WO2009084472A1 - Method for detecting intractable epilepsy developed in neonatal period and infancy - Google Patents
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- WO2009084472A1 WO2009084472A1 PCT/JP2008/073164 JP2008073164W WO2009084472A1 WO 2009084472 A1 WO2009084472 A1 WO 2009084472A1 JP 2008073164 W JP2008073164 W JP 2008073164W WO 2009084472 A1 WO2009084472 A1 WO 2009084472A1
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- the present invention relates to a method for detecting refractory epilepsy from onset of neonatal period to infancy.
- Neonatal-infant-onset intractable epilepsy includes early myoclonic encephalopathy (earlyEmyoclonic encephalopathy: EME), Otawara syndrome (early infantile epileptic encephalopathy with suppression burst: EIEE), and West syndrome (temporal epilepsy).
- EME early myoclonic encephalopathy
- Otawara syndrome early infantile epileptic encephalopathy with suppression burst: EIEE
- West syndrome temporary epilepsy.
- Many of early myoclonic encephalopathy and Otawara syndrome develop early in infancy, and are characterized by severe psychomotor developmental delay in addition to epileptic seizures and marked suppressionsupburst on the electroencephalogram.
- West syndrome is well known for its series-forming spasms and hypusarismia on the electroencephalogram.
- Non-patent Document 1 Non-patent Document 1
- an object of the present invention is to provide a novel means capable of diagnosing refractory epilepsy onset in neonates and infants.
- the present invention is a method performed on a sample isolated from a living body, and uses as an index whether or not the STXBP1 gene is deleted and / or whether or not a gene encoding an abnormal STXBP1 exists.
- the present invention provides a method for detecting intractable epilepsy in neonatal to infancy.
- a method for detecting the disease which enables a definitive diagnosis of intractable epilepsy that begins in the neonatal period to infancy.
- a definitive diagnosis of the disease becomes possible, and individualization and optimization of treatment including gene therapy is expected.
- the present invention reports for the first time the involvement of the STXBP1 gene in the disease. It has already been clarified that the gene is involved in the transport / release of synaptic vesicles, but there are no reports of genes involved in the transport / release of synaptic vesicles in general epilepsy. With the disclosure of the present invention, it is considered that elucidation of the pathophysiology of intractable epilepsy onset in neonatal period to infancy progresses at a stretch and leads to the development of effective management and treatment methods.
- FIG. 5 is a diagram showing an alignment of the sequence analysis data of base substitution sites found in patients 2 to 5 and the amino acid sequence of the corresponding region in each species. Four base substitutions cause amino acid substitutions within the functional domain.
- the method for detecting refractory epilepsy in the neonatal period to infancy period of the present invention is a method performed on a sample isolated from a living body, and whether or not the STXBP1 gene is deleted and / or encodes abnormal STXBP1 Whether the gene is present or not is used as an index to determine whether or not the living body suffers from or is likely to develop the intractable epilepsy.
- the intractable epilepsy should be detected by examining at least one of (1) whether the STXBP1 gene is deleted or (2) the presence of a gene encoding an abnormal STXBP1. Can do.
- STXBP1 is a known protein and is known to be involved in the transport and release of synaptic vesicles.
- two types of variant mRNAs are generated from the STXBP1 gene genome sequence shown in SEQ ID NO: 65.
- the sequences of these variants are registered in GenBank as accession numbers NM_003165 (variant 1 / isoform a) and NM_001032221 (variant 2 / isoform b).
- the amino acid sequences of isoforms a and b are shown in SEQ ID NOs: 2 and 4
- the cDNA sequences are shown in SEQ ID NOs: 1 and 3, respectively.
- SEQ ID NO: 2 and SEQ ID NO: 4 have the same amino acid sequence in the region from the 1st to the 575th amino acids, and SEQ ID NO: 1 and SEQ ID NO: 3 are the 1st to 1703th bases.
- the base sequence is the same in this region.
- SEQ ID NOs: 45 to 64 are excerpts of each exon of the STXBP1 gene and a 300 bp intron region before and after that from the base sequence shown in SEQ ID NO: 65.
- the positions of exons, coding regions and UTR regions in each sequence are shown in Table 1 below.
- the notation “301-452nt” represents the region from the 301st base to the 452nd base counted from the first base in the corresponding SEQ ID NO.
- Exon 19 is the final coding exon of isoform a
- isoform b is exon 19 skipped and exon 20 becomes the final coding exon.
- normal STXBP1 includes the above-mentioned two types of isoforms that are wild-type STXBP1, and in addition to these, if there is a natural mutant that exhibits the same physiological activity as the isoform. They are also included.
- abnormal STXBP1 refers to a natural variant of STXBP1, in which the activity as STXBP1 is changed or eliminated. Examples of such anomalous STXBP1 include the four types of missense mutations described below.
- STXBP1 includes both normal STXBP1 and abnormal STXBP1, unless it is clear from the context.
- mutations that cause abnormal STXBP1 expression include, but are not limited to, missense mutations and nonsense mutations.
- missense mutations that can be used as an index in the present invention include the following mutations (1) to (4) identified in the following Examples.
- the position of the mutation is expressed based on the base sequence and amino acid sequence of isoform a, which is one of wild-type STXBP1, that is, SEQ ID NOs: 1 and 2, and “aa84” is represented in SEQ ID NO: 2.
- the 84th amino acid, “251nt” represents the 251st base in SEQ ID NO: 1.
- Aa84 changes from valine to aspartic acid (251 nt changes from t to a)
- Aa180 changes from cysteine to tyrosine (539nt changes from g to a)
- Aa443 changes from methionine to arginine (1328nt changes from t to g)
- Mutation that aa544 changes from glycine to aspartic acid (mutation that changes 1631nt from g to a)
- these mutations are found, it is considered that the living body suffers from or is likely to develop refractory epilepsy that begins in the neonatal period to infancy, but the mutation that can be used as an index in the present invention Is not limited to these. That is, as described in the Examples below, these missense mutations occur in the amino acid located inside the folded structure in the three-dimensional structure of wild type STXBP1 (Protein Data Bank ID, 1DN1). Similarly, even if it is a site other than the above, if substitution occurs in the amino acid at a position important for maintaining the three-dimensional structure such as the inside of the folding structure, the activity may be changed or lost, and abnormal STXBP1 may be obtained. It can be an indicator in the method.
- the sequence of the coding region and the genome sequence of the wild type STXBP1 gene are shown in SEQ ID NOs: 1 and 3 and SEQ ID NO: 65, and are known as registered in GenBank. Also, codons encoding amino acids are known. Therefore, the base substitution that causes such a missense mutation is not limited to the base substitution identified in the following Examples.
- a method of examining at least one of (1) whether the STXBP1 gene is deleted or (2) the presence of a gene encoding an abnormal STXBP1 For example, as described below, a method of analyzing a genomic DNA sample and a method of analyzing an mRNA sample can be mentioned. It is also possible to encode STXBP1 gene deletion or abnormal STXBP1 by examining whether the STXBP1 protein is deleted or whether abnormal STXBP1 protein is present in protein samples isolated from living organisms. The presence of a gene can be examined. Among these methods, the method of the present invention is preferably a method for examining the presence of a gene encoding STXBP1 gene deletion or abnormal STXBP1 using a genomic DNA sample.
- Examples of the method performed using a genomic DNA sample include, but are not limited to, the following methods (a) to (d).
- A In situ hybridization method
- Cells are collected from the target organism and a chromosome sample is prepared.
- a probe is prepared by labeling DNA that specifically hybridizes with the STXBP1 gene region, and the probe is hybridized with the chromosome specimen. Deletion of the STXBP1 gene can be detected by examining the presence or absence of a signal from the probe.
- the labeling of DNA is not particularly limited, but is usually performed using a radioisotope or a fluorescent dye (Cy5, Cy3, FITC, etc.), and a fluorescent dye is more commonly used. When a fluorescently labeled probe is used, this method is called FISH method.
- a DNA probe that specifically hybridizes to the STXBP1 gene region can be easily prepared by those skilled in the art with reference to the STXBP1 gene genome sequence shown in SEQ ID NO: 65 of the Sequence Listing. Specifically, for example, by preparing a primer capable of amplifying a desired region in SEQ ID NO: 65 and performing PCR using a genomic DNA containing a normal STXBP1 gene as a template, DNA used for the probe can be obtained. Further, as described in the Examples below, clones such as BAC clones containing the STXBP1 gene region can be labeled and used as probes. Clones such as BAC clones containing human genomic DNA are also commercially available and are readily available. The DNA used for the probe may cover the entire coding region of the STXBP1 gene or may cover only a part of the coding region.
- (B) Southern hybridization method A genomic DNA sample obtained from a target organism is cleaved with an arbitrary restriction enzyme and then electrophoresed on an agarose gel or the like to transfer the DNA onto a membrane. On this membrane, a DNA probe prepared by labeling a DNA that specifically hybridizes with the STXBP1 gene region is hybridized. Deletion of the STXBP1 gene can be detected by examining the presence or absence of the detected band. For example, even a mutation such as a point mutation can be detected by this method because the size of the detected band changes when the mutation causes a change in the restriction enzyme site.
- the labeling of the probe is not particularly limited, but is usually performed using a hapten such as a radioisotope or digoxigenin.
- the preparation method of DNA used as a probe is the same as described in (a).
- a method for detecting heteroduplexes using denaturing high performance liquid chromatography (dHPLC) and a High Resolution Melt method specifically described in the following examples are known.
- the High Resolution Melt method is the melting of double-stranded DNA using a fluorescent dye (SYTO (registered trademark) 9, LC Green (registered trademark), EvaGreen (registered trademark), etc.) that binds to double-stranded DNA at high density.
