Evaluation of Integrative Diagnostic Algorithm for AAT Deficiency Detection
An integrative diagnostic algorithm for α1-antitrypsin (AAT) deficiency testing in the clinical laboratory was developed and evaluated. A novel rapid LightCycler (Roche, Indianapolis, IN) molecular assay was used to detect the common S and Z deficiency allelic variants. However, use of such molecular assays for these variants also can result in the misclassification of significant numbers of "at-risk" patient samples containing other rare AAT deficiency alleles. In the diagnostic algorithm presented herein, patient samples with selected genotypes that exhibit abnormally low AAT concentrations by immunoassay are phenotyped by isoelectric focusing. To test the efficacy of this algorithm, we retrospectively evaluated a data set of 50,020 serum samples for which protein phenotype and AAT concentration had been determined. This algorithm can successfully detect the majority of at-risk samples containing rare deficiency alleles.
α1-Antitrypsin (AAT) deficiency (OMIM 107400) is one of the most common serious hereditary disorders. This disease is inherited in an autosomal recessive manner and results in low serum and lung levels of AAT. This AAT insufficiency leads to uncontrolled neutrophil elastase activity, which targets the structural protein elastin, particularly in the lungs. This results in high risk for the development of emphysema in the third to fifth decade of life. Other AAT deficiency disorders, most notably an increased risk for the development of liver disease, can also be attributed to the presence of specific sequence variation in the AAT gene.
The AAT gene, located on chromosome 14 at region 14q32.1, consists of 1 untranslated exon followed by 4 translated exons. To date, at least 130 unique variant alleles of the AAT gene have been identified. Deficiency variant alleles can lead to reduced levels of circulating AAT in the serum or a defective ability to inhibit protease. Patients exhibiting 2 AAT deficiency alleles are generally considered to be at risk, whereas patients with a single AAT deficiency allele are considered to be at low risk for the development of AAT deficiency-related symptoms and are denoted as carriers in this work. Although AAT deficiency is considered to be underdiagnosed, the progression of AAT deficiency-related disorders can be dramatically slowed through lifestyle changes and protein replacement therapy.
The M allele is considered the native variant and results in normal levels of AAT protein production and resulting serum concentrations (100-250 mg/dL [18.4-46.0 µmol/L]), whereas the Z and S deficiency alleles account for the majority of the deleterious variants found in patients with AAT deficiency and are caused by single base substitutions that substantially reduce the concentration of circulating AAT. The homozygous ZZ protein phenotype is considered to cause the most severe phenotype and often results in the development of severe lung and liver problems. The SZ and SS protein phenotypes can also lead to severe pulmonary problems, particularly in people who smoke. The variation for the S deficiency allele is located in exon 3 (c.791A>T, GenBank accession No. K02212), causing an amino acid alteration of Glu264Val. The variation for the Z deficiency allele is located in exon 5 (c.1024G>A), causing a glutamine change to lysine at codon 342. Only about 15% of the normal amount of AAT is found in the serum of patients with homozygous ZZ protein phenotypes, whereas approximately 60% of normal concentrations are observed in people with the SS protein phenotype. Furthermore, the null allele is defined as having no protein detectable by isoelectric focusing (IEF) and a serum concentration of less than 30 mg/dL (5.5 μmol/L) by immunoturbidimetric assay.
World Health Organization guidelines recommend screening for AAT deficiency at least once in all patients with chronic obstructive pulmonary disease and in adults and children with asthma. The diagnosis of patients at risk for the development of AAT deficiency is often by IEF and concordant serum concentration determination by immunoturbidimetric assay of the AAT protein. The use of real-time polymerase chain reaction (PCR) analysis to detect the S and Z deficiency alleles in a large number of patient samples is potentially a more rapid and less technically demanding method than IEF for AAT analysis. Real-time PCR assays also have the advantage that a wide variety of sample types can be used. We validated a LightCycler assay (Roche, Indianapolis, IN) for S and Z deficiency allele detection using rapid PCR technology and FRET probes. This AAT genotyping assay quickly detected the S and Z deficiency alleles from peripheral blood, serum, and amniocyte samples. DNA extracted from amniocytes is useful for prenatal diagnosis because serum samples are not readily available for prenatal protein phenotype testing.
A large number of rare AAT variant alleles (other than the S and Z alleles) have been described and are potential deficiency alleles. The efficacy of using an assay that detects only the 2 most common AAT deficiency alleles (S and Z) for identifying all patients who have 2 deficiency alleles and who are at risk for the development of AAT deficiency-related symptoms is unknown. Patients could potentially be misclassified as carriers instead of at risk if an S or Z allele is paired with another rare deficiency allele. Furthermore, in rare cases, genotyping screens for only the more common deficiency alleles would miss patients with 2 rare deficiency alleles.
A diagnostic algorithm was developed that uses a combined approach consisting of an initial genotypic screen for S and Z deficiency alleles with concurrent AAT serum protein concentration testing. This step is followed by protein phenotyping by means of IEF for patient samples with abnormally low AAT serum concentrations who do not have the ZZ, SS, or SZ genotypes as assayed by the LightCycler AAT genotyping assay Figure 1. A retrospective database consisting of the protein phenotypes as determined by IEF and concurrent serum AAT concentrations of 50,025 clinical samples was used to evaluate the efficacy of this algorithm, incorporating genetic screening for the S and Z variants in identifying at-risk and carrier patient samples in a large clinical population.
(Enlarge Image)
Figure 1.
