Analysis of the &lt;FONT FACE=Symbol&gt;D&lt;/font&gt;F508 mutation in a Brazilian cystic fibrosis population: comparison of pulmonary status of homozygotes with other patients

Maróstica, P.J.C.; Raskin, S.; Abreu-e-Silva, F.A.

doi:10.1590/S0100-879X1998000400009

Abstract

Sixty-one cystic fibrosis patients admitted for check-up or antibiotic treatment were enrolled for genetic and clinical evaluation. Genetic analysis was performed on blood samples stored on neonatal screening cards using PCR techniques to determine the presence of DF508 mutations. Clinical evaluation included Shwachman and Chrispin-Norman scores, age at onset of symptoms and diagnosis, spirometry, awake and sleep pulse oximetry, hyponychial angle measurement and presence of chronic Pseudomonas aeruginosa colonization. Eighteen patients (29.5%) were homozygous for the DF508 mutation, 26 (42.6%) had one DF508 mutation and 17 (27.9%) were noncarriers, corresponding to a 50.8% prevalence of the mutation in the whole population. Analysis by the Kruskal-Wallis test for comparison of genetic status with continuous variables or by the chi-square test and logistic regression for dichotomous variables showed no significant differences between any two groups for a = 0.05. We conclude that genetic status in relation to the DF508 mutation is not associated with pulmonary status as evaluated by the above variables

cystic fibrosis; DF508 gene mutation; genotype; phenotype

Braz J Med Biol Res, April 1998, Volume 31(4) 529-532

Analysis of the DF508 mutation in a Brazilian cystic fibrosis population: comparison of pulmonary status of homozygotes with other patients

P.J.C. Maróstica¹, S. Raskin² and F.A. Abreu-e-Silva¹

¹Unidade de Pneumologia Pediátrica, Hospital de Clínicas de Porto Alegre,

Porto Alegre, RS, Brasil

²Division of Genetics, Pediatrics Department, Vanderbilt University, Nashville, TN, USA and Departamento de Genética, Universidade Federal do Paraná, Curitiba, PR, Brasil

References

Abstract

Introduction

Material and Methods

Results

Correspondence and Footnotes ^{Correspondence and Footnotes} Correspondence and Footnotes

Sixty-one cystic fibrosis patients admitted for check-up or antibiotic treatment were enrolled for genetic and clinical evaluation. Genetic analysis was performed on blood samples stored on neonatal screening cards using PCR techniques to determine the presence of DF508 mutations. Clinical evaluation included Shwachman and Chrispin-Norman scores, age at onset of symptoms and diagnosis, spirometry, awake and sleep pulse oximetry, hyponychial angle measurement and presence of chronic Pseudomonas aeruginosa colonization. Eighteen patients (29.5%) were homozygous for the DF508 mutation, 26 (42.6%) had one DF508 mutation and 17 (27.9%) were noncarriers, corresponding to a 50.8% prevalence of the mutation in the whole population. Analysis by the Kruskal-Wallis test for comparison of genetic status with continuous variables or by the chi-square test and logistic regression for dichotomous variables showed no significant differences between any two groups for a = 0.05. We conclude that genetic status in relation to the DF508 mutation is not associated with pulmonary status as evaluated by the above variables.

Key words: cystic fibrosis, DF508 gene mutation, genotype, phenotype

Cystic fibrosis (CF) is the most common autosomal recessive disorder among Caucasians. Its incidence is around 1 per 2000 live births in most white populations (1). Since the discovery of the gene responsible for coding the protein involved, a chloride channel called cystic fibrosis transmembrane conductance regulator, many mutations causing the disease have been described. The most common among them is DF508, which consists of a three-base pair deletion leading to the loss of one phenylalanine at residue number 508 of the involved protein (2-4).

Many studies have been performed so far to analyze DNA from CF patients and to compare homozygotes for this mutation with heterozygotes and with carriers of two different mutations. Most results have been consistent with respect to the status of pancreatic sufficiency, showing a greater prevalence of insufficiency among homozygotes, but they have diverged as to the evaluation of pulmonary involvement (5-14). For this reason, it is important to study pulmonary involvement in every CF group to search for correlations between genotype and phenotype.

In the present study we compared the pulmonary involvement of cystic fibrosis patients having or not a deletion of phenylalanine at residue 508.

