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WO2020013233A1 - Als biomarker and als diagnosis method - Google Patents

Als biomarker and als diagnosis method Download PDF

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Publication number
WO2020013233A1
WO2020013233A1 PCT/JP2019/027355 JP2019027355W WO2020013233A1 WO 2020013233 A1 WO2020013233 A1 WO 2020013233A1 JP 2019027355 W JP2019027355 W JP 2019027355W WO 2020013233 A1 WO2020013233 A1 WO 2020013233A1
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adar2
nucleic acid
acid sequence
seq
mrna
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PCT/JP2019/027355
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French (fr)
Japanese (ja)
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伸 郭
雄也 山下
孝史 保坂
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国立大学法人東京大学
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Definitions

  • the present invention relates to a biomarker for diagnosing ALS and a method for diagnosing ALS using the biomarker.
  • ALS Amyotrophic lateral sclerosis
  • AMPA ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
  • RNA editing related to ALS is a deamination reaction in which adenosine is converted to inosine (hereinafter referred to as “AI RNA editing”), and unedited GluA2 is expressed.
  • AI RNA editing a deamination reaction in which adenosine is converted to inosine
  • GluA2 is expressed.
  • the expression of abnormally high AMPA receptors and the fact that GluA2 is unedited are due to decreased expression (down-regulation) of the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2).
  • Non-Patent Document 1 Non-Patent Document 2, and Non-Patent Document 3
  • glutamine (Q) is present at the site facing the lumen of the ion channel pore (Q / R site), but in edited GluA2, it is converted to arginine (R) by AI RNA editing .
  • the Q / R site of the edited GluA2 prevents calcium ion inflow due to the positively charged R, whereas the unedited GluA2 exhibits calcium permeability due to the neutrally charged Q at the site.
  • ADAR2 conditional knockout mice (ADAR2 flox / flox / VAChT-Cre.Fast; AR2 mice) exhibit progressive motor dysfunction and skeletal muscle denervation, but these symptoms are unedited at the Q / R site. It has been reported that this is due to the disappearance of lower motor neurons caused by calcium permeability of AMPA receptor having type GluA2 subunit (Non-Patent Document 4). Furthermore, the abnormal intracellular localization of TDP-43 (transactive response DNA-binding protein of 43 kDa) in motor neurons, which is a neuropathological feature of sporadic ALS patients, is associated with sporadic ALS patients. ADAR2 has been confirmed without exception in down-regulated motoneurons (Non-Patent Document 5). As described above, it was revealed that down-regulation of ADAR2 is closely related to motor neuron death in ALS patients.
  • Non-Patent Document 6 Development of biomarkers for early diagnosis of ALS is needed to enable effective treatment before such many motor neurons disappear. As described above, down-regulation of ADAR2 is closely related to the development of ALS, so if ADAR2 activity can be examined in a bodily fluid that can be collected, it will be possible to diagnose the presence or absence of ALS early. . However, at present, there are no reliable biomarkers that allow such diagnosis.
  • an object of the present invention is to provide a biomarker useful for diagnosing or determining a therapeutic effect of ALS, and to provide a method for diagnosing ALS and a method for determining a therapeutic effect using the biomarker. .
  • the present inventors focused on ADAR2-dependent AI RNA editing as an index of ADAR2 down-regulation that causes ALS development, and searched for RNA having this AI RNA editing site. Ten sites were found in linear RNA and one in circular RNA. Furthermore, they found that the variation in the editing rate of these ADAR2-dependent AI RNA editing sites was equivalent between intracellular RNA and extracellular RNA. As described above, the present inventors have found for the first time that extracellular (ie, in body fluid) RNA having an AI RNA editing site whose editing rate fluctuates in an ADAR2 activity-dependent manner can be used as a biomarker for ALS. Completed the invention.
  • the present invention provides the following (1) to (10).
  • the ADAR2-dependent AI RNA editing site is the nucleic acid of the ADAR2 pre-mRNA represented by SEQ ID NO: 1 Position 178 in the sequence, position 191 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, position 192 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, Position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, position 28 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO:
  • a biomarker for diagnosing the pathology of ALS which comprises one or more RNA molecules having an ADAR2-dependent AI RNA editing site.
  • the ADAR2-dependent AI RNA editing site is located at position 178 in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1, and in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1.
  • Position 191 position 192 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, represented by SEQ ID NO: 3
  • Position 28 in the nucleic acid sequence of GluA2 mRNA position 498 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3
  • the ADAR2-dependent AI RNA editing site is located at position 178 in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1, and in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1.
  • Position 191 position 192 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, represented by SEQ ID NO: 3
  • Position 28 in the nucleic acid sequence of GluA2 mRNA position 498 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3
  • a kit used for diagnosing the pathology of ALS comprising at least an element for identifying a nucleotide at an ADAR2-dependent AI RNA editing site.
  • Circular junction bonding position by back splicing ADAR2-dependent AI RNA editing site of RNA expressed in HeLa cells.
  • A Changes in the expression level of ADAR2 mRNA due to ADAR2 overexpression.
  • mock cells transfected with pCl (empty vector).
  • OE cells transfected with ADAR2 / pCl.
  • a and B Changes in the expression level of ADAR2 mRNA due to ADAR2 overexpression and changes in editing rate at 12 editing sites having homologous sites with mice.
  • Mock cells transfected with pCl (empty vector)
  • OE cells transfected with ADAR2 / pCl. Shows the edit rate of Mock transfected cells (white) and ADAR2 / pCl transfected cells (black) (Mann-Whitney U-test; * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001).
  • (A) shows the editing rate of 10 ADAR2-dependent AI RNA editing in the culture supernatant of Mock-transfected cells (white) and the culture supernatant of ADAR2 / pCl-transfected cells (dot) (Mann -Whitney U-test; * P ⁇ 0.05, ** P ⁇ 0.01).
  • (B) In culture supernatant of cells transfected with scramble siRNA (white), culture supernatant of cells transfected with siADAR2-1 (dot), and culture supernatant of cells transfected with siADAR2-2 (hatched). 5 shows the editing rates of the five ADAR2-dependent AI RNA editing sites (Mann-Whitney U-test; * P ⁇ 0.05).
  • Circ GRIA2 (hsa_circ_0125620) in SH-SY5Y cells and their culture supernatant.
  • A Electrophoresis of circ GRIA2 (hsa_circ_0125620) and Sanger sequencing. PCR product of circ GRIA2 in SH-SY5Y cells (arrow). N: Negative control. The junction of back splicing from exon 13 to exon 11 is shown. The circled arrow indicates the Q / R site of circ GRIA2 , and ADAR2 knockdown reduced the editing rate (G (sequencing by a sequencer determines inosin (I) as guanine (G) because the cDNA sequence is determined). Identified) and the peak of A increases).
  • FIG. 2 A conceptual diagram of a method for detecting the editing ratio at the Q / R site of circ GRIA2 and an example of measuring the editing ratio using the 2100 Bioanalyzer.
  • Fragment obtained PCR product circ GRIA2 with edit type Q / R site was cut with HpyCH4V is, 76 bp, a 160bp and 196 bp.
  • fragment obtained by digesting the PCR product circ GRIA2 with unedited form of Q / R site in HpyCH4V is, 24 bp, 76 bp, a 160bp and 172 bp.
  • the 24 bp fragment is not detected because it overlaps the peak of the low molecular marker (LMM).
  • HMM High molecular marker.
  • a first embodiment of the present invention is a method for diagnosing the pathology of ALS, comprising the step of identifying the nucleotide of an ADAR2-dependent AI RNA editing site present in a diagnostic sample.
  • the method for diagnosing the condition of ALS according to the first embodiment of the present invention (hereinafter, also referred to as “the ALS diagnostic method of the present invention”) can be used to determine whether a diagnosis target has developed ALS. Of course, it can also be used to determine the course of treatment for patients who have already developed ALS. Therefore, the first embodiment of the present invention is also a “method of assisting diagnosis of a pathological condition of ALS”.
  • the “ALS” in the present embodiment includes not only sporadic ALS but also familial ALS as long as the ALS2 pathology is caused by ADAR2 down-regulation of motor neurons. More specifically, the ALS diagnostic method of the present invention is a method for diagnosing the condition of ALS, and comprises the following steps (a) to (d).
  • step (A) identifying the nucleotides at the ADAR2-dependent AI RNA editing site of the RNA present in the diagnostic sample, (B) calculating the ratio of the nucleotide identified in step (a) to adenine (A) or inosine (I); and (c) calculating the ratio of A or I calculated in step (b) Comparing the ratio of nucleotides of the ADAR2-dependent AI RNA editing site to A or I, respectively.
  • the diagnostic sample refers to a biological sample derived from a subject (a person who has developed ALS or a person who is suspected of developing ALS, hereinafter also referred to as a “subject”) for performing a pathological diagnosis of ALS.
  • a subject a person who has developed ALS or a person who is suspected of developing ALS, hereinafter also referred to as a “subject” for performing a pathological diagnosis of ALS.
  • body fluids such as blood and cerebrospinal fluid.
  • Extraction of RNA from a diagnostic sample can be performed by a method well-known to those skilled in the art, and can be easily performed using a commercially available RNA extraction kit or the like.
  • the term “ADAR2-dependent AI RNA editing” as used herein means that adenosine (A) in RNA after transcription is converted to inosine (I) by ADAR2 enzyme activity in motor neurons.
  • ADAR2-dependent AI RNA editing refers to the position of A that is converted to I by AD
  • ADAR2 functions normally in motoneurons of healthy individuals ("healthy” in the present specification means a state in which ALS has not developed), so that ADAR2-dependent AI RNA editing sites of healthy individuals
  • the nucleotide is usually expected to be I (sequencing by a sequencer is identified as guanine (G) to sequence the cDNA).
  • the nucleotide at the ADAR2-dependent AI-RNA editing site in ALS patients may be A (since ADAR2 is down-regulated). It has been confirmed that ADAR1 functions normally in motor neurons of ALS patients (Non-Patent Document 3).
  • the ADAR2-dependent AI RNA editing site can be searched for as follows. Compare the RNA sequence of motoneurons from healthy subjects and the motoneurons from ALS patients. It can be an RNA editing site. Alternatively, the RNA sequence of a motor neuron derived from a healthy non-human animal is compared with the RNA sequence of a motor neuron derived from an ALS-model non-human animal (for example, AR2 mouse) knocked down by ADAR2. A site where the editing rate is reduced in RNA derived from an ALS model non-human animal as compared to RNA is detected, and if a homologous site is detected in human, it may be used as an ADAR2-dependent AI RNA editing site.
  • the following search method can be exemplified.
  • Determine the sequence of RNA extracted from motor neurons of a healthy human or non-human animal compare the obtained sequence information with the genome sequence information of the animal (genome sequence information registered in a data bank, etc.),
  • the position detected as G on the RNA derived from motoneurons is A
  • the position detected as G on the motor neuron-derived RNA is set as a candidate site for the ADAR2-dependent AI RNA editing site.
  • a position on the human genome homologous to the candidate site for the non-human animal may be used as a candidate site for a human ADAR2-dependent AI RNA editing site.
  • the search for an AI RNA editing site in human motoneurons may be performed by referring to, for example, Picardi et al., Sci Rep. 5: 14941 DOI: 10.1038 / srep14941 2015.
  • the obtained candidate site also includes an ADAR1 edited site.
  • Whether or not a candidate site is actually a site that has been edited by ADAR2 can be determined, for example, by the AI editing rate of the candidate site in motoneurons derived from ALS patients or ALS model non-human animals in which ADAR2 has been knocked down (A Efficiency of conversion to I. For example, the ratio of the RNA at the candidate site to the total of the RNA at the site A and the RNA at the site I) is compared with the AI editing rate of the candidate site derived from a healthy person or animal. Can be confirmed.
  • the AI editing rate of a candidate site derived from a cell line in which ADAR2 is overexpressed and / or a cell line in which ADAR2 is knocked down, and a control cell line (cells in which ADAR2 is not overexpressed or knocked down) can be confirmed by comparing the AI editing rates of the candidate sites derived from.
  • a candidate site where the AI editing rate is obviously changed due to a change in ADAR2 activity can be determined as “ADAR2-dependent AI RNA editing”.
  • the type of nucleotide at the ADAR2-dependent AI RNA editing site can be detected based on the sequence information of the RNA region containing the AI RNA editing site. More specifically, for example, the ADAR2-dependent AI RNA editing site, The 178th position in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, Position 191 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, The 192nd position in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, Position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, Position 28 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3, Position 498 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3, Position 202 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, Position 672 in the nucleic acid sequence
  • RNA region containing the AI RNA editing site was amplified under the PCR conditions shown in Table 2 and the amplified product was sequenced to confirm what the nucleotide of the AI RNA editing site was.
  • SEQ ID NOs: 1 to 6 show the sequences of the respective mRNAs.
  • circ GRIA2 has_circ_0125620
  • FIG. 1 shows an unedited sequence and a binding position by back splicing.
  • the primer pairs shown in Table 1 and the PCR conditions shown in Table 2 are examples, and are not limited to the primer pairs and PCR conditions shown in these tables.
  • the recognition sequence of a restriction enzyme present near the editing site changes depending on the presence or absence of AI RNA editing
  • the PCR product containing the AI ⁇ RNA editing site is cleaved with the restriction enzyme to determine whether the restriction enzyme is cleaved. From this, it can be confirmed whether the AI RNA editing site is A or I.
  • Step (b) is a step of calculating the AI editing rate at the AI RNA editing site in the diagnostic sample.
  • the ratio of the nucleotide of the ADAR2-dependent AI RNA editing site of the healthy subject to A or I is determined by comparing the “diagnostic sample” in the steps (a) and (b) with a sample derived from a healthy subject ( It can be calculated by substituting a biological sample (eg, blood, cerebrospinal fluid, etc.) derived from a person who does not have ALS, and performing each step. Alternatively, if the AI editing rate data of the ADAR2-dependent AI RNA editing site of a healthy person already exists, the data may be compared with the data.
  • a biological sample eg, blood, cerebrospinal fluid, etc.
  • step (c) when the AI editing rate (the rate of conversion to I) of the ADAR2-dependent AI RNA editing site in the diagnostic sample is significantly lower than the AI editing rate in the sample derived from a healthy subject
  • the subject can be determined to have, or may have, developed ALS.
  • the AI editing rates of a plurality of ADAR2-dependent AI RNA editing sites may be determined to make a comprehensive judgment.
  • the second embodiment of the present invention is a biomarker for diagnosing the condition of ALS, which is a biomarker comprising one or more RNA molecules having an ADAR2-dependent AI RNA editing site.
  • the biomarker in the second embodiment (hereinafter also referred to as “biomarker of the present invention”) may be a linear molecule if it is an RNA molecule having the “ADAR2-dependent AI RNA editing site” described in the first embodiment. Although it may be any of a round RNA and a circular RNA, a circular RNA that exists relatively stably in a body fluid is more preferable.
  • the second embodiment of the present invention is also a “biomarker for assisting in the diagnosis of a pathological condition of ALS”.
  • RNA molecule having the ADAR2-dependent AI RNA editing site examples include, but are not limited to, ADAR2 pre-mRNA containing the nucleic acid sequence represented by SEQ ID NO: 1, CYFIP2 mRNA containing the nucleic acid sequence represented by SEQ ID NO: 2 GluA2 mRNA containing the nucleic acid sequence represented by SEQ ID NO: 3, SON mRNA containing the nucleic acid sequence represented by SEQ ID NO: 4, TMEM63B mRNA containing the nucleic acid sequence represented by SEQ ID NO: 5, and represented by SEQ ID NO: 6
  • a circular circ GRIA2 (has_circ_0125620) containing a nucleic acid sequence can be mentioned.
  • a third embodiment of the present invention is the use of one or more RNA molecules having an ADAR2-dependent AI RNA editing site as ALS biomarkers.
  • one or more RNA molecules having an ADAR2-dependent AI RNA editing site are used for diagnosing ALS (or assist in diagnosing ALS).
  • a fourth embodiment of the present invention relates to a kit used for diagnosing the condition of ALS (or a kit used for assisting the diagnosis of the condition of ALS), which comprises at least an ADAR2-dependent AI RNA editing site. It is a kit containing elements for identifying nucleotides.
  • elements for identifying nucleotides of ADAR2-dependent AI RNA editing refers to those necessary for identifying nucleotides of ADAR2-dependent AI RNA editing sites present in a sample.
  • kits may further include reagents used to identify nucleotides at the ADAR2-dependent AI RNA editing site, and instructions for use (including those stored on an electromagnetic recording medium). Good.
  • mice used in the experiments were homozygous conditional ADAR2 knockout mice (ADAR2 flox / flox / VAChT-Cre.Fast; AR2 mice), which are Cre targets whose expression is regulated by the VAChT (vesicular acetylcholine transporter) promoter. It has a floxed ADARB1 allele (Hideyama et al., J Neurosci, 30, 11917-11925 2010).
  • a wild-type mouse of the same age as the knockout mouse C57BL / 6J mice; Oriental Yeast Co., Ltd.
  • All animal experiments were approved by the University of Tokyo Animal Care Committee and were performed in accordance with the Animal Experiment Guidelines of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
  • RNA extraction and sequencing From motor neuron samples, RNA extraction and DNaseI treatment were performed using the RNeasy micro Kit. The extracted total RNA was treated with RiboGone mammalian kit (Takara Bio) to remove ribosomal RNA. A cDNA library for RNA sequencing was synthesized from total RNA using the SMARTer Stranded RNA-Seq Kit (Takara Bio) and sequenced using Illumina HiSeq TM 2000 (Illumina, Inc.).
  • RNA sequence information of 15 or more reads in 2 or more animals was mapped to a mouse genome sequence (NCBI37 / mm9). As a result of mapping, 215 sites where A on the mouse genome sequence was G in the determination of RNA derived from motor neurons were identified and designated as a mouse AI RNA editing site.
  • HeLa cells and SH-SY5Y cells were obtained from MEM ⁇ -medium (WAKO) supplemented with 10% FBS (Thermo Fisher Scientific), 100 U / ml penicillin and 100 ⁇ g / ml streptomycin (Thermo Fisher Scientific). The medium was cultured under the conditions of 37 ° C. and 5% CO 2 . 1-5-1.
  • Overexpression of human ADAR2 in HeLa cells HeLa cells were seeded at a density of 1.0 ⁇ 10 4 cells / cm 2 in 6-well plates. 24 hours after the start of the culture, 2.5 ⁇ g of human ADAR2 vector (hADAR2 / pCI) (Yamashita et al., Embo Mol.
  • RNA extraction and reverse transcription from cells were performed on HeLa cells and SH-SY5Y cells using an RNA spin Mini Kit (GE Healthcare).
  • RNA spin Mini Kit GE Healthcare
  • cDNA was synthesized using ReverTra ACE qPCR-RT Master Mix Kit (TOYOBO) containing oligoDT primer and randam primer.
  • TOYOBO ReverTra ACE qPCR-RT Master Mix Kit
  • For the extraction of circular RNA 20 ⁇ g of total RNA was treated twice with 1 U / ⁇ g of RNaseR (Epicentre) (37 ° C., 15 minutes), and then purified with the RNeasy micro Kit.
  • cDNA was synthesized using ReverTra ACE qPCR-RT Master Mix Kit (TOYOBO).
  • qPCR Real-time quantitative PCR Quantitative PCR
  • PCR amplification of AI RNA editing site and its analysis PCR was performed under the conditions shown in Table 2 using the primer pairs shown in Table 1.
  • PCR products were purified using MiniElute PCR purification Kit (QIAGEN) or MiniElute Gel Extraction Kit (QIAGEN).
  • AI in GluA2 mRNA and circ GRIA2 (hsa_circ_0125620) Q / R site, CYFIP2 (cytoplasmic fragile X mental retardation interacting protein 2) mRNA lysine / glutamate (K / E) site and TMEM63B (transmembrane protein 63B) mRNA Q / R site
  • the degree of editing was calculated by treating the PCR product with a restriction enzyme, and determining the cleavage with the editing-dependent restriction enzyme by quantitative analysis using Bioanalyzer 2100 (Agilent Technology).
  • GluA2 mRNA, CYFIP2 mRNA, TMEM63B mRNA and circ GRIA2 derived PCR products were cut with Bbvl , Msel , HpaII and HpyCH4V (New England Biolabs), respectively.
  • the editing rate was shown by the molar ratio of the band of the edited RNA to the sum of the bands derived from the edited RNA (edited RNA) and the unedited RNA (unedited RNA) (Table 5).
  • the PCR products are sequenced with a 3100 Genetic Analyzer sequencer (Applied Biosystems), and then, each of the AI RNA editing sites The editing rate was calculated by using the ab1 Peak Reporter tool (https://apps.thermofisher.com/ab1peakreporter) (Thermo Fisher Scientifics) as the signal intensity ratio of the guanosine peak to the sum of the adenosine peak and the guanosine peak. .
  • ADAR2 was overexpressed in cultured cells, and changes in the editing rate at the 28 sites described above were examined.
  • ADAR2 was overexpressed in HeLa cells (FIG. 2A) and SH-SY5Y cells (FIG.
  • ADAR2 of SH-SY5Y cells was knocked down with siRNA, and the change in editing rate at these 10 sites was examined.
  • the expression level of ADAR2 mRNA in SH-SY5Y cells transfected with siADAR2-1 or siADAR2-2 was significantly reduced (FIG. 3C).
  • the editing rate was reduced at all 10 sites, but the site where the degree of the reduction reached a statistically significant level was 4 sites, that is, K / E of CYFIP2 mRNA.
  • Site, Q / R site of GluA2 mRNA, R / G site of GluA2 mRNA, and R / G site of SON mRNA (FIG. 3D).
  • RNA with ADAR2-dependent AI RNA editing site present in culture supernatant Next, we examined whether mRNAs containing 10 ADAR2-dependent AI RNA editing sites (ADAR2, CYFIP2, GluA2, SON and TMEM63B mRNAs) were secreted from the cells. The culture supernatant of SH-SY5Y cells The presence of all five types of mRNA was confirmed. Therefore, the editing rate of the ADAR2-dependent AI RNA editing site of mRNA secreted into the culture supernatant of SH-SY5Y cells was examined.
  • ADAR2-dependent AI RNA editing site of mRNA secreted into the culture supernatant of SH-SY5Y cells was examined.
  • circ GRIA2 (hsa_circ_0125620) in SH-SY5Y cells and the culture supernatant thereof was transferred to circBase (Glazar et al., RNA 20, 1666-1670 2014).
  • circ GRIA2 (hsa_circ_0125620, hsa_circ_0125618, and hsa_circ_0125619), which is a transcript of GRIA2 containing a Q / R site, was found.
  • RNA 19, 141- 157 2013 the amount of RNaseR-resistant RNA is about 1% of the total RNA extracted before RNaseR treatment, and the ratio has been reported so far (Jeck et al., RNA 19, 141- 157 2013). .
  • circ GRIA2 hsa_circ_0125620
  • hsa_circ_0125620 was detected reproducibly in SH-SY5Y cells (FIG. 5A), but hsa_circ_0125618. And hsa_circ_0125619 could not be detected.
  • the Q / R site has two HpyCH4V recognition sites in the PCR product derived from the edited hsa_circ_0125620, whereas the Q / R site has the unedited hsa_circ_0125620 in the PCR product derived from the hsa_circ_0125620. There is one more (three in total) recognition sites (FIG. 5B).
  • the editing rate (95% on average) at the Q / R site of hsa_circ_0125620 is higher than the editing rate of GluA2 mRNA (linear mRNA) (87% on average), which is significant due to ADAR2 knockdown (Fig. 5C)
  • hsa_circ_0125620 in the culture supernatant of SH-SY5Y cells was examined.
  • a culture supernatant of 160 ml or more was required.
  • Most of the Q / R site of hsa_circ_012562 in the culture supernatant of SH-SY5Y cells was in an edited state even after ADAR2 knockdown.
  • a close look at the chromatography revealed a small but 172 bp fragment in the knockdown.

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Abstract

Provided are a biomarker for ALS diagnosis and an ALS diagnosis method using said biomarker. Specifically, the present invention is a method for diagnosing ALS pathology, includes a step in which the type of nucleotide at an ADAR2-dependent A–I RNA editing site present in a diagnostic sample is identified, and, more specifically, is characterized by including steps (a)–(d). (a) A step in which a nucleotide at an ADAR2-dependent A–I RNA editing site present in the diagnostic sample is identified; (b) a step in which the ratio at which the nucleotide identified in step (a) is adenine (A) or inosine (I) is calculated; and (c) a step in which the A or I ratio calculated in step (b) and the ratio at which the nucleotide at the ADAR2-dependent A–I RNA editing site in a healthy person is A or I are each compared.

Description

ＡＬＳのバイオマーカーおよびＡＬＳの診断方法Biomarkers for ALS and methods for diagnosing ALS
　本発明は、ALSの診断用バイオマーカーおよび該バイオマーカーを用いたALSの診断方法に関する。 The present invention relates to a biomarker for diagnosing ALS and a method for diagnosing ALS using the biomarker.
　筋萎縮性側索硬化症（ALS）は、上位および下位運動ニューロンの変性により細胞死を起こす成人発症の運動ニューロン疾患である。ALSを発症すると、運動ニューロンの喪失に起因する進行性の筋力低下や筋萎縮が生じ、大多数の患者は数年内に呼吸筋麻痺により死に至る。
　ALSでは神経伝達に関わるグルタミン受容体の１つであるAMPA（α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid）受容体の異常が運動ニューロン死の原因であることが突き止められている。
　具体的には、ALS患者の運動ニューロンでは、AMPA受容体のカルシウム透過性を規定するサブユニットであるGluA2に本来生ずべきRNA編集（DNAがRNAに転写された後、RNA塩基に変化が生じる現象のこと。ALSに関わるRNA編集は、アデノシンがイノシンへ変換される脱アミノ基反応（以下「A-I RNA編集」）である。）が起こらず未編集型GluA2が発現するため、カルシウム透過性が異常に高いAMPA受容体が発現していること、そして、GluA2が未編集型となるのは、RNA編集酵素であるadenosine deaminase acting on RNA 2（ADAR2）酵素の発現低下（ダウンレギュレーション）によることなどが明らかにされた（非特許文献１、非特許文献２および非特許文献３）。
　未編集型GluA2には、イオンチャネルポアの内腔に面する部位（Q/R部位）にグルタミン（Q）が存在するが、編集型GluA2では、A-I RNA編集によってアルギニン（R）に変換される。編集型GluA2のQ/R部位は、陽性電荷のRのため、カルシウムイオンの流入を妨げるのに対し、未編集型GluA2ではこの部位が中性電荷のQのため、カルシウム透過性を示す。 Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease that causes cell death by degeneration of upper and lower motor neurons. The onset of ALS results in progressive weakness and atrophy due to loss of motor neurons, and most patients die from respiratory muscle paralysis within a few years.
In ALS, abnormalities in AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), one of the glutamine receptors involved in neurotransmission, were found to be the cause of motor neuron death. ing.
Specifically, in ALS patients' motor neurons, GluA2, a subunit that regulates the calcium permeability of the AMPA receptor, undergoes RNA editing that occurs naturally (changes in RNA bases after DNA is transcribed into RNA) RNA editing related to ALS is a deamination reaction in which adenosine is converted to inosine (hereinafter referred to as “AI RNA editing”), and unedited GluA2 is expressed. The expression of abnormally high AMPA receptors and the fact that GluA2 is unedited are due to decreased expression (down-regulation) of the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2). (Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3).
In unedited GluA2, glutamine (Q) is present at the site facing the lumen of the ion channel pore (Q / R site), but in edited GluA2, it is converted to arginine (R) by AI RNA editing . The Q / R site of the edited GluA2 prevents calcium ion inflow due to the positively charged R, whereas the unedited GluA2 exhibits calcium permeability due to the neutrally charged Q at the site.
