Subjects

The clinical data and blood samples were obtained from three Han Chineses families with histories of ARCA that included 6 affected individuals. A cohort of 36 additional unrelated ARCA pedigrees and 196 sporadic ataxia patients were chosen for subsequent mutational screening analyses. These analyses included gene mutation screenings for, and exclusion of subjects with the following: Friedreich's ataxia, ataxia with vitamin E deficiency, ataxia plus oculomotor apraxia type 1, ataxia plus oculomotor apraxia type 2 and so on. We also screened for, and excluded subjects with the following more frequent subtypes of autosomal dominant cerebellar ataxia: nucleotide expansion mutations of the ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, ATXN8OS, ATXN10, PPP2R2B, TBP, NOP56 and atrophin-1 genes in the SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA8, SCA10, SCA12, SCA17, SCA36 and DRPLA, point mutations of KCNC3, PRKCG, KCND3, PDYN, FGF14, AFG3L2, and TGM6 gene in the SCA13, SCA14, SCA19, SCA23, SCA27, and SCA28 substypes, and insertion and deletion mutations of the SPTBN2, TTBK2, KCND3, ITPR1 and BEAN genes in the SCA5, SCA11, SCA22, SCA15/16/29 and SCA31 subtypes. The analyses also included 500 unaffected healthy individuals who were matched for geographical ancestry as controls. Written informed consent was obtained from each subject or their guardian. This study was approved by the Ethic Committee of the Xiangya Hospital of Central South University in China (equivalent to an Institutional Review Board).

Exome sequencing, copy number variations (CNVs), and linkage analysis.

Genomic DNA was extracted from whole peripheral blood using standard methods (QIAGEN, Valencia, CA). Qualified genomic DNA extracted from two affected individuals in family 1 (Samples II:2 and II:3) was sheared by sonication and then hybridized to the SureSelect Biotiny-lated RNA Library for enrichment according to the manufacturer’s instructions. The enriched library targeting the exome was sequenced on the HiSeq 2000 platform to acquire paired-end reads with read length of 90 base pair. The sequenced reads were aligned to the human genome reference (UCSC hg 18 version) using SOAPaligner [15]. Next, those reads that were aligned in the desired target regions were collected for SNP calling and subsequent analysis. We estimated quality scores and made the consensus SNP callings using SOAPsnp (v 1.03) (Li et al., 2010). The low quality variations were filtered out based on the following criteria: (i) consensus quality score <20; (ii) average copy number at the allele site >=2; (iii) distance of two adjacent SNPs <5 bp; and (iv) sequencing depth <4 or >500. For indels in the targeted exome regions, we aligned the reads to the reference genome using BWA (http://bio-bwa.sourceforge.net). The alignment results were used to identify the breakpoints by gatk. Finally, we annotated the genotypes of insertions and deletions [16]. We have shared our deep-sequencing data in NIH Short Read Archive and the accession number is SRA105955.

To exclude CNVs in the critical interval, whole genome CNV analyses of seven samples (I:1, I:2 and II:1-5) were performed using the Illumina HumanHap660 BeadChip. CnvPartition for GenomeStudio was used to call CNVs, and only samples with call rates > 98% were included. To localize the disease-causing gene, we also carried out linkage analyses of whole-genome SNPs. The genotype assignments were determined using GenomeStudio genotyping module software (Illumina). Two-point logarithm of odds scores were calculated using the MERLIN linkage program version 1.01 [17]. Marker allele frequencies were estimated from the founders of the pedigree via MERLIN, and the disease was considered to be autosomal recessive with a frequency of 0.0001 and a penetrance of 95%.

Plasmids

Full-length human CHIP cDNA was amplified by PCR using primers W1/W2 from the human fetal brain cDNA library as the template and inserted in-frame into p3xFlag-CMV-24 (Sigma, USA) at EcoRI/SalI sites. Mutant CHIP^N130I, CHIP^W147C, CHIP^L165F, CHIP^Y207X, and CHIP^S236T were generated with QuikChange site-directed mutagenesis protocol (Stratagene, La Jolla, CA, USA) with M1/M2, M3/M4, M5/M6, M7/M8, and M9/M10 primers, respectively. The primers used in this study are shown in Table S1 of File S1. Full-length human NR2A cDNA and Fbx2 cDNA were artificially synthesized by the TaKaRa Biotechnology Company (Dalian, China) and cloned into the pcDNA3.1-myc-his-B(-) vector (Invitrogen, USA) at EcoR I/BamH I sites and pKH3-HA at BamH I/Xho I sites, respectively. All constructs were confirmed by sequencing.

Cell culture and transfections

Human Embryonic Kidney 293 cells were grown at 37°C under 5% CO2 in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 100 U/ml of penicillin/streptomycin. Expression plasmids were transfected into cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.

