Dipl. Biol. Martina Veith, PD Dr. Timm Greulich

It has now been 59 years since Laurell and Eriksson described the first cases of alpha-1 antitrypsin deficiency (AATD). Since then, much has been learned about the disease (1). AATD is the most common inherited disorder in adults (2), and although approximately 120,000 people in Europe carry the Pi*ZZ genotype (Pi*ZZ is a common mutation in AATD), only a small proportion of these are diagnosed and treated (3). However, early diagnosis is very important so that doctors can take preventive measures and, if necessary, initiate appropriate treatment.

The Alpha-1 Antitrypsin Laboratory at the University of Marburg was founded in 2003 and is one of the leading laboratories in Europe for the diagnosis of Alpha-1 Antitrypsin Deficiency. Most specialized laboratories have developed their own diagnostic algorithm for AATM detection, and therefore the sequence of diagnostic procedures varies from laboratory to laboratory.

The diagnosis of AATD should generally begin with determining the AAT concentration in the blood. If AATD is suspected, for example, if the patient has a persistent cough, sputum production, or shortness of breath, the general practitioner or pulmonologist should order a serum alpha-1-antitrypsin (AAT) test, usually at a local, commercial laboratory. If the serum level is less than 90 mg/dL (0.9 g/L), molecular genetic analysis should then be initiated to detect various mutations and confirm a possible AAT deficiency at the DNA level.

The information for the development and function of living organisms is encoded in DNA (deoxyribonucleic acid). Each segment of DNA that codes for a specific protein is called a gene. DNA is therefore the blueprint (genetic code) for protein production. A deficiency of AAT is caused by mutations (changes) in the genetic code for AAT, specifically the SERPINA1 gene (serine protease inhibitor A1 gene). This segment of DNA is the one that is examined for mutations.

The Alpha-1-Antitrypsin Laboratory in Marburg offers a service for free molecular genetic analysis, supported by Grifols.

Two testing methods have been available for this purpose since June 2021:

1. The AlphaKit® from the company Grifols (DNA is extracted from dried blood; see also the article "What happens to my sample in the lab") and
2. A new testing procedure using the so-called "AlphaID®" (Grifols) is available. This involves a simple cheek swab (Figure 1). A sterile sponge is used to collect a sample of cheek mucosa (epithelial cells). The sample is then registered on a web platform by the physician using a barcode. Each AlphaID® has a unique barcode. The sample, along with the barcode, is then sent by mail to the Alpha-1 Antitrypsin Laboratory in Marburg.

DNA can be extracted from the epithelial cells of this sample, and within a few hours, a multiplex polymerase chain reaction (PCR) allows for the parallel detection of the 14 most common AAT mutations (Table 1). It is important, however, that the donor does not eat, drink (including water!), smoke, or chew gum for 30 minutes before the buccal swab is taken.

The physician can access the results of the AATM test using the AlphaID® via the web portal a few days after submitting the sample. Only the physician can link the results to the patient's data using the sample barcode.

For results that cannot be explained by PCR or in cases of suspected rare mutations, further analyses may be necessary. One option is to sequence the genetic code for alpha-1-antitrypsin. This involves determining the sequence of the building blocks (bases) in the DNA and then comparing it to a template (reference sequence).

One advantage of AlphaID® is that no second blood sample is required (non-invasive test) and the application is very simple. This makes the test suitable for infants, toddlers, and anxious individuals.

Depending on the type and location of the mutation on the SERPINA1 gene, the activity of the AAT protein is slightly reduced, severely reduced, or completely absent. Consequently, the protective function of AAT (enzymatic breakdown of lung tissue) is either no longer present or only partially functional.

Table 1: The 14 mutations that can be directly detected with the A1AT Genotyping Test from Progenika Biopharma (Grifols).

