The IDDEA project: a strategy for the detection of alpha-1 antitrypsin deficiency in COPD patients in the primary care setting

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1 Therapeutic Advances in Respiratory Disease Original Research The IDDEA project: a strategy for the detection of alpha-1 antitrypsin deficiency in COPD patients in the primary care setting Ther Adv Respir Dis (2011) 5(4) DOI: / ! The Author(s), Reprints and permissions: journalspermissions.nav Jesús Molina, Xavier Flor, Rosa García, Rosario Timiraos, Gema Tirado-Conde and Marc Miravitlles Abstract: Objective: Primary care provides the main route for access to health care for patients with common chronic illnesses such as chronic obstructive pulmonary disease (COPD). Alpha-1 antitrypsin (AAT) deficiency is an underdiagnosed pathology associated with COPD risk which has a very low prevalence. The Information and Detection of the Deficiency of AAT (IDDEA) project was developed to identify AAT-deficient patients at primary care centres by providing adequate diagnostic tools to family doctors. Methods: Patients with COPD were identified and registered on a specially designed website. Dried blood samples were collected on filter papers and sent to the laboratory for AAT levels and AAT deficiency-related genotype determinations. Results were uploaded to the website and analysed. Results: Between January 2008 and April 2009, 596 patients were identified by 90 participating physicians. The number of patients who did not have AAT deficiency (serum concentrations 60 mg AAT/dl) was 549 (98.9%). Nineteen patients (3.2%) were carriers of the allelic variant Pi*Z among which two were homozygous PiZZ (one of them was an index case) and one was heterozygous PiSZ. These three newly detected cases were registered in the Spanish Registry of Patients with AAT Deficiency. An estimate of the gene frequency of the S allele was 7.65% and the severe deficiency Z allele was 1.76%. Conclusions: Results confirm that ATT deficiency is still underdiagnosed. The IDDEA system appears to be a useful tool for the detection of AAT deficiency in the primary care setting. Keywords: alpha-1 antitrypsin deficiency, chronic obstructive, genetic carrier detection, primary care, pulmonary disease Introduction Alpha-1 antitrypsin (AAT) is the most prevalent human protease inhibitor in serum with a normal concentration of mg/dl. Defective production of this protein is known as AAT deficiency, one of the most common human hereditary disorders. Depending on genetic and external factors, AAT deficiency can lead to compromised pulmonary protection with increased risk of early onset pulmonary emphysema and liver disease [Stoller and Aboussouan, 2005; American Thoracic Society/European Respiratory Society, 2003; World Health Organization, 1997; Cox, 1995; Seersholm et al. 1994]. More than 100 genetic variants of the gene encoding for AAT have been identified and have been classified from A to Z [DeMeo and Silverman, 2004]. Disease occurs when two protease inhibitor (Pi) deficient alleles are inherited from the AAT gene locus. The predominant normal phenotype is MM (PiMM) while most subjects with severe AAT deficiency are homozygous ZZ (PiZZ) [Blanco et al. 2006; de Serres, 2002; Kamboh, 1985]. Z variants result in normal rates of AAT synthesis but improper folding. As a consequence, only a fraction of synthesized AAT is released into the circulation. The S allele (Pi*S) is more common than Pi*Z in some countries such as Spain and Portugal and its Correspondence to: Jesús Molina Médico de Familia, Centro de Salud Francia I, Calle de Francia, S/N, Fuenlabrada, Spain jmolinaparis@gmail.com Xavier Flor Centre d Atenció Primària Chafarinas, Barcelona, Spain and Grupo de Respiratorio de semfyc (Sociedad Española de Medicina de Familia y Comunitaria) Rosa García Rosario Timiraos Centro de Salud de Culleredo, Culleredo, Spain and Grupo de Respiratorio de semfyc (Sociedad Española de Medicina de Familia y Comunitaria) Gema Tirado-Conde Fundació Clínic, Institut d investigació Biomèdica August Pi i Sunyer (IDIBAPS) Hospital Clínic, Barcelona, Spain Marc Miravitlles Grupo de Respiratorio de semfyc (Sociedad Española de Medicina de Familia y Comunitaria) 237

