Study of α1-antitrypsin Phenotypes Frequencies in Patients with Primary Antibody Deficiency

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1 ORIGINAL ARTICLE Iran J Allergy Asthma Immunol June 2006; 5(2): Study of α1-antitrypsin Phenotypes Frequencies in Patients with Primary Antibody Deficiency Mohammad Reza Fazlollahi 1,2, Asghar Aghamohammadi 1, Reza Farid Hosseini 2, Abbas Sahebghadam Lotfi 3, Alireza Khoshdel 3, Abolhassan Farhoudi 1, Masoud Movahedi 1, Mohammad Gharagozlou 1, Habibeh Mozaffari 1, Fariborz Zandieh 1, Mahboubeh Mansouri 1, Javad Ghaffari 4, and Nima Rezaei 1 1 Department of Allergy and Clinical Immunology, Children Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Allergy and Clinical Immunology, Mashhad University of Medical Sciences, Mashhad, Iran 3 Department of Clinical Biochemistry, Tarbiat Modarres University, Tehran, Iran 4 Department of Pediatrics, Mazandaran University of Medical Sciences, Sari, Iran Received: 9 July 2005; Received in revised form: 11 March 2006; Accepted: 27 April 2006 ABSTRACT Primary antibody deficiencies are the most frequent primary immunodeficiency disorders. Bronchiectasis as a feature of these disorders may be developed due to some factors such α-1- antitrypsin deficiency. In order to determine the prevalence of two common α-1-antitrypsin deficiency alleles (PI*Z and PI*S) in Iranian patients with antibody deficiency, this study was performed. The prevalence of PI*M, PI*S, and PI*Z allele combinations was determined in 40 patients with primary antibody deficiency (with and without bronchiectasis) and compared with 60 healthy control subjects. Phenotyping was performed by isoelectric focusing. The phenotype frequencies among patients were as follow: M in 92.5%, S in 2.5% and Z in 5%. There was not any significant difference in distribution of alleles or phenotypes between patients and control subjects. Moreover, no significant difference was found between patients with and without bronchiectasis. We did not find evidence to support an association between α-1-antitrypsin phenotypes and primary antibody deficiencies in a small, controlled study. Larger studies will be required to clarify the relationship between α-1-antitrypsin genotype and susceptibility to bronchiectasis in patients with antibody deficiency. Key words: Allele; Alpha-1-Antitrypsin; Antibody Deficiency; Bronchiectasis INTRODUCTION Chronic lung disease, particularly the development of bronchiectasis, is a common and serious medical problem of patients with primary antibody deficiency, Corresponding Author: Asghar Aghamohammadi, MD; Department of Allergy and Clinical Immunology, Children Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran. Tel: (+98 21) , Fax: (+98 21) , aghamohammadi@iranianpia.org which may necessitate frequent hospitalizations and in some, could lead to severe respiratory impairment. 1 If diagnosis and/or optimal treatment are delayed, bronchiectasis may develop up to 50% of patients. 2 In addition to infections, some factors such as specific antibody deficiency and α-1-antitrypsin (AAT) deficiency may be important causes of developing bronchiectasis. The major function of AAT in the lungs is to protect the connective tissue from human neutrophil elastase (HNE) released from triggered neutrophils. 3-7 Copyright 2006, IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY. All rights reserved. 69

2 M.R. Fazlollahi, et al. Despite the well recognized association between AAT deficiency and the early development of emphysema, a number of studies have indicated that bronchiectasis results from AAT deficiency However, the role of α- 1- antitrypsin deficiency in the development of bronchiectasis is controversial. 9 AAT deficiency has been reported in some patients with primary antibody deficiency In a study on 43 patients with common variable immunodeficiency, the patients were classified according to AAT phenotype; although a Z allele frequency in the patients with bronchiectasis was 0 077, the results were inconclusive. 