Genetic interaction of GSH metabolic pathway genes in cystic fibrosis

Background Cystic 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. Method A 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. Results The 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. Conclusion Polymorphisms in genes associated with metabolism of GSH act on the CF’s severity.


Background
Cystic Fibrosis (CF) is a common disease in Caucasian population, with a prevalence of 1:2,500 live births [1]. CF shows an autosomal recessive pattern of inheritance [2], which is caused by mutations in Cystic Fibrosis Transmembrane Regulator (CFTR) gene. More than 1,900 mutations in CFTR gene have been associated to CF clinical severity [3][4][5][6]. However, in the pulmonary disease, which is the main cause of CF morbidity and mortality, several studies have demonstrated that clinical variability is influenced by modifier genes and environmental factors [2,7].
In our referral center, we have observed clinical variability among CF's patients. However, the patients present similar socioeconomic status, none severe malnutrition, mutations of classes I, II and/or III, similar support of the Parents Association (www.fibrocis.org.br), and receive free medication by the government. Therefore, we postulate that clinical variability could be associated with interaction of polymorphisms in modifier genes. In this context, we aim to analyze polymorphisms in GCLC and GST genes (GCLC -129C>T and -3506A>G; GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G) associating to clinical markers of CF severity.

Ascertainment of patients
It was conducted a cross-sectional study and patients were selected during the period of 2010 and 2011, in a university center for CF patients care. This study was approved by Ethics Research Board of the Faculty of Medical Sciences at University of Campinas -São Paulo -Brazil (#528/ 2008). The study was in accordance to the Helsinki declaration and all patients signed a consent form before beginning the study. For patients below the age of 18 the consent was granted by their parents or guardians.
Diagnosis of CF was confirmed in patients with two doses of sodium and chloride from the sweat with values greater than 60 mEq/L. In a group of patients we identified two CFTR gene mutations. No patient had received neonatal screening test performed for CF. A total of 215 patients were selected for the study. Among them, 35 patients without clinical data for statistical analysis and those who did not sign the consent form were excluded from the study, and 180 CF patients were included at the study.

Clinical variables
The polyps, osteoporosis, meconium ileus, diabetes mellitus and pancreatic insufficiency (PI). The clinical score evaluation was performed by two specialists in double-blind analyses, and in discordance case, another one was consulted. The Shwachman-Kulczycki score compares clinical manifestations among patients, detect treatment effects and aid in the determination of diagnostic criteria. To that end, the system evaluates 4 major parameters: general activity, nutrition, chest radiographic findings and physical examination. The score for each parameter ranges from 5 to 25. Lower score's values are associated with CF severity [29]. The BS is a tomographic scoring system to assess pulmonary involvement, determine therapeutic effects and aid selection of patients for transplants, with small variation between the scores given by the various examiners and the score proved to have good reproducibility and high correlation with pulmonary function test results. A total of 9 categories, worth 3 points each, are scored, and a maximal score equals a high degree of severity. The final score must be subtracted from 25. Lower score's values are associated with CF severity [29]. Kanga score is a system designed to assess acute exacerbations of the disease, to predict improvement or worsening of pulmonary function and to evaluate therapeutic effects with little variability between examiners and correlated significantly with pulmonary function (FEV 1 and forced vital capacity) test results. The system helps identify daily clinical changes and includes 5 common symptoms (cough, fluid secretion, appetite loss, dyspnea and frailty) and 5 physical signs (temperature, weight, respiratory frequency, wheezing and respiratory sounds). Each criterion is worth 1 to 5 points. Higher score's values are associated with CF severity [29].
Spirometry was performed in patients older than 7 years, using the CPFS/D spirometer (MedGraphics, Saint Paul, Minnesota, USA). Data was recorded by the PF BREEZE software version 3.8B for Windows 95/98/NT [32] and the following markers were included: forced Table 2 Genotypic characteristic of gene polymorphisms at GCLC, GSTM1, GSTT1, and GSTP1 genes and CFTR gene mutation among cystic fibrosis patients   DNA extraction and polymorphisms genotyping DNA sample was obtained from peripheral blood using phenol-chloroform standard procedure. In order to evaluate DNA concentration, we quantified the entire sample using GE NanoVue™ Spectrophotometer (GE Healthcare Biosciences, Pittsburgh, USA).
The CFTR gene mutations were investigated by PCR technique (F508del) and the restriction fragment length polymorphism (RFLP) method (G542X, R1162X, R553X, G551D and N1303K). Some mutations in CF patients were obtained by sequencing or MLPA (Multiplex Ligationdependent Probe Amplification) analysis: S4X, 2183A>G, 1717-G>A and I618T. For sequencing and MLPA, we used the same MegaBace1000® (GE Healthcare Biosciences, Pittsburgh, USA) [33]. The CFTR genotype separated the patients in 3 groups: (1) patients with two mutations identified classes I, II and/or III, (2) patients with one mutations identified classes I, II or III, (3) patients with no identified mutation. All mutations identified were included in the class one, two or three of the CFTR gene. Other identified mutations, class IV (P205S e R334W) were not included in the statistical analysis.
For the GSTM1 and GSTT1 genes deletion, a multiplex PCR reaction was performed and the CYP1A1 Table 3 Genetic interaction of polymorphisms and CFTR mutations in association with cystic fibrosis clinical variables gene was included as an internal reaction control [34]. In the assay tests for the wild type allele, the samples for which no signal is obtained are homozygous for the deletion. Heterozygotes and homozygotes for the wild-type allele cannot be differentiated by the multiplex technique used. As GSTM1 and GSTT1 deletion analyze not provide complete genotypes, minor allele frequencies and Hardy-Weinberg Equilibrium cannot be evaluated for these loci in our study. By the method realized we have two groups of GSTM1 and GSTT1 gene: (i) patients homozygous to gene deletion; (2) patients with at least an allele expressed. The RFLP was performed for polymorphisms GCLC*-129C>T, GCLC*-3506A>G [10,15] and GSTP1*+313A>G [28].
Other genes were studied in the same CF reference center with same population as previously published [11,13,14]. The GSTM1 and GSTT1 genes were studied in our center considering other CF patients as recent published data showed [12]. The patient's studied by Lima et al. (2012) [12] did not include the same CF patients, and in the previously study, only the follow clinical variables were employed: age, sex, ethnic and Shwachman-Kulczycki.