„"What happens to my sample in the laboratory?"“

Dipl. Biol. Martina Veith, PD Timm Greulich

Alpha-1-antitrypsin (AAT) is primarily produced in the liver and is found in virtually all body tissues. It acts as a protective protein, serving as an important antagonist to endogenous substances that can break down tissue. A deficiency of alpha-1-antitrypsin (AAT) is caused by mutations in the genetic code for AAT and results in reduced and/or defective synthesis and release of AAT molecules, which can lead to diseases such as pulmonary emphysema or liver disease.

A general practitioner or pulmonologist can order a blood test if there is a well-founded suspicion of AAT deficiency, for example, if the patient has a persistent cough, sputum production, or shortness of breath. The first diagnostic step is the quantitative determination of the AAT level in the serum or using the AlphaKit Quick Screen (Grifols). The AAT level is usually measured in a local, commercial laboratory. If a reduced serum level is detected (< 90 mg/dL), molecular genetic analysis should then be initiated to detect various mutations in order to confirm the AAT deficiency at the DNA level and to draw conclusions for an appropriate therapy. To date, over 100 genetic variants of the AAT protein are known. Diagnostic procedures are available at both the DNA and protein levels to identify these different variants.

For the analysis, blood is taken from the fingertip/earlobe and placed on a special filter paper (AlphaKit® from Grifols), which is then sent to the Alpha-1 Center in Marburg. The AlphaKits® are carefully checked in the laboratory for completeness (name, date of birth, doctor's address, and signature) and entered into a database. All samples are genotypically analyzed (at the DNA level) for specific mutations responsible for AATM.

Since June 2016, the Marburg Alpha-1 Center, together with Grifols and its subsidiary Progenika, has developed a new method based on xMAP technology (Luminex®) to perform genotypic analysis in a more specific and time-saving manner (Veith et al., 2019 (DOI: 10.2147/COPD.S224221)). Typically, in commercial laboratories, DNA is tested for two mutations using polymerase chain reaction (PCR): the Z mutation and the S mutation.
The new method now allows for the simultaneous testing for 14 different mutations using multiplex PCR. The Luminex® technology is based on microscopic polystyrene beads, which serve as a carrier material for biochemical detection reactions. Currently, 100 different bead types are available, distinguished by their fluorescent color. Each bead type can be loaded with different detection reagents (e.g., DNA probes). By combining different beads in a single test, up to 100 different detection reactions can be performed simultaneously in a very small sample volume.

The DNA is isolated from the AlphaKit® and the DNA fragments to be analyzed are amplified using multiplex PCR and bound to the bead-coupled probes. For example, the Z mutation is located at position 1096 on the AAT gene. Normally, guanine is found at this position, but in the Z mutation, adenine is found instead (adenine and guanine are two of the four different universal DNA building blocks). This point mutation later causes the amino acid lysine to be used instead of glutamine at position 366 during protein assembly, which in turn leads to misfolding of the alpha-1-antitrypsin protein and thus to agglomeration in the liver.

The "bead" is coupled to a specific DNA probe, namely the counterpart of the mutation being investigated. After PCR, the single strands to be analyzed can then be added to the "bead" probes. These single strands were labeled with biotin during the amplification process (PCR). Biotin, together with a dye (SAPE), serves to generate a detectable signal. If a mutation is present at the site being investigated, the single strand will bind to the probe with the exchanged base; otherwise, it will bind to the probe without the mutation.

The analysis and evaluation of the bead-based tests is performed using the Luminex® analysis system. It is based on the method of flow cytometry using two different lasers. A red laser identifies the color code of the "beads," while simultaneously a second, green laser performs the quantitative detection.

In addition to genotypic analysis, phenotypic analysis (analysis at the protein level) is always performed at the Marburg Alpha-1 Center to confirm the presence of a mutation. For this purpose, the protein is purified from the filter paper and separated using a method called isoelectric focusing (IEF). IEF is a biochemical technique for separating proteins according to their acidic and basic amino acid content.

In rare cases, further analyses may be necessary. To detect these as well, we have the capability to sequence the genetic code for alpha-1-antitrypsin. This involves determining the sequence of bases in the DNA and then comparing it to a template.

Thus, the Marburg Alpha-1 Center has several methods available to make a reliable diagnosis of AATM.