Fernando Augusto de Lima Marson

Saliva as a potential tool for cystic fibrosis diagnosis

BackgroundSaliva and sweat are modified by cystic fibrosis (CF). In both cases the chloride and sodium ion concentrations for healthy subjects and CF patients differ, this representing a possible alternative tool for CF diagnosis. In this context, the aim of this study was to compare the concentrations of these ions in saliva samples taken from CF patients and healthy subjects.MethodsA case–control study was carried out at a university CF center, in which the saliva samples were analyzed on an ABL 835 Radiometer® to determine the ion concentration.ResultsFor the CF patients (n = 80) the values for the biochemical parameters of chloride, potassium and sodium ion concentration were higher (p < 0.009) and the volume and pH of the saliva were lower than in the case of healthy subjects (p < 0.009). For the healthy subjects group (n = 84) versus CF patients, according to the ROC curve, the values for sodium were: cutoff: 13.5 mmol/L, sensitivity: 73.4%, specificity: 70.6%; and for chloride: cutoff: 20 mmol/L, sensitivity: 68.1%, specificity: 72.9%.ConclusionsThe chloride and sodium concentrations in the saliva samples were higher for CF patients in comparison with healthy subjects. Thus, saliva as a tool for CF diagnosis can be considered a new challenge, and a population study including patients in all age classes needs to be performed, in different countries over the world, to extend the database to include a broad spectrum of information in order to identify normal ion concentration ranges for CF patients according to age, genotype and environment.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2614233148750145

Asthma: Gln27Glu and Arg16Gly polymorphisms of the beta2-adrenergic receptor gene as risk factors

BackgroundAsthma is caused by both environmental and genetic factors. The ADRB2 gene, which encodes the beta 2-adrenergic receptor, is one of the most extensively studied genes with respect to asthma prevalence and severity. The Arg16Gly (+46A > G) and Gln27Glu (+79C > G) polymorphisms in the ADRB2 gene cause changes in the amino acids flanking the receptor ligand site, altering the response to bronchodilators and the risk of asthma through complex pathways. The ADRB2 polymorphisms affect beta-adrenergic bronchodilator action and are a tool to identify at-risk populations.ObjectiveTo determine the frequency of these two polymorphisms in allergic asthma patients and healthy subjects and to correlate these data with the occurrence and severity of asthma.MethodsEighty-eight allergic asthma patients and 141 healthy subjects were included in this study. The ADRB2 polymorphisms were analyzed using the amplification-refractory mutation system – polymerase chain reaction (ARMS-PCR) technique. The statistical analysis was performed with the SPSS 21.0 software using the Fisher’s Exact and χ2 tests.ResultsThe ADRB2 polymorphisms were associated with asthma occurrence. The Arg16Arg, Gln27Gln and Gln27Glu genotypes were risk factors; the odds ratios were 6.782 (CI = 3.07 to 16.03), 2.120 (CI = 1.22 to 3.71) and 8.096 (CI = 3.90 to 17.77), respectively. For the Gly16Gly and Glu27Glu genotypes, the odds ratios were 0.312 (CI = 0.17 to 0.56) and 0.084 (CI = 0.04 to 0.17), respectively. The haplotype analysis showed that there were associations between the following groups: Arg16Arg-Gln27Gln (OR = 5.108, CI = 1.82 to 16.37), Gly16Gly-Glu27Glu (OR = 2.816, CI = 1.25 to 6.54), Arg16Gly-Gln27Glu (OR = 0.048, CI = 0.01 to 0.14) and Gly16Gly-Gln27Glu (OR = 0.1036, CI = 0.02 to 0.39). The polymorphism Gln27Glu was associated with asthma severity, as the Gln27Gln genotype was a risk factor for severe asthma (OR = 2.798, CI = 1.099 to 6.674) and the Gln27Glu genotype was a protective factor for mild (OR = 3.063, CI = 1.037 to 9.041) and severe (OR = 0.182, CI = 0.048 to 0.691) asthma.ConclusionsThe Arg16Gly and Gln27Glu polymorphisms in the ADRB2 gene are associated with asthma presence and severity.

