JP2013034451A

JP2013034451A - Method for predicting severe moyamoya disease by using rnf213 genetic polymorphism

Info

Publication number: JP2013034451A
Application number: JP2011175013A
Authority: JP
Inventors: Naomichi Matsumoto; 直通松本; Satoko Miyatake; 聡子宮武
Original assignee: Yokohama National University NUC; Yokohama City University
Current assignee: Yokohama National University NUC; Yokohama City University
Priority date: 2011-08-10
Filing date: 2011-08-10
Publication date: 2013-02-21
Anticipated expiration: 2031-08-10
Also published as: JP5822293B2

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of predicting the risk of onset of moyamoya disease and its degree of severity more in detail than conventional methods.SOLUTION: This method for examining the moyamoya disease is provided by detecting c.14576G>A polymorphism in the RNF213 gene of a subject and in case that it has a homozygous A/A polymorphism, determining the subject to be of a severe type of poor prognosis, or to have a high onset risk.

Description

本発明は、RNF213遺伝子多型による重症もやもや病の予測方法に関し、より詳細には、もやもや病の疾患感受性遺伝子であるRNF213遺伝子多型（c.14576G>A）をホモ接合性で有する症例は重症型もやもや病を呈すると予測する方法に関する。 The present invention relates to a method for predicting severe moyamoya disease by RNF213 gene polymorphism, and more specifically, a case in which RNF213 gene polymorphism (c.14576G> A), a disease susceptibility gene for moyamoya disease, is homozygous. The present invention relates to a method for predicting that type moyamoya disease is present.

もやもや病は、ウィリス動脈輪部の狭窄性変化と脳底部の異常血管網を呈する原因不明の疾患で、若年性の脳虚血発作や脳出血の原因として頻度の高い疾患の一つである（非特許文献[1]）。慢性に進行し繰り返す脳虚血や脳出血による神経学的機能障害や知能低下をきたし一般に自然予後は不良である（非特許文献[2]）。特に、発症年齢が早く初発時より重篤な脳梗塞を呈し、急速に進行し、著しく予後不良の重症群が存在することが臨床的に知られていた（非特許文献[3]）。早期の外科的な血行再建を行うことで機能障害を残すことなく治療を行うことが可能と考えられるが、若年者への血行再建術はそれ自体が周術期の脳卒中のリスクとなりうるため、早急な手術適応のある症例を早期に抽出する指標が求められている（非特許文献[4]）。 Moyamoya disease is a disease of unknown cause that presents with stenotic changes in the circle of Willis and an abnormal vascular network in the base of the brain, and is one of the most frequent causes of juvenile cerebral ischemic attacks and cerebral hemorrhage (non Patent document [1]). Neurological dysfunction and intelligence decline due to cerebral ischemia and cerebral hemorrhage that progresses and repeats chronically, and the natural prognosis is generally poor (Non-patent Document [2]). In particular, it has been clinically known that there is a severe group with early onset age, severe cerebral infarction from the first onset, progressing rapidly, and remarkably poor prognosis (Non-patent Document [3]). Although early surgical revascularization is thought to allow treatment without leaving any functional impairment, revascularization for young people can itself be a risk of perioperative stroke, There is a demand for an index for early extraction of cases with immediate surgical indication (Non-Patent Document [4]).

もやもや病は約10％に家族内発症がみられること（非特許文献[5]）、一卵性双生児でのもやもや病発症率は90%、同胞再発率が一般集団の発症頻度の42倍と高率であること（非特許文献[6]）、発症頻度に人種差があり、東アジア、特に日本においては有病率が欧米の約10倍であること（有病率 10万人当たり3.16）（非特許文献[7-9]）などから、発症に何らかの遺伝学的因子が関与していることが想定されてきた。2010年にKamadaらが初のもやもや病の疾患感受性遺伝子としてRNF213遺伝子を報告し、（非特許文献[10]）もやもや病発病リスクの診断方法を開発した（特許文献[1]）。 Moyamoya disease occurs in the family in about 10% (Non-patent document [5]), Moyamoya disease incidence in identical twins is 90%, sibling recurrence rate is 42 times the incidence of the general population High rate (Non-patent document [6]), racial differences in incidence, and prevalence is about 10 times that of Europe and the US in East Asia, especially Japan (prevalence 3.16 per 100,000 people) (Non-patent document [7-9]) and the like, it has been assumed that some genetic factors are involved in the onset. In 2010, Kamada et al. Reported the RNF213 gene as the first disease susceptibility gene for Moyamoya disease (Non-patent Document [10]) and developed a method for diagnosing the risk of Moyamoya disease (Patent Document [1]).

