Mutations in CIZ1 cause adult onset primary cervical dystonia

Abstract

Objective: Primary dystonia is usually of adult onset, can be familial, and frequently involves the cervical musculature. Our goal was to identify the causal mutation in a family with adult onset, primary cervical dystonia.

Methods: Linkage and haplotype analyses were combined with solution-based whole-exome capture and massively parallel sequencing in a large Caucasian pedigree with adult onset, primary cervical dystonia to identify a cosegregating mutation. High-throughput screening and Sanger sequencing were completed in 308 Caucasians with familial or sporadic adult onset cervical dystonia and matching controls for sequence variants in this mutant gene.

Results: Exome sequencing led to the identification of an exonic splicing enhancer mutation in exon 7 of CIZ1 (c.790A>G, p.S264G), which encodes CIZ1, Cip1-interacting zinc finger protein 1. CIZ1 is a p21(Cip1/Waf1) -interacting zinc finger protein expressed in brain and involved in DNA synthesis and cell-cycle control. Using a minigene assay, we showed that c.790A>G altered CIZ1 splicing patterns. The p.S264G mutation also altered the nuclear localization of CIZ1. Screening in subjects with adult-onset cervical dystonia identified 2 additional CIZ1 missense mutations (p.P47S and p.R672M).

Interpretation: Mutations in CIZ1 may cause adult onset, primary cervical dystonia, possibly by precipitating neurodevelopmental abnormalities that manifest in adults and/or G1/S cell-cycle dysregulation in the mature central nervous system.

FIGURE 1

Family A pedigree and linkage analysis. (A) Filled symbols, definitely affected. Half-filled symbols, possibly affected. Symbols with central dots, unaffected carriers. Arrow, proband. CIZ1 genotypes: wild-type (+/+) and heterozygous c.790A>G (+/−). (B) Multipoint HLOD scores for Family A generated under an autosomal dominant model with disease allele frequency set to 0.0001 and penetrance of 0.5. Marker positions are plotted on the x-axis and vertical dashed lines delimit the 22 autosomes.

FIGURE 2

Organization of CIZ1 gene, transcripts, and full-length protein. (A) Structure of CIZ1 on the reverse strand of Chr. 9 presented in the 5' to 3' direction showing the location of three identified mutations (c.139C>T, c.790A>G, and c.2015G>T) in probands with CD. (B) In brain, CIZ1 is transcribed into two major isoforms that differ at variable Exon 8. (C) Missense mutations in highly conserved regions of CIZ1 are shown in relationship to functional domains: QD1, glutamine-rich domain 1; NLS, putative nuclear localization sequence (259–264 aa); QD2, glutamine-rich domain 2; ZF, zinc finger domain; AD, acidic domain; MH3, matrin (MATR3)-homologous domain 3. Other putative NLSs (NLStradamus prediction cutoff of 0.05) are not shown (240–246 aa, 695–723 aa, 832–839 aa, 875–898 aa). (D) Protein sequence analysis with ClustalW2 shows that P47, S264, and R672 are located within highly conserved domains of CIZ1. *Mutation in Family A

FIGURE 3

Minigene assay of CIZ1 splicing. (A) Segment of CIZ1 containing Exons 6, 7, 8, and 9 were cloned into the multiple cloning site (MCS) of the pET01 exontrap vector, which contains a short stretch of a eukaryotic phosphatase gene (E) 3' to the eukaryotic strong long terminal repeater promoter of the Rous Sarcoma Virus (RSV). A second segment of E is followed by a polyadenylation site (pA). The c.790A>G mutation at the exonic splicing enhancer is located 2 nucleotides from the Exon 7–Intron 7 splice site. (B) The results of four wild-type and four c.790A>G transfections showed that the A>G mutation exerted significant effects on splicing patterns in HEK 293T cells (*P < 0.05).

FIGURE 4

Cellular localization of wild-type and mutant CIZ1 (S264G) in transiently transfected HEK 293T cells. CIZ1 was visualized with a GFP tag or rabbit anti-CIZ1 antibody. In comparison to wild-type CIZ1, average particle size was larger and the number of particles tended to be smaller in cells expressing S264G. β-tubulin, red; CIZ1, green; and DAPI, violet. Scale bar, 10 μm.