This study answered several questions relating to frequency, nature, recurrence, and phenotype/genotype correlations in mutations of the KCNC3 (Kv3.3) ion channel. We found that KCNC3 mutations were relatively rare, but that two mutations were recurring and involved the S4 domain. Specific disease causing mutations were predictive of the clinical phenotype with little variability within and between families.
Overall, the number of variants discovered after complete sequencing of the four coding exons and adjacent exon-intron junctions was small. Although we sequenced close to 700 alleles, many DNA variants were only observed a single time, either in controls or in affected index cases. These were located in the 5’UTR, in exons as synonymous or non-synonymous variants, and in the 3’UTR. The exception was one SNP in intron 3 g.13858G>A that reached a frequency of 7.3% in ataxia patients, but had a frequency of 0.70% in controls. As our study was not designed to examine the significance of allele frequency differences between cases and control, the biologic consequences of variants seen in cases and controls remain unknown at this time. Even though functional effects cannot be immediately gleaned from the sequence changes, it is possible that these variants could contribute to development or progression of disease.
In addition to rare non-coding variants, we identified three amino acid changing DNA variants. Of 260 index cases with a family history consistent with autosomal dominant inheritance, only five carried a mutation in the KCNC3
gene. Thus, it is highly unlikely that KCNC3
mutations explain a large proportion of the 30–40% of SCA families that have no identified mutations (Pulst, 2001
The two recurring mutations and the variant of unknown significance seen in a single sample occurred in highly conserved arginines. The arginines at positions 366, 420 and 423 are virtually invariant in all voltage-gated potassium channels. Arg423 is one of several arginine residues in the S4 transmembrane segment that sense and respond to changes in voltage (Aggarwal and MacKinnon, 1996
; Seoh, et al., 1996
). The positively charged residues in S4, including Arg420 and Arg423, are responsible for voltage-dependent conformational changes that result in channel opening or closing.
The p.Arg420His mutation, originally described in a Filipino pedigree (Waters, et al., 2006a
), was identified in three additional pedigrees from different European countries. These mutations had arisen independently as shown by haplotype analysis. The p.Arg420His mutation results in a non-functional subunit with dominant-negative properties on channel expression. In all four pedigrees, the p.Arg420His mutation was associated with late-onset progressive ataxia and cerebellar atrophy. Age of onset was highly variable within and between pedigrees and ranged from 25 to 51 in this study () and from 22 to more than 60 years of age in the previously reported Filipino pedigree.
The variant p.Arg366His is located at the intracellular end of the S2 transmembrane segment in the voltage sensor domain. Recently reported X-ray structures of voltage-gated potassium channels indicate that this positively-charged residue is involved in a network of electrostatic interactions that likely stabilize the voltage sensor domain (Long, et al., 2005a
; Long, et al., 2005b
). The p.Arg366His variant, which was seen in one individual with late onset, also resulted in a non-functional subunit with dominant-negative effects. When wild type and mutant subunits were expressed in Xenopus oocytes at a 1:1 expression ratio, current amplitudes were reduced to 50% (). Despite the likely importance of Arg366 for channel function, we cannot designate the p.Arg366His variant as disease-causing due to its lack of recurrence and absence of segregation in the family. The possibility remains, however, that the affected nephew without the p.Arg366His change represents a phenocopy.
A novel p.Arg423His mutation was observed in two pedigrees. The p.Arg423His mutation was associated with delayed development of motor milestones and lifelong, relatively stable ataxia, and thus resembled the phenotype observed in the large French pedigree segregating the p.Phe448Leu mutation. In contrast to presence of mild mental retardation in all carriers of the p.Phe448Leu mutation and seizures in some, none of the four individuals with the p.Arg423His mutation showed mental impairment or seizures.
Despite the shared early-onset phenotype, the effects of the two mutations in a heterologous expression system were distinct. The p.Arg423His mutation generated non-functional subunits with a strong dominant negative effect on channel expression, whereas the p.Phe448Leu mutation increased the stability of the open state of the channel. Therefore, the initial notion that gain-of-function mutations in KCNC3 would be associated with early-onset and dominant-negative mutations with late-onset and progression is not supported. It is currently unknown why the two dominant-negative mutations, p.Arg420His and p.Arg423His, have such different phenotypes. It remains possible that p.Arg423His has gain of function effects that were not detected in our experiments. For instance, mutations in conserved S4 arginine residues of the Nav1.4 voltage-gated Na+
channel associated with hypokalemic periodic paralysis generate aberrant leak currents through putative ‘gating pores’ in the voltage sensor domain that may contribute to the disease phenotype (Sokolov, et al., 2007
; Struyk, et al., 2008
This study extends the mutational and phenotypic spectrum of SCA13 and confirms the importance of voltage-gated potassium channels in cerebellar development and function. The p.Arg420His, p.Arg423His, and the previously identified p.Phe448Leu changes meet accepted criteria for mutations by segregation in pedigrees, recurrence, change of evolutionarily conserved amino acids, lack in control samples, and abnormal function in vitro
. The p.Arg366His variant meets some of these criteria, but has not been observed a second time. Therefore, despite the significant effects on channel function, its status as a disease-causing mutation should be interpreted with caution. The previously reported p.Phe448Leu mutation (Herman-Bert, et al., 2000
;) remains the only KCNC3 mutations with a cellular gain-of-function phenotype. Our study suggests that SCA13 will likely be a relatively rare cause of autosomal dominant ataxia.