Loss-of-function of the voltage-gated sodium channel NaV1.5 (channelopathies) in patients with irritable bowel syndrome

Abstract

Background & aims: SCN5A encodes the α-subunit of the voltage-gated sodium channel NaV1.5. Many patients with cardiac arrhythmias caused by mutations in SCN5A also have symptoms of irritable bowel syndrome (IBS). We investigated whether patients with IBS have SCN5A variants that affect the function of NaV1.5.

Methods: We performed genotype analysis of SCN5A in 584 persons with IBS and 1380 without IBS (controls). Mutant forms of SCN5A were expressed in human embryonic kidney-293 cells, and functions were assessed by voltage clamp analysis. A genome-wide association study was analyzed for an association signal for the SCN5A gene, and replicated in 1745 patients in 4 independent cohorts of IBS patients and controls.

Results: Missense mutations were found in SCN5A in 13 of 584 patients (2.2%, probands). Diarrhea-predominant IBS was the most prevalent form of IBS in the overall study population (25%). However, a greater percentage of individuals with SCN5A mutations had constipation-predominant IBS (31%) than diarrhea-predominant IBS (10%; P < .05). Electrophysiologic analysis showed that 10 of 13 detected mutations disrupted NaV1.5 function (9 loss-of-function and 1 gain-of-function function). The p. A997T-NaV1.5 had the greatest effect in reducing NaV1.5 function. Incubation of cells that expressed this variant with mexiletine restored their sodium current and administration of mexiletine to 1 carrier of this mutation (who had constipation-predominant IBS) normalized their bowel habits. In the genome-wide association study and 4 replicated studies, the SCN5A locus was strongly associated with IBS.

Conclusions: About 2% of patients with IBS carry mutations in SCN5A. Most of these are loss-of-function mutations that disrupt NaV1.5 channel function. These findings provide a new pathogenic mechanism for IBS and possible treatment options.

Trial registration: ClinicalTrials.gov NCT01717404.

Keywords: GI Motility; Genetics; Polymorphism; Voltage-Gated Sodium Channel.

Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Voltage-gated Na+ ion channel NaV1.5 topology with missense mutations in SCN5A identified in a cohort with IBS. DI - DIV are homologous 6 transmembrane helix domains 1 through 4.

Figure 2

Electrophysiologic abnormalities in NaV1.5 mutations. A, Representative whole cell Na+ current traces (step to -30 mV) from HEK293 cells transfected with wild-type (control, black) or A997T (blue). Dotted line = 0 pA/pF. B, NaV1.5 current-voltage plot showing shifts in voltage-dependence of inactivation for T220I (maroon) and A997T (blue) and positive shifts in voltage-dependence of activation for G615E (orange) and A997T (blue). C, Window currents for T220I, G615E, A997 are the shaded areas under the intersecting current-voltage curves. D&E show representative single traces (step to -30 mV, peaks normalized to 100%) of whole cell Na+ voltage-dependent current for mutations compared to wild-type (controls) for in the activation (D) and inactivation kinetics (E). D, Activation kinetics were altered for mutations G615E (orange), A997T (blue), and G1158S (violet) compared to wild-type, (control, black). Inset, G615E, A997T, and G1158S activate slower than wild-type. E, Inactivation kinetics were altered for mutations T630M (maize), P648L (green), or E1780G (purple) compared to wild-type (control, black). Inset, mutations T630M, P648L, or E1780G inactivate faster than wild-type.

Figure 3

Distribution of the electrophysiologic abnormalities across all mutations. A, Shown in black are gain-of-function (GOF, 2/18) and in grey are loss-of-function (LOF, 16/18) parameters for all IBS cases (IBS-D, IBS-C and IBS-M). B, Activation and inactivation LOF abnormalities are shown in IBS-D (4/18, blue), IBS-C (8/16, red) and IBS-M (1/16, yellow).

Figure 4

Loss-of-function phenotype in the mutant sodium channel p.A997T-NaV1.5 with partial rescue by mexiletine. A, Representative Na+ current traces (step to -30 mV) from wild-type (black trace) or p.A997T-NaV1.5 (grey trace) without (left) or with (right) mexiletine (10μM). Inset shows the same traces scaled to peak. B, Averages of peak current densities. *P<0.05 to wild-type and †P<0.05 to no drug by a one-way parametric ANOVA with Bonferroni's posttest (n = 9-25). C, Voltage dependence of steady-state activation and inactivation of wild-type (filled symbols) or A997T-NaV1.5 (empty symbols) previously exposed to no drug (left) or mexiletine (right). Boltzmann function fits for wild-type NaV1.5 (black lines) or A997T-NaV1.5 (grey lines). D, Averages of V1/2 of activation. *P<0.05 to wild-type by a oneway parametric ANOVA with Bonferroni's posttest (n = 9-25).

Figure 5

Bowel movement frequency was improved in the A997T-NaV1.5 patient after mexiletine treatment. Complete spontaneous (dark grey) and small hard (light grey) bowel movements for the patient for 3.5 weeks preceding and 5 weeks following a 5 day treatment with mexiletine showing normalization of bowel habits with mexiletine.