Identified SNPs and distribution of rs4567. (A) Illustration of the positions of the six SNPs identified in the ERManI gene. (B) Genotypes of rs4567 among PIZZ individuals that developed end-stage liver disease, requiring transplantation at different ages. The age at which each patient was placed on the transplant list (age at listing) was used as an indicator for age of onset. TX, transplant.

rs4567(A) suppresses the translation of transfected ERManI in HeLa cells. (A) The translational efficiency of transfected ERManI (during a 20 minute pulse with ³⁵S-methionine) in HeLa cells, with or without co-transfected Pi Z, were evaluated by a combination of immunoprecipitation, SDS-PAGE, autoradiography and quantified by PhosphoImager analysis. The error bars represent standard error of the mean. (B) The concentration of transfected ERManI (from above) under steady-state conditions was evaluated by Western blotting of cell lysates. The relative ERManI concentrations, as compared with control, were quantified in five separate experiments. The error bars represent a standard error of the mean.

Overexpression of mutant or wild type AAT induces rs4567(A)-mediated translational suppression of ERManI. The translational efficiency of ERManI (A) or ERManI (G) in the presence of co-transfected Z or wildtype AAT were evaluated by metabolic radiolabeling and quantified by PhosphoImager analysis. The p-value was calculated by the Student's t-test.

Percentage of identified SNPs in different groups. (A) Percentage of allelic genotypes of each SNP in the indicated groups. P values are calculated with the χ² statistic with 2 df. (B) Frequency of the rs4567 genotypes in the Caucasian population and in 200 ZZ individuals without liver disease.

rs4567 does not affect transcription or turnover rate of ERManI and its expression is not affected by serum. (A) HeLa cells were either not transfected [C], or cotransfected with ERManI(A) or ERManI(G) plus empty vector or Pi Z. 24hr after transfection. Cells were lysed and total mRNA was extracted and separated on agarose gels, and then subjected to Northern Blotting with ERManI-or Actin-specific probes. The bands were quantified using NIH Image J, and the ratio between ERManI and Actin was calculated. (B) HeLa cells co-transfected with ERManI(A) or ERManI(G) plus empty vector or Pi Z. 48 hr after transfection, cells were metabolically labeled with ³⁵S-methionine and chased for 0.5, 1, and 2 hr. At each time point, cells were lysed and subjected to immunoprecipitation of ERManI. The immunoprecipitates were resolved by SDS-PAGE and detected by autoradiography. After quantification by PhosphoImager analysis, the percentage of ERManI left at each chase time point was calculated and plotted. Error bars represent standard deviation from three independent experiments. (C) HeLa cells were co-transfected with ERManI(A) plus empty vector or Pi Z , and then metabolically labeled with ³⁵S-methionine in the presence 10% fetal bovine serum for 20 min. Cells were then lysed followed by the immunoprecipitation of ERManI. The immunoprecipitates were resolved by SDS-PAGE and detected by autoradiography. Quantification was performed by PhosphoImager analysis. The percentage of ERManI level in the presence of Z versus in the absence of Z was calculated with error bars representing the standard error of the mean.

Predicted binding of miR-205 to rs4567 in ERManI. The predicted binding pattern between mRNA sequences of ERManI is shown. The nucleotide at rs4567 (red) and flanking sequences (black) are shown, as is the sequence for mature human miR-205 (blue). The seed region and predicted 3' compensatory sequences that contribute to hybridization are underlined.