- SYTO registered trademark
- LC Green registered trademark
- EvaGreen registered trademark
- these known methods can be used as a method for examining whether or not there is a polynucleotide that cannot express normal STXBP1.
- These methods for detecting heteroduplexes are particularly preferred methods for detecting point mutations.
- those skilled in the art can easily prepare primers to be used with reference to the nucleotide sequences shown in SEQ ID NOs: 45 to 64 or SEQ ID NO: 65. Primers consisting of the base sequences shown in SEQ ID NOs: 5-44 used in the examples can be preferably used.
- (D) Base sequence analysis In order to examine gene mutations in detail, it is desirable to perform base sequence analysis. Mutations can be identified in detail by determining the base sequence of the STXBP1 gene on the target biological genomic DNA and comparing it with the wild-type STXBP1 gene sequence. By analyzing the determined base sequence using known software such as SeqScape (registered trademark), mutation detection and profiling can be easily performed.
- SeqScape registered trademark
- the methods (a) to (d) described above can be combined as appropriate. For example, first, by (a) and / or (b), it is examined whether or not the STXBP1 gene region is present in the genomic DNA sample. If it exists, perform (d) to check for mutations in the STXBP1 gene region. The inspection can be performed more efficiently by narrowing down the region where the base sequence should be determined by (c) and performing (d).
- examples of a method for carrying out the present invention using an mRNA sample include, but are not limited to, the methods described below.
- the presence or absence of STXBP1 mRNA can be easily examined, for example, by performing a Northern hybridization method or RT-PCR method using a probe or primer prepared based on the nucleotide sequence shown in SEQ ID NO: 1 or 3. If STXBP1 mRNA is not detected, it is determined that the STXBP1 gene has been deleted, and it can be determined that intractable epilepsy has been detected.
- mutations can be identified in detail by analyzing the base sequence of cDNA obtained by reverse transcription reaction from mRNA samples as described in (d) above.
- the method (c) above may be performed on the STXBP1 cDNA
- the cDNA of STXBP1 is relatively short as 2 kbp or less as shown in SEQ ID NOS: 1 and 3, so the method (c) above is performed.
- the necessity to narrow down the region for determining the base sequence is low.
- Northern hybridization and RT-PCR itself are well-known conventional methods, and those skilled in the art can easily prepare probes and primers based on the nucleotide sequence shown in SEQ ID NO: 1 or 3.
- CGH comparative genomic hybridization
- Chromosome specimens are prepared by collecting peripheral blood leukocytes from patient 1 and their parents, and a probe prepared by labeling multiple BAC clones located in the region of 9q33.3-q34.11 is hybridized to the specimen. Observed under a fluorescence microscope. As a result, when BAC clones 516d6, 936o12, 24k1, 152i20, and 99f23 were used as probes, no signal was detected in one of the two homologous chromosomes, and the fine deletion was a region of about 2.1 Mb. Was found (Fig. 1B). This microdeletion was not found in the parental chromosomes. That is, the chromosomal microdeletion observed in the patient was a de novo mutation (new mutation), strongly suggesting that it may cause intractable epilepsy.
- STXBP1 syntaxin binding protein 1
- STXBP1 also known as Munc18-1, GeneBank accession number NM_003165 (variant 1) NM_001032221 (variant 2)
- STXBP1 also known as Munc18-1
- NM_003165 variant 1
- NM_001032221 variant 2
- mutation analysis was performed as follows in 58 patients with intractable epilepsy that developed from neonatal to infancy.
- Genomic DNA was collected from peripheral blood leukocytes obtained from patients. Genomic DNA was subjected to genome-wide amplification using Genomiphi version 2 (GE healthcare), and mutation analysis was performed using this amplified DNA. Mutation analysis at the coding exon and exon-intron boundary of the STXBP1 gene (exon 1-20) was performed using the High resolution melt method. This is because the fluorescent dye that binds to the fluorescent dye double-stranded DNA is used to treat the melting (thermal denaturation) process of the PCR product as a change in fluorescence intensity, and temperature-fluorescence data is acquired and analyzed at high density. This is a method for detecting heteroduplexes rapidly and with high sensitivity.
- the reaction was performed with the composition of 1 ⁇ GC buffer II, 0.4 mM each dNTP, 0.2 ⁇ M each primer, 1 ⁇ l LCGreen Plus (Idaho Technology), and 0.5 U LA Taq polymerase (TAKARA).
- the reaction conditions were a two-step cycle of 95 ° C for 10 seconds and 62 ° C for 30 seconds after heat denaturation at 95 ° C for 1 minute. The number of cycles was determined as appropriate by monitoring real-time PCR.
- Table 2 shows primer base sequences and reaction temperatures.
- the upper row of each exon amplification primer described in Table 2 is the sense primer, and the lower row is the antisense primer.
- the numbers described under each exon number indicate the sequence numbers describing the sequences of each exon and the 300 bp intron around it.
- PCR products were purified using ExoSAP-IT (GE health care), and then cycle sequencing was performed using BigDye Terminator chemistry version 3 (Applied Biosystems).
- the reaction product was purified by gel filtration using Sephadex G-50 (GE healthcare) and Multiscreen-96 (Millipore), and a sequence was obtained using ABI Genetic Analyzer 3100 (Applied Biosystems). The obtained sequences were analyzed for the presence or absence of mutation using SeqScape version 2.1.1 software (Applied Biosystems).
- mutation analysis was performed again using genomic DNA that was not amplified as a whole, and mutations on the genomic DNA were confirmed.
- STXBP1 is a soluble protein and does not have a localization signal in its amino acid sequence (Schutz, D., et al., A dual function for Munc-18 in exocytosis of PC12 cells. Eur J Neurosci, 2005. 21 (9): p. 2419-32.).
Abstract
Disclosed is a novel method for detecting intractable epilepsy developed in the neonatal period and infancy. The method of the invention is performed for a sample separated from the living body and uses whether or not syntaxin-binding protein 1 (STXBP1) gene is deleted and/or whether or not a gene encoding abnormal STXBP1 is present as an index. According to the invention, definitive diagnosis of the disease can be performed and individualization and optimization of therapy including gene therapy is expected. Association of STXBP1 gene with the disease is first reported by the invention.
Description
本発明は、新生児期～乳児期発症の難治性てんかんの検出方法に関する。
The present invention relates to a method for detecting refractory epilepsy from onset of neonatal period to infancy.
新生児～乳児期発症の難治性てんかんには、早期ミオクロニー脳症（early myoclonic encephalopathy：EME）、大田原症候群（early infantile epileptic encephalopathy with suppression burst：EIEE）、West症候群（点頭てんかん）がある。早期ミオクロニー脳症や大田原症候群の多くは乳児期早期に発症し、てんかん発作に加えて重度の精神運動発達遅滞と、脳波上顕著なsuppression burstを認めるのが特徴である。West症候群はシリーズ形成性のスパズムと脳波上のヒプスアリスミアがよく知られている。脳形成異常、染色体異常、周産期低酸素性脳障害などが原因として知られているが、明らかな原因がない特発性のものがあり遺伝的な素因が存在する（非特許文献１）。
Neonatal-infant-onset intractable epilepsy includes early myoclonic encephalopathy (earlyEmyoclonic encephalopathy: EME), Otawara syndrome (early infantile epileptic encephalopathy with suppression burst: EIEE), and West syndrome (temporal epilepsy). Many of early myoclonic encephalopathy and Otawara syndrome develop early in infancy, and are characterized by severe psychomotor developmental delay in addition to epileptic seizures and marked suppressionsupburst on the electroencephalogram. West syndrome is well known for its series-forming spasms and hypusarismia on the electroencephalogram. It is known as a cause of brain dysplasia, chromosomal abnormality, perinatal hypoxic encephalopathy, etc., but there are idiopathic things with no obvious cause, and there is a genetic predisposition (Non-patent Document 1).
EIEEとEMEの多くはWest症候群に移行することから、これら3疾患には共通の遺伝背景が存在することが示唆されていた。今までに同定された疾患責任遺伝子としては、家系例の解析からX染色体上に位置する2つの遺伝子ARX (aristaless related homeobox), CDKL5 (cyclin-dependent kinase-like 5) が報告されている（非特許文献２、３）。孤発例においても、男児のEIEE（非特許文献４）およびWest症候群（非特許文献１、５）においてARX変異が、女児のWest症候群においてCDKL5変異（非特許文献６）が報告されている。しかしながら、同定された責任遺伝子変異で原因が説明できない症例が多く、これらの遺伝子のみに着目して新生児～乳児期発症の難治性てんかんの確定診断を行なうことはできない。他の遺伝子の関与が示唆されているものの、確定診断に有用な新たな責任遺伝子は報告されていない。
多 く Many of EIEE and EME migrated to West syndrome, suggesting that these three diseases have a common genetic background. As a disease-responsible gene identified so far, two genes ARXista (aristaless related homeobox) and CDKL5 (cyclin-dependent-kinase-like 5) on the X chromosome have been reported from the analysis of family cases (non- Patent Documents 2 and 3). In isolated cases, ARX mutations have been reported in EIEE (Non-Patent Document 4) and West syndrome (Non-Patent Documents 1 and 5) in boys, and CDKL5 mutation (Non-Patent Document 6) in West syndrome in girls. However, there are many cases in which the cause cannot be explained by the identified responsible gene mutation, and it is not possible to make a definitive diagnosis of intractable epilepsy with neonatal-infant onset by focusing only on these genes. Although the involvement of other genes has been suggested, no new responsible gene useful for definitive diagnosis has been reported.