A cross-sectional study was conducted on CF patients seen at Hospital de Clínicas de Porto Alegre, Brazil, during their hospital stay for antibiotic treatment or check-up. The diagnosis had been previously confirmed by two sweat tests and clinical evidence of disease. The patients were invited to enter the study and their parents signed a written consent. The study was approved by the Ethics Committee of the hospital.

Sixty-one patients (38 boys and 23 girls aged 4 months to 17 years) were studied over a period of 24 months.

Blood samples (three drops) were collected on neonatal screening cards, stored in plastic bags and mailed to the Division of Genetics of Vanderbilt University for genetic analysis. Blood was then extracted and DNA amplified by the polymerase chain reaction (PCR), followed by gel electrophoresis for identification of the DF508 mutation.

Patients were evaluated in terms of Shwachman score as modified by Doershuk et al. (15) and Chrispin-Norman score (16), age at onset of symptoms and at diagnosis, spirometry, pulse oximetry while sleeping and awake, hyponychial angle measurement and colonization with Pseudomonas aeruginosa. Only patients aged 6 years or older were eligible for pulmonary function tests. Vital capacity, forced expiratory volume in one second (FEV1), and forced expiratory flow rate in the middle half of expiration (FEF 25-75%) were determined. Awake oximetry was performed with an Ohmeda 37OO pulse oximeter measuring hemoglobin saturation with the patient at rest; when saturation was evaluated during sleep, at night, mean value and percentage of total study time under 91 or 86% were obtained by transferring results from the device eight-hour memory to a personal computer. Hyponychial angle measurement was performed by the shadowgram technique that employs a light source and the projection of finger edge onto a white surface for angle measurement (17). All evaluations were made during a three-day period just prior to patient discharge, when clinical conditions were considered to be best.

The patient would be rated as chronically colonized by P. aeruginosa when at least three sputum samples were positive for this microorganism for at least 3 months and at the moment of evaluation, as also done by others (18).

For statistical analysis we employed SAS and SPSS programs; methods included the Kruskal-Wallis test for continuous variables or the chi-square test and logistic regression for dichotomous ones, with the level of significance set at a = 0.05. The power of the sample size was 90%, except for spirometry which only 28 patients were able to perform (65% power).

General findings

Sixty-one patients of a total of 86 under treatment at the center during the study period were included. There were 38 males and 23 females (chi-square = 3.69, P = 0.05) ranging in age from 4 months to 17.6 years. All were white (Table 1).

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Eighteen patients (29.5%) had 2 DF508 mutations, 26 (42.6%) had one DF508 mutation and 17 (27.9%) were noncarriers, reflecting a 50.8% prevalence of this mutation in the population as a whole.

Comparison of the three groups showed no difference between them for any of the variables studied (Table 2). Pseudomonas aeruginosa colonization was more frequent among homozygotes for the DF508 mutation and this difference almost reached significance (P = 0.08, logistic regression). When age was included in the regression model, the difference was shown to be related to this variable rather than to genetic group (odds ratio 1.034, 95% confidence interval 1.017-1.051) (Table 3).

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As was the case for most other studies, we were unable to show any differences in pulmonary involvement when only the DF508 mutation was considered (11,19-24). This is a simplification of the issue linking genotype and phenotype since many different mutations are included in the heterozygote and noncarrier groups and also because other genetic and environmental factors may play a part. Even so, we thought it would be important to perform this study because results have been variable depending on the population analyzed. Also we might find a predictable pattern of pulmonary disease among the groups studied, at least for the homozygotes, as is the case for pancreatic involvement (5-12).

Although some investigators have found differences between DF508 groups with respect to some variables of pulmonary involvement and P. aeruginosa colonization (11,13,25-27), we did not. When we first analyzed the results, we found a close to significant difference in bacterial colonization between homozygotes and noncarriers of the mutation. We thought that this may have happened because of the small sample size and that increasing the number of patients might give us significant results. But when age was included in the logistic regression model we found that this was a confounding variable. The lack of analysis of other variables involved may have led to divergent results by different authors. Also, most studies do not standardize time of data collection which may oscillate significantly when patients with unstable pulmonary conditions are included in the studies.

No significant differences were found between genetic groups when only the DF508 mutation was analyzed. Thus, information about patient genetic status in relation to this mutation alone does not allow any conclusion with respect to pulmonary status.