Suzuki J, Takaku A (1969) Cerebrovascular"moyamoya" disease. Disease showing abnormal net-like vessels in baseof brain. Arch Neurol 20 (3):288-299Suzuki J, Takaku A (1969) Cerebrovascular "moyamoya" disease. Disease showing abnormal net-like vessels in baseof brain. Arch Neurol 20 (3): 288-299 Kuroda S, Houkin K (2008) Moyamoya disease:current concepts and future perspectives. Lancet Neurol 7 (11):1056-1066.doi:S1474-4422(08)70240-0 [pii] 10.1016/S1474-4422(08)70240-0Kuroda S, Houkin K (2008) Moyamoya disease: current concepts and future perspectives.Lancet Neurol 7 (11): 1056-1066.doi: S1474-4422 (08) 70240-0 [pii] 10.1016 / S1474-4422 (08) 70240-0 Kim SK, Seol HJ, Cho BK, Hwang YS, Lee DS, WangKC (2004) Moyamoya disease among young patients: its aggressive clinical courseand the role of active surgical treatment. Neurosurgery 54 (4):840-844;discussion 844-846Kim SK, Seol HJ, Cho BK, Hwang YS, Lee DS, WangKC (2004) Moyamoya disease among young patients: its aggressive clinical courseand the role of active surgical treatment. Neurosurgery 54 (4): 840-844; discussion 844-846 Kim SK, Cho BK, Phi JH, Lee JY, Chae JH, KimKJ, Hwang YS, Kim IO, Lee DS, Lee J, Wang KC (2010) Pediatric moyamoya disease:An analysis of 410 consecutive cases. Ann Neurol 68 (1):92-101.doi:10.1002/ana.21981Kim SK, Cho BK, Phi JH, Lee JY, Chae JH, KimKJ, Hwang YS, Kim IO, Lee DS, Lee J, Wang KC (2010) Pediatric moyamoya disease: An analysis of 410 consecutive cases.Ann Neurol 68 (1 ): 92-101.doi: 10.1002 / ana.21981 Yamauchi T, Houkin K, Tada M, Abe H (1997)Familial occurrence of moyamoya disease. Clin Neurol Neurosurg 99 Suppl2:S162-167Yamauchi T, Houkin K, Tada M, Abe H (1997) Family occurrence of moyamoya disease.Clin Neurol Neurosurg 99 Suppl2: S162-167 N.Kanai (1992) A Genetic Study of SpontaneousOcculusion of the Circle of Willis (Moyamoya Disease). Journal of Tokyo Women'sMedical University 62 (11):1227-1258N. Kanai (1992) A Genetic Study of Spontaneous Occulusion of the Circle of Willis (Moyamoya Disease). Journal of Tokyo Women's Medical University 62 (11): 1227-1258 Graham JF, Matoba A (1997) A survey of moyamoyadisease in Hawaii. Clin Neurol Neurosurg 99 Suppl 2:S31-35.doi:S0303846797000371 [pii]Graham JF, Matoba A (1997) A survey of moyamoyadisease in Hawaii. Clin Neurol Neurosurg 99 Suppl 2: S31-35.doi: S0303846797000371 [pii] Uchino K, Johnston SC, Becker KJ, Tirschwell DL(2005) Moyamoya disease in Washington State and California. Neurology 65(6):956-958. doi:65/6/956 [pii]10.1212/01.wnl.0000176066.33797.82Uchino K, Johnston SC, Becker KJ, Tirschwell DL (2005) Moyamoya disease in Washington State and California. Neurology 65 (6): 956-958.doi: 65/6/956 [pii] 10.1212 / 01.wnl.0000176066.33797. 82 Yonekawa Y, Ogata N, Kaku Y, Taub E, Imhof HG(1997) Moyamoya disease in Europe, past and present status. Clin NeurolNeurosurg 99 Suppl 2:S58-60. doi:S0303846797000425 [pii]Yonekawa Y, Ogata N, Kaku Y, Taub E, Imhof HG (1997) Moyamoya disease in Europe, past and present status.Clin NeurolNeurosurg 99 Suppl 2: S58-60.doi: S0303846797000425 [pii] Kamada F, Aoki Y, Narisawa A, Abe Y,Komatsuzaki S, Kikuchi A, Kanno J, Niihori T, Ono M, Ishii N, Owada Y, FujimuraM, Mashimo Y, Suzuki Y, Hata A, Tsuchiya S, Tominaga T, Matsubara Y, Kure S(2011) A genome-wide association study identifies RNF213 as the first Moyamoyadisease gene. J Hum Genet 56 (1):34-40. doi:jhg2010132 [pii]10.1038/jhg.2010.132Kamada F, Aoki Y, Narisawa A, Abe Y, Komatsuzaki S, Kikuchi A, Kanno J, Niihori T, Ono M, Ishii N, Owada Y, FujimuraM, Mashimo Y, Suzuki Y, Hata A, Tsuchiya S, Tominaga T, Matsubara Y, Kure S (2011) A genome-wide association study identifies RNF213 as the first Moyamoyadisease gene. J Hum Genet 56 (1): 34-40. Doi: jhg2010132 [pii] 10.1038 / jhg.2010.132

特開2010-259390 号公報JP 2010-259390 A

しかし、特開2010-259390 号公報にもとづく方法では、もやもや病の発症のリスクは予想できるものの、診療上外科的加療を検討するうえで必要不可欠な重症度に関する情報は得られなかった。またもやもや病は浸透率が低い疾患であることから、リスクが予想されても、実際に発症する確率に関する情報は得られなかった。 However, although the method based on Japanese Patent Application Laid-Open No. 2010-259390 can predict the risk of moyamoya disease, information on the severity that is indispensable for examining surgical treatment in clinical practice has not been obtained. Moyamoya disease is a disease with low penetrance, so information on the probability of actual onset could not be obtained even if the risk was expected.

本発明は、この現状に基づき、従来法よりもより詳細に発症のリスクを予想すること、およびその重症度を予測することを可能とする方法を提供することを目的とする。 An object of the present invention is to provide a method that makes it possible to predict the risk of onset in more detail than the conventional method and to predict the severity of the risk based on this current situation.

