Citation and Contact

Brunklaus A, Feng T, Brünger T, Perez-Palma E, Heyne H, Matthews E, Semsarian C, Symonds JD, Zuberi SM, Lal D, and Schorge S. Gene variant effects across sodium channelopathies predict function and guide precision therapy. In revision.

andreas.brunklaus@glasgow.ac.uk eduardoperez@udd.cl dlal@broadinstitute.org

Abstract

Introduction

Pathogenic variants in the voltage-gated sodium channel gene family (SCNs) 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.

Methods

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 characterised 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.

Results

We detected 38 missense variant pairs with an identical disease-associated variant in a different sodium channel gene. 35 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% CI = 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% CI = 10.9 to 34.4; P<0.001). Pore-loop regions were frequently associated with loss-of-function (LoF) variants, whereas inactivation sites were associated with gain-of-function (GoF; odds ratio = 42.1, 95% CI = 14.5 to 122.4; P<0.001), whilst variants occurring in voltage-sensing regions comprised a range of gain- and loss-of-function effects.

Conclusions

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 GoF versus LoF 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).

SCN viewer main features

The SCN viewer enables the exploration of functional readouts from pathogenic variants within genes of the voltage-gated sodium channel family. Ten sodium channel genes are included: SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A, SCN11A. The tool shows 369 variants which have been electrophysiologically characterized for Gain of Function (GoF), Loss of Function (LoF) and Mixed effects. Functional readouts for each variant were aggregated from more than 400 references. After alignment, functional effects are often similar between analogous positions across different gene family members. Thus the SCN-viewer is a useful tool to aid variant interpretation.

1) Burden analysis view: The SCN viewer webtool first shows a burden analysis across the alignment of all ten members of the voltage-gated sodium channel gene family (Length = 2472 amino acids). The amino acid alignment allows for data aggregation from multiple genes into a single index sequence. We carried out a burden analysis to account for the rate of Gain of Function (GoF, orange area in plot), Loss of Function (LoF, blue area in plot) and Mixed effects (yellow area in plot) observed across the alignment. The user can explore and zoom into multiple regions. For comparison, we also provide the possibility to view the pathogenic variant burden observed in the ClinVar database (purple area) and general population variant burden from the genome aggregation database (gnomAD, green area). The user can select different burden tracks to plot and compare over the alignment (Figure 1).

Figure 1

2) Table view: Immediately below the plot, the tool shows the same alignment in a table format. This enables the user to directly explore the analyzed sodium channel variants and connect to the associated literature. Again, the user can explore Gain of Function (GoF, orange rows), Loss of Function (LoF, blue rows) and Mixed effects (yellow rows) variants observed across the alignment using the same color code as the plot. Phenotypes observed in the ClinVar database are provided in the “ClinVar phenotype reports” for comparison, although the ClinVar database does not provide functional readouts. The user can switch between the burden analysis view and table view. For example, selecting a region of interest in the burden plot will highlight the specific amino acids involved in the table view below. Lastly, users interested in a single amino acid in a specific gene, can search for it in the corresponding gene column (Figure 2). To search, simply type the Reference amino acid (one letter code) + underscore + the position in the protein (e.g., I_774).

Figure 2

video-tutorial