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Jornal de Pediatria
ISSN: 0021-7557
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Sociedade Brasileira de Pediatria
Brasil
Bieger, Andréia Marisa; de Lima Marson, Fernando Augusto; Bertuzzo, Carmen Sílvia
Prevalence of F508 mutation in the cystic fibrosis transmembrane conductance regulator gene among
cystic fibrosis patients from a Brazilian referral center
Jornal de Pediatria, vol. 88, núm. 6, noviembre-diciembre, 2012, pp. 531-534
Sociedade Brasileira de Pediatria
Porto Alegre, Brasil
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Jornal de Pediatria
Original Article
Copyright © by Sociedade Brasileira de Pediatria
Prevalence of ΔF508 mutation in the cystic fibrosis
transmembrane conductance regulator gene among cystic
fibrosis patients from a Brazilian referral center
Andréia Marisa Bieger,1 Fernando Augusto de Lima Marson,2 Carmen Sílvia Bertuzzo3
Abstract
Objective: To verify the presence of ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator
gene among patients with cystic fibrosis diagnosed by the sweat test for sodium and chlorine and followed at the
Pediatric Pneumology Outpatient Clinic of Universidade Estadual de Campinas, Brazil, a referral center for the
treatment of cystic fibrosis.
Methods: The study analyzed 167 DNA samples from cystic fibrosis patients. Patients’ genotype was determined
by polymerase chain reaction, and allele and genotype frequencies of ΔF508 mutation were calculated.
Results: The genotype frequencies found for -/-, ΔF508/-, and ΔF508/ΔF508 genotypes were respectively:
43.7% (73 patients), 32.9% (55 patients), and 23.4% (39 patients). Of the 334 alleles analyzed, we observed a
frequency of 201 (60.18%) alleles for the absence of ΔF508 mutation and of 133 (39.82%) for the presence of
ΔF508 mutation. Hardy-Weinberg equilibrium was calculated, obtaining a chi-square value = 16.34 (p ≤ 0.001).
The study population was out of equilibrium. The expected values for -/-, ΔF508/-, and ΔF508/ΔF508 genotypes
were respectively: 32.22% (60.48 patients), 47.93% (80.04 patients), and 15.86% (26.48 patients).
Conclusions: In the analyzed population, ΔF508 mutation was less prevalent than the allele without this
mutation. The frequency observed in this study was similar to that from other areas in Brazil and in the world,
mainly due to the predominantly Caucasian origin of the population included in the study.
J Pediatr (Rio J). 2012;88(6):531-4: Mucoviscidosis, ΔF508, genotype.
Introduction
Cystic fibrosis (CF) is the most frequent recessive
region, cause loss of function of the CFTR protein, which
autosomal disease in the population, especially that of
in normal conditions acts as a chloride channel,2-4 leading
Caucasians. CF incidence varies according to ethnicity and,
to CF.
in Brazil, its prevalence is estimated to be from 1/3,500
More than 1,900 mutations in the CFTR gene were
to 1/10,000 live births, depending on the geographical
identified, but the first to be identified and also the most
area.1
frequent is the ∆F508 mutation, present in nearly 70% of
Mutations in the cystic fibrosis transmembrane
CF cases, depending on the analyzed population.5 In some
conductance regulator (CFTR) gene, located at the 7q31
populations, it is possible to easily identify all mutations in
1. Biomedical scientist. Specialist, Genética Molecular e Citogenética, Faculdade de Ciências Médicas (FCM), Universidade Estadual de Campinas (UNICAMP),
Campinas, SP, Brazil. Departamento de Genética Médica, UNICAMP, Campinas, SP, Brazil.
2. MSc, Saúde da Criança e do Adolescente. PhD candidate, Saúde da Criança e do Adolescente, Departamento de Pediatria, FCM, UNICAMP, Campinas, SP,
Brazil.
3. PhD. Professor, Departamento de Genética Médica, FCM, UNICAMP, Campinas, SP, Brazil.
No conflicts of interest declared concerning the publication of this article.
Financial support: Fundação de Apoio ao Ensino, à Pesquisa e à Extensão (FAEPEX).
Suggested citation: Bieger AM, Marson FA, Bertuzzo CS. Prevalence of ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator gene among
cystic fibrosis patients from a Brazilian referral center. J Pediatr (Rio J). 2012;88(6):531-4.
