Short communicationMisclassification of an apparent alpha 1-antitrypsin “Z” deficiency variant by melting analysis
Introduction
Alpha-1 antitrypsin (AAT) deficiency is one of the most common potentially lethal genetic diseases among Caucasian adults [1], [2]. As a ubiquitously expressed member of the SERPINA family of protease inhibitors, AAT accomplishes its most important function by coating the lungs to inhibit neutrophil elastase. Thus, a deficiency in AAT results in the aberrant proteolysis of the connective tissue matrix of the lungs, leading to progressive pulmonary damage and eventually emphysema in the fourth or fifth decade of life. In addition to the loss of anti-protease activity, certain AAT variants, such as the Z variant, can polymerize in hepatocytes, resulting in an amyloid-like effect that may progress to liver dysfunction in childhood [3]. AAT deficiency is treated using recombinant protein therapy [4]. Severe liver disease may require transplantation, although promising pharmaceuticals are currently being developed [5].
AAT is coded by the SERPINA1 gene which is highly polymorphic with over 100 alleles documented in the literature [1]. AAT variants are traditionally designated as letters from B-Z often with additional subscripts to denote subtypes. The nomenclature was designed to indicate the migration pattern of an AAT variant when analyzed using isoelectricfocusing electrophoresis, with B migrating towards the anode and Z towards the cathode. Many of these alleles produce functional proteins, the most common being the M variants. A number of deficiency alleles which reduce circulating AAT concentration and/or AAT activity have also been described. Of the deficiency alleles, S and Z are the most common and are caused by single nucleotide substitutions in exons 3 and 5, respectively [1].
Potential AAT deficiency is investigated by quantifying serum AAT protein concentration, determining AAT phenotype, and molecular genotyping to identify specific DNA mutations [6], [7]. AAT phenotype determined by isoelectricfocusing electrophoresis is considered the gold standard for identifying AAT variants. Molecular genotyping is less laborious and more cost efficient, and can be utilized to identify individuals with S or Z alleles. This can be accomplished using melt-curve analysis with fluorescence resonance energy transfer (FRET) probes specific to the region of the gene where the mutation is located [8], [9]. Since the S and Z mutations are located on different exons, different PCR primers and probe pairs are used to identify them. Published algorithms have been proposed which integrate serum AAT concentration with S and Z genotyping results [6], [7]. In our laboratory, AAT phenotyping is performed on non-S, non-Z, or heterozygous S or Z individuals with an AAT concentration < 100 mg/dL. In contrast, if the total AAT is ≥ 100 mg/dL only S and Z genotyping is performed to ascertain S or Z carrier status in the patient and phenotyping is not performed. Phenotype testing for individuals with a total AAT concentration that is < 100 mg/dL is necessary for two reasons. First, it confirms the presence of the deleterious S or Z mutation(s), and second it can be used to detect any rare AAT variants. Genotype analysis only differentiates the specific allele being test for. Thus, any allele that does not provide an appropriate signal using the S or Z probes may be an M, but might also be a rare benign or pathogenic mutation. Here we describe a patient with a total AAT of 100 mg/dL that was determined to be a Z heterozygote using a routine genotype assay (Fig. 1B). Phenotype testing was performed due to an AAT concentration near the decision threshold and was determined to be MP (Fig. 2).
Section snippets
Total AAT
Serum AAT was quantified using an immunoturbidimetric assay on the Roche Modular Analytics P.
Genotype analysis
The genotype assay was performed using a LightCycler V2.0 (Roche Molecular Biosystems, Indianapolis, IN) instrument and previously published probes specific for the wild-type AAT sequence at the Z and S loci [6], [10]. A detailed description of the assay has been described previously [6], [7], [10]. If the Z or S allele is present, the probe will melt at a lower temperature (5.4 °C) than the wild-type.
Results and discussion
Repeating the tests for AAT genotype and phenotype did not resolve the discrepancy. Further investigation of the genotype analysis showed no difference in the delta Tm of the patient results compared to the Z heterozygote control. A delta Tm is used to differentiate target from non-target mutation in a heterozygote [8], [9]. The value is calculated by subtracting the temperature of the highest melting peak from the peak of interest. An acceptable delta Tm would vary less than 0.2 °C when
Acknowledgements
We would like to thank Matt Campbell for his technical assistance.
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2014, Clinica Chimica ActaCitation Excerpt :However, the IEF method is laborious and time consuming. Molecular genotyping is effective, more cost efficient and can be utilized to identify individuals with S or Z mutants [10]. Conventional molecular genotyping methods to detect signal nucleotide polymorphisms (SNPs), including restriction fragment length polymorphism (RFLP) [11], single strand conformation polymorphism (SSCP) [12] and melt-curve analysis with fluorescence resonance energy transfer (FRET) probes using two fluorescent dyes specific to the region of the gene where the mutation is located [13] are all limited by cumbersome protocols.
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2019, ERS MonographReal-Time PCR to Detect α-1 Antitrypsin S and Z Alleles in Formalin-Fixed Paraffin-Embedded Tissue
2018, Journal of Applied Laboratory MedicineCOPD in individuals with the piMZ alpha-1 antitrypsin genotype
2017, European Respiratory Review