Pilot Alpha-1 Antitrypsin Deficiency Screening Programme to determine the Prevalence of Alpha-1 Antitrypsin Deficiency in Ireland

We recently undertook a research project to identify the incidence of AATD in a representative sample of the general population of Ireland. This involved screening 1000 anonymised DNA samples for the presence of the S and Z mutations and was undertaken in collaboration with Dr. Joe McPartlin of the Trinity College Biobank. The gene frequencies revealed for both the S and Z mutation were higher than anticipated based on studies in other European populations.

For the purpose of this study the randomised nationally-based buccal swab collection from the Trinity Biobank was investigated. Ethical approval for the collection was granted by the joint Hospital Ethics Committee of Tallaght and St. James’s Hospitals. The confidentiality of the participant’s data and samples was respected and ensured by irreversible anonymisation. DNA was extracted from buccal swabs according to the Biobank standard protocols. AAT deficient individuals were identified through separate genotyping assays for the S and Z mutations using Real-Time PCR technology and melt curve analysis on a Roche Lightcycler. A Z genotyping assay was performed on 1095 DNA samples provided by the Biobank. This revealed 46 MZ or carrier individuals in the population. The frequency of the Z gene in this population was, therefore, 0.022. An S assay, carried out on 960 biobank samples, revealed 98 MS carriers and 1 SS (homozygote) individual. The frequency of the S gene in this population was, therefore, 0.053. In total, between the two assays, 3 AAT deficient individuals were identified, constituting 2 SZ and 1 SS genotype. A total of 140 AATD carriers were detected, constituting 46 MZ and 98 MS individuals.

Deficiency Phenotype No. Detected/No. Screened
MZ 46/1095
MS 98/960
SS 1/960
SZ 2/2055

Table 1. Number of Deficiency Phenotypes Detected in Population Screened

The percentage of deficiency alleles detected was higher than anticipated from studies in other populations. The allele frequencies for S and Z in Ireland were previously estimated at between 0.02-0.04 and 0.005-0.015 (Luisetti et al, Thorax 2004). The S variant, thought common to the Iberian Peninsula, was detected with unusually high frequency in the Irish population. Our pilot study shows S and Z alleles occur at frequencies of 0.053 and 0.022 respectively in the Irish population. As the random sample was from the 32 counties, and extrapolating from a population of 6 million on the island of Ireland this would suggest there are approximately 2,900 ZZ and 14,000 SZ AAT deficient individuals and over 200,000 MZ carriers on the island of Ireland. Compared to the gene frequencies in our targeted detection programme the Z mutation would appear to be much more clinically significant with a higher penetrance than S in the two populations we have evaluated. The prospect that alpha-1 antitrypsin deficiency is much more common in Ireland than previously thought will help highlight the fact that all COPD, non-responsive asthma and cryptogenic liver disease patients should be tested for AATD. Increased awareness and understanding of AATD is vital to prevent the continuing under-diagnosis of this condition. Early diagnosis of AATD, with appropriate medical follow-up and lifestyle changes, can prevent, or at least postpone, AATD complications.

Adoption of New Sebia Phenotyping Method:

In the last year we have acquired a new piece of equipment for the Targeted Detection Programme. This has allowed us to implement a more accurate method of phenotyping. We can now identify the different alpha-1 antitrypsin phenotypes with an increased sensitivity of detection. The Sebia Hydragel 18 AAT Isofocusing kit is designed for the qualitative detection and identification of the different phenotypes of alpha-1 antitrypsin (AAT) in the electrophoretic patterns of human sera. The procedure includes isoelectricfocusing on agarose gel, performed on the semi-automatic HYDRASYS system, followed by immunofixation with anti-alpha-1 antitrypsin antiserum. The assay is carried out in two stages. Firstly, isoelectrofocusing on agarose gel is used to fractionate the proteins in the serum samples. This is followed by immunofixation with enzyme (peroxidase)-labelled anti-alpha-1 antitrypsin antiserum to identify the various phenotypes of alpha-1 antitrypsin.

To summarise, we have found this new phenotyping method to be highly specific, rapid and simple to perform. It represents a more accurate method of screening for alpha-1 antitrypsin deficiency and improves the identification of not only the most common but also various rare AAT phenotypes.


National Alpha-1 Antitrypsin Deficiency Targeted Detection Programme

Alpha-1 antitrypsin (AAT) is an antiprotease produced chiefly by the liver. Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder characterised by low serum levels of AAT and is associated with lung and liver disease.

In May 2004 a national targeted detection programme for alpha-1 antitrypsin deficiency was established in Beaumont Hospital. Funded directly by the Irish Government's Department of Health, the screening programme provides free testing to patients with chronic obstructive pulmonary disease (COPD), non-responsive asthma, cryptogenic liver disease and to relatives of AATD patients.