本発明者らは、上記課題を解決するために、もやもや病患者204名について、RNF213遺伝子の特定の変異（c.14576G>A）を解析したところ、家族歴のあるもやもや病症例の95％、孤発性症例の80％にこの変異がみられることがわかった。また一般日本人集団の1.8％が同様の変異を有するキャリアであることがわかった。一般にヒトは23対46本の染色体上に約2万個の対になった遺伝子を持つ。そこでこの遺伝子変異を有する症例について詳細にみると、この変異を１つ持つもの（ヘテロ接合性）、２つ持つもの（ホモ接合性）の２通りが存在することが分かった。そこで２通りの変異のパターンによって、臨床症状が規定されるかどうかを、統計学的に解析した。一般集団にはヘテロ接合例は存在するが、ホモ接合例は存在しなかった。ホモ接合例群では、ヘテロ接合群に比べ発症リスクが極端に大きく、その発症確率が78％以上であることがわかった。さらにホモ接合では、発症年齢が有意に早期で、初発症状が重篤な脳梗塞であるなど、従来臨床で経験的に知られていた重症型に一致することが判明した。本発明は、これらの知見に基づいて完成された。 In order to solve the above problems, the present inventors analyzed a specific mutation (c.14576G> A) of the RNF213 gene for 204 moyamoya patients, and found that 95% of Moyamoya disease cases with a family history, This mutation was found in 80% of sporadic cases. In addition, 1.8% of the general Japanese population was found to be carriers with similar mutations. In general, humans have about 20,000 pairs of genes on 23 to 46 chromosomes. Therefore, when the cases having this gene mutation were examined in detail, it was found that there were two types of one having this mutation (heterozygosity) and two having this mutation (homozygosity). Therefore, it was statistically analyzed whether clinical symptoms are defined by two patterns of mutations. There were heterozygous cases in the general population, but no homozygous cases. In the homozygous case group, the onset risk was extremely large compared to the heterozygous group, and the onset probability was found to be 78% or more. Furthermore, homozygosity was found to be consistent with a severe type that has been empirically known in clinical practice, such as the onset age being significantly early and the initial symptom being severe cerebral infarction. The present invention has been completed based on these findings.

本発明の要旨は以下の通りである。
（１）被験者のRNF213遺伝子におけるc.14576G>A多型を検出し、ホモ接合性の多型A/Aを持つ場合に、予後不良の重症型である、あるいは発症リスクが高いと判定する、もやもや病の検査方法。
（２）c.14576G>A多型をゲノムDNAで解析して検出する（１）記載の検査方法。
（３）c.14576G>A多型をRNAレベルで解析して検出する（１）記載の検査方法。
（４）c.14576G>A多型をタンパク質レベルで解析して検出する（１）記載の検査方法。 The gist of the present invention is as follows.
(1) When a c.14576G> A polymorphism in the RNF213 gene of a test subject is detected and has a homozygous polymorphism A / A, it is determined that the prognosis is severe or the risk of onset is high. Test method for moyamoya disease.
(2) The test method according to (1), wherein c.14576G> A polymorphism is analyzed by genomic DNA and detected.
(3) The test method according to (1), wherein c.14576G> A polymorphism is analyzed and detected at the RNA level.
(4) The test method according to (1), wherein the c.14576G> A polymorphism is analyzed and detected at the protein level.

本発明の方法によりRNF213遺伝子の多型を検出することで、その変異のパターン（ヘテロ接合性・ホモ接合性）によって、従来法より詳細な発症リスクと、いわゆる重症型であるかの予測が可能となる。これにより、発症早期の症例においては、早急に手術が適応であるかの判断において有用なバイオマーカーとなりうる。また未発症症例においては、発症確率などのより詳細なリスクの評価とその予想される重症度に関する情報が提供される。これらの情報は、もやもや病による脳血管イベントの予防に有用であると考えられる。 By detecting the polymorphism of the RNF213 gene by the method of the present invention, it is possible to predict whether it is so-called severe type with more detailed onset risk than conventional methods by the mutation pattern (heterozygous / homozygous) It becomes. As a result, it can be a useful biomarker in the early determination of whether or not surgery is indicated for cases. In the case of undeveloped cases, more detailed risk assessment such as onset probability and information on the expected severity are provided. Such information is considered useful for preventing cerebrovascular events caused by moyamoya disease.

またMRIや血管造影などの高額、侵襲的な検査を要さないことから、日本においては、保因者が比較的多く予後不良の当該疾患の発症前スクリーニング法としての意義もある。 In addition, since expensive and invasive examinations such as MRI and angiography are not required, it is significant in Japan as a pre-onset screening method for the disease with a relatively large number of carriers and poor prognosis.