Immunoblot analysis

Proteins were separated by 6%-10% SDS–PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, USA). The following primary antibodies were used: monoclonal anti-Flag antibody (Sigma, USA); polyclonal anti-Myc antibody (Cell Signaling Technology, USA); and monoclonal anti-HA antibody (Millipore, USA). Sheep anti-rabbit antibody (Amersham Pharmacia Biotech) was used as the secondary antibody. Detection was performed using the ECL plus Western blotting detection system (GE Healthcare, USA) according to manufacturer’s instruction. Bands were scanned and quantified by densitometric analysis with the NIH ImageJ software and normalized to β-actin as a loading control.

Mouse brain immunofluorescence

C57BL/6 mice (8-10 w) were used for these studies. After fixation in 4% phosphate-buffered paraformaldehyde, mouse brains were frozen and cut coronally into 15 μm thick slices. After pre-incubation, the slices were incubated at 4°C for 48 h with a rabbit anti-CHIP antibody (Santa Cruz, USA) then visualized with Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch laboratories, USA). For the double immunofluorescence staining, the brain sections were treated with the rabbit anti-CHIP antibody and a mouse anti-NR2A monoclonal antibody (Millipore, USA). A mixture of Cy3-conjugated AffiniPure Goat Anti-Rabbit IgG and Dylight-488 conjugated AffiniPure Goat anti-Mouse IgG (Jackson ImmunoResearch laboratories, USA) was employed as a secondary antibody. Finally, images were acquired with a a confocal laser microscope (Leica, Germany). All procedures regarding the care and use of animals were approved by the Institutional Animal Care and Use Committee of Central South University of China, and all methods used in the experiments were in accordance with institutional regulations and the guidelines of the Hunan Animal Care and Use Committee.

The clinical characteristics of families with ARCA

A two-generation ARCA family (family 1) with four affected individuals was recruited from the Jiangxi province in China. Another two families (family 2, 3) were all from the Hunan province in China. The diagnosis of hereditary ataxia was determined according to the criteria of Harding [18]. The proband (II:5) in famlily 1 was 34 years old, and suffered from progressive ataxia beginning at the age of 21, and presented with severe atrophy in the cerebellum as determined by brain MRI (Figure 1B). The detailed clinical features at the time of initial clinical assessment in the three families are summarized in Table 1. All patients showed features of cerebellar ataxia associated with obvious cerebellar atrophy. The latest clinical assessment on the three patients (II:2, II:3, II:5) in family 1 indicated that the ICARS and SARA scores were significantly increased (Table S2 in File S1 and Text S1). Interestingly, after ~20 years with ARCA, two patients in family 1 (patient II:2 and II:3) were found to have cognitive impairment, although they did not show obvious atrophy in the cortex and hippocampus (Table S2, S3 in File S1 and Figure S1, S2). In addition, the nerve conduction velocity, electromyogram, visual evoked response, auditory evoked potential, somatosensory evoked potential in three patients (II:2, II:3, II:5) from family 1 were partly abnormal, indicating that some pathological changes occurred in not only the central nervous system but also the peripheral nerves (Table S4 in File S1). No mutation in known hereditary ataxia-linked genes was identified in these patients.

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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.

https://doi.org/10.1371/journal.pone.0081884.g001

Exome sequencing combined with linkage analysis identified carboxyl terminus of the Hsc70-interacting protein gene (CHIP) as the candidate gene

To search for the candidate gene, we performed whole-exome sequencing of the DNA from two affected individuals (II:2 and II:3) of family 1 (Figure 1A) using the Illumina Genome Analyser II platform (Table S5 in File S1). Sequencing data were then aligned to the human genome reference (UCSC hg 18 version). After calling of single-nucleotide polymorphisms and insertions or deletions, we removed non-synonymous mutations, splice acceptor and donor site mutations, and indels variants that have been reported in the dbSNP129, the ‘HapMap 8’, the SNP dataset of the 1000 Genome Project and 800 additional normal subjects (data not shown). As ARCAs are inherited in an autosomal recessive fashion, we focused on homozygous and compound heterozygous mutations. After filtering (Table 2), only the CHIP gene remained as common in both II:2 and II:3 subjects. Sanger sequencing indicated that the patients (II:1, 2, 3 and 5) were homozygous at c.493CT (p.L165F) of CHIP, whereas their parents (I:1 and I:2) and normal sibling II:4 were heterozygous (Figure 1C). These findings suggest that c.493CT (p.L165F) of CHIP completely co-segregated with the disease phenotype within family 1. The p.L165F variant was located in a highly conserved position of CHIP (Figure 1D), however, it was not identified in any of the 500 unaffected controls matched for geographical ancestry.

