Prevalence of F508del Mutation and Evaluation of Molecular Screening for Cystic Fibrosis in Brazil

Prevalence of F508del Mutation and Evaluation of Molecular Screening for Cystic Fibrosis in Brazil

Corresponding author: Dr. Sabrina Gonçalves, Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Departamento de Análises Clínicas, Campus Universitário – Trindade. Florianópolis – SC, Brazil. Zip code: 88010- 970, Tel: +55 (48) 3721 9712;

Fax: +55 (48) 3721 9542; E-mail:


Introduction: Cystic fibrosis (CF) is considered the most common lethal inherited disease among children and young people of Caucasian origin and it is a consequence of mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). As established by the Brazilian Health Ministry Unified Health System, people who have a positive immunoreactive trypsin (IRT) result test and a positive sweat test are referred for molecular investigation of the F508del mutation in the CFTR gene.

Objective: To verify the frequency of the F508del mutation in the state of Santa Catarina, Brazil, and to identify the requirement for a broader molecular screening to confirm or exclude the diagnosis of cystic fibrosis.

Methods: Descriptive, retrospective, cross-sectional study of patients followed at the center of excellence for the treatment of the cystic fibrosis in the state of Santa Catarina between 2002 and 2015.

Results: Ninety-four patients with mean age of 7.62 ± 4.08 years were studied; 55.32% of whom male and 94.68% Caucasian. Of the 188 alleles analyzed, a frequency of 92 (48.94%) alleles was observed for the F508del mutation and 96 (51.06%) for other mutations. The genotype frequencies found for F508del/F508del, F508del/another mutation, and another mutation/another mutation genotypes were, respectively: 28.72% (27 patients), 40.43% (38 patients), and 30.85% (29 patients). Hardy-Weinberg equilibrium was calculated and yielded a chi-square value = 3.43 (p = 0.06). The study population was in equilibrium.

Conclusions: According to the guidelines of the Brazilian neonatal screening program, the molecular diagnosis be only be con- firmed with the presence of two disease-causing mutations in the CFTR gene, one in each allele. In our study, we observed that only 28.72% of patients had a confirmed molecular diagnosis, which allows us to infer that the molecular diagnosis strategy for cystic fibrosis must be restructured in Brazil to provide a diagnostic contribution to the sweat test.

Keywords: Cystic Fibrosis; Genotype; Mutation; Prevalence


CF: Cystic Fibrosis;

CFTR: Cystic Fibrosis Transmembrane Conductance Regulator; IRT: immunoreactive trypsin


Cystic fibrosis (CF) is considered the most common lethal in- herited disease among children and teenagers of Caucasian origin and it is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene [1,2]. Since this gene was discovered, over 2,000 different mutations have been described [3]. Such mutations vary among the pop- ulations due to ethnical and geographic differences. However, the F508del mutation is the most common and is present in approximately 70% of the world population [4,5].

In Brazil, the national neonatal screening program for CF is performed by dosage of immunoreactive trypsin (IRT) from a serum sample. In order to confirm a positive result test, the workflow is the measurement of electrolyte concentration in sweat (sweat test) and molecular testing for the most common mutation, F508del [6]. Nonetheless, studies have shown that the frequency of the F508del mutation is low in Brazil, likely due to racial miscegenation and to particular mutation profiles in different regions of the country [4,7,8]. Moreover, an esti- mated 73% of mutant alleles of CF in Brazil remain non-char- acterized, which indicates other mutations may be involved on those profiles [7].

The direct detection of mutations, through molecular method- ologies, is an important strategy to help the early diagnosis of CF since it is able to raise the predictive value of the neona- tal screening test, based on IRT dosage, and of the sweat test (confirmation test for CF) [7,9]. In this regard, it is important to point out that molecular diagnosis has been proved to be useful to clarify atypical CF cases, which may have inconclusive or normal sweat test results [10]. In addition, with the intro- duction of new drugs that enable correcting the CFTR protein affected by certain mutations [11], molecular detection has become a key tool to be used in individualized therapies for specific mutations.

In that context, the present study aimed to investigate the fre- quency of the F508del mutation in the state of Santa Catarina, Brazil by analyzing patients followed by the center of excel- lence in CF treatment in the state (Children’s Hospital Joana de Gusmão) in order to evaluate the need for broadening molecu- lar screening to include mutations other than F508del aiming to aid in confirming or excluding the diagnosis.