RNF213蛋白とRNF213遺伝子の構造図。図の上から下に向かって、３つのドメインを持つRNF213蛋白の構造、今回の研究で発明者たちが発見した新規の遺伝子変異によって生じるアミノ酸置換、RNF213遺伝子の２つのアイソフォーム、を図示した。Structure diagram of RNF213 protein and RNF213 gene. From the top to the bottom of the figure, the structure of the RNF213 protein with three domains, the amino acid substitution caused by the novel gene mutation discovered by the inventors in this study, and the two isoforms of the RNF213 gene are shown. c.14576G>A 多型と発症年齢の相関についての図。図(A)はc.14576G>Aの遺伝型がホモ接合性(AA)、ヘテロ接合性(GA)、野生型(GG)である３つの患者グループについて、それぞれの発症年齢の分布について箱ひげ図を示した。(○) は軽度外れ値、(Δ) は高度の外れ値を意味する。図(B)は３つの患者群におけるもやもや病の累積発症率のグラフである。c.14576G> A Diagram of correlation between polymorphism and age at onset. Figure (A) shows the box-whisker distribution of the age at onset for three patient groups with c.14576G> A genotype homozygous (AA), heterozygous (GA), and wild type (GG). The figure is shown. (○) means a slight outlier and (Δ) means a high outlier. FIG. (B) is a graph of the cumulative incidence of moyamoya disease in the three patient groups. c.14576G>A多型と臨床症状の相関についての図。図(A)は204名のもやもや病患者において、c.14576G>A多型がホモ接合性(AA)、ヘテロ接合性(GA)、野生型(GG)である３つの患者グループにおける様々な臨床症候の頻度を棒グラフで示した。各々の臨床症候があるかないかについて、その臨床記録が存在する患者の合計数を棒グラフの下段に（）で示し、各患者群において各々の症候を有する患者の数を各々の棒グラフの上に示した。Infarct:脳梗塞, TIA:一過性脳虚血, ICH/IVH:脳出血/脳室内出血,FH:家族歴あり, Intel:知的機能の低下, Epi:てんかん, PCA:後大脳動脈の障害合併, Bil:両側性の血管病変。図(B)は１５歳未満で発症した患者について、図(A)と同様の内容を棒グラフで提示した。The figure about the correlation of c.14576G> A polymorphism and a clinical symptom. Figure (A) shows various clinical studies in 204 patients with moyamoya disease in three patient groups with the c.14576G> A polymorphism being homozygous (AA), heterozygous (GA), and wild type (GG). The frequency of symptoms was shown as a bar graph. For each clinical symptom, the total number of patients whose clinical records exist is indicated by () at the bottom of the bar graph, and the number of patients with each symptom in each patient group is indicated above each bar graph. It was. Infarct: Cerebral infarction, TIA: Transient cerebral ischemia, ICH / IVH: Cerebral hemorrhage / intraventricular hemorrhage, FH: Family history, Intel: Decreased intellectual function, Epi: Epilepsy, PCA: Post-cerebral artery complication , Bil: Bilateral vascular lesions. In FIG. (B), the same contents as those in FIG.

以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.

本発明は、被験者のRNF213遺伝子におけるc.14576G>A多型を検出し、ホモ接合性の多型A/Aを持つ場合に、予後不良の重症型である、あるいは発症リスクが高いと判定する、もやもや病の検査方法を提供する。RNF213遺伝子はヒト17番染色体上に存在する。RNF213遺伝子におけるc.14576G>A多型の変異は、エキソン61に存在し、p.Arg4859Lys（4859番目のR（アルギニン）がK（リジン）に置換）のミスセンス変異をもたらす。 The present invention detects a c.14576G> A polymorphism in the RNF213 gene of a subject and determines that it has a severe prognosis or a high risk of developing when it has a homozygous polymorphism A / A. Provide a method for testing moyamoya disease. The RNF213 gene is present on human chromosome 17. The c.14576G> A polymorphism mutation in the RNF213 gene is present in exon 61, resulting in a missense mutation in p.Arg4859Lys (4859th R (arginine) is replaced with K (lysine)).

RNF213がコードするタンパクはAAA (ATPases associated with variety of a cellular activities) ドメインをもつRINGフィンガータンパクであり、E3 ユビキチンリガーゼ活性とエネルギー依存性アンフォルダーゼ活性を有する。また機能はよく知られていないが、2A1904 domainの存在も想定されている。この遺伝子には少なくとも２つのアイソフォームが存在することが知られている。Isoform 1は61個のエキソンからなり、5256個のアミノ酸から成るタンパク質をコードする。このタンパク質には前述の３つのドメインの存在が想定されている。Isoform 2 は17個のエキソンからなり、1063個のアミノ酸から成るタンパク質をコードする。このタンパク質には2A1904 domainの存在が想定されている。C.14576G>A多型は isoform 1のみに存在する。 The protein encoded by RNF213 is a RING finger protein having an AAA (ATPases associated with variety of a cellular activities) domain, and has E3 ubiquitin ligase activity and energy-dependent unfoldase activity. The function is not well known, but the existence of 2A1904 domain is also assumed. This gene is known to have at least two isoforms. Isoform 1 consists of 61 exons and encodes a protein of 5256 amino acids. This protein is assumed to have the aforementioned three domains. Isoform 2 consists of 17 exons and encodes a protein consisting of 1063 amino acids. This protein is assumed to have a 2A1904 domain. The C.14576G> A polymorphism exists only in isoform 1.

配列情報は、NCBI Reference Sequences NM020914 (isoform1), NM020954 (isoform2)に従っている。 The sequence information conforms to NCBI Reference Sequences NM020914 (isoform1) and NM020954 (isoform2).

