From the sample of 180 analyzed patients, 90 (50%) were male, 165 (91.7%) were European-Caucasian derived and 15 (8.3%) were African-derived individuals. The patients’ CFTR
genotypes were: 44 patients (24.44%) without identified mutation, 51 (28.33%) with one identified mutation (25% F508del/-, 2.78% G542X/-, 0.56% R1162X/-) and 85 (47.22%) patients with two identified mutations (31.67% F508del/F508del, 6.67% F508del/G542X, 2.78% F508del/R1162X, 2.22% F508del/N1303K, 0.56% F508del/R553X, 0.56% F508del/S4X, 0.56% F508del/1717-1
A, 0.56% G542X/R1162X, 0.56% G542X/I618T, 0.56% G542X/2183A
G and 0.56% R1162X/R1162X).
The spectrum of isolated Bacteria in secretion was: 76 (42.2%) with mucoid and 101 (56.1%) with non-mucoid P. aeruginosa
; 141 (78.3%), S. aureus;
25 (13.9%), B. cepacia;
and 18 (10%), A. xylosoxidans
. Comorbidities associated with CF severity were: 143 (79.4%) with pancreatic insufficiency; 33 (18.3%), nasal polyps; 33 (18.3%), diabetes mellitus; 29 (16.1%), osteoporosis; and 27 (15%), meconium ileus. For the variables with their numerical distribution, see data listed in Table
Description of quantitative variables (in months) of CF patients treated at the pediatric clinic at UNICAMP
gene D/I polymorphism showed a higher frequency for ACE*
D (228/360 alleles) compared with ACE*
I (132/360 alleles). The genotype frequencies were: 72 (40.0%) with D/D; 84 (46.67%) with D/I; and 24 (13.3%) with I/I. The population is in Hardy-Weinberg equilibrium (p
0.05). Analysis of 70 healthy control subjects in UNICAMP demonstrated the genotype frequency: 20 (29%) with D/D, 37 (53%) of D/I, and 13 (18%) I/I [23
]. There was no difference in frequency of genotypes in relation to our study (p
0.210). The analyses of the ACE
gene D/I polymorphism with the clinical variables are denoted in Table
, where every association possible between the clinical trial, CFTR
mutation identified and ACE
gene D/I polymorphism can be observed.
Association ofACEgene D/I polymorphism with variables used as markers of severity of CF, patients followed at the pediatric center in UNICAMP distributed byCFTRgene mutation identified divided into cohorts
gene D/I polymorphism was associated with the onset of clinical manifestations (Table
), in the subgroup of patients with one identified CFTR
mutation. We observed that patients with I/I genotype had OR: 0.297 (0.084 – 0.995), as protection factor, and the ones with D/D genotype had OR: 1.519 (1.074 to 2.146), as a severity factor.
Association ofACEgene D/I polymorphism with onset of clinical symptoms of patients in months considering the cohorts toCFTRmutation
D is associated with a higher gene expression and, consequently, promotes a greater inflammatory response in the body, leading to early symptoms [8
]. The earliest onset of signs and symptoms are accompanied by early onset of inflammation and deterioration of lung and pancreatic functions. These symptoms are characteristic of severe patients.
An association of the infection/colonization by B. cepacia
gene D/I polymorphism was identified for patients without taking the CFTR
mutation into account, OR: 4.509 (1.513 - 10.89), and for patients with one CFTR
mutation identified to class I, II or III, OR: (1.43 - 40.38), for the D/D genotype (Table
Association of theACEgene D/I polymorphism, withoutCFTRgenotype distribution and presence ofB. cepacia(BC)
In the analysis of the BS and ACE
gene D/I polymorphism, an association was found when no grouping by CFTR
genotype occurred (p
0.015), as well in the subgroup of patients for whom one class I, II and/or III mutation have been identified (p
0.038), and in the subgroup of patients for whom two class I, II and/or III mutation have been identified (p
0.042) (Figure ). There was no difference between BS and the age of patients after categorization. Younger patients (≤ 154
months) had the same distribution of BS as older patients (> 154
0.761). Age is not a variable that contributes to the association between the ACE
gene D/I polymorphism and BS. The analysis of an association between the BS and the age of patients with CF was performed in order to show that age had no influence on the score value analysis. We can conclude that the ACE
gene D/I polymorphism acts in genetic modulation by association with BS. The BS is a computed tomography score, which measures pulmonary involvement, therapeutic effects and selection of patients for transplantation, which detects anatomical changes of the lung parenchyma [15
]. The BS has low variation between examiners, good reproducibility, high sensitivity and specificity, and high correlation with pulmonary function test [15
]. The values obtained in the score 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.