Manuscript submitted Feb 29 2012, accepted for publication Jun 06 2012.
http://dx.doi.org/10.2223/JPED.2225
531
532 Jornal de Pediatria - Vol. 88, No. 6, 2012
ΔF508 mutation in the CFTR gene in cystic fibrosis - Bieger AM et al.
the CFTR gene present in the patients; however, in mixed
genotype frequency of ΔF508 mutation found in the study
populations, such as the Brazilian one, it is not feasible
of Bernardino et al.9 and that of the present analysis was
yet. The ∆F508 mutation occurs due to a deletion of three
assessed with the Statistical Package for the Social Sciences
bases in exon 10, resulting in the loss of the amino acid
v.17.0 software.10
phenylalanine at position 508, which leads to a deficiency
in CFTR folding and subsequently to a degradation in the
rough endoplasmic reticulum.6
The project was approved by the research ethics
committee of the institution according to judgment
#528/2008.
The CFTR protein promotes chlorine reabsorption in sweat
glands, but in CF the CFTR is absent in the epithelium or
presents qualitative or quantitative changes in its level of
Results
expression; thus, chlorine is not reabsorbed, causing high
A total of 167 samples were analyzed. Of these, the
concentrations of ions in sweat. This dysfunction can affect
samples that had the ΔF508 mutation were amplified again
several organs, particularly those which secrete mucus,
to confirm the result. The results of the descriptive statistical
including upper and lower airways, pancreas, biliary tract,
analysis of genotypes with ΔF508 mutation are shown in Table
male genitalia, intestine, and sweat glands.7 The main
1. Hardy-Weinberg equilibrium was calculated, obtaining a
morbidity and mortality factor for CF is accumulation of
chi-square value = 16.34 (p ≤ 0.001). The study population
secretion in the lungs, leading to their obstruction. Clinical
was out of equilibrium. The expected values for -/-, ΔF508/-,
presentation, disease severity, and rate of CF progression
and ΔF508/ΔF508 genotypes were respectively: 32.22%
vary considerably, and some variations may occur due to
(60.48 patients), 47.93% (80.04 patients), and 15.86%
presence of different combinations of mutations in the CFTR
(26.48 patients).
gene. Among homozygous patients for ∆F508 mutation,
the severity of the pulmonary disease is variable, and the
reasons for the low pulmonary correlation between genotype
Discussion
and phenotype are not clear; however, this mutation in
Among the 167 patients analyzed, 73 (43.7%) did not
homozygosis, or combined with another severe mutation,
have the ΔF508 mutation; 55 (32.9%) were heterozygous
leads to the classical picture of CF.8
who had a mutated allele for ΔF508, being thus compound
Considering the importance of ∆F508 mutation in CF,
due to its high frequency and severity, the aim of this
study was to verify the presence of ΔF508 mutation in
heterozygous; and 39 (23.4%) were homozygous, with two
mutated alleles for ΔF508. All patients had CF previously
diagnosed by the sweat test for sodium and chlorine.
the CFTR gene in patients diagnosed with CF by the sweat
The calculation of Hardy-Weinberg equilibrium performed
test for sodium and chlorine and followed at the Pediatric
in this study showed that the genotype distribution of this
Pneumology Outpatient Clinic of Universidade Estadual
mutation in our sample is not in agreement with Hardy-
de Campinas (UNICAMP), Brazil, a referral center for the
Weinberg’s law. This may be happening because many
treatment of CF.
homozygous individuals for this mutation presented with a
more dramatic disease picture, having thus a higher chance
Methods
of being diagnosed, while other genotypes associated
with more benign mutations would have a late diagnosis.
We analyzed 167 peripheral blood DNA samples from
Moreover, in this population there is a strong environmental
patients diagnosed with CF coming from the Pediatric
influence on the severity of clinical manifestations, which
Pneumology Outpatient Clinic of Hospital de Clínicas of
could interfere in Hardy-Weinberg equilibrium.
UNICAMP who showed abnormal values (above 60 mEq/L) in
The allele frequency of ΔF508 mutation was 39.82%
the sweat test for sodium and chlorine. DNA was extracted
in the analyzed population. In a study conducted in São
using the phenol-chloroform technique.