　また、ADAR2のコンディショナルノックアウトマウス（ADAR2flox/flox/VAChT-Cre.Fast；AR2マウス）は、進行性運動障害および骨格筋の脱神経症状を呈するが、これらの症状はQ/R部位未編集型GluA2サブユニットを有するAMPA受容体のカルシウム透過性によって惹起される下位運動ニューロンの消失によるものであることが報告されている（非特許文献４）。
　さらに、孤発性ALS患者の神経病理的特徴である運動ニューロン中のTDP-43（transactive response DNA-binding protein of 43kDa）の細胞内局在異常（TDP-43病理）が、孤発性ALS患者のADAR2がダウンレギュレートされた運動ニューロンにおいて例外なく確認されている（非特許文献５）。
　以上のように、ADAR2のダウンレギュレーションがALS患者の運動ニューロン死と密接に関連していることが明らかにされた。 In addition, ADAR2 conditional knockout mice (ADAR2 flox / flox / VAChT-Cre.Fast; AR2 mice) exhibit progressive motor dysfunction and skeletal muscle denervation, but these symptoms are unedited at the Q / R site. It has been reported that this is due to the disappearance of lower motor neurons caused by calcium permeability of AMPA receptor having type GluA2 subunit (Non-Patent Document 4).
Furthermore, the abnormal intracellular localization of TDP-43 (transactive response DNA-binding protein of 43 kDa) in motor neurons, which is a neuropathological feature of sporadic ALS patients, is associated with sporadic ALS patients. ADAR2 has been confirmed without exception in down-regulated motoneurons (Non-Patent Document 5).
As described above, it was revealed that down-regulation of ADAR2 is closely related to motor neuron death in ALS patients.
　ALSでは、明らかな筋力低下または筋萎縮の症状が出た時点で、約30%以上の前角ニューロンがすでに変性しているとの報告がある（非特許文献６）。このような多くの運動ニューロンが消失する前に効果的な治療を可能にするためには、早期にALSを診断するためのバイオマーカーの開発が必要である。上述のように、ADAR2のダウンレギュレーションがALSの発症と密接に関連していることから、ADAR2活性を採取可能な体液中で調べることができれば早期にALS発症の有無を診断することが可能になる。しかしながら、現在のところ、このような診断を可能にする信頼性の高いバイオマーカーは存在しない。 In ALS, it is reported that about 30% or more of the anterior horn neurons have already been degenerated at the time when obvious muscle weakness or muscle atrophy symptoms appear (Non-Patent Document 6). Development of biomarkers for early diagnosis of ALS is needed to enable effective treatment before such many motor neurons disappear. As described above, down-regulation of ADAR2 is closely related to the development of ALS, so if ADAR2 activity can be examined in a bodily fluid that can be collected, it will be possible to diagnose the presence or absence of ALS early. . However, at present, there are no reliable biomarkers that allow such diagnosis.
　上記事情に鑑み、本発明は、ALSの診断または治療効果の判定に有用なバイオマーカーの提供、並びに、該バイオマーカーを用いたALSの診断方法および治療効果の判定方法の提供を解決課題とする。 In view of the above circumstances, an object of the present invention is to provide a biomarker useful for diagnosing or determining a therapeutic effect of ALS, and to provide a method for diagnosing ALS and a method for determining a therapeutic effect using the biomarker. .
　本発明者らは、ALS発症の原因であるADAR2ダウンレギュレーションの指標として、ADAR2依存性A-I RNA編集に着目し、このA-I RNA編集部位を有するRNAを検索したところ、ADAR2活性依存的なA-I RNA編集部位を直鎖状RNA中に10カ所、環状RNA中に1カ所見出した。さらに、これらのADAR2依存性A-I RNA編集部位の編集率の変動は、細胞内のRNAと細胞外のRNAで同等であることも併せて見出した。
　以上のように、本発明者らは、ADAR2活性依存的にその編集率が変動するA-I RNA編集部位を有する細胞外（すなわち、体液中）のRNAがALSのバイオマーカーとして利用できることを初めて見出し本発明を完成させた。 The present inventors focused on ADAR2-dependent AI RNA editing as an index of ADAR2 down-regulation that causes ALS development, and searched for RNA having this AI RNA editing site. Ten sites were found in linear RNA and one in circular RNA. Furthermore, they found that the variation in the editing rate of these ADAR2-dependent AI RNA editing sites was equivalent between intracellular RNA and extracellular RNA.
As described above, the present inventors have found for the first time that extracellular (ie, in body fluid) RNA having an AI RNA editing site whose editing rate fluctuates in an ADAR2 activity-dependent manner can be used as a biomarker for ALS. Completed the invention.
　すなわち、本発明は、以下の（１）～（１０）である。
（１）ALSの病態を診断する方法であって、診断サンプル中に存在するADAR2依存性A-I RNA編集部位のヌクレオチドを同定する工程を含む、前記方法。
（２）ALSの病態を診断する方法であって、以下の（ａ）～（ｄ）の工程を含むことを特徴とする上記（１）に記載の方法。
（ａ）診断サンプル中に存在するRNAのADAR2依存性A-I RNA編集部位のヌクレオチドを同定する工程、
（ｂ）工程（ａ）で同定したヌクレオチドがアデニン（A）またはイノシン（I）である割合を算出する工程、および
（ｃ）工程（ｂ）で算出したAまたはIの割合と、健常者のADAR2依存性A-I RNA編集部位のヌクレオチドがAまたはIである割合とを、各々、比較する工程
（３）前記ADAR2依存性A-I RNA編集部位が、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第178番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第191番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第192番目の位置、配列番号２で表されるCYFIP2 mRNAの核酸配列中第171番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第28番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第498番目の位置、配列番号４で表されるSON mRNAの核酸配列中第202番目の位置、配列番号４で表されるSON mRNAの核酸配列中第672番目の位置、配列番号４で表されるSON mRNAの核酸配列中第720番目の位置、配列番号５で表されるTMEM63B mRNAの核酸配列中第157番目の位置および／または配列番号６で表されるcircGRIA2（has_circ_0125620）の核酸配列中第409番目であることを特徴とする上記（１）または（２）に記載の方法。
（４）前記診断サンプルが体液由来であることを特徴とする上記（１）ないし（３）のいずれかに記載の方法。
（５）前記体液が、髄液または血液のいずれかであることを特徴とする上記（４）に記載の方法。
（６）ALSの病態を診断するためのバイオマーカーであって、ADAR2依存性A-I RNA編集部位を有する１または複数のRNA分子からなるバイオマーカー。
（７）前記ADAR2依存性A-I RNA編集部位が、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第178番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第191番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第192番目の位置、配列番号２で表されるCYFIP2 mRNAの核酸配列中第171番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第28番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第498番目の位置、配列番号４で表されるSON mRNAの核酸配列中第202番目の位置、配列番号４で表されるSON mRNAの核酸配列中第672番目の位置、配列番号４で表されるSON mRNAの核酸配列中第720番目の位置、配列番号５で表されるTMEM63B mRNAの核酸配列中第157番目の位置および／または配列番号６で表されるcircGRIA2（has_circ_0125620）の核酸配列中第409番目であることを特徴とする上記（６）に記載のバイオマーカー。
（８）ADAR2依存性A-I RNA編集部位を有する１または複数のRNA分子のALSバイオマーカーとしての使用。
（９）前記ADAR2依存性A-I RNA編集部位が、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第178番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第191番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第192番目の位置、配列番号２で表されるCYFIP2 mRNAの核酸配列中第171番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第28番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第498番目の位置、配列番号４で表されるSON mRNAの核酸配列中第202番目の位置、配列番号４で表されるSON mRNAの核酸配列中第672番目の位置、配列番号４で表されるSON mRNAの核酸配列中第720番目の位置、配列番号５で表されるTMEM63B mRNAの核酸配列中第157番目の位置および／または配列番号６で表されるcircGRIA2（has_circ_0125620）の核酸配列中第409番目であることを特徴とする上記（８）に記載の使用。
（１０）ALSの病態を診断するために使用するキットであって、少なくともADAR2依存性A-I RNA編集部位のヌクレオチドを同定するための要素を含むキット。 That is, the present invention provides the following (1) to (10).
(1) A method for diagnosing the condition of ALS, comprising the step of identifying a nucleotide at an ADAR2-dependent AI RNA editing site present in a diagnostic sample.
(2) The method according to the above (1), which is a method for diagnosing a pathological condition of ALS, comprising the following steps (a) to (d).
(A) identifying the nucleotides at the ADAR2-dependent AI RNA editing site of the RNA present in the diagnostic sample,
(B) calculating the ratio of the nucleotide identified in step (a) to adenine (A) or inosine (I); and (c) calculating the ratio of A or I calculated in step (b) Comparing the ratio of the nucleotide of the ADAR2-dependent AI RNA editing site to A or I, respectively (3) the ADAR2-dependent AI RNA editing site is the nucleic acid of the ADAR2 pre-mRNA represented by SEQ ID NO: 1 Position 178 in the sequence, position 191 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, position 192 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, Position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, position 28 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3, nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3 Position 498 in the middle, position 202 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, SEQ ID NO: At position 672 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, position 720 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, and position 157 in the nucleic acid sequence of the TMEM63B mRNA represented by SEQ ID NO: 5 The method according to the above (1) or (2), which is the 409th position in the nucleic acid sequence of the circ GRIA2 (has_circ_0125620) represented by the third position and / or SEQ ID NO: 6.
(4) The method according to any one of the above (1) to (3), wherein the diagnostic sample is derived from a body fluid.
(5) The method according to (4), wherein the bodily fluid is either cerebrospinal fluid or blood.
(6) A biomarker for diagnosing the pathology of ALS, which comprises one or more RNA molecules having an ADAR2-dependent AI RNA editing site.
(7) The ADAR2-dependent AI RNA editing site is located at position 178 in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1, and in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1. Position 191, position 192 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, represented by SEQ ID NO: 3 Position 28 in the nucleic acid sequence of GluA2 mRNA, position 498 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3, and position 202 in the nucleic acid sequence of SON mRNA represented by SEQ ID NO: 4. Position, position 672 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, position 720 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, and position of the TMEM63B mRNA represented by SEQ ID NO: 5 # 409 th der nucleic acid sequence of circ GRIA2 (has_circ_0125620) represented by the 157th position and / or SEQ ID NO: 6 in a nucleic acid sequence Biomarkers described above (6), wherein the.
(8) Use of one or more RNA molecules having an ADAR2-dependent AI RNA editing site as an ALS biomarker.
(9) The ADAR2-dependent AI RNA editing site is located at position 178 in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1, and in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1. Position 191, position 192 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, represented by SEQ ID NO: 3 Position 28 in the nucleic acid sequence of GluA2 mRNA, position 498 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3, and position 202 in the nucleic acid sequence of SON mRNA represented by SEQ ID NO: 4. Position, position 672 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, position 720 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, and position of the TMEM63B mRNA represented by SEQ ID NO: 5 # 409 th der nucleic acid sequence of circ GRIA2 (has_circ_0125620) represented by the 157th position and / or SEQ ID NO: 6 in a nucleic acid sequence Use according to the above (8), characterized in that.
(10) A kit used for diagnosing the pathology of ALS, the kit comprising at least an element for identifying a nucleotide at an ADAR2-dependent AI RNA editing site.
　本発明にかかるバイオマーカーを使用することで、ALSの早期診断および治療効果の判定が可能となる。 早期 By using the biomarker according to the present invention, early diagnosis of ALS and determination of the therapeutic effect can be performed.