15 Therefore, because the development of bronchiectasis is a frequent occurrence in the patients with antibody deficiency, we have used a phenotyping assay to investigate a possible association between AAT deficiency and antibody deficiency with bronchiectasis. MATERIALS AND METHODS Patients The study comprised forty patients with primary antibody deficiency, meeting international diagnostic criteria, 17,18 These patients were followed up in the immunodeficiency clinic at the Children Hospital Medical Center in Tehran. The immunodeficient patients were subdivided into two groups of with and without bronchiectasis, based on High Resolution Computerized Tomography (HRCT) of lung. HRCT was performed in the patients, who clinically indicated due to recurrent chest infections, or plain chest X ray (CXR) appearance. Sixty normal healthy nonimmunodeficient adults were also included in this study as control subjects. The study was approved by the Research and Ethics Committees of Tehran University of Medical Science. Methods Blood specimens were collected by venopuncture during a stable phase of the disease; Separated sera were stored at -70 C. Serum immunoglobulins were assessed by nephelometry. α 1-Antitripsin Phenotyping AAT phenotypes have been determined in all patients and control subjects by Iso Electric Focusing (IEF) of blood samples. IEF was performed essentially according to the standard procedure described by Jeppsson et al. 19 Briefly, IEF was performed on a multiphor apparatus (Model LKB 2117; Pharmacia) connected to a power supply (Model LKB 2103; Pharmacia). Each gel was made to a final concentration of acrylamide 7.5% (W/V), carrier on ampholytes 1% (W/V) with narrow range of ph and glycerol 16% (W/V). Polymerization of gel was achieved with ammonium persulfate and TEMED. Samples were applied by means of 8 4 No.3 filter paper pad (Watman; Clifton, NJ). Prefocusing was performed in 750 volt/hour following focusing in 2000 volt/hour at constant current. Fixation, staining and destaining of the gels were carried out. Then the sample profiles were compared with standard serum phenotypes that we obtained from AAT laboratory, Professor MR. Fagerhol, Ullevaal University Hospital, Oslo, Norway. Statistical Analysis The χ2 test was used for statistical analysis of the PI phenotype frequencies in the patient's group and control populations. RESULTS The study population involved 40 patients (28 male and 12 female). In all 40 patients bronchiectasis was confirmed or excluded by CT scan or chest X-ray. Ten patients had bronchiectasis and 30 patients had not any clinical evidence of bronchiectasis. Diagnosis of bronchiectasis was assessed by CT scan alone in 9 patients, and by CT scan and bronchography in 1 patient. Table 1 shows features of patients; including the age at presentation, the blood serum presentation levels of IgG, IgA, IgM, and evidences of autoimmunity or liver disease. A comparison of the proportion of AAT phenotype between subjects with immunodeficiency and in healthy control subjects is presented in Table 2. Z heterozygote alleles (PI MZ) were observed in 1.7% of healthy subjects and 5% of immunodeficient patients. However, this difference was not statistically significant (p= 0.56). The distributions of AAT phenotypes among patients with and without bronchiectasis and control subjects are presented in Table 3. Although the Z allele frequency was increased in the patients with bronchiectasis when compared to controls, patients with and without bronchiectasis demonstrated no significant difference for either Z or S alleles in the patient compared to the controls (Table 3). 70/ IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 5, No. 2, June 2006

3 α-1-antitrypsin in Primary Antibody Deficiency N0 Sex Diagnosis Table 1. Characteristics of patients with primary antibody deficiency Immunoglobulin (mg/dl) Radiological Autoimmunity Liver Disease IgG IgM IgA Method Bronchiectasis Phenotyping 1 F CVID NO NO CXR NO SS 2 M CVID < NO NO CXR NO MM 3 M CVID < <10 NO NO CXR NO MM 4 F CVID NO NO CXR NO MM 5 F CVID NO NO CXR NO MM 6 M CVID NO NO HRCT NO MZ 7 F CVID 325 <20 <10 NO NO HRCT NO MM 8 M CVID NO NO HRCT NO MM 9 M CVID YES NO HRCT NO MM 10 F CVID YES NO HRCT NO MM 11 F CVID <100 UND UND NO NO HRCT NO MM 12 F CVID 350 <10 <10 NO NO HRCT NO MM 13 M CVID UND NO NO HRCT NO MM 14 F CVID 17 UND UND YES NO HRCT NO MM 15 M CVID UND YES NO HRCT YES MM 16 M CVID UND NO NO HRCT YES MM 17 M CVID UND NO NO HRCT YES MM 18 M CVID < <10 NO YES HRCT YES MM 19 M CVID 75 UND UND NO NO HRCT YES MZ 20 M CVID 300 <10 <10 NO NO HRCT YES MM 21 F CVID YES NO HRCT YES MM 22 M CVID <100 <10 <10 NO NO HRCT YES MM 23 M CVID <100 <10 <10 NO NO HRCT YES MM 24 M CVID UND 48 5 YES NO HRCT YES MM 25 F HIGM 200 >400 <10 NO NO HRCT NO MM 26 F HIGM UND >400 UND NO NO CXR NO MM 27 F HIGM 10 >480 5 NO NO CXR NO MM 28 M SAb NO NO CXR NO MM 29 M XLA 10 UND UND NO NO CXR NO MM 30 M XLA < <10 NO NO CXR NO MM 31 M XLA 180 UND UND NO NO CXR NO MM 32 M XLA 67 UND UND NO NO CXR NO MM 33 M XLA <100 <10 <10 NO NO CXR NO MM 34 M XLA < <10 NO NO CXR NO MM 35 M XLA 260 <10 <10 NO NO CXR NO MM 36 M XLA NO NO CXR NO MM 37 M XLA < <10 NO NO HRCT NO MM 38 M XLA <100 <10 <10 NO NO HRCT NO MM 39 M XLA <100 <10 <20 NO NO HRCT NO MM 40 M XLA 35 UND UND NO NO HRCT NO MM F: Female; M: Male; UND: Undetectable; CVID: Common Variable Immunodeficiency; XLA: X-Linked Agammaglobulinemia; SAb: Specific Antibody Deficiency; HIgM: Hyper IgM Syndrome; CXR: Chest X-Ray; HRCT: High Resolution Computed Tomography Vol. 5, No. 2, June 2006 IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY /71