Address for correspondence: P.J.C. Maróstica, Unidade de Pneumologia Pediátrica, Hospital das Clínicas de Porto Alegre, Av. Ramiro Barcelos, 2350, Sala 1131, 90035-003 Porto Alegre, RS, Brasil. E-mail: pmarostica@nutecnet.com.br

Received June 4, 1997. Accepted January 15, 1998.

Discussion

1. Mearns MB (1993). Cystic fibrosis: the first 50 years, a review of the clinical problems and their management. In: Dodge JA, Brock DJH & Widdicombe JH (Editors), Cystic Fibrosis Current Topics John Wiley and Sons, Chichester.
2. Riordan JR, Rommens JM, Kerem BS, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok L, Plavsik N, Chou JL, Drumm ML, Ianuzzi MC, Collins FS & Tsui LC (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science, 245: 1066-1073.
3. Kerem BS, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M & Tsui LC (1989). Identification of the cystic fibrosis gene: genetic analysis. Science, 245: 1073-1080.
4. Rommens JM, Ianuzzi MC, Kerem BS, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole JL, Kennedy D, Hidaka N, Zsiga M, Buchwald M, Riordan JR, Tsui LC & Collins F (1989). Identification of the cystic fibrosis gene: chromosome walking and jumping. Science, 245: 1059-1065.
5. Hamosh A & Cutting GR (1993). Genotype/phenotype relationships in cystic fibrosis. In: Dodge JA, Brock DJH & Widdicombe JH (Editors), Cystic Fibrosis Current Topics John Wiley and Sons, Chichester.
6. Pignatti PF (1991). Cystic fibrosis gene mutations and correlation with clinical manifestations. Pathologie Biologie, 39: 582-584.
7. Santis G, Osborne L, Knight RA & Hodson ME (1990). Linked marker haplotypes and the DF508 mutation in adults with mild pulmonary disease and cystic fibrosis. Lancet, 335: 1426-1429.
8. Lanng S, Schwartz M, Thorsteinsson B & Koch C (1991). Endocrine and exocrine pancreatic function and the DF508 mutation in cystic fibrosis. Clinical Genetics, 40: 345-348.
9. Kerem E, Corey M, Kerem BS, Rommens J, Markiewicz D, Levison H, Tsui LC & Durie P (1990). The relation between genotype and phenotype in cystic fibrosis - analysis of the most common mutation (DF508). New England Journal of Medicine, 323: 1517-1522.
10. Santis G, Osborne L, Knight R & Hodson ME (1990). Independent genetic determinants of pancreatic and pulmonary status in cystic fibrosis. Lancet, 336: 1081-1084.
11. The Cystic Fibrosis Genotype-Phenotype Consortium (1993). Correlation between genotype and phenotype in patients with cystic fibrosis. New England Journal of Medicine, 329: 1308-1313.
12. Rosenstein BJ (1994). Genotype-phenotype correlations in cystic fibrosis. Lancet, 343: 746-747.
13. Gan K-H, Veeze HJ, Ouweland MW, Halley DJJ, Scheffer H, Hout A, Overbeek SE, Jongste JC, Bakker W & Heijerman HGM (1995). A cystic fibrosis mutation associated with mild lung disease. New England Journal of Medicine, 333: 95-99.
14. Alton EWFW (1996). A mild variant of cystic fibrosis. Thorax, 51 (Suppl 2): S51-S54.
15. Doershuk CF, Matthews LW, Tucker AS, Nudelman H, Eddy G, Wise M & Spector S (1964). A 5 year clinical evaluation therapeutic program for patients with cystic fibrosis. Journal of Pediatrics, 65: 677-693.
16. Chrispin AR & Norman AP (1974). The systematic evaluation of the chest radiograph in cystic fibrosis. Pediatric Radiology, 2: 101-106.
17. Sinniah D & Omar A (1979). Quantitation of digital clubbing by shadowgram technique. Archives of Diseases in Childhood, 54: 145-146.
18. Wilmott RW, Tyson SL & Matthew DJ (1985). Cystic fibrosis survival rates. The influences of allergy and Pseudomonas aeruginosa American Journal of Diseases in Children, 139: 669-671.
19. Campbell III PW & Phillips III JA (1992). The cystic fibrosis gene and relationships to clinical status. Seminars in Respiratory Infections, 7: 150-157.
20. Campbell III PW, Phillips III JA, Krishnamani MRS, Maness KJ & Hazinski TA (1991). Cystic fibrosis: relationship between clinical status and F508 deletion. Journal of Pediatrics, 118: 239-241.
21. Burke W, Aitken ML, Chen SH & Scott R (1992). Variable severity in pulmonary disease in adults with identical cystic fibrosis mutations. Chest, 102: 506-509.
22. Al-Jader LN, Meredith AL, Ryley HC, Cheadle JP, Maguire S, Owen G, Goodchild MC & Harper PS (1992). Severity of chest disease in cystic fibrosis patients in relation to their genotypes. Journal of Medical Genetics, 29: 883-887.
23. O'Rawe A, McIntosh I, Dodge JA, Brock DJH, Redmond AOB, Ward R & MacPherson JS (1992). Increased energy expenditure in cystic fibrosis is associated with specific mutations. Clinical Science, 82: 71-76.
24. Packe GE, Miedzybrodska ZH, Russel G, Friend JAR, Haites NE, Kelly KF & Dean JCS (1992). Comparison of clinical features in cystic fibrosis patients with different genotypes. Thorax, 47: 866 (Abstract).
25. Johansen HK, Nir M, Hřfiby N, Koch C & Schwarts M (1991). Severity of cystic fibrosis in patients homozygous and heterozygous for delta F508 mutation. Lancet, 337: 631-634.
26. Liechti-Gallati S, Bonsall I, Malik N, Schneider V, Kraemer LG, Ruedeberg A, Moser H & Kraemer R (1992). Genotype/phenotype association in cystic fibrosis: analysis of the delta F508, R553X and 3905insT mutations. Pediatric Research, 32: 175-178.
27. Borgo G, Gasparini P, Bonizzato A, Cabrini G, Mastella G & Pignatti PF (1993). Cystic fibrosis: the delta F508 mutation does not lead to an exceptionally severe phenotype. A cohort study. European Journal of Pediatrics, 152: 1006-1011.

