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2019 - The patient-independent human iPSC model – a new tool for rapid determination of genetic variant pathogenicity in long QT syndrome

Icon CE   CardioExcyte 96 publication in Heart Rhythm (2019)

Authors:
Chavali, N.V., Kryshtal, D.O., Parikh, S.S., Wang, L., Glazer, A.M., Blackwell, D.J., Kroncke, B.M., Shoemaker, M.B., Knollmann, B.C.

Journal:
Heart Rhythm (2019) DOI: https://doi.org/10.1016/j.hrthm.2019.04.031


Abstract:

Background

Commercial genetic testing for Long QT Syndrome (LQTS) has rapidly expanded, but the inability to accurately predict whether a rare variant is pathogenic has limited its clinical benefit. Novel missense variants are routinely reported as “Variant of Unknown Significance (VUS)” and cannot be used to screen family members at-risk for sudden cardiac death. Better approaches to determine pathogenicity of VUS are needed.

Objective

To rapidly determine the pathogenicity of a CACNA1C variant reported by commercial genetic testing as a VUS using a patient-independent induced pluripotent stem cell (hiPSC) model.

Methods

Using CRISPR/Cas9 genome editing, CACNA1C-p.N639T was introduced into a previously-established hiPSC from an unrelated healthy volunteer, thereby generating a patient-independent hiPSC model. Three independent heterozygous N639T hiPSC lines were generated and differentiated into cardiomyocytes (CM). Electrophysiological properties of N639T hiPSC-CM were compared to those of isogenic and population control hiPSC-CM by measuring the extracellular field potential (EFP) of 96-well hiPSC-CM monolayers, and by patch-clamp.

Results

Significant EFP prolongation was observed only in optically-stimulated but not in spontaneously-beating N639T hiPSC-CM. Patch clamp studies revealed that N639T prolonged the ventricular action potential by slowing voltage-dependent inactivation of CaV1.2 currents. Heterologous expression studies confirmed the effect of N639T on CaV1.2 inactivation.

Conclusion

The patient-independent hiPSC model enabled rapid generation of functional data to support reclassification of a CACNA1C VUS to “likely pathogenic”, thereby establishing a novel LQTS type 8 mutation. Furthermore, our results indicate the importance of controlling beating rates to evaluate functional significance of LQTS VUS in high-throughput hiPSC-CM assays.


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