Gene variant effects across sodium channelopathies predict function and guide precision therapy

Abstract

Pathogenic variants in the voltage-gated sodium channel gene family lead to early onset epilepsies, neurodevelopmental disorders, skeletal muscle channelopathies, peripheral neuropathies and cardiac arrhythmias. Disease-associated variants have diverse functional effects ranging from complete loss-of-function to marked gain-of-function. Therapeutic strategy is likely to depend on functional effect. Experimental studies offer important insights into channel function but are resource intensive and only performed in a minority of cases. Given the evolutionarily conserved nature of the sodium channel genes, we investigated whether similarities in biophysical properties between different voltage-gated sodium channels can predict function and inform precision treatment across sodium channelopathies. We performed a systematic literature search identifying functionally assessed variants in any of the nine voltage-gated sodium channel genes until 28 April 2021. We included missense variants that had been electrophysiologically characterized in mammalian cells in whole-cell patch-clamp recordings. We performed an alignment of linear protein sequences of all sodium channel genes and correlated variants by their overall functional effect on biophysical properties. Of 951 identified records, 437 sodium channel-variants met our inclusion criteria and were reviewed for functional properties. Of these, 141 variants were epilepsy-associated (SCN1/2/3/8A), 79 had a neuromuscular phenotype (SCN4/9/10/11A), 149 were associated with a cardiac phenotype (SCN5/10A) and 68 (16%) were considered benign. We detected 38 missense variant pairs with an identical disease-associated variant in a different sodium channel gene. Thirty-five out of 38 of those pairs resulted in similar functional consequences, indicating up to 92% biophysical agreement between corresponding sodium channel variants (odds ratio = 11.3; 95% confidence interval = 2.8 to 66.9; P < 0.001). Pathogenic missense variants were clustered in specific functional domains, whereas population variants were significantly more frequent across non-conserved domains (odds ratio = 18.6; 95% confidence interval = 10.9-34.4; P < 0.001). Pore-loop regions were frequently associated with loss-of-function variants, whereas inactivation sites were associated with gain-of-function (odds ratio = 42.1, 95% confidence interval = 14.5-122.4; P < 0.001), whilst variants occurring in voltage-sensing regions comprised a range of gain- and loss-of-function effects. Our findings suggest that biophysical characterisation of variants in one SCN-gene can predict channel function across different SCN-genes where experimental data are not available. The collected data represent the first gain- versus loss-of-function topological map of SCN proteins indicating shared patterns of biophysical effects aiding variant analysis and guiding precision therapy. We integrated our findings into a free online webtool to facilitate functional sodium channel gene variant interpretation (http://SCN-viewer.broadinstitute.org).

Keywords: SCN1A; SCN2A; SCN4A; SCN5A; SCN8A.

Figure 1

SCN protein structure and position of disease-causing missense versus gnomAD variants. (A) and (B) SCN protein structure from side and top view. (C) Patient variants shown in red. (D) GnomAD variants shown in blue.

Figure 2

Enrichment analysis of gnomAD versus pathogenic variants and LoF versus GoF variants. Enrichment analysis of (A) gnomAD versus pathogenic and (B) LoF versus GoF variants across different protein parts including four homologous domains (D1–D4), each consisting of six transmembrane segments (S1–S6) and large cytoplasmic loops.

Figure 3

2D representation of pathogenic SCN variants with functional effects. 2D representation of the SCN protein. The alpha subunit consists of four homologous domains (D1–4) each formed of six transmembrane segments (S1–S6). Segment 4 represents the voltage sensor and S5–6 the pore region. Individual missense variants are displayed as different coloured bars. Blue denotes GoF, red LoF and yellow mixed function. Analogue missense pairs are displayed as circles with amino acid details.

Figure 4

3D illustration comparing GoF with LoF locations across the SCN protein. (A) GoF variants are illustrated in blue. (B) LoF variants are illustrated in red.

Figure 5

In-silico prediction versus reported biophysical SCN variant effects. Prediction agreement [as a percentage (%)] detailed according to different protein parts including four homologous domains (D1–D4), each consisting of six transmembrane segments (S1–S6) and large cytoplasmic loops. In silico prediction was performed according to ‘funNCion’ (http://funNCion.broadinstitute.org).

Figure 6

2D representation of SCN variants with functional effects according to single sodium channels. 2D representation of different SCN proteins. The alpha subunit consists of four homologous domains (D1–4) each formed of six transmembrane segments (S1–S6). Segment 4 represents the voltage sensor and segments S5–6 the pore region. Individual missense variants are displayed as different coloured circles. Blue denotes GoF, red LoF, yellow mixed function and purple similar-to-wild-type (STW). Benign variants are illustrated in pale colours.