Effect of exercise test on pulmonary function of obese adolescents

OBJECTIVE to investigate the pulmonary response to exercise of non-morbidly obese adolescents, considering the gender. METHODS a prospective cross-sectional study was conducted with 92 adolescents (47 obese and 45 eutrophic), divided in four groups according to obesity and gender. Anthropometric parameters, pulmonary function (spirometry and oxygen saturation [SatO2]), heart rate (HR), blood pressure (BP), respiratory rate (RR), and respiratory muscle strength were measured. Pulmonary function parameters were measured before, during, and after the exercise test. RESULTS BP and HR were higher in obese individuals during the exercise test (p = 0.0001). SatO2 values decreased during exercise in obese adolescents (p = 0.0001). Obese males had higher levels of maximum inspiratory and expiratory pressures (p = 0.0002) when compared to obese and eutrophic females. Obese males showed lower values of maximum voluntary ventilation, forced vital capacity, and forced expiratory volume in the first second when compared to eutrophic males, before and after exercise (p = 0.0005). Obese females had greater inspiratory capacity compared to eutrophic females (p = 0.0001). Expiratory reserve volume was lower in obese subjects when compared to controls (p ≤ 0,05). CONCLUSION obese adolescents presented changes in pulmonary function at rest and these changes remained present during exercise. The spirometric and cardiorespiratory values were different in the four study groups. The present data demonstrated that, in spite of differences in lung growth, the model of fat distribution alters pulmonary function differently in obese female and male adolescents.

Genetic interaction of GSH metabolic pathway genes in cystic fibrosis

BackgroundCystic fibrosis (CF) is a monogenic disease caused by CFTR gene mutations, with clinical expression similar to complex disease, influenced by genetic and environmental factors. Among the possible modifier genes, those associated to metabolic pathways of glutathione (GSH) have been considered as potential modulators of CF clinical severity. In this way it is of pivotal importance investigate gene polymorphisms at Glutamate-Cysteine Ligase, Catalytic Subunit (GCLC), Glutathione S-transferase Mu 1 (GSTM1), Glutathione S-transferase Theta 1 (GSTT1), and Glutathione S-transferase P1 (GSTP1), which have been associated to the GSH metabolic pathway and CF clinical severity.MethodA total of 180 CF’s patients were included in this study, which investigated polymorphisms in GCLC and GST genes (GCLC -129C>T and -3506A>G; GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G) by PCR and PCR-RFLP associating to clinical variables of CF severity, including variables of sex, clinical scores [Shwachman-Kulczycki, Kanga e Bhalla (BS)], body mass index, patient age, age for diagnosis, first clinical symptoms, first colonization by Pseudomonas aeruginosa, sputum’s microorganisms, hemoglobin oxygen saturation in the blood, spirometry and comorbidities. The CFTR genotype was investigated in all patients, and the genetic interaction was performed using MDR2.0 and MDRPT0.4.7 software.ResultsThe analysis of multiple genes in metabolic pathways in diseases with variable clinical expression, as CF disease, enables understanding of phenotypic diversity. Our data show evidence of interaction between the GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G polymorphism with CFTR gene mutation classes, and BS (Balance testing accuracy= 0.6824, p= 0.008), which measures the commitment of bronchopulmonary segments by tomography.ConclusionPolymorphisms in genes associated with metabolism of GSH act on the CF’s severity.

CFTR genotype and clinical outcomes of adult patients carried as cystic fibrosis disease

BACKGROUND There are nearly 2000 cystic fibrosis transmembrane regulator (CFTR) mutations that cause cystic fibrosis (CF). These mutations are classified into six classes; on the one hand, the first three classes cause severe disease involvement in early childhood, on the other hand, the Class IV, V and VI mutations cause minor severe disease in the same age. Nowadays, with therapeutic advances in CF management and competence of pediatricians, physicians of adults have to deal with two groups of CF patients: (i) adults diagnosed in childhood with severe mutations and (ii) adults who initiated symptoms in adulthood and with Class IV, V and VI mutations. The aim of this study was to analyze adults from a clinical center, treated as CF disease, screening the CFTR genotype and evaluating the clinical characteristics. METHODS Thirty patients followed as CF disease at the University Hospital were enrolled. After a complete molecular CFTR negative screening and sweat test levels between 40 and 59mEq/L, five patients were characterized as non-CF disease and were excluded. Molecular screening was performed by CFTR gene sequencing/MLPA or by specific mutation screening. Clinical data was obtained from medical records. The patients were divided into three groups: (1) patients with Class I, II and III mutations in two CFTR alleles; (2) genotype with at least one allele of Class IV, V or VI CFTR mutations and, (3) non-identified CFTR mutation+one patient with one allele with CFTR mutation screened (Class I). RESULTS There was an association of CFTR class mutation and sodium/chloride concentration in the sweat test (sodium: p=0.040; chloride: p=0.016), onset of digestive symptoms (p=0.012), lung function parameter (SpO2 - p=0.016), Bhalla score (p=0.021), age at diagnosis (p=0.008) and CF-related diabetes (p=0.029). There was an association between Pseudomonas aeruginosa chronic colonization (as clinical marker for the lung disease status) and lung impairment (FEV1% - p=0.027; Bhalla score - p=0.021), CF-related diabetes (p=0.040), chloride concentration in the sweat test (p=0.040) and chronic infection by microorganisms (Staphylococcus aureus - p=0.039; mucoid P. aeruginosa - p=0.001). There is no positive association with the status of other clinical markers and the CFTR genotype groups. For clinical association with pancreatic insufficiency (as clinical marker for digestive symptoms), no association was related. CONCLUSION The adults with CF diagnosed by sweat test have specific clinical and genotypic characteristics, being a population that should be studied to cause better future management. Some patients treated as CF disease by clinical symptoms, showed no disease, taking into account the sweat test and complete exon sequencing/MLPA screening.