従って、本発明の目的は、新生児～乳児期発症の難治性てんかんを診断できる新規な手段を提供することにある。
Therefore, an object of the present invention is to provide a novel means capable of diagnosing refractory epilepsy onset in neonates and infants.
本願発明者らは、鋭意研究の結果、新生児～乳児期発症の難治性てんかんの女児症例において第９番染色体上の微細欠失を同定した。次いで、該欠失領域中に存在するシンタキシン結合タンパク質１（syntaxin binding protein 1; STXBP1）に着目して、新生児～乳児期発症の難治性てんかん患者58名において変異解析を行なった結果、4名の患者において健常者には認められないミスセンス変異を見出し、本願発明を完成した。
As a result of diligent research, the inventors of the present application have identified a fine deletion on chromosome 9 in a girl case of intractable epilepsy with onset of neonatal to infancy. Then, focusing on syntaxin binding protein 1 (syntaxin binding protein 1; STXBP1) present in the deletion region, mutation analysis was conducted in 58 patients with intractable epilepsy with neonatal-infant onset. The present inventors completed the present invention by finding a missense mutation that is not recognized by healthy individuals in patients.
すなわち、本発明は、生体から分離した試料に対して行なう方法であって、STXBP1遺伝子が欠失しているか否か及び／又は異常型STXBP1をコードする遺伝子が存在するか否かを指標とする、新生児期～乳児期発症の難治性てんかんの検出方法を提供する。
That is, the present invention is a method performed on a sample isolated from a living body, and uses as an index whether or not the STXBP1 gene is deleted and / or whether or not a gene encoding an abnormal STXBP1 exists. The present invention provides a method for detecting intractable epilepsy in neonatal to infancy.
本発明により、新生児期～乳児期発症の難治性てんかんの確定診断が可能な該疾患の検出方法が提供された。本発明によれば、該疾患の確定診断が可能となり、遺伝子治療を含めた治療の個別化・至適化が期待される。該疾患へのSTXBP1遺伝子の関与は本発明が初めて報告するものである。該遺伝子はシナプス小胞の輸送・放出に関わることが既に明らかになっているが、てんかん全般においてもシナプス小胞の輸送・放出に関わる遺伝子の報告はない。本発明の開示により、新生児期～乳児期発症の難治性てんかんの病態生理の解明が一気に進み、有効な管理・治療法の開発につながると考えられる。
According to the present invention, there is provided a method for detecting the disease, which enables a definitive diagnosis of intractable epilepsy that begins in the neonatal period to infancy. According to the present invention, a definitive diagnosis of the disease becomes possible, and individualization and optimization of treatment including gene therapy is expected. The present invention reports for the first time the involvement of the STXBP1 gene in the disease. It has already been clarified that the gene is involved in the transport / release of synaptic vesicles, but there are no reports of genes involved in the transport / release of synaptic vesicles in general epilepsy. With the disclosure of the present invention, it is considered that elucidation of the pathophysiology of intractable epilepsy onset in neonatal period to infancy progresses at a stretch and leads to the development of effective management and treatment methods.
本発明の新生児期～乳児期発症の難治性てんかんの検出方法は、生体から分離した試料に対して行なう方法であり、STXBP1遺伝子が欠失しているか否か及び／又は異常型STXBP1をコードする遺伝子が存在するか否かを指標として、該生体が上記難治性てんかんを罹患しているか否か又は発症するおそれがあるか否かを判断する。下記実施例にある通り、該難治性てんかん患者の中には、STXBP1遺伝子を完全に欠失している者又は異常型STXBP1をコードする変異STXBP1遺伝子を有する者が認められ、これらの遺伝子異常は健常者には認められない。従って、(1)STXBP1遺伝子が欠失しているか否か、(2)異常型STXBP1をコードする遺伝子が存在するか否か、の少なくともいずれかを調べることにより、上記難治性てんかんを検出することができる。
The method for detecting refractory epilepsy in the neonatal period to infancy period of the present invention is a method performed on a sample isolated from a living body, and whether or not the STXBP1 gene is deleted and / or encodes abnormal STXBP1 Whether the gene is present or not is used as an index to determine whether or not the living body suffers from or is likely to develop the intractable epilepsy. As shown in the following examples, among the patients with refractory epilepsy, those who have completely deleted the STXBP1 gene or those who have a mutant STXBP1 gene encoding an abnormal type STXBP1, are recognized. Not accepted by healthy individuals. Therefore, the intractable epilepsy should be detected by examining at least one of (1) whether the STXBP1 gene is deleted or (2) the presence of a gene encoding an abnormal STXBP1. Can do.
STXBP1は公知のタンパク質であり、シナプス小胞の輸送・放出に関与することが知られている。ヒトにおいては、配列番号６５に示すSTXBP1遺伝子ゲノム配列から2種類のバリアントmRNAが生成される。これらのバリアントの配列はGenBankにアクセッション番号NM_003165（バリアント１／アイソフォームa）、NM_001032221（バリアント２／アイソフォームb）として登録されている。アイソフォームa及びbのアミノ酸配列を配列表の配列番号２及び４に、cDNA配列を配列番号１及び３にそれぞれ示す。なお、配列番号２と配列番号４とは、第1番～第575番アミノ酸の領域においてアミノ酸配列が同一であり、また、配列番号１と配列番号３とは、第1番～第1703番塩基の領域において塩基配列が同一である。配列番号４５～６４は、配列番号６５に示す塩基配列から、STXBP1遺伝子の各エクソン及びその前後300bpのイントロンの領域を抜粋して示したものである。各配列中のエクソン、コード領域及びUTR領域の位置を下記表１に示す。表中、「301-452nt」という表記は、該当する配列番号中の第１番目の塩基から数えて301番目の塩基から452番目の塩基までの領域を表す。エクソン19はアイソフォームaの最終コーディングエクソンであり、アイソフォームbはエクソン19は飛ばしてエクソン20が最終コーディングエクソンとなる。
STXBP1 is a known protein and is known to be involved in the transport and release of synaptic vesicles. In humans, two types of variant mRNAs are generated from the STXBP1 gene genome sequence shown in SEQ ID NO: 65. The sequences of these variants are registered in GenBank as accession numbers NM_003165 (variant 1 / isoform a) and NM_001032221 (variant 2 / isoform b). The amino acid sequences of isoforms a and b are shown in SEQ ID NOs: 2 and 4, and the cDNA sequences are shown in SEQ ID NOs: 1 and 3, respectively. SEQ ID NO: 2 and SEQ ID NO: 4 have the same amino acid sequence in the region from the 1st to the 575th amino acids, and SEQ ID NO: 1 and SEQ ID NO: 3 are the 1st to 1703th bases. The base sequence is the same in this region. SEQ ID NOs: 45 to 64 are excerpts of each exon of the STXBP1 gene and a 300 bp intron region before and after that from the base sequence shown in SEQ ID NO: 65. The positions of exons, coding regions and UTR regions in each sequence are shown in Table 1 below. In the table, the notation “301-452nt” represents the region from the 301st base to the 452nd base counted from the first base in the corresponding SEQ ID NO. Exon 19 is the final coding exon of isoform a, and isoform b is exon 19 skipped and exon 20 becomes the final coding exon.
本発明において、「正常型STXBP1」としては、野生型STXBP1である上記２種類のアイソフォームがあり、これらの他にも、該アイソフォームと同様の生理活性を示す天然の変異体が存在すればそれらも包含される。一方、「異常型STXBP1」とは、STXBP1の天然の変異体であって、STXBP1としての活性が変化又は消失したものをいう。そのような異常型STXBP1の例としては、後述する４種類のミスセンス変異を挙げることができ、これらのほかにも、例えば、短縮型の（truncated）STXBP1、活性に重要なアミノ酸に置換又は欠失を生じたSTXBP1、活性に重要な領域にアミノ酸の挿入が生じたSTXBP1等の天然の変異体が存在すればそれらも包含される。なお、本明細書及び特許請求の範囲において、単に「STXBP1」と言った場合には、文脈からそうではないことが明らかな場合を除き、正常型STXBP1と異常型STXBP1との両者を包含するものとする。
In the present invention, “normal STXBP1” includes the above-mentioned two types of isoforms that are wild-type STXBP1, and in addition to these, if there is a natural mutant that exhibits the same physiological activity as the isoform. They are also included. On the other hand, “abnormal STXBP1” refers to a natural variant of STXBP1, in which the activity as STXBP1 is changed or eliminated. Examples of such anomalous STXBP1 include the four types of missense mutations described below. Besides these, for example, truncated STXBP1, substitution or deletion of amino acids important for activity If natural mutants such as STXBP1 that produces amino acids, STXBP1 in which amino acid insertion occurs in a region important for activity, these are also included. In the present specification and claims, the term “STXBP1” includes both normal STXBP1 and abnormal STXBP1, unless it is clear from the context. And
異常型STXBP1の発現をもたらす変異としては、例えば、ミスセンス変異やナンセンス変異が挙げられるが、これらに限定されない。本発明で指標とし得るミスセンス変異の例としては、下記実施例で同定された以下(1)～(4)の変異が挙げられる。なお、変異の位置は、野生型STXBP1の１つであるアイソフォームaの塩基配列及びアミノ酸配列、すなわち配列番号１及び２を基準として表したものであり、「aa84」とは配列番号２中の第84番アミノ酸、「251nt」とは配列番号１中の第251番塩基を表す。
(1) aa84がバリンからアスパラギン酸になる変異（251ntがtからaに変異）
(2) aa180がシステインからチロシンになる変異（539ntがgからaになる変異）
(3) aa443がメチオニンからアルギニンになる変異（1328ntがtからgになる変異）
(4) aa544がグリシンからアスパラギン酸になる変異（1631ntがgからaになる変異） Examples of mutations that cause abnormal STXBP1 expression include, but are not limited to, missense mutations and nonsense mutations. Examples of the missense mutation that can be used as an index in the present invention include the following mutations (1) to (4) identified in the following Examples. The position of the mutation is expressed based on the base sequence and amino acid sequence of isoform a, which is one of wild-type STXBP1, that is, SEQ ID NOs: 1 and 2, and “aa84” is represented in SEQ ID NO: 2. The 84th amino acid, “251nt”, represents the 251st base in SEQ ID NO: 1.
