Elsevier

Clinica Chimica Acta

Volume 412, Issues 15–16, 15 July 2011, Pages 1454-1456
Clinica Chimica Acta

Short communication
Misclassification of an apparent alpha 1-antitrypsin “Z” deficiency variant by melting analysis

https://doi.org/10.1016/j.cca.2011.03.032Get rights and content

Abstract

Background

Alpha-1 antitrypsin (AAT) is a protease inhibitor that protects the lungs from degradation by neutrophil elastase. AAT deficiency is associated with both lung and liver diseases. AAT deficiency is diagnosed using a combination of genetic and biochemical tests. Here we demonstrate how polymorphisms in the AAT gene can lead to genotype/phenotype discrepancies in common AAT assays.

Methods

Total AAT was measured using an immunoturbidimetric assay. AAT phenotype was determined using isoelectric focusing. Genotypic identification of Z and S AAT alleles was performed by melt curve analysis using a LightCycler. Genotype/phenotype discrepancy was amended using gene sequencing.

Results

Genotype and phenotype analysis produced conflicting results as a consequence of a polymorphism located in the probe designed to detect the Z allele. Sequencing revealed that the polymorphism had previously been reported as a rare P allele. The probe that caused the discrepancy was designed to match the WT sequence. A new probe was designed to specifically detect the Z allele, eliminating the possibility of future discordance.

Conclusions

Laboratories utilizing melt-curve analysis to diagnose patients should be aware of the potential for false positive results caused by polymorphisms located in the binding region of the genotyping probes. Alternatively, the probes should be designed to be specific to the mutation, rather than to the WT sequence.

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.

References (12)

  • J.K. Stoller et al.

    Alpha1-antitrypsin deficiency

    Lancet

    (2005)
  • N.A. Kalsheker

    Alpha1-Antitrypsin deficiency: best clinical practice

    J Clin Pathol

    (2009)
  • D.H. Perlmutter

    Alpha-1-antitrypsin deficiency: biochemistry and clinical manifestations

    Ann Med

    (1996)
  • I. Nita et al.

    Prolastin, a pharmaceutical preparation of purified human alpha1-antitrypsin, blocks endotoxin-mediated cytokine release

    Respir Res

    (2005)
  • T. Hidvegi et al.

    An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis

    Science

    (2010)
  • J.A. Bornhorst et al.

    Evaluation of an integrative diagnostic algorithm for the identification of people at risk for alpha1-antitrypsin deficiency

    Am J Clin Pathol

    (2007)
There are more references available in the full text version of this article.

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