Mutações do gene cystic fibrosis transmembrane conductance regulator e deleções dos genes glutationa S-transferase em pacientes com fibrose cística no Brasil

OBJECTIVE To determine the effects that mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and deletion of the glutathione S-transferase (GST) genes mu-1 (GSTM1) and theta-1 (GSTT1) have on the clinical course of cystic fibrosis (CF) in patients residing in the southeastern region of Brazil. METHODS The study sample consisted of all consecutive CF patients treated at the Hospital de Clínicas School of Medical Sciences of the State University at Campinas between March of 2002 and March of 2005. We included 66 CF patients. Genomic DNA was analyzed by polymerase chain reaction and restriction endonuclease digestion for the identification of the genotypes. RESULTS The DF508 mutation of the CFTR gene was found in 44 patients (66.7%). The null genotypes GSTM1, GSTT1 and GSTM1/GSTT1 were found in 40.9%, 15.2%, and 3.0% of the patients, respectively. The DF508 CFTR mutation was more common in patients diagnosed with CF before 2.5 years of age than in those diagnosed later (75.5% vs. 41.2%; p = 0.008). The frequency of the DF508 CFTR mutation, as well as of the GSTM1 and GSTT1 genotypes, was not found to be associated with gender, ethnicity, pulmonary disease status, or pancreatic disease status. CONCLUSIONS When the patients were stratified by clinical and epidemiological features, the frequencies of the GSTM1 and GSTT1 null genotypes were similar, suggesting that the inherited absence of these enzymatic pathways does not alter the course of CF. However, the high frequency of the DF508 CFTR mutation found in younger children suggests that it influences the age at diagnosis of CF in this region of Brazil.

Association between the IVS4G>T mutation in the TCF7L2 gene and susceptibility to diabetes in cystic fibrosis patients

BackgroundClinical complications appear to be a decisive factor for the prognosis of patients. Diabetes is an important complication of cystic fibrosis(CF). In our study we evaluated the association between the IVS4G>T mutation in the TCF7L2 gene with the presence of diabetes in patients with CF.FindingsWe evaluated 145 patients with CF in relation to the genotype of the IVS4G>T mutation. For this, the PCR method associated with specific enzyme digestion was used. The genotypes G/G, G/T and T/T were observed to have frequencies of 54 (37.2%), 78 (53.8%) and 13 (9%), respectively. There was no association between genotype and the occurrence of diabetes among patients.ConclusionsIn our sample, no association was found between the IVS4G>T mutation in the TCF7L2 gene and diabetes.

IL8 gene as modifier of cystic fibrosis: unraveling the factors which influence clinical variability

The severity of cystic fibrosis (CF) is associated with classes of mutations in the CFTR gene (cystic fibrosis transmembrane regulator), physical environment and modifier genes interaction. The IL8 gene (interleukin 8), according to its respective polymorphisms, influences inflammatory responses. This study analyzed IL8 gene polymorphisms (rs4073, rs2227306 and rs2227307), by means of PCR/RFLP, and their association with pulmonary function markers and clinical severity scores in 186 patients with CF, considering the CFTR genotype. There was an association between rs2227307 and precocity of the disease. The severity of lung disease was associated with the following markers: transcutaneous arterial hemoglobin oxygen saturation (SaO2) (regardless of CFTR genotype, for the polymorphisms rs4073, rs2227306 and rs2227307); mucoid Pseudomonas aeruginosa (regardless of CFTR genotype, for the polymorphisms rs2227306 and rs2227307). Pulmonary function markers (SaO2 and spirometric variables) and clinical severity scores were also associated with IL8 gene polymorphisms. This study identified the IL8 gene, represented by rs4073 and rs2227306 polymorphisms, and particularly the rs2227307 polymorphism, as potentiating factors for the degree of variability in the severity of CF, especially in pulmonary clinical manifestation correlated with increased morbidity and mortality.