(1) Aa84 changes from valine to aspartic acid (251 nt changes from t to a)
(2) Aa180 changes from cysteine to tyrosine (539nt changes from g to a)
(3) Mutation that aa443 changes from methionine to arginine (1328nt changes from t to g)
(4) Mutation that aa544 changes from glycine to aspartic acid (mutation that changes 1631nt from g to a)
(1) aa84がバリンからアスパラギン酸になる変異（251ntがtからaに変異）
(2) aa180がシステインからチロシンになる変異（539ntがgからaになる変異）
(3) aa443がメチオニンからアルギニンになる変異（1328ntがtからgになる変異）
(4) aa544がグリシンからアスパラギン酸になる変異（1631ntがgからaになる変異） Examples of mutations that cause abnormal STXBP1 expression include, but are not limited to, missense mutations and nonsense mutations. Examples of the missense mutation that can be used as an index in the present invention include the following mutations (1) to (4) identified in the following Examples. The position of the mutation is expressed based on the base sequence and amino acid sequence of isoform a, which is one of wild-type STXBP1, that is, SEQ ID NOs: 1 and 2, and “aa84” is represented in SEQ ID NO: 2. The 84th amino acid, “251nt”, represents the 251st base in SEQ ID NO: 1.
(1) Aa84 changes from valine to aspartic acid (251 nt changes from t to a)
(2) Aa180 changes from cysteine to tyrosine (539nt changes from g to a)
(3) Mutation that aa443 changes from methionine to arginine (1328nt changes from t to g)
(4) Mutation that aa544 changes from glycine to aspartic acid (mutation that changes 1631nt from g to a)
これらのうちの少なくともいずれか１つの変異が見つかれば、該生体は新生児期～乳児期発症の難治性てんかんを罹患している又は発症するおそれがあると考えられるが、本発明で指標とし得る変異はこれらに限定されない。すなわち、下記実施例に記載される通り、これらのミスセンス変異は、野生型STXBP1の立体構造（Protein Data Bank ID, 1DN1）において、折りたたまれた構造の内部に位置するアミノ酸で生じているが、上記以外の部位であっても、同様に折りたたみ構造内部等の立体構造の維持に重要な位置のアミノ酸に置換が生じれば、活性が変化又は消失して異常型STXBP1となり得るため、本発明の検出方法において指標とし得る。野生型STXBP1遺伝子のコード領域の配列及びゲノム配列は、配列番号１及び３並びに配列番号６５に示され、GenBankにも登録されている通り公知である。また、アミノ酸をコードするコドンも公知である。従って、このようなミスセンス変異を生じる塩基置換は、下記実施例で同定された塩基置換に限定されない。
If at least one of these mutations is found, it is considered that the living body suffers from or is likely to develop refractory epilepsy that begins in the neonatal period to infancy, but the mutation that can be used as an index in the present invention Is not limited to these. That is, as described in the Examples below, these missense mutations occur in the amino acid located inside the folded structure in the three-dimensional structure of wild type STXBP1 (Protein Data Bank ID, 1DN1). Similarly, even if it is a site other than the above, if substitution occurs in the amino acid at a position important for maintaining the three-dimensional structure such as the inside of the folding structure, the activity may be changed or lost, and abnormal STXBP1 may be obtained. It can be an indicator in the method. The sequence of the coding region and the genome sequence of the wild type STXBP1 gene are shown in SEQ ID NOs: 1 and 3 and SEQ ID NO: 65, and are known as registered in GenBank. Also, codons encoding amino acids are known. Therefore, the base substitution that causes such a missense mutation is not limited to the base substitution identified in the following Examples.
生体から分離した試料を用いて、(1)STXBP1遺伝子が欠失しているか否か、(2)異常型STXBP1をコードする遺伝子が存在するか否か、の少なくともいずれかを調べる方法としては、例えば、以下に記載するとおり、ゲノムDNA試料を解析する方法やmRNA試料を解析する方法が挙げられる。また、生体から分離したタンパク質試料について、STXBP1タンパク質が欠失しているか否かや、異常型STXBP1タンパク質が存在するか否かを調べることによっても、STXBP1遺伝子の欠失や異常型STXBP1をコードする遺伝子の存在を調べることができる。これらの方法のうち、本発明の方法としては、ゲノムDNA試料を用いてSTXBP1遺伝子の欠失や異常型STXBP1をコードする遺伝子の存在を調べる方法が好ましい。
Using a sample isolated from a living body, as a method of examining at least one of (1) whether the STXBP1 gene is deleted or (2) the presence of a gene encoding an abnormal STXBP1, For example, as described below, a method of analyzing a genomic DNA sample and a method of analyzing an mRNA sample can be mentioned. It is also possible to encode STXBP1 gene deletion or abnormal STXBP1 by examining whether the STXBP1 protein is deleted or whether abnormal STXBP1 protein is present in protein samples isolated from living organisms. The presence of a gene can be examined. Among these methods, the method of the present invention is preferably a method for examining the presence of a gene encoding STXBP1 gene deletion or abnormal STXBP1 using a genomic DNA sample.
ゲノムDNA試料を用いて実施する方法としては、例えば以下の(ア)～(エ)の方法を挙げることができるが、これらに限定されない。
Examples of the method performed using a genomic DNA sample include, but are not limited to, the following methods (a) to (d).
(ア) in situハイブリダイゼーション法
対象生体から細胞を採取し、染色体標本試料を調製する。STXBP1遺伝子領域と特異的にハイブリダイズするDNAを標識してプローブを作製し、該プローブを上記染色体標本とハイブリダイズさせる。プローブからのシグナルの有無を調べることにより、STXBP1遺伝子の欠失を検出することができる。DNAの標識は、特に限定されないが、通常、ラジオアイソトープ又は蛍光色素（Cy5、Cy3、FITC等）を用いて行なわれ、蛍光色素がより一般的に用いられている。蛍光標識プローブを用いる場合、この手法はFISH法と呼ばれる。STXBP1遺伝子領域と特異的にハイブリダイズするDNAプローブは、当業者であれば、配列表の配列番号６５に示すSTXBP1遺伝子ゲノム配列を参照して容易に調製することができる。具体的には、例えば配列番号６５中の所望の領域を増幅できるプライマーを調製し、正常なSTXBP1遺伝子を含むゲノムDNAを鋳型としてPCRを行なうことにより、プローブに用いるDNAを得ることができる。また、下記実施例に記載されるように、STXBP1遺伝子領域を含むBACクローン等のクローンを標識してプローブとして用いることもできる。ヒトゲノムDNAを含むBACクローン等のクローンは市販もされており、入手は容易である。プローブに用いるDNAは、STXBP1遺伝子のコード領域の全領域をカバーするものであってもよいし、コード領域の一部のみをカバーするものであってもよい。 (A) In situ hybridization method Cells are collected from the target organism and a chromosome sample is prepared. A probe is prepared by labeling DNA that specifically hybridizes with the STXBP1 gene region, and the probe is hybridized with the chromosome specimen. Deletion of the STXBP1 gene can be detected by examining the presence or absence of a signal from the probe. The labeling of DNA is not particularly limited, but is usually performed using a radioisotope or a fluorescent dye (Cy5, Cy3, FITC, etc.), and a fluorescent dye is more commonly used. When a fluorescently labeled probe is used, this method is called FISH method. A DNA probe that specifically hybridizes to the STXBP1 gene region can be easily prepared by those skilled in the art with reference to the STXBP1 gene genome sequence shown in SEQ ID NO: 65 of the Sequence Listing. Specifically, for example, by preparing a primer capable of amplifying a desired region in SEQ ID NO: 65 and performing PCR using a genomic DNA containing a normal STXBP1 gene as a template, DNA used for the probe can be obtained. Further, as described in the Examples below, clones such as BAC clones containing the STXBP1 gene region can be labeled and used as probes. Clones such as BAC clones containing human genomic DNA are also commercially available and are readily available. The DNA used for the probe may cover the entire coding region of the STXBP1 gene or may cover only a part of the coding region.
