DGGE banding patterns representing four controls (lanes 2–5) heterozygous for the M3 mutation (E376D), while our patient (lane 1), although also heterozygous for the M3 variant also presents with a shifted (faster migrating) banding depicting the novel deletion mutation (T379Δ). B. Sanger sequencing defines the deleted base pairs. The predicted amino acid sequence resulting from the novel 49 bp deletion (denoted by * on lower chromatogram) observed in our patient, results in the replacement of 16 amino acids and the addition of 24 amino acids through partial translation of the 3′ UTR.

^Δ379 Mutant. (A) Immunoblot (anti-GFP) detection of α₁AT-GFP fusion protein (C-terminal tag) in whole-cell lysate and concentrated conditioned media (ie secreted) from HEK293T cells transfected with plasmids expressing either wild-type or Δ379 mutant α₁AT-GFP. Red arrow denotes position of ∼75 kDa α₁AT-GFP band, note the absence of this band in conditioned media from cells transfected with Δ379 mutant, indicating impaired secretion of mutant protein; (B) Immunoblot (anti-α₁AT) detection of α₁AT-GFP fusion protein (C-terminal tag) in whole-cell lysate, or following immunprecipitation from conditioned media (i.e. secreted) from HEK293T cells transfected with plasmids expressing either wild-type or Δ379 mutant α₁AT-GFP; (C) Transfection of either wild-type or Δ379 mutant α₁AT with an N-terminal EGFP fusion into HEK293T cells clearly indicated normal proteolytic processing of the secretion signal peptide. Both ∼75 kDA and ∼27 kDA bands are visible, representing full-length and processed (i.e. signal peptide cleaved) α₁AT-GFP fusion protein respectively; (D) At higher expression levels, accumulation of insoluble Δ379 mutant α₁AT was observed in HEK293T cells, clearly denoted by the presence of a darker band in the insoluble fraction from cells transfected with Δ379 mutant; (E) Detection of soluble (whole-cell lysate), insoluble and secreted (concentrated conditioned media) α₁AT in HeLa cells transfected with either wild-type or Δ379 mutant α₁AT-GFP. Red arrow denotes position of ∼75 kDa α₁AT-GFP band. Note the absence of this band in conditioned media from cells transfected with Δ379 mutant, indicating impaired secretion of mutant protein; (E) Fluorescent micrographs of HEK293T cells following transfection with either wild-type or Δ379 mutant α₁AT-GFP expression plasmids. Increased intracellular aggregation of mutant protein is clearly visible. NB: Loading controls represent α-tubulin immunoblot or PonceauS staining in lysate or secreted (conditioned media) samples, respectively.