Brazilian Journal of Pulmonology

ISSN (on-line): 1806-3756 | ISSN (printed): 1806-3713


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Current Issue: 2012 - Volume 38 - Number 1 (January/February)


Cystic fibrosis transmembrane conductance regulator gene mutations and glutathione S-transferase null genotypes in cystic fibrosis patients in Brazil

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


Carmen Silvia Passos Lima; Manoela Marques Ortega; Fernando Augusto Lima Marson; Roberto Zulli; Antônio Fernando Ribeiro; Carmen Silvia Bertuzzo



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 F508 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 F508 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 F508 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 F508 CFTR mutation found in younger children suggests that it influences the age at diagnosis of CF in this region of Brazil.



Objetivo: Determinar os efeitos que a mutação do gene cystic fibrosis transmembrane conductance regulator (CFTR) e da deleção dos genes glutationa S-transferase (GST) mu-1 (GSTM1) e teta-1 (GSTT1) têm na evolução clínica da fibrose cística (FC) em pacientes da região sudeste do Brasil. Métodos: Entre março de 2002 e março de 2005, incluímos no estudo todos os pacientes com FC atendidos consecutivamente no Departamento de Pediatria do Hospital de Clínicas da Faculdade de Ciências Médicas da Universidade Estadual de Campinas. O DNA genômico de 66 pacientes com FC foi analisado por PCR e digestão com endonuclease de restrição para a identificação dos genótipos. Resultados: A mutação F508 do gene CFTR foi identificada em 44 (66,7%) pacientes. As deleções dos genes GSTM1, GSTT1 e da combinação nula GSTM1/GSTT1 foram identificadas em 40,9%, 15,2% e 3,0% dos pacientes, respectivamente. A mutação F508 do gene CFTR foi mais comum em pacientes diagnosticados com FC antes dos 2,5 anos de idade que naqueles diagnosticados mais tarde (75,5% vs. 41,2%; p = 0,008). Conclusões: Foram observadas frequências similares da mutação F508 do gene CFTR e dos genótipos GSTM1 e GSTT1 nos pacientes, independentemente do sexo, etnia ou status da doença pulmonar ou pancreática. Quando os pacientes foram estratificados por aspectos clínicos e epidemiológicos, as frequências dos genótipos GSTM1 e GSTT1 nulos foram semelhantes, sugerindo que a ausência herdada dessas vias enzimáticas não altera o curso da FC. Em contraste, a alta frequência da mutação F508 no gene CFTR encontrada em pacientes mais jovens sugere que essa mutação influencia a idade no momento do diagnóstico de FC nessa região do país.



Keywords: : Cystic fibrosis; Cystic fibrosis transmembrane conductance regulator; Glutathione transferase.


Palavras-chave: Fibrose cística; Regulador de condutância transmembrana em fibrose cística; Glutationa transferase.




Cystic fibrosis (CF) is one of the most common severe autosomal recessive genetic diseases among populations of northern European descent. The major clinical manifestations of the disorder are chronic sinopulmonary disease and pancreatic exocrine insufficiency.(1)

There is considerable heterogeneity among individuals with CF in terms of the severity of the disease. Attempts to link phenotypes to specific CF transmembrane conductance regulator (CFTR) gene mutations have been successful, particularly for pancreatic disease status.(2-5) This correlation, however, has not completely held for pulmonary disease, which can vary markedly among patients with the same CFTR gene mutation.(6) The severity of pulmonary disease in CF seems to be influenced by other mutations located out of the CFTR gene locus,(7,8) such as the those occurring in the glutathione S-transferase (GST) enzymes.(9-13) Glutathione is a major local pulmonary antioxidant that is present in the epithelial lining fluid. The GST enzymes detoxify harmful organic hydroperoxides that are formed as a result of exposure to oxidant stress, such as those found in the lungs of patients with CF,(14) by conjugating them with glutathione, thereby potentially preventing further pulmonary damage.

The genes GST mu-1 (GSTM1) and GST theta-1 (GSTT1) are polymorphic in humans and are absent or homozygous null, resulting in a lack of active proteins, in approximately 40% and 20% of normal individuals, respectively.(15) In CF, there is no consensus regarding the roles of the GSTM1 and GSTT1 null genotypes in the severity of lung disease(9-13) and pancreatic disease.(9)

Among Whites in Brazil, CF is one of the most common severe inherited diseases.(16) Short survival due to pulmonary disease has been described in CF patients treated at our hospital.(16) However, the modifiers of disease severity, if any, are still unknown. Therefore, we considered it necessary to identify the CFTR mutation, as well as the GSTM1 and GSTT1 genotypes, in CF patients in Brazil, in order to determine whether these genetic factors influence the clinical course of the disease in the southeastern region of the country.