Association between genetic polymorphisms in DNA mismatch repair-related genes with risk and prognosis of head and neck squamous cell carcinoma

We examined the influence of MLH1 c.−93G>A, MSH2 c.211 + 9C>G, MSH3 c.3133G>A and EXO1 c.1765G>A polymorphisms, involved in DNA mismatch repair (MMR), on head and neck (HN) squamous cell carcinoma (SCC) risk and prognosis. Aiming to identify genotypes, DNA from 450 HNSCC patients and 450 controls was analyzed by PCR‐RFLP or real time PCR. MSH2 GG plus MSH3 GG (31.7% vs. 18.7%, p = 0.003) genotypes were higher in laryngeal SCC (LSCC) patients than in controls. Carriers of the respective combined genotype were under a 3.69 (95% CI: 1.54–8.81)‐fold increased risk of LSCC. Interactions of tobacco and tobacco plus all the above‐mentioned polymorphisms on HNSCC and LSCC risk were also evident in study (p = 0.001). At 60 months of follow‐up, relapse‐free survival (RFS) was shorter in patients with EXO1 GG genotype (54.8% vs. 61.1%, p = 0.03) and overall survival (OS) was shorter in patients with MSH3 GG genotype (42.8% vs. 52.5%, p = 0.02) compared to those with other genotypes, respectively. After multivariate Cox analysis, patients with EXO1 GG and MSH3 GG genotypes had worst RFS (HR: 1.50, 95% CI: 1.03–2.20, p = 0.03) and OS (HR: 1.59, 95% CI: 1.19–2.13, P = 0.002) than those with the remaining genotypes, respectively. Our data present, for the first time, evidence that inherited MLH1 c.‐93G>A, MSH2 c.211 + 9C>G, MSH3 c.3133G>A, and EXO1 c.1765G>A abnormalities of DNA MMR pathway are important determinants of HNSCC, particularly among smokers, and predictors of patient outcomes.

Association of clinical severity of cystic fibrosis with variants in the SLC gene family (SLC6A14, SLC26A9, SLC11A1 and SLC9A3)

INTRODUCTION Cystic fibrosis (CF) manifests with clinical and histopathological variability depending on environmental and genetic factors. Moreover, the genes encoding ion channels[rs3788766(SLC6A14), rs7512462(SLC26A9), rs17235416(SLC11A1) and rs17563161(SLC9A3)] have been insufficiently studied as modifier genes. Then, our objective was associate the variants in the genes of SLC family with 43 CF severity markers. METHODS The variants were identified by real-time-PCR in 188 CF patients considering the CFTR genotype. Statistical analyses were performed by parametric and nonparametric tests. The correction by multiple testing was performed by the False Rate Discovery test, alpha=0.05. RESULTS Depending on the CFTR mutations, we found association of: (i) rs3788766*CC with mucoid Pseudomonas aeruginosa (OR=0.171; 95%CI=0.029-0.696), non-mucoid P. aeruginosa (OR=0.283; 95%CI=0.094-0.853) and Staphyloccocus aureus (OR=4.443; 95%CI=1.019-40.64), largest FEFmax(p=0.041) and best response to bronchodilator for FEF50%(p=0.033) and FEV1/FVC(p=0.044); (ii) rs3788766*CT with early start of pulmonary symptom (OR=3.524; 95%CI=1.229-10.1) and osteoporosis (OR=0.203; 95%CI=0.022-0.883); (iii) rs3788766*TT with lowest body mass index (OR=4.242; 95%CI=1.505-11.95), presence of mucoid P. aeruginosa (OR=3.176; 95%CI=1.29-7.819) and S. aureus (OR=0.116; 95%CI=0.004-0.881), highest Bhalla score (p=0.047) and lowest FEFmax(p=0.028) and FEF25%(p=0.031) values; (iv) rs7512462*CC with highest Shwachman-Kulczycki score (p=0.019), FVC(p=0.043), FEV1(p=0.047), FEV1/FVC(p=0.022), FEF50%(p=0.038) and FEF25-75%(p=0.016); (v) rs7512462*CT with lowest values of FVC(p=0.034), FEV1(p=0.047), FEV1/FVC(p=0.022), FEF25%(p=0.012), FEF50%(p=0.038), FEF75%(p=0.008), FEF25-75%(p=0.016) and ERV(p=0.023); (vi) rs7512462*TT with best response to the inhaled bronchodilator for FEV1(p=0.011), FEF50%(p=0.019), FEF75%(p=0.036) and FEF25-75%(p=0.008); (vii) rs17234516*Normal allele with lowest value of SaO2 (p=0.010) and S. aureus (OR=3.333; 95%CI=1.085-10.24); (viii) rs17563161*GG with lowest age for onset of digestive symptoms (OR=2.564; 95%CI=1.234-5.33). CONCLUSIONS The clinical and laboratory variability of CF were associated with the variants in the genes of SLC family in our sample.