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Jornal de Pediatria ISSN: 0021-7557 [email protected] 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 Available in: http://www.redalyc.org/articulo.oa?id=399738193014 How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Non-profit academic project, developed under the open access initiative 0021-7557/12/88-06/531 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, References 1. Okay TS, Oliveira WP, Raiz-Júnior R, Rodrigues JC, Del Negro GM. Frequency of the deltaF508 mutation in 108 cystic fibrosis patients in Sao Paulo: comparison with reported Brazilian data. Clinics (Sao Paulo). 2005;60:131-4. 2. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989;245:1066‑73. 3. Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science. 1989;245:1059-65. 4. Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, et al. Identification of the cystic fibrosis gene: genetic analysis. Science. 1989;245:1073-80. 5. Cystic Fibrosis Mutation Database (online). www.genet.sickkids. on.ca/cftr/. Access: 20/11/2011. 534 Jornal de Pediatria - Vol. 88, No. 6, 2012 6. Ko YH, Thomas PJ, Delannoy MR, Pedersen PL. The cystic fibrosis transmembrane conductance regulator. Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein. J Biol Chem. 1993;268:24330-8. 7. Alvarez AE, Ribeiro AF, Hessel G, Bertuzzo CS, Ribeiro JD. Fibrose cística em um centro de referência no Brasil: características clínicas e laboratoriais de 104 pacientes e sua associação com o genótipo e a gravidade da doença. J Pediatr (Rio J). 2004;80:371-9. 8. Mickle JE, Cutting GR. Genotype-phenotype relationships in cystic fibrosis. Med Clin North Am. 2000;84:597-607. 9. Bernardino AL, Ferri A, Passos-Bueno MR, Kim CE, Nakaie CM, Gomes CE, et al. Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations. Genet Test. 2000;4:69‑74. 10.SPSS 17.0 for Windows (computer program). Statistical Package for Social Science (SPSS). Release Version 17.0.1. Chicago, IL: SPSS Incorporation; 2011. ΔF508 mutation in the CFTR gene in cystic fibrosis - Bieger AM et al. 11.Martins CS, Ribeiro F, Costa FF. Frequency of the cystic fibrosis delta F 508 mutation in a population from São Paulo State, Brazil. Braz J Med Biol Res. 1993;26:1037-40. 12.Vidigal PV, Reis FJ, Boson WL, De Marco LA, Brasileiro-Filho G. p.F508del in a heterogeneous cystic fibrosis population from Minas Gerais, Brazil. Braz J Med Biol Res. 2008;41:643-7. 13.Oliveira YA. Fibrose quística [dissertação]. Covilhã, Portugal: Universidade da Beira Interior; 2008. 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]