Mutations in ANO3 cause dominant craniocervical dystonia: ion channel implicated in pathogenesis

Abstract

In this study, we combined linkage analysis with whole-exome sequencing of two individuals to identify candidate causal variants in a moderately-sized UK kindred exhibiting autosomal-dominant inheritance of craniocervical dystonia. Subsequent screening of these candidate causal variants in a large number of familial and sporadic cases of cervical dystonia led to the identification of a total of six putatively pathogenic mutations in ANO3, a gene encoding a predicted Ca(2+)-gated chloride channel that we show to be highly expressed in the striatum. Functional studies using Ca(2+) imaging in case and control fibroblasts demonstrated clear abnormalities in endoplasmic-reticulum-dependent Ca(2+) signaling. We conclude that mutations in ANO3 are a cause of autosomal-dominant craniocervical dystonia. The locus DYT23 has been reserved as a synonym for this gene. The implication of an ion channel in the pathogenesis of dystonia provides insights into an alternative mechanism that opens fresh avenues for further research.

Figure 1

Family Pedigrees (A) Pedigree showing the structure of the index family. Definitely affected family members are represented by filled symbols, and definitely unaffected family members are represented by empty symbols. The affectation status of family member III-2, represented by a circle with a question mark in the center, was uncertain. DNA was available as marked. Sequencing findings for the ANO3 c.1480A>T (p.Arg494Trp) mutation are indicated above and to the left of each symbol. Individuals marked with an asterisk were clinically examined. The following abbreviations are used: WT, homozygous wild-type alleles; and M, heterozygous mutation carrier. (B) Structure of the second phenotypically similar family, which carries a second, different mutation (c.1470G>C [p.Trp490Cys]) in the same exon of ANO3. DNA availability, examined individuals, and mutational status are marked as in (A). (C) Structure of a third family affected by a mutation (c.2053A>G [p.Ser685Gly]) in exon 21 of ANO3. Individuals I-2, II-1, and III-1 exhibited onset of dystonic tremor of the head, upper limbs, and larynx in the first decade of life. Individual II-4 developed laryngeal dystonia in her late twenties. DNA availability, examined individuals, and mutational status are marked as in (A).

Figure 2

Mutations in Exon 15 of ANO3 Diagram showing complete conservation of protein sequence and almost complete conservation of amino acid sequence across species in the region of exon 15 of ANO3, in which two disease-segregating mutations (c.1480A>T [p.Arg494Trp] and c.1470G>C [p.Trp490Cys]) were found (the affected bases and codons are shown in boldface and red, respectively). At the bottom, aligned electropherograms show normal and mutated sequences.

Figure 3

Graphical Summary of Expression Data (A) Box plot of ANO3 mRNA expression levels in 12 CNS regions. The expression levels are based on exon array experiments and are plotted on a log2 scale (y axis). This plot shows significant variation in ANO3 transcript expression across the 12 CNS regions analyzed: putamen (PUTM, n = 121), frontal cortex (FCTX, n = 122), temporal cortex (TCTX, n = 114), hippocampus (HIPP, n = 114), cervical spinal cord (SPCO, n = 13), substantia nigra (SNIG, n = 96), hypothalamus (HYPO, n = 13), medulla (specifically inferior olivary nucleus, MEDU, n = 109), intralobular white matter (WHMT, n = 120), thalamus (THAL, n = 107), and cerebellar cortex (CRBL, n = 129). ANO3 mRNA expression is significantly higher in the putamen than in all other brain regions. Whiskers extend from the box to 1.5× the interquartile range. (B) Graph to show ANO3 mRNA expression levels in six brain regions during the course of human brain development. The expression levels are based on exon array experiments and are plotted on a log2 scale. The brain regions analyzed are the striatum (STR), amygdala (AMY), neocortex (NCX), hippocampus (HIP), mediodorsal nucleus of the thalamus (MD), and cerebellar cortex (CBC). This plot shows increasing expression of ANO3 mRNA during human brain development, particularly in the striatum, from the early midfetal period to adolescence.

Figure 4

Graphical Summary of Fibroblast Functional Studies (A) Typical trace of [Ca²⁺]_c, as measured by fura-2, in control (ctrl 1 and ctrl 2) and mutation-bearing (ANO3 c.1470G>C [p.Trp490Cys]) fibroblasts in response to the application of 100 μM ATP. (B) A histogram shows a significantly decreased change in [Ca²⁺]_c in response to 100 μM ATP (black bars) in mutation-bearing fibroblasts and no change in [Ca²⁺]_c in response to 50 mM KCl (blue bars). Error bars represent the SEM, and the asterisk indicates p < 0.05. (C) Mean trace of [Ca²⁺]_c in response to thapsigargin (1 μM) and subsequent Ca²⁺ (2 mM) challenge (arrows). Error bars represent the SEM. (D) Histograms demonstrate a significant difference in ER calcium pool in ANO3-mutant cells compared to controls in response to thapsigargin (black bars) but no changes in the activation of store-operated calcium channels in response to the subsequent calcium challenge (blue bars). Error bars represent the SEM, and the double asterisks indicate p < 0.001.