Correspondence and Footnotes

Publication Dates

Publication in this collection
06 Oct 1998
Date of issue
Apr 1998

History

Accepted
15 Jan 1998
Received
04 June 1997

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] 1. Mearns MB (1993). Cystic fibrosis: the first 50 years, a review of the clinical problems and their management. In: Dodge JA, Brock DJH & Widdicombe JH (Editors), Cystic Fibrosis Current Topics John Wiley and Sons, Chichester.

[2] 2. Riordan JR, Rommens JM, Kerem BS, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok L, Plavsik N, Chou JL, Drumm ML, Ianuzzi MC, Collins FS & Tsui LC (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science, 245: 1066-1073.

[3] 3. Kerem BS, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M & Tsui LC (1989). Identification of the cystic fibrosis gene: genetic analysis. Science, 245: 1073-1080.

[4] 4. Rommens JM, Ianuzzi MC, Kerem BS, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole JL, Kennedy D, Hidaka N, Zsiga M, Buchwald M, Riordan JR, Tsui LC & Collins F (1989). Identification of the cystic fibrosis gene: chromosome walking and jumping. Science, 245: 1059-1065.

[5] 5. Hamosh A & Cutting GR (1993). Genotype/phenotype relationships in cystic fibrosis. In: Dodge JA, Brock DJH & Widdicombe JH (Editors), Cystic Fibrosis Current Topics John Wiley and Sons, Chichester.

[6] 6. Pignatti PF (1991). Cystic fibrosis gene mutations and correlation with clinical manifestations. Pathologie Biologie, 39: 582-584.

[7] 7. Santis G, Osborne L, Knight RA & Hodson ME (1990). Linked marker haplotypes and the DF508 mutation in adults with mild pulmonary disease and cystic fibrosis. Lancet, 335: 1426-1429.

[8] 8. Lanng S, Schwartz M, Thorsteinsson B & Koch C (1991). Endocrine and exocrine pancreatic function and the DF508 mutation in cystic fibrosis. Clinical Genetics, 40: 345-348.