対象生体から細胞を採取し、染色体標本試料を調製する。STXBP1遺伝子領域と特異的にハイブリダイズするDNAを標識してプローブを作製し、該プローブを上記染色体標本とハイブリダイズさせる。プローブからのシグナルの有無を調べることにより、STXBP1遺伝子の欠失を検出することができる。DNAの標識は、特に限定されないが、通常、ラジオアイソトープ又は蛍光色素（Cy5、Cy3、FITC等）を用いて行なわれ、蛍光色素がより一般的に用いられている。蛍光標識プローブを用いる場合、この手法はFISH法と呼ばれる。STXBP1遺伝子領域と特異的にハイブリダイズするDNAプローブは、当業者であれば、配列表の配列番号６５に示すSTXBP1遺伝子ゲノム配列を参照して容易に調製することができる。具体的には、例えば配列番号６５中の所望の領域を増幅できるプライマーを調製し、正常なSTXBP1遺伝子を含むゲノムDNAを鋳型としてPCRを行なうことにより、プローブに用いるDNAを得ることができる。また、下記実施例に記載されるように、STXBP1遺伝子領域を含むBACクローン等のクローンを標識してプローブとして用いることもできる。ヒトゲノムDNAを含むBACクローン等のクローンは市販もされており、入手は容易である。プローブに用いるDNAは、STXBP1遺伝子のコード領域の全領域をカバーするものであってもよいし、コード領域の一部のみをカバーするものであってもよい。 (A) In situ hybridization method Cells are collected from the target organism and a chromosome sample is prepared. A probe is prepared by labeling DNA that specifically hybridizes with the STXBP1 gene region, and the probe is hybridized with the chromosome specimen. Deletion of the STXBP1 gene can be detected by examining the presence or absence of a signal from the probe. The labeling of DNA is not particularly limited, but is usually performed using a radioisotope or a fluorescent dye (Cy5, Cy3, FITC, etc.), and a fluorescent dye is more commonly used. When a fluorescently labeled probe is used, this method is called FISH method. A DNA probe that specifically hybridizes to the STXBP1 gene region can be easily prepared by those skilled in the art with reference to the STXBP1 gene genome sequence shown in SEQ ID NO: 65 of the Sequence Listing. Specifically, for example, by preparing a primer capable of amplifying a desired region in SEQ ID NO: 65 and performing PCR using a genomic DNA containing a normal STXBP1 gene as a template, DNA used for the probe can be obtained. Further, as described in the Examples below, clones such as BAC clones containing the STXBP1 gene region can be labeled and used as probes. Clones such as BAC clones containing human genomic DNA are also commercially available and are readily available. The DNA used for the probe may cover the entire coding region of the STXBP1 gene or may cover only a part of the coding region.
(イ) サザンハイブリダイゼーション法
対象生体から得たゲノムDNA試料を任意の制限酵素で切断後、アガロースゲル等で電気泳動し、メンブレンにDNAを転写する。このメンブレン上で、STXBP1遺伝子領域と特異的にハイブリダイズするDNAを標識して調製したDNAプローブをハイブリダイズさせる。検出されるバンドの有無を調べることにより、STXBP1遺伝子の欠失を検出することができる。また、例えば点変異のような変異であっても、制限酵素部位に変化を生じる変異である場合には、検出されるバンドのサイズが変化するため、該方法で検出し得る。プローブの標識は、特に限定されないが、通常、ラジオアイソトープやジゴキシゲニン等のハプテンを用いて行なわれる。ここでプローブとして用いるDNAの調製方法等は(ア)における説明と同様である。 (B) Southern hybridization method A genomic DNA sample obtained from a target organism is cleaved with an arbitrary restriction enzyme and then electrophoresed on an agarose gel or the like to transfer the DNA onto a membrane. On this membrane, a DNA probe prepared by labeling a DNA that specifically hybridizes with the STXBP1 gene region is hybridized. Deletion of the STXBP1 gene can be detected by examining the presence or absence of the detected band. For example, even a mutation such as a point mutation can be detected by this method because the size of the detected band changes when the mutation causes a change in the restriction enzyme site. The labeling of the probe is not particularly limited, but is usually performed using a hapten such as a radioisotope or digoxigenin. Here, the preparation method of DNA used as a probe is the same as described in (a).
対象生体から得たゲノムDNA試料を任意の制限酵素で切断後、アガロースゲル等で電気泳動し、メンブレンにDNAを転写する。このメンブレン上で、STXBP1遺伝子領域と特異的にハイブリダイズするDNAを標識して調製したDNAプローブをハイブリダイズさせる。検出されるバンドの有無を調べることにより、STXBP1遺伝子の欠失を検出することができる。また、例えば点変異のような変異であっても、制限酵素部位に変化を生じる変異である場合には、検出されるバンドのサイズが変化するため、該方法で検出し得る。プローブの標識は、特に限定されないが、通常、ラジオアイソトープやジゴキシゲニン等のハプテンを用いて行なわれる。ここでプローブとして用いるDNAの調製方法等は(ア)における説明と同様である。 (B) Southern hybridization method A genomic DNA sample obtained from a target organism is cleaved with an arbitrary restriction enzyme and then electrophoresed on an agarose gel or the like to transfer the DNA onto a membrane. On this membrane, a DNA probe prepared by labeling a DNA that specifically hybridizes with the STXBP1 gene region is hybridized. Deletion of the STXBP1 gene can be detected by examining the presence or absence of the detected band. For example, even a mutation such as a point mutation can be detected by this method because the size of the detected band changes when the mutation causes a change in the restriction enzyme site. The labeling of the probe is not particularly limited, but is usually performed using a hapten such as a radioisotope or digoxigenin. Here, the preparation method of DNA used as a probe is the same as described in (a).
(ウ) ヘテロ二本鎖の検出による遺伝子変異スクリーニング
de novoで生じる点変異等の突然変異は通常ヘテロ接合体の形で見られるため、ゲノムDNA試料を熱変性後に再会合させることにより、正常型DNAと変異DNAとがハイブリダイズしたヘテロ二本鎖が生じる。ヘテロ二本鎖は、(1)非変性ポリアクリルアミドゲル中で異なる移動度を示す、(2)ミスマッチ部分の塩基は化学物質や酵素による切断を受けやすい、(3)変性の際に異なる変性温度を示す、といった特性を有する。これらの特性を利用してヘテロ二本鎖を検出する方法がこの分野において公知であり、変異の検査方法として実用化もされている。具体的には、例えば、変性高速液体クロマトグラフィー（dHPLC）を用いてヘテロ二本鎖を検出する方法や、下記実施例に具体的に記載されるHigh Resolution Melt法が知られている。High Resolution Melt法とは、二本鎖DNAに高密度で結合する蛍光色素（SYTO(登録商標)9, LC Green(登録商標), EvaGreen(商標)等）を用いて、二本鎖DNAの融解（熱変性）の過程を蛍光強度の変化としてとらえ、ヘテロ二本鎖を検出する方法である。すなわち、二本鎖DNAに高密度で結合する蛍光色素を用いて二本鎖DNAを染色すると、該二本鎖DNAを融解（熱変性）させたとき、二本鎖が解離した部位から蛍光色素が脱落するため、二本鎖DNAからの蛍光シグナルの量が減少する。従って、そのような蛍光色素を用いることで、二本鎖DNAの熱変性の過程を蛍光強度の変化として視覚的にとらえることができる。温度－蛍光のデータを高密度で取得し解析することで、ヘテロ二本鎖の検出を迅速に高感度で行うことができる。本発明においても、正常型STXBP1を発現できないポリヌクレオチドが存在するか否かを調べる手法として、これらの公知の方法を用いることができる。ヘテロ二本鎖を検出するこれらの方法は、点変異を検出する方法として特に好ましい方法である。本発明においてHigh Resolution Melt法を用いる場合には、使用するプライマーは、当業者であれば配列番号４５～６４又は配列番号６５に示す塩基配列を参照して容易に調製可能であり、例えば下記実施例で用いられている配列番号５～４４に示される塩基配列から成るプライマーを好ましく用いることができる。 (C) Genetic mutation screening by detection of heteroduplexes Since mutations such as point mutations that occur in de novo are usually seen in the form of heterozygotes, normal DNA can be obtained by reassociating genomic DNA samples after heat denaturation. A heteroduplex in which DNA and mutant DNA are hybridized is generated. Heteroduplexes (1) show different mobility in non-denaturing polyacrylamide gels, (2) mismatched bases are susceptible to cleavage by chemicals and enzymes, (3) different denaturation temperatures during denaturation It has the characteristic that shows. Methods for detecting heteroduplexes using these characteristics are known in this field, and have been put into practical use as mutation testing methods. Specifically, for example, a method for detecting heteroduplexes using denaturing high performance liquid chromatography (dHPLC) and a High Resolution Melt method specifically described in the following examples are known. The High Resolution Melt method is the melting of double-stranded DNA using a fluorescent dye (SYTO (registered trademark) 9, LC Green (registered trademark), EvaGreen (registered trademark), etc.) that binds to double-stranded DNA at high density. This is a method of detecting heteroduplexes by treating the process of (thermal denaturation) as a change in fluorescence intensity. That is, when a double-stranded DNA is stained with a fluorescent dye that binds to the double-stranded DNA at a high density, when the double-stranded DNA is melted (thermally denatured), the fluorescent dye is drawn from the site where the double strand is dissociated Will drop off, reducing the amount of fluorescent signal from double-stranded DNA. Therefore, by using such a fluorescent dye, the process of heat denaturation of double-stranded DNA can be visually recognized as a change in fluorescence intensity. By acquiring and analyzing temperature-fluorescence data at high density, heteroduplex detection can be performed quickly and with high sensitivity. Also in the present invention, these known methods can be used as a method for examining whether or not there is a polynucleotide that cannot express normal STXBP1. These methods for detecting heteroduplexes are particularly preferred methods for detecting point mutations. When using the High Resolution Melt method in the present invention, those skilled in the art can easily prepare primers to be used with reference to the nucleotide sequences shown in SEQ ID NOs: 45 to 64 or SEQ ID NO: 65. Primers consisting of the base sequences shown in SEQ ID NOs: 5-44 used in the examples can be preferably used.
