The Cystic Fibrosis Gene Is Not Likely to be Involved in Chronic Obstructive Pulmonary Disease P. Gasparini, A. Savoia, M. Luisetti, V. Peona, and P. F. Pignatti Institute of Biological Sciences, University of Verona, Verona, and Institute of Respiratory Diseases, University of Pavia, Pavia, Italy
The etiology of chronic obstructive pulmonary disease (COPD) is still unknown, and both genetic and environmental factors may playa role. Some clinical aspects of COPD occur similarly in cystic fibrosis (CF), particularly in adult patients. A DNA polymorphic marker in strong linkage disequilibrium with the CF locus has been used to check the hypothesis that mutations of this locus might be involved in COPD. Its allele frequencies have been found to be in equilibrium in COPD patients as well as in appropriate control subjects. The determinants possibly involved in genetic predisposition to COPD therefore do not seem to comprise CF gene mutations.
Chronic obstructive pulmonary disease (COPD) is a condition characterized by chronic airways limitation to airflow, due to chronic bronchitis andlor emphysema. Chronic bronchitis is a condition associated with excessive tracheobronchial mucus production, while emphysema shows a permanent enlargement of the air spaces distal to the terminal bronchiole, accompanied by the destruction of their walls. In many cases, bronchiectasis is also present, which is permanent abnormal dilatation of one or more large bronchi because of the destruction of the elastic and muscular components of the bronchial wall. COPD onset is usually between the fifth and the sixth decade of life. Familial aggregation of COPD has been demonstrated (1, 2), although the observations may also be related to indoor pollution generated by smoking family members. Even if the influence of environmental factors appears to be strong, several studies have suggested a genetic predisposition to the development of the disease. In particular, lung disease is associated with the alpha-l-proteinase inhibitor (aIPI) phenotype ZZ: homozygotes have a risk of developing COPD at least 15 times higher than other individuals. o.Pl deficiency could explain the mechanism of lung damage, as the organism could not respond adequately to alveolar milieu changes (3). Other genetic factors possibly involved are still uncertain. All the above-described manifestations, particularly bronchiectasis, can be present in CF patients, including adult
cases (4,5). CF is the most frequent lethal autosomal recessive disease in Caucasoid populations. The molecular defect is still unknown, but it may be related to a defect in the regulation of chloride channel permeability in sweat gland ducts and airway epithelia (6), as confirmed by the recent discovery of the gene (7-9). Several DNA markers in linkage disequilibrium with the CF locus are known (10), located on chromosome 7 (11). CF can therefore be investigated in families with tightly linked probes. Pancreatic insufficiency in CF patients was found to be associated to DNA marker haplotypes (12, 13). In a recent study, we have determined CF genotypes (14) with the MP6d-9 probe, which is the most closely linked marker to the CF gene presently available (15), and have related them to the clinical course of the patients (16). Patients with the 2/2 genotype were significantly more severely affected than those with the 1/2 genotype. No patient homozygous for allele 1 was observed. We therefore hypothesized that an individual with the III genotype would not present with classic CF, but possibly with a related, less severe, or later-occurring affection such as COPD (16). For all these reasons, CF could be a candidate gene for genetic predisposition to COPD development. In this study, we check the hypothesis that a milder CF mutation, linked to MP6d-9 allele 1, might be associated with the development of COPD, by studying the MP6d-9 genotypes of 16 patients affected by COPD.
Key HiJrds: pulmonary emphysema, chronic bronchitis, bronchiectasis, DNA analysis, restriction fragment length polymorphism
Materials and Methods
(Received in original form August 22, 1989 and in revised form November 15, 1989) Address correspondence to: Professor Pier Franco Pignatti, Istituto di Scienze Biologiche, Universita di Verona, Strada Le Grazie, 37134 Verona, Italy. Abbreviations: alpha-l-proteinase inhibitor, aIPI; cystic fibrosis, CF; chronic obstructive pulmonary disease, COPO. Am. J. Respir, Cell Mol. BioI. Vol. 2. pp. 297-299, 1990
We studied 16 patients affected by COPD, five of them with evidence of bronchiectasis and negative sweat test. There were 8 males and 8 females, with ages ranging from 25 to 80 yr, with a mean age of 60 yr. A family history for the disease was present in 4 cases. Eight patients were previous smokers; two of them were smoking at present. One patient was a CF gene obligate carrier. Control subjects were represented by 16 age- and sex-matched patients, affected by other
298
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990
a
b
c
d 13 kb
.- 8.5 kb
Figure 1. Autoradiogram of cystic fibrosislinked MP6d-9/MspI DNA restriction fragment length polymorphism in 4 patients with chronic obstructive pulmonary disease. The numbers indicate fragment size (kb). The 13 kb fragment corresponds to allele 1; the 2 fragments generated by the presence of the restriction site (8.5 kb and 4.5 kb) correspond to allele 2.