4 M.R. Fazlollahi, et al. Table 2. AAT phenotypes distribution in patients and control group AAT Phenotypes Patients (n=40) Healthy Controls (n=60) No. % No. % P- value MM SS MZ Table 3. Distribution of AAT phenotypes in patients, sub grouped according to evidence of bronchiectasis AAT Phenotypes Patients with Bronchiectas is (n=10) Patients without Bronchiectasis (n=30) P- value No. % No. % MM SS MZ DISCUSSION Bronchiectasis is a pathologic description of lung damage characterized by inflamed and dilated thickwalled bronchi. These features may result from a number of possible causes and these may influence treatment and prognosis. Relevant factors were identified; cystic fibrosis, cilliary dysfunction, congenital defect, rheumatoid arthritis, early childhood pneumonia, pertussis, or measles immune defects and AAT deficiency. 5,20 The major physiological role of AAT is to protect the supporting elastic tissue in the lung alveoli from hydrolysis by neutrophil elastase that ultimately predisposes these individuals to the development of emphysema, infections leading to respiratory complications and bronchiectasis. 21 This view is generally held that AAT is extremely rare in the Asian groups but the database in the recent study demonstrated AAT is one of the most serious hereditary disorders that affect individuals in all racial subgroup worldwide. 7 In addition to number of case reports about description of patients with primary immune deficiency and AAT deficiency 11-14,some evidences are available that AAT deficiency might be related to primary antibody deficiencies, including the proximity of the AAT gene (Pi) to heavy chain gene of immunoglobulin (Gm), 15,22 and occurrence of pulmonary diseases (emphysema and bronchiectasis) both in affected patients with AAT deficiency and CVID. 15 In the present study, we found no statistically significant difference in the proportion of any a1- antitrypsin phenotype found in the disease and control populations. A small increase in the absolute number of deficient allele (PI*Z) in the diseased group (2/40) compared to the control group (1/60) was noted (Table 2). Thus, we could not find evidence to support an association between PI*alleles and primary antibody deficiency patients in a rather small, controlled study. We observed the presence of one PI*Z heterozygous individual in the subgroup of bronchiectatic patients as well as non bronchiectatic patients. Our study indicates that AAT phenotype distribution is not different between patients with bronchiectasis and non bronchiectatic subjects (Table 3). The most of previous studies were reported IgA deficiency and IgG subclass deficiency in patients with hypogammaglobulinemia11-16 and a connection between antibody deficiency and α-1-antitrypsin deficiency was not obvious. Only in a recent controlled study, has been demonstrated sub grouping of CVID patients had a Z allele frequency of in those patients with bronchiectasis, which was higher than in normal controls; however, statistical analysis revealed this was not significant. In present study the results indicated a tendency towards higher Z allele frequency of α-1- antitrypsin in patients; but it does not support a pathophysiologic implication of AAT deficiency in development of bronchiectasis in patients with primary antibody deficiency. The relative lack of AAT could theoretically also promote the development of autoimmunity due to the exposure of normally intracellular antigens. 4 Heterozygote for the Z allele has been reported to be at increased risk of developing anterior uveitis and rheumatoid arthritis as well as a variety of collagen vascular diseases. 5 In our study, six patients had autoimmunity and vasculitis manifestations but their phenotypes were normal (MM). Overall, these associations appear weak and often controversial and it seems to lack impact on clinical management. This study has a number of limitations. Because of unavailability of polymerase chain reaction techniques in our laboratory, we used isoelectric focusing for gene determination, however, as previously reported, 15 IEF phenotyping showed excellent correlation with genotyping technique. Ideally, all patients should be 72/ IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 5, No. 2, June 2006

