Expression-System Biases Compromise the Functional Assessment of KCNH2 Variants and Clinical Risk Assessment

At a 1:1 WT-to-T74R expression ratio in hERG channels, current amplitude drops to 31.0% of WT, consistent with dominant-negative loss when multiple T74R subunits incorporate.

• Why it matters: Functional testing of KCNH2 variants for long QT syndrome type 2 depends on allele-balanced expression, yet standard dual-expression constructs may skew subunit ratios seen in heterozygous patients.
• What they did: Allele-specific KCNH2 expression was quantified from two human left ventricular RNA-seq datasets, then T74R effects were calibrated and tested using automated patch clamp with IRES, P2A, and dCMV dual-expression strategies versus WT/variant co-electroporation.
• The result: All dual-expression strategies produced substantial expression imbalances (IRES most asymmetric), and P2A altered T74R trafficking/biophysics via an added N-terminal proline; co-electroporation best controlled relative expression for heterozygous functional phenotyping.

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