 2 diabetes in resistant hypertensive patients

Background: Hypertension, diabetes and obesity are not isolated findings, but a series of interacting interactive physiologic derangements. Taking into account genetic background and lifestyle behavior, AI (autonomic imbalance) could be a common root for RHTN (resistant hypertension) or RHTN plus type 2 diabetes (T2D) comorbidity development. Moreover, circadian disruption can lead to metabolic and vasomotor impairments such as obesity, insulin resistance and resistant hypertension. In order to better understand the triggered emergence of obesity and T2D comorbidity in resistant hypertension, we investigated the pattern of autonomic activity in the circadian rhythm in RHTN with and without type 2 diabetes (T2D), and its relationship with serum adiponectin concentration. Methods: Twenty five RHTN patients (15 non-T2D and 10 T2D, 15 males, 10 females; age range 34 to 70 years) were evaluated using the following parameters: BMI (body mass index), biochemical analysis, serum adiponectinemia, echocardiogram and ambulatory electrocardiograph heart rate variability (HRV) in time and frequency domains stratified into three periods: 24 hour, day time and night time. Results: Both groups demonstrated similar characteristics despite of the laboratory analysis concerning T2D like fasting glucose, HbA1c levels and hypertriglyceridemia. Both groups also revealed disruption of the circadian rhythm: inverted sympathetic and parasympathetic tones during day (parasympathetic > sympathetic tone) and night periods (sympathetic > parasympathetic tone). T2D group had increased BMI and serum triglyceride levels (mean 33.7 +/- 4.0 vs 26.6 +/- 3.7 kg/m(2) - p = 0.00; 254.8 +/- 226.4 vs 108.6 +/- 48.7 mg/dL - p = 0.04), lower levels of adiponectin (6729.7 +/- 3381.5 vs 10911.5 +/- 5554.0 ng/mL - p = 0.04) and greater autonomic imbalance evaluated by HRV parameters in time domain compared to non-T2D RHTN patients. Total patients had HRV correlated positively with serum adiponectin (r = 0.37 [95% CI - 0.04 - 1.00] p = 0.03), negatively with HbA1c levels (r = -0.58 [95% CI -1.00 - -0.3] p = 0.00) and also adiponectin correlated negatively with HbA1c levels (r = -0.40 [95% CI -1.00 - -0.07] p = 0.02). Conclusion: Type 2 diabetes comorbidity is associated with greater autonomic imbalance, lower adiponectin levels and greater BMI in RHTN patients. Similar circadian disruption was also found in both groups indicating the importance of lifestyle behavior in the genesis of RHTN.

Reduced transcription of TCOF1 in adult cells of Treacher Collins syndrome patients

Background: Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder caused by frameshift deletions or duplications in the TCOF1 gene. These mutations cause premature termination codons, which are predicted to lead to mRNA degradation by nonsense mediated mRNA decay (NMD). Haploinsufficiency of the gene product (treacle) during embryonic development is the proposed molecular mechanism underlying TCS. However, it is still unknown if TCOF1 expression levels are decreased in postembryonic human cells. Methods: We have estimated TCOF1 transcript levels through real time PCR in mRNA obtained from leucocytes and mesenchymal cells of TCS patients (n = 23) and controls (n = 18). Mutational screening and analysis of NMD were performed by direct sequencing of gDNA and cDNA, respectively. Results: All the 23 patients had typical clinical features of the syndrome and pathogenic mutations were detected in 19 of them. We demonstrated that the expression level of TCOF1 is 18-31% lower in patients than in controls (p < 0.05), even if we exclude the patients in whom we did not detect the pathogenic mutation. We also observed that the mutant allele is usually less abundant than the wild type one in mesenchymal cells. Conclusions: This is the first study to report decreased expression levels of TCOF1 in TCS adult human cells, but it is still unknown if this finding is associated to any phenotype in adulthood. In addition, as we demonstrated that alleles harboring the pathogenic mutations have lower expression, we herein corroborate the current hypothesis of NMD of the mutant transcript as the explanation for diminished levels of TCOF1 expression. Further, considering that TCOF1 deficiency in adult cells could be associated to pathologic clinical findings, it will be important to verify if TCS patients have an impairment in adult stem cell properties, as this can reduce the efficiency of plastic surgery results during rehabilitation of these patients.