The methodology is based on a descriptive, retrospective, cross-sectional study based on information from patient re- cords, genetic information reports, and interviews with the legal caretakers of patients followed in the CF outpatient clinic of the Children’s Hospital Joana de Gusmão of Santa Catarina, Brazil. The records, from 2002 to 2015, were individually ac- cessed over the hospital’s intranet. The following information was collected: patient age, sex, ethnicity, and result of the ge- netic assay for CF.

For clinical-demographic categorical variables, such as sex and ethnicity, the absolute and percentage values were used. For age, mean and standard deviation were used. The descriptive analysis was performed by calculating the frequency of the al- lele and genotype of the F508del mutation in the CFTR gene of the patients analyzed. Hardy-Weinberg equilibrium was calculated using the software BioEstat version 5.3 [12] aiming to verify whether the population analyzed was in equilibrium. Chi-squared test (χ2), calculated in the same statistical soft- ware, was employed in order to verify the association of the allelic frequency of F508del mutation observed in other stud- ies with the present analysis. Finally, p values below 0.05 were considered statistically significant.

This project was approved by the institution’s Research Ethics Committee (CONEP 789.932/2014).


The population consisted of 94 patients (188 alleles), 52

(55.32%) males and 42 (44.68%) females (χ2 = 1.06; p = 0.30), with clinical findings of CF who had previously undergone IRT dosage and sweat test. Of those, 94.68% were of Caucasian or- igin.

The mean age of the patients included in the study was 7.62 ±

4.08 years, ranging from one month to 15 years.

The results found in the analysis of the descriptive statistics for the genotypes with the F508del mutation are presented in Table 1. Of the 188 alleles analyzed, the frequencies of 92 (48.94%) alleles for the F508del mutation and of 96 (51.06%) for other mutations were observed. Hardy-Weinberg equilib- rium calculation yielded χ2(1) = 3.43 (p = 0.06). The values expected for the respective genotypes were: F508del/F508del (22.51 patients), F508del/another mutation (46.98 patients), and another mutation/another mutation (24.51 patients). Thus, the values observed and expected for the genotypes were not significantly different (p > 0.05). That allows concluding that the population-based sample adopted is in equilibrium and, as such, is representative of the overall population, which makes it adequate for the goals of the present study.

Percentage of genotypes Genotype frequencies (n) Genotypes

F508del/F508del 27 28,72%

F508del/another mutation 38 40,43%

Another mutation/another mutation 29 30,85%

Total 94 100,00%

n = sample size in absolute number of patients.

Table 1. Distribution of genotypes for the F508del mutation.


CF is an autosomal recessive genetic disease, therefore, equi- librium is expected in the prevalence between sexes. Howev- er, the present study found a slight predominance of males (55.32%), although the distribution by sex showed no statisti- cally significant difference. This peculiarity was also observed by Streit et al. [13] and Maróstica et al. [14], who found values of 61% and 62.3% for males, respectively. According to the hy- pothesis raised by Coutinho et al. [15], the greater deteriora- tion in pulmonary function observed in girls during puberty could explain the slight predominance of CF among males.

Regarding the Caucasian origin of the patients in the study, the prevalence of 94.68% matches the data found in studies car- ried out in the states of Rio Grande do Sul [14] (100%) and São Paulo [15] (94.29%). However, studies in the state of Ba- hia reported 28.7% Caucasoid patients with CF [16], which shows that the variation in prevalence found among states may be due to the greater European ancestry in the South and Southeast regions of Brazil and greater African ancestry in the Northeast region [2,4,17]. On the other hand, the present study also found low prevalence (48.94%) of the F508del mutation among patients of Caucasian origin, which contrasts with the literature that shows a prevalence of approximately 70 to 88% of the F508del mutation in the Caucasoid population [3,18,19]. Possible mistakes by the legal caretakers during the interview regarding the ethnical group the patients belonged to might explain the discrepancy in that finding.