ヒトRNF213遺伝子(isoform1)のゲノム配列を配列表の配列番号１に示す。配列番号１に示すゲノム配列中、エキソンの位置は以下の通りである。（以下の番号はmRNAの始まりを1としたときの、exonに当たる塩基の番号を記したものである。ゲノムのphysical positionの番号ではmRNAの1にあたるのが、78234667 になる。http://www.ncbi.nlm.nih.gov/nuccore/NC_000017.10?from=78234667&to=78370086&report=genbank より）
mRNA join(1..35,2707..2911,12374..12537,17897..18043, 26948..27496,27766..27888,28792..28970,29703..29861, 30761..30960,33853..34136,34691..34947,37455..37652, 45386..45602,46262..46335,48152..48305,52146..52301, 56322..56411,58324..58446,64164..64332,66950..67123, 67462..67611,71140..71765,73239..73404,75295..75512, 76720..76860,76967..77131,78335..79490,82266..82459, 82991..83149,83812..87420,88904..89040,89435..89530, 90819..90937,92074..92193,92646..92800,93153..93300, 93575..93710,97422..97613,99195..99340,100868..101022, 102236..102448,102743..102924,103567..103687, 106882..106982,107095..107277,108636..108805, 108902..109001,111008..111119,111655..111868, 112109..112299,113592..113723,114894..115004, 115435..115677,116016..116097,116896..116930, 118754..118848,119965..120121,120681..120861, 122113..122204,122811..123062,124173..124305, 124672..124755,125384..125564,125824..126025, 127746..127823,128307..128501,128962..129051, 129146..129330,132479..135420) The genomic sequence of the human RNF213 gene (isoform1) is shown in SEQ ID NO: 1 in the sequence listing. In the genome sequence shown in SEQ ID NO: 1, the positions of exons are as follows. (The numbers below indicate the number of the base corresponding to exon when the start of mRNA is 1. The physical position number of the genome is 78234667, which corresponds to mRNA 1) http: // www .ncbi.nlm.nih.gov / nuccore / NC_000017.10? from = 78234667 & to = 78370086 & report = genbank)
mRNA join (1..35,2707..2911,12374..12537,17897..18043, 26948..27496,27766..27888,28792..28970,29703..29861, 30761..30960,33853. .34136,34691..34947,37455..37652, 45386..45602,46262..46335,48152..48305,52146..52301, 56322..56411,58324..58446,64164..64332,66950. .67123, 67462..67611,71140..71765,73239..73404,75295..75512, 76720..76860,76967..77131,78335..79490,82266..82459, 82991..83149,83812. .87420,88904..89040,89435..89530, 90819..90937,92074..92193,92646..92800,93153..93300, 93575..93710,97422..97613,99195..99340,100868. .101022, 102236..102448,102743..102924,103567..103687, 106882..106982,107095..107277,108636..108805, 108902..109001,111008..111119,111655..111868, 112109. .112299,113592..113723,114894..115004, 115435..115677,116016..116097,116896..116930, 118754..118848,119965..120121,120681..120861, 122113..122204,122811. .123062,124173..124305, 124672..124755,125384..125564,125824..126025, 127746..127823,128307..128501,128962..129051, 129146..129330,132479..135420)

ヒトRNF213遺伝子(isoform1)のmRNA配列及びこれがコードするRNF213蛋白のアミノ酸配列をそれぞれ配列番号２及び３に示す。 The mRNA sequence of the human RNF213 gene (isoform 1) and the amino acid sequence of the RNF213 protein encoded by this are shown in SEQ ID NOs: 2 and 3, respectively.

RNF213遺伝子(isoform1)のエキソン61に存在するc.14576G>A（p.Arg4859Lys）の変異を配列表の配列で説明すると、以下の通りである。 The mutation of c.14576G> A (p.Arg4859Lys) present in exon 61 of the RNF213 gene (isoform1) is described as follows in the sequence listing.

・配列番号１に示すヒトRNF213遺伝子のゲノム配列中の124279番目のGがAに置換。（ゲノムのphysical positionの番号では1=78234667 になる。）
・配列番号２に示すヒトRNF213遺伝子のmRNA配列中の14719番目のGがAに置換。(coding DNA の14576番目である。)
・配列番号３に示すヒトRNF213遺伝子のmRNAがコードするRNF213蛋白のアミノ酸配列中の4859番目のArgがLysに置換。 -The 124279th G in the genome sequence of the human RNF213 gene shown in SEQ ID NO: 1 was replaced with A. (The number of the physical position of the genome is 1 = 78234667.)
-G of the 14719th position in the mRNA sequence of the human RNF213 gene shown in SEQ ID NO: 2 was replaced with A. (It is 14576th of coding DNA.)
-The 4859th Arg in the amino acid sequence of the RNF213 protein encoded by the mRNA of the human RNF213 gene shown in SEQ ID NO: 3 was replaced with Lys.

ヒトRNF213遺伝子(isoform2)のゲノム配列を配列表の配列番号４に示す。配列番号４に示すゲノム配列中、エキソンの位置は以下の通りである。（以下の番号はmRNAの始まりを1としたときの、exonに当たる塩基の番号を記したものである。ゲノムのphysical positionの番号ではmRNAの1にあたるのが、78234667 になる。http://www.ncbi.nlm.nih.gov/nuccore/NC_000017.10?from=78234667&to=78370086&report=genbank より）
mRNA join(1..35,2707..2911,12374..12537,26948..27496, 27766..27888,28792..28970,29703..29861,30761..30960, 33853..34136,34691..34947,37455..37652,45386..45602, 46262..46335,48152..48305,52146..52301,56322..56411, 58324..60597) The genomic sequence of the human RNF213 gene (isoform 2) is shown in SEQ ID NO: 4 in the sequence listing. In the genome sequence shown in SEQ ID NO: 4, the positions of exons are as follows. (The numbers below indicate the number of the base corresponding to exon when the start of mRNA is 1. The physical position number of the genome is 78234667, which corresponds to mRNA 1) http: // www .ncbi.nlm.nih.gov / nuccore / NC_000017.10? from = 78234667 & to = 78370086 & report = genbank)
mRNA join (1..35,2707..2911,12374..12537,26948..27496, 27766..27888,28792..28970,29703..29861,30761..30960, 33853..34136,34691. .34947,37455..37652,45386..45602, 46262..46335,48152..48305,52146..52301,56322..56411, 58324..60597)

