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. 2013 Dec 2;8(12):e81884.
doi: 10.1371/journal.pone.0081884. eCollection 2013.

Identification of CHIP as a novel causative gene for autosomal recessive cerebellar ataxia

Yuting Shi  1 , Junling Wang, Jia-Da Li, Haigang Ren, Wenjuan Guan, Miao He, Weiqian Yan, Ying Zhou, Zhengmao Hu, Jianguo Zhang, Jingjing Xiao, Zheng Su, Meizhi Dai, Jun Wang, Hong Jiang, Jifeng Guo, Yafang Zhou, Fufeng Zhang, Nan Li, Juan Du, Qian Xu, Yacen Hu, Qian Pan, Lu Shen, Guanghui Wang, Kun Xia, Zhuohua Zhang, Beisha Tang
Affiliations

Identification of CHIP as a novel causative gene for autosomal recessive cerebellar ataxia

Yuting Shi et al. PLoS One. .

Abstract

Autosomal recessive cerebellar ataxias are a group of neurodegenerative disorders that are characterized by complex clinical and genetic heterogeneity. Although more than 20 disease-causing genes have been identified, many patients are still currently without a molecular diagnosis. In a two-generation autosomal recessive cerebellar ataxia family, we mapped a linkage to a minimal candidate region on chromosome 16p13.3 flanked by single-nucleotide polymorphism markers rs11248850 and rs1218762. By combining the defined linkage region with the whole-exome sequencing results, we identified a homozygous mutation (c.493CT) in CHIP (NM_005861) in this family. Using Sanger sequencing, we also identified two compound heterozygous mutations (c.389AT/c.441GT; c.621C>G/c.707GC) in CHIP gene in two additional kindreds. These mutations co-segregated exactly with the disease in these families and were not observed in 500 control subjects with matched ancestry. CHIP colocalized with NR2A, a subunit of the N-methyl-D-aspartate receptor, in the cerebellum, pons, medulla oblongata, hippocampus and cerebral cortex. Wild-type, but not disease-associated mutant CHIPs promoted the degradation of NR2A, which may underlie the pathogenesis of ataxia. In conclusion, using a combination of whole-exome sequencing and linkage analysis, we identified CHIP, encoding a U-box containing ubiquitin E3 ligase, as a novel causative gene for autosomal recessive cerebellar ataxia.

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Conflict of interest statement

Competing Interests: The authors including Jianguo Zhang, Jingjing Xiao, Zheng Su, Meizhi Dai and Jun Wang are employed by the company of BGI-Shenzhen. These authors have declared that no competing interests exist in this paper, including paid employment, consultancy, patents applications and products in development or marketed products etc. And this does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. The pedigrees, brain MRIs, and CHIP mutations identified.
(A) The pedigree of family 1 with autosomal-recessive spinocerebellar ataxia. (B) The brain MRI of II-5 in family 1. Panel (left): axial T1-weighted image showing atrophy of the cerebellar vermis. Panel (right): midline sagittal T1-weighted image showing cerebellar atrophy, particularly evident in the superior vermis, with enlargement of the fourth ventricle. (C) Sanger sequencing results of codons 164–166 in exon 1 of the CHIP gene in a WT subject (left), an individual carrying the heterozygous variant (middle), and an individual carrying the homozygous c.493C>T (p.L165F) mutation (right). (D) The L165F missense mutation occurred at an evolutionarily conserved amino acid (in red) in the CHIP. (E and F) The pedigrees of families 2 and 3. Sanger sequencing results of the members of these two families. The red arrows indicate the mutation sites.
Figure 2
Figure 2. Genomic organization of the human CHIP gene and the domain structure of the CHIP protein.
The CHIP gene consists of seven exons. The CHIP protein has two key domains: the TPR domains and the one U-box domain. The five mutations identified in CHIP are indicated with arrows. Three mutations [c.493 CγT (p.L165F); c.389A>T (p.N130I); c.441G>T (p.W147C); c.707G>C (p.S236T)] are were located between the third TPR domain and the second low complexity segment. The c.621C>G (p.Y207X) mutation encodes a truncated protein without a U-box domain and the S236T mutation is located in the U-box domain.
Figure 3
Figure 3. Expression of CHIP in the mouse brain and the effect of ARCA-associcated mutations on its ability to promote the degradation of NR2A.
(A) The expression of CHIP in the cerebellum (Cb), hippocampus (Hip), cerebral cortex (Ctx), pons (PN) and medulla oblongata (MO) of mouse brain as analyzed with immunohistochemistry. (B) Co-localization of CHIP (red) and calcium-binding protein calbindin D-28K (green) in Purkinje cells. (C) Co-localization of CHIP (red) and NR2A (green) in Cb, PN and MO. (D) Coexpression of flag-tagged WT, but not ARCA-associated CHIP mutants (CHIPN130I, CHIPW147C, CHIPL165F, CHIPY207X, CHIPS236T), with HA-Fbx2 promoted the degradation of NR2A. Expression vectors for CHIP, Fbx2 and NR2A were transfected into Human Embryonic Kidney 293 cells. At 36 h after transfection, cells were treated with cycloheximide (CHX, 100 μg/ml) and chased for different time periods. NR2A was detected with western blot using the Myc antibody. Quantitative analysis was performed using NIH ImageJ analysis software. Values represent the mean ± S.D. of three independent experiments.
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