Skip to main content
. 2017 Mar 22;25(6):669–679. doi: 10.1038/ejhg.2017.32

Figure 2.

Figure 2

Follow-up studies on variants in PIGN and PIGT. (a) PIGN-knockout HEK293 cells were generated and transfected with human wild-type or p.(L311W) mutant PIGN cDNA cloned into pME or pTK expression vectors. Restoration of the cell surface expression of CD59 was evaluated by flow cytometry. The mutant construct using the pME promoter did not rescue CD59 surface expression as efficiently as the wild-type construct, indicating that the variant results in reduced PIGN activity. (b) Levels of expressed wild-type and p.(L311W) mutant HA-tagged PIGN in pME-vector transfected cells were analysed by western blotting using an anti-HA antibody. After normalisation with luciferase activity and GAPDH, expression of the mutant protein appeared to be reduced by only ~10% compared with the wild-type protein. (c) PIGT-knockout HEK293 cells were transfected with wild-type or mutant PIGT cDNA cloned into pME or pTK expression vectors. Restoration of the cell surface expression of CD59 was evaluated by flow cytometry. The mutant constructs using the pTK promoter did not rescue CD59 surface expression as efficiently as the wild-type construct, indicating that the variants result in reduced PIGT activity. (d) Levels of expressed wild-type and mutant FLAG-tagged PIGT in pME-vector transfected cells were analysed by western blotting. After normalisation, expression of the mutant protein appeared to be reduced only for the p.(L578fs*35) variant. (e) Allelic ratio plots along chromosome 20 (for high confidence SNVs only) showed that the PIGT variant shared in 270250 and 270306 lies within a large region of autozygosity.