Mutational analysis of ITPR1 in a Taiwanese cohort with cerebellar ataxias

Background The inositol 1,4,5-triphosphate (IP3) receptor type 1 gene (ITPR1) encodes the IP3 receptor type 1 (IP3R1), which modulates intracellular calcium homeostasis and signaling. Mutations in ITPR1 have been implicated in inherited cerebellar ataxias. The aim of this study was to investigate the role of ITPR1 mutations, including both large segmental deletion and single nucleotide mutations, in a Han Chinese cohort with inherited cerebellar ataxias in Taiwan. Methodology and principal findings Ninety-three unrelated individuals with molecularly unassigned spinocerebellar ataxia selected from 585 pedigrees with autosomal dominant cerebellar ataxias, were recruited into the study with elaborate clinical evaluations. The quantitative PCR technique was used to survey large segmental deletion of ITPR1 and a targeted sequencing approach was applied to sequence all of the 61 exons and the flanking regions of ITPR1. A novel ITPR1 mutation, c.7721T>C (p.V2574A), was identified in a family with dominantly inherited cerebellar ataxia. The proband has an adult-onset non-progressive pure cerebellar ataxia and her daughter is afflicted with a childhood onset cerebellar ataxia with intellectual sub-normalities. Conclusion ITPR1 mutation is an uncommon cause of inherited cerebellar ataxia, accounting for 0.2% (1/585) of patients with dominantly inherited cerebellar ataxias in Taiwan. This study broadens the mutational spectrum of ITPR1 and also emphasizes the importance of considering ITPR1 mutations as a potential cause of inherited cerebellar ataxias.


Methodology and principal findings
Ninety-three unrelated individuals with molecularly unassigned spinocerebellar ataxia selected from 585 pedigrees with autosomal dominant cerebellar ataxias, were recruited into the study with elaborate clinical evaluations. The quantitative PCR technique was used to survey large segmental deletion of ITPR1 and a targeted sequencing approach was applied to sequence all of the 61 exons and the flanking regions of ITPR1. A novel ITPR1 mutation, c.7721T>C (p.V2574A), was identified in a family with dominantly inherited cerebellar ataxia. The proband has an adult-onset non-progressive pure cerebellar ataxia and her daughter is afflicted with a childhood onset cerebellar ataxia with intellectual subnormalities.

Conclusion
ITPR1 mutation is an uncommon cause of inherited cerebellar ataxia, accounting for 0.2% (1/585) of patients with dominantly inherited cerebellar ataxias in Taiwan. This study broadens the mutational spectrum of ITPR1 and also emphasizes the importance of considering ITPR1 mutations as a potential cause of inherited cerebellar ataxias. PLOS
There is a wide phenotypic diversity linked to ITPR1 missense mutations. Two ITPR1 missense mutations (p.N587D and p.S1487D, reference sequence NM_001168272) were recognized as a cause of ataxic cerebral palsy [21], while an ITPR1 missense mutation (p.I2583N, reference sequence NM_001168272) was recently identified to result in severe pontine and cerebellar hypoplasia [22]. Homozygous, compound heterozygous and heterozygous mutations in ITPR1 could also cause Gillespie syndrome, which features a non-progressive cerebellar ataxia, aniridia, congenital hypotonia, and intellectual sub-normality (MIM #206700) [23,24]. The important role played by ITPR1 in the cerebellum is also demonstrated by the following facts. First, the ataxin-1, ataxin-2 and ataxin-3 are important players in the IP3 pathway and aberrations in the IP3 pathway have been shown in patients with SCA1, SCA2 or SCA3 [25]. Second, carbonic anhydrase 8 (CA8) is capable of inhibiting IP3 binding to IP3R1. CA8 mutations disrupt the formation of synapses of the Purkinje cells and are associated with an autosomal dominant spinocerebellar ataxia plus mental retardation [26]. Furthermore, autoantibody to IP3R1 may induce autoimmune cerebellar ataxia in the humans [27,28]. All these evidences support the association between ITPR1 mutations and cerebellar ataxia.
Although ITPR1 mutations have been identified in a small number of cases with inherited cerebellar ataxia, the relevant research in Han Chinese populations remains limited. The aim of this study was to investigate the role of ITPR1 mutations, including both large deletion and single nucleotide mutations, in a Han Chinese cohort with inherited cerebellar ataxias in Taiwan.

