alpha 1-antitrypsin gene mutation hot spot associated with the formation of a retained and degraded null variant

Null alpha 1-antitrypsin (alpha 1AT) alleles represent the end of a continuum of variants associated with profound alpha 1AT deficiency and an increased risk of emphysema. This study characterizes the molecular basis of QOclayton, a new example of an alpha 1AT null allele arising from a mutational hot spot in the alpha 1AT gene. The QOclayton allele is identical to the normal M1(V213) alpha 1AT allele except for an insertion of a cytosine. This insertion occurs in the alpha 1AT sequence which normally has seven cytosines corresponding to amino acid residues 360 to 362. The QOclayton mutation is located in the same reiterated DNA sequence as the alpha 1AT QObolton deletion mutation and the insertion mutation allele QOsaarbruecken. The QOclayton cytosine insertion causes a 3' frameshift and results in the formation of a termination codon at residue 376, the same consequence as the alpha 1AT QOmattawa mutation (L353 T-insertion with a 3' frameshift). To determine the molecular mechanisms responsible for the absence of alpha 1AT associated with the QOclayton gene, an in vitro model of QOclayton was established using Chinese hamster ovary cells (CHO) transfected with the QOclayton gene. These cells were evaluated for alpha 1AT mRNA expression, protein synthesis and secretion. Although the QOclayton gene expresses a similar amount of alpha 1AT mRNA as compared with the normal alpha 1AT gene, no QOclayton protein is secreted. Protein trafficking and double-label immunofluorescence demonstrate that the QOclayton protein is retained in the rough endoplasmic reticulum or pre-Golgi compartment and is degraded (t(1/2) = 6.5 h). Since QOmattawa, QObolton, and QOsaarbruecken have similar termination sites in the alpha 1AT mRNA, they may share a similar intracellular fate.