JP2013000084A - Method for detecting autosomal recessive spinocerebellar degenerations - Google Patents

Abstract

Disclosed is a novel disease-responsible gene for spinocerebellar degeneration.
A test method for autosomal recessive hereditary spinocerebellar degeneration, which comprises detecting a mutation in the SYT14 gene encoding Synaptotagmin XIV (Syt14). Also provided is a method of immunostaining Purkinje cells using an antibody against Syt14 protein; an immunostaining reagent comprising an antibody against Syt14 protein.
[Selection] Figure 5

Description

  The present invention relates to a method for detecting autosomal recessive hereditary spinocerebellar degeneration.

  Autosomal recessive hereditary cerebellar ataxia (ARCA) is a general term for heterogeneous diseases including non-progressive cerebellar hypoplasia and progressive spinocerebellar degeneration (SCA). In addition, it is accompanied by various neurological symptoms and non-neurological symptoms, and often develops at a young age of 20 years or younger, and elderly ARCA is rare (Non-Patent Documents 1 to 3). More than 20 types of ARCA responsible genes have been identified so far, and the frequency of Friedrich ataxia is the highest in Western countries (Non-Patent Documents 4 to 6), but the causative gene was still found in about half of patients. (Non-Patent Document 3). There are no reports of Friedrich ataxia in Japan, and there are no statistics on the frequency of the gene responsible for ARCA.

Fogel, B.L., and Perlman, S. (2007). Clinical features and molecular geneticsof autosomal recessive cerebellar ataxias. Lancet Neurol 6, 245-257. Embirucu, E.K., Martyn, M.L., Schlesinger, D., and Kok, F. (2009) .Autosomalrecessive ataxias: 20 types, and counting.Arq Neuropsiquiatr 67,1143-1156. Anheim, M., Fleury, M., Monga, B., Laugel, V., Chaigne, D., Rodier, G., Ginglinger, E., Boulay, C., Courtois, S., Drouot, N., et al. (2010) .Epidemiological, clinical, paraclinical and molecular study of a cohort of102 patients affected with autosomal recessive progressive cerebellarataxia from Alsace, Eastern France: implications for clinical management.Neurogenetics 11, 1-12. Palau, F., and Espinos, C. (2006). Autosomal recessive cerebellar ataxias.Orphanet J Rare Dis 1, 47. Manto, M., and Marmolino, D. (2009). Cerebellar ataxias. Curr Opin Neurol 22,419-429. Vermeer, S., Hoischen, A., Meijer, RP, Gilissen, C., Neveling, K., Wieskamp, N., De Brouwer, A., Koenig, M., Anheim, M., Assoum, M., et al. (2010) .Targeted next-generation sequencing of a 12.5 Mb homozygous regionreveals ANO10 mutations in patients with autosomal-recessive cerebellarataxia. Am J Hum Genet 87, 813-819.

  An object of the present invention is to provide a novel disease-responsible gene for autosomal recessive spinal cerebellar degeneration.

  In this case, we experienced an older onset SCA sibling with mental retardation that was found in a Japanese family with consanguineous marriage. As a result, we identified SYT14 as a novel disease-responsible gene for autosomal recessive hereditary spinocerebellar degeneration. Two homozygous mutations of the same gene were identified in two SCA brother patients, and the gene was confirmed to be expressed in cerebellar Purkinje cells. The present invention has been completed based on these findings.

The gist of the present invention is as follows.
(1) A method for examining autosomal recessive hereditary spinocerebellar degeneration, comprising detecting a mutation in the SYT14 gene encoding SynaptotagminXIV (Syt14).
(2) The test method according to (1), wherein a mutation in the SYT14 gene is detected for both alleles.
(3) The test method according to (1) or (2), wherein a mutation in the SYT14 gene is analyzed by genomic DNA and detected.
(4) The test method according to (1) or (2), wherein the mutation of the SYT14 gene is analyzed and detected at the RNA level.
(5) The test method according to (1) or (2), wherein a mutation in the SYT14 gene is analyzed and detected at the protein level.
(6) A method of immunostaining Purkinje cells using an antibody against Syt14 protein.
(7) An immunostaining reagent containing an antibody against Syt14 protein.
(8) The reagent according to (7) for immunostaining Purkinje cells.

  By establishing an efficient screening system for responsible genes, genetic diagnosis of this disease is performed, and individualization of treatment including gene therapy is expected. In addition, since it has already been clarified that a similar protein (SynaptotagminI) encoded by this gene exists in synaptic vesicles that regulate secretion from neurons, elucidation of the pathophysiology of this disease has progressed rapidly. This is thought to lead to the development of effective management and treatment methods.

  One type of missense mutation was identified in the SYT14 gene in adult-onset recessive hereditary spinocerebellar degeneration (ARCA). By analyzing the SYT14 gene, it becomes possible to make a definitive diagnosis of ARCA disease and detect carriers.

SCA family tree, MRI with SYT14 homozygous mutation c.1451G> A. Left: Family tree of the subject family with autosomal recessive inherited SCA. The asterisk is the subject whose DNA was obtained. Affected individuals are IV-3 and IV-4. Right: Brain MRI and T1-weighted images of IV-3 and IV-4. Horizontal image (upper) and sagittal image (lower). In both cases, cerebellar atrophy can be confirmed. Confirmation and preservation of SYT14 mutation. Upper left: Confirmation of SYT14 c.1451G> A mutation by the Sanger method. It was confirmed that the mutation was heterozygous in non-affected individuals (III-1, IV-1, IV-2) and homozygous in affected individuals (V-3, IV-4). Upper right: Syt14 domain structure schema. The p.Gly484Asp mutation (dotted) is present in the C2B domain. Bottom: Mutant amino acids are highly conserved from humans to flies. Expression analysis of SYT14 by TaqManTM quantitative PCR. Analysis of SYT14 expression by TaqManTM quantitative PCR method using first strand cDNAs from adult tissues (upper left), fetal tissues (upper right), mouse tissues (lower left), and mouse brain sites (lower right) as templates SYT14 expression was analyzed by TaqManTM quantitative PCR. In human tissues, it was confirmed that the expression was strong in the brain. Expression in the brain was also observed in the mouse, and it was confirmed that the expression was strong in the cerebellum by brain region. Intracellular distribution of wild type and mutant type (p.Gly484Asp). COS-1 cells were transfected with a v5 / His-tagged Syt14 expression vector (pEF-DEST51), and anti-v5 antibody (green: using Alexa488-labeled secondary antibody) and anti-protein disulfide isomerase (PDI, endoplasmic reticulum) Marker) antibody (red: using Alexa546-labeled secondary antibody) was double-stained. Nuclei were stained with 4 ', 6-diamidino-2-phenylindole (DAPI). Scale bar is 10μm. In the wild type and the mutant type, the intracellular distribution was clearly different, and it was found that the mutant type accumulated in the endoplasmic reticulum. Mouse cerebellum and human cerebellum immunostained with anti-Syt14 antibody (Ab-Syt14) Upper: Mouse cerebellar Ab-Syt14 immunostained (green: using Alexa488-labeled secondary antibody), nucleus stained with DAPI . Scale bar is 100 μm. It was confirmed that it was strongly expressed in cerebellar Purkinje cells. The figure on the right shows strong magnification (scale bar is 10 μm). Bottom: immunostaining with Ab-Syt14 of human cerebellum (using diaminobenzidine as substrate). Nuclei were stained with hematoxylin. Scale bar is 100 μm. In humans, it was confirmed that expression was strong in cerebellar Purkinje cells. The right figure shows a strong enlargement (scale bar is 20μm).

  Hereinafter, the present invention will be described in detail.

  The present invention provides a method for testing autosomal recessive hereditary spinocerebellar degeneration, which comprises detecting a mutation in the SYT14 gene encoding SynaptotagminXIV (Syt14).

  Autosomal recessive hereditary spinocerebellar degeneration (ARCA) targeted by the test method of the present invention is a general term for heterogeneous diseases that exhibit autosomal recessive inheritance and clinically show ataxia. In addition to ataxic symptoms, various neurological and non-neurological symptoms such as ataxia, peripheral neuropathy, pyramidal tract disorder, extrapyramidal dysfunction, etc., often occur in younger than 20 years. To date, more than 20 ARCA responsible genes have been identified, and the frequency of Friedrich ataxia is the highest in Western countries, but there is an epidemiological survey that the causative gene is unknown in about half of the patients, There may be many patients who are not known. There are no statistics on the frequency of ARCA causative genes in Japan, but there are no reported cases of Friedrich ataxia, which is the most frequent in the United States and Europe, and the composition of the causative gene is different from Western countries. However, as in Europe and the United States, it is estimated that there are many ARCA patients whose causative genes are not known. Currently, ARCA is diagnosed comprehensively based on family history, clinical symptoms, and imaging findings. For gene diagnosis, base sequence determination (direct sequence) and mutation search are performed by the Sanger method for individual genes that have been identified as causative genes.

Synaptotagmin XIV (Syt14; Synaptotagmin 14) is one of 17 types of Synaptotagmin (Syt) known so far. The structural features of Syt have one transmembrane region (TM) on the N-terminal side and two C2 domains known as Ca2 + binding motifs on the C-terminal side (derived from the C2 regulatory region of protein kinase C) Ca ²⁺ , phospholipids, proteins, etc. bind as ligands in the motif.) There are points with C2A and C2B, and it is also called C-type tandem C2 protein. SynaptotagminI (Syt1) is the best studied among Syts, and is known to act as a Ca ²⁺ sensor on synaptic vesicles and promote the fusion of synaptic vesicles with presynaptic membranes. The SYT14 gene was cloned by Fukuda M in 2003 as a molecule conserved from Drosophila to humans (Fukuda, M. (2003) Molecular cloning, expression, and characterization of a novel class of synaptotagmin (Syt XIV) conserved from Drosophila to humans J Biochem 133, 641-649.) Is a molecule that was initially studied by qPCR and mainly expressed in the testis, heart, and kidney, with no expression in the brain. . There are four types of isoforms in Syt14, and all isoforms have TM, C2A, and C2B domains. However, C2 domains are different from Syt1 in that they do not have Ca ²⁺ binding ability. In 2007, Quintero-Rivera F et al. Found a de novo balanced translocation t (1; 3) (q32.1; q25.1) in a 12-year-old girl with mental retardation, and SYT14 was the breakpoint. (Quintero-Rivera, F., Chan, A., Donovan, DJ, Gusella, JF, Ligon, AH (2007). Disruption of a synaptotagmin (SYT14) associated with neurodevelopmental abnormalities. Am J Med Genet A 143, 558-563.). They have shown in Northern blots that SYT14 mRNA expression is seen in the human brain, suggesting that Syt14 may function in the brain.

