Mutations in KCND3 cause spinocerebellar ataxia type 22

Abstract

Objective: To identify the causative gene in spinocerebellar ataxia (SCA) 22, an autosomal dominant cerebellar ataxia mapped to chromosome 1p21-q23.

Methods: We previously characterized a large Chinese family with progressive ataxia designated SCA22, which overlaps with the locus of SCA19. The disease locus in a French family and an Ashkenazi Jewish American family was also mapped to this region. Members from all 3 families were enrolled. Whole exome sequencing was performed to identify candidate mutations, which were narrowed by linkage analysis and confirmed by Sanger sequencing and cosegregation analyses. Mutational analyses were also performed in 105 Chinese and 55 Japanese families with cerebellar ataxia. Mutant gene products were examined in a heterologous expression system to address the changes in protein localization and electrophysiological functions.

Results: We identified heterozygous mutations in the voltage-gated potassium channel Kv4.3-encoding gene KCND3: an in-frame 3-nucleotide deletion c.679_681delTTC p.F227del in both the Chinese and French pedigrees, and a missense mutation c.1034G>T p.G345V in the Ashkenazi Jewish family. Direct sequencing of KCND3 further identified 3 mutations, c.1034G>T p.G345V, c.1013T>C p.V338E, and c.1130C>T p.T377M, in 3 Japanese kindreds. Immunofluorescence analyses revealed that the mutant p.F227del Kv4.3 subunits were retained in the cytoplasm, consistent with the lack of A-type K(+) channel conductance in whole cell patch-clamp recordings.

Interpretation: Our data identify the cause of SCA19/22 in patients of diverse ethnic origins as mutations in KCND3. These findings further emphasize the important role of ion channels as key regulators of neuronal excitability in the pathogenesis of cerebellar degeneration.

Figure 1

Pedigree charts of the Families A, B, C, D, E and F. The gender of the family members is obscured for privacy. The proband is denoted by an arrow. Filled diamonds represent affected members, grayed diamonds represent members with information suggesting spinocerebellar ataxia but not confirmed, open diamonds indicate unaffected individuals, and those with a dot within an open diamond denote at-risk individuals. / = deceased; *= members who underwent whole exome sequencing, # = individuals included in linkage analysis. + = wildtype allele, del = allele with F227del, V= allele with G345V, E= allele with V338E, M= allele with T377M.

Figure 2

KCND3 mutations and Kv4.3 membrane topology. (A-D) The electrophoregrams of KCND3 heterozygous mutations. (E) Predicted Kv4.3 topology and locations of four mutations: red triangle- c.679_681delTTC (p.F227del), blue circles- c.1013T>C p.V338E, c.1034G>T p. G345V and c.1130C>T p.T377M. The mutated residues are evolutionarily conserved, as shown by aligning protein sequences of Kv4.3 orthologs in various organisms.

Figure 3

Confocal images demonstrating impaired cell surface expression and cytoplasmic retention of the mutant F227del Kv4.3. HEK-293T cells were transiently transfected with wild-type (A), p.F227del mutant (B) human Kv4.3 or empty vector (C) and immunostained with Kv4.3-specific antibody and green fluorophore-labeled secondary antibodies. Green fluorophore-labeled wild-type Kv4.3 (D) was expressed in the plasma membrane (E), colocalizing with DsRed-labeled membrane protein P0 (F). The spatial profile of fluorescent intensity of wild-type (wt) Kv4.3 (green) and P0 (red) along the arrow imposed on the images is shown in (G). The x axis displays the distance relative to the start point of the arrow and the y axis displays the fluorescence intensity. F227del Kv4.3 (H) was deficient in targeting to the plasma membrane (I, J). The spatial profile of fluorescent intensity of F227del Kv4.3 (green) and P0 (red) along the arrow imposed on the images is shown in (K). Instead, F227del Kv4.3 was retained in the cytoplasm (L) and colocalized with an ER-specific marker (M, N). The ratio of cell surface expression for the p.F227del Kv4.3 was significantly lower (O) than that observed in the wild type Kv4.3 (mean ± s.e.m.; p.F227del: 0.28 ± 0.04, n = 10; wild type Kv4.3: 0.61 ± 0.06, n = 10; p = 0.0014, Paired t test). Scale bar: 10 μm.

Figure 4

Electrophysiological recordings of the Kv4.3 channels. Whole-cell patch-clamp recordings revealed endogenous currents in un-transfected cells (A), large rapidly inactivating A-type potassium current in wild-type Kv4.3-transfected cells (B), and reduced current amplitudes in p.F227del Kv4.3-transfected cells (C). WT (black circles) had a significantly larger current density than p.F227del (red circles) (***P < 0.0005) and the current density of p.F227del was similar to that of Ctrl (filled circles) (D).