A novel mutation in the glycogen synthase 2 gene in a child with glycogen storage disease type 0

Background: Glycogen storage disease type 0 is an autosomal recessive disease presenting in infancy or early childhood and characterized by ketotic hypoglycemia after prolonged fasting and postprandial hyperglycemia and hyperlactatemia. Sixteen different mutations have been identified to date in the gene which encodes hepatic glycogen synthase, resulting in reduction of glycogen storage in the liver. Case Presentation: Biochemical evaluation as well as direct sequencing of exons and exon-intron boundary regions of the GYS2 gene were performed in a patient presenting fasting hypoglycemia and postprandial hyperglycemia and her parents. The patient was found to be compound heterozygous for one previously reported nonsense mutation (c. 736 C>T; R243X) and a novel frameshift mutation (966_967delGA/insC) which introduces a stop codon 21 aminoacids downstream from the site of the mutation that presumably leads to loss of 51% of the COOH-terminal part of the protein. The glycemia and lactatemia of the parents after an oral glucose tolerance test were evaluated to investigate a possible impact of the carrier status on the metabolic profile. The mother, who presented a positive family history of type 2 diabetes, was classified as glucose intolerant and the father, who did not exhibit metabolic changes after the glucose overload, had an antecedent history of hypoglycemia after moderate alcohol ingestion. Conclusion: The current results expand the spectrum of known mutations in GYS2 and suggest that haploinsufficiency could explain metabolic abnormalities in heterozygous carriers in presence of predisposing conditions.

Phenotypic variation among four family members with congenital hypothyroidism caused by two distinct thyroglobulin gene mutations

Background: Thyroglobulin (Tg) is a large glycoprotein that is intimately involved in the biosynthesis of thyroxine and triiodothyronine. At least 38 mutations have been described in the Tg gene that are associated with varying degrees of hypothyroidism. We studied the Tg gene in four related subjects with congenital hypothyroidism. Summary: We found a novel compound heterozygous constellation (IVS30 + 1G>T/A2215D) in a brother and sister and one previously described related mutation (IVS30+1G>T) in their two sibling second degree cousins. The brother with the IVS30 + 1G>T/A2215D mutation and the two siblings with the IVS30+1G>T mutation had fetal or neonatal goiter and all had hypothyroidism. Conclusions: This study further confirms the association of the IVS30+1G>T mutation of the Tg gene with hypothyroidism. Computer analysis predicts that the A2215D mutation, first reported here, should cause structural instability of Tg but when present as a compound heterozygous mutation with IVS30+G>T/A its effect is unclear but is likely to be influenced by iodine intake.

The C242T polymorphism of the p22-phox gene (CYBA) is associated with higher left ventricular mass in Brazilian hypertensive patients

Background: Reactive oxygen species have been implicated in the physiopathogenesis of hypertensive end-organ damage. This study investigated the impact of the C242T polymorphism of the p22-phox gene (CYBA) on left ventricular structure in Brazilian hypertensive subjects. Methods: We cross-sectionally evaluated 561 patients from 2 independent centers [Campinas (n = 441) and Vitoria (n = 120)] by clinical history, physical examination, anthropometry, analysis of metabolic and echocardiography parameters as well as p22-phox C242T polymorphism genotyping. In addition, NADPH-oxidase activity was quantified in peripheral mononuclear cells from a subgroup of Campinas sample. Results: Genotype frequencies in both samples were consistent with the Hardy-Weinberg equilibrium. Subjects with the T allele presented higher left ventricular mass/height(2.7) than those carrying the CC genotype in Campinas (76.8 +/- 1.6 vs 70.9 +/- 1.4 g/m(2.7); p = 0.009), and in Vitoria (45.6 +/- 1.9 vs 39.9 +/- 1.4 g/m(2.7); p = 0.023) samples. These results were confirmed by stepwise regression analyses adjusted for age, gender, blood pressure, metabolic variables and use of anti-hypertensive medications. In addition, increased NADPH-oxidase activity was detected in peripheral mononuclear cells from T allele carriers compared with CC genotype carriers (p = 0.03). Conclusions: The T allele of the p22-phox C242T polymorphism is associated with higher left ventricular mass/height(2.7) and increased NADPH-oxidase activity in Brazilian hypertensive patients. These data suggest that genetic variation within NADPH-oxidase components may modulate left ventricular remodeling in subjects with systemic hypertension.