2 Therapeutic Advances in Respiratory Disease 5 (4) presence causes a moderately low level of AAT [Blanco et al. 2006]. It is estimated that AAT deficiency may cause from 1% to 4% of all chronic obstructive pulmonary disease (COPD) cases. Although detection programs are available, AAT deficiency is still underdiagnosed [de la Roza et al. 2006; de Serres, 2003] and delayed diagnosis is common [Campbell, 2000]. Because primary care provides the main route for accessing health care for patients with common chronic illnesses such as COPD, the IDDEA (Information and Detection of the Deficiency of AAT) program was launched in the primary care setting. Material and methods Aim and study design The primary objective of this study was to develop a tool for the efficient detection of AAT deficiency in COPD patients in the primary care setting to reduce underdiagnosis. The secondary objective was to identify patients with AATdeficient genotypes and compare the prevalence with previous studies. To achieve these objectives, family practice physicians from primary care centres in Spain contributed to the study by recruiting patients with COPD. Participating physicians were provided with the following diagnostic resources: information about AAT deficiency and the importance of its early detection, simple sampling methods, and an Internet-based tool specifically developed to coordinate the flow of data between the primary care centre, the analysis laboratory and the pneumologist supervisor of the project. The study was conducted in accordance with the Declaration of Helsinki of the 18th World Medical Assembly and approved by the ethics committee of the Institute of Research and Investigation in Primary Care (IDIAP), Jordi Gol, Barcelona, Spain. Patients provided written informed consent to participate in the study. The database generated was under the responsibility of the Spanish Registry of Patients with Alpha- 1-Antitrypsin Deficiency (REDAAT) of the Spanish Society of Pneumology and Thoracic Surgery (SEPAR). Study population Participant patients were recruited at the primary care centres during regular visits. Using the medical records of the patients, individuals fulfilling the following selection criteria were included in the study: previously diagnosed with COPD (postbronchodilator forced expiratory volume in one second [FEV 1 ]/forced vital capacity [FVC] <70%) [Levy et al. 2006] and age >30 years old. The main exclusion criteria were being diagnosed with any other respiratory disease (i.e. asthma, bronchiectasis, acute bronchitis, chronic bronchitis with normal pulmonary function) and being diagnosed with pneumonia or other active inflammatory processes. IDDEA project description and development The detection program began in January 2008 and ended in April A website application IDDEA project tool was developed to assist with study conduct. The project was presented to the coordinators of the Respiratory Group of the Spanish Society of Family and Community Medicine (semfyc), who then invited the 140 family doctors who were members of the group to participate. Physicians who were interested in participating registered in an anonymous database on the project website. These physicians were sent the informed consent, patient educational material and supplies for sample collection and analysis. For targeted detection, previously described testing procedures and algorithms were followed [Miravitlles et al. 2010]. Figure 1 shows the flowchart of events related to the project development. Blood sampling Sampling was performed on recruited patients by the dried blood spot (DBS) method [Ferrarotti et al. 2007]. Briefly, drops of fresh blood collected by fingertip puncture were allowed to thoroughly saturate the three dashed-line, 13-mm printed circles on the filter paper (903; Schleicher & Schuell, Dassel, Germany). The filter was allowed to air dry for at least 12 h at room temperature and then was sent in a Tyvek Õ envelope to the analysis laboratory. Laboratory analysis and validation AAT levels in DBS samples were determined by an immunonephelometric method [Costa et al. 2000]. According to the detection threshold 238