Methods and subjects Patients and ethics statement
Ninety-three individuals with molecularly unassigned SCA, selected from 585 unrelated patients with dominantly inherited cerebellar ataxias, after excluding SCA1, 2, 3, 6, 7, 8, 10, 12, 17, 31, 35, 36 and dentatorubral pallidoluysian atrophy (DRPLA) [29][30][31], were enrolled. Clinical evaluations included standard neurological examination, Scale for the Assessment and Rating of Ataxia (SARA) [32,33], as well as nerve conduction studies and electromyography. Brain magnetic resonance image (MRI) and magnetic resonance spectroscopy (MRS), focusing on the cerebellar hemispheres and vermis, were also performed to assess the cerebellar atrophy and metabolite changes. The protocol for this study was approved by the institutional review board of Taipei Veterans General Hospital and all the participants had given written informed consents.

Detection of large segmental deletions in ITPR1
Genomic DNA was extracted from the white blood cells in the peripheral blood with standard protocols. We looked for large segmental deletions in ITPR1 utilizing the TaqMan 1 Copy Number Assays kit on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). Eight TaqMan 1 detecting probes were selected with reference to those reported previously [34]. All of the detecting probes used in this study are listed in the S1 Table. Targeted sequencing and variant calling to detect single nucleotide or oligonucleotide variants Targeted sequencing covering the coding exons and flanking introns of ITPR1 (reference sequence: NM_001168272) was carried out by using SeqCap EZ Choice Enrichment Kits (Roche/Nimblegen) according to the manufacturer's protocol. The enriched DNA library was subjected to massively parallel sequencing using HiSeq2000 platform (Illumina 1 ) with 100-bp paired-end reads. The generated FASTQ files were aligned to the GRCh38 reference sequence using Novoalign V.2.07.19 and the PICARD tool MarkDuplicates. Calling was performed using SAMtools V.0.18. The resulting calls were annotated with ANNOVAR. Only variants with the following functional consequences: frameshift, stop gain, stop loss, non-synonymous missense or splice site variants and a global minor allele frequency (MAF) < 0.001 (based on the single nucleotide polymorphism database (dbSNP Build 144, http://www.ncbi.nlm.nih. gov/snp/) were kept. Sanger sequencing was used to further validate variants identified by targeted sequencing.

General information
For the 93 patients with molecularly unassigned autosomal dominant cerebellar ataxia recruited in this study, the average age at symptom onset was 37.2 ± 18.1 years (range from 1 to 74 years). The common symptoms shared by most patients included gait disturbance (87.1%), dysarthria (52.7%) and appendicular incoordination (50.5%). Tremor was observed in only 19.4% of the subjects, and cognitive impairment in 15.1% of the patients. The flow chart outlining selection of the study cohort and demographics of the study cohort could be found in supplementary S1 Fig and S2 Table.