de novoで生じる点変異等の突然変異は通常ヘテロ接合体の形で見られるため、ゲノムDNA試料を熱変性後に再会合させることにより、正常型DNAと変異DNAとがハイブリダイズしたヘテロ二本鎖が生じる。ヘテロ二本鎖は、(1)非変性ポリアクリルアミドゲル中で異なる移動度を示す、(2)ミスマッチ部分の塩基は化学物質や酵素による切断を受けやすい、(3)変性の際に異なる変性温度を示す、といった特性を有する。これらの特性を利用してヘテロ二本鎖を検出する方法がこの分野において公知であり、変異の検査方法として実用化もされている。具体的には、例えば、変性高速液体クロマトグラフィー（dHPLC）を用いてヘテロ二本鎖を検出する方法や、下記実施例に具体的に記載されるHigh Resolution Melt法が知られている。High Resolution Melt法とは、二本鎖DNAに高密度で結合する蛍光色素（SYTO(登録商標)9, LC Green(登録商標), EvaGreen(商標)等）を用いて、二本鎖DNAの融解（熱変性）の過程を蛍光強度の変化としてとらえ、ヘテロ二本鎖を検出する方法である。すなわち、二本鎖DNAに高密度で結合する蛍光色素を用いて二本鎖DNAを染色すると、該二本鎖DNAを融解（熱変性）させたとき、二本鎖が解離した部位から蛍光色素が脱落するため、二本鎖DNAからの蛍光シグナルの量が減少する。従って、そのような蛍光色素を用いることで、二本鎖DNAの熱変性の過程を蛍光強度の変化として視覚的にとらえることができる。温度－蛍光のデータを高密度で取得し解析することで、ヘテロ二本鎖の検出を迅速に高感度で行うことができる。本発明においても、正常型STXBP1を発現できないポリヌクレオチドが存在するか否かを調べる手法として、これらの公知の方法を用いることができる。ヘテロ二本鎖を検出するこれらの方法は、点変異を検出する方法として特に好ましい方法である。本発明においてHigh Resolution Melt法を用いる場合には、使用するプライマーは、当業者であれば配列番号４５～６４又は配列番号６５に示す塩基配列を参照して容易に調製可能であり、例えば下記実施例で用いられている配列番号５～４４に示される塩基配列から成るプライマーを好ましく用いることができる。 (C) Genetic mutation screening by detection of heteroduplexes Since mutations such as point mutations that occur in de novo are usually seen in the form of heterozygotes, normal DNA can be obtained by reassociating genomic DNA samples after heat denaturation. A heteroduplex in which DNA and mutant DNA are hybridized is generated. Heteroduplexes (1) show different mobility in non-denaturing polyacrylamide gels, (2) mismatched bases are susceptible to cleavage by chemicals and enzymes, (3) different denaturation temperatures during denaturation It has the characteristic that shows. Methods for detecting heteroduplexes using these characteristics are known in this field, and have been put into practical use as mutation testing methods. Specifically, for example, a method for detecting heteroduplexes using denaturing high performance liquid chromatography (dHPLC) and a High Resolution Melt method specifically described in the following examples are known. The High Resolution Melt method is the melting of double-stranded DNA using a fluorescent dye (SYTO (registered trademark) 9, LC Green (registered trademark), EvaGreen (registered trademark), etc.) that binds to double-stranded DNA at high density. This is a method of detecting heteroduplexes by treating the process of (thermal denaturation) as a change in fluorescence intensity. That is, when a double-stranded DNA is stained with a fluorescent dye that binds to the double-stranded DNA at a high density, when the double-stranded DNA is melted (thermally denatured), the fluorescent dye is drawn from the site where the double strand is dissociated Will drop off, reducing the amount of fluorescent signal from double-stranded DNA. Therefore, by using such a fluorescent dye, the process of heat denaturation of double-stranded DNA can be visually recognized as a change in fluorescence intensity. By acquiring and analyzing temperature-fluorescence data at high density, heteroduplex detection can be performed quickly and with high sensitivity. Also in the present invention, these known methods can be used as a method for examining whether or not there is a polynucleotide that cannot express normal STXBP1. These methods for detecting heteroduplexes are particularly preferred methods for detecting point mutations. When using the High Resolution Melt method in the present invention, those skilled in the art can easily prepare primers to be used with reference to the nucleotide sequences shown in SEQ ID NOs: 45 to 64 or SEQ ID NO: 65. Primers consisting of the base sequences shown in SEQ ID NOs: 5-44 used in the examples can be preferably used.
(エ) 塩基配列解析
遺伝子変異を詳細に調べるためには、塩基配列の解析を行なうことが望ましい。対象生体ゲノムDNA上のSTXBP1遺伝子の塩基配列を決定し、これを野生型STXBP1遺伝子配列と比較することにより、変異を詳細に同定できる。決定した塩基配列は、例えばSeqScape (登録商標) 等の公知のソフトウェアを用いて解析することにより、変異の検出やプロファイリングを容易に行うことができる。 (D) Base sequence analysis In order to examine gene mutations in detail, it is desirable to perform base sequence analysis. Mutations can be identified in detail by determining the base sequence of the STXBP1 gene on the target biological genomic DNA and comparing it with the wild-type STXBP1 gene sequence. By analyzing the determined base sequence using known software such as SeqScape (registered trademark), mutation detection and profiling can be easily performed.
遺伝子変異を詳細に調べるためには、塩基配列の解析を行なうことが望ましい。対象生体ゲノムDNA上のSTXBP1遺伝子の塩基配列を決定し、これを野生型STXBP1遺伝子配列と比較することにより、変異を詳細に同定できる。決定した塩基配列は、例えばSeqScape (登録商標) 等の公知のソフトウェアを用いて解析することにより、変異の検出やプロファイリングを容易に行うことができる。 (D) Base sequence analysis In order to examine gene mutations in detail, it is desirable to perform base sequence analysis. Mutations can be identified in detail by determining the base sequence of the STXBP1 gene on the target biological genomic DNA and comparing it with the wild-type STXBP1 gene sequence. By analyzing the determined base sequence using known software such as SeqScape (registered trademark), mutation detection and profiling can be easily performed.
上記した(ア)～(エ)の方法は、適宜組み合わせて行なうことができる。例えば、まず(ア)及び／又は(イ)により、ゲノムDNA試料中にSTXBP1遺伝子領域が存在するかどうかを調べる。存在する場合には(エ)を行なってSTXBP1遺伝子領域中の変異の有無を調べる。 (ウ)により塩基配列を決定すべき領域を絞り込んで(エ)を行なうとより効率的に検査が可能である。
The methods (a) to (d) described above can be combined as appropriate. For example, first, by (a) and / or (b), it is examined whether or not the STXBP1 gene region is present in the genomic DNA sample. If it exists, perform (d) to check for mutations in the STXBP1 gene region. The inspection can be performed more efficiently by narrowing down the region where the base sequence should be determined by (c) and performing (d).
また、mRNA試料を用いて本発明を実施する方法としては、例えば以下に述べる方法が挙げられるが、これらに限定されない。STXBP1のmRNAの有無は、例えば、配列番号１又は３に示す塩基配列を基に作製したプローブやプライマーを用いてノーザンハイブリダイゼーション法やRT-PCR法を行なうことで容易に調べることができる。STXBP1のmRNAが検出されない場合にはSTXBP1遺伝子が欠失していると判断され、難治性てんかんが検出されたと判断できる。また、mRNA試料から逆転写反応により得たcDNAの塩基配列を上記(エ)に述べたように解析することで、変異を詳細に同定することができる。STXBP1のcDNAに対して上記(ウ)の方法を行なっても差し支えないが、STXBP1のcDNAは、配列番号１及び３に示す通り2kbp以下と比較的短いため、上記(ウ)の方法を行なって塩基配列を決定する領域を絞り込む必要性は低い。ノーザンハイブリダイゼーションやRT-PCR自体は周知の常法であり、当業者であれば配列番号１又は３に示す塩基配列をもとに容易にプローブやプライマーを作製することができる。
In addition, examples of a method for carrying out the present invention using an mRNA sample include, but are not limited to, the methods described below. The presence or absence of STXBP1 mRNA can be easily examined, for example, by performing a Northern hybridization method or RT-PCR method using a probe or primer prepared based on the nucleotide sequence shown in SEQ ID NO: 1 or 3. If STXBP1 mRNA is not detected, it is determined that the STXBP1 gene has been deleted, and it can be determined that intractable epilepsy has been detected. In addition, mutations can be identified in detail by analyzing the base sequence of cDNA obtained by reverse transcription reaction from mRNA samples as described in (d) above. Although the method (c) above may be performed on the STXBP1 cDNA, the cDNA of STXBP1 is relatively short as 2 kbp or less as shown in SEQ ID NOS: 1 and 3, so the method (c) above is performed. The necessity to narrow down the region for determining the base sequence is low. Northern hybridization and RT-PCR itself are well-known conventional methods, and those skilled in the art can easily prepare probes and primers based on the nucleotide sequence shown in SEQ ID NO: 1 or 3.
以下、本発明を実施例に基づきより具体的に説明する。
Hereinafter, the present invention will be described more specifically based on examples.
１．新生児期～乳児期発症の難治性てんかんに合併する染色体異常のスクリーニング
全ゲノム解析用の4200個のBACクローンを搭載したアレイを用いたCGH（comparative genomic hybridization）法により、乳児期に難治性てんかんを発症した女児患者（患者１）について染色体異常の解析を行なった。患者又は健常者から得た末梢血白血球よりゲノムDNAを採取し、(1)患者DNAをCy5標識し健常者DNAをCy3標識（CGH1）、(2)患者DNAをCy3標識し健常者DNAをCy5標識（CGH2）の2通りでプローブを調製した。これらのプローブを用いて本願発明者らの研究室で作成した4200BACアレイCGHを行い、GenePix4000B(AXON社製)でスキャン後、GenePixPro6.0(AXON社製)で数値化を行い、常法により解析を行なった。その結果、図1Aに示す通り、該患者１において第９番染色体の9q33.3-q34.11の領域にかけて微細欠失があることが判明した。 1. Screening for chromosomal abnormalities associated with intractable epilepsy from neonatal to infancy The CGH (comparative genomic hybridization) method using an array of 4200 BAC clones for genome-wide analysis is used to treat intractable epilepsy during infancy Chromosome abnormalities were analyzed for the onset girl patient (patient 1). Genomic DNA is collected from peripheral blood leukocytes obtained from patients or healthy subjects. (1) Patient DNA is Cy5 labeled and healthy subject DNA is Cy3 labeled (CGH1), (2) Patient DNA is Cy3 labeled and healthy subject DNA is Cy5 Probes were prepared in two ways, labeled (CGH2). Using these probes, we performed 4200BAC array CGH created in our laboratory, scanned with GenePix4000B (AXON), digitized with GenePixPro6.0 (AXON), and analyzed by conventional methods Was done. As a result, as shown in FIG. 1A, it was found that there was a fine deletion in the region of 9q33.3-q34.11 of chromosome 9 in the patient 1.
