Using the genome aggregation database, computational pathogenicity prediction tools, and patch clamp heterologous expression studies to demote previously published long QT syndrome type 1 mutations from pathogenic to benign

Abstract

Background: Mutations in the KCNQ1-encoded Kv7.1 potassium channel cause long QT syndrome (LQTS) type 1 (LQT1). It has been suggested that ∼10%-20% of rare LQTS case-derived variants in the literature may have been published erroneously as LQT1-causative mutations and may be "false positives."

Objective: The purpose of this study was to determine which previously published KCNQ1 case variants are likely false positives.

Methods: A list of all published, case-derived KCNQ1 missense variants (MVs) was compiled. The occurrence of each MV within the Genome Aggregation Database (gnomAD) was assessed. Eight in silico tools were used to predict each variant's pathogenicity. Case-derived variants that were either (1) too frequently found in gnomAD or (2) absent in gnomAD but predicted to be pathogenic by ≤2 tools were considered potential false positives. Three of these variants were characterized functionally using whole-cell patch clamp technique.

Results: Overall, there were 244 KCNQ1 case-derived MVs. Of these, 29 (12%) were seen in ≥10 individuals in gnomAD and are demotable. However, 157 of 244 MVs (64%) were absent in gnomAD. Of these, 7 (4%) were predicted to be pathogenic by ≤2 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.

Conclusion: This study offers preliminary evidence for the demotion of 32 (13%) previously published LQT1 MVs. Of these, 29 were demoted because of their frequent sighting in gnomAD. Additionally, in silico analysis and in vitro functional studies have facilitated the demotion of 3 ultra-rare MVs (F127L, P477L, L619M).

Keywords: Arrhythmia; Genetics; Heart arrest; KCNQ1; Long QT syndrome; Pediatrics.

Figure 1.. Breakdown of KCNQ1 case-derived missense variants in gnomAD and after in silico pathogenicity analysis.

Each of 244 unique KCNQ1 case-derived MVs were assessed for rarity using gnomAD. Variants were considered “ultra-rare” if completely absent (allele count = 0), “rare” if seen 1 to 9 times, or “common” if seen in ≥10 individuals within gnomAD. In silico analysis was conducted using eight publically available tools to assess the predicted pathogenicity of all KCNQ1 case-derived MVs. Each variant was placed into one of three categories based on how many tools predicted that variant to be pathogenic: likely benign (≤ 2 tools), uncertain significance (3-5 tools), or likely pathogenic (≥ 6 tools).

Figure 2.. KCNQ1-F127L, -P477L, and -L619M missense variants did not significantly alter I_Ks current density in heterologous TSA 201 cells.

(A) Depicted is a schematic representation of the KCNQ1-encoded potassium channel alpha subunit (Kv7.1) with KCNQ1-F127L, -P477L, and -L619M variants. (B) Whole cell I_Ks current representative tracings from non-transfected TSA201 cells and TSA201 cells expressing KCNQ1-WT, -F127L, -P477L, and -L619M determined from a holding potential of −80 mV and testing potentials from −40 mV to +80 mV in 10 mV increments with 4s duration. (C) Current-voltage relationship for I_Ks KCNQ1-WT, -F127L, -P477L, and -L619M missense variants and for non-transfected cells. All values represent mean ± SEM. A p<0.05 was considered to be significant.