  The genomic sequence of the human SYT14 gene is shown in SEQ ID NO: 1 in the sequence listing. In the genome sequence shown in SEQ ID NO: 1, the positions of exons are as follows.

・ Isoform 1
Exon1: 1-85 (59-coding)
Exon2: 14517-14564
Exon3: 27701-27821
Exon4: 75441-75605
Exon5: 82847-83062
Exon6: 156130-156401
Exon7: 161820-162269
Exon8: 217529-217718
Exon9: 221229-221285
Exon10: 222537-222850
・ Isoform 2
Exon1: 1-85 (59-coding)
Exon2: 14517-14564
Exon3: 27701-27821
Exon4: 75441-75605
Exon5: 82847-83062
Exon6: 156130-156401
Exon7: 161820-162269
Exon8: 217529-217718
Exon9: 222537-222850
Isoform 2 is deficient in exon9 compared to isoform 1.

・ Isoform 3
Exon1: 1-85 (73-coding)
Exon2: 14517-14564
Exon3: 75441-75605
Exon4: 82847-83062
Exon5: 156130-156401
Exon6: 161820-162269
Exon7: 217529-217718
Exon8: 221229-221285
Exon9: 222537-222850
Isoform 3 differs from isoform 1 in the exon1 start codon and lacks exon3.

・ Isoform 4
Exon1: 1-85 (73-coding)
Exon2: 14517-14564
Exon3: 75441-75605
Exon4: 82847-83062
Exon5: 156130-156401
Exon6: 161820-162269
Exon7: 217529-217718
Exon8: 222537-222850
Isoform 4 differs from isoform 1 in the exon1 start codon and lacks exon3 and exon9.

  The mRNA sequence of transcript variant 1 of human SYT14 gene and the amino acid sequence of Syt14 protein (isoform 1) encoded by this are shown in SEQ ID NOs: 2 and 3, respectively.

  The mRNA sequence of transcript variant 2 of the human SYT14 gene and the amino acid sequence of the Syt14 protein (isoform 2) encoded by this are shown in SEQ ID NOs: 4 and 5, respectively.

  The mRNA sequence of transcript variant 3 of human SYT14 gene and the amino acid sequence of Syt14 protein (isoform 3) encoded by this are shown in SEQ ID NOs: 6 and 7, respectively.

  The mRNA sequence of transcript variant 4 of human SYT14 gene and the amino acid sequence of Syt14 protein (isoform 4) encoded by this are shown in SEQ ID NOs: 8 and 9, respectively.

  From the analysis results of the present inventors, it was found that the mutation of SYT14 gene is a pathological mutation of ARCA. ARCA patients can have mutations in the SYT14 gene either homozygous or heterozygous. Examples of mutations in the SYT14 gene include, but are not limited to, deletion, point mutation, missense mutation, nonsense mutation, and frameshift mutation. In the examples described below, a missense mutation p.Gly484Asp [c.1451G> A] found in exon 8 of the SYT14 gene was identified, and when this mutation was homozygous, morbidity was confirmed, In the case of heterozygosity, no morbidity was confirmed. The missense mutation p.Gly484Asp [c.1451G> A] can be present in any of isoforms 1-4 of the Syt14 protein.

  The missense mutation p.Gly484Asp [c.1451G> A] found in exon 8 of the SYT14 gene is described as follows in the sequence listing.

-The 217680th G in the genome sequence of the human SYT14 gene shown in SEQ ID NO: 1 was replaced with A.
-The 1509th G in the mRNA sequence of human SYT14 transcript variant 1 shown in SEQ ID NO: 2 was replaced with A.
-Asp was replaced with 484th Gly in the amino acid sequence of Syt14 protein (isoform 1) encoded by mRNA of human SYT14 transcript variant 1 shown in SEQ ID NO: 3.
-The 1509th G in the mRNA sequence of human SYT14 transcript variant 2 shown in SEQ ID NO: 4 was replaced with A.
-Asp was replaced with 484th Gly in the amino acid sequence of Syt14 protein (isoform 2) encoded by mRNA of human SYT14 transcript variant 2 shown in SEQ ID NO: 5.
-The 1388th G in the mRNA sequence of human SYT14 transcript variant 3 shown in SEQ ID NO: 6 was replaced with A.
-439th Gly in the amino acid sequence of Syt14 protein (isoform 3) encoded by mRNA of human SYT14 transcript variant 3 shown in SEQ ID NO: 7 was replaced with Asp.
-The 1388th G in the mRNA sequence of human SYT14 transcript variant 4 shown in SEQ ID NO: 8 was replaced with A.
-439th Gly in the amino acid sequence of Syt14 protein (isoform 4) encoded by mRNA of human SYT14 transcript variant 4 shown in SEQ ID NO: 9 was replaced with Asp.