全ゲノム解析用の4200個のBACクローンを搭載したアレイを用いたCGH（comparative genomic hybridization）法により、乳児期に難治性てんかんを発症した女児患者（患者１）について染色体異常の解析を行なった。患者又は健常者から得た末梢血白血球よりゲノムDNAを採取し、(1)患者DNAをCy5標識し健常者DNAをCy3標識（CGH1）、(2)患者DNAをCy3標識し健常者DNAをCy5標識（CGH2）の2通りでプローブを調製した。これらのプローブを用いて本願発明者らの研究室で作成した4200BACアレイCGHを行い、GenePix4000B(AXON社製)でスキャン後、GenePixPro6.0(AXON社製)で数値化を行い、常法により解析を行なった。その結果、図1Aに示す通り、該患者１において第９番染色体の9q33.3-q34.11の領域にかけて微細欠失があることが判明した。 1. Screening for chromosomal abnormalities associated with intractable epilepsy from neonatal to infancy The CGH (comparative genomic hybridization) method using an array of 4200 BAC clones for genome-wide analysis is used to treat intractable epilepsy during infancy Chromosome abnormalities were analyzed for the onset girl patient (patient 1). Genomic DNA is collected from peripheral blood leukocytes obtained from patients or healthy subjects. (1) Patient DNA is Cy5 labeled and healthy subject DNA is Cy3 labeled (CGH1), (2) Patient DNA is Cy3 labeled and healthy subject DNA is Cy5 Probes were prepared in two ways, labeled (CGH2). Using these probes, we performed 4200BAC array CGH created in our laboratory, scanned with GenePix4000B (AXON), digitized with GenePixPro6.0 (AXON), and analyzed by conventional methods Was done. As a result, as shown in FIG. 1A, it was found that there was a fine deletion in the region of 9q33.3-q34.11 of chromosome 9 in the patient 1.
次いで、この微細欠失の領域をより詳細に調べるため、白血球染色体標本を用いたin situハイブリダイゼーション法を行なった。患者１及びその両親から末梢血白血球を採取して染色体標本を作製し、9q33.3-q34.11の領域に位置する複数のBACクローンを標識して調製したプローブを該標本にハイブリダイズさせて蛍光顕微鏡下で観察した。その結果、BACクローン516d6、936o12、24k1、152i20、及び99f23をプローブとした場合には、2本ある相同染色体のうち一方においてシグナルが検出されず、微細欠失は約2.1Mbの領域であることが判明した（図1B）。両親の染色体ではこの微細欠失は認められなかった。すなわち、該患者で認められた染色体微細欠失はde novo変異（新生突然変異）であり、難治性てんかんの原因となっている可能性が強く示唆された。
Next, in situ hybridization using a leukocyte chromosome specimen was performed in order to examine the region of this fine deletion in more detail. Chromosome specimens are prepared by collecting peripheral blood leukocytes from patient 1 and their parents, and a probe prepared by labeling multiple BAC clones located in the region of 9q33.3-q34.11 is hybridized to the specimen. Observed under a fluorescence microscope. As a result, when BAC clones 516d6, 936o12, 24k1, 152i20, and 99f23 were used as probes, no signal was detected in one of the two homologous chromosomes, and the fine deletion was a region of about 2.1 Mb. Was found (Fig. 1B). This microdeletion was not found in the parental chromosomes. That is, the chromosomal microdeletion observed in the patient was a de novo mutation (new mutation), strongly suggesting that it may cause intractable epilepsy.
２．STXBP1遺伝子の変異解析
上記欠失領域には脳で発現している遺伝子が複数あったが、そのうち、シンタキシン結合タンパク質１（syntaxin binding protein 1; STXBP1、別名Munc18-1、GeneBankアクセッション番号NM_003165（バリアント１）、NM_001032221（バリアント２）)を候補遺伝子として、新生児～乳児期発症の難治性てんかん患者58名で以下のとおり変異解析を行った。 2. Mutation analysis of STXBP1 gene There were several genes expressed in the brain in the above deletion region. Among them, syntaxin binding protein 1 (STXBP1, also known as Munc18-1, GeneBank accession number NM_003165 (variant 1) NM_001032221 (variant 2)) was used as a candidate gene, and mutation analysis was performed as follows in 58 patients with intractable epilepsy that developed from neonatal to infancy.
上記欠失領域には脳で発現している遺伝子が複数あったが、そのうち、シンタキシン結合タンパク質１（syntaxin binding protein 1; STXBP1、別名Munc18-1、GeneBankアクセッション番号NM_003165（バリアント１）、NM_001032221（バリアント２）)を候補遺伝子として、新生児～乳児期発症の難治性てんかん患者58名で以下のとおり変異解析を行った。 2. Mutation analysis of STXBP1 gene There were several genes expressed in the brain in the above deletion region. Among them, syntaxin binding protein 1 (STXBP1, also known as Munc18-1, GeneBank accession number NM_003165 (variant 1) NM_001032221 (variant 2)) was used as a candidate gene, and mutation analysis was performed as follows in 58 patients with intractable epilepsy that developed from neonatal to infancy.
患者から得た末梢血白血球よりゲノムDNAを採取した。ゲノムDNAは、Genomiphi version 2 (GE healthcare)を用いて全ゲノム増幅させ、この増幅DNAを用いて変異解析を行なった。STXBP1遺伝子（エクソン1-20）のコーディングエクソンおよびエクソン－イントロン境界における変異解析はHigh resolution melt法を用いて行った。これは、蛍光色素二本鎖DNAに結合する蛍光色素を用いてPCR産物の融解（熱変性）の過程を蛍光強度の変化としてとらえ、温度－蛍光のデータを高密度で取得し解析することで、ヘテロ二本鎖の検出を迅速に高感度で行う方法である。リアルタイムPCRおよび引き続いてのHigh resolution melt解析はRoterGene-6000 (Corbett Life Science) を用いて12-μlの反応系で行った。エクソン2から20までは, 1×ExTaq buffer, 0.2 mM each dNTP, 0.2 μM each primer, 1 μl DMSO, 1 μl LCGreen Plus (Idaho Technology), 0.25 U Ex TaqHS polymerase (TAKARA)の組成で反応を行った。反応条件は、95℃1分の熱変性後、95℃10秒、アニーリング20秒、伸長20秒のサイクルとした。エクソン1に関しては, 1×GC buffer II, 0.4 mM each dNTP, 0.2 μM each primer, 1 μl LCGreen Plus (Idaho Technology), 0.5 U LA Taq polymerase (TAKARA)の組成で反応を行った。反応条件は、95℃1分の熱変性後、95℃10秒、62℃30秒の2ステップのサイクルとした。サイクル数はリアルタイムＰＣＲをモニターして適宜決定した。プライマーの塩基配列と反応温度を表２に示す。表２中に記載される各エクソン増幅用プライマーの上段がセンスプライマー、下段がアンチセンスプライマーである。また、各エクソン番号の下に記載した数字は、各エクソン及びその前後300bpのイントロンの配列を記載した配列番号を示している。
Genomic DNA was collected from peripheral blood leukocytes obtained from patients. Genomic DNA was subjected to genome-wide amplification using Genomiphi version 2 (GE healthcare), and mutation analysis was performed using this amplified DNA. Mutation analysis at the coding exon and exon-intron boundary of the STXBP1 gene (exon 1-20) was performed using the High resolution melt method. This is because the fluorescent dye that binds to the fluorescent dye double-stranded DNA is used to treat the melting (thermal denaturation) process of the PCR product as a change in fluorescence intensity, and temperature-fluorescence data is acquired and analyzed at high density. This is a method for detecting heteroduplexes rapidly and with high sensitivity. Real-time PCR and subsequent high-resolution melt analysis were performed in a 12-μl reaction system using RoterGene-6000 (Corbett Life Science). Exons 2 to 20 were reacted with the composition of 1 × ExTaq buffer, 0.2 mM each dNTP, 0.2 μM each primer, 1 μl DMSO, 1 μl LCGreen Plus (Idaho Technology), 0.25 U Ex TaqHS polymerase (TAKARA) . The reaction conditions were 95 ° C for 1 minute after heat denaturation, 95 ° C for 10 seconds, annealing for 20 seconds, and extension for 20 seconds. For exon 1, the reaction was performed with the composition of 1 × GC buffer II, 0.4 mM each dNTP, 0.2 μM each primer, 1 μl LCGreen Plus (Idaho Technology), and 0.5 U LA Taq polymerase (TAKARA). The reaction conditions were a two-step cycle of 95 ° C for 10 seconds and 62 ° C for 30 seconds after heat denaturation at 95 ° C for 1 minute. The number of cycles was determined as appropriate by monitoring real-time PCR. Table 2 shows primer base sequences and reaction temperatures. The upper row of each exon amplification primer described in Table 2 is the sense primer, and the lower row is the antisense primer. The numbers described under each exon number indicate the sequence numbers describing the sequences of each exon and the 300 bp intron around it.
