title: Alpha 1-antitrypsin nonsense mutation associated with a retained truncated protein and reduced mRNA.
abstract: alpha 1-Antitrypsin (alpha 1AT) provides the major protection in the lung against neutrophil elastase-mediated proteolysis. Inheritance of alpha 1AT deficiency alleles is associated with an increased risk of emphysema and liver disease. alpha 1AT null alleles cause the total absence of serum alpha 1AT and represent the ultimate in a continuum of alleles associated with alpha 1AT deficiency. The molecular mechanisms responsible for absence of serum alpha 1AT include splicing abnormalities, deletion of alpha 1AT coding exons, and premature stop codons. We identified an Italian individual with asthma, emphysema, and a very low level of serum alpha 1AT. DNA sequencing demonstrated the Mprocida deficiency allele and a novel null allele, QOtrastevere (c654 G-->A, W194Z), a nonsense mutation near the intron 2 (IVS2) splice acceptor site. To determine the molecular basis of QOtrastevere and specifically to evaluate whether this nonsense mutation interfered with mRNA processing by altered splicing, we used a Chinese hamster ovary cell line permanently transfected with QOtrastevere or normal M alpha 1AT with and without IVS2. Northern blot analysis demonstrated that the normal M construct, with or without IVS2, expressed alpha 1AT mRNA of a similar size. The nonsense mutation was associated with moderately reduced alpha 1AT mRNA regardless of the presence or absence of IVS2. Reduction in alpha 1AT mRNA regardless of the opportunity for splicing supports a translational-translocation model as the cause of reduced alpha 1AT mRNA rather than the nuclear scanning model. Pulse-chase studies followed by immunoprecipitation demonstrated an endoplasmic reticulum-retained 31 kDa QOtrastevere alpha 1AT, which was rapidly degraded. Although mRNA content was moderately reduced, retention and rapid intracellular degradation of the truncated form are the major mechanisms for the absence of secreted alpha 1AT.
authors.affiliation: Clinical Studies Section, Pulmonary-Critical Care Medicine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1590, USA.
authors.forename: J
authors.initials: J
authors.lastname: Lee
authors.forename: N
authors.initials: N
authors.lastname: Novoradovskaya
authors.forename: B
authors.initials: B
authors.lastname: Rundquist
authors.forename: J
authors.initials: J
authors.lastname: Redwine
authors.forename: C
authors.initials: C
authors.lastname: Saltini
authors.forename: M
authors.initials: M
authors.lastname: Brantly
mesh_terms: D000483:Alleles; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004252:DNA Mutational Analysis; D005838:Genotype; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008297:Male; D010375:Pedigree; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D012333:RNA, Messenger; D000515:alpha 1-Antitrypsin; C114212:alpha 1-antitrypsin QOtrastevere