circGRIA2（has_circ_0125620）のアミノ酸配列およびQ/R部位（未編集状態）を示す。Circular junction: バックスプライシングによる結合位置circ shows the amino acid sequence of GRIA2 (has_circ_0125620) and the Q / R site (unedited). Circular junction: bonding position by back splicing HeLa細胞で発現するRNAのADAR2依存性A-I RNA編集部位。（A）ADAR2過剰発現によるADAR2 mRNAの発現量の変化。mock：pCl（空のベクター）をトランスフェクションした細胞。OE：ADAR2/pClをトランスフェクションした細胞。βアクチン mRNAの発現量に対して標準化したADAR2 mRNAの発現レベルを示す（Mann-Whitney U-test;***P<0.001）。データは、平均値±SEMで示す（n=6）。（B）マウス脊髄運動ニューロンのA-I RNA編集部位と相同部位をもつヒトのA-I RNA編集部位における、ADAR2過剰発現後の編集率の変化。pCl（白）またはADAR2/pClをトランスフェクションした細胞（黒）中での編集率を、平均値±SEM（n=6、Mann-Whitney U-test;*P<0.05、**P<0.01）で示す。CYFIP2、cytoplasmic fragile X mental retardation protein interacting protein 2； OPTN、optineurin；SON、SON；deoxyribonucleic acid binding protein；TMEM63B、transmembrane protein 63B；+10ADAR2-dependent AI RNA editing site of RNA expressed in HeLa cells. (A) Changes in the expression level of ADAR2 mRNA due to ADAR2 overexpression. mock: cells transfected with pCl (empty vector). OE: cells transfected with ADAR2 / pCl. The expression level of ADAR2 mRNA normalized to the expression amount of β-actin mRNA is shown (Mann-Whitney U-test; *** P <0.001). Data are shown as mean ± SEM (n = 6). (B) Changes in the editing rate after ADAR2 overexpression in a human AI RNA editing site having a homologous site to the mouse spinal motor neuron AI RNA editing site. The edit rate in cells transfected with pCl (white) or ADAR2 / pCl (black) was expressed as mean ± SEM (n = 6, Mann-Whitney U-test; * P <0.05, ** P <0.01) Indicated by CYFIP2, cytoplasmic fragile X mental retardation protein interacting protein 2; OPTN, optineurin; SON, SON; deoxyribonucleic acid binding protein; TMEM63B, transmembrane protein 63B; +10 SH-SY5Y細胞で発現するADAR2依存性A-I RNA編集部位。ADAR2 mRNAの発現量はβ-アクチン mRNAの発現に対して標準化し、データは平均値±SEM（n=6）で示す。（AおよびB）ADAR2過剰発現によるADAR2 mRNAの発現量の変化および12カ所のマウスと相同部位を持つ編集部位における編集率の変化。Mock：pCl（空のベクター）をトランスフェクションした細胞、OE：ADAR2/pClをトランスフェクションした細胞。Mockをトランスフェクションした細胞（白）およびADAR2/pClをトランスフェクションした細胞（黒）の編集率を示す（Mann-Whitney U-test;*P<0.05、**P<0.01、***P<0.001）。（CおよびD）ADAR2ノックダウン後のADAR2 mRNAの発現レベルの変化および10カ所のADAR2依存性A-I RNA編集部位における編集率の変化。Scr：scramble siRNAをトランスフェクションした細胞、KD-1： siADAR2-1をトランスフェクションした細胞、KD-2：siADAR2-2をトランスフェクションした細胞。scramble siRNA（白）、siADAR2-1（黒）またはsiADAR2-2（斜線）をトランスフェクションした細胞の編集率を示す（Mann-Whitney U-test;*P<0.05、**P<0.01、***P<0.001）。ADAR2-dependent AI RNA editing site expressed in SH-SY5Y cells. The expression level of ADAR2 mRNA was normalized to the expression of β-actin mRNA, and the data are shown as mean ± SEM (n = 6). (A and B) Changes in the expression level of ADAR2 mRNA due to ADAR2 overexpression and changes in editing rate at 12 editing sites having homologous sites with mice. Mock: cells transfected with pCl (empty vector), OE: cells transfected with ADAR2 / pCl. Shows the edit rate of Mock transfected cells (white) and ADAR2 / pCl transfected cells (black) (Mann-Whitney U-test; * P <0.05, ** P <0.01, *** P < 0.001). (C and D) Changes in ADAR2 mRNA expression levels after ADAR2 knockdown and changes in editing rates at 10 ADAR2-dependent AI RNA editing sites. Scr: cells transfected with scramble siRNA, KD-1: cells transfected with siADAR2-1, KD-2: cells transfected with siADAR2-2. Shows the edit rate of cells transfected with scramble siRNA (white), siADAR2-1 (black) or siADAR2-2 (hatched) (Mann-Whitney U-test; * P <0.05, ** P <0.01, ** * P <0.001). SH-SY5Y細胞の培養上清中のADAR2依存性A-I RNA編集部位。各実験値は、平均値±SEMで示す（n=6）。（A）Mockをトランスフェクションした細胞の培養上清（白）およびADAR2/pClをトランスフェクションした細胞の培養上清（ドット）中の10カ所のADAR2依存性A-I RNA編集の編集率を示す（Mann-Whitney U-test;*P<0.05、**P<0.01）。（B）scramble siRNAをトランスフェクションした細胞の培養上清（白）、siADAR2-1をトランスフェクションした細胞の培養上清（ドット）およびsiADAR2-2をトランスフェクションした細胞の培養上清（斜線）中の5カ所のADAR2依存性A-I RNA編集部位の編集率を示す（Mann-Whitney U-test;*P<0.05）。ADAR2-dependent AI RNA editing site in culture supernatant of SH-SY5Y cells. Each experimental value is shown as mean ± SEM (n = 6). (A) shows the editing rate of 10 ADAR2-dependent AI RNA editing in the culture supernatant of Mock-transfected cells (white) and the culture supernatant of ADAR2 / pCl-transfected cells (dot) (Mann -Whitney U-test; * P <0.05, ** P <0.01). (B) In culture supernatant of cells transfected with scramble siRNA (white), culture supernatant of cells transfected with siADAR2-1 (dot), and culture supernatant of cells transfected with siADAR2-2 (hatched). 5 shows the editing rates of the five ADAR2-dependent AI RNA editing sites (Mann-Whitney U-test; * P <0.05). SH-SY5Y細胞およびその培養上清中のcircGRIA2（hsa_circ_0125620）。（A）circGRIA2（hsa_circ_0125620）の電気泳動およびサンガーシークエンス。SH-SY5Y細胞中のcircGRIA2のPCR産物（矢印）。N：ネガティブコントロール。エクソン13からエクソン11に繋がるバックスプライシングの連結部を示す。丸く囲み矢印で示す箇所がcircGRIA2のQ/R部位で、ADAR2ノックダウンにより編集率が減少した（G（シークエンサーによる配列決定ではcDNAの配列を決定するためイノシン（I）はグアニン（G）として同定される）のピークが減少し、Aのピークが増加）。（B）circGRIA2のQ/R部位における編集率の検出方法の概念図と2100 Bioanalyzerを用いた編集率の測定例。編集型のQ/R部位を持つcircGRIA2のPCR産物をHpyCH4Vで切断して得られる断片は、76bp、160bpおよび196bpである。これに対し、未編集型のQ/R部位を持つcircGRIA2のPCR産物をHpyCH4Vで切断して得られる断片は、24bp、76bp、160bpおよび172bpである。なお、24bpの断片は、低分子マーカー（LMM）のピークと重なるため検出されない。HMM：高分子マーカー。（C）ADAR2の発現量およびcircGRIA2とGluA2 mRNAのQ/R部位における編集率。Scr：scramble siRNAをトランスフェクションした細胞、KD-1：siADAR2-1をトランスフェクションした細胞、KD-2：siADAR2-2をトランスフェクションした細胞。各実験値は、平均値±SEMで示す（n=6）。scramble siRNA（白）、siADAR2-1（ドット）またはsiADAR2-2（斜線）をトランスフェクションした細胞中の編集率を示す（Mann-Whitney U-test;***P<0.001）。 Circ GRIA2 (hsa_circ_0125620) in SH-SY5Y cells and their culture supernatant. (A) Electrophoresis of circ GRIA2 (hsa_circ_0125620) and Sanger sequencing. PCR product of circ GRIA2 in SH-SY5Y cells (arrow). N: Negative control. The junction of back splicing from exon 13 to exon 11 is shown. The circled arrow indicates the Q / R site of circ GRIA2 , and ADAR2 knockdown reduced the editing rate (G (sequencing by a sequencer determines inosin (I) as guanine (G) because the cDNA sequence is determined). Identified) and the peak of A increases). (B) A conceptual diagram of a method for detecting the editing ratio at the Q / R site of circ GRIA2 and an example of measuring the editing ratio using the 2100 Bioanalyzer. Fragment obtained PCR product circ GRIA2 with edit type Q / R site was cut with HpyCH4V is, 76 bp, a 160bp and 196 bp. In contrast, fragment obtained by digesting the PCR product circ GRIA2 with unedited form of Q / R site in HpyCH4V is, 24 bp, 76 bp, a 160bp and 172 bp. The 24 bp fragment is not detected because it overlaps the peak of the low molecular marker (LMM). HMM: High molecular marker. (C) Expression level of ADAR2 and circ GRIA2 and GluA2 mRNA editing rates at Q / R sites. Scr: cells transfected with scramble siRNA, KD-1: cells transfected with siADAR2-1, KD-2: cells transfected with siADAR2-2. Each experimental value is shown as mean ± SEM (n = 6). Shows the editing rate in cells transfected with scramble siRNA (white), siADAR2-1 (dot) or siADAR2-2 (hatched) (Mann-Whitney U-test; *** P <0.001). 培養上清中のcircGRIA2由来のPCR産物の解析結果。HpyCH4Vで処理したPCR産物を2100 Bioanalyzerで解析した結果を示す。挿入図は、クロマトグラフィー中の丸く囲んだ部分を拡大したものである。Analysis results of PCR product derived from circ GRIA2 in culture supernatant. The results of analyzing the PCR product treated with HpyCH4V with 2100 Bioanalyzer are shown. The inset is an enlargement of the circled part in the chromatography.
　本発明の第１の実施形態は、ALSの病態を診断する方法であって、診断サンプル中に存在するADAR2依存性A-I RNA編集部位のヌクレオチドを同定する工程を含む、前記方法である。
　本発明の第１の実施形態のALSの病態の診断方法（以下「本発明のALS診断方法」とも記載する）は、診断対象者がALSを発症しているかどうかを判断するために使用できることはもちろんのこと、すでにALSを発症した患者の治療経過を判断するためにも使用することができる。従って、本発明の第１の実施形態は、「ALSの病態の診断を補助する方法」でもある。また、本実施形態における「ALS」には、運動ニューロンのADAR2ダウンレギュレーションが原因でALSの病態を発症するものであれば、孤発性ALSのみならず家族性ALSも含まれる。
　本発明のALS診断方法は、より具体的には、ALSの病態を診断する方法であって、以下の（ａ）～（ｄ）の工程を含むことを特徴とする診断方法である。
（ａ）診断サンプル中に存在するRNAのADAR2依存性A-I RNA編集部位のヌクレオチドを同定する工程、
（ｂ）工程（ａ）で同定したヌクレオチドがアデニン（A）またはイノシン（I）である割合を算出する工程、および
（ｃ）工程（ｂ）で算出したAまたはIの割合と、健常者のADAR2依存性A-I RNA編集部位のヌクレオチドがAまたはIである割合とを、各々、比較する工程 A first embodiment of the present invention is a method for diagnosing the pathology of ALS, comprising the step of identifying the nucleotide of an ADAR2-dependent AI RNA editing site present in a diagnostic sample.
The method for diagnosing the condition of ALS according to the first embodiment of the present invention (hereinafter, also referred to as “the ALS diagnostic method of the present invention”) can be used to determine whether a diagnosis target has developed ALS. Of course, it can also be used to determine the course of treatment for patients who have already developed ALS. Therefore, the first embodiment of the present invention is also a “method of assisting diagnosis of a pathological condition of ALS”. The “ALS” in the present embodiment includes not only sporadic ALS but also familial ALS as long as the ALS2 pathology is caused by ADAR2 down-regulation of motor neurons.
More specifically, the ALS diagnostic method of the present invention is a method for diagnosing the condition of ALS, and comprises the following steps (a) to (d).
(A) identifying the nucleotides at the ADAR2-dependent AI RNA editing site of the RNA present in the diagnostic sample,
(B) calculating the ratio of the nucleotide identified in step (a) to adenine (A) or inosine (I); and (c) calculating the ratio of A or I calculated in step (b) Comparing the ratio of nucleotides of the ADAR2-dependent AI RNA editing site to A or I, respectively.
　工程（ａ）において、診断サンプルとは、ALSの病態診断を行う対象者（ALSを発症している者またはALSの発症が疑われる者、以下「被験者」とも記載する）に由来する生体サンプルのことで、例えば、血液、脳脊髄液などの体液のことである。診断サンプル中のRNAの抽出は当業者において周知の方法によって実施することが可能で、市販のRNA抽出キットなどを使用することで容易に行うことができる。
　また、本明細書中の「ADAR2依存性A-I RNA編集」とは、運動ニューロン中において、転写後のRNA中のアデノシン（A）がADAR2酵素活性によりイノシン（I）に変換されることを意味し、「ADAR2依存性A-I RNA編集」とは、ADAR2酵素活性によりIに変換されるAの位置のことをいう。 In the step (a), the diagnostic sample refers to a biological sample derived from a subject (a person who has developed ALS or a person who is suspected of developing ALS, hereinafter also referred to as a “subject”) for performing a pathological diagnosis of ALS. For example, it refers to body fluids such as blood and cerebrospinal fluid. Extraction of RNA from a diagnostic sample can be performed by a method well-known to those skilled in the art, and can be easily performed using a commercially available RNA extraction kit or the like.
The term “ADAR2-dependent AI RNA editing” as used herein means that adenosine (A) in RNA after transcription is converted to inosine (I) by ADAR2 enzyme activity in motor neurons. The term “ADAR2-dependent AI RNA editing” refers to the position of A that is converted to I by ADAR2 enzyme activity.
　健常者（本明細書中において「健常」とはALSを発症していない状態を意味する）の運動ニューロン中ではADAR2が正常に機能しているため、健常者のADAR2依存性A-I RNA編集部位のヌクレオチドは、通常、I（シークエンサーによる配列決定ではcDNAの配列を決定するためグアニン（G）として同定される）であると予想される。これに対しALS患者のADAR2依存性A-I RNA編集部位のヌクレオチドはAである場合が生じる（ADAR2がダウンレギュレーションされているため）。なお、ALS患者の運動ニューロンにおいてADAR1は正常に機能していることが確認されている（非特許文献３）。 ADAR2 functions normally in motoneurons of healthy individuals ("healthy" in the present specification means a state in which ALS has not developed), so that ADAR2-dependent AI RNA editing sites of healthy individuals The nucleotide is usually expected to be I (sequencing by a sequencer is identified as guanine (G) to sequence the cDNA). In contrast, the nucleotide at the ADAR2-dependent AI-RNA editing site in ALS patients may be A (since ADAR2 is down-regulated). It has been confirmed that ADAR1 functions normally in motor neurons of ALS patients (Non-Patent Document 3).
　そこで、ADAR2依存性A-I RNA編集部位は次のようにして検索することができる。
 健常者由来の運動ニューロンのRNA配列とALS患者由来の運動ニューロンのRNA配列を比較し、健常者由来のRNAに比べ、ALS患者由来のRNAでは編集率が低下している部位をADAR2依存性A-I RNA編集部位とすることができる。あるいは、健常な非ヒト動物由来の運動ニューロンのRNA配列とADAR2をノックダウンしたALSモデル非ヒト動物（例えば、AR2マウスなど）由来の運動ニューロンのRNA配列を比較し、健常な非ヒト動物由来のRNAに比べALSモデル非ヒト動物由来のRNAで編集率が低下している部位を検出し、ヒトにおいて相同部位が認められたらADAR2依存性A-I RNA編集部位としてもよい。 Thus, the ADAR2-dependent AI RNA editing site can be searched for as follows.