ヒトRNF213遺伝子(isoform2)のmRNA配列及びこれがコードするRNF213蛋白のアミノ酸配列をそれぞれ配列番号５及び６に示す。 The mRNA sequence of the human RNF213 gene (isoform 2) and the amino acid sequence of the RNF213 protein encoded by this are shown in SEQ ID NOs: 5 and 6, respectively.

RNF213遺伝子におけるc.14576G>A多型は、G/G（正常型）、G/A（ヘテロ接合性；1アレルだけ多型有）及びA/A（ホモ接合性；2アレルとも多型有）に分類される。 C.14576G> A polymorphism in RNF213 gene is G / G (normal), G / A (heterozygous; only one allele is polymorphic) and A / A (homozygous; both alleles are polymorphic) )are categorized.

本発明者らの解析結果により、RNF213遺伝子におけるc.14576G>A多型をヘテロ接合性(G/A)で持つ人とホモ接合性(A/A)で持つ人では、発症リスクに違いがあることがわかった。後述の実施例では、ヘテロ接合性では236倍罹患しやすく、ホモ接合性では、変異のない人に比べ罹患リスクは計算上無限大になった。これを統計学的に計算すると、ホモ接合性変異を持っている人がもやもや病と診断される状態になる確率は78−100％と計算される。特開2010-259390号公報の方法では、ヘテロ接合性とホモ接合性の発症リスクの違いは考慮されておらず、ヘテロ接合性とホモ接合性を同等の遺伝学的効果をもつものと扱ってリスクを計算している。（後述の実施例においても仮に同様に考えて計算すると260倍と計算されるので、特開2010-259390号公報の方法で得られた数値よりも高い。）また特開2010-259390号公報の方法では、発明に至る研究の対象となったサンプルサイズも比較的小さい。以上から、本発明で提供されるデータのほうがより正確なものと考えられる。 According to the analysis results of the present inventors, there is a difference in risk of onset between those who have the c.14576G> A polymorphism in the RNF213 gene as heterozygous (G / A) and those who are homozygous (A / A). I found out. In the examples described below, heterozygosity is 236 times more likely to be affected, and homozygosity is a computationally infinite risk compared to those without mutations. If this is calculated statistically, the probability that a person with a homozygous mutation will be diagnosed with moyamoya disease is calculated as 78-100%. The method disclosed in Japanese Patent Application Laid-Open No. 2010-259390 does not consider the difference in risk of heterozygosity and homozygosity, and treats heterozygosity and homozygosity as having equivalent genetic effects. Risk is calculated. (In the examples described later, if it is calculated in the same way, it is calculated as 260 times, which is higher than the value obtained by the method of Japanese Patent Application Laid-Open No. 2010-259390). In the method, the sample size that has been the subject of research leading to the invention is also relatively small. From the above, it is considered that the data provided by the present invention is more accurate.

また、本発明者らの解析結果により、ホモ接合性変異を持つ場合、統計学的な有意差を持って、発症年齢が若く（4才未満）、初発時に神経学的後遺症を残す脳梗塞を起こす可能性が高く、また通常障害されない後大脳動脈の病変を合併しやすく、障害される血管の分布も典型例に比べ広範になることがわかった。臨床の現場において、重症で神経学的予後不良群と認識されてきた群の特徴に一致している。このような群も早期のうちに外科的加療を行うことによって、神経学後遺症を少なくすることが可能であり、また本発明の方法を利用することで、場合によっては、ホモ接合性変異を持つ人に対して重篤な初発症状が起こる前に予防的手術を行いうる可能性もある。よって、上記変異をヘテロ接合性／ホモ接合性に分けて解析する本発明の方法は臨床において非常に有用な情報を提供するものと考える。このような情報は、本発明に至る研究で解析した多数のサンプルの遺伝型と臨床症状の相関を統計学的に解析したことで明らかになった。特開2010-259390号公報の方法では、臨床症状との相関は検討されていないため、重症度に関する情報が得られなかったと考える。 In addition, according to the analysis results of the present inventors, when there is a homozygous mutation, cerebral infarction having a statistically significant difference, younger onset age (under 4 years old), and leaving a neurological sequelae at the first onset It was found that the lesions of the cerebral arteries, which are highly likely to occur and are usually not damaged, are likely to be complicated, and the distribution of damaged blood vessels is wider than that of the typical case. This is consistent with the characteristics of a group that has been recognized in the clinical setting as a severe and poor neurological prognosis group. Such a group can also reduce the neurological sequelae by performing surgical treatment in an early stage, and in some cases, it has a homozygous mutation by using the method of the present invention. There is a possibility that preventive surgery can be performed before serious initial symptoms occur in a person. Therefore, it is considered that the method of the present invention for analyzing the above-mentioned mutation by dividing it into heterozygous / homozygous provides very useful information in clinical practice. Such information has been clarified by statistically analyzing the correlation between the genotype and clinical symptoms of many samples analyzed in the study leading to the present invention. In the method disclosed in Japanese Patent Application Laid-Open No. 2010-259390, the correlation with clinical symptoms has not been studied, so it is considered that information on the severity could not be obtained.

