A population-based study of KCNH7 p.Arg394His and bipolar spectrum disorder

Abstract

We conducted blinded psychiatric assessments of 26 Amish subjects (52 ± 11 years) from four families with prevalent bipolar spectrum disorder, identified 10 potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger Amish Study of Major Affective Disorder (ASMAD) cohort, and studied mutant potassium channels in neurons. Fourteen of 26 Amish had bipolar spectrum disorder. The only candidate allele shared among them was rs78247304, a non-synonymous variant of KCNH7 (c.1181G>A, p.Arg394His). KCNH7 c.1181G>A and nine other potentially pathogenic variants were subsequently tested within the ASMAD cohort, which consisted of 340 subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar 1 disorder, bipolar spectrum disorder and any major affective disorder). KCNH7 c.1181G>A had the highest enrichment among individuals with bipolar spectrum disorder (χ(2) = 7.3) and the strongest family-based association with bipolar 1 (P = 0.021), bipolar spectrum (P = 0.031) and any major affective disorder (P = 0.016). In vitro, the p.Arg394His substitution allowed normal expression, trafficking, assembly and localization of HERG3/Kv11.3 channels, but altered the steady-state voltage dependence and kinetics of activation in neuronal cells. Although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of HERG-type potassium channels, electrophysiological data) implicates neuronal HERG3/Kv11.3 potassium channels in the pathophysiology of bipolar spectrum disorder. Such a finding, if corroborated by future studies, has implications for mental health services among the Amish, as well as development of drugs that specifically target HERG3/Kv11.3.

Figure 1.

A (Upper panel): 26 individuals from four families underwent blinded, independent psychiatric assessments using the Structured Clinical Interview for DSM-IV (SCID), Research Version. Exome sequencing was done on subjects designated with a red asterisk. Families A–C (blue enclosures) were interviewed during the first phase of the study and Family D (green enclosure) was recruited later. Black symbols indicate individuals who met DSM-IV-TR criteria for at least two of three symptom clusters—mania, major depression, psychosis—and were considered multidomain affected with bipolar spectrum disorder. Gray symbols indicate individuals who met diagnostic criteria for depressive illness (recurrent or single episode) uncomplicated by mania or psychosis. The ‘‡’symbol indicates subjects who were unavailable for interviews or declined to participate. B (Lower panel): during the second phase of the study, 340 samples from the ASMAD were used to test associations of exome variants with bipolar spectrum disorder (eighteen ASMAD samples were individuals from Families A and C and thus excluded from the replication analysis). All ASMAD subjects were genotyped for 10 candidate exome variants and categorized as unaffected (N = 247) or affected (N = 93) by major affective illness; the latter category was then subdivided into the increasingly restrictive designations of bipolar spectrum disorder (N = 78) and bipolar 1 disorder (N = 63).

Figure 2.

Among seven Amish individuals with bipolar spectrum disorder, we identified a total of 83 668 exome variants, 17 609 of which remained after filtering out synonymous and intronic changes. Focusing on low-frequency alleles with potentially high pathogenicity, we excluded exome variants with minor allele frequency (MAF) >10% among population-specific control exomes. Only 10 of these variants were present in all seven individuals. These 10 ‘candidate’ alleles were then used to test for associations with bipolar spectrum disorder and broader diagnostic categories within the extended core pedigree (Families A–D, N = 26) and the larger ASMAD cohort (N = 340), respectively.

Figure 3.

Testing for the association of 10 rare candidate alleles with bipolar 1 (BP1, circles), bipolar spectrum (BPS, squares), and any major affective disorder (any Aff, triangles) among 340 subjects from the Amish Study of Major Affective Disorder cohort. FBAT P-values (abscissa) and χ² distribution (ordinate) were calculated for each of the 10 rare candidate gene variants detected by exome sequencing. Nine of these variants (ALDH9A1, XIRP2, MUC4, ALG10B, CCDC65, CSRNP2, KRT75, UTP14C and NEK5) are plotted in gray. KCNH7 c.1181G>A, represented with red symbols, shows the strongest association with affective disorders and shows an unusual distribution behavior among the 10 variants. For graphical clarity, FBAT is transformed to the −log10; dotted lines indicate arbitrary thresholds of P ≤ 0.5 and X² ≥ 4 for FBAT and chi-square testing, respectively.

Figure 4.

Left panel: localization of overexpressed KCNH7 wild-type and Arg394His in Neuro-2a cells immunostained under non-permeabilizing conditions (see Materials and Methods) with mouse monoclonal anti-V5 IgG_2a (1:500), followed by AlexaFluor 488-conjugated goat anti-mouse IgG_2a (1:400). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, 1.5 μg/ml) (blue fluorescent signal). (A and B) KCNH7 wild-type and Arg394His with the V5 epitope tag inserted in the S1–S2 extracellular loop localize to the plasma membrane in non-permeabilized Neuro-2a cells (single confocal images). (C and D) Maximum projection z-stack images of the cells shown in A and B. (E) Left—confocal image of Neuro-2a cells transiently overexpressing Arg394His S1-V5-S2-KCNH7. Right—Orthogonal projection of a section through the cell in the center of the left image demonstrating membrane localization for Arg394His S1-V5-S2-KCNH7. (F) Western blot of transiently overexpressed wild-type and Arg394His S1-V5-S2-KCNH7 fusion proteins in Neuro-2a cells from the same transfections used for A–D. S1-V5-S2-KCNH7 fusion proteins migrated as a core glycosylated and mature glycosylated doublet at ∼140 kDa. β-Actin was labeled as a loading control. Primary antibodies: anti-V5 mouse monoclonal IgG_2a (1:5000) and anti-β actin (1:1 000 000). Secondary antibody: goat anti-mouse IgG HRP-conjugated (1:1500). Data are representative of four independent transfections. Right panel: electrophysiological characteristics of wild-type (WT) and Arg394His HERG3 (KCNH7) currents. (G) Representative currents from a Neuro-2a cell transiently expressing WT HERG3 channels. (H) Representative currents from a Neuro-2a cell transiently expressing Arg394His HERG3 channels. (I) Scaled peak current–voltage (I/I_max) curves for WT (blue) and Arg394His (red) channels. The results are normalized to the maximal current size in each cell. The data points are connected by lines for an illustrative purpose only. n = 12 and 9 for WT and Arg394His, respectively. (J) Normalized conductance (G/G_max) as a function of voltage for WT (blue) and Arg394His (red). The half-activation voltage (V_0.5) and the apparent equivalent charge movement were –7.5 ± 1.1 mV and 4.7 ± 0.53e₀ for WT and 4.5 ± 1.1 mV and 3.8 ± 0.50e₀ for Arg394His. n = 12 and 9 for WT and Arg394His, respectively. The V_0.5 values for Arg394His are statistically different from those for WT (P < 1 × 10^–5). The equivalent charge numbers are indistinguishable between the groups (P = 0. 129). The smooth curves are Boltzmann fits to the pooled results. Kinetics of ionic currents at 20 mV (K) and –120 mV (L). Currents are scaled to facilitate comparison. The sweep width represents the mean ± SEM. In (K), n = 7 and 8 for WT and Arg394His, respectively. In (L), n = 5 and 4 for WT and Arg394His, respectively.