.- 4.5 kb
pulmonary disease (lung cancer,S; thoracic sarcoid, 4; tuberculosis, 3; pneumonia, 2; idiopathic pulmonary fibrosis, 1; and collagen vascular disorder, 1) and treated in the same clinic, and by 12 healthy blood donors. A total of 53 CF patients from the Cystic Fibrosis Center of Verona, and their parents, partially corresponding to those utilized in a previous study (14), were analyzed. We collected 20 ml of fresh whole blood from each individual by venipuncture. DNA was obtained with 2 phenol-chloroform extractions according to standard procedures. After DNA restriction with the MspI endonuclease in the conditions recommended by the suppliers (I.B.I., New Haven, CT; Promega Co., Madison, WI), DNA fragments were separated by agarose-gel electrophoresis and transferred onto nitrocellulose membrane (Hybond C-E). Hybridization was performed with the oligolabeled probe MP6d-9 (Multiprime Amersham International, Buckinghamshire, UK) as described (14). Autoradiograms were developed after an exposure for 24 h at -80 0 C. COPD diagnosis was done according to ATS statement (17). CF diagnosis was confirmed by at least 2 positive sweat tests (18). The chi-square test was used to estimate the difference of proportion. Linkage was calculated with the standardized disequilibrium coefficient, delta (19).
as that of the normal control subjects and of the normal chromosomes of the CF gene carriers (X2 = 1.03, P = NS), whereas it was different from CF patients (X2 = 22.72, P < 0.001). The data also show equilibrium between COPD and normal control gene frequencies (delta = -0.039). Strong linkage disequilibrium of the marker locus alleles in CF patients was instead found (delta = 0.40), as expected (14). The genotypes of COPD patients, as well as those of normal control subjects, were distributed according to Hardy-Weinberg equilibrium. Twenty-five percent (4 of 16) MP6d-9 1/1 homozygotes were found in COPD patients, whereas none were observed in CF patients. It remains to be seen if MP6d-9 1/1 homozygotes have a distinct clinical condition.
Discussion In this report, we have checked if the CF gene could be involved in the etiology and the pathogenesis of COPD by analyzing the frequency of a CF DNA polymorphism in COPD patients and in control subjects. TABLE 1
Distribution of the alleles of a cystic fibrosislinked DNA polymorphism in patients with chronic obstructive pulmonary disease
Results Figure 1 shows an example of the MP6d-9 DNA polymorphism (15) determination in 4 different individuals. The heterozygotes (a and c), and both types of homozygotes (2/2, b; and 1/1, d), are easily distinguished. The results obtained with the DNA analysis of COPD patients, other pulmonary disease patients, and CF subjects and their parents are summarized in Table 1. The distribution of the MP6d-9 alleles among COPD patients was the same
MP6d-9 Polymorphism
COPD Patients
17 (53%) Allele 1 15 (47%) Allele 2 Total number of chromosomes investigated 32 (100%)
Controls
Non-CF Chromosomes of CF Carriers
CF Patients
32 (57%) 24 (43%)
66 (63%) 39 (37%)
14 (13%) 92 (87%)
56 (100%)
105 (100%)
106 (100%)
Gasparini, Savoia, Luisetti et ai.: CF Gene Is Not Likely to be Involved in COPD
That diseases of differing clinical severity, or even formerly classified as different nosologic entities, could be related to mutations in the same gene, has been demonstrated in several cases, for example in the hemoglobinopathies (20), and more recently in Duchenne and Becker muscular dystrophies (21) and in inherited collagen disorders (22). The CF locus was candidate for investigation because pulmonary involvement, probably due to the presence of an abnormal respiratory tract fluid, is very common. In the series of patients here and previously described, it was a constant feature. Pseudomonas colonization, nasal polyposis, pancreatic insufficiency, meconium ileus, and other symptoms are also usually present, which all perhaps may depend on the defective regulation of ion transport in CF epithelial cells. The CF gene has recently been cloned (7,8), and one mutation present in 68 % of CF chromosomes in the Canadian population identified (9). The mutation is associated with MP6d-9 allele 2. These observations confirmed the presence of different mutations that were previously supposed on the basis of the correlation between patient phenotype and genotype (12, 13), and the different frequencies of CF haplotypes in different populations (10, 14). Homozygotes for the mutation are more severely affected than heterozygotes (9); therefore, if the gene were involved in COPD, different