A man was evaluated for alpha-1 antitrypsin (A1AT) deficiency (A1AD) after a history of abnormal liver function tests. The patient’s brother was previously diagnosed with A1AD with a ZZ phenotype and had a recent liver transplant. Other family history is unknown. The patient was originally phenotyped as MZ at an outside institution, but his A1AT levels were lower than expected for the MZ phenotype. Samples were sent for evaluation of an uncommon A1AT variant.
The correct answer is ...
The correct answer is: Isoelectric Focusing (IEF) Gel Electrophoresis.
Alpha-1 antitrypsin (A1AT) is a serine protease inhibitor encoded by the SERPINA1 gene on chromosome 14 and produced in hepatocytes. The main function of A1AT is to protect the lungs from neutrophil elastase, an enzyme that is produced by white blood cells for the purpose of degrading bacterial outer membranes. The wild-type allele (M) codes for functionally expressed A1AT protein, while certain single nucleotide polymorphisms (SNPs) can cause the resulting protein to be expressed at lower concentrations. The two most common alleles associated with A1AT deficiency are S and Z. In the context of deficiency, A1AT is not present in high enough concentrations to inhibit neutrophil elastase in the lungs, leading to degradation of elastin within the alveoli, resulting in loss of elasticity and structural integrity. In addition to losing protection against neutrophil elastase in the lungs, certain A1AT variants polymerize in hepatocytes, causing liver damage. Since A1AT alleles are inherited in an autosomal codominant pattern, which is referred to as a phenotype, people can have varying A1AT expression levels with homozygous MM being fully expressed and homozygous ZZ being the most deleterious and most frequently associated with clinical manifestations of deficiency. Determining a patient’s A1AT concentration and phenotype is important in determining what treatment is needed.
Quantitation by nephelometry is a technique used to determine the amount of circulating A1AT (1). It works by adding antibodies to A1AT to the patient’s sample, and measuring the scatter observed in the solution. This scatter is proportional to the concentration of A1AT, calculated by using a standard curve. While determining the amount of A1AT a patient has is important for their diagnosis of A1AD, this technique does not provide any information about the individual’s phenotype.
Genotyping by melt curve analysis uses real-time PCR to determine if the patient has particular SNPs in exon 3 (S variant) or exon 5 (Z variant) of the SERPINA1 gene (2). The technique uses PCR to amplify a DNA fragment around each SNP and uses Förster resonance energy transfer (FRET) probes to determine the presence or absence of S or Z alleles. This technique does not give any information about the presence of other alleles and does not directly detect the wild-type M allele.
Proteotyping by liquid chromatography tandem mass spectrometry (LC-MS/MS) is a technique that can be used to identify the presence of A1AT S and Z variant proteins (3,4). Patient serum samples are digested with trypsin, and the resulting A1AT peptide fragments are separated by LC followed by MS/MS. Similar to genotyping, this technique does not specifically detect the M allele or other variant proteins.
Phenotyping by isoelectric focusing (IEF) gel electrophoresis separates out the distinct A1AT proteins in agarose gels by their isoelectric point (5). The proteins will migrate to the point on the gel at which it has no net charge. The protein variants are identified based on the distinct migration pattern when compared to the protein derived from the M allele. This technique can be used to detect the wild-type M allele, the common S and Z deficiency alleles, and rare variants, as each will show a distinct band on the IEF gel.
Nicholas Larkey, Ph.D.
Fellow, Clinical Chemistry
Melissa Snyder, Ph.D., DABCC
Division Chair, Clinical Biochemistry
Associate Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science