  A mutation in the SYT14 gene may be detected for both alleles. Moreover, the mutation of SYT14 gene may be analyzed with genomic DNA, or may be analyzed with RNA level or protein level. In the case of analyzing with genomic DNA, a mutation in the SYT14 gene can be detected using a biological sample such as a biopsy tissue such as blood, saliva, brain, skin, kidney, or pancreas collected from a subject. For example, nucleic acid samples such as genomic DNA, mRNA or cDNA synthesized from mRNA are isolated or prepared from peripheral blood leukocytes, and if necessary, the region containing the SYT14 gene mutation site is amplified to mutate the SYT14 gene. Can be detected.

  In order to analyze the mutation of the SYT14 gene with genomic DNA, for example, a coding region including the mutation site of the SYT14 gene may be amplified from the genomic DNA using an appropriately designed primer, and direct sequencing may be performed. Using commercially available software such as SeqScape (registered trademark), mutation detection and profiling can be easily performed. Whether the mutation is homozygous or heterozygous can be confirmed from the waveform data of the sequence. In addition to direct sequencing, methods such as PCR-RFLP method, sequence-specific oligo probe method (SSCP method), SSOP method, and allele-specific amplification method (MASA method) may be used.

  To analyze mutations in the SYT14 gene at the RNA level, methods such as the Northern hybridization method, Southern hybridization method, DNA chip or DNA array analysis method, in situ hybridization method, RT-PCR method, etc. Use it. “Analyzing mutations in the SYT14 gene at the RNA level” is a concept that includes analyzing cDNA synthesized from mRNA.

  To analyze mutations in the SYT14 gene at the protein level, Western blotting using anti-Syt14 antibody is used to check for changes in protein molecular weight, and immunostaining is performed on cells and tissue samples using anti-Syt14 antibody. Thus, a method for examining the intracellular localization of Syt14 is conceivable.

  The present invention also provides a method of immunostaining Purkinje cells using an antibody against Syt14 protein.

  The present invention also provides an immunostaining reagent comprising an antibody against Syt14 protein. Purkinje cells can be immunostained with the reagent of the present invention.

  The antibody against Syt14 protein may be one that specifically binds to Syt14 protein, and may be either a polyclonal antibody or a monoclonal antibody.

  In order to produce a polyclonal antibody, it can be produced according to a known method or a method analogous thereto. For example, an immunizing antigen (which may be a full-length Syt14 protein or a partial peptide containing an epitope of Syt14 protein (for example, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 or 13)) is administered to an animal ( Immunization), and an antibody-containing substance against the antigen is collected from the immunized animal, and the antibody is separated and purified. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually carried out about twice, once every about 2 to 6 weeks. Polyclonal antibodies can be collected from blood, ascites, etc., preferably from blood of immunized animals. Polyclonal antibodies can be separated and purified by immunoglobulin separation and purification methods (for example, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, adsorption / desorption method using ion exchanger, ultracentrifugation method, gel filtration method, A specific purification method in which only an antibody is collected using an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the antibody is obtained by dissociating the binding.

  Monoclonal antibodies can be prepared by the hybridoma method of G. Koehler and C. Milstein described in Nature (1975) 256: 495, Science (1980) 208: 692-. That is, after immunizing an animal, antibody-producing cells are isolated from the spleen of the immunized animal and fused with myeloma cells to prepare monoclonal antibody-producing cells (hybridoma). Furthermore, it specifically reacts with Syt14 protein (which may be full-length Syt14 protein or a partial peptide containing an epitope of Syt14 protein (for example, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 or 13)). However, cell lines that produce antibodies that do not substantially cross-react with other antigenic proteins (or peptides) may be isolated. This cell line can be cultured, and the desired monoclonal antibody can be obtained from the culture. Purification of the monoclonal antibody can be performed according to the above-described method for separating and purifying immunoglobulin.

  In immunostaining, there are methods such as autoradiography, gold colloid method, fluorescent antibody method, and enzyme antibody method to visualize antigen-antibody reaction, and antibodies against Syt14 protein are radioactive isotopes, gold particles, fluorescent dyes, It may be labeled with an enzyme or the like. In addition, the antigen-antibody reaction may be performed by either a direct method or an indirect method.

  The present invention provides a Syt14 protein mutant (hereinafter, p.Gly484Asp mutant) having a missense mutation p.Gly484Asp [c.1451G> A] found in exon 8 of the SYT14 gene. By using the p.Gly484Asp mutant, it will be possible to elucidate the pathophysiology of ARCA disease, leading to the development of effective disease management and treatment methods.

