Antonio Fernando Ribeiro

Fibrose cística em um centro de referência no Brasil: características clínicas e laboratoriais de 104 pacientes e sua associação com o genótipo e a gravidade da doença

OBJECTIVE To identify the clinical, laboratory and radiographic characteristics of the cystic fibrosis patients under care at Universidade Estadual de Campinas (UNICAMP) in the last decade of the twentieth century, and to investigate the association of these characteristics with genotype and severity of the disease as measured by the Shwachman score. METHODS Descriptive, retrospective and cross-sectional study of the patients assisted at UNICAMP hospitals Cystic Fibrosis Clinic from July 1990 to July 2000. RESULTS One hundred and four patients were studied; 53.8% male; 93.3% Caucasian; 89.4% presented with respiratory symptoms; 59.6% presented with digestive symptoms; 5.8% had meconium ileus; 4.8% had diabetes. The mean age at onset of symptoms was 3 months, and the mean age at diagnosis was 2 years and 4 months. At diagnosis, 69.9 and 56.6% of the patients had weight and height below 10th percentile, respectively; in 10.6%, sweat chloride was < 60 mEq/l. Staphylococcus aureus was found in 80.2%, Pseudomonas aeruginosa in 76%, and Burkholderia cepacia in 5.2%. DeltaF508 homozygosis was observed in 18.75%, whereas 62.50% of the patients were DeltaF508 heterozygous. A moderate/severe Shwachman score was found in 15.7%. Eighteen patients died in that period (17.3%). The mean age at death was 7 years and 8 months; median survival after diagnosis was 18 years and 4 months. Patients who have at least one DeltaF508 mutation have more frequent alterations in fecal fat levels when compared to patients who do not have this mutation (p < 0.05). There were no differences in any parameter between DeltaF508 homozygous and heterozygous patients. CONCLUSIONS The clinical and laboratory characteristics of the 104 patients studied were similar to the characteristics described for patients in other countries. Exceptions are the higher age at diagnosis and lower survival. Our results support the recommendation for early diagnosis and the need for more treatment opportunities in the population of cystic fibrosis patients.OBJECTIVE: To identify the clinical, laboratory and radiographic characteristics of the cystic fibrosis patients under care at Universidade Estadual de Campinas (UNICAMP) in the last decade of the twentieth century, and to investigate the association of these characteristics with genotype and severity of the disease as measured by the Shwachman score. METHODS: Descriptive, retrospective and cross-sectional study of the patients assisted at UNICAMP hospitals Cystic Fibrosis Clinic from July 1990 to July 2000. RESULTS: One hundred and four patients were studied; 53.8% male; 93.3% Caucasian; 89.4% presented with respiratory symptoms; 59.6% presented with digestive symptoms; 5.8% had meconium ileus ; 4.8% had diabetes. The mean age at onset of symptoms was 3 months, and the mean age at diagnosis was 2 years and 4 months. At diagnosis, 69.9 and 56.6% of the patients had weight and height below 10th percentile, respectively; in 10.6%, sweat chloride was < 60 mEq/l. Staphylococcus aureus was found in 80.2%, Pseudomonas aeruginosa in 76.0%, and Burkholderia cepacia in 5.2%. DF508 homozygosis was observed in 18.75%, whereas 62.50% of the patients were DF508 heterozygous. A moderate/severe Shwachman score was found in 15.7%. Eighteen patients died in that period (17.3%). The mean age at death was 7 years and 8 months; median survival after diagnosis was 18 years and 4 months. Patients who have at least one DF508 mutation have more frequent alterations in fecal fat levels when compared to patients who do not have this mutation (p < 0.05). There were no differences in any parameter between DF508 homozygous and heterozygous patients. CONCLUSIONS: The clinical and laboratory characteristics of the 104 patients studied were similar to the characteristics described for patients in other countries. Exceptions are the higher age at diagnosis and lower survival. Our results support the recommendation for early diagnosis and the need for more treatment opportunities in the population of cystic fibrosis patients.

Controvérsias na fibrose cística: do pediatra ao especialista

Objetivos: nos ultimos 70 anos, a fibrose cistica emergiu da obscuridade para o reconhecimento como a mais importante doenca hereditaria, potencialmente letal, incidente na raca branca. Embora seja uma doenca genetica, na qual o defeito basico acomete celulas de varios orgaos, nem todos os individuos expressam respostas clinicas na mesma intensidade. Varias manifestacoes clinicas, principalmente pulmonares e digestivas, podem ocorrer durante a vida dos pacientes fibrocisticos. O objetivo deste artigo e propiciar ao pediatra geral uma visao atualizada dos principais assuntos referentes a fibrose cistica. Fontes dos dados: revisao sistematica e atualizada em fonte de dados oficial (Medline). Sintese dos dados: foram revisados 79 artigos sobre fibrose cistica, de periodicos internacionais, colocando, de modo atual e critico, os principais eventos relacionados com a incidencia, a fisiopatogenia, as manifestacoes clinicas, o diagnostico e o tratamento da fibrose cistica. Conclusoes: apesar de nao existir cura para essa doenca, muitos conhecimentos novos sobre a etiologia e a fisiopatologia, adquiridos nas duas ultimas decadas, propiciaram uma nova abordagem para o tratamento da fibrose cistica. A compreensao dos mecanismos basicos da doenca pulmonar, bem como das manifestacoes digestivas na fibrose cistica, decorrente dos conhecimentos de pesquisas recentes, tem sido a chave para o aumento da sobrevida e a melhora da qualidade de vida dos pacientes.