本発明の方法において、RNF213遺伝子の変異は、両アリルについて検出するとよい。また、RNF213遺伝子の変異は、ゲノムDNAで解析してもよいし、RNAレベル又はタンパク質レベルで解析してもよい。ゲノムDNAで解析する場合には、RNF213遺伝子の変異は、被験者から採取した血液、唾液、脳、皮膚、腎臓、膵臓などの生検組織などの生体試料を用いて、検出することができる。例えば、末梢血白血球から、ゲノムDNA、mRNA又はmRNAから合成したcDNAなどの核酸試料を分離あるいは調製し、必要に応じて、RNF213遺伝子の変異部位を含む領域を増幅して、RNF213遺伝子の変異を検出することができる。 In the method of the present invention, mutations in the RNF213 gene may be detected for both alleles. Further, the mutation of the RNF213 gene may be analyzed with genomic DNA, or may be analyzed with RNA level or protein level. In the case of analyzing with genomic DNA, a mutation in the RNF213 gene can be detected using a biological sample such as biopsy tissue such as blood, saliva, brain, skin, kidney and pancreas collected from a subject. For example, a nucleic acid sample such as genomic DNA, mRNA or cDNA synthesized from mRNA is isolated or prepared from peripheral blood leukocytes, and if necessary, a region containing the mutation site of RNF213 gene is amplified to mutate RNF213 gene. Can be detected.

RNF213遺伝子の変異をゲノムDNAで解析するには、例えば、適切に設計したプライマーを用いて、ゲノムDNAからRNF213遺伝子の変異部位を含むコーディング領域を増幅し、ダイレクトシークエンスするとよい。SeqScape（登録商標）などの市販のソフトウェアを用いれば、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。変異がホモ接合性かヘテロ接合性かについては、シークエンスの波形データから確認することができる。ダイレクトシークエンス以外にも、PCR-RFLP法、配列特異的オリゴプローブ法（SSOP法）、一本鎖コンフォメーション多型解析法（SSCP法）、アレル特異的増幅法（MASA法）などの方法を用いてもよい。 In order to analyze the mutation of the RNF213 gene with genomic DNA, for example, a coding region including the mutation site of the RNF213 gene may be amplified from the genomic DNA using an appropriately designed primer, and direct sequencing may be performed. Using commercially available software such as SeqScape (registered trademark), mutation detection and profiling can be easily performed. Whether the mutation is homozygous or heterozygous can be confirmed from the waveform data of the sequence. In addition to direct sequencing, PCR-RFLP method, sequence-specific oligo probe method (SSOP method), single-strand conformation polymorphism analysis method (SSCP method), allele-specific amplification method (MASA method), etc. are used. May be.

RNF213遺伝子の変異をRNAレベルで解析するには、ノーザンハイブリダイゼーション法、サザンハイブリダイゼーション法、DNAチップ又はDNAアレイ解析法、in situハイブリダイゼーション法などのハイブリダイゼーション法、RT-PCR法などの方法を用いるとよい。「RNF213遺伝子の変異をRNAレベルで解析する」とは、mRNAから合成されるcDNAを解析することも含む概念である。 To analyze mutations in the RNF213 gene at the RNA level, methods such as Northern hybridization, Southern hybridization, DNA chip or DNA array analysis, in situ hybridization, and RT-PCR are used. Use it. “Analyzing mutations in the RNF213 gene at the RNA level” is a concept that includes analyzing cDNA synthesized from mRNA.

RNF213遺伝子の変異をタンパク質レベルで解析するには、抗RNF213蛋白抗体を使用したウェスタンブロット法により、タンパク質分子量の変化の有無をみる方法や細胞、組織検体に対して抗RNF213蛋白抗体を使用した免疫染色を行ってRNF213蛋白の細胞内局在をみる方法が考えられる。 To analyze mutations in the RNF213 gene at the protein level, Western blotting using anti-RNF213 protein antibodies can be used to check for changes in the molecular weight of the protein, and immunization using anti-RNF213 protein antibodies against cell and tissue samples. A method of examining the intracellular localization of RNF213 protein by staining is conceivable.

RNF213遺伝子の変異解析に使用できるプライマーの配列の一例を以下に示すが、これに限定されるわけではない。また、PCRの条件は適宜設定することができる。 Examples of primer sequences that can be used for mutation analysis of the RNF213 gene are shown below, but are not limited thereto. PCR conditions can be set as appropriate.

CTCGCAGCCAGTCTCAAAGT（フォワードプライマー）（配列番号７）
TCCCCTATGCAGTGATCCTT（リバースプライマー）（配列番号８） CTCGCAGCCAGTCTCAAAGT (forward primer) (SEQ ID NO: 7)
TCCCCTATGCAGTGATCCTT (reverse primer) (SEQ ID NO: 8)

以下、実施例に基づいて本発明を具体的に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to these Examples.