  As a p.Gly484Asp variant, the 484th Gly in the amino acid sequence of Syt14 protein (isoform 1) shown in SEQ ID NO: 3 is substituted with Asp, and the amino acid sequence of human Syt14 protein (isoform 2) shown in SEQ ID NO: 5 Among them, the 484th Gly substituted with Asp, the 439th Gly substituted with Asp in the amino acid sequence of the Syt14 protein (isoform 3) shown in SEQ ID NO: 7, the human SYT14 transcript variant 4 shown in SEQ ID NO: 9 In which the 439th Gly in the amino acid sequence of the Syt14 protein (isoform 4) encoded by mRNA is substituted with Asp. Further, in the amino acid sequences of these proteins, an organism having an amino acid sequence in which one or several amino acids other than the amino acid at the site having a pathological mutation are deleted, substituted or added, and equivalent to the p.Gly484Asp mutant It may be a protein having a biological activity (for example, antigenicity, immunostaining, ability to regulate secretion from nerve cells, etc.). Furthermore, the p.Gly484Asp variant is encoded by DNA that hybridizes under stringent conditions with DNA complementary to DNA consisting of the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8, and p.Gly484Asp variant May have a biological activity equivalent to that of the protein (eg, antigenicity, immunostaining, ability to regulate secretion from nerve cells, etc.). Hybridization is performed under stringent conditions. The stability of the nucleic acid duplex or hybrid is expressed by the melting temperature Tm (the temperature at which the probe dissociates from the target DNA). This melting temperature is used to define stringent conditions. Assuming that the Tm drops by 1 ° C. due to a 1% mismatch, the temperature of the final wash of the hybridization reaction must be lowered. For example, when a sequence having 95% or more identity with a probe is required, the final washing temperature must be lowered by 5 ° C. In fact, for 1% mismatch, the Tm will vary between 0.5 and 1.5 ° C. An example of stringent conditions is to hybridize in 5x SSC / 5x Denhardt solution / 1.0% SDS at 68 ° C and wash in 0.2x SSC / 0.1% SDS at room temperature. An example of moderately stringent conditions is a wash at 42 ° C. in 3 × SSC. The salt concentration or temperature can be varied to achieve an optimal level of identity between the probe and the target nucleic acid. For further guidance on such conditions, see Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (Eds.), 1995, Current Protocols in Molecular Biology, (John See Wiley & Sons. NY) at Unit 2.10.

  The p.Gly484Asp mutant may be a human-derived wild-type Sty14 protein mutant or a non-human animal-derived wild-type Syt14 protein mutant.

  The p.Gly484Asp mutant can be produced by a known method. For example, the p.Gly484Asp mutant protein is obtained by obtaining DNA encoding the p.Gly484Asp mutant protein, incorporating the obtained DNA into an appropriate expression vector, introducing it into an appropriate host, and producing it as a recombinant protein. Proteins can be produced (for example, Satoshi Saigo, Yoko Sano, CURRENT PROTOCOLS Compact Edition, Molecular Biology Experiment Protocol, I, II, III, Maruzen Co., Ltd .: Original, Ausubel, FM, etc., Short (See Protocols in Molecular Biology, Third Edition, John Wiley & Sons, Inc., New York). Alternatively, it can also be produced according to a known peptide synthesis method.

  An isolated DNA encoding the p.Gly484Asp mutant protein can be produced, for example, as follows.

  MRNA is extracted from healthy human peripheral blood leukocytes, and cDNA is synthesized using reverse transcriptase and oligo dT primers. The coding region of human Syt14 is amplified by PCR. The obtained PCR product is DNA encoding the wild type Syt14 protein. The amino acid sequence of the wild-type Syt14 protein is shown in SEQ ID NOs: 3, 5, 7, and 9, and the nucleotide sequence of the DNA encoding it is shown in SEQ ID NOs: 2, 4, 6, and 8.

  DNA encoding the p.Gly484Asp mutant protein can be prepared by mutating the coding region of SYT14 by point mutagenesis. The mutated SYT14 coding region is amplified by PCR. The obtained PCR product is DNA encoding the p.Gly484Asp mutant protein. Examples of DNA encoding the p.Gly484Asp mutant protein include those in which the 1509th G in the mRNA sequence of human SYT14 transcript variant 1 shown in SEQ ID NO: 2 is replaced with A, human SYT14 transcript variant 2 shown in SEQ ID NO: 4 In which the 1509th G in the mRNA sequence is replaced with A, the 1388th G in the mRNA sequence of the human SYT14 transcript variant 3 shown in SEQ ID NO: 6 is replaced with A, the human shown in SEQ ID NO: 8 Examples include those in which the 1388th G in the mRNA sequence of SYT14 transcript variant 4 is replaced with A.

  A recombinant vector containing DNA encoding the p.Gly484Asp mutant protein can be obtained by a known method (for example, the method described in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). It can be obtained by inserting DNA encoding the p.Gly484Asp mutant protein into an appropriate expression vector.

  As expression vectors, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, etc. Bacteriophages, animal viruses such as retroviruses and vaccinia viruses, insect pathogenic viruses such as baculoviruses, and the like can be used.

  A promoter, enhancer, splicing signal, poly A addition signal, selection marker, SV40 replication origin, and the like may be added to the expression vector.

  The expression vector may be a fusion protein expression vector. Various fusion protein expression vectors are commercially available: pGEX series (Amersham Pharmacia Biotech), pET CBD Fusion System 34b-38b (Novagen), pET Dsb Fusion Systems 39b and 40b (Novagen), pET GST Fusion System 41 and 42 (Novagen).