The study sample consisted of all consecutive CF patients treated in the Hospital de Clinicas of the State University of Campinas, located in the city of Campinas, Brazil, between March of 2002 and March of 2005. The diagnosis of CF was based on signs and symptoms, such as failure to thrive, meconium ileus, vomiting, abdominal pain, diarrhea, steatorrhea, and recurrent pulmonary infections. Two tests of chloride concentration in sweat, with results equal to or greater than 60 mEq/L and performed by the classical pilocarpine test,(17) were required for confirmation of the diagnosis. We considered the age at diagnosis of CF, as well as the current severity of the pulmonary and pancreatic diseases, as measured by the Shwachman score.(18) All procedures were carried out in accordance with the ethical principles of the institutional guidelines, and all patients or their relatives gave written informed consent.

Genomic DNA of peripheral blood was used for the genotyping of all CF patients. The F508 CFTR mutation was detected directly by polymerase chain reaction (PCR; Figure 1a).(19) The G542X, G551D, R553X, R1162X, W1282X, and N1303K mutations of the CFTR gene were analyzed by restriction endonuclease digestion of the PCR products.(20) The genes GSTM1, GSTT1 and -globin (as a reaction control) were amplified by multiplex-PCR (Figure 1b).(21)

The patients were stratified by the pattern of CFTR gene mutation (homozygous F508 mutation, heterozygous F508 mutation, and other or unknown CFTR mutations) and GSTM1 and GSTT1 gene mutations (present or with homozygous deletion). We used the chi-square test or Fisher's exact test to calculate the statistical significance of differences in age (< 1.0 year vs. ≥ 1.0 year; and < 2.5 years vs. ≥ 2.5 years); gender (male vs. female); ethnic origin (White vs. African-Brazilian); and lung and pancreatic exocrine status (excellent/good/mild vs. moderate/severe); and GSTM1 and GSTT1 genotypes (present vs. null), isolated or in combination. The associations of genotypes and clinical variables were also evaluated by multivariate analysis using a logistic regression model. Factors with a p ≤ 0.05 were considered statistically significant.

All analyses were performed using the statistical package SAS System for Windows, version 8.1 (SAS Institute, Cary, NC, USA).


The study involved 66 patients with CF (Table 1). The median age of the patients at diagnosis was 1.0 year (range, 0.1-15.0 years). At the time of CF diagnosis, 27 of the patients were younger than 1.0 year of age, 39 were at least 1.0 year old, 49 were under 2.5 years of age, and 17 were over 2.5 years of age. Males and females were similarly represented in the study. Almost all individuals were White. Moderate and severe forms of CF were found in 57.6% of the patients. The prevalences of these forms of the disease were similar in patients under and over 1.0 year of age (59.2% and 56.4%, respectively; p = 1.00), as well as in those under and over 2.5 years of age (61.2% and 47.1%, respectively; p = 0.40).

The F508 CFTR mutation was identified in 44 of the 66 patients (66.7%). The homozygous and heterozygous mutations were seen in 17 (25.8%) and in 27 (40.9%), respectively. The G542X, R1162X, and N1303K mutations of the CFTR gene were found in heterozygosis in 8 (12.1%), 2 (3.0%), and 1 (1.5%) of the patients, respectively. The G551D, R553X, and W1282X mutations were not found in any of our patients. We found that 19 of the patients did not have any of the mutations studied. The allelic frequencies of the F508, G542X, R1162X, and N1303K mutations of the CFTR gene were 0.462, 0.061, 0.015, and 0.008, respectively. The frequencies of the homozygous and heterozygous F508 CFTR mutations were similar in patients under and over 1.0 year of age (34.8% and 31.8%, respectively; p = 0.18). However, such mutations were more common in patients under 2.5 years of age than in the older patients (75.5% vs. 41.2%; p = 0.008). No significant differences regarding gender, ethnic origin, or lung/exocrine pancreatic disease status were observed among the CF patients in relation to the distinct patterns of the CFTR mutation. Similar frequencies of the G542X, R1162X, and N1303K mutation patterns of the CFTR gene (data not shown), as well as of the GSTM1, GSTT1, and GSTM1/GSTT1 null genotypes, were seen in patients under and over 1.0 year of age (data not shown). The same was true for patients under and over 2.5 years of age at diagnosis, regardless of gender, ethnic origin, or lung/exocrine pancreatic disease status (Table 1). Similar frequencies of the distinct patterns of the CFTR mutations in combination with the genotypes GSTM1 null, GSTT1 null, and GSTM1/GSTT1 null were seen in patients under and over 1.0 year of age (data not shown). After multivariate analysis, the frequencies of the genotypes were also similar among patients stratified by age (< 2.5 and ≥ 2.5 years of age), gender, ethnic origin, and lung/exocrine pancreatic disease status (Table 2). No differences in age, gender, ethnic origin, or lung/exocrine pancreatic disease status were seen among the patients with the G542X, R1162X, or N1303K mutations in the CFTR gene combined with the GSTM1, GSTT1, or GSTM1/GSTT1 null genotypes (data not shown).