3 J Molina, X Flor et al. 1. Registration form Physician 2. Welcome + password 3. Folder with material mail Website 4. Envelope with patient samples Coordinator pneumologist 6. New results 7. Weekly update 5. Results form fulfillment PO Box Laboratory Figure 1. Flowchart of events in the IDDEA Project. After registration (1), the physician gained access to their personal area of the website (2) and was sent the materials for participation (3). Patients dried blood spot samples were sent by mail to the analysis laboratory for AAT levels and possible genotype determination (4). Results were posted on the website (5), where they were interpreted and validated by the pneumologist supervisor. A weekly with updated patient results was sent to the physician (6 and 7). predetermined by the laboratory and the guidelines for the treatment and diagnosis of AAT deficiency [Vidal et al. 2006], AAT levels were considered: normal if they were at least 110 mg/ dl; intermediate if they ranged between 60 and less than 110 mg/dl; and deficient if they were less than 60 mg/dl. Samples in which detected AAT levels were below normal (110 mg/dl) or were insufficient to allow AAT measurement, were analysed for genotype through real-time polymerase chain reaction (PCR) DNA sequencing according to the method described by Rodriguez and colleagues [Rodriguez et al. 2002]. Absence of S and Z in the presence of normal concentrations was interpreted as the presence of the most frequent normal allele M. The possible presence of other extremely infrequent alleles was not included in this analysis. In the case of AAT deficiency detection, diagnosis confirmation by whole blood analysis was recommended, as well as performance of AAT testing of any of the patient s siblings. Analysis of the results To assess the adequacy of the IDDEA system as a reliable tool for the detection of AAT deficiency in the primary care centre setting, the number of participating physicians as well as patient recruitment were examined. Data collected from the laboratory were analysed and classified with respect to AAT levels as well as to the number and percentage of samples with AAT-deficiencyassociated genotype. Results Project evolution The study started in January 2008 with 67 participating physicians (out of the 140 invited to participate) and 15 recruited patients. By April 2009, 90 physicians from 25 primary care centres had participated and enrolled 596 COPD patients. The ratio of patients recruited per physician progressively increased to 6.6 by the end of the study. The progression of recruitment over time is shown in Figure 2. Patients The majority of recruited patients were men (n ¼ 482; 80.9%), with a mean age of 61.0 years, ranging from 30 to 83 years and with a standard deviation (SD) of 8.3 years. Average FEV 1 was 61.3% (SD 17.8%). Almost all patients were either current (50.2%) or exsmokers (43.6%). Detailed smoking habits and the respective respiratory function data are summarized in Table 1. Analysis performed Normal AAT levels (110 mg/dl) were observed in 352 samples (63.4%); intermediate levels (60 to <110 mg/dl) in 197 (35.5%); and deficient levels (<60 mg/dl) in six (1.1%)

4 Therapeutic Advances in Respiratory Disease 5 (4) Physicians Patients Ratio patients/physician Individuals recruited Ratio Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Figure 2. Numbers of participating physicians and recruited patients from January 2008 to April Table 1. Patients smoking habits and lung function (mean ± SD). Smokers (n ¼ 299) Exsmokers (n ¼ 260) Nonsmokers (n ¼ 37) Cigarettes (packs/year) 55.5± ±47.1 FEV 1 (%) 61.6± ± ±18.3 FVC (%) 79.0± ± ±18.6 FEV 1 /FVC (%) 60.3± ± ±10.7 FEV 1, forced expiratory volume in one second; FVC, forced vital capacity. Of the genotyping results corresponding to intermediate or low AAT serum concentrations in dried blood spots, 19 patients (3.19%) were carriers of the severe deficient allele Z. Out of these 19 patients, 2 were diagnosed with severe homozygous PiZZ deficiency (0.34% of the total). One of the two was an index case and the other was a relative of a known index case not included in this study. One patient had the PiSZ genotype. The remaining 16 individuals had only one Z allele and intermediate AAT serum concentrations (probably PiMZ genotype). A total of 91 individuals were carriers of a deficient allele S. One was the aforementioned PiSZ; 10 (1.7%) had the PiSS genotype and the remaining 80 (13.4%) cases had only one S allele and mostly intermediate AAT concentrations (probably PiMS genotype). There were two atypical cases with deficient AAT levels (<60 