  A transformant can be obtained by introducing a recombinant vector containing DNA encoding the p.Gly484Asp mutant protein into a host.

  Hosts include bacterial cells (eg, Escherichia, Bacillus, Bacillus, etc.), fungal cells (eg, yeast, Aspergillus, etc.), insect cells (eg, S2 cells, Sf cells, etc.), animal cells (eg, CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, etc.), plant cells and the like.

  In order to introduce a recombinant vector into a host, the method described in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 (for example, calcium phosphate method, DEAE-dextran method, transfection method, Injection method, lipofection method, electroporation method, transduction method, scrape loading method, shotgun method, etc.) or infection.

  The transformant can be cultured in a medium, and p.Gly484Asp mutant protein can be collected from the culture. When the p.Gly484Asp mutant protein is secreted into the medium, the medium may be recovered, and the p.Gly484Asp mutant protein may be separated from the medium and purified. When the p.Gly484Asp mutant protein is produced in the transformed cell, the cell may be lysed, and the p.Gly484Asp mutant protein may be separated from the lysate and purified.

  When the p.Gly484Asp mutant protein is expressed in the form of a fusion protein with another protein (functioning as a tag), the fusion protein should be isolated and purified, and then treated with FactorXa or an enzyme (enterokinase) Can cleave another protein to obtain the desired p.Gly484Asp mutant protein.

  Separation and purification of the p.Gly484Asp mutant protein can be performed by known methods. Known separation and purification methods include molecular methods such as salting out and solvent precipitation methods that utilize differences in solubility, dialysis methods, ultrafiltration methods, gel filtration methods, and SDS-polyacrylamide gel electrophoresis methods. Method utilizing difference, method utilizing charge difference such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing hydrophobic difference such as reverse phase high performance liquid chromatography A method using a difference in isoelectric point, such as isoelectric focusing, is used.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to these Examples.

[Example 1]
(result)
Fig. 1 shows the family tree and MRI image of the family analyzed in this study. In the subject family, two children (IV-3 and IV-4) of a cousin's married couple had an older onset SCA with mental retardation, and cerebellar atrophy was also confirmed on the image. For III-1, IV-1, IV-2, IV-3, all using Affymetrix's Human Mapping 10K Array Xba 142 2.0 (10K array) and Genome-Wide Human SNP Array 6.0 (SNP 6.0 array) Genome SNP typing and Allegro version 2 software (Gudbjartsson, DF, T. Thorvaldsson, et al. (2005). "Allegro version 2." Nat Genet 37 (10): 1015-1016) Was used to calculate the LOD score assuming autosomal recessive inheritance (calculated with the disease allele frequency set to 0.001). As a result, 20 regions with a LOD score exceeding 1.5, a total of 63.22 Mb, were identified as candidate regions (Table 1, maximum LOD score 2.0516). Furthermore, for the same region, SNP 6.0 array data confirmed whether it was a homozygous region, and SNP 6.0 array also confirmed that it was a homozygous region of 1.0 Mb or more, a total of 11.92 Mb. Was a candidate region for the disease locus (Table 2). Since all patients were male, the LOD score assuming an X-chromosomal recessive inheritance format was also calculated (calculated with the disease allele frequency set to 0.001). Results The LOD score was positive in 4 areas, totaling 74.56 Mb (maximum LOD score 0.9031).

  In order to identify the responsible gene mutation from the above candidate region, exon capture using Agilent Technologies SureSelect Human All Exon Kit v.1 was performed on 3 μg genomic DNA of IV-3 and IV-4. DNA was analyzed using Illumina next-generation sequencer GAIIx. The resulting sequence of approximately 71 million reads (IV-3) and approximately 148 million reads (IV-4) was mapped and assembled with Qualities (MAQ) (Li, H., J. Ruan, et al (2008). “Mapping short DNA sequencing reads and calling variants using mapping quality scores.” Genome Res 18 (11): 1851-1858.) And NextGENe software v2.00 (SoftGenetics) were used for analysis. In MAQ, 59,491,138 reads and 126,159,746 reads are mapped onto the genome, respectively, and 65.0% (IV-3) and 71.3% (IV-4) of all genes that covered one or more complete coding sequences (CDS). ), 77.7% (IV-3) and 80.3% (IV-4) had 90% or more CDS covered by 1 lead or more.

