Abstract 19352: Utilizing the Genome Aggregation Database, in silico Tools, and Patch Clamp Heterologous Expression Studies to Demote Previously Published Type 1 Long QT Syndrome Mutations From Pathogenic to Benign

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Introduction: Loss of function mutations in the KCNQ1-encoded Kv7.1 potassium channel cause type 1 long QT syndrome (LQT1). To date, hundreds of unique KCNQ1 missense variants (MSVs) have been published as “pathogenic”. However, a 1% background rate of rare KCNQ1 MSVs in the healthy population exists. It has been suggested that up to 10-20% of rare LQTS case-derived variants in the literature may be false positives. Here, we set out to determine which previously published KCNQ1 case variants are likely false positives.

Methods: Derived from six large compendia, a list of all published, case-derived KCNQ1 MSVs was compiled. The occurrence of each MSV within the Genome Aggregation Database (gnomAD) comprising 141,352 individuals was assessed. Eight in silico tools were used to predict each variant’s putative pathogenicity. Case-derived variants that either i) were too frequently found in gnomAD or ii) were absent in gnomAD but predicted to be pathogenic by ≤ 2 tools were considered as the short list for potential false positives. Three of these variants (F127L, P477L, and L619M) were characterized functionally using whole cell patch clamp technique.

Results: Overall, there were 244 KCNQ1 case-derived MSVs. Of these, 29 (12%) were seen in ≥ 10 individuals in gnomAD and can be demoted. However, 157/244 (64%) MSVs were absent in gnomAD. Of these, 7 (4%) were predicted to be pathogenic by ≤ 2 in silico tools, 3 of which we characterized functionally. There was no significant difference in current density between heterozygous KCNQ1-F127L, -P477L, or -L619M variant-containing channels compared to KCNQ1-WT.

Conclusions: Using a combination of gnomAD, in silico tools, and a functional validation assay, we offer preliminary evidence for the demotion of 32 (13%) previously published LQT1 MSVs. Of these, 29 MSVs were demoted because of their frequent sighting in gnomAD. The greater challenge is the identification of the erroneous LQT1 classifications when the variant of interest is absent from gnomAD. Here, in silico analysis and in vitro functional studies have facilitated the demotion of three ultra-rare MSVs (F127L, P477L, and L619M).

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