Genetic analyses
We did not identify any individual carrying a large segmental deletion of ITPR1 (S2 Fig). The targeted sequencing panel, which covered the sequences of all 61 exons and exon-intron boundary regions of ITPR1, had a full coverage of the targeted regions and an average depth of 485X for each targeted base. Only one novel missense variant, c.7721T>C (p.V2574A), in exon 58 of ITPR1 was identified to be a potential causal variant. Sanger sequencing confirmed the variant in the proband (II-3) with dominantly inherited cerebellar ataxia and further revealed that the variant was present in her affected daughter (III-1) and absent in her unaffected brother (II-1) (Fig 1). The variant is not present in 1000 Genomes Project data (1000G, http://www.1000genomes.org/), the single nucleotide polymorphism database (dbSNP Build 144, http://www.ncbi.nlm.nih.gov/snp/) or the Genome Aggregation Database (gnomAD) (http://gnomad.broadinstitute.org).
Probing the pathogenicity of the ITPR1 sequence variant c.7721T>C through bioinformatics analyses In silico analysis with MutationTaster predicted this novel variant c.7721T>C (p.V2574A) as disease-causing with a high probability value of 0.999, indicating a high security of this prediction [35]. SIFT predicted c.7721T>C (p.V2574A) as damaging with a score of 0.01 and the threshold of SIFT score for pathogenicity was ≦ 0.05 [36]. The Combined Annotation Dependent Depletion (CADD) program estimated the Phred score of this sequence variant as 27.2, predicting that ITPR1 p.V2574A was the top 0.19% most deleterious variant in the genome [37]. All of the bioinformatics predictions unanimously supported the pathogenicity of the c.7721T>C (p.V2574A) in ITPR1 ( Table 1). The V2574 residue was highly conserved from human to honeybee, which further supported the pathogenic role of ITPR1 p.V2574A ( Fig  2B). The sequence change was not observed in 1,062 ethnically matched control chromosomes either.

Clinical characteristics of patients with the ITPR1 mutation
The pedigree of our SCA patients with ITPR1 p.V2574A mutation is shown in Fig 1A. The clinical characteristics of the index case and her offspring were summarized in Table 2. The proband (II-3, Fig 1A) had experienced an insidious and non-progressive gait disturbance since age 30. Upon examination at age 35 years, she was found to have fine tremors over the head and hands, especially when getting nervous, and her hand-writing was sloppy. Mild dysarthria and dysphagia were also noticed. She had normal cognitive functions. The eye movements were full in all directions but with mild saccadic pursuits. There was no focal weakness and the muscle tone was normal. Diffusely decreased deep tendon reflexes were found with flexor plantar responses. There was mild unsteadiness on tandem walking. The latest SARA score was 4.5 at the tenth year after the disease onset (S1 Video). Nerve conduction studies and electromyography were unremarkable. Brain MRI at the tenth year after disease onset demonstrated a few tiny non-specific hyperintense spots in the white matter of cerebral hemispheres on T2-weighted images and a mild atrophy of the cerebellar hemispheres and vermis on T1-weighted images (Fig 3). The MRS revealed NAA/Cr ratios of 1.16, 1.02 and 0.87 in the right cerebellar hemisphere, left cerebellar hemisphere and cerebellar vermis, respectively, indicating a very mild biochemical abnormality.
The proband's daughter (III-1) had experienced a childhood-onset non-progressive cerebellar ataxia and cognitive impairment since age of 7 years. Mild slurred speech, saccadic pursuits and unsteady tandem walking on neurological examination, a SARA score of 5.5 (S2 Video) and mini-mental state examination (MMSE) score of 25 were documented at the 13 th year after the symptom onset. The nerve conduction studies were normal. Brain MRI featured a mild atrophy of the cerebellar hemispheres and vermis on the T1-weighted images (Fig 3). There was no obvious biochemical abnormality in the right cerebellar hemisphere, left cerebellar hemisphere or vermis (NAA/Cr ration of 1.55, 1.25 and 1.02, respectively) on MRS at the 13 th year after the onset.