  Among the mutations or polymorphisms detected by MAQ and NextGENe, those that exist outside the above candidate regions, and polymorphisms registered in dbSNP130 were excluded. Among the remaining mutations or polymorphisms, those detected as homozygous mutations and present in the coding region or intron region within 50 bp of the coding region are in SYT14 (1q32.2) exon8 encoding SynaptotagminXIV (Syt14) Only the missense mutation p.Gly484Asp [c.1451G> A] observed was observed (Table 3). In the Sanger sequence, c.1451G> A mutation was homozygous in IV-3 and IV-4, and heterozygous in III-1, IV-1, and IV-2 (Fig. 2). This missense mutation is an amino acid mutation that exists on the C2 (C2B) domain of Syt14 and is highly conserved from human to flies (Fig. 2), and was confirmed not to be observed in the Japanese normal control 576 allele. The mutation was consistently predicted to be a pathological mutation with different mutation effect prediction software (Table 4). No pathological mutation was detected in the candidate region assuming X-chromosomal recessive inheritance. Syt14 was first reported as a non-expressed molecule in the mouse brain (Fukuda, M. (2003). "Molecular cloning, expression, and characterization of a novel class of synaptotagmin (Syt XIV) conserved from Drosophila to Humans. "J Biochem 133 (5): 641-649.), TaqManTM quantitative PCR confirms that SYT14 is strongly expressed in the brain in human tissues and SYT14 is also expressed in the mouse brain. It was confirmed that its expression was strong in the cerebellum (Fig. 3).

  In the forced expression experiment of Syt14 using COS-1 cells, wild-type Syt14 was localized around the nucleus and in the vicinity of the cell membrane, whereas the p.Gly484Asp mutant was not observed in the vicinity of the cell membrane, The distribution was confirmed by immunostaining, and the reticulated distribution observed in the mutants was confirmed to co-localize with protein disulfide isomerase (PDI), an endoplasmic reticulum protein (Fig. 4). From the difference in distribution from this wild type, it was considered that the p.Gly484Asp mutant was very likely to be pathological. Furthermore, when a rabbit polyclonal antibody (Ab-Syt14) against Syt14 was prepared, it was confirmed that Syt14 was specifically expressed in Purkinje cells in mouse and human cerebellum (FIG. 5). This result further firmly confirmed that the Syt14 mutation causes SCA.

(Materials and methods)
・ Case A single Japanese family with a clinically diagnosed case of SCA was included. In total, genomic DNA was extracted from peripheral blood leukocytes from 2 SCA cases and 3 unaffected individuals and used for analysis.

・ Homozygous mapping and haplotype analysis
Whole genome SNP typing was performed using 10K and SNP 6.0 arrays. Typing was performed according to Affymetrix recommended protocol. Based on the data of 10K array, using Allegro version 2 software (Gudjartsson, DF, T. Thorvaldsson, et al. (2005). "Allegro version 2." Nat Genet 37 (10): 1015-1016.) Point linkage analysis was performed, and an LOD score was calculated assuming an autosomal recessive inheritance format (calculated with the disease allele frequency set to 0.001). For areas where the LOD score exceeded 1.5, the SNP 6.0 array data confirmed whether it was a homozygous area, and the SNP 6.0 array also confirmed that it was a homozygous area of 1.0 Mb or higher. Was a candidate region for the disease locus.

・ Mutation analysis
Exon capture using Agilent Technologies SureSelect Human All Exon Kit v.1 was performed on 3 μg genomic DNA of IV-3 and IV-4, and the captured DNA was analyzed using Illumina next-generation sequencer GAIIx. The resulting sequence was mapped and assembled with Qualities (MAQ) (Li, H., J. Ruan, et al. (2008). "Mapping short DNA sequencing reads and calling variants using mapping quality scores." Genome Res 18 (11): 1851-1858.) And NextGENe software v2.00 (SoftGenetics). Among the mutations or polymorphisms detected by MAQ and NextGENe, those that exist outside the above candidate regions, and polymorphisms registered in dbSNP130 were excluded. Among the remaining mutations or polymorphisms, those detected as homozygous mutations and present in the coding region or intron region within 50 bp from the coding region were used as candidate genes.

  In the analysis of Japanese normal controls, the exon 8 and exon intron border regions of the SYT14 protein translation region were amplified by PCR, and after purification, the nucleotide sequence was determined by direct sequencing. The PCR reaction solution has a composition of 1 × ExTaq buffer, 0.2 mM each dNTP, 0.5 μM each primer, 0.125 U Ex TaqHS polymerase (TAKARA). Reaction conditions and primer sequences used are shown in Table 5. The PCR product was purified by ExoSAP-IT (GE healthcare) and then subjected to cycle sequence reaction using BigDye Terminator chemistry version 3.1 (Applied Biosystems). The reaction product was purified by gel filtration using Sephadex G-50 (GE healthcare) and Multiscreen-96 (Millipore), and a sequence was obtained using ABI Genetic Analyzer 3100 or 3500 (Applied Biosystems). The obtained sequences were analyzed for the presence or absence of mutations using Sequencher software ver. 4.10.1 (Gene Codes Corporation).

The nucleotide sequences of the forward and reverse primers in Table 5 are shown in SEQ ID NOs: 10 and 11, respectively.