The distribution of the patients enrolled in the study by age at diagnosis, gender, ethnic origin, and severity of lung/exocrine pancreatic disease status showed that our CF patients were similar to those in other countries.(1) The F508 CFTR gene mutation was the most common mutation found in our study, which corroborates other reports from the same region of Brazil,(16,22-25) as well as international reports.(26,27) Other mutations of the CFTR gene, such as R1162X and N1303K, were less common in our CF patients,(22,24,28) as has been reported for CF patients in other parts of the world.(27) The frequencies of the GSTM1 and GSTT1 null genotypes in the CF patients in our study were similar to those found in healthy individuals, in Brazil(29) and elsewhere.(15) Therefore, our sample seems to be representative of CF patients throughout the world.

We found a high frequency of the F508 CFTR mutation in patients diagnosed with the disease before 2.5 years of age in comparison with those diagnosed later. However, we found no difference in the frequencies of the gene mutation in patients stratified by lung/pancreatic disease status. Because the frequencies of the moderate and severe forms of CF were similar in the patients under and over 2.5 years of age, the combination of the F508 CFTR gene mutation and the diagnosis of the disease at a younger age suggests that the mutation provokes more symptoms or makes symptoms more persistent, which might prompt the parents of patients with this mutation to seek medical advice sooner.

The F508 mutation results in the production of an abnormally folded CFTR that is not trafficked normally to the apical cell membrane.(30) In fact, there is evidence that the gene abnormality alone does not affect the severity of the lung disease.(6,16,26) This seems to be influenced by other mutations located out of the CFTR locus. In addition, the association between the F508 mutation and the severity of pancreatic exocrine insufficiency in CF patients has been consistently demonstrated.(2,5) Therefore, it is noteworthy that our sample size (n = 66) might not have been sufficient to detect associations between the F508 mutation in the CFTR gene and the clinical features, and that the method used for determining the lung/pancreatic disease status (the Shwachman score) might have been inappropriate for the purposes of this study. In addition, a second mild allele accompanying the F508 mutation might have protected our patients against severe pancreatic disease, as previously described.(2,5) It is also possible that our patients did not survive long enough to present pancreatic dysfunction.

We found no differences between the patients with the GSTM1 or GSTT1 genes and those without in terms of age, gender, ethnic origin, or lung/pancreatic disease status, suggesting that the GST enzymes do not alter the clinical characteristics of CF patients in our region. The GST multigene family of detoxifying enzymes is involved in protecting various tissue types from oxidative damage. The homozygous GSTM1 null genotype has been associated with greater lung disease severity and decreased survival in CF patients,(9-11,13) although there are conflicting data.(12) In addition, we found that the inherited absence of the GSTM1(9) and GSTT1(11,12) enzymes was not associated with the severity of pancreatic or lung disease. Although the absence of these GST proteins might have altered the clinical function of our CF patients, their influence might not have been strong enough to be detected in our sample. Furthermore, the F508 CFTR mutation combined with the GSTM1 or GSTT1 null genotypes were also equally distributed among our CF patients, stratified by the clinical characteristics. However, our sample size was too small to permit consistent conclusions to be drawn regarding the association of these genotypes with the clinical manifestations of the disease.

In conclusion, our data provide preliminary evidence that the GST detoxifying enzymes do not influence the course of CF, but that the F508 mutation alters the age at diagnosis of the disease. However, epidemiological studies involving larger samples of patients should be carried out in order to clearly identify the roles that these mutations play in the population of CF patients in Brazil.


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Study carried out at the State University at Campinas School of Medical Sciences, Campinas, Brazil.
Correspondence to: Carmen Silvia Passos Lima. Department of Internal Medicine, State University at Campinas School of Medical Sciences, Rua Alexander Fleming, 181, Cidade Universitária Zeferino Vaz, CEP 13083-970, Campinas, SP, Brasil.
Tel/Fax: 55 19 3521-7496. E-mail:
Financial support: This study received financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo Research Foundation).
Submitted: 19 September 2011. Accepted, after review: 31 October 2011.
**A versão completa em português deste artigo está disponível em

About the authors

Carmen Silvia Passos Lima
Professor. State University at Campinas School of Medical Sciences, Campinas (SP) Brazil.

Manoela Marques Ortega
Researcher. State University at Campinas School of Medical Sciences, Campinas (SP) Brazil.

Fernando Augusto Lima Marson
Researcher. State University at Campinas School of Medical Sciences, Campinas (SP) Brazil.

Roberto Zulli
Statistician. State University at Campinas School of Medical Sciences, Campinas (SP) Brazil.

Antônio Fernando Ribeiro
Researcher. State University at Campinas School of Medical Sciences, Campinas (SP) Brazil.

Carmen Silvia Bertuzzo
Professor. State University at Campinas School of Medical Sciences, Campinas (SP) Brazil.



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