Discussion
We identified a novel missense ITPR1 mutation, c.7721T>C (p.V2574A), in a patient with a slowly progressive cerebellar ataxia from 93 unrelated patients with molecularly unassigned SCA. Since these 93 patients were selected from a cohort of 585 unrelated patients with dominantly inherited cerebellar ataxias after a comprehensive genetic testing for SCA, mutation in ITPR1 accounts for 0.2% (1/585) of dominantly inherited cerebellar ataxias in Taiwan. Several lines of evidence support the pathogenicity of this ITPR1 mutation, namely, p.V2574A. First, the sequence change was absent in 1,062 ethnically matched control chromosomes. Second, the variant was absent in several genetic polymorphism databases, including dbSNP, 1000 Genomes Project and gnomAD database. Third, the variant occurs at an evolutionarily conserved amino acid residue of the IP3R1 protein (Fig 2) and a deleterious effect was unanimously predicted by several bioinformatics programs, including Muta-tionTaster [35], SIFT [36] and CADD [37] (Table 1). Furthermore, this variant has a perfect genotype-phenotype co-segregation in the pedigree (Fig 1). These evidences substantially support the pathogenicity of this mutation and classify it as a "likely pathogenic variant" according to the guideline for the interpretation of sequence variants recommended by the American College of Medical Genetics and Genomics and the Association of Molecular Pathology [38].
So far, there have been 16 mutations in ITPR1 associated with autosomal dominant cerebellar ataxia and 5 with dominantly inherited Gillespie syndrome (Table 3 and Fig 2A). Most of the mutations locate in the IP3 binding domain and the coupling/regulatory region of IP3R1. Mutations localized in the coupling/regulatory domain of IP3R1, such as ITPR1 p.P1068L (originally annotated as p.P1059L, reference sequence NM_002222 [6]), might affect the binding affinity and transmission of the IP3 signaling which further disrupts the calcium influx, resulting in cerebellar ataxia [11]. Similar to ITPR1 p.G2580A (annotated as p.G2547A, reference sequence NM_001099952, in the original literature [17]) and p.I2583N (annotated as p.I2550N, reference sequence NM_001099952, in the original literature [22]) mutations, the p.V2574A mutation identified in this study is also situated in the sixth transmembrane domain (S6) adjacent to the S5-S6 pore-loop. As a result, these three mutations might alter the structure of the calcium-selective channel and affect the calcium influx through the channel. Moreover, since the functional IP3R1 is a tetrameric structure [39,40], mutations in the transmembrane domain might disrupt the assembly conformation of the functional channels which is essential for the intracellular calcium release (Fig 2 and S3 Fig).
The clinical features of the individuals with ITPR1-associated cerebellar ataxia are quite heterogeneous. Tremors and vermis atrophy are common but not pathognomonic. The onset of ataxia could be in the infantile stage, early childhood [14,15,[17][18][19]21], or adulthood [6,20], and intellectual impairment were frequently found in the individuals with an earlier age at The proband (II-3) presented with an adult-onset ataxia but her offspring (III-1) featured a childhood-onset ataxia and intellectual sub-normalities. Both cases demonstrated non-progressive cerebellar ataxia and mild atrophy of the vermis. These inter-familial and intra-familial heterogeneities suggest that there might be modifying factors underlying the pathogenesis of ITPR1-associated cerebellar ataxia.
There are several validated methods to detect copy number variation (CNV) in the genome, including multiplex ligation-dependent probe amplification (MLPA) [12,41], array comparative genomic hybridization (CGH) [6], qPCR [9,10,34] and fine sequence array. We first looked for CNV utilizing the qPCR method with multiple probes covering most of the deletion hot spots but failed to find any ITPR1 large deletions in our cohort. We further surveyed ITPR1 mutations with high throughput targeted sequencing and found an ITPR1 missense   mutation. Our findings emphasize the importance of using multiple diagnostic tools to detect both CNV and single nucleotide variants in ITPR1. ITPR1 missense mutations-associated cerebellar ataxia might have been under-estimated prior to the high-throughput sequencing era. Given that IP3R1 consists of 2,743 amino acids and the full-length ITPR1 gene is composed of 10,197 base pairs and 61 exons, it is technically challenging and time-and cost-consuming to perform genetic screening in such a large gene. The diagnostic proficiency has significantly improved with the next generation sequencing technology. Researches focusing on the missense mutation in ITPR1 might be much feasible from now on. ITPR1 missense mutation is an uncommon but unneglectable cause of cerebellar ataxia in Taiwan. Cases with non-progressive cerebellar ataxia and vermis atrophy might be candidates with mutations in ITPR1. Our findings have broadened the mutational spectrum of ITPR1 and emphasized the role of ITPR1 in cerebellar function.
Supporting information S1