Molecular characterization of a new alpha-1-antitrypsin M variant allele, Mwhitstable: implications for DNA-based
H J Ambrose1, S M Chambers, G Mieli-Vergani
1AstraZeneca Diagnostics, Northwich, Cheshire, UK.
Abstract:
The mother and second child from a family, already with one PI ZZ child, were typed PI MZ by isoelectric focusing and unexpectedly as PI ZZ using a commercial alpha-1-antitrypsin genotyping kit. Both methods typed the father and first child as PI MZ and PI ZZ, respectively. DNA sequence analysis identified a 26-base pair (bp) deletion and 2-bp insertion in intron IV of the normal PI*M allele from both the mother and second child. The majority of the binding site for an amplification primer of the genotyping kit was absent in the variant deletion-insertion allele. The apparent PI*Z/PI*Z genotype of the mother and second child therefore arose from amplification of the PI*Z allele alone. Two hundred random DNA samples were subsequently examined and 5 of these were found to be heterozygous for the same deletion-insertion allele. The authors have designated the previously undescribed PI*M allele that harbors this benign polymorphism PI*Mwhitstable. The genotyping kit has been redesigned and revalidated, and its performance is not affected by the presence of the PI*Mwhitstable allele. The Gen Bank accession number for the nucleotide sequence described is AF159454.
Insights
A novel PI*Mwhitstable allele in alpha-1-antitrypsin genotyping can lead to false PI*Z/PI*Z results. Redesigning the genotyping kit resolved this issue, ensuring accurate alpha-1-antitrypsin (AAT) testing.
Area of Science:
- Genetics
- Molecular Biology
- Clinical Diagnostics
Background:
- Alpha-1-antitrypsin (AAT) deficiency is a genetic disorder.
- Accurate genotyping is crucial for diagnosing and managing AAT deficiency.
- Existing genotyping kits may be susceptible to errors from genetic variations.
Purpose of the Study:
- To investigate unexpected alpha-1-antitrypsin (AAT) genotyping results in a family.
- To identify the genetic basis for discrepancies between isoelectric focusing and genotyping methods.
- To characterize a novel AAT allele and its impact on diagnostic assays.
Main Methods:
- Isoelectric focusing (IEF) for AAT phenotyping.
- Commercial AAT genotyping kit analysis.
- DNA sequence analysis of the PI*M allele.
- Screening of random DNA samples for the novel allele.
Main Results:
- A family showed discrepant AAT genotypes between IEF and a commercial kit.
- DNA sequencing revealed a novel PI*Mwhitstable allele with a deletion-insertion in intron IV.
- This variant disrupted primer binding, causing false PI*Z/PI*Z results due to preferential amplification of the PI*Z allele.
- The PI*Mwhitstable allele was found in 5 out of 200 random DNA samples.
Conclusions:
- The PI*Mwhitstable allele is a previously undescribed benign polymorphism.
- This allele can cause false-positive PI*Z/PI*Z genotypes in specific AAT genotyping kits.
- The genotyping kit was redesigned and revalidated to accurately detect the PI*Mwhitstable allele.
- Accurate AAT genotyping is essential for correct diagnosis and patient management.