Abstract and Introduction
Abstract
An integrative diagnostic algorithm for α1-antitrypsin (AAT) deficiency testing in the clinical laboratory was developed and evaluated. A novel rapid LightCycler (Roche, Indianapolis, IN) molecular assay was used to detect the common S and Z deficiency allelic variants. However, use of such molecular assays for these variants also can result in the misclassification of significant numbers of "at-risk" patient samples containing other rare AAT deficiency alleles. In the diagnostic algorithm presented herein, patient samples with selected genotypes that exhibit abnormally low AAT concentrations by immunoassay are phenotyped by isoelectric focusing. To test the efficacy of this algorithm, we retrospectively evaluated a data set of 50,020 serum samples for which protein phenotype and AAT concentration had been determined. This algorithm can successfully detect the majority of at-risk samples containing rare deficiency alleles.
Introduction
α1-Antitrypsin (AAT) deficiency (OMIM 107400) is one of the most common serious hereditary disorders. This disease is inherited in an autosomal recessive manner and results in low serum and lung levels of AAT. This AAT insufficiency leads to uncontrolled neutrophil elastase activity, which targets the structural protein elastin, particularly in the lungs. This results in high risk for the development of emphysema in the third to fifth decade of life. Other AAT deficiency disorders, most notably an increased risk for the development of liver disease, can also be attributed to the presence of specific sequence variation in the AAT gene.
The AAT gene, located on chromosome 14 at region 14q32.1, consists of 1 untranslated exon followed by 4 translated exons. To date, at least 130 unique variant alleles of the AAT gene have been identified. Deficiency variant alleles can lead to reduced levels of circulating AAT in the serum or a defective ability to inhibit protease. Patients exhibiting 2 AAT deficiency alleles are generally considered to be at risk, whereas patients with a single AAT deficiency allele are considered to be at low risk for the development of AAT deficiency-related symptoms and are denoted as carriers in this work. Although AAT deficiency is considered to be underdiagnosed, the progression of AAT deficiency-related disorders can be dramatically slowed through lifestyle changes and protein replacement therapy.
The M allele is considered the native variant and results in normal levels of AAT protein production and resulting serum concentrations (100-250 mg/dL [18.4-46.0 µmol/L]), whereas the Z and S deficiency alleles account for the majority of the deleterious variants found in patients with AAT deficiency and are caused by single base substitutions that substantially reduce the concentration of circulating AAT. The homozygous ZZ protein phenotype is considered to cause the most severe phenotype and often results in the development of severe lung and liver problems. The SZ and SS protein phenotypes can also lead to severe pulmonary problems, particularly in people who smoke. The variation for the S deficiency allele is located in exon 3 (c.791A>T, GenBank accession No. K02212), causing an amino acid alteration of Glu264Val. The variation for the Z deficiency allele is located in exon 5 (c.1024G>A), causing a glutamine change to lysine at codon 342. Only about 15% of the normal amount of AAT is found in the serum of patients with homozygous ZZ protein phenotypes, whereas approximately 60% of normal concentrations are observed in people with the SS protein phenotype. Furthermore, the null allele is defined as having no protein detectable by isoelectric focusing (IEF) and a serum concentration of less than 30 mg/dL (5.5 μmol/L) by immunoturbidimetric assay.
World Health Organization guidelines recommend screening for AAT deficiency at least once in all patients with chronic obstructive pulmonary disease and in adults and children with asthma. The diagnosis of patients at risk for the development of AAT deficiency is often by IEF and concordant serum concentration determination by immunoturbidimetric assay of the AAT protein. The use of real-time polymerase chain reaction (PCR) analysis to detect the S and Z deficiency alleles in a large number of patient samples is potentially a more rapid and less technically demanding method than IEF for AAT analysis. Real-time PCR assays also have the advantage that a wide variety of sample types can be used. We validated a LightCycler assay (Roche, Indianapolis, IN) for S and Z deficiency allele detection using rapid PCR technology and FRET probes. This AAT genotyping assay quickly detected the S and Z deficiency alleles from peripheral blood, serum, and amniocyte samples. DNA extracted from amniocytes is useful for prenatal diagnosis because serum samples are not readily available for prenatal protein phenotype testing.
A large number of rare AAT variant alleles (other than the S and Z alleles) have been described and are potential deficiency alleles. The efficacy of using an assay that detects only the 2 most common AAT deficiency alleles (S and Z) for identifying all patients who have 2 deficiency alleles and who are at risk for the development of AAT deficiency-related symptoms is unknown. Patients could potentially be misclassified as carriers instead of at risk if an S or Z allele is paired with another rare deficiency allele. Furthermore, in rare cases, genotyping screens for only the more common deficiency alleles would miss patients with 2 rare deficiency alleles.
A diagnostic algorithm was developed that uses a combined approach consisting of an initial genotypic screen for S and Z deficiency alleles with concurrent AAT serum protein concentration testing. This step is followed by protein phenotyping by means of IEF for patient samples with abnormally low AAT serum concentrations who do not have the ZZ, SS, or SZ genotypes as assayed by the LightCycler AAT genotyping assay Figure 1. A retrospective database consisting of the protein phenotypes as determined by IEF and concurrent serum AAT concentrations of 50,025 clinical samples was used to evaluate the efficacy of this algorithm, incorporating genetic screening for the S and Z variants in identifying at-risk and carrier patient samples in a large clinical population.
(Enlarge Image)
Figure 1.
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