mg/dl) in which no deficient Z allele was detected. The first was an 81-year-old man, exsmoker, with lung function slightly below normal limits (FEV 1 /FVC ¼ 68%). The second was a 61-year-old man, also an exsmoker, with lung function slightly below normal (FEV 1 / FVC ¼ 69%) and with an S allele. Since not all of the samples were genotyped, it is impossible to establish the absolute frequency of the S and Z allele variants in our study population. However, based on the genotyping results of the AAT intermediate and deficient samples, an estimate of the gene frequency of the S allele is 7.65% and the severe deficiency Z allele is 1.76%. Table 2 summarizes the possible genotypes and the ranges of AAT serum levels measured in all of the samples. Table 3 summarizes the AAT serum values measured in the Z and S carriers. Discussion In this pilot study we took advantage of current clinical and medical informatics [Smith et al. 2009] to engage primary care physicians in the 240

5 J Molina, X Flor et al. Table 2. Possible genotype and AAT levels measured in all samples. Genotype AAT concentration Normal (110 g/dl) Intermediate (60 to <110 mg/dl) Deficient (<60 mg/dl) NA Total NT No S, no Z Pi*S Pi*Z PiSS PiSZ 1 1 PiZZ 2 2 NA 1 1 Total AAT, alpha-1 antitrypsin; NA, not available (insufficient or defective sample for analysis); NT, not tested (samples with normal AAT levels in which genotype was not analysed according to detection thresholds predetermined by the laboratory and the guidelines for the treatment and diagnostic of AAT deficiency); Pi, Protease inhibitor; Pi*S, Pi with a single S allele; PiSS, Pi homozygous S; PiSZ, Pi heterozygous SZ; Pi*Z, Pi with a single Z allele; PiZZ, Pi homozygous Z. Table 3. AAT serum values (mg/dl) in the Z and S carriers. Mean SD Range Pi*S (n ¼ 80) Pi*Z (n ¼ 16) PiSS (n ¼ 10) PiSZ (n ¼ 1) 84.9 PiZZ (n ¼ 2) AAT, alpha-1 antitrypsin; Pi, Protease inhibitor; Pi*S, Pi with a single S allele; PiSS, Pi homozygous S; PiSZ, Pi heterozygous SZ; Pi*Z, Pi with a single Z allele; PiZZ, Pi homozygous Z. screening and diagnosis of a disease with very low prevalence (AAT deficiency) that is related to one with a high prevalence (COPD). The first interesting result of the study was that the ratio of patients per physician increased substantially as the number of participating physicians progressively increased. The IDDEA project tool not only facilitated collaboration between the primary care physician and the pneumologist but proved useful for the detection of AAT deficiency in the primary care setting and thus potentially helpful in reducing the underdiagnosis of this disease [de Serres, 2003]. Although this study was not powered to provide pharmacoeconomic data, it is not unreasonable to suggest that reduction of intermediate steps (i.e. standard clinical visits to the pneumologist and standard analysis procedures) could have a positive impact on resource utilization. Regarding the results of the analysis, we observed that 98.9% of the tested samples showed normal or intermediate AAT levels. In our study, subjects with normal serum AAT levels were considered not to be carriers of the homozygous PiZZ genotype. For this reason, the estimated gene frequencies obtained may be less than the actual frequencies, since the presence of S or Z alleles in the unanalysed samples with normal AAT concentrations was not considered. In our study we found 16 cases of heterozygous Z and one case of PiSZ. Importantly, we found 2 cases (0.34%) of severe AAT deficiency homozygous PiZZ, one being an index case. Our percentage of homozygous PiZZ cases was similar to that found in two other Spanish studies performed in the pneumology setting in COPD patients [de la Roza et al. 2005; Blanco et al. 2004]. Moreover, the allelic frequency found for the Pi*Z allele (1.76%) was also similar to that found in one of the aforementioned studies [de la Roza et al. 2005]. These data indicate that our patient sample was not biased, which supports the reliability of the screening for AAT deficiency performed in the primary care setting with the IDDEA tool