Measurements of CFTR-Mediated Cl- Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis

Background Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl−) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. Methodology/Principal Findings To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl− secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n = 51), individuals with clinical CF suspicion (n = 49) and age-matched non-CF controls (n = 18). Conclusive measurements were obtained for 96% of cases. Patients with “Classic CF”, presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl− secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl− secretion (10–57%) and non-CF controls show CFTR-mediated Cl− secretion ≥30–35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in “CF suspicion” individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl− secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. Conclusions/Significance Determination of CFTR-mediated Cl− secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.

Polymorphisms in ADRB2 gene can modulate the response to bronchodilators and the severity of cystic fibrosis

BackgroundThe most common cystic fibrosis (CF) manifestation is the progressive chronic obstructive pulmonary disease caused by deficiency, dysfunction, or absence of the CFTR (Cystic Fibrosis Transmembrane Regulator) protein on the apical surface of the cells in the respiratory tract. The use of bronchodilators (BD), and inhaled corticosteroids (IC) have been suggested for the management of airway inflammation in CF. The effectiveness of BD and IC have been verified, proven in laboratory and in the clinical treatment for asthma patients. However, in CF, the effectiveness of these drugs is controversial. The extent of asthma’s response to BD depends on the presence of polymorphisms in the ADRB2 gene. In contrast, in CF, little is known about the response to the BD and the association of CF´s severity with the different polymorphisms in ADRB2 gene. In this context, our objective was to verify whether the Arg16Gly and Glu27Gln polymorphisms in ADRB2 gene are associated with severity and with the bronchodilator response in CF patients.MethodCross-sectional study of 122 CF patients subjected to analysis of mutations in the CFTR gene, polymorphisms in ADRB2 gene, along with clinical and laboratorial characteristics of severity.ResultThe Arg16Gly polymorphism in ADRB2 gene was associated with pancreatic insufficiency(p:0.009), Bhalla score(p:0.039), forced expiratory volume in the first second[FEV1(%)](p:0.003), forced expiratory flow between 25 and 75% of the forced vital capacity-FVC[FEF25-75(%)](p:0.008) and lower age at the first isolation of the Pseudomonas aeruginosa(p:0.012). The response to the BD spirometry was associated with clinical severity markers, FEV1(%)(p:0.011) and FEF25-75(%)(p:0.019), for the Arg16Gly polymorphism in the ADRB2 gene. The haplotype analysis showed association with the FEV1/FVC marker from the spirometry test, before and after using the BD, with higher values in the group with Gly/Gly and Glu/Glu, respectively, for the Arg16Gly and Gln27Glu polymorphisms. The analysis by MDR2.0 software, showed association with FEF25-75%; the response to Arg16Gly was respondent by 17.35% and Gln27Glu by 6.8% in variation found.ConclusionThere was an association between the Arg16Gly and Gln27Glu polymorphisms in ADRB2 gene with CF´s severity and bronchodilator response.

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

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.

The ACE gene D/I polymorphism as a modulator of severity of cystic fibrosis

BackgroundCystic Fibrosis (CF) is a monogenic disease with complex expression because of the action of genetic and environmental factors. We investigated whether the ACE gene D/I polymorphism is associated with severity of CF.MethodsA cross-sectional study was performed, from 2009 to 2011, at University of Campinas – UNICAMP. We analyzed 180 patients for the most frequent mutations in the CFTR gene, presence of the ACE gene D/I polymorphism and clinical characteristics of CF.ResultsThere was an association of the D/D genotype with early initiation of clinical manifestations (OR: 1.519, CI: 1.074 to 2.146), bacterium Burkholderia cepacia colonization (OR: 3.309, CI: 1.476 to 6.256) and Bhalla score (BS) (p = 0.015). The association was observed in subgroups of patients which were defined by their CFTR mutation genotype (all patients; subgroup I: no mutation detected; subgroup II: one CFTR allele identified to mutation class I, II or III; subgroup III: both CFTR alleles identified to mutation class I, II and/or III).ConclusionAn association between the D allele in the ACE gene and the severity of CF was found in our study.

Associação entre os polimorfismos dos genes MBL2, TGF-β1 e CD14 com a gravidade da doença pulmonar na fibrose cística

Objective: To identify associations between genetic polymorphisms (in the MBL2, TGF-β1 and CD14 genes) and the severity of the lung disease in patients with cystic fibrosis (CF), as well as between the presence of ΔF508 alleles and lung disease severity in such patients. Methods: This was a cross-sectional cohort study, based on clinical and laboratory data, involving 105 patients with CF treated at a university hospital in the 2005-2006 period. We included 202 healthy blood donors as controls for the determination of TGF-β1 and CD14 gene polymorphisms. Polymorphisms in the MBL2 and TGF-β1 genes at codon 10, position +869, were genotyped using the allelespecific PCR technique. The C-159T polymorphism in the CD14 gene was genotyped using PCR and enzymatic digestion. Results: Of the 105 CF patients evaluated, 67 presented with severe lung disease according to the Shwachman score. The MBL2 gene polymorphisms were not associated with disease severity in the CF patients. Analysis of the T869C polymorphism in the TGF-β1 gene showed an association only between TC heterozygotes and mild pulmonary disease. Although patients presenting the TT genotype of the C159T polymorphism in the CD14 gene predominated, there was no significant difference regarding lung disease severity. Conclusions: There was an association between the TC genotype of the T869C polymorphism (TGF-β1) and mild pulmonary disease in CF patients. In the CD14 gene, the TT genotype seems to be a risk factor for pulmonary disease but is not a modulator of severity. We found no association between being a ΔF508 homozygote and presenting severe lung disease.

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.