CYP2C19 and ABCB1 gene polymorphisms are differently distributed according to ethnicity in the Brazilian general population

Background: Recent studies have reported the clinical importance of CYP2C19 and ABCB1 polymorphisms in an individualized approach to clopidogrel treatment. The aims of this study were to evaluate the frequencies of CYP2C19 and ABCB1 polymorphisms and to identify the clopidogrel-predicted metabolic phenotypes according to ethnic groups in a sample of individuals representative of a highly admixtured population. Methods: One hundred and eighty-three Amerindians and 1,029 subjects of the general population of 4 regions of the country were included. Genotypes for the ABCB1c.C3435T (rs1045642), CYP2C19*2 (rs4244285), CYP2C19*3 (rs4986893), CYP2C19*4 (rs28399504), CYP2C19*5 (rs56337013), and CYP2C19*17 (rs12248560) polymorphisms were detected by polymerase chain reaction followed by high resolution melting analysis. The CYP2C19*3, CYP2C19*4 and CYP2C19*5 variants were genotyped in a subsample of subjects (300 samples randomly selected). Results: The CYP2C19*3 and CYP2C19*5 variant alleles were not detected and the CYP2C19*4 variant allele presented a frequency of 0.3%. The allelic frequencies for the ABCB1c.C3435T, CYP2C19*2 and CYP2C19*17 polymorphisms were differently distributed according to ethnicity: Amerindian (51.4%, 10.4%, 15.8%); Caucasian descent (43.2%, 16.9%, 18.0%); Mulatto (35.9%, 16.5%, 21.3%); and African descent (32.8%, 20.2%, 26.3%) individuals, respectively. As a result, self-referred ethnicity was able to predict significantly different clopidogrel-predicted metabolic phenotypes prevalence even for a highly admixtured population. Conclusion: Our findings indicate the existence of inter-ethnic differences in the ABCB1 and CYP2C19 variant allele frequencies in the Brazilian general population plus Amerindians. This information could help in stratifying individuals from this population regarding clopidogrel-predicted metabolic phenotypes and design more cost-effective programs towards individualization of clopidogrel therapy.

Evaluating gene by sex and age interactions on cardiovascular risk factors in Brazilian families

Background: In family studies, it is important to evaluate the impact of genes and environmental factors on traits of interest. In particular, the relative influences of both genes and the environment may vary in different strata of the population of interest, such as young and old individuals, or males and females. Methods: In this paper, extensions of the variance components model are used to evaluate heterogeneity in the genetic and environmental variance components due to the effects of sex and age (the cutoff between young and old was 43 yrs). The data analyzed were from 81 Brazilian families (1,675 individuals) of the Baependi Family Heart Study. Results: The models allowing for heterogeneity of variance components by sex suggest that genetic and environmental variances are not different in males and females for diastolic blood pressure, LDL-cholesterol, and HDL-cholesterol, independent of the covariates included in the models. However, for systolic blood pressure, fasting glucose and triglycerides, the evidence for heterogeneity was dependent on the covariates in the model. For instance, in the presence of sex and age covariates, heterogeneity in the genetic variance component was suggested for fasting glucose. But, for systolic blood pressure, there was no evidence of heterogeneity in any of the two variance components. Except for the LDL-cholesterol, models allowing for heterogeneity by age provide evidence of heterogeneity in genetic variance for triglycerides and systolic and diastolic blood pressure. There was evidence of heterogeneity in environmental variance in fasting glucose and HDL-cholesterol. Conclusions: Our results suggest that heterogeneity in trait variances should not be ignored in the design and analyses of gene-finding studies involving these traits, as it may generate additional information about gene effects, and allow the investigation of more sophisticated models such as the model including sex-specific oligogenic variance components.

249 TP53 mutation has high prevalence and is correlated with larger and poorly differentiated HCC in Brazilian patients

Background: Ser-249 TP53 mutation (249(Ser)) is a molecular evidence for aflatoxin-related carcinogenesis in Hepatocellular Carcinoma (HCC) and it is frequent in some African and Asian regions, but it is unusual in Western countries. HBV has been claimed to add a synergic effect on genesis of this particular mutation with aflatoxin. The aim of this study was to investigate the frequency of 249Ser mutation in HCC from patients in Brazil. Methods: We studied 74 HCC formalin fixed paraffin blocks samples of patients whom underwent surgical resection in Brazil. 249Ser mutation was analyzed by RFLP and DNA sequencing. HBV DNA presence was determined by Real-Time PCR. Results: 249Ser mutation was found in 21/74 (28%) samples while HBV DNA was detected in 13/74 (16%). 249Ser mutation was detected in 21/74 samples by RFLP assay, of which 14 were confirmed by 249Ser mutant-specific PCR, and 12 by nucleic acid sequencing. All HCC cases with p53-249ser mutation displayed also wild-type p53 sequences. Poorly differentiated HCC was more likely to have 249Ser mutation (OR = 2.415, 95% CI = 1.001 - 5.824, p = 0.05). The mean size of 249Ser HCC tumor was 9.4 cm versus 5.5 cm on wild type HCC (p = 0.012). HBV DNA detection was not related to 249Ser mutation. Conclusion: Our results indicate that 249Ser mutation is a HCC important factor of carcinogenesis in Brazil and it is associated to large and poorly differentiated tumors.