milder mutations still to be determined should be sought. They are predicted to be associated with MP6d-9 allele 1. For the purpose of this investigation, we have utilized a recently described DNA marker, very tightly associated with the CF locus on chromosome 7. We have determined the distribution of CF genotypes in COPD patients, compared these data with those obtained in normal control subjects and in CF patients, and calculated linkage. Our results show (1) the lack of association between the CF gene and COPD, as the CF genotypes are distributed in the COPD patients similarly to the control subjects and differently from the CF patients; and (2) that no linkage disequilibrium of the CF locus marker alleles with a hypothetical COPD gene can be detected. Therefore, COPD, even if it may show clinical symptoms similar to CF, can be related neither to a molecular alteration of the same gene, nor to a simple Mendelian inheritance of a second gene located within detectable distance of the CF locus. The possibility cannot be ruled out that CF gene-linked mutations may contribute to COPD development, if they were randomly distributed on the chromosomes tested, but it is unlikely. In conclusion, our data agree with the exclusion of the involvement of the CF gene or of CF locus-linked mutations in the determination or progression of COPD. Further genetic studies are therefore necessary to map and to identify COPD susceptibility genes.
NOTE ADDED IN PRESS: The COPD patients were analyzed for the presence of the gene mutation recently described for CF (9). No COPD patient chromosome had the
299
mutation. No control patient or normal subject had it either, whereas it was present in Italian CF patients. This observation is in accord with the conclusions of this work and with the discussion. Acknowledgments: We thank all patients and their families, Professor G. Mastella, Director of the Cystic Fibrosis Center of Verona, and Professor C. Grassi, Director of the Institute of Respiratory Diseases Clinics of the University of Pavia, for their collaboration; Drs. L. Pasturenzi and A. Bulgheroni for their interest; and Mrs. R. Galavotti for technical assistance. This work was supported by grants from the Italian Ministry of Education (M.P.I.) and from the Progetto Finalizzato Biotecnologie of the Consiglio Nazionale delle Ricerche. Drs. Gasparini and Savoia gratefully acknowledge fellowships from the Cystic Fibrosis Center of Verona.
References I. Higgins, M., and J. Keller. 1975. Familial occurrence of chronic respiratory disease and familial resemblance in ventilatory capacity. J. Chronic Dis. 28:239-251. 2. Lebowitz, M. D., R. J. Knudson, and B. Burrows. 1984. Family aggregation of pulmonary function measurements. Am. Rev. Respir. Dis. 129: 8-11. 3. Vogel, F., and A. G. Motulsky. 1986. Human Genetics. Springer-Verlag, Berlin. 4. Boye, N. P., I. J. K. Skarpaos, and O. Fausa. 1980. Cystic fibrosis in adult patients. Eur. J. Respir. Dis. 61 :227-232. 5. Wanner, A., and M. A. Sackner. 1983. Pulmonary Disease. Little, Brown, Boston. 6. Welsh, M. J., and C. M. Liedtke. 1986. Chloride and potassium channels in cystic fibrosis airway epithelia. Nature 322:467-470. 7. Rommens, J. M., M. C. Iannuzzi, B. Kerem et al. 1989. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059-1065. 8. Riordan, J. R., J. M. Rommens, B. Kerem et al. 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073. 9. Kerem, 8., J. M. Rommens, J. A. Buchanan et al. 1989. Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1080. 10. Tsui, L.-C. 1989. Tracing the mutations in cystic fibrosis by means of closely linked DNA markers. Am. J. Hum. Genet. 44:303-306. II. Duncan, A. M. V., M. Buchwald, and L.-c. Tsui. 1988. In situ hybridization of two cloned chromosome 7 sequences tightly linked to the cystic fibrosis gene. Cytogenet. Cell Genet. 49:309-310. 12. Kerem, B., J. Buchanan, P. Durie etal. 1989. DNA marker haplotype association with pancreatic sufficiency in cystic fibrosis. Am. J. Hum. Genet. 44:827-834. 13. Ferrari, M., M. Antonelli, F. Bellini et al. 1990. Genetic differences in cystic fibrosis patients with and without pancreatic insufficiency-an Italian collaborative study. Hum. Genet. (In press) 14. Estivill, X., P. Gasparini, G. Novelli et al. 1989. Linkage disequilibrium for DNA haplotypes near the cystic fibrosis locus in two South European populations. Hum. Genet. 83:175-178. 15. Estivill, X., C. McLean, V. Nunes et al. 1989. Isolation of a new DNA marker in linkage disequilibrium with cystic fibrosis, situated between 13.11 and IRP. Am. J. Hum. Genet. 