[9] 9. Kerem E, Corey M, Kerem BS, Rommens J, Markiewicz D, Levison H, Tsui LC & Durie P (1990). The relation between genotype and phenotype in cystic fibrosis - analysis of the most common mutation (DF508). New England Journal of Medicine, 323: 1517-1522.

[10] 10. Santis G, Osborne L, Knight R & Hodson ME (1990). Independent genetic determinants of pancreatic and pulmonary status in cystic fibrosis. Lancet, 336: 1081-1084.

[11] 11. The Cystic Fibrosis Genotype-Phenotype Consortium (1993). Correlation between genotype and phenotype in patients with cystic fibrosis. New England Journal of Medicine, 329: 1308-1313.

[12] 12. Rosenstein BJ (1994). Genotype-phenotype correlations in cystic fibrosis. Lancet, 343: 746-747.

[13] 13. Gan K-H, Veeze HJ, Ouweland MW, Halley DJJ, Scheffer H, Hout A, Overbeek SE, Jongste JC, Bakker W & Heijerman HGM (1995). A cystic fibrosis mutation associated with mild lung disease. New England Journal of Medicine, 333: 95-99.

[14] 14. Alton EWFW (1996). A mild variant of cystic fibrosis. Thorax, 51 (Suppl 2): S51-S54.

[15] 15. Doershuk CF, Matthews LW, Tucker AS, Nudelman H, Eddy G, Wise M & Spector S (1964). A 5 year clinical evaluation therapeutic program for patients with cystic fibrosis. Journal of Pediatrics, 65: 677-693.

[16] 16. Chrispin AR & Norman AP (1974). The systematic evaluation of the chest radiograph in cystic fibrosis. Pediatric Radiology, 2: 101-106.

[17] 17. Sinniah D & Omar A (1979). Quantitation of digital clubbing by shadowgram technique. Archives of Diseases in Childhood, 54: 145-146.

[18] 18. Wilmott RW, Tyson SL & Matthew DJ (1985). Cystic fibrosis survival rates. The influences of allergy and Pseudomonas aeruginosa American Journal of Diseases in Children, 139: 669-671.

[19] 19. Campbell III PW & Phillips III JA (1992). The cystic fibrosis gene and relationships to clinical status. Seminars in Respiratory Infections, 7: 150-157.

[20] 20. Campbell III PW, Phillips III JA, Krishnamani MRS, Maness KJ & Hazinski TA (1991). Cystic fibrosis: relationship between clinical status and F508 deletion. Journal of Pediatrics, 118: 239-241.

[21] 21. Burke W, Aitken ML, Chen SH & Scott R (1992). Variable severity in pulmonary disease in adults with identical cystic fibrosis mutations. Chest, 102: 506-509.

[22] 22. Al-Jader LN, Meredith AL, Ryley HC, Cheadle JP, Maguire S, Owen G, Goodchild MC & Harper PS (1992). Severity of chest disease in cystic fibrosis patients in relation to their genotypes. Journal of Medical Genetics, 29: 883-887.

[23] 23. O'Rawe A, McIntosh I, Dodge JA, Brock DJH, Redmond AOB, Ward R & MacPherson JS (1992). Increased energy expenditure in cystic fibrosis is associated with specific mutations. Clinical Science, 82: 71-76.

[24] 24. Packe GE, Miedzybrodska ZH, Russel G, Friend JAR, Haites NE, Kelly KF & Dean JCS (1992). Comparison of clinical features in cystic fibrosis patients with different genotypes. Thorax, 47: 866 (Abstract).

[25] 25. Johansen HK, Nir M, Hřfiby N, Koch C & Schwarts M (1991). Severity of cystic fibrosis in patients homozygous and heterozygous for delta F508 mutation. Lancet, 337: 631-634.

[26] 26. Liechti-Gallati S, Bonsall I, Malik N, Schneider V, Kraemer LG, Ruedeberg A, Moser H & Kraemer R (1992). Genotype/phenotype association in cystic fibrosis: analysis of the delta F508, R553X and 3905insT mutations. Pediatric Research, 32: 175-178.

[27] 27. Borgo G, Gasparini P, Bonizzato A, Cabrini G, Mastella G & Pignatti PF (1993). Cystic fibrosis: the delta F508 mutation does not lead to an exceptionally severe phenotype. A cohort study. European Journal of Pediatrics, 152: 1006-1011.