Paulo, Brazil, Okay et al.2 found an allele frequency of
DNA amplification to identify ΔF508 mutation was
44.5% for ΔF508 mutation, higher to that observed in the
performed in the 167 samples using the polymerase chain
present study. Another study of allele frequency in Minas
reaction (PCR) technique, and PCR results were visualized
Gerais, Brazil, and São Paulo showed lower values than
by 12% denaturing polyacrylamide gel electrophoresis.
those of the present study, 21.7 and 33% respectively.11,12
Descriptive statistical analysis was performed by calculating
In comparison, the work of Bernardino et al.,9 in which 160
allele and genotype frequencies of ΔF508 mutation in the
samples of CF patients were analyzed for several mutations
CFTR gene for the study sample. Hardy-Weinberg equilibrium
in the CFTR gene, found a genotype frequency of ΔF508
was calculated using the Hardy-Weinberg Equilibrium
mutation of respectively 47 (29.4%), 61 (38.1%), and 52
Calculator – Online Encyclopedia for Genetic Epidemiology
(32.5%) for the genotypes ΔF508/ΔF508, ΔF508/-, and
Studies software (2008) to verify if the genotype distribution
for patients with two unidentified mutations. Comparing
of ΔF508 mutation is balanced. The association between the
our data with those from the survey of Bernardino et al.,9
Jornal de Pediatria - Vol. 88, No. 6, 2012 533
ΔF508 mutation in the CFTR gene in cystic fibrosis - Bieger AM et al.
Table 1 -
Calculation of genotype frequency for ΔF508 mutation
Genotypes
Genotype
frequency (n)
Percentage
of genotypes
Cumulative
percentage
-/-
73
43.7
43.7
ΔF508/-
55
32.9
76.6
ΔF508/ΔF508
39
23.4
100.0
167
100.0
Total
(-) = absence of ΔΔF508 allele; n = sample size in absolute number of patients.
there was no statistically significant difference (p = 0.109).
and 39 (23.4%) homozygous patients, with two mutated
Anyway, the values obtained were close, and the small
alleles for ΔF508. These data corroborate the importance
variation observed between the studies can be attributed
of using ΔF508 mutation as a diagnostic tool and mainly as
to the ethnic variation in each microregion.
a factor to be considered for a better genetic counseling.
Because CF is a recessive autosomal genetic disease with
high morbidity and mortality rates, the inclusion of testing
for CF in the neonatal screening has become extremely
And finally, with the development of new specific therapies
for each mutation, screening for ΔF508 will be important,
due to its high frequency in the population.
important. Molecular analysis is essential in order to obtain
To determine patient’s genotype for other mutations in
an accurate diagnosis. The molecular test performed in
the CFTR gene associated with CF, the analyzed population
this study is a worldwide spread exam that, combined with
needs to be assessed by molecular analysis for other changes
neonatal screening, confers better treatment conditions
in the gene using different molecular techniques, such as
and allows for genetic counseling for parents before a
sequencing and other methods of gene analysis.
new pregnancy occurs.6 Therefore, molecular analysis
should begin with ΔF508 mutation, and when the patient
is negative for this mutation, molecular analysis becomes
complex due to the high number of existing mutations in
the CFTR gene.
The molecular characterization of the disease enables
genetic counseling and appropriate pulmonary surveillance,
which may become even more important as therapeutic
Acknowledgements
The authors would like to thank the staff of the Molecular
Genetics Laboratory from the School of Medicine of UNICAMP
for their support in this study and the interdisciplinary group
of the Pediatric Outpatient Clinic of UNICAMP.
advances improve prognosis and allow for the development
of new pharmaceutical methodologies that could play
a role in the correction of CF phenotype, emphasizing
the importance of the genotyping of each patient during
diagnosis. CFTR pharmacotherapy aims to improve
intracellular transportation, its expression and function;
therefore, these treatments are directed to a determined
class of specific mutation or to only one mutation.13
In conclusion, the study population showed that ΔF508
mutation was less prevalent compared with the sum of the
other mutant alleles. The frequency was found to be close
to that of other regions in Brazil and in the world, mainly
due to the predominantly Caucasian origin of the population
included in the study. In this study, allele frequency for
ΔF508 mutation was 39.82%, with 55 heterozygous
patients (32.9%), showing a mutated allele for ΔF508,
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Correspondence:
Fernando Augusto de Lima Marson
Departamento de Pediatria
Universidade Estadual de Campinas
Rua Tessália Vieira de Camargo, 126
Cidade Universitária, Zeferino Vaz
CEP 13083-887 - Campinas, SP - Brazil
Tel.:+55 (19) 3521.8902
E-mail: [email protected]