44:704-710. 16. Gasparini, P., G. Novelli, X. Estivill et al. 1990. The genotype of a new linked DNA marker, MP6d-9, is related to the clinical course of cystic fibrosis. J. Med. Genet. 27: 17-20. 17. American Thoracic Society. 1987. Standards for diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am. Rev. Respir. Dis. 136:225-244. 18. Gibson, L. E., and R. E. Cooke. 1959. A test for concentration of electrolytes in sweat in cystic fibrosis of pancreas utilizing pilocarpine by iontophoresis. Pediatrics 23:545-551. 19. Ott, J. 1985. Analysis of Human Genetic Linkage. Johns Hopkins University Press, Baltimore. 20. Stomatoyannopulos, G., A. W. Nieuhnis, P. Leder, and P. W. Majerus. 1987. The Molecular Basis of Blood Disease. W. B. Saunders, Philadelphia. 21. Love, D. R., and K. E. Davies. 1989. Duchenne muscular dystrophy: the gene and the protein. Mol. Bioi. Med. 6:7-17. 22. Sykes, B. 1989. Inherited collagen disorders. Mol. BioI. Med. 6: 19-26.
The etiology of chronic obstructive pulmonary disease (COPD) is still unknown, and both genetic and environmental factors may playa role. Some clinical aspects of COPD occur similarly in cystic fibrosis (CF), particularly in adult patients. A DNA polymorphic marker in strong linkage disequilibrium with the CF locus has been used to check the hypothesis that mutations of this locus might be involved in COPD. Its allele frequencies have been found to be in equilibrium in COPD patients as well as in appropriate control subjects. The determinants possibly involved in genetic predisposition to COPD therefore do not seem to comprise CF gene mutations.
Chronic obstructive pulmonary disease (COPD) is a condition characterized by chronic airways limitation to airflow, due to chronic bronchitis andlor emphysema. Chronic bronchitis is a condition associated with excessive tracheobronchial mucus production, while emphysema shows a permanent enlargement of the air spaces distal to the terminal bronchiole, accompanied by the destruction of their walls. In many cases, bronchiectasis is also present, which is permanent abnormal dilatation of one or more large bronchi because of the destruction of the elastic and muscular components of the bronchial wall. COPD onset is usually between the fifth and the sixth decade of life. Familial aggregation of COPD has been demonstrated (1, 2), although the observations may also be related to indoor pollution generated by smoking family members. Even if the influence of environmental factors appears to be strong, several studies have suggested a genetic predisposition to the development of the disease. In particular, lung disease is associated with the alpha-l-proteinase inhibitor (aIPI) phenotype ZZ: homozygotes have a risk of developing COPD at least 15 times higher than other individuals. o.Pl deficiency could explain the mechanism of lung damage, as the organism could not respond adequately to alveolar milieu changes (3). Other genetic factors possibly involved are still uncertain. All the above-described manifestations, particularly bronchiectasis, can be present in CF patients, including adult
cases (4,5). CF is the most frequent lethal autosomal recessive disease in Caucasoid populations. The molecular defect is still unknown, but it may be related to a defect in the regulation of chloride channel permeability in sweat gland ducts and airway epithelia (6), as confirmed by the recent discovery of the gene (7-9). Several DNA markers in linkage disequilibrium with the CF locus are known (10), located on chromosome 7 (11). CF can therefore be investigated in families with tightly linked probes. Pancreatic insufficiency in CF patients was found to be associated to DNA marker haplotypes (12, 13). In a recent study, we have determined CF genotypes (14) with the MP6d-9 probe, which is the most closely linked marker to the CF gene presently available (15), and have related them to the clinical course of the patients (16). Patients with the 2/2 genotype were significantly more severely affected than those with the 1/2 genotype. No patient homozygous for allele 1 was observed. We therefore hypothesized that an individual with the III genotype would not present with classic CF, but possibly with a related, less severe, or later-occurring affection such as COPD (16). For all these reasons, CF could be a candidate gene for genetic predisposition to COPD development. In this study, we check the hypothesis that a milder CF mutation, linked to MP6d-9 allele 1, might be associated with the development of COPD, by studying the MP6d-9 genotypes of 16 patients affected by COPD.