To exclude the possible contribution of CNVs, which cannot be determined by exome sequencing, we surveyed the whole genomes of seven subjects (I:1, I:2, and II:1-5) in family 1 for CNVs using the Illumina HumanHap660 Bead Chip. No CNVs co-segregated with the phenotype. Further, we carried out linkage analysis of whole-genome SNPs. Interestingly, we mapped a linkage to a minimal candidate region of 8.55cM on chromosome 16p13.3 flanked by SNP markers rs11248850 and rs1218762, where the CHIP gene is located. The multi-point logarithm of the odds score was ascertained as 1.917 (Table S6 in File S1).

Mutations in the CHIP gene

CHIP, also known as the STIP1 homology and U box-containing protein 1 (STUB1), is a multifunctional E3 ubiquitin ligase. As shown in Figure 2, the CHIP gene consists of seven exons, and its protein harbors three domains: an N-terminal three-tetratricopeptide repeat domain, a highly charged middle domain, and a carboxyl-terminal U-box domain [19] (Figure 2). The TPR domain of CHIP serves as protein-protein interaction domain that has traditionally been thought to mediate interactions with heat shock proteins [20], while the U-box domain of CHIP acts as an ubiquitin ligase [21]. The amino acid sequence of human CHIP has 97% identity and 98% similarity with the mouse Chip. The c.493CT (p.L165F) mutation we found in family 1 was a missense mutation in the third exon of CHIP.

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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.

https://doi.org/10.1371/journal.pone.0081884.g002

To identify additional deleterious variants among other families with ARCA, we performed Sanger sequencing to screen the exons and flanking introns of the CHIP gene in an additional 36 families with histories of ARCA and 196 sporadic individuals affected with ataxia; individuals with the common cerebellar ataxia-linked genes were excluded. CHIP mutations were found in two additional ARCA families. One compound heterozygous mutation [c.389AT (p.N130I) and c.441GT (p.W147C)] was identified in family 2 (Figure 1E), and another compound heterozygous mutation [c.621CG (p.Y207X) and c.707GC (p. S236T)] was identified in family 3 (Figure 1F). The c.621CG (p.Y207X) mutation is a non-sense mutation that was found in the fifth exon of CHIP; this mutation substitutes the codon for Y207 (TAC) with a stop codon (TAG) and generates a truncated protein with only 206 amino acids. The other mutant forms including c.389AT (p.N130I), c.441GT (p.W147C), and c.707GC (p. S236T) are missense mutations. These mutations completely co-segregated with the phenotype in these two families and were not detected in 500 controls who were matched for geographic origin. All the five variants (c.389AT, c.441GT, c.493CT, c.621CG and c.707GC) were predicted to be probably damaging (Table S7 in File S1).

The distribution of CHIP in the mouse brain

Using immunofluorescence, we showed that CHIP was highly expressed in the cerebellum, pons, medulla oblongata, hippocampus and cerebral cortex (Figure 3A). Moreover, we demonstrated that CHIP co-localized with the calcium-binding protein calbindin D-28K in Purkinje cells (Figure 3B), which is an essential determinant of normal motor coordination and sensory integration in the cerebellum [22].

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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 (CHIP^N130I, CHIP^W147C, CHIP^L165F, CHIP^Y207X, CHIP^S236T), 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.

https://doi.org/10.1371/journal.pone.0081884.g003

Characterization of the activity of mutant CHIPs

N-methyl-D-aspartate receptors (NMDARs) are mainly composed of a structural NR1 subunit and a NR2 subunit (NR2A-D), which modulate the biophysical properties of the NMDARs [23-25]. Nelson et al reported that CHIP functions with Fbx2 to promote the ubiquitination and degradation of NMDARs [26]. Indeed, the levels of NR2B (the embryonic counterpart of NR2A) are significantly elevated in cultured cortical neurons from embryonic CHIP -/- mice [26]. NMDARs play a fundamental role in excitatory neurotransmission and cerebellar-dependent motor coordination [27]; thus the alterations of NMDAR function in CHIP knockout mice may underlie the pathogenesis of ataxia.

To characterize the ubiquitin ligase activity of mutant CHIPs, we chose NR2A as a test substrate. In the mouse brain, we observed that CHIP co-localized with NR2A in the cerebellum, pons, and medulla oblongata (Figure 3C). Coexpression of WT CHIP and Fbx2 significantly increased the degradation of NR2A, whereas WT CHIP or Fbx2 alone did not produce significant effects on the degradation of NR2A. In contrast, none of the ARCA-associated CHIP mutants effectively promoted the degradation of NR2A (Figure 3D).