Of the 188 alleles analyzed, the F508del mutation was identi- fied in 92 (48.94%). Raskin et al. [20], in a similar study also in Santa Catarina, analyzed 48 alleles and found frequency of 27% for the F508del mutation, lower than in the present study (χ2(1) = 7.39; p < 0.01). However, another research [21] also in Santa Catarina analyzed 96 alleles and observed frequency of 55.21% for the F508del mutation, which is similar to that found in the present study (χ2(1) = 1.00; p = 0.32). The low fre- quency observed for the F508del mutation in that study may derive from the small number of alleles analyzed, which makes

the sample non-representative of the population studied.

Other studies on allelic frequency carried out in Brazil also reported variations in the prevalence of F508del, as observed by Streit et al. in the state of Rio Grande do Sul [13], Raskin et al. in Paraná [21], Coutinho et al. in São Paulo [15], Perone et al. in Minas Gerais [22], Cabello et al. in Rio de Janeiro [23] and Araujo et al. in Pará [24]. Those studies analyzed, respec- tively, 154, 100, 140, 222, 148, and 66 alleles and reported

frequencies of 48.7% (χ2(1) = 0.002; p = 0.96), 39% (χ2(1) =

2.60; p = 0.11), 50% (χ2(1) = 0.04; p = 0.85), 48.2% (χ2(1) =

0.02; p = 0.88), 25.68% (χ2(1) = 18.88; p < 0.01), and 22.7%

(χ2(1) = 13.76; p < 0.01). Besides the F508del mutation having different prevalences among states, its frequency was also low compared to worldwide rates and in North American countries (around 66% of cases), likely due to the ethnical variation in each region [4,8,25].

Although F508del is the most common mutation and is recom- mended by the Brazilian national neonatal screening program for diagnosis of CF [5,6], other mutations may play a role in the disease. In the present study, the rate of detection was below 50% when analyzing a single mutation. This way, using strat- egies based on the analysis of different mutations of the CFTR gene enables increasing the accuracy of the diagnosis of CF.

Moreover, according to guidelines of the Brazilian neonatal screening program, only the identification of two pathogenic mutations of the CFTR gene, one in each allele, confirms the molecular diagnosis [26]. Therefore, using only the F508del mutation as screening, the present study found that 30.85% of the patients did not have definitive molecular diagnosis al- though all of them carried the characteristic clinical criteria of the disease; 40.43% of the patients had inconclusive diagnosis, in which the F508del mutation could only be detected in one of the alleles, and only 28.72% of the patients had confirmed molecular diagnosis.

In face of that, the molecular diagnostic strategy contemplated by the Brazilian neonatal screening program has caused some controversy, particularly due to the low percentage of patients whose genetic test was confirmed by two mutations, as ob- served in the present study and in researches carried out in the states of Rio Grande do Sul, São Paulo [15], Minas Gerais

[22] and Rio de Janeiro [4], where homozygote patients for the F508del mutation accounted for, respectively, 31.2%, 30%, 26.13%, and 10.81% of the CF cases. Furthermore, in Brazil, mutation screening for CF is performed using imported mo- lecular diagnostic kits, which are highly specific but little sen- sitive, i.e., they detect, at most, 80 to 85% of alleles in patients with CF [26]. The main reason for the low sensitivity of the method is racial miscegenation since the mutations that cause CF in European countries are not the same found in Brazil [27]. Another relevant fact that speaks against the investigation of a single mutation is the progress of CF therapeutics since new medications have been successfully used to modulate the CFTR protein in a mutation-specific way [28]. Two drugs are avail- able for the F508del mutation in the international market [11], however, as observed in the present study, over half (51.06%) of the alleles analyzed had no F508del mutation, which indi- cates the spectrum of mutations detected must be broadened to provide the patients with innovative and promising treat- ment options that are also available for other mutations.


Therefore, the molecular diagnostic strategy for CF clearly must be restructured to provide a diagnostic contribution to the sweat test, enable the genetic counseling of couples and family members, investigate individualized therapies for cer- tain mutations with the drugs available and those being re- searched, and develop new strategies for the management and treatment of patients. In this sense, knowing the mutational profile of each population is the key principle for the design and application of any new strategy towards advances in di- agnostic and therapy. However, in face of the lack of previous molecular studies, the mutational profile of the CFTR gene is completely unknown in some states of Brazil, which prevents the implementation or improvement of any strategies based on the genotype-phenotype relation.

Acknowledgements: We acknowledge to CAPES for the finan- cial support during the doctorate.

Disclosure: The authors declare that they have no conflicts of interest.

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