Key HiJrds: pulmonary emphysema, chronic bronchitis, bronchiectasis, DNA analysis, restriction fragment length polymorphism
Materials and Methods
(Received in original form August 22, 1989 and in revised form November 15, 1989) Address correspondence to: Professor Pier Franco Pignatti, Istituto di Scienze Biologiche, Universita di Verona, Strada Le Grazie, 37134 Verona, Italy. Abbreviations: alpha-l-proteinase inhibitor, aIPI; cystic fibrosis, CF; chronic obstructive pulmonary disease, COPO. Am. J. Respir, Cell Mol. BioI. Vol. 2. pp. 297-299, 1990
We studied 16 patients affected by COPD, five of them with evidence of bronchiectasis and negative sweat test. There were 8 males and 8 females, with ages ranging from 25 to 80 yr, with a mean age of 60 yr. A family history for the disease was present in 4 cases. Eight patients were previous smokers; two of them were smoking at present. One patient was a CF gene obligate carrier. Control subjects were represented by 16 age- and sex-matched patients, affected by other
298
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990
a
b
c
d 13 kb
.- 8.5 kb
Figure 1. Autoradiogram of cystic fibrosislinked MP6d-9/MspI DNA restriction fragment length polymorphism in 4 patients with chronic obstructive pulmonary disease. The numbers indicate fragment size (kb). The 13 kb fragment corresponds to allele 1; the 2 fragments generated by the presence of the restriction site (8.5 kb and 4.5 kb) correspond to allele 2.
.- 4.5 kb
pulmonary disease (lung cancer,S; thoracic sarcoid, 4; tuberculosis, 3; pneumonia, 2; idiopathic pulmonary fibrosis, 1; and collagen vascular disorder, 1) and treated in the same clinic, and by 12 healthy blood donors. A total of 53 CF patients from the Cystic Fibrosis Center of Verona, and their parents, partially corresponding to those utilized in a previous study (14), were analyzed. We collected 20 ml of fresh whole blood from each individual by venipuncture. DNA was obtained with 2 phenol-chloroform extractions according to standard procedures. After DNA restriction with the MspI endonuclease in the conditions recommended by the suppliers (I.B.I., New Haven, CT; Promega Co., Madison, WI), DNA fragments were separated by agarose-gel electrophoresis and transferred onto nitrocellulose membrane (Hybond C-E). Hybridization was performed with the oligolabeled probe MP6d-9 (Multiprime Amersham International, Buckinghamshire, UK) as described (14). Autoradiograms were developed after an exposure for 24 h at -80 0 C. COPD diagnosis was done according to ATS statement (17). CF diagnosis was confirmed by at least 2 positive sweat tests (18). The chi-square test was used to estimate the difference of proportion. Linkage was calculated with the standardized disequilibrium coefficient, delta (19).
as that of the normal control subjects and of the normal chromosomes of the CF gene carriers (X2 = 1.03, P = NS), whereas it was different from CF patients (X2 = 22.72, P < 0.001). The data also show equilibrium between COPD and normal control gene frequencies (delta = -0.039). Strong linkage disequilibrium of the marker locus alleles in CF patients was instead found (delta = 0.40), as expected (14). The genotypes of COPD patients, as well as those of normal control subjects, were distributed according to Hardy-Weinberg equilibrium. Twenty-five percent (4 of 16) MP6d-9 1/1 homozygotes were found in COPD patients, whereas none were observed in CF patients. It remains to be seen if MP6d-9 1/1 homozygotes have a distinct clinical condition.
