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{"project":"2_test","denotations":[{"id":"17590087-10742102-85657859","span":{"begin":253,"end":255},"obj":"10742102"},{"id":"17590087-6587361-85657860","span":{"begin":483,"end":485},"obj":"6587361"},{"id":"17590087-12112377-85657861","span":{"begin":4059,"end":4061},"obj":"12112377"},{"id":"T7451","span":{"begin":253,"end":255},"obj":"10742102"},{"id":"T31814","span":{"begin":483,"end":485},"obj":"6587361"},{"id":"T31713","span":{"begin":4059,"end":4061},"obj":"12112377"}],"text":"Materials and Methods\n\nGenome-wide linkage in mice.\nOne hundred and twenty DNA fragments were amplified across the genome, each selected to contain one or more strain-specific SNPs that would differentiate between C57BL/6J and 129x1/SvJ inbred strains [12]. Each fragment was initially amplified in 11 affected mice and nine unaffected mice; genotype calling was performed by dye-terminator sequencing of these fragments. Linkage analysis using these data was performed using mlink [13], which revealed a positive linkage at Chromosome 6qE1, on the 129x1/SvJ background (two-point LOD score 5.13 at marker 20.MMHAP85FLG2). In an attempt to narrow the disease interval we performed backcross experiments that resulted in the generation of three additional affected mice. Genotyping of all affected mice across the disease-segregating interval revealed flanking recombinants and a candidate region of ~5 Mb, between markers D6Mit37 and 44.MMHAP85FLG5 (Figure S1). This region contains 16 genes and predicted transcripts.\n\nIdentification of the underlying genetic lesion in mice.\nIdentification of similar phenotypes in mice linked to the 6qE1 interval was performed by literature searches. This revealed the Itpr1opt/opt mouse, in which disease is caused by homozygous deletion mutation of exons 43 and 44 of Itpr1. Primer pairs were designed to sequence each of the coding exons and at least 50 bp of each flanking intronic sequence of Itpr1. PCR amplification of each exon was performed using DNA from two affected mice as templates. The Itpr1Δ18/Δ18 mutation was confirmed by sequencing in all affected mice (Figure S2).\nBreeding experiments were performed between two female mice heterozygous for the current mutation (Itpr1wt/Δ18) and a male mouse heterozygous for the Itpr1opt mutation (Itpr1wt/opt). This resulted in two litters of mice with a total of four affected Itpr1opt/Δ18 pups (from a total of 15; two of seven from first mating; two of eight from the second mating) with a phenotype indistinguishable from that of the Itpr1Δ18/Δ18 and Itpr1opt /opt mice.\n\nAnalysis of Itpr1 protein in mice.\nWe performed Western blot analyses using standard techniques with ECL detection kits (Amersham, http://www.amersham.com). Briefly, dissected whole brains from postnatal day 21 littermates were homogenized in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and a cocktail of protease inhibitors (Roche, http://www.roche.com). Homogenates were diluted appropriately, mixed with 4× reducing sample buffer, and loaded onto 4%–12% precast gradient gels (Novex, http://www.invitrogen.com) for SDS-PAGE and immunoblotting. The antibodies to Itpr1 (1:2,000) and Actb (1:5,000) were used as recommended by manufacturers.\n\nImmunohistochemistry.\nBrains were isolated from 21-d-old mice, perfused with 4% paraformaldehyde in PBS, and post-fixed overnight in the same fixative. Brains were embedded in gelatin, and 35-μm sagittal sections were cut using a sliding microtome (NeuroScience Associates, http://www.neuroscienceassociates.com). Sections from wild-type, heterozygote, and homozygous brains were placed in the MultiBrain template. Sections were washed in 1× PBS prior to 1 h of incubation in block solution containing 1× PBS with 20% normal goat serum and 0.3% Triton X-100 (pH 7.4). Sections were incubated overnight at 4 °C in primary antibodies: affinity purified polyclonal Itpr1 antibody (1:2,000, Chemicon International, http://www.chemicon.com) and monoclonal anti-Calb1 antibody (1:6,000, Sigma-Aldrich, http://www.sigmaaldrich.com) diluted in carrier solution. Following extensive washes (in 6.0 ml of PBS, three times), sections were incubated with appropriate secondary antibodies (Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG [Invitrogen, http://www.invitrogen.com]) for 1 h at room temperature. Sections were washed and mounted on glass slides in a buffered medium containing Mowiol (Calbiochem, http://www.emdbiosciences.com) as described earlier [14]. Sections were imaged using a laser scanning confocal microscope (LSM 510; Zeiss, http://www.zeiss.com). Imaging parameters (pinhole, detector gain, laser power) were optimized, and were kept constant for the wild-type, heterozygous, and homozygous mutant brains. Specificity of the Itpr1 antibodies was verified by preabsorption control experiments. Antibody dilutions were incubated for 24 h at 4 °C with the immunizing peptide. Tissue sections were incubated with the preabsorbed antibodies and processed as described above. Under these conditions, no staining above autofluorescence was detected.