**4.4 Genotyping of AAT**

Genotyping assays are typically performed using PCR-based restriction fragment length polymorphism (RFLP) analysis or by melting curve analysis on real-time PCR instruments with specific primers and probes designed for the Z and S mutations (Ferrarotti et al. 2004; Rodriguez et al. 2002). RFLP methods, although cheaper, are time-consuming and have been superseded by the faster and more efficient melting curve methods (Aslanidis, Nauck, and Schmitz 1999; Bartels et al. 2009).

The advantage of genotyping assays is that they allow the rapid screening of DNA collected by dried blood spots and are not as prone to errors in interpretation as the IEF method. The inherent limitation of the genotyping assay is that most laboratories include only primers for the deficient mutation. This means that in an assay for the Z mutation, a rare mutation will

Alpha-1 Antitrypsin Deficiency – A Genetic Risk Factor for COPD 203

The Irish National Targeted Detection Programme for Alpha-1 Antitrypsin Deficiency is supported by funding from the Irish Government Department of Health and Children. We would like to thank Geraldine O'Brien in the Alpha One Foundation (Ireland) for helpful discussions and John Walsh and Angela McBride of the Alpha-1 Foundation (USA) for support and advice. We wish to thank Pat O'Brien and Eric Mahon in the Biochemistry Department, Beaumont Hospital for invaluable discussions and help with patient sampling and electrophoresis and nephelometry techniques, Professor Maurizio Luisetti and Dr. Ilaria Ferrarotti at the University of Pavia in Italy for sequencing of rare SERPINA1 mutations, and Dr. Marc Miravitlles in the Department of Pneumology at the Hospital Clinic of Barcelona, Spain for help with the genotyping assay. We are grateful to Professor Dermot Kenny and the RCSI Clinical Research Centre in Beaumont Hospital for the ongoing use of their facilities. We would finally like to thank all the members of the Respiratory Research laboratory in the Department of Medicine, and AATD patients attending Beaumont

Abboud, R. T., and S. Vimalanathan. 2008. Pathogenesis of COPD. Part I. The role of protease-antiprotease imbalance in emphysema. *Int J Tuberc Lung Dis* 12 (4):361-7. Aboussouan, L. S., and J. K. Stoller. 2009. Detection of alpha-1 antitrypsin deficiency: a

Alexander, R. L., Jr. 1980. Comparison of radial immunodiffusion and laser nephelometry

Alpha 1-antitrypsin deficiency: memorandum from a WHO meeting. 1997. *Bull World Health* 

for quantitating some serum proteins. *Clin Chem* 26 (2):314-7.

Fig. 11. Model for diagnosis of COPD and AATD.

**6. Acknowledgement** 

Hospital for their continuing help.

*Organ* 75 (5):397-415.

review. *Respir Med* 103 (3):335-41.

**7. References** 

be mistakenly classified as M, unless specific primers for M AAT are used. This rare mutation should of course be termed "non-Z" but this important distinction is not always made. To avoid any errors in diagnosis, any genotype result should be correlated to the AAT concentration. For example, a Null/Z individual will be classified MZ on a typical genotyping assay, however, consideration of the AAT concentration in this individual should prompt further investigation by sequencing.

Fig. 10. Genotyping assay for the Z mutation by melting curve analysis on a real-time PCR system (Roche LightCycler 480).