Discussion In this report, we have checked if the CF gene could be involved in the etiology and the pathogenesis of COPD by analyzing the frequency of a CF DNA polymorphism in COPD patients and in control subjects. TABLE 1
Distribution of the alleles of a cystic fibrosislinked DNA polymorphism in patients with chronic obstructive pulmonary disease
Results Figure 1 shows an example of the MP6d-9 DNA polymorphism (15) determination in 4 different individuals. The heterozygotes (a and c), and both types of homozygotes (2/2, b; and 1/1, d), are easily distinguished. The results obtained with the DNA analysis of COPD patients, other pulmonary disease patients, and CF subjects and their parents are summarized in Table 1. The distribution of the MP6d-9 alleles among COPD patients was the same
MP6d-9 Polymorphism
COPD Patients
17 (53%) Allele 1 15 (47%) Allele 2 Total number of chromosomes investigated 32 (100%)
Controls
Non-CF Chromosomes of CF Carriers
CF Patients
32 (57%) 24 (43%)
66 (63%) 39 (37%)
14 (13%) 92 (87%)
56 (100%)
105 (100%)
106 (100%)
Gasparini, Savoia, Luisetti et ai.: CF Gene Is Not Likely to be Involved in COPD
That diseases of differing clinical severity, or even formerly classified as different nosologic entities, could be related to mutations in the same gene, has been demonstrated in several cases, for example in the hemoglobinopathies (20), and more recently in Duchenne and Becker muscular dystrophies (21) and in inherited collagen disorders (22). The CF locus was candidate for investigation because pulmonary involvement, probably due to the presence of an abnormal respiratory tract fluid, is very common. In the series of patients here and previously described, it was a constant feature. Pseudomonas colonization, nasal polyposis, pancreatic insufficiency, meconium ileus, and other symptoms are also usually present, which all perhaps may depend on the defective regulation of ion transport in CF epithelial cells. The CF gene has recently been cloned (7,8), and one mutation present in 68 % of CF chromosomes in the Canadian population identified (9). The mutation is associated with MP6d-9 allele 2. These observations confirmed the presence of different mutations that were previously supposed on the basis of the correlation between patient phenotype and genotype (12, 13), and the different frequencies of CF haplotypes in different populations (10, 14). Homozygotes for the mutation are more severely affected than heterozygotes (9); therefore, if the gene were involved in COPD, different milder mutations still to be determined should be sought. They are predicted to be associated with MP6d-9 allele 1. For the purpose of this investigation, we have utilized a recently described DNA marker, very tightly associated with the CF locus on chromosome 7. We have determined the distribution of CF genotypes in COPD patients, compared these data with those obtained in normal control subjects and in CF patients, and calculated linkage. Our results show (1) the lack of association between the CF gene and COPD, as the CF genotypes are distributed in the COPD patients similarly to the control subjects and differently from the CF patients; and (2) that no linkage disequilibrium of the CF locus marker alleles with a hypothetical COPD gene can be detected. Therefore, COPD, even if it may show clinical symptoms similar to CF, can be related neither to a molecular alteration of the same gene, nor to a simple Mendelian inheritance of a second gene located within detectable distance of the CF locus. The possibility cannot be ruled out that CF gene-linked mutations may contribute to COPD development, if they were randomly distributed on the chromosomes tested, but it is unlikely. In conclusion, our data agree with the exclusion of the involvement of the CF gene or of CF locus-linked mutations in the determination or progression of COPD. Further genetic studies are therefore necessary to map and to identify COPD susceptibility genes.
