Fig. 3

Schematic diagram of integrated approach to VUS evaluation, incorporating functional evaluation of recombinant channels. Clinical evaluation, establishing a clear clinical phenotype is the key driver of clinical management. Where the clinical phenotype suggests LQTS, interrogation of known LQTS genes proceeds through clinical genetics. The flow chart assumes that results of such analysis yields a VUS in KCNQ1, KCNH2, or SCN5A. For all 3 genes, previously uncharacterized variants can be investigated using whole-cell patch clamp measurements of current carried by recombinant channels heterologously expressed in a mammalian expression system. At the minimum, experiments should involve multiple replicate recordings from WT and variant (V) channels separately and with equal co-expression of WT + V to mimic the heterozygous state. Key measurements are of current size (which can be normalized to cell surface area, as cell capacitance, to facilitate pooling of data from the different experiments) and time-course. Differing conventional square-wave voltage clamp protocols can be used to enable quantification of amplitude and kinetics. Action potential voltage clamp will give a direct ‘snapshot’ of effect of the variant on current profile during a physiological waveform. For KCNQ1 and KCNH2 variants, a complementary trafficking assay should be performed allowing quantification of expressed mature/surface channel protein. Such a trafficking assay should use the same range of WT and variant expression conditions as patch clamp, ideally also including vector only control and known LQTS variants as controls. Biophysical modelling may not be required where variants produce a large dominant negative effect on channel expression, but it is useful in all cases and necessary where the predominant effects under patch clamp are on current kinetics. At the minimum, such modelling should investigate variant effects on AP duration in a human ventricle model. More sophisticated multicellular modelling can investigate effects on simulated QT interval. Modelling should include conditions to mimic heterozygous inheritance. The integrated results from functional evaluation can inform overall variant classification of pathogenicity and inform clinical management