\n\nAnalysis of ITPR1 in SCA15 patients.\nDNA was extracted from EBV immortalized lymphocytes, derived from family members. The coding exons and at least 50 bp of flanking introns of ITPR1 were PCR amplified and sequenced using dye-terminator sequencing (BigDye version 3.1; Applied Biosystems, http://www.appliedbiosystems.com). Sequence reactions were run on an ABI3730XP automated sequencer as per the manufacturer's instructions (Applied Biosystems). This analysis was performed in all three affected family members for whom genomic DNA was available (members 6, 7, and 19). Primer sequences and conditions are available upon request. Sequence data were analyzed using Sequencher (Gene Codes Corporation, http://www.genecodes.com). Genome-wide SNP genotyping was performed using Infinium HumanHap550 SNP genotyping chips as per the manufacturer's protocol (Illumina, http://www.illumina.com). This product assays 555,352 unique SNPs. Data were collected using the Illumina BeadStation scanner and data collection software. Genotypes were produced using the genotyping module of BeadStudio (version 2.3.25; Illumina), and log R ratio and B allele frequency were visualized using the genome viewer tool within this package. In order to rule out the possibility that the observed deletion within ITPR1 was a benign copy number variant we examined log R ratio and B allele frequency metrics of HumanHap550 genotyping data at this locus from 577 individuals of Northern European descent from North America and Europe, produced by us as a part of an ongoing study.\nIn an attempt to narrow the unknown intervals flanking the deletion observed in family AUS1, we designed primers for 30 PCR amplifications that would generate overlapping fragments across the two bordering regions (primer sequence and conditions available upon request). There were ten primer pairs in the telomeric flanking region and 20 pairs in the centromeric flanking region (Figure S3). On average each product was ~750 bp in size, and amplifications were performed using genomic DNA from each of the three affected individuals (family members 6, 7, and 19). Dye-terminator sequencing of each product was performed using the forward and reverse primers designed for amplification; running and analysis of each fragment was performed as described above. Amplification of a fragment from a normal diploid genome was denoted by the presence of a heterozygous polymorphism; amplification of a fragment from a region of the genome harboring a heterozygous genomic deletion was inferred when homozygosity for the major allele and the minor allele were noted among the three affected family members (i.e., this is inconsistent with Mendelian inheritance in related individuals known to share a common haplotype).\nUsing the data from the experiments described above we were able to limit the size of unknown regions flanking the deletion to ~4 kb on the telomeric side and 7 kb on the centromeric side. All combinations of forward primers from the newly defined region flanking the deletion on the telomeric side with reverse primers from the newly defined region flanking the deletion on the centromeric side were used in PCR amplification reactions performed with DNA from the three affected family members and single unaffected family members. This experiment was performed in an attempt to amplify across the deleted fragment and define the exact breakpoint. A single fragment was obtained from the third forward primer from the telomeric side (T3f 5′-TGAATGCTCAATTTTCCAGC-3′) with the 11th reverse primer from the centromeric side (C11r 5′-GGGAAAATGGATAGAGGGTG-3′). The fragment, which is 953 bp in size, was sequenced as described above and compared to the current build of the human genome. A similar series of experiments was performed to identify the deletion breakpoints in families H27 and H33; we were able to amplify a 369-bp PCR product across the breakpoint found in affected members of family H27 using primer pair H27-11F 5′-GACCTCAAGAAGGCATGAATAC-3′ and H27-3R 5′-ATGGTGGCCAGGTACACAAG-3′ (Figure S4), but to date we have been unable to identify the breakpoint in family H33.\n\nWestern blot analysis in SCA15 patients.\nEBV immortalized lymphoblasts from three affected family members who carry the deletion and one family member without the mutation were used as a readily accessible source of protein; all samples came from members of family AUS1. Protein extraction was performed using lysis buffer containing 1× TBS, 1% Triton X-100, and a cocktail of protease inhibitors (Roche) with overnight lysis at −80 °C. Homogenates were diluted appropriately, mixed with 4× reducing sample buffer, and loaded onto 4%–12% precast gradient gels (NuPAGE, Invitrogen) for SDS-PAGE and immunoblotting. The antibodies to ITPR1 (1:1,000) and ACTB (1:5,000) were used as recommended by manufacturers."}

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PMC:1892049 / 19166-28690 JSON TXT