NOTE ADDED IN PRESS: The COPD patients were analyzed for the presence of the gene mutation recently described for CF (9). No COPD patient chromosome had the
299
mutation. No control patient or normal subject had it either, whereas it was present in Italian CF patients. This observation is in accord with the conclusions of this work and with the discussion. Acknowledgments: We thank all patients and their families, Professor G. Mastella, Director of the Cystic Fibrosis Center of Verona, and Professor C. Grassi, Director of the Institute of Respiratory Diseases Clinics of the University of Pavia, for their collaboration; Drs. L. Pasturenzi and A. Bulgheroni for their interest; and Mrs. R. Galavotti for technical assistance. This work was supported by grants from the Italian Ministry of Education (M.P.I.) and from the Progetto Finalizzato Biotecnologie of the Consiglio Nazionale delle Ricerche. Drs. Gasparini and Savoia gratefully acknowledge fellowships from the Cystic Fibrosis Center of Verona.
References I. Higgins, M., and J. Keller. 1975. Familial occurrence of chronic respiratory disease and familial resemblance in ventilatory capacity. J. Chronic Dis. 28:239-251. 2. Lebowitz, M. D., R. J. Knudson, and B. Burrows. 1984. Family aggregation of pulmonary function measurements. Am. Rev. Respir. Dis. 129: 8-11. 3. Vogel, F., and A. G. Motulsky. 1986. Human Genetics. Springer-Verlag, Berlin. 4. Boye, N. P., I. J. K. Skarpaos, and O. Fausa. 1980. Cystic fibrosis in adult patients. Eur. J. Respir. Dis. 61 :227-232. 5. Wanner, A., and M. A. Sackner. 1983. Pulmonary Disease. Little, Brown, Boston. 6. Welsh, M. J., and C. M. Liedtke. 1986. Chloride and potassium channels in cystic fibrosis airway epithelia. Nature 322:467-470. 7. Rommens, J. M., M. C. Iannuzzi, B. Kerem et al. 1989. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059-1065. 8. Riordan, J. R., J. M. Rommens, B. Kerem et al. 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073. 9. Kerem, 8., J. M. Rommens, J. A. Buchanan et al. 1989. Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1080. 10. Tsui, L.-C. 1989. Tracing the mutations in cystic fibrosis by means of closely linked DNA markers. Am. J. Hum. Genet. 44:303-306. II. Duncan, A. M. V., M. Buchwald, and L.-c. Tsui. 1988. In situ hybridization of two cloned chromosome 7 sequences tightly linked to the cystic fibrosis gene. Cytogenet. Cell Genet. 49:309-310. 12. Kerem, B., J. Buchanan, P. Durie etal. 1989. DNA marker haplotype association with pancreatic sufficiency in cystic fibrosis. Am. J. Hum. Genet. 44:827-834. 13. Ferrari, M., M. Antonelli, F. Bellini et al. 1990. Genetic differences in cystic fibrosis patients with and without pancreatic insufficiency-an Italian collaborative study. Hum. Genet. (In press) 14. Estivill, X., P. Gasparini, G. Novelli et al. 1989. Linkage disequilibrium for DNA haplotypes near the cystic fibrosis locus in two South European populations. Hum. Genet. 83:175-178. 15. Estivill, X., C. McLean, V. Nunes et al. 1989. Isolation of a new DNA marker in linkage disequilibrium with cystic fibrosis, situated between 13.11 and IRP. Am. J. Hum. Genet. 44:704-710. 16. Gasparini, P., G. Novelli, X. Estivill et al. 1990. The genotype of a new linked DNA marker, MP6d-9, is related to the clinical course of cystic fibrosis. J. Med. Genet. 27: 17-20. 17. American Thoracic Society. 1987. Standards for diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am. Rev. Respir. Dis. 136:225-244. 18. Gibson, L. E., and R. E. Cooke. 1959. A test for concentration of electrolytes in sweat in cystic fibrosis of pancreas utilizing pilocarpine by iontophoresis. Pediatrics 23:545-551. 19. Ott, J. 1985. Analysis of Human Genetic Linkage. Johns Hopkins University Press, Baltimore. 20. Stomatoyannopulos, G., A. W. Nieuhnis, P. Leder, and P. W. Majerus. 1987. The Molecular Basis of Blood Disease. W. B. Saunders, Philadelphia. 21. Love, D. R., and K. E. Davies. 1989. Duchenne muscular dystrophy: the gene and the protein. Mol. Bioi. Med. 6:7-17. 22. Sykes, B. 1989. Inherited collagen disorders. Mol. BioI. Med. 6: 19-26.