A range of methods are used to diagnose AATD including phenotyping by isoelectric focussing and genotyping by RT-PCR. Upon diagnosis the Alpha One Foundation also provides a range of ancillary services to patients including counselling, expert advice, information packs and leaflets, and opportunities to enrol in clinical trials and to join the Alpha-1 patient support group. Alpha-1 antitrypsin (AAT) is a 52kDa glycosylated protein. Produced in the liver and secreted into the blood, AAT diffuses into the lungs where it functions as an antiprotease. Antiproteases regulate and inactivate protein-splitting enzymes such as neutrophil elastase, an enzyme capable of destroying alveolar wall connective tissue. AAT is the most abundant antiprotease in the lung and therefore plays a major role in maintaining a healthy, functioning lung. Alpha-1 antitrypsin deficiency (AATD) is a hereditary autosomal codominant disorder caused by mutations in the AAT gene located on chromosome 14. Genetic variants of the AAT gene are characterised by their electrophoretic mobilities as medium (M), slow (S) or very slow (Z). The most common variants associated with disease are the S (Glu264Val) and Z (Glu342Lys) mutations, caused by a single amino acid replacement of glutamic acid at positions 264 and 342 of the polypeptide respectively. Both mutations result in decreased levels of circulating AAT due to retention of the aberrantly folded protein in the liver, and classically result in liver disease in children, and early onset emphysema or occasionally liver disease in adults. Moreover, the small amount of AAT that reaches the lung in AATD patients is inactivated by cigarette smoke. Smoking is the single biggest risk factor for the development of emphysema in AATD patients, and individuals with AATD who smoke develop severe, early-onset emphysema. The most commonly observed genotypes are MM (normal), MS, MZ (heterozygotes), SZ (compound heterozygote) and SS or ZZ (homozygotes). It is unclear, as yet, whether the carrier status (MS or MZ) confers an increased risk of disease.

AATD is under-diagnosed with prolonged delays in diagnosis common. In addition, the majority of AATD individuals with emphysema are misdiagnosed as COPD patients. A recent US study showed it takes an average 5.6 years from the time symptoms first appear to accurate diagnosis. Increased awareness and understanding of AATD is therefore vital to prevent the continuing under-diagnosis of this condition. To this end, we have launched a national registry of AATD patients and a website (www.alpha1.ie) providing a resource for doctors, patients, and the general public. All patients diagnosed through our targeted detection programme are offered a variety of services including counselling, expert advice, information packs/leaflets, and opportunities to enrol in clinical trials and to join the Alpha-1 patient support group. Based on studies in other European countries it is estimated that 1,200 Irish citizens have AATD and up to 200,000 Irish citizens are carriers, yet only 110 individuals with AATD have been identified in Ireland to date. A research project recently undertaken in our laboratory screened 1,000 anonymised DNA samples provided by the Trinity College Biobank for the presence of the S and Z mutations. This investigation of a sample Irish population revealed a gene frequency of 0.05 for the S mutation and 0.022 for the Z mutation, which is higher than anticipated based on studies in other European populations.

   Phenotype  AAT (%)
 What does it mean?
 MM  100  No altered AAT gene so no risk of disease 
 MS  80  Carries altered AAT gene but evidence suggests no increased risk of disease


 MZ  60  Carries altered AAT gene & may develop disease (particularly if smoking)
Mild Deficiency
 SS  60  Carries two altered AAT genes but evidence suggests no increased risk of disease
Severe Deficiency
 SZ/ZZ  10 - 30  Carries two altered AAT genes and will probably develop disease (particularly if smoking)

Table 1: Explanation of the various AAT phenotypes and their clinical consequences.

In summary, AATD is more prevalent in Ireland than previously thought, even allowing for the targeted, symptomatic population investigated in this programme. The advantages of early and accurate diagnosis of AATD are manifold and include (1) closer observation and management of affected individuals, especially regarding pulmonary and liver health, (2) family member testing, at least some of whom may have lung or liver complications, (3) aggressive smoking cessation efforts, which have been associated with lower rates of smoking among AAT-deficient individuals, and (4) consideration of occupational hazards and environment as exposures to some occupational dusts and vapours can accelerate pulmonary decline. Once identified, AATD patients have the opportunity to enrol in clinical trials currently taking place in Beaumont Hospital, such as the AAT augmentation therapy clinical trial for ZZ individuals, and the MZ family study which is attempting to fully clarify the risk of COPD in MZ individuals. To conclude, the importance of an early diagnosis of AATD cannot be over-emphasised as the resulting appropriate medical follow-up and lifestyle changes can help prevent or at least postpone the development of AATD-related lung and liver disease.


What is Alpha-1 Antitrypsin?
Alpha-1 antitrypsin is a vital protein produced by the liver to protect the lungs. It provides protection from the harmful effects of infections and inhaled irritants, particularly tobacco smoke. It can be easily measured by a simple blood test. 
What is Alpha-1 Antitrypsin Deficiency?
Alpha-1 antitrypsin deficiency (Alpha-1) is a genetic condition which, after cystic fibrosis, is the commonest genetic disorder in Ireland. It severely affects more than 15,000 people, with another 250,000 carriers also at risk of lung and liver disease on the island of Ireland. It is a proven genetic risk factor for chronic obstructive pulmonary disease (COPD).
How Do I Get Tested?
The Alpha One Foundation provides free testing for Alpha-1 as part of a national screening programme which is funded by the HSE. For more details ring 01-8093871 or email alpha1@rcsi.ie.