5 α-1-antitrypsin in Primary Antibody Deficiency examined for bronchiectasis by high resolution CT scan; however, the diagnosis of bronchiectasis was assessed by method other than CT scan (chest radiography) in 16 patients, because CT scan has been performed only if indicated clinically due to recurrent infections. We did not determine plasma AAT levels in this study, because AAT serum concentrations are not accurate factor for screening the patients with intermediate AAT deficiency, moreover AAT is an acute phase reactant that may augment the steady state plasma AAT levels in Z heterozygotes. 5 In conclusion, the low prevalence of Pi deficiency phenotypes in our patients suggests a lack of contribution of AAT deficiency to the pathogenesis of pulmonary complications in patients with Primary Antibody Deficiencies. These findings could describe the reason of pulmonary function abnormalities in only some patients, despite adequate immunologlobulin replacement therapy. 21,23 the data cannot rule out the possibility of this association. In order to reliably and definitively determine the relation between AAT deficiency and primary antibody deficiency, large-scale studies will be required. ACKNOWLEDGEMENT This project has been performed by the grant from Tehran University of Medical Sciences and Health Services. REFERENCES 1. Cunningham-Rundles C, Bodian C. Common variable immunodeficiency: clinical and immunological features of 248 patients. Clin Immunol 1999; 92(1): Ochs HD, Stiem ER, Winkelstien JA.Antibody deficiencies. In: Stiehm ER, Ochs HD, Winkelstein JA, editors. Immunologic disorders in infants and children. Philadelphia: Elsevier Saunders, 2003: Barker A, Brantly M, Campbell E. Alpha 1-antitrypsin deficiency: memorandum from a WHO meeting. Bull World Health Organ. 1997; 75(5): Carrell RW, Lomas DA. Alpha1-antitrypsin deficiency--a model for conformational diseases. N Engl J Med 2002; 3,346(1): Stoller JK, Snider GL, Brantly ML, Fallat RJ, Stockley RA, Turino GM, et al. American Thoracic Society/ European Respiratory Society Statement: Standards for the diagnosis and management of individuals with alpha-1 antitrypsin deficiency. Pneumologie 2005; 59(1): Luisetti M, Seersholm N. Alpha1-antitrypsin deficiency. 1: epidemiology of alpha1-antitrypsin deficiency. Thorax 2004; 59(2): de Serres FJ. Worldwide racial and ethnic distribution of alpha1-antitrypsin deficiency: summary of an analysis of published genetic epidemiologic surveys. Chest 2002; 122(5): Rodriguez-Cintron W, Guntupalli K, Fraire AE. Bronchiectasis and homozygous (P1ZZ) alpha 1- antitrypsin deficiency in a young man. Thorax 1995; 50(4): Cuvelier A, Muir JF, Hellot MF, Benhamou D, Martin JP, Benichou J, Sesboue R. Distribution of alpha(1)- antitrypsin alleles in patients with bronchiectasis. Chest 2000; 117(2): King MA, Stone JA, Diaz PT, Mueller CF, Becker WJ, Gadek JE. Alpha 1-antitrypsin deficiency: evaluation of bronchiectasis with CT. Radiology 1996; 199(1): Osergaard PA. Alpha-1-antitrypsin levels and clinical symptoms in forty-eight children with selective IgA deficiency. Eur J Pediatr 1984; 142(4): Gelfand EW, Cox DW, Lin MT, Dosch HM. Severe combined immune-deficiency disease in patient with alpha 1-antitrypsin deficiency. Lancet 1979; 28,2(8135): Phung ND, Kubo RT, Spector SL. Alpha 1-antitrypsin deficiency and common variable hypogammaglobulinemia in a patient with asthma. Chest 1982; 81(1): Phung ND, Harbeck RJ, Helbling-Muntges C. Familial hypogammaglobulinemia. Genetic linkage with alpha 1- antitrypsin deficiency. Arch Intern Med 1983; 143(3): Sansom ME, Ferry BL, Sherrell ZP, Chapel HM. A preliminary assessment of alpha-1 antitrypsin S and Z deficiency allele frequencies in common variable immunodeficiency patients with and without bronchiectasis. Clin Exp Immunol 2002; 130(3): Barth J, Winkler I, Schmidt EW, Braun BE, Mollmann HW, Skvaril F. IgG subclass distribution in the serum of patients with homozygous alpha1-antitrypsin deficiency. Pneumologie 1990; 44(Suppl 1): Notarangelo L, Casanova JL, Fischer A, Puck J, Rosen F, Seger R, et al. International Union of Immunological Societies Primary Immunodeficiency diseases classification committee. Primary immunodeficiency diseases: an update. J Allergy Clin Immunol 2004; 114(3): Vol. 5, No. 2, June 2006 IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY /73

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