6 Therapeutic Advances in Respiratory Disease 5 (4) We observed two patients with deficient AAT levels who were not carriers of the severe deficient Z allele. In our study, only the S and Z alleles were identified by genotyping, therefore other rare deficient variants could not be detected. In these cases, additional blood samples were requested from the attending physicians for further investigation. The three newly detected ATT deficient cases (two PiZZ and one PiSZ) were registered in the REDAAT [Lara et al. 2007; Miravitlles et al. 1998], which currently includes information on nearly 400 individuals with severe deficiency, and together with other national registries forms the Alpha One International Registry (AIR) [Stockley et al. 2007]. Conclusion During the development of this pilot study, family practice physicians were progressively recruited to participate in the screening and detection of AAT deficiency in COPD patients by using the IDDEA web tool. A considerable number of carriers of the severe AAT deficiency allelic variant Pi*Z were detected. This result not only confirms that the disease is still underdiagnosed but highlights the IDDEA tool as a useful instrument for the detection of this pathology in the primary care setting. Moreover, the possible adaptation for use in detection programs for other underdiagnosed diseases in the primary care setting is appealing. Acknowledgments The authors are grateful to all colleagues and researchers from the participating primary care centres and to the Laboratorio di Biochimica e Genetica (Centro di Riferimento per la Diagnosi del Deficit Ereditario di Alfa 1-Antitripsina, Pavia, Italy) where the analyses were performed. All authors contributed equally to the study. Funding This study was sponsored by Grifols. Conflict of interest statement There are no other conflicts of interest. References American Thoracic Society/European Respiratory Society. (2003) Standards for the diagnosis and management of individuals with alpha-1 antitrypsin deficiency. Am J Respir Crit Care Med 168: Blanco, I., de Serres, F.J., Fernandez-Bustillo, E., Lara, B. and Miravitlles, M. (2006) Estimated numbers and prevalence of PI*S and PI*Z alleles of alpha1-antitrypsin deficiency in European countries. Eur Respir J 27: Blanco, I., Fernandez-Bustillo, E., de Serres, F.J., Alkassam, D. and Rodriguez, M.C. (2004) [PI*S and PI*Z alpha 1-antitrypsin deficiency: estimated prevalence and number of deficient subjects in Spain]. Med Clin (Barc) 123: Campbell, E.J. (2000) Alpha1-antitrypsin deficiency: incidence and detection program. Respir Med 94(Suppl. C): S18 S21. Costa, X., Jardi, R., Rodriguez, F., Miravitlles, M., Cotrina, M., Gonzalez, C. et al. (2000) Simple method for alpha1-antitrypsin deficiency screening by use of dried blood spot specimens. Eur Respir J 15: Cox, D.W. (1995) Alpha-1 antitrypsin deficiency, In: Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds). The Molecular and Metabolic Basis of Inherited Disease, 7th edn, McGraw-Hill: New York. de la Roza, C., Lara, B., Vila, S. and Miravitlles, M. (2006) [Alpha1-antitrypsin deficiency: situation in Spain and development of a screening program]. Arch Bronconeumol 42: de la Roza, C., Rodriguez-Frias, F., Lara, B., Vidal, R., Jardi, R. and Miravitlles, M. (2005) Results of a casedetection programme for alpha1-antitrypsin deficiency in COPD patients. Eur Respir J 26: de Serres, F.J. (2002) Worldwide racial and ethnic distribution of alpha1-antitrypsin deficiency: summary of an analysis of published genetic epidemiologic surveys. Chest 122: de Serres, F.J. (2003) Alpha-1 antitrypsin deficiency is not a rare disease but a disease that is rarely diagnosed. Environ Health Perspect 111: DeMeo, D.L. and Silverman, E.K. (2004) Alpha1- antitrypsin deficiency. 2: genetic aspects of alpha(1)-antitrypsin deficiency: phenotypes and genetic modifiers of emphysema risk. Thorax 59: Ferrarotti, I., Scabini, R., Campo, I., Ottaviani, S., Zorzetto, M., Gorrini, M. et al. (2007) Laboratory diagnosis of alpha1-antitrypsin deficiency. Transl Res 150: Kamboh, M.I. (1985) Biochemical and genetic aspects of human serum a-1 proteinase inhibitor protein. Dis Markers 3: 135. Lara, B., de la Roza, C., Vila, S., Vidal, R. and Miravitlles, M. (2007) Development and results of the Spanish registry of patients with alpha-1-antitrypsin deficiency. Int J Chron Obstruct Pulmon Dis 2: Levy, M.L., Fletcher, M., Price, D.B., Hausen, T., Halbert, R.J. and Yawn, B.P. (2006) International Primary Care Respiratory Group (IPCRG) 242

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