title: Molecular confirmation of alpha 1-antitrypsin genotypes in newborn dried blood specimens.
abstract: Deficiency of alpha 1-antitrypsin (alpha 1AT), 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, PiS and PiS, 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 alpha 1AT activity using a fluorometric elastase inhibition assay. A second DBS from specimens determined to be alpha 1AT 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-bp fragment. The single base substitution in PiS 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 PiS 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Genetics and IVF Institute, Fairfax, Virginia 22031-4609.
authors.forename: W C
authors.initials: WC
authors.lastname: Spence
authors.forename: J E
authors.initials: JE
authors.lastname: Morris
authors.forename: K
authors.initials: K
authors.lastname: Pass
authors.forename: P D
authors.initials: PD
authors.lastname: Murphy
mesh_terms: D000595:Amino Acid Sequence; D004587:Electrophoresis, Agar Gel; D005838:Genotype; D006801:Humans; D007231:Infant, Newborn; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000097:blood; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase