Molecular Confirmation of α1-Antitrypsin Genotypes in Newborn Dried Blood Specimens

Abstract

Deficiency of α₁-antitrypsin (α₁AT), a common hereditary disorder of Caucasians, is associated with an increased risk for early-onset chronic obstructive pulmonary disease and childhood liver dysfunction. The two most common deficiency variants, Pi^S and Pi^Z, are both single base-pair substitutions causing amino acid modifications, although neither mutation creates or destroys a naturally occurring restriction site. Dried blood specimens (DBS) submitted to the New York State Department of Health for mandated newborn screening tests were tested for α₁AT activity using a fluorometric elastase inhibition assay. A second DBS from specimens determined to be α₁AT deficient was phenotyped on an agarose isoelectric focusing gel. Genotypic confirmation was performed by amplifying, directly from a DBS, the regions of the DNA containing the S and Z mutation. The Z mutation was analyzed with a modified primer designed to create an artificial restriction site in the normal allele. TaqI digestion produces two bands, a 157- and a 22-hp fragment. The single base substitution in Pi^Z individuals eliminates this TaqI restriction site, thus showing the same 179-bp fragment before and after digestion. A primer mismatch placed close to the S mutation creates a restriction site in the normal allele, producing a 100-bp product after TaqI digestion. The restriction site is abolished in individuals that carry the S mutation, with a 121-bp product observed before and after digestion. Of 11,081 specimens screened, 3 Pi^Z neonates, all Caucasian, were detected by these methodologies for an estimated incidence of 1:2019 in the Caucasian or 1:3694 in the general population in New York State. Additionally, 5 Pi^Sz, 59 Pi^Mz, 1 Pi^S, and 10 Pi^ms newborns were detected.