Compare the RNA sequence of motoneurons from healthy subjects and the motoneurons from ALS patients. It can be an RNA editing site. Alternatively, the RNA sequence of a motor neuron derived from a healthy non-human animal is compared with the RNA sequence of a motor neuron derived from an ALS-model non-human animal (for example, AR2 mouse) knocked down by ADAR2. A site where the editing rate is reduced in RNA derived from an ALS model non-human animal as compared to RNA is detected, and if a homologous site is detected in human, it may be used as an ADAR2-dependent AI RNA editing site.
　また、他の方法として、次のような検索方法も例示できる。
　健常なヒトまたは非ヒト動物の運動ニューロンから抽出したRNAの配列を決定し、得られた配列情報と当該動物のゲノム配列情報（データバンク等に登録されているゲノム配列情報）とを比較し、ゲノム配列情報ではAであるのに対し運動ニューロン由来のRNA上ではGと検出された位置をADAR2依存性A-I RNA編集集部位の候補部位とする。ADAR2依存性A-I RNA編集部位の候補部位を非ヒト動物において同定した場合は、非ヒト動物の候補部位と相同なヒトゲノム上の位置を、ヒトのADAR2依存性A-I RNA編集部位の候補部位としてもよい。また、ヒト運動ニューロン中のA-I RNA編集部位の検索については、例えば、Picardi et al., Sci Rep. 5:14941 DOI: 10.1038/srep14941 2015などを参照して行ってもよい。
　得られた候補部位には、ADAR1による編集部位も含まれている。候補部位のうち実際にADAR2による編集を受けている部位であるかどうかは、例えば、ALS患者またはADAR2をノックダウンしたALSモデル非ヒト動物に由来する運動ニューロンにおける候補部位のA-I編集率（AがIに変換される効率。例えば、候補部位のヌクレオチドがAであるRNAとIであるRNAの総和に対するIであるRNAの割合）と健常者または健常動物に由来する候補部位のA-I編集率を比較することで確認できる。あるいは、ADAR2を過剰発現させた細胞株および／またはADAR2をノックダウンした細胞株等に由来する候補部位のA-I編集率とコントロールの細胞株（ADAR2の過剰発現またはノックダウンが行われていない細胞）に由来する候補部位のA-I編集率を比較することで確認できる。以上の確認の結果、ADAR2活性の変化によりA-I編集率が明らかに変動している候補部位を「ADAR2依存性A-I RNA編集」とすることができる。 Further, as another method, the following search method can be exemplified.
Determine the sequence of RNA extracted from motor neurons of a healthy human or non-human animal, compare the obtained sequence information with the genome sequence information of the animal (genome sequence information registered in a data bank, etc.), In the genome sequence information, the position detected as G on the RNA derived from motoneurons is A, whereas the position detected as G on the motor neuron-derived RNA is set as a candidate site for the ADAR2-dependent AI RNA editing site. When a candidate site for an ADAR2-dependent AI RNA editing site is identified in a non-human animal, a position on the human genome homologous to the candidate site for the non-human animal may be used as a candidate site for a human ADAR2-dependent AI RNA editing site. . The search for an AI RNA editing site in human motoneurons may be performed by referring to, for example, Picardi et al., Sci Rep. 5: 14941 DOI: 10.1038 / srep14941 2015.
The obtained candidate site also includes an ADAR1 edited site. Whether or not a candidate site is actually a site that has been edited by ADAR2 can be determined, for example, by the AI editing rate of the candidate site in motoneurons derived from ALS patients or ALS model non-human animals in which ADAR2 has been knocked down (A Efficiency of conversion to I. For example, the ratio of the RNA at the candidate site to the total of the RNA at the site A and the RNA at the site I) is compared with the AI editing rate of the candidate site derived from a healthy person or animal. Can be confirmed. Alternatively, the AI editing rate of a candidate site derived from a cell line in which ADAR2 is overexpressed and / or a cell line in which ADAR2 is knocked down, and a control cell line (cells in which ADAR2 is not overexpressed or knocked down) Can be confirmed by comparing the AI editing rates of the candidate sites derived from. As a result of the above confirmation, a candidate site where the AI editing rate is obviously changed due to a change in ADAR2 activity can be determined as “ADAR2-dependent AI RNA editing”.
　工程（ａ）において、ADAR2依存性A-I RNA編集部位のヌクレオチドの種類は、当該A-I RNA編集部位を含むRNA領域の配列情報に基づいて検出することができる。
　より具体的には、例えば、ADAR2依存性A-I RNA編集部位が、
配列番号１で表されるADAR2 pre-mRNAの核酸配列中第178番目の位置、
配列番号１で表されるADAR2 pre-mRNAの核酸配列中第191番目の位置、
配列番号１で表されるADAR2 pre-mRNAの核酸配列中第192番目の位置、
配列番号２で表されるCYFIP2 mRNAの核酸配列中第171番目の位置、
配列番号３で表されるGluA2 mRNAの核酸配列中第28番目の位置、
配列番号３で表されるGluA2 mRNAの核酸配列中第498番目の位置、
配列番号４で表されるSON mRNAの核酸配列中第202番目の位置、
配列番号４で表されるSON mRNAの核酸配列中第672番目の位置、
配列番号４で表されるSON mRNAの核酸配列中第720番目の位置、
配列番号５で表されるTMEM63B mRNAの核酸配列中第157番目の位置または、
配列番号６で表されるcircGRIA2（has_circ_0125620）の核酸配列中第409番目である場合には、診断サンプルから抽出したRNAから調製したcDNAを鋳型として、各々、例えば、表１に示すプライマーペアを用いて、表２に示すPCR条件にてこれらのA-I RNA編集部位を含むRNA領域を増幅し、その増幅産物のシークエンスを行うことでA-I RNA編集部位のヌクレオチドが何であるかを確認することができる（なお、配列番号１～６は、各mRNAの配列を示している）。circGRIA2（has_circ_0125620）は環状RNAであるため、図１に未編集の配列とともに、バックスプライシングによる結合位置を示した。
　なお、表１に示すプライマーペアおよび表２に示すPCR条件は１例でありこれらの表に示されるプライマーペアおよびPCR条件に限定されるものではない。 In step (a), the type of nucleotide at the ADAR2-dependent AI RNA editing site can be detected based on the sequence information of the RNA region containing the AI RNA editing site.
More specifically, for example, the ADAR2-dependent AI RNA editing site,
The 178th position in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1,
Position 191 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1,
The 192nd position in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1,
Position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2,
Position 28 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3,
Position 498 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3,
Position 202 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4,
Position 672 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4,
Position 720 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4,
Position 157 in the nucleic acid sequence of TMEM63B mRNA represented by SEQ ID NO: 5, or
In the case of the 409th position in the nucleic acid sequence of circ GRIA2 (has_circ_0125620) represented by SEQ ID NO: 6, a primer pair shown in Table 1 is used as a template, using cDNA prepared from RNA extracted from a diagnostic sample as a template. The RNA region containing the AI RNA editing site was amplified under the PCR conditions shown in Table 2 and the amplified product was sequenced to confirm what the nucleotide of the AI RNA editing site was. (Note that SEQ ID NOs: 1 to 6 show the sequences of the respective mRNAs). Since circ GRIA2 (has_circ_0125620) is a circular RNA, FIG. 1 shows an unedited sequence and a binding position by back splicing.
In addition, the primer pairs shown in Table 1 and the PCR conditions shown in Table 2 are examples, and are not limited to the primer pairs and PCR conditions shown in these tables.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　また、A-I RNA編集の有無によって、当該編集部位付近に存在する制限酵素の認識配列が変わる場合には、A-I RNA編集部位を含むPCR産物を、当該制限酵素で切断することで、その切断の有無から当該A-I RNA編集部位がAであるか、Iであるかを確認することができる。 When the recognition sequence of a restriction enzyme present near the editing site changes depending on the presence or absence of AI RNA editing, the PCR product containing the AI 切断 RNA editing site is cleaved with the restriction enzyme to determine whether the restriction enzyme is cleaved. From this, it can be confirmed whether the AI RNA editing site is A or I.
　工程（ｂ）は、診断サンプル中のA-I RNA編集部位におけるA-I編集率を算出する工程で、配列決定を行った全RNA量に対し、A-I RNA編集部位がAであるRNAまたはIであるRNA量の割合を算出する工程である。
　工程（ｃ）において、健常者のADAR2依存性A-I RNA編集部位のヌクレオチドがAまたはIである割合は、工程（ａ）および工程（ｂ）の「診断サンプル」を健常者由来のサンプル（すなわち、ALSを発症していない者に由来する体液（例えば、血液、脳脊髄液など）などの生体サンプル）に置き換えて、各工程を実施することで算出できる。あるいは、健常者のADAR2依存性A-I RNA編集部位のA-I編集率データがすでに存在する場合には、そのデータと比較してもよい。工程（ｃ）における比較の結果、診断サンプル中のADAR2依存性A-I RNA編集部位のA-I編集率（Iに変換される率）が健常者由来のサンプル中の当該A-I編集率よりも有意に低い場合、被験者はALSを発症しているか、または、発症している可能性があると判断することができる。
　なお、ALSの診断にあたっては、複数のADAR2依存性A-I RNA編集部位のA-I編集率を求めて総合的に判断してもよい。 Step (b) is a step of calculating the AI editing rate at the AI RNA editing site in the diagnostic sample. The amount of RNA where the AI RNA editing site is A or the amount of RNA where the AI RNA editing site is I based on the total RNA amount subjected to sequencing. This is the step of calculating the ratio of.
In the step (c), the ratio of the nucleotide of the ADAR2-dependent AI RNA editing site of the healthy subject to A or I is determined by comparing the “diagnostic sample” in the steps (a) and (b) with a sample derived from a healthy subject ( It can be calculated by substituting a biological sample (eg, blood, cerebrospinal fluid, etc.) derived from a person who does not have ALS, and performing each step. Alternatively, if the AI editing rate data of the ADAR2-dependent AI RNA editing site of a healthy person already exists, the data may be compared with the data. As a result of the comparison in step (c), when the AI editing rate (the rate of conversion to I) of the ADAR2-dependent AI RNA editing site in the diagnostic sample is significantly lower than the AI editing rate in the sample derived from a healthy subject The subject can be determined to have, or may have, developed ALS.
In diagnosing ALS, the AI editing rates of a plurality of ADAR2-dependent AI RNA editing sites may be determined to make a comprehensive judgment.
　本発明の第２の実施形態は、ALSの病態を診断するためのバイオマーカーであって、ADAR2依存性A-I RNA編集部位を有する１または複数のRNA分子からなるバイオマーカーである。
　第２の実施形態におけるバイオマーカー（以下「本発明のバイオマーカー」とも記載する）は、第１の実施形態で説明した「ADAR2依存性A-I RNA編集部位」を有するRNA分子であれば、直鎖状RNA、環状RNAのいずれであってもよいが、体液中において比較的安定に存在する環状RNAがより好ましい。本発明の第２の実施形態は、「ALSの病態の診断を補助するためのバイオマーカー」でもある。
　ADAR2依存性A-I RNA編集部位を有するRNA分子としては、限定はしないが、例えば、配列番号１で表される核酸配列を含むADAR2 pre-mRNA、配列番号２で表される核酸配列を含むCYFIP2 mRNA、配列番号３で表される核酸配列を含むGluA2 mRNA、配列番号４で表される核酸配列を含むSON mRNA、配列番号５で表される核酸配列を含むTMEM63B mRNAおよび配列番号６で表される核酸配列を含む環状のcircGRIA2（has_circ_0125620）を挙げることができる。 The second embodiment of the present invention is a biomarker for diagnosing the condition of ALS, which is a biomarker comprising one or more RNA molecules having an ADAR2-dependent AI RNA editing site.
The biomarker in the second embodiment (hereinafter also referred to as “biomarker of the present invention”) may be a linear molecule if it is an RNA molecule having the “ADAR2-dependent AI RNA editing site” described in the first embodiment. Although it may be any of a round RNA and a circular RNA, a circular RNA that exists relatively stably in a body fluid is more preferable. The second embodiment of the present invention is also a “biomarker for assisting in the diagnosis of a pathological condition of ALS”.
Examples of the RNA molecule having the ADAR2-dependent AI RNA editing site include, but are not limited to, ADAR2 pre-mRNA containing the nucleic acid sequence represented by SEQ ID NO: 1, CYFIP2 mRNA containing the nucleic acid sequence represented by SEQ ID NO: 2 GluA2 mRNA containing the nucleic acid sequence represented by SEQ ID NO: 3, SON mRNA containing the nucleic acid sequence represented by SEQ ID NO: 4, TMEM63B mRNA containing the nucleic acid sequence represented by SEQ ID NO: 5, and represented by SEQ ID NO: 6 A circular circ GRIA2 (has_circ_0125620) containing a nucleic acid sequence can be mentioned.
　本発明の第３の実施形態は、ADAR2依存性A-I RNA編集部位を有する１または複数のRNA分子のALSバイオマーカーとしての使用である。
　本発明の第３の実施形態にかかるALSバイオマーカーとしての使用には、ADAR2依存性A-I RNA編集部位を有する１または複数のRNA分子をALSの診断を行うための使用（またはALSの診断を補助するための使用）、ALSの病態解明のための使用など、ALSに関連する情報を取得するためのあらゆる使用が含まれる。 A third embodiment of the present invention is the use of one or more RNA molecules having an ADAR2-dependent AI RNA editing site as ALS biomarkers.
For use as an ALS biomarker according to the third embodiment of the present invention, one or more RNA molecules having an ADAR2-dependent AI RNA editing site are used for diagnosing ALS (or assist in diagnosing ALS). Use for obtaining information related to ALS, including use for elucidating the pathology of ALS.
