Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans

Abstract

We observed a severe autosomal recessive movement disorder in mice used within our laboratory. We pursued a series of experiments to define the genetic lesion underlying this disorder and to identify a cognate disease in humans with mutation at the same locus. Through linkage and sequence analysis we show here that this disorder is caused by a homozygous in-frame 18-bp deletion in Itpr1 (Itpr1(Delta18/Delta18)), encoding inositol 1,4,5-triphosphate receptor 1. A previously reported spontaneous Itpr1 mutation in mice causes a phenotype identical to that observed here. In both models in-frame deletion within Itpr1 leads to a decrease in the normally high level of Itpr1 expression in cerebellar Purkinje cells. Spinocerebellar ataxia 15 (SCA15), a human autosomal dominant disorder, maps to the genomic region containing ITPR1; however, to date no causal mutations had been identified. Because ataxia is a prominent feature in Itpr1 mutant mice, we performed a series of experiments to test the hypothesis that mutation at ITPR1 may be the cause of SCA15. We show here that heterozygous deletion of the 5' part of the ITPR1 gene, encompassing exons 1-10, 1-40, and 1-44 in three studied families, underlies SCA15 in humans.

Figure 1. Immunohistochemistry and Western Blot Analysis of ITPR1 Protein Levels in Mouse Cerebellum

(A–F) Immunohistochemistry of cerebellum from a wild-type mouse (A and D), a mouse heterozygous for the Itpr1 18-bp deletion (B and E), and a mouse homozygous for the 18-bp Itpr1 deletion (C and F). (A–C) Immunohistochemistry using polyclonal Itpr1 anti-rabbit antibody (1:2,000; Alexa Fluor 555); (D–F) immunohistochemistry using monoclonal Calb1 anti-mouse antibody (1:6,000; Alexa Fluor 488). Scale bars denote 100 μm. As previously described, Iptr1 is highly expressed in the Purkinje cells. Notably, there appears to be decreased immunoreactivity to Itpr1 in the heterozygous and homozygous mutant mice. (G) Western blot performed to examine Itpr1 levels in whole brain from wild-type, Itpr1^wt/Δ18, and Itpr1^Δ18/Δ18 mice; this clearly shows a reduction of Itpr1 in brain tissue from Itpr1^wt/Δ18 mice and a greater reduction of Itpr1 in Itpr1^Δ18/Δ18 mice.

Figure 2. Metrics Derived from Analysis of DNA from Affected Family Member 7 Using Illumina Infinium HumanHap550 Genotyping Chips

The upper and lower plots are log R ratio and B allele frequency, respectively, at an ~800-kb segment on the p arm of Chromosome 3. Log R ratio is the ratio of normalized, observed R to expected R for each SNP (each SNP is a blue dot) and thus serves as a surrogate of copy number at each locus. B allele frequency is a measure of the number of times the A or B alleles are detected at each locus (each SNP is denoted by a blue dot). Thus, SNPs with a B allele frequency of one are apparent B/B homozygotes, SNPs with a B allele frequency of 0.5 are apparent A/B heterozygotes, and those with a B allele frequency of zero are apparent A/A homozygotes. Clearly, these plots show a contiguous region ~200 kb long with decreased copy number and apparent homozygosity (bounded by a red box). As we have demonstrated previously, this is indicative of a heterozygous genomic deletion [15]. Below these plots is a schematic of the two known genes affected by this deletion, ITPR1 and SUMF1.

Figure 3. Mutation Analysis in the Australian SCA15 Family

(Top) Pedigree of kindred. Filled symbols denote affected individuals; open symbols, unaffected individuals; grey symbol denotes unknown disease status; bulls-eye symbol denotes obligate carrier. w/w, wild-type at ITPR1; w/m, heterozygous carrier of the ITPR1 deletion. (Middle) Schematic of primer pairs used to narrow the unknown regions between known deleted sequence and known diploid sequence at the SCA15 locus. Nine primer pairs (T1–T9) were used to amplify across the unknown region telomeric to the known deleted region; 19 primer pairs (C1–C19) were used to amplify across the unknown region centromeric to the known deleted region. All PCRs were carried out in the three affected family members. Analysis of these data narrowed the unknown region, and ultimately we were able to use primer T3f and C11r to amplify across the deletion breakpoint in the three affected family members, producing a fragment of 953 bp in affected individuals. (Bottom) Gel showing amplification product using primer pair T3f and C11r from affected pedigree members 6, 7, and 19; in pedigree member 23, with unknown disease affection status; in a neurologically normal control (C); and in a no template control (NC).

Figure 4. Western Blot Analysis of ITPR1 Protein Levels in EBV Immortalized Lymphoblasts from AUS1 Family Members

Western blot performed to examine ITPR1 levels in EBV immortalized lymphocytes from AUS1 affected family members carrying the ITPR1 deletion and from an AUS1 family member of unknown disease status who does not carry the deletion. Notably the samples from patients with ITPR1 deletion show a dramatic decrease in ITPR1 levels. To demonstrate equal loading, these samples were diluted one in five, and the Western blot was repeated using an antibody against ACTB.