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. Author manuscript; available in PMC: 2022 Jul 7.
Published in final edited form as: Genet Med. 2022 Mar 16;24(6):1238–1248. doi: 10.1016/j.gim.2022.02.010

Figure 1. Measurement of loss-of-function homozygous and heterozygous peak current.

Figure 1.

a For homozygous experiments, 1 copy of SCN5A were inserted into engineered HEK293 LP cells. The Landing Pad (LP) comprises an AttP and BFP locus, and allows insertion of a single insert per cell.

b Representative raw peak current densities in a WT and p.Ala735Glu cell. Inset: voltage protocol used.

c Measurement of homozygous peak current density in 35 SCN5A missense variants and one nonsense variant (normalized to WT). Mean ± standard errors. 11-67 cells were studied per variant.

d For heterozygous experiments, we used a combination of LP and SB systems. First, a SB plasmid bearing a WT copy of SCN5A was randomly inserted into the genome. A clone of these cells was identified that has an equal level of NaV1.5 in patch clamp experiments to typical LP expression. Next, a second copy of SCN5A bearing WT or variant was incorporated through the LP system.

e Representative raw peak current densities in a single transfected WT, dually integrated WT+WT, and WT+p.Ala735Glu cell.

f Peak current density measurements for 35 SCN5A missense variants and one nonsense variant in expression with WT SCN5A (normalized to single WT). Mean ± standard errors. 27-164 cells were studied per variant.