Figure 4

Reporter gene activity assay of variants at the 5′ regulatory region and exon 1a untranslated region found in this study. (a) The mean ratio and standard deviation of firefly luciferase activity to Renilla luciferase activity of each construct expressed in HEK293 cell line. No variant construct showed significant differences compared to both wild type constructs TGC and CAG simultaneously. (b) The mean ratio and standard deviation of firefly luciferase activity to Renilla luciferase activity of each construct expressed in SKNSH cell line. *indicates four mutation constructs including g-1528T > C (1.79 ± 0.60), g-1442G > A (1.99 ± 0.65), g.-142G > T (1.99 ± 0.65), and g-140A > T (1.62 ± 0.56) that showed significant elevation of reporter gene activity compared to two wild type constructs TGC (0.37 ± 0.66) and CAG (0.66 ± 0.50) simultaneously (P < 0.05). (c) The mean ratio and standard deviation of firefly luciferase activity to Renilla luciferase activity of the variant at exon 1a untranslated region measured in HEK293 cells. The mutant allele had significant elevated reporter gene activity (1.34 ± 0.16) compared to the wild type (1.09 ± 0.16; P < 0.05). (d) Similarly, the mutant allele showed significant reporter gene activity (4.18 ± 0.35) compared to the wild type (3.63 ± 0.28; P < 0.05) in SKNSH cells. MM indicates a multiple mutation that was found in one patient. TGC and CAG are two major haplotypes of the 5′ regulatory region found in this study.