Figure 1 Association of clinical data, with numerical distribution, withACEgene D/I polymorphism and subgroups ofCFTRmutations. a Bloxplot denoting the association of the ACE gene D/I polymorphism in patients without taking CFTR gene into account. There were (more ...)
Evolution of CF is secondary to mutation class in the CFTR
gene and environment factors. Many studies have correlated mutations, polymorphisms and clinical variables to CF [5
]. Association studies commonly face the problem of having insufficient sample size for the number of mutations in the CFTR
gene to achieve a homogeneous population and characterize the follow-up of chronic and persistent lung disease [27
Unlike other genetic diseases such as asthma, CF is monogenic. It was expected that mutations in the CFTR
gene would determine the CF severity. Patients with mutations of classes I, II and III have more severe clinical forms than those with mutations IV, V and VI. However, we can observe changes in severity of CF in patients with identical mutations in the CFTR
]. Our study allowed us to characterize the association between the CFTR
gene, the environment and one possible CF modifier gene in patients of a Reference University Center, using a statistical method of gene association versus clinical markers.
The main environmental factor in the clinical variability of CF is the patients ´ access to treatment [28
]. At our center, treatment is guaranteed by the public health system, which allows equal access for all patients included in the study, and it is not an additional factor in the analysis of data, which is not true in all CF centers in Brazil. Unlike the U.S. where the private system ensures better treatment in CF [29
], in Brazil, the public health system is the reference.
Some review articles have suggested a possible modulation of the CF severity by the ACE
gene. This fact is based on the proinflammatory property of the ACE protein [2
]. To the best of our knowledge, few studies had characterized the ACE
gene as a potential factor in the clinical CF severity [8
]. Bartlett et al
. (2009) [31
], in a multicenter study, studied the same polymorphism in relation to the propensity for liver disease in patients with CF. They genotyped 124 patients with CF and liver disease and 843 patients with CF and no liver disease. In addition to this polymorphism, four other genes, and their polymorphisms, were analyzed. The polymorphism D/I in the ACE
gene was not associated with the presence of liver disease in CF patients, OR: 1.11 (0.85 to 1.44).
Finally, the presence of B. cepacia
complex increases inflammation, favoring the exacerbation of immune response, further deterioration of the bronchopulmonary structure and causing rapid deterioration of lung function [32
]. More studies to determine whether the presence of the D/D genotype causes increased gene expression and, therefore, facilitates chronic infection by different bacteria are needed. The D/D genotype of the ACE
gene D/I polymorphism was significantly associated with higher values of BS. Higher values on the BS are associated with greater clinical severity [15
]. Patients with the D/D genotype had higher severity, when compared to patients with the I/I genotype. This data confirms that higher gene expression, given by the D allele, leads to a change in the structure of the lung parenchyma, with subsequent increases in the value of the score.
Our data suggest that ACE gene should be studied in other populations, principally in populations with high prevalence of chronic pulmonary infection by B. cepacia, early onset of clinical manifestations and early onset of severe lung disease (showed by BS).
Patient’s subgroups that were defined on the basis of CFTR mutation analysis may also to be different in comorbidities which may unmask the role of the ACE gene as modifier that was studied in this study, being a limitation of our work.