　本発明の第４の実施形態は、ALSの病態を診断するために使用するキット（またはALSの病態の診断を補助するために使用するキット）であって、少なくともADAR2依存性A-I RNA編集部位のヌクレオチドを同定するための要素を含むキットである。
　第４の実施形態において、「ADAR2依存性A-I RNA編集のヌクレオチドを同定するための要素」とは、サンプル中に存在するADAR2依存性A-I RNA編集部位のヌクレオチドを同定するために必要なもののことで、限定はしないが、例えば、当該A-I RNA編集部位を含むRNAから調製したcDNAを鋳型として、当該A-I RNA編集部位に対応する部位を含むcDNA領域をPCR増幅するためのプライマーペア、当該A-I RNA編集部位付近に認識配列を持つ制限酵素であってA-I編集によってその認識配列が消失または出現する制限酵素などを挙げることができる。当該キットには、さらに、ADAR2依存性A-I RNA編集部位のヌクレオチドを同定するために使用する試薬や、使用説明書（電磁的記録媒体に保存されている場合を含む）などが含まれていてもよい。 A fourth embodiment of the present invention relates to a kit used for diagnosing the condition of ALS (or a kit used for assisting the diagnosis of the condition of ALS), which comprises at least an ADAR2-dependent AI RNA editing site. It is a kit containing elements for identifying nucleotides.
In the fourth embodiment, “elements for identifying nucleotides of ADAR2-dependent AI RNA editing” refers to those necessary for identifying nucleotides of ADAR2-dependent AI RNA editing sites present in a sample. Although not limited, for example, using a cDNA prepared from RNA containing the AI RNA editing site as a template, a primer pair for PCR-amplifying a cDNA region containing a site corresponding to the AI RNA editing site, the AI RNA editing Restriction enzymes having a recognition sequence near the site, wherein the recognition sequence disappears or appears due to AI editing, and the like. The kit may further include reagents used to identify nucleotides at the ADAR2-dependent AI RNA editing site, and instructions for use (including those stored on an electromagnetic recording medium). Good.
　本明細書が英語に翻訳されて、単数形の「a」、「an」、および「the」の単語が含まれる場合、文脈から明らかにそうでないことが示されていない限り、単数のみならず複数のものも含むものとする。
　以下に実施例を示してさらに本発明の説明を行うが、本実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。 Where the specification is translated into English and includes the words "a", "an", and "the" in the singular, the singular as well as the singular unless the context clearly indicates otherwise. It shall include a plurality.
Hereinafter, the present invention will be further described with reference to examples. However, these examples are merely examples of the embodiments of the present invention, and do not limit the scope of the present invention.
１．材料と実験方法
１－１．動物
　実験に使用したマウスは、ホモ接合性コンディショナルADAR2ノックアウトマウス（ADAR2flox/flox/VAChT-Cre.Fast；AR2マウス）で、VAChT（vesicular acetylcholine transporter）プロモーターで発現制御されるCreのターゲットであるfloxed ADARB1アリルを有する（Hideyama et al., J Neurosci, 30, 11917-11925 2010）。コントロールとしてノックアウトマウスと同系統（C57BL/6J mice；Oriental Yeast Co., Ltd.）、同週齢の野生型マウスを使用した。全ての動物実験は、東京大学動物取扱委員会により承認され、日本国文部科学省の動物実験ガイドラインに従って行った。 1. Materials and experimental method 1-1. Animals The mice used in the experiments were homozygous conditional ADAR2 knockout mice (ADAR2 flox / flox / VAChT-Cre.Fast; AR2 mice), which are Cre targets whose expression is regulated by the VAChT (vesicular acetylcholine transporter) promoter. It has a floxed ADARB1 allele (Hideyama et al., J Neurosci, 30, 11917-11925 2010). As a control, a wild-type mouse of the same age as the knockout mouse (C57BL / 6J mice; Oriental Yeast Co., Ltd.) and the same age were used. All animal experiments were approved by the University of Tokyo Animal Care Committee and were performed in accordance with the Animal Experiment Guidelines of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
１－２．マウス脊髄からの単一運動ニューロンの単離
　7週齢のマウス脊髄由来の凍結切片（20 μm）をcryostat（Model CM1850；Leica Microsystems GmbH）で調製し、スライドグラス（MATSUNAMI）に静置した。静置した凍結切片を100 %メタノールで40秒間固定した後、0.05 %のトルイジンブルーで1分間染色し、70 %のエタノールで4回リンスしたのち、完全に乾燥させた。運動ニューロンは、マイクロダイセクションシステム（Laser Micro Dissector 7000；Leica Microsystems GmbH）を用いて前角から切り出し、RNeasy micro Kit（QIAGEN）に添付のRLTバッファー（200 μl）を入れたチューブに回収した。切り出した単一運動ニューロンの数は、1,912～2,214であった。全てのサンプルは、使用時まで-80℃で保存した。 1-2. Isolation of single motoneurons from mouse spinal cord Frozen sections (20 μm) from 7-week-old mouse spinal cord were prepared with cryostat (Model CM1850; Leica Microsystems GmbH) and placed on a slide glass (MATSUNAMI). The fixed frozen section was fixed with 100% methanol for 40 seconds, stained with 0.05% toluidine blue for 1 minute, rinsed four times with 70% ethanol, and completely dried. Motor neurons were excised from the anterior horn using a microdissection system (Laser Micro Dissector 7000; Leica Microsystems GmbH) and collected in a tube containing RLT buffer (200 μl) attached to the RNeasy micro Kit (QIAGEN). The number of excised single motoneurons ranged from 1,912 to 2,214. All samples were stored at -80 ° C until use.
１－３．RNAの抽出および配列決定
　運動ニューロンサンプルから、RNeasy micro Kitを用いてRNA抽出およびDNaseI処理を行った。リボソームRNAを除くために、抽出した総RNAをRiboGone mammalian kit（Takara Bio）で処理した。RNAシークエンス用のcDNAライブラリーは、SMARTer Stranded RNA-Seq Kit（Takara Bio）を使用して総RNAから合成し、Illumina HiSeqTM2000（Illumina, Inc.）を用いてシークエンスを行った。 1-3. RNA extraction and sequencing From motor neuron samples, RNA extraction and DNaseI treatment were performed using the RNeasy micro Kit. The extracted total RNA was treated with RiboGone mammalian kit (Takara Bio) to remove ribosomal RNA. A cDNA library for RNA sequencing was synthesized from total RNA using the SMARTer Stranded RNA-Seq Kit (Takara Bio) and sequenced using Illumina HiSeq ™ 2000 (Illumina, Inc.).
１－４．ヒト－マウス間で相同なA-I RNA編集部位の検索
　野生型マウス（n=3）およびAR2マウス（n=3）から調製した運動ニューロン由来のRNAの配列決定を行い、野生型マウス、AR2マウスそれぞれ2匹以上で15リード以上あったRNA配列情報をマウスゲノム配列（NCBI37/mm9）にマッピングした。マッピングの結果、マウスゲノム配列上のAが運動ニューロン由来のRNAの配列決定ではGとなっている部位215箇所を同定し、マウスA-I RNA編集部位とした。次に、マウスで同定した215箇所のA-I RNA編集部位と相同なヒトゲノム配列（GRCh37/hg19）上の部位を、UCSC liftOver tool（http://genome.ucsc.edu/cgi-bin/hgLiftOver)（Hinrichs et al., Nucleic Acids Res, 34, D590-598 2006）を用いて同定した。 1-4. Search for AI RNA editing sites homologous between human and mouse Sequence of RNAs derived from motoneurons prepared from wild-type mice (n = 3) and AR2 mice (n = 3) were determined. The RNA sequence information of 15 or more reads in 2 or more animals was mapped to a mouse genome sequence (NCBI37 / mm9). As a result of mapping, 215 sites where A on the mouse genome sequence was G in the determination of RNA derived from motor neurons were identified and designated as a mouse AI RNA editing site. Next, the site on the human genomic sequence (GRCh37 / hg19) that is homologous to the 215 AI RNA editing sites identified in the mouse was converted to the UCSC liftOver tool (http://genome.ucsc.edu/cgi-bin/hgLiftOver) ( Hinrichs et al., Nucleic Acids Res, 34, D590-598 2006).
１－５．培養細胞へのトランスフェクション
　HeLa細胞およびSH-SY5Y細胞は、10 % FBS（Thermo Fisher Scientific）、100 U/ml ペニシリンおよび100 μg/ml ストレプトマイシン（Thermo Fisher Scientific）を添加したMEM α-培地（WAKO）中、37℃、5 % CO2の条件で培養した。
１－５－１．HeLa細胞内でのヒトADAR2の過剰発現
　HeLa細胞は、6-ウェルプレートに1.0×104細胞/cm2の密度で播種した。培養開始から24時間後、Lipofectamine 3000 transfection reagent（lnvitrogen）を用いて、2.5 μgのヒトADAR2ベクター（hADAR2/pCI）（Yamashita et al., Embo Mol. Med., 5 1710-1709 2013）を細胞にトランスフェクションした後、48時間培養を行った。
１－５－２．SH-SY5Y細胞内でのヒトADAR2の過剰発現
　SH-SY5Y細胞は、10-cmディッシュに7.6×104細胞/cm2の密度で播種した。培養開始から24時間後、2.0 μgのhADAR2/pCIをLipofectamine 3000 transfection reagentを用いてトランスフェクションした。トランスフェクション後、培地を無血清培地に換えて48時間培養した。
１－５－３．SH-SY5Y細胞のヒトADAR2のノックダウン
　SH-SY5Y細胞は、10-cmディッシュに7.6×104細胞/cm2の密度で播種した。培養開始から24時間後、30 nMの各種siRNA（表３）をLipofectamine RNAiMAX transfection reagent（lnvitrogen）を用いて1回目のトランスフェクションを行った。48時間培養したのち、1回目と同様に2回目のトランスフェクションを行った。2回目のトランスフェクション時に、培地を無血清培地に換えて48時間培養した。 1-5. Transfection into cultured cells HeLa cells and SH-SY5Y cells were obtained from MEM α-medium (WAKO) supplemented with 10% FBS (Thermo Fisher Scientific), 100 U / ml penicillin and 100 μg / ml streptomycin (Thermo Fisher Scientific). The medium was cultured under the conditions of 37 ° C. and 5% CO 2 .
1-5-1. Overexpression of human ADAR2 in HeLa cells HeLa cells were seeded at a density of 1.0 × 10 4 cells / cm 2 in 6-well plates. 24 hours after the start of the culture, 2.5 μg of human ADAR2 vector (hADAR2 / pCI) (Yamashita et al., Embo Mol. Med., 5 1710-1709 2013) was added to the cells using Lipofectamine 3000 transfection reagent (lnvitrogen). After transfection, the cells were cultured for 48 hours.
1-5-2. Overexpression of human ADAR2 in SH-SY5Y cells SH-SY5Y cells were seeded at a density of 7.6 × 10 4 cells / cm 2 in 10-cm dishes. Twenty-four hours after the start of culture, 2.0 μg of hADAR2 / pCI was transfected using Lipofectamine 3000 transfection reagent. After transfection, the medium was changed to a serum-free medium and cultured for 48 hours.
1-5-3. Knockdown of human ADAR2 in SH-SY5Y cells SH-SY5Y cells were seeded at a density of 7.6 × 10 4 cells / cm 2 in a 10-cm dish. Twenty-four hours after the start of the culture, the first transfection of 30 nM of various siRNAs (Table 3) was performed using Lipofectamine RNAiMAX transfection reagent (lnvitrogen). After culturing for 48 hours, a second transfection was performed in the same manner as the first transfection. At the time of the second transfection, the medium was changed to a serum-free medium and cultured for 48 hours.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
１－６．細胞からのRNA抽出および逆転写
　HeLa細胞およびSH-SY5Y細胞から、RNA spin Mini Kit （GE Healthcare）を用いてRNA抽出およびDNaseI処理を行った。1μgの総RNAを鋳型にして、oligoDTプライマーおよびrandamプライマーを含んでいるReverTra ACE qPCR-RT Master Mix Kit（TOYOBO）を使用し、cDNAを合成した。
　環状RNAの抽出に関しては、20 μgの総RNAを1 U/μgのRNaseR（Epicentre）で2回処理（37℃、15分間）した後、RNeasy micro Kitで精製した。RNaseRで処理した総RNAを鋳型にしてReverTra ACE qPCR-RT Master Mix Kit（TOYOBO）を用いてcDNAを合成した。 1-6. RNA extraction and reverse transcription from cells RNA extraction and DNaseI treatment were performed on HeLa cells and SH-SY5Y cells using an RNA spin Mini Kit (GE Healthcare). Using 1 μg of total RNA as a template, cDNA was synthesized using ReverTra ACE qPCR-RT Master Mix Kit (TOYOBO) containing oligoDT primer and randam primer.
For the extraction of circular RNA, 20 μg of total RNA was treated twice with 1 U / μg of RNaseR (Epicentre) (37 ° C., 15 minutes), and then purified with the RNeasy micro Kit. Using total RNA treated with RNaseR as a template, cDNA was synthesized using ReverTra ACE qPCR-RT Master Mix Kit (TOYOBO).
１－７．培養上清からの直鎖状RNAおよび環状RNAの抽出
　FBS中のRNAのコンタミネーションを避けるために、SH-SY5Y細胞を無血清培地で48時間培養した後、10 mlの培地を回収した。この時、細胞は、10-cmディッシュでコンフルエントの状態であった。
　直鎖状RNAは4 mlの培養上清から抽出した。細胞成分の混入を防ぐために、培養上清を室温にて15分間（3,000 g）遠心した後、総RNAをTRIzol LS Reagent（Thermo Fisher Scientifics）を用いて抽出した。
　環状RNAの抽出に関しては、160 mlから400 mlの培養上清を回収し、細胞成分の混入を防ぐために、3,000 gで15分間、室温で遠心した後、lyophilizer（FZ-2.5CS, LABCONCO, Kansas City, USA）で凍結乾燥し、総RNAをTRIzol LS Reagent （Thermo Fisher Scientifics）用いて抽出した。得られた総RNAをDNaseI（RNase-free DNase Set, QIAGEN）で処理し、RNeasy micro Kitを用いて精製した。総RNAはBioanalyzer 2100（Agilent Technology, Santa Clara, CA）で定量し、これを鋳型にしてReverTra ACE qPCR-RT Master Mix Kitを用いて逆転写を行った。
　ADAR2過剰発現およびノックダウンのコントロールとして、各々、pCI（空のベクター）またはランダムsiRNA（siRNAの混合物）（Stealth RNAiTMsiRNA Negative Control, Thermo Fisher Scientifics）をトランスフェクションし、同じ処理を行ったSH-SY5Y細胞の培養上清を使用した。 1-7. Extraction of linear RNA and circular RNA from culture supernatant To avoid contamination of RNA in FBS, SH-SY5Y cells were cultured in a serum-free medium for 48 hours, and 10 ml of the medium was collected. At this time, the cells were confluent on a 10-cm dish.