Genetic Diversity on the Integrase Region of the pol Gene among HIV Type 1-Infected Patients Naive for Integrase Inhibitors in Sao Paulo City, Brazil

The presence of mutations associated with integrase inhibitor (INI) resistance among INI-naive patients may play an important clinical role in the use of those drugs Samples from 76 HIV-1-infected subjects naive to INIs were submitted to direct sequencing. No differences were found between naive (25%) subjects and subjects on HAART (75%). No primary mutation associated with raltegravir or elvitegravir resistance was found. However, 78% of sequences showed at least one accessory mutation associated with resistance. The analysis of the 76 IN sequences showed a high polymorphic level on this region among Brazilian HIV-1-infected subjects, including a high prevalence of aa substitutions related to INI resistance. The impact of these findings remains unclear and further studies are necessary to address these questions.

Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

Background: The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGFbeta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods: Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V ( 25 mu g/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05. Results: IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 +/- 0.118 vs. 0.874 +/- 0.282, p < 0.001), bronchioles (0.294 +/- 0.139 vs. 0.646 +/- 0.172, p < 0.001) and in the septal interstitium (0.027 +/- 0.014 vs. 0.067 +/- 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 +/- 0.07 vs. 1.0 +/- 0.528, p = 0.002) and V (1.12 +/- 0.42 vs. 4.74 +/- 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression ( p < 0.0001). Conclusions: Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 mu g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c(+), CD11b(+) and CD19(+) cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

rst Transcriptional Activity Influences kirre mRNA Concentration in the Drosophila Pupal Retina during the Final Steps of Ommatidial Patterning

Background: Drosophila retinal architecture is laid down between 24-48 hours after puparium formation, when some of the still uncommitted interommatidial cells (IOCs) are recruited to become secondary and tertiary pigment cells while the remaining ones undergo apoptosis. This choice between survival and death requires the product of the roughest (rst) gene, an immunoglobulin superfamily transmembrane glycoprotein involved in a wide range of developmental processes. Both temporal misexpression of Rst and truncation of the protein intracytoplasmic domain, lead to severe defects in which IOCs either remain mostly undifferentiated and die late and erratically or, instead, differentiate into extra pigment cells. Intriguingly, mutants not expressing wild type protein often have normal or very mild rough eyes. Methodology/Principal Findings: By using quantitative real time PCR to examine rst transcriptional dynamics in the pupal retina, both in wild type and mutant alleles we showed that tightly regulated temporal changes in rst transcriptional rate underlie its proper function during the final steps of eye patterning. Furthermore we demonstrated that the unexpected wild type eye phenotype of mutants with low or no rst expression correlates with an upregulation in the mRNA levels of the rst paralogue kin-of-irre (kirre), which seems able to substitute for rst function in this process, similarly to their role in myoblast fusion. This compensatory upregulation of kirre mRNA levels could be directly induced in wild type pupa upon RNAi-mediated silencing of rst, indicating that expression of both genes is also coordinately regulated in physiological conditions. Conclusions/Significance: These findings suggest a general mechanism by which rst and kirre expression could be fine tuned to optimize their redundant roles during development and provide a clearer picture of how the specification of survival and apoptotic fates by differential cell adhesion during the final steps of retinal morphogenesis in insects are controlled at the transcriptional level.

Differential gene expression profiling in mucus glands of honey bee (Apis mellifera) drones during sexual maturation

The mating sign that each drone leaves when mating with a queen essentially consists of mucus gland proteins. We employed a Representational Difference Analysis (RDA) methodology to identify genes that are differentially expressed in mucus glands during sexual maturation of drones. The RDA library for mucus glands of newly emerged drones was more complex than that of 8 day-old drones, with matches to 20 predicted genes. Another 26 reads matched to the Apis genome but not to any predicted gene. Since these ESTs were located within ORFs they may represent novel honey bee genes, possibly fast evolving mucus gland proteins. In the RDA library for mucus glands of 8 day-old drones, most reads corresponded to a capsid protein of deformed wing virus, indicating high viral loads in these glands. The expression of two genes encoding venom allergens, acid phosphatase-1 and hyaluronidase, in drone mucus glands argues for their homology with the female venom glands, both associated with the reproductive system.

A score system for quality evaluation of RNA sequence tags: an improvement for gene expression profiling

Background: High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) or Sequencing-by-Synthesis (SBS) represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. Results: This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. Conclusion: These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at http://gdm.fmrp.usp.br/s3t/.S3T source code and datasets can also be downloaded from the aforementioned website.

The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n=18) presented lower levels of CEBPA expression compared to healthy controls (n=5), but higher levels than those in acute myeloid leukemia with t(8;21) (n=9) and with inv(16) (n=5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved.