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Jornal de Pediatria

Print version ISSN 0021-7557On-line version ISSN 1678-4782

J. Pediatr. (Rio J.) vol.81 no.6 Porto Alegre Nov./Dec. 2005 



Association between alpha 1 antitrypsin deficiency and cystic fibrosis severity



Elisangela Jacinto de FariaI; Isabel Cristina Jacinto de FariaI; Alfonso Eduardo AlvarezII; José Dirceu RibeiroIII; Antônio Fernando RibeiroIV; Carmen Silvia BertuzzoV

IMSc. (Biology), Departamento de Genética, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil
IIMSc. Pediatric pulmonologist, Faculdade de Ciências Médicas, UNICAMP, Campinas, SP, Brazil
IIIPhD. Pediatric pulmonologist. Professor, Departamento de Pediatria, Faculdade de Ciências Médicas, UNICAMP, Campinas, SP, Brazil
IVPhD. Pediatric Gastroenterologist. Professor, Faculdade de Ciências Médicas, UNICAMP, Campinas, SP, Brazil
VPhD. Geneticist. Professor, Faculdade de Ciências Médicas, UNICAMP, Campinas, SP, Brazil





OBJECTIVE: To ascertain the distribution of alpha 1 antitrypsin genotypes and correlate it with the severity of pulmonary disease in patients with cystic fibrosis
METHOD: A clinical and laboratory cross sectional study of 70 patients at the Universidade Estadual de Campinas teaching hospital. Cystic fibrosis diagnoses was confirmed by both clinical and laboratory methods. The severity of cystic fibrosis was evaluated by Shwachman score. All the patients were tested for the presence of S and Z alleles for alpha 1 antitrypsin deficiency using polymerase chain reaction.
RESULTS: Nine (12.8%) patients were heterozygous for S or Z alleles or the heterozygote compound (SZ). No significant differences were found in clinical severity of Cystic fibrosis between genotypes of alpha 1 antitrypsin. No significant differences were found when the patients were divided according to the presence or absence of the DF508 mutation.
CONCLUSION: In this study, the first undertaken in Brazil into the association of alpha 1 antitrypsin deficiency and cystic fibrosis, we did not find an association between the deficiency and cystic fibrosis severity.

Key words: Cystic fibrosis, alpha 1 antitrypsin.




Cystic fibrosis (CF) is the most common and lethal autosomal recessive genetic diseases and affects one in 2,500 Caucasians. The risk of its heterozygote in the general population is one to 25. More than 1,000 mutations have been identified to the CFTR gene1, which codes for a protein containing 1,489 amino acids.

Cystic Fibrosis is characterized by abnormal chlorine flow at the apical membrane of epithelial cells, causing diverse clinical manifestations including pancreatic insufficiency, lung disease, meconium ileus, elevated sweat chlorine levels and obstruction of the vas deferens.

The extreme diversity of CF phenotypes is probably influenced by other genetic areas, distant from the CFTR locus. Many of the genes that are studied nowadays as modifiers of CF, particularly among those that influence the severity of the lung disease, are involved in controlling infection, immunity and inflammation. Some of these include the class II HLA antigens, mannose-binding lectin and alpha 1 antitrypsin2.

Alpha 1 antitrypsin (A1AT) is a highly polymorphic glycoprotein that is synthesized by hepatocytes and alveolar macrophages. It is a member of the serine family of protease inhibitors and protects tissues from proteolytic attack by leukocyte proteases, such as elastase, cathepsin and trypsin, during inflammatory reactions3. The protein is coded by a single gene (Pi: protease inhibitor) in the 14q31-32.2 chromosome4. Around 90 alleles have been identified5, although not all are associated with low or undetectable serum A1AT levels. The normal variant is PiM. Currently the most common deficient alleles are PiZ (Z) and PiS (S). The Z allele causes a single point mutation which changes a guanine to an adenine guanine adenine on exon 5 of the gene, and results in the substitution of glutamic acid to lysine in A1AT.

Homozygotes for Z have 15% of normal A1AT levels and their risk of developing pulmonary emphysema are higher 6, and less often, liver diseases in newborns. The S allele is more common than the Z allele results in a single point mutation where adenine is swapped for timine at exon 3, resulting in the substitution of glutamic acid by valine. Levels of A1AT are reduced in SS homozygotes (60% of normal). With SZ, the quantity of A1AT (37% of normal) is low enough to put affected individuals at risk of chronic lung disease6.

With relation to the progression of pulmonary status in CF, it is known that there is noteworthy proteases-antiproteases imbalance in favor of neutrophil elastase within the lungs. These proteases have deleterious effects, destroying elastin in the lungs, stimulating mucus production and cleaving immunoglobulins and fibronectin7. In patients with CF and alpha 1 antitrypsin deficiency (DA1AT), this imbalance could be exacerbated and pulmonary damage be even greater.

Studies into the association between DA1AT and CF are rare and have returned controversial results.

In contrast with what might be expected, one study of CF patients demonstrated that those with the association with DA1AT exhibited better pulmonary function parameters than individuals CF without DA1AT (VEF1 62.55% of normal and VEF 51.15% of normal, respectively)8,9. Another study with a significant patient sample, did not find any association between the gravity of CF and the presence of DA1AT10. Bearing in mind the elevated quantity of elastase from dead neutrophils and present in the airway secretion of CF sufferers, it would be expected that an association between DA1AT and CF would confer a more severe phenotype on CF patients. Our study objective was to investigate whether the presence of S and Z alleles of A1AT was related to the severity of lung disease at a reference center for CF treatment in Brazil.


Patients and methods

This was a clinical and laboratory based, cross-sectional study of patients from the Cystic Fibrosis Clinic at the Hospital de Clínicas of the Universidade Estadual de Campinas (UNICAMP) from March 2000 to August 2002. All patients were recruited if they were on outpatients treatment during this period, had a Cystic Fibrosis diagnosis confirmed by compatible clinical history and at least two sweat test results with chlorine levels greater than or equal to 60 mEq/l, performed by iontophoresis sweat stimulation with pilocarpine according to the method described by Gibson & Cooke11.

Those responsible for the patients signed informed consent before the study began.

The study was approved by the Committee for Ethics in Research at the Medical Sciences Faculty/UNICAMP.

The chi-square test was used to compare categorical variables. Fisher's exact test was applied if one of the 2 X 2 table's cell values less than or equal to 5. Significance was set at 5%.

The clinical criteria analyzed included pulmonary manifestations, digestive manifestations and Shwachman score 12. The laboratory evaluation included pulmonary function tests, sweat sodium and chlorine, chest x-ray and computerized tomography of the thorax. The Shwachman score assesses physical activity, the physical examination, nutrition and x-ray findings. For each item the maximum score is 25 points and the lower the score, the worse the clinical status of the patient. The score is graded from excellent86-100 , through good71-85, medium 56-70, moderate41-55 and severe (40 or less), according to total number of points. The Shwachman score is in Table 1.

All of the patients had been genotyped previously for the CFTR gene by the tem at the Molecular Genetics Laboratory at UNICAMP. The patients' DNA was extracted and, by means of polymerase chain reaction, specific regions were amplified to be analyzed for the five mutations in question: ΔDF508, G542X, N1303K, G551D and R553X.

All patients were analyzed for A1AT deficiency S and Z alleles using polymerase chain reaction (PCR), which creates sites recognized by the restriction enzymes XmnI (S allele) and TaqI (Z allele)13 . The two amplifications were optimized so that they could be performed on the same PCR program. The reaction was conducted in a volume of 100 µl, with the following reagent concentrations: 100 µM of each deoxyribonucleotide triphosphate (dATP, dTTP, dGTP, dCTP); 40 pmoles of each initiator; 2.5 units of Taq DNA polymerase (Centbio-UFRGS); 1.5 mM de MgCl2 present in the enzyme-specific buffer, and 1 µg of genomic DNA.

The amplification reaction was performed in a temperature cycler and consisted of an initial denaturing at 94 °C for 7 minutes followed by a 95 °C for 5 minutes; 50 °C for 1 minute (annealing) and 74 °C for 2 minutes (extension). Next followed 34 cycles at 95 °C for 1 minute; 50 °C for 1 minute and 74 °C for 2 minutes and, finally, one cycle at 74 °C for 9 minutes (termination).

The 15 µl amplification sample for the S mutation was digested for 12 hours at 37 °C in a total volume of 50 µl containing 2 µl of restriction enzyme (XmnI) and 5 µl of the buffer recommended by the supplier and the final volume was made up with 30 µl of water. After digestion, the sample was subjected to polyacrylamide gel electrophoresis at 14 % (3.75 ml TBE 10X; 14.75 ml of H2O; 12.5 ml of Bis acrylamide; 200 µl of ammonium persulphate 10% and 40 ml of Temed). The gel was then stained with ethidium bromide for viewing the bands.

The 15 µl sample of amplified DNA for the Z mutation was digested for 12 hours at 65 °C in a total volume total of 50 µl containing 1 µl of restriction enzyme (TaqI) and 5 µl of the buffer recommended by the supplier and the final volume was made up with 29 µl of H2O.

After digestion samples were subjected to polyacrylamide gel electrophoresis at 14%.



Seventy cystic fibrosis patients were analyzed. There were 36 males (51%) and 34 females (49%), with a mean age of 10.2 and standard deviation of 9.44 years. Ninety-seven percent of the patients were Caucasoids and 3% Negroids. Nine patients with cystic fibrosis (12.8%) were heterozygotes for the S or Z allele or composite heterozygotes (SZ). No patient was homozygous for either S or Z alleles . In Brazil, the normal frequency for S heterozygotes is 9.2% and for Z heterozygotes it is 5.5% (14). No statistically significant difference was found in clinical severity between A1AT genotypes (MSΧ c2(1) = 0.0487- 0.80 < p < 0.90; MZ c2(1) = 2.1228- 0.10 < p < 0.20; SZ c2(1) = 2.9719, 0.05 < p < 0.10) (Table 2). No statistically significant difference was found when patients were separated according to presence or absence of the ΔDF508 mutation (Table 3). The age groups of the patients and the severity of pulmonary status can be observed in Table 4.








This study evaluated children and adults suffering from cystic fibrosis. It was confirmed that there was a great majority of Caucasoids, which was expected, despite the elevated number of people of mixed race in Brazil.

The Shwachman score was used to evaluate the severity of pulmonary status12. In earlier studies carried out at Unicamp, it was confirmed that the score exhibited a statistically significant correlation with colonization by Pseudomonas aeruginosa, colonization by Pseudomonas aeruginosa mucosa, forced vital capacity, first second forced expiratory volume, transcutaneous hemoglobin saturation, number of infectious exacerbations in the previous year, indications for Dornase Alpha, indications for regular respiratory physiotherapy and indications for home oxygen therapy15,16. The Shwachman score is therefore a good indicator of general severity.

Proteases have potentially deleterious effects such as the destruction of elastin in the lungs, stimulation of mucus production and cleavage of immunoglobulins and fibronectin 7,17.

In cases with bacterial colonization, the deleterious effect of this imbalance has been well studied. Thus, with infectious processes, as dos neutrophils are lysed, elastase could be liberated into the extracellular medium. This elevated quantity of liberated protease could attack the elastin and cause pulmonary damage. In cases where patients have CF and DA1AT concomitantly, this imbalance would be exacerbated and pulmonary damage could be greater. Based on this, clinical trials of the use of neutrophilic elastase inhibitors, such as alpha 1 antitrypsin aerosol for the treatment of CF, have been performed and achieved some success18.

Nevertheless, published literature shows that the genotypes that result in mild to moderate A1AT deficiency, MS, SS and MZ were not associated with aggravation of the lung disease of cystic fibrosis patients10,19,20 . In contrast, they postulated that the association of these two alterations could exhibit a significant improvement in pulmonary function8,9 because the neutrophil elastase is a powerful proteolytic enzyme capable of lysing bacteria such as Pseudomonas aeruginosa7,20,21. It was suggested that the neutrophil elastase and other proteases are not exclusively destructive, but could also exhibit beneficial effects and that they are important to lyse bacteria and could reduce the regulation of inflammation, by inhibiting neutrophil activation, by cleavage of immunoglobulin , neutrophil and complement receptors and by inducing apoptosis19,22 . Overall, it appears that the final result of the action of elastase on the pulmonary epithelium depends on the balance between the positive and negative effects of elastase. Furthermore, in the case of those with alpha 1 antitrypsin deficiency, this effect will depend on the variation in enzyme activity.

It is apt to note that the severity of the pulmonary condition involves other variables such as environmental factors: age, pancreatic status, nutritional status 23,24 and genetic factors such as: variations in DNA in introns, unidentified genes distant from the CF locus, A second mutation on the same allele which attenuates the effect of the primary mutation25-28. It is clear, therefore, that the pulmonary manifestation of CF are multifactorial.

In this study, the first performed in Brazil into the association between A1AT deficiency and CF did not find any significant relation between the pulmonary status of CF patients and the distribution pf alleles for alpha 1 antitrypsin deficiency. When the patients were split according to the presence of the two alleles, one allele or no alleles for the ΔDF508 mutation and compared them according to pulmonary status, no statistical difference was observed, proving there is no relation between the presence of the DF508, mutation, alpha 1 antitrypsin deficiency and pulmonary status.

It is important to emphasize that only one patient with the SZ allele was detected, having a moderate enzyme deficiency; the remainder were all heterozygotes for the S or Z alleles. Therefore, since the frequency of homozygotes for the S and Z mutations and the composite mutation SZ was low in the population of cystic fibrosis patients studied, it was concluded that A1AT deficiency did not perform a significant role in the pulmonary manifestations of cystic fibrosis because this was a small sample.

It is worth pointing out that nothing can be stated on the subject of the SS, ZZ or SZ genotypes since none were identified in the group studied. Therefore the sample should be amplified for further analysis with multicenter studies.



This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (Foundation for the Support of Research for the State of São Paulo - Fapesp ).



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Carmen Sílvia Bertuzzo
Shigeo Mori, 1710, Barão Geraldo
CEP 13082-084 - Campinas, SP, Brazil
Tel.: +55 (19) 3788.8907 Fax: +55 (19) 3788.8909
E-mail: or

Manuscript received Jul 21 2004, accepted for publication Jul 27 2005.

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