Statistical analysis
To develop statistical analyses, patients were divided into two subgroups according to clinical variables distribution. We evaluate minor allele frequency (MAF) and Hardy-Weinberg equilibrium (HWE) using OEGE software (Online Encyclopedia for Genetic Epidemiology studies, 2008). In order to evaluate genetic interaction among the polymorphisms in our sample, we used the Multifactor Dimensionality Reduction (MDR) model, which is a nonparametric and genetic model-free data mining for nonlinear interaction identification among genetic and environmental attributes [35][36][37]. To adjust results for multiple comparisons, we performed a MDR permutation test in our sample, totalizing 100,000 permutations.

Results and discussion
The CF is a disease with a high phenotypic variability, resulting from the interaction between genetic and environmental factors, which could affect differential expression between patients with similar CFTR gene mutations [2]. Despite CF's severity is difficult to characterize, we describe 28 clinical variables (Table 1), consistent with the phenotype feature of the disease.
The polymorphism GCLC-129C>T is not in Hardy-Weinberg equilibrium. The MAF observed in present data is 0.012, and the data achieved at National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/), by HapMap project, shows a MAF of 0.076. The observed difference can be a strong factor to consider the polymorphism as an important modifier of CF severity, but we did not find in our data genotype-phenotypeassociation. Maybe, with a bigger population and other clinical variables can be possible to determine the polymorphism influence. Another factor should be considered that is the Brazilian population is an admixture population, what can provide changes in the clinical associations.
The MDR analysis showed evidence of interaction of GSTM1 and GSTT1 genes deletion, GSTP1*+313A>G, and CFTR mutations (p= 0.008) and BS clinical score (Table 3). All data was previously associated with a point-point analysis considering which gene polymorphism in association with one clinical manifestation at a time by CFTR groups, and after Bonferroni correction, we did not observed positives p-value  Figure 2 Dendrogram and graph of entropy with the genes interaction and their polymorphisms in response to Bhalla score. A. Dendrogram with the genes interaction and their polymorphisms in response to Bhalla score (BS). The stronger interaction of response to Bhalla clinical score is given by CFTR mutations and GSTM1 and GSTT1 genes deletion. Secondly, the polymorphism GSTP1*+313A> G was associated with the clinical variable. Polymorphisms GCLC*-129C>T and GCLC*-350A>G were not associated with the BS. B. Graph of entropy measuring the power of different genotypes and the interaction between them, for the genes analyzed in the gene-gene interaction with BS. The CFTR mutation gene, GSTP1*+313A>G and GSTM1 and GSTT1 deletion polymorphisms showed significant association with BS. The CFTR mutation showed influence of 3.38% for BS, the GSTM1 and GSTT1 deletion together, showed 2.82% of influence, while the GSTP1*+313A>G showed influence of 0.5%. The CFTR mutation showed a value of 4.15% for the interaction between the deletion polymorphism with GSTM1 and GSTT1 genes and 3.57% for the association with GSTP1*+313A>G. The (Additional file 1: Table S4, Additional file 2: Table S5,  Additional file 3: Table S6, Additional file 4: Table S7, Additional file 5: Table S8). In this context, the data by gene-gene interaction provides a better tool to contribute in the clinical manifestation association. The association observed cannot identify if those patients who carry risk alleles at all four loci will have the most severe clinical phenotype reflected by the BS. Genotype combinations of the genes that show association with BS clinical score are demonstrated in Figure 1. The largest association regarding to BS clinical response was given by the interaction of GSTM1 and GSTT1 genes deletion, and CFTR mutations. The polymorphism GSTP1*+313A>G presented entropy with BS clinical score, but with minor intensity (Figure 2).
In the study, different clinical markers were analyzed and the association has found with BS clinical score. The BS clinical score is a computed tomography, which measures pulmonary involvement, therapeutic effects and selection of patients for transplantation, which detects anatomical changes of the lung parenchyma [29,38,39]. The BS has low variation between examiners, good reproducibility, high sensitivity and specificity, and high correlation with pulmonary function test [29]. Values obtained in the BS can predict severity associated with deterioration of the structure of the lung parenchyma, which later in clinical evolution can be observed by other variables such as BMI and lung function.
The most important cause of mortality and morbidity is the increase of oxidizing reactions; in this way, understand the mechanisms associated with this process is important to the clinical dynamics of the disease and to comprehend the mechanisms by which the disease shows a variability in its severity among patients with similar CFTR genotype. The association of clinical, laboratory, and genetic markers, such as modifier genes, with CF's severity can provide better understanding of clinical variability finding among these patients being important in the clinical prognosis. In addition, further studies should be developed to investigate the possibility to use products related to GSH pathway as a possible target of new drugs to CF and also to understand the oxidative process which takes place in the lungs of these patients.