High resolution melt解析でヘテロ二本鎖と判定したサンプルに関しては、ExoSAP-IT (GE healthcare)でPCR産物を精製後、BigDye Terminator chemistry version 3 (Applied Biosystems)を用いてサイクルシークエンス反応を行った。反応物はSephadex G-50 (GE healthcare) とMultiscreen-96 (Millipore)を用いてゲル濾過にて精製し、ABI Genetic Analyzer 3100 (Applied Biosystems)でシークエンスを得た。得られたシークエンスは、SeqScape version 2.1.1 software (Applied Biosystems)を用いて変異の有無について解析を行った。変異が認められたサンプルに関しては、全ゲノム増幅させないゲノムDNAを鋳型とした変異解析を再度行い、ゲノムDNA上での変異を確認した。
For samples determined to be heteroduplex by High resolution melt analysis, PCR products were purified using ExoSAP-IT (GE health care), and then cycle sequencing was performed using BigDye Terminator chemistry version 3 (Applied Biosystems). The reaction product was purified by gel filtration using Sephadex G-50 (GE healthcare) and Multiscreen-96 (Millipore), and a sequence was obtained using ABI Genetic Analyzer 3100 (Applied Biosystems). The obtained sequences were analyzed for the presence or absence of mutation using SeqScape version 2.1.1 software (Applied Biosystems). For samples with mutations, mutation analysis was performed again using genomic DNA that was not amplified as a whole, and mutations on the genomic DNA were confirmed.
その結果、4名の患者でミスセンス変異を認めた：患者2が251T>A, アミノ酸V84D変異, 患者3が539G>A, アミノ酸C180Y変異, 患者4が1328T>G, アミノ酸M443R変異, 患者5が1631G>A, アミノ酸G544D変異（図1B, 2）。これらの変異は対照の健常者250名に認められず、3つの変異については両親のゲノムDNAには見られないde novo変異であることを確認した。1例（患者５）については、父親が既に死亡していたため、母親のみ同変異がないことを確認した。変異はすべて、多種間で高度に保存されたアミノ酸で起こっていた（図2A）。また、Protein Data Bank ID, 1DN1に登録のSTXBP1の3次元立体構造を参照すると、上記変異により置換されるアミノ酸は、構造的に折りたたまれたタンパク質の内部に位置していた。これらのことから、上記した置換がタンパク質の安定性を著しく低下させることが予想された。変異が見つかった患者１ないし５の臨床情報を表３に示す。
As a result, 4 patients had missense mutations: patient 2 was 251T> A, amino acid V84D mutation, patient 3 was 539G> A, amino acid C180Y mutation, patient 4 was 1328T> G, amino acid M443R mutation, patient 5 was 1631G> A, amino acid G544D mutation (Fig. 1B, 2). These mutations were not observed in 250 healthy controls, and three mutations were confirmed to be de novo mutations not found in the parents' genomic DNA. In one case (patient 5), since the father had already died, only the mother confirmed that there was no mutation. All mutations occurred at amino acids that were highly conserved across species (Figure 2A). Moreover, referring to the three-dimensional structure of STXBP1 registered in Protein Data Bank ID, 1DN1, the amino acid substituted by the mutation was located inside the structurally folded protein. From these facts, it was predicted that the above-described substitution significantly reduced the stability of the protein. Table 3 shows clinical information of patients 1 to 5 in which mutations were found.
３．生細胞における変異STXBP1タンパク質の局在
次に我々は、患者で見られたSTXBP1変異の生物学的意義について、マウス神経芽細胞腫であるNeuroblastoma 2A細胞に対する一過性発現系を用いて検討を行った。STXBP1は可溶性蛋白であり、そのアミノ酸配列中に局在シグナルを有していない（Schutz, D., et al., A dual function for Munc-18 in exocytosis of PC12 cells. Eur J Neurosci, 2005. 21(9): p. 2419-32.）。蛍光蛋白であるEGFP蛋白とSTXBP1のキメラ蛋白を作成し、GFPの蛍光をもとに細胞内局在を検討したところ、予想通り核と細胞膜を除く細胞内への分布が認められた（図3）。興味深いことに、患者で認められた4種類の変異についてそれぞれ変異蛋白を作成したところ、一部の細胞で細胞質内での変異蛋白の凝集が認められた（図3）。このことから、新生児～乳児期発症の難治性てんかん患者で認められた変異は蛋白の安定性を低下させ、結果として凝集を引き起こすことが強く示唆された。 3. Localization of mutant STXBP1 protein in living cells Next, we investigated the biological significance of STXBP1 mutations found in patients using a transient expression system for neuroblastoma 2A cells, a mouse neuroblastoma. It was. STXBP1 is a soluble protein and does not have a localization signal in its amino acid sequence (Schutz, D., et al., A dual function for Munc-18 in exocytosis of PC12 cells. Eur J Neurosci, 2005. 21 (9): p. 2419-32.). A chimeric protein of EGFP protein and STXBP1, which are fluorescent proteins, was prepared, and the intracellular localization was examined based on the fluorescence of GFP. As expected, distribution into the cells excluding the nucleus and cell membrane was observed (Fig. 3). ). Interestingly, when mutant proteins were prepared for each of the four types of mutations observed in patients, aggregation of the mutant proteins in the cytoplasm was observed in some cells (Figure 3). This strongly suggests that mutations found in patients with intractable epilepsy from neonatal to infancy reduced protein stability, resulting in aggregation.
次に我々は、患者で見られたSTXBP1変異の生物学的意義について、マウス神経芽細胞腫であるNeuroblastoma 2A細胞に対する一過性発現系を用いて検討を行った。STXBP1は可溶性蛋白であり、そのアミノ酸配列中に局在シグナルを有していない（Schutz, D., et al., A dual function for Munc-18 in exocytosis of PC12 cells. Eur J Neurosci, 2005. 21(9): p. 2419-32.）。蛍光蛋白であるEGFP蛋白とSTXBP1のキメラ蛋白を作成し、GFPの蛍光をもとに細胞内局在を検討したところ、予想通り核と細胞膜を除く細胞内への分布が認められた（図3）。興味深いことに、患者で認められた4種類の変異についてそれぞれ変異蛋白を作成したところ、一部の細胞で細胞質内での変異蛋白の凝集が認められた（図3）。このことから、新生児～乳児期発症の難治性てんかん患者で認められた変異は蛋白の安定性を低下させ、結果として凝集を引き起こすことが強く示唆された。 3. Localization of mutant STXBP1 protein in living cells Next, we investigated the biological significance of STXBP1 mutations found in patients using a transient expression system for neuroblastoma 2A cells, a mouse neuroblastoma. It was. STXBP1 is a soluble protein and does not have a localization signal in its amino acid sequence (Schutz, D., et al., A dual function for Munc-18 in exocytosis of PC12 cells. Eur J Neurosci, 2005. 21 (9): p. 2419-32.). A chimeric protein of EGFP protein and STXBP1, which are fluorescent proteins, was prepared, and the intracellular localization was examined based on the fluorescence of GFP. As expected, distribution into the cells excluding the nucleus and cell membrane was observed (Fig. 3). ). Interestingly, when mutant proteins were prepared for each of the four types of mutations observed in patients, aggregation of the mutant proteins in the cytoplasm was observed in some cells (Figure 3). This strongly suggests that mutations found in patients with intractable epilepsy from neonatal to infancy reduced protein stability, resulting in aggregation.
Claims (5)
- 生体から分離した試料に対して行なう方法であって、STXBP1遺伝子が欠失しているか否か及び／又は異常型STXBP1をコードする遺伝子が存在するか否かを指標とする、新生児期～乳児期発症の難治性てんかんの検出方法。 A method performed on a sample isolated from a living body, wherein the index indicates whether or not the STXBP1 gene is deleted and / or whether or not a gene encoding an abnormal STXBP1 exists. A method for detecting refractory epilepsy.
- 正常型STXBP1は配列表の配列番号２又は４に示されるアミノ酸配列から成る請求項１記載の方法。 The method according to claim 1, wherein normal STXBP1 comprises the amino acid sequence shown in SEQ ID NO: 2 or 4 in the sequence listing.
- 前記試料がゲノムDNAである請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the sample is genomic DNA.
- 前記異常型STXBP1をコードする遺伝子は、ミスセンス変異を有するSTXBP1遺伝子である請求項１ないし３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the gene encoding the abnormal STXBP1 is a STXBP1 gene having a missense mutation.
- 前記ミスセンス変異は少なくとも以下のいずれか一つである請求項４記載の方法。
(1) 野生型STXBP1中のaa84のバリンがアスパラギン酸になる変異、
(2) 野生型STXBP1中のaa180のシステインがチロシンになる変異、
(3) 野生型STXBP1中のaa443のメチオニンがアルギニンになる変異、
(4) 野生型STXBP1中のaa544のグリシンがアスパラギン酸になる変異。 The method according to claim 4, wherein the missense mutation is at least one of the following.
(1) Mutation in which avain valine in wild-type STXBP1 becomes aspartic acid,
(2) Mutation in which cysteine of aa180 in wild-type STXBP1 becomes tyrosine,
(3) Mutation in which methionine of aa443 in wild-type STXBP1 becomes arginine,
(4) Mutation in which aa544 glycine in wild-type STXBP1 becomes aspartic acid.
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| US11891605B2 (en) | 2014-06-16 | 2024-02-06 | University Of Southampton | Reducing intron retention |
| US10538764B2 (en) | 2014-06-16 | 2020-01-21 | University Of Southampton | Reducing intron retention |
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| US10941405B2 (en) | 2015-10-09 | 2021-03-09 | University Of Southampton | Modulation of gene expression and screening for deregulated protein expression |
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