・ Expression analysis
TaqManTM quantitative PCR was performed for the expression analysis of Syt14. CDNA templates for adult tissues (Human MTCTM Panel I, # 636742), fetal tissues (Human Fetal MTCTM Panel, # 636747), and mouse tissues (Mouse MTCTM Panel I, # 636745) were purchased from Clontech and used for mouse brain. Site-specific cDNA (GSMBRSET) was purchased from Nippon Genetics. As probes used for TaqManTM quantitative PCR, human SYT14 (Hs00950169_m1) for human SYT14 expression analysis, human β-actin (ACTB, 4326315E) as an internal standard, mouse Syt14 (Mm00805319_m1) for mouse SYT14 expression analysis, Mouse Actb (43522341E) was purchased from Applied Biosystems as an internal standard and used. 20 μl of quantitative PCR reaction solution is composed of 10 μl of TaqMan® Gene Expression Master Mix (Applied Biosystems), 1 μl of TaqManTM probe for ACTB and SYT14, 1 μl of cDNA (1 ng of Clontech cDNA, cDNA of Nippon Genetics Co., Ltd.) Contained 25 ng). The PCR protocol was Rotor-Gene Q (QIAGEN) and the following protocol was used. 50 ° C, 2 minutes -95 ° C, 10 minutes-(95 ° C, 15 seconds-60 ° C, 1 minute) x 40 cycles. The expression level was relatively quantified by the 2- ^ΔΔCt method using Rotor-Gene Q Series Software (QIAGEN). ΔCt was (cycling threshold (Ct) of SYT14-Ct of ACTB), and ΔΔCt was (control sample ΔCt-ΔCt of sample). The relative amount was calculated as 2− ^ΔΔCt .

Antibody Antibody 106-120 amino acids (LGSEYSTRKNSQDKI) (SEQ ID NO: 12) and 250-264 amino acids (CPSEGSTGHEIESFH) (SEQ ID NO: 13) of human Syt14 were used as antigens and prepared using one rabbit, and CNBr-activated Sepharose 4B And purified.

・ Web software The following web software was used as the mutation effect prediction software.

PolyPhen, http://genetics.bwh.harvard.edu/pph/
PolyPhen2, http://genetics.bwh.harvard.edu/pph2/
SIFT, http://blocks.fhcrc.org/sift/SIFT.html
Align GCGD, http://agvgd.iarc.fr/

  The present invention can be used for genetic diagnosis. That is, a definitive diagnosis of ARCA can be performed by examining the nucleotide sequence of the SYT14 gene.

  The present invention can also be used to study new mechanisms of neurodegeneration. That is, it is known that a similar protein of this protein (such as Syt I) is involved in regulatory secretion such as neurotransmitter release at the synapse. Although the function of this protein (Syt14 protein) itself has not yet been elucidated, it has been identified as a disease-responsible gene for ARCA, and it is considered that research on the mechanism by which regulatory secretion disorders cause neurodegeneration will progress.

<SEQ ID NO: 1>
SEQ ID NO: 1 shows the genomic sequence of the human SYT14 gene.
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the mRNA sequence of human SYT14 transcript variant 1.
<SEQ ID NO: 3>
SEQ ID NO: 3 shows the amino acid sequence of Syt14 protein (isoform 1) encoded by human SYT14 transcript variant 1 mRNA.
<SEQ ID NO: 4>
SEQ ID NO: 4 shows the mRNA sequence of human SYT14 transcript variant 2.
<SEQ ID NO: 5>
SEQ ID NO: 5 shows the amino acid sequence of Syt14 protein (isoform 2) encoded by mRNA of human SYT14 transcript variant 2.
<SEQ ID NO: 6>
SEQ ID NO: 6 shows the mRNA sequence of human SYT14 transcript variant 3.
<SEQ ID NO: 7>
SEQ ID NO: 7 shows the amino acid sequence of Syt14 protein (isoform 3) encoded by mRNA of human SYT14 transcript variant 3.
<SEQ ID NO: 8>
SEQ ID NO: 8 shows the mRNA sequence of human SYT14 transcript variant 4.
<SEQ ID NO: 9>
SEQ ID NO: 9 shows the amino acid sequence of Syt14 protein (isoform 4) encoded by mRNA of human SYT14 transcript variant 4.
<SEQ ID NO: 10>
SEQ ID NO: 10 shows the nucleotide sequence of the forward primer for amplifying the exon 8 and exon intron border region of the protein translation region of SYT14.
<SEQ ID NO: 11>
SEQ ID NO: 11 shows the nucleotide sequence of the reverse primer for amplifying the exon 8 and exon intron border region of the protein translation region of SYT14.
<SEQ ID NO: 12>
SEQ ID NO: 12 shows the sequence of 106-120 amino acids of human Syt14 protein.
<SEQ ID NO: 13>
SEQ ID NO: 13 shows the 250-264 amino acid sequence of human Syt14 protein.

Claims (8)

A method for testing autosomal recessive hereditary spinocerebellar degeneration, comprising detecting a mutation in the SYT14 gene encoding SynaptotagminXIV (Syt14). The test method according to claim 1, wherein a mutation of the SYT14 gene is detected for both alleles. The test method according to claim 1 or 2, wherein the mutation of the SYT14 gene is detected by analyzing the genomic DNA. The test method according to claim 1 or 2, wherein a mutation of the SYT14 gene is analyzed and detected at the RNA level. The test method according to claim 1 or 2, wherein a mutation in the SYT14 gene is detected by analysis at a protein level. A method of immunostaining Purkinje cells using an antibody against Syt14 protein. An immunostaining reagent comprising an antibody against Syt14 protein. The reagent according to claim 7 for immunostaining Purkinje cells.