Linear RNA was extracted from 4 ml of culture supernatant. To prevent the contamination of cell components, the culture supernatant was centrifuged at room temperature for 15 minutes (3,000 g), and then the total RNA was extracted using TRIzol LS Reagent (Thermo Fisher Scientifics).
For the extraction of circular RNA, collect the culture supernatant from 160 ml to 400 ml, centrifuge at 3,000 g for 15 minutes at room temperature to prevent contamination with cell components, and then use lyophilizer (FZ-2.5CS, LABCONCO, Kansas City, USA) and total RNA was extracted using TRIzol LS Reagent (Thermo Fisher Scientifics). The obtained total RNA was treated with DNaseI (RNase-free DNase Set, QIAGEN) and purified using the RNeasy micro Kit. Total RNA was quantified using Bioanalyzer 2100 (Agilent Technology, Santa Clara, Calif.), And using this as a template, reverse transcription was performed using ReverTra ACE qPCR-RT Master Mix Kit.
SH-SY5Y cells transfected with pCI (empty vector) or random siRNA (mixture of siRNA) (Stealth RNAiTM siRNA Negative Control, Thermo Fisher Scientifics) and subjected to the same treatment as ADAR2 overexpression and knockdown controls, respectively Was used.
１－８．リアルタイム定量PCR
　定量PCR（qPCR）は、Light Cycler System（Roche Diagnostics）を用い、表４に示すqPCRプライマーおよびプローブを用いて、Yamashita et al., Neurosci Res, 73, 42-48 2012に記載されたPCR条件に従って行った。
Figure JPOXMLDOC01-appb-T000004
 1-8. Real-time quantitative PCR
Quantitative PCR (qPCR) was performed according to the PCR conditions described in Yamashita et al., Neurosci Res, 73, 42-48 2012 using the Light Cycler System (Roche Diagnostics) and the qPCR primers and probes shown in Table 4. went.
Figure JPOXMLDOC01-appb-T000004
１－９．A-I RNA編集部位のPCR増幅およびその解析
　PCRは、表１に示すプライマーペアを用いて、表２に示す条件で行った。PCR産物は、MiniElute PCR purification Kit（QIAGEN）またはMiniElute Gel Extraction Kit（QIAGEN）を用いて精製した。GluA2mRNAおよびcircGRIA2（hsa_circ_0125620）のQ/R部位、CYFIP2（cytoplasmic fragile X mental retardation interacting protein 2）mRNAのリジン／グルタミン酸（K/E）部位およびTMEM63B（transmembrane protein 63B）mRNAのQ/R部位におけるA-I編集の程度は、PCR産物を制限酵素で処理し、編集依存的な制限酵素による切断を、Bioanalyzer 2100（Agilent Technology）を用いた定量解析で算出した。GluA2 mRNA、CYFIP2 mRNA、TMEM63B mRNAおよびcircGRIA2由来のPCR産物は、各々、Bbvl、Msel、HpaIIおよびHpyCH4V（New England Biolabs）で切断した。編集率は、編集されたRNA（編集RNA）と編集されていないRNA（未編集RNA）に由来するバンドの和に対する、編集RNAのバンドのモル比で示した（表５）。他方、編集RNAと未編集RNAに由来するPCR産物を異なる様式で切断する制限酵素が無い場合、そのPCR産物を3100 Genetic Analyzer sequencer（Applied Biosystems）で配列を決定した後、各A-I RNA編集部位における編集率は、アデノシンのピークとグアノシンのピークの和に対するグアノシンのピークのシグナル強度比をab1 Peak Reporter tool（https://apps.thermofisher.com/ab1peakreporter）（Thermo Fisher Scientifics）を使用して算出した。 1-9. PCR amplification of AI RNA editing site and its analysis PCR was performed under the conditions shown in Table 2 using the primer pairs shown in Table 1. PCR products were purified using MiniElute PCR purification Kit (QIAGEN) or MiniElute Gel Extraction Kit (QIAGEN). AI in GluA2 mRNA and circ GRIA2 (hsa_circ_0125620) Q / R site, CYFIP2 (cytoplasmic fragile X mental retardation interacting protein 2) mRNA lysine / glutamate (K / E) site and TMEM63B (transmembrane protein 63B) mRNA Q / R site The degree of editing was calculated by treating the PCR product with a restriction enzyme, and determining the cleavage with the editing-dependent restriction enzyme by quantitative analysis using Bioanalyzer 2100 (Agilent Technology). GluA2 mRNA, CYFIP2 mRNA, TMEM63B mRNA and circ GRIA2 derived PCR products were cut with Bbvl , Msel , HpaII and HpyCH4V (New England Biolabs), respectively. The editing rate was shown by the molar ratio of the band of the edited RNA to the sum of the bands derived from the edited RNA (edited RNA) and the unedited RNA (unedited RNA) (Table 5). On the other hand, when there is no restriction enzyme that cuts the PCR products derived from the edited RNA and the unedited RNA in a different manner, the PCR products are sequenced with a 3100 Genetic Analyzer sequencer (Applied Biosystems), and then, each of the AI RNA editing sites The editing rate was calculated by using the ab1 Peak Reporter tool (https://apps.thermofisher.com/ab1peakreporter) (Thermo Fisher Scientifics) as the signal intensity ratio of the guanosine peak to the sum of the adenosine peak and the guanosine peak. .
Figure JPOXMLDOC01-appb-T000005
 １－１０．統計分析
　統計分析は、JMP Pro 13 softwareを用いて行った。2群の統計的比較には、マン・ホイットニーのU検定を用いた。P<0.05の時に有意差有りとした。
Figure JPOXMLDOC01-appb-T000005
 1-10. Statistical analysis Statistical analysis was performed using JMP Pro 13 software. Mann-Whitney U test was used for statistical comparison of the two groups. When P <0.05, there was a significant difference.
２．結果
２－１．野生型マウスおよびAR2マウスの運動ニューロンにおけるA-I RNA編集の比較
　3匹の野生型マウスおよび3匹のAR2マウスの脊髄運動ニューロンから抽出したRNAの配列を決定し、その配列をNCBI37/mm9にマッピングして、215カ所のA-I RNA編集部位を同定した。編集率が50%未満のA-I RNA編集部位の数は、野生型マウスよりもAR2マウスの運動ニューロンにおいて多かったことから、AR2マウスと野生型マウス間の各A-I RNA編集部位における編集効率を比較することで、ADAR2依存性A-I RNA編集部位の検出可能であることが示唆された。215カ所のA-I RNA編集部位のうち、102部位おいて、AR2マウスにおける編集効率が野生型マウスにおける編集効率よりも2%低かった。そこで、UCSC liftOver toolを使用して、ヒトゲノム配列（GRCh37/hg19）上で、マウスの215カ所のA-I RNA編集部位と相同部位を検索したところ、ヒトRNAとマウスRNA間で28カ所の相同部位を同定した。
　そこで、これら28カ所のA-I RNA編集部位（表６）が、ADAR2活性依存的に編集率が変化する編集部位なのかどうかを、ヒト由来の培養細胞を用いて検討した。 2. Result 2-1. Comparison of AI RNA editing in motoneurons of wild-type and AR2 mice RNAs extracted from spinal motoneurons of three wild-type mice and three AR2 mice were sequenced, and the sequences were mapped to NCBI37 / mm9. As a result, 215 AI RNA editing sites were identified. Compare the editing efficiency at each AI RNA editing site between AR2 and wild-type mice, since the number of AI RNA editing sites with an editing rate of less than 50% was greater in motoneurons of AR2 mice than in wild-type mice This suggested that the ADAR2-dependent AI RNA editing site could be detected. Of the 215 AI RNA editing sites, at 102 sites, the editing efficiency in AR2 mice was 2% lower than the editing efficiency in wild-type mice. Therefore, using the UCSC liftOver tool, we searched for 215 AI RNA editing sites and homologous sites in the human genome sequence (GRCh37 / hg19), and found 28 homologous sites between human RNA and mouse RNA. Identified.
Therefore, it was examined using human-derived cultured cells whether or not these 28 AI RNA editing sites (Table 6) are editing sites in which the editing rate changes depending on ADAR2 activity.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
２－２．ヒト培養細胞におけるADAR2依存性A-I RNA編集部位の検索
　培養細胞中でADAR2を過剰発現させ、上記28の部位における編集率の変化を調べた。HeLa細胞（図２A）およびにSH-SY5Y 細胞（図３A）にADAR2を過剰発現させると、28のうち10の部位（ADAR2 pre-mRNAのintron +10、ADAR2 pre-mRNAのintron +23、ADAR2 pre-mRNAのintron +24、CYFIP2 mRNAのK/E部位、GluA2 mRNAのQ/R部位、GluA2 mRNAのR/G部位、SON mRNAのT/A部位-1、SON mRNAのR/G部位、SON mRNAのL/L部位およびTMEM63B mRNAのQ/R部位）において、編集率が有意に増加した（図２Bおよび図３B）。
　次に、SH-SY5Y細胞のADAR2をsiRNAでノックダウンした後、これら10の部位における編集率の変化を調べた。siADAR2-1またはsiADAR2-2をトランフェクションしたSH-SY5Y細胞中のADAR2 mRNAの発現量は、有意に減少していた（図３C）。ADAR2をノックダウンすると、10カ所全ての部位において編集率が減少していたが、その減少の程度が統計的に有意なレベルに達している部位は、4カ所、すなわち、CYFIP2 mRNAのK/E部位、GluA2 mRNAのQ/R部位、GluA2 mRNAのR/G部位およびSON mRNAのR/G部位であった（図３D）。他の6カ所については、ADAR2をノックダウンしない状態の元々の編集率が低いため、ノックダウンによる編集率の変化を検出できなかった可能性が考えられる。
　SH-SY5Y細胞でADAR2過剰発現させた場合とADAR2ノックダウンした場合の各A-I RNA編集部位の編集効率の変動は、矛盾するものではなく、上記10カ所のA-I RNA編集部位でADAR2がA-I変換を行っていることが示唆された。なお、上記10の部位のうち、SON mRNAのR/G部位は、これまでにADAR2依存性A-I RNA編集部位であるとの報告はない。 2-2. Search for ADAR2-Dependent AI RNA Editing Sites in Cultured Human Cells ADAR2 was overexpressed in cultured cells, and changes in the editing rate at the 28 sites described above were examined. When ADAR2 was overexpressed in HeLa cells (FIG. 2A) and SH-SY5Y cells (FIG. 3A), 10 out of 28 sites (ADAR2 pre-mRNA intron +10, ADAR2 pre-mRNA intron +23, ADAR2 Intron +24 of pre-mRNA, K / E site of CYFIP2 mRNA, Q / R site of GluA2 mRNA, R / G site of GluA2 mRNA, T / A site of SON mRNA-1, R / G site of SON mRNA, In the L / L site of SON mRNA and the Q / R site of TMEM63B mRNA), the editing rate was significantly increased (FIGS. 2B and 3B).
Next, ADAR2 of SH-SY5Y cells was knocked down with siRNA, and the change in editing rate at these 10 sites was examined. The expression level of ADAR2 mRNA in SH-SY5Y cells transfected with siADAR2-1 or siADAR2-2 was significantly reduced (FIG. 3C). When ADAR2 was knocked down, the editing rate was reduced at all 10 sites, but the site where the degree of the reduction reached a statistically significant level was 4 sites, that is, K / E of CYFIP2 mRNA. Site, Q / R site of GluA2 mRNA, R / G site of GluA2 mRNA, and R / G site of SON mRNA (FIG. 3D). At the other six locations, it is possible that the change in the editing rate due to the knockdown could not be detected because the original editing rate without ADAR2 knockdown was low.
Variations in the editing efficiency of each AI RNA editing site between ADAR2 overexpression and ADAR2 knockdown in SH-SY5Y cells are not inconsistent, and ADAR2 converts AI conversion at the above 10 AI RNA editing sites. It was suggested that they did. In addition, among the above 10 sites, the R / G site of SON mRNA has not been reported to be an ADAR2-dependent AI RNA editing site.
２－３．培養上清中に存在するADAR2依存性A-I RNA編集部位を有するRNA
　次に、10カ所のADAR2依存性A-I RNA編集部位を持つmRNA（ADAR2、CYFIP2、GluA2、SONおよびTMEM63BのmRNA）が細胞から分泌されるかどうかを調べたところ、SH-SY5Y細胞の培養上清中にこれら5種類全てのmRNAの存在が確認された。そこで、SH-SY5Y細胞の培養上清中に分泌されたmRNAのADAR2依存性A-I RNA編集部位の編集率を調べた。
　SH-SY5Y細胞でADAR2を過剰発現させた場合、細胞外RNAの10カ所のADAR2依存性A-I RNA編集部位の編集率は、細胞内RNAの編集率と同じ様に変動する傾向にあった。ADAR2 pre-mRNA のintron position +10、CYFIP2 mRNA のK/E部位、SON mRNAのT/A部位とR/G部位および TMEM63B mRNAのQ/R部位においては、その編集率が有意に増加した（図４A）。ADAR2をノックダウンした細胞では、培地中のRNAのCYFIP2 mRNAのK/E部位およびGluA2 mRNAのQ/R部位における編集率が有意に減少していた（図４B）。 2-3. RNA with ADAR2-dependent AI RNA editing site present in culture supernatant
Next, we examined whether mRNAs containing 10 ADAR2-dependent AI RNA editing sites (ADAR2, CYFIP2, GluA2, SON and TMEM63B mRNAs) were secreted from the cells. The culture supernatant of SH-SY5Y cells The presence of all five types of mRNA was confirmed. Therefore, the editing rate of the ADAR2-dependent AI RNA editing site of mRNA secreted into the culture supernatant of SH-SY5Y cells was examined.
When ADAR2 was overexpressed in SH-SY5Y cells, the editing rate of the 10 ADAR2-dependent AI RNA editing sites of extracellular RNA tended to fluctuate similarly to the editing rate of intracellular RNA. In the intron position +10 of ADAR2 pre-mRNA, K / E site of CYFIP2 mRNA, T / A site and R / G site of SON mRNA, and Q / R site of TMEM63B mRNA, the editing rate was significantly increased ( FIG. 4A). In the cells in which ADAR2 was knocked down, the editing rate at the K / E site of CYFIP2 mRNA and the Q / R site of GluA2 mRNA of the RNA in the medium was significantly reduced (FIG. 4B).
２－４．SH-SY5Y細胞およびその培養上清中のcircGRIA2（hsa_circ_0125620）のADAR2依存性A-I RNA編集部位
　ADAR2依存性A-I RNA編集部位を有する環状RNAを circBase（Glazar et al., RNA 20, 1666-1670 2014）で検索した結果、Q/R部位を含むGRIA2の転写産物であるcircGRIA2（hsa_circ_0125620、hsa_circ_0125618およびhsa_circ_0125619）を見出した。SH-SY5Y細胞由来の総RNA20μg中、RNaseR耐性RNAの量は、RNaseR処理前の抽出総RNAの約1%程度であり、その割合はこれまでの報告（Jeck et al., RNA 19, 141-157 2013）と一致していた。。RNaseR耐性RNAの中から、circGRIA2（hsa_circ_0125620）を検出するためにdivergent primerを用いてPCRを行ったところ、SH-SY5Y細胞中において、hsa_circ_0125620は再現性をもって検出できたが（図５A）、hsa_circ_0125618とhsa_circ_0125619は検出できなかった。Q/R部位が編集型のhsa_circ_0125620に由来するPCR産物には、2カ所のHpyCH4V認識部位が存在しているのに対し、Q/R部位が未編集型のhsa_circ_0125620に由来するPCR産物には、さらに1カ所（合計3カ所）の認識部位が存在している（図５B）。
　SH-SY5Y細胞内では、hsa_circ_0125620のQ/R部位における編集率（平均で95%）は、GluA2 mRNA（直鎖状mRNA）の編集率（平均で87%）よりも高く、ADAR2ノックダウンにより有意に減少した（図５C） 2-4. The ADAR2-dependent AI RNA editing site of circ GRIA2 (hsa_circ_0125620) in SH-SY5Y cells and the culture supernatant thereof was transferred to circBase (Glazar et al., RNA 20, 1666-1670 2014). As a result, circ GRIA2 (hsa_circ_0125620, hsa_circ_0125618, and hsa_circ_0125619), which is a transcript of GRIA2 containing a Q / R site, was found. In 20 μg of total RNA derived from SH-SY5Y cells, the amount of RNaseR-resistant RNA is about 1% of the total RNA extracted before RNaseR treatment, and the ratio has been reported so far (Jeck et al., RNA 19, 141- 157 2013). . When PCR was performed using divergent primers to detect circ GRIA2 (hsa_circ_0125620) from among the RNaseR-resistant RNAs, hsa_circ_0125620 was detected reproducibly in SH-SY5Y cells (FIG. 5A), but hsa_circ_0125618. And hsa_circ_0125619 could not be detected. The Q / R site has two HpyCH4V recognition sites in the PCR product derived from the edited hsa_circ_0125620, whereas the Q / R site has the unedited hsa_circ_0125620 in the PCR product derived from the hsa_circ_0125620. There is one more (three in total) recognition sites (FIG. 5B).
In SH-SY5Y cells, the editing rate (95% on average) at the Q / R site of hsa_circ_0125620 is higher than the editing rate of GluA2 mRNA (linear mRNA) (87% on average), which is significant due to ADAR2 knockdown (Fig. 5C)
　次に、SH-SY5Y細胞の培養上清中におけるhsa_circ_0125620について検討を行った。hsa_circ_0125620を検出するために、160 ml以上の培養上清が必要であった。SH-SY5Y細胞の培養上清中のhsa_circ_012562のQ/R部位は、ADAR2のノックダウン後であっても、ほとんどが編集された状態であった。しかし、クロマトグラフィーをよく見てみると（図６の挿入図）、ノックダウンの方には、僅かではあるが172bpのフラグメントが確認できた。このことから、細胞外に分泌されたcircGRIA2のQ/R部位についても、細胞内のADAR2がノックダウンされた場合には、培養上清中にQ/R部位が未編集型のcircGRIA2が分泌されていることが示唆される。本実験では、ADAR2ノックダウンという人為的な状況で検討を行っており、ADAR2ノックダウン前にすでに存在していたQ/R部位編集型のcircGRIA2が未だ存在しており、それらが細胞外に分泌されたために、細胞外で検出されたcircGRIA2のほとんどがQ/R部位編集型である可能性が考えられる。環状RNAは安定で長期間存在し得ること（Bachmayr-Heyda et al., Sci Rep, 5, 8057 2015）を考慮するとこのような可能性も十分あり得る。すなわち、実際のALS患者由来の細胞外（体液中の）circGRIA2はQ/R部位が未編集型であるものが分泌されている可能性が高く、健常者と比較してcircGRIA2のQ/R部位の編集率が低下している可能性が高い。 Next, hsa_circ_0125620 in the culture supernatant of SH-SY5Y cells was examined. To detect hsa_circ_0125620, a culture supernatant of 160 ml or more was required. Most of the Q / R site of hsa_circ_012562 in the culture supernatant of SH-SY5Y cells was in an edited state even after ADAR2 knockdown. However, a close look at the chromatography (inset in FIG. 6) revealed a small but 172 bp fragment in the knockdown. From this, regarding the Q / R site of circ GRIA2 secreted extracellularly, if ADAR2 in the cell was knocked down, circ GRIA2 whose Q / R site was unedited was found in the culture supernatant. It is suggested that it is secreted. In this experiment, we are investigating the artificial situation of ADAR2 knockdown, and the Q / R site-editing type circ GRIA2, which already existed before ADAR2 knockdown, still exists, and these were extracellular. It is possible that most of the extracellularly detected circ GRIA2 was Q / R site-edited due to secretion. Considering that circular RNA is stable and can exist for a long period of time (Bachmayr-Heyda et al., Sci Rep, 5, 8057 2015), such a possibility is quite possible. That is, the actual ALS from patients (in body fluids) the extracellular circ GRIA2 the Q / R site likely is secreted those which are unedited form, the circ GRIA2 compared to healthy persons Q / There is a high possibility that the editing rate of the R region has dropped.

Claims (10)

  1. 　ALSの病態を診断する方法であって、診断サンプル中に存在するADAR2依存性A-I RNA編集部位のヌクレオチドを同定する工程を含む、前記方法。 方法 A method for diagnosing the pathology of ALS, comprising the step of identifying the nucleotide of an ADAR2-dependent AI-RNA editing site present in a diagnostic sample.
  2. 　ALSの病態を診断する方法であって、以下の（ａ）～（ｄ）の工程を含むことを特徴とする請求項１に記載の方法。
    （ａ）診断サンプル中に存在するRNAのADAR2依存性A-I RNA編集部位のヌクレオチドを同定する工程、
    （ｂ）工程（ａ）で同定したヌクレオチドがアデニン（A）またはイノシン（I）である割合を算出する工程、および
    （ｃ）工程（ｂ）で算出したAまたはIの割合と、健常者のADAR2依存性A-I RNA編集部位のヌクレオチドがAまたはIである割合とを、各々、比較する工程     The method according to claim 1, which is a method for diagnosing a pathological condition of ALS, comprising the following steps (a) to (d).
    (A) identifying the nucleotides at the ADAR2-dependent AI RNA editing site of the RNA present in the diagnostic sample,
    (B) calculating the ratio of the nucleotide identified in step (a) to adenine (A) or inosine (I); and (c) calculating the ratio of A or I calculated in step (b) Comparing the ratio of nucleotides of the ADAR2-dependent AI RNA editing site to A or I, respectively.
  3. 　前記ADAR2依存性A-I RNA編集部位が、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第178番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第191番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第192番目の位置、配列番号２で表されるCYFIP2 mRNAの核酸配列中第171番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第28番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第498番目の位置、配列番号４で表されるSON mRNAの核酸配列中第202番目の位置、配列番号４で表されるSON mRNAの核酸配列中第672番目の位置、配列番号４で表されるSON mRNAの核酸配列中第720番目の位置、配列番号５で表されるTMEM63B mRNAの核酸配列中第157番目の位置および／または配列番号６で表されるcircGRIA2（has_circ_0125620）の核酸配列中第409番目であることを特徴とする請求項１または２に記載の方法。 The ADAR2-dependent AI RNA editing site is the 178th position in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1, and the 191st position in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1. Position, position 192 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, GluA2 represented by SEQ ID NO: 3 Position 28 in the nucleic acid sequence of mRNA, position 498 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3, position 202 in the nucleic acid sequence of SON mRNA represented by SEQ ID NO: 4 Position 672 in the nucleic acid sequence of the SON mRNA represented by No. 4, position 720 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, in the nucleic acid sequence of TMEM63B mRNA represented by SEQ ID NO: 5 157 th position and / or in the nucleic acid sequence of circ GRIA2 represented by SEQ ID NO: 6 (has_circ_0125620) a first position 409 The method according to claim 1 or 2, characterized.
  4. 　前記診断サンプルが体液由来であることを特徴とする請求項１ないし３のいずれかに記載の方法。 方法 The method according to any one of claims 1 to 3, wherein the diagnostic sample is derived from a body fluid.
  5. 　前記体液が、髄液または血液のいずれかであることを特徴とする請求項４に記載の方法。 The method according to claim 4, wherein the body fluid is either cerebrospinal fluid or blood.
  6. 　ALSの病態を診断するためのバイオマーカーであって、ADAR2依存性A-I RNA編集部位を有する１または複数のRNA分子からなるバイオマーカー。 {A biomarker for diagnosing the pathology of ALS, which is composed of one or more RNA molecules having an ADAR2-dependent AI editing site.
  7. 　前記ADAR2依存性A-I RNA編集部位が、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第178番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第191番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第192番目の位置、配列番号２で表されるCYFIP2 mRNAの核酸配列中第171番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第28番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第498番目の位置、配列番号４で表されるSON mRNAの核酸配列中第202番目の位置、配列番号４で表されるSON mRNAの核酸配列中第672番目の位置、配列番号４で表されるSON mRNAの核酸配列中第720番目の位置、配列番号５で表されるTMEM63B mRNAの核酸配列中第157番目の位置および／または配列番号６で表されるcircGRIA2（has_circ_0125620）の核酸配列中第409番目であることを特徴とする請求項６に記載のバイオマーカー。 The ADAR2-dependent AI RNA editing site is the 178th position in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1, and the 191st position in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1. Position, position 192 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, GluA2 represented by SEQ ID NO: 3 Position 28 in the nucleic acid sequence of mRNA, position 498 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3, position 202 in the nucleic acid sequence of SON mRNA represented by SEQ ID NO: 4 Position 672 in the nucleic acid sequence of the SON mRNA represented by No. 4, position 720 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, in the nucleic acid sequence of TMEM63B mRNA represented by SEQ ID NO: 5 157 th position and / or in the nucleic acid sequence of circ GRIA2 represented by SEQ ID NO: 6 (has_circ_0125620) a first position 409 Biomarkers of claim 6, wherein.
  8. 　ADAR2依存性A-I RNA編集部位を有する１または複数のRNA分子のALSバイオマーカーとしての使用。 {ADAR2-dependent AI} Use of one or more RNA molecules having an RNA editing site as an ALS biomarker.
  9. 　前記ADAR2依存性A-I RNA編集部位が、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第178番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第191番目の位置、配列番号１で表されるADAR2 pre-mRNAの核酸配列中第192番目の位置、配列番号２で表されるCYFIP2 mRNAの核酸配列中第171番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第28番目の位置、配列番号３で表されるGluA2 mRNAの核酸配列中第498番目の位置、配列番号４で表されるSON mRNAの核酸配列中第202番目の位置、配列番号４で表されるSON mRNAの核酸配列中第672番目の位置、配列番号４で表されるSON mRNAの核酸配列中第720番目の位置、配列番号５で表されるTMEM63B mRNAの核酸配列中第157番目の位置および／または配列番号６で表されるcircGRIA2（has_circ_0125620）の核酸配列中第409番目であることを特徴とする請求項８に記載の使用。 The ADAR2-dependent AI RNA editing site is the 178th position in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1, and the 191st position in the nucleic acid sequence of the ADAR2 pre-mRNA represented by SEQ ID NO: 1. Position, position 192 in the nucleic acid sequence of ADAR2 pre-mRNA represented by SEQ ID NO: 1, position 171 in the nucleic acid sequence of CYFIP2 mRNA represented by SEQ ID NO: 2, GluA2 represented by SEQ ID NO: 3 Position 28 in the nucleic acid sequence of mRNA, position 498 in the nucleic acid sequence of GluA2 mRNA represented by SEQ ID NO: 3, position 202 in the nucleic acid sequence of SON mRNA represented by SEQ ID NO: 4 Position 672 in the nucleic acid sequence of the SON mRNA represented by No. 4, position 720 in the nucleic acid sequence of the SON mRNA represented by SEQ ID NO: 4, in the nucleic acid sequence of TMEM63B mRNA represented by SEQ ID NO: 5 157 th position and / or in the nucleic acid sequence of circ GRIA2 represented by SEQ ID NO: 6 (has_circ_0125620) a first position 409 Use according to claim 8, characterized.
  10. 　ALSの病態を診断するために使用するキットであって、少なくともADAR2依存性A-I RNA編集部位のヌクレオチドを同定するための要素を含むキット。 (4) A kit used for diagnosing the pathological condition of ALS, the kit comprising at least an element for identifying a nucleotide at an ADAR2-dependent AI-RNA editing site.
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