〔実施例１〕
RNF213遺伝子におけるホモ接合性のc.14576G>A多型は、早期発症で重症型もやもや病の強力な予知因子である
抄録
背景最近もやもや病 (MMD)の疾患感受性遺伝子としてRNF213遺伝子が報告された。私たちは、RNF213遺伝子の遺伝型ともやもや病の臨床型についての相関を明らかにすることにした。 [Example 1]
Abstract Background The homozygous c.14576G> A polymorphism in the RNF213 gene is a powerful predictor of early-onset and severe moyamoya disease Abstract Background Recently, the RNF213 gene has been reported as a disease susceptibility gene for moyamoya disease (MMD). We decided to clarify the correlation between the genotype of the RNF213 gene and the clinical type of Moyamoya disease.

方法もやもや病患者204名について、RNF213遺伝子の全コーディング領域のＤＮＡ配列を決定し、検出された多型は、62組の患者の両親、13人の母親、4人の父親、及び283人の健常日本人にも見られるかどうか検索した。患者の臨床情報は、主治医の記録から収集した。遺伝型―臨床型連関は統計学的に解析した。 Methods For 204 moyamoya patients, the DNA sequence of the entire coding region of the RNF213 gene was determined, and the detected polymorphisms were found in 62 patients' parents, 13 mothers, 4 fathers, and 283 healthy individuals We searched whether it was seen by Japanese. Patient clinical information was collected from the attending physician's records. Genotype-clinical type association was analyzed statistically.

結果 c.14576G>A 多型は、家族性もやもや病患者の95.1%, 孤発性もやもや病患者の79.2%, 健常日本人の1.8%に見られた。この多型をヘテロ接合性もしくはホモ接合性に持つ場合のOdds比は259(P<0.001)で、もやもや病との強い相関が確定された。この多型をホモ接合性に持つ患者が15人見られたが、健常日本人や、非罹患の患者の両親には1人も確認されなかった。ホモ接合性にこの多型を持つ場合の、もやもや病発症確率は少なくとも78％と計算された。ホモ接合性多型を有する患者は、それ以外の患者に比べ、有意に発症年齢が若かった（ホモ接合性患者の発症年齢中央値＝３歳、ヘテロ接合性患者の発症年齢中央値＝7歳、多型をもたない患者の発症年齢中央値＝8歳）、ホモ接合性患者の60％は4歳未満でもやもや病と診断されており、その全症例の初発症状は脳梗塞であった。初発時の症状が脳梗塞であること、後大脳動脈(PCA)にも病変が及んでいること、はどちらも、もやもや病の予後不良因子として知られているが、これらは、ホモ接合性患者で有意に頻度が高かった。C.14576GF>A多型以外の変異は臨床病型との相関は明らかではなかった。 Results c.14576G> A polymorphism was found in 95.1% of familial moyamoya disease patients, 79.2% of sporadic moyamoya disease patients, and 1.8% of healthy Japanese. When this polymorphism is heterozygous or homozygous, the Odds ratio was 259 (P <0.001), confirming a strong correlation with Moyamoya disease. Fifteen patients who were homozygous for this polymorphism were found, but none was found in the parents of healthy Japanese or unaffected patients. The probability of developing moyamoya disease with this polymorphism in homozygosity was calculated to be at least 78%. Patients with homozygous polymorphisms were significantly younger than others (median onset of homozygous patients = 3 years, median onset of heterozygous patients = 7 years) , Median age of onset of patients without polymorphism = 8 years), 60% of homozygous patients were diagnosed with Moyamoya disease even under 4 years of age, the first symptom of all cases was cerebral infarction . The initial symptoms are cerebral infarction and the posterior cerebral artery (PCA) also has lesions, both of which are known as poor prognostic factors for Moyamoya disease. The frequency was significantly higher. Mutations other than the C.14576GF> A polymorphism were not clearly correlated with clinical disease types.

結論 RNF213遺伝子のc.14576G>A多型は、もやもや病発症と強い相関があった。さらに重要なことは、ホモ接合性のc.14576G>A多型は、早期の外科的治療が必要な、いわゆる重症型のもやもや病のよいバイオマーカーになりうるということである。 Conclusion The c.14576G> A polymorphism of the RNF213 gene was strongly correlated with the development of moyamoya disease. More importantly, the homozygous c.14576G> A polymorphism can be a so-called severe moyamoya disease biomarker that requires early surgical treatment.

本文
はじめに
もやもや病は、まれな脳血管の病気で、進行性に、内頸動脈の終末部とそこから枝分かれする主幹動脈の狭窄もしくは閉塞をきたす。血管の閉塞がおこると、代償性に異常血管網が形成される、これが‘もやもや血管’と呼ばれるもので、血管造影での所見が‘たばこの煙’のように見えることから名づけられた。¹ もやもや病は世界中で見られているが、東アジア、とりわけ日本と韓国での有病率が高い。^2,3 年間発症率は日本で0.35-0.54/100000人年であるが、^4,5 ヨーロッパではその10分の1である。^6,7 好発年齢は2峰性のピークを示す。すなわち10歳未満の層に大きなピークがあり、30-40歳代に小さなピークがある。⁴ 小児期発症の場合、一過性脳虚血(TIA)もしくは脳梗塞(infarction)が、もっとも典型的な症状であるが、成人発症では脳出血がより高頻度に見られる。もやもや病は小児の脳卒中の最も頻度の高い原因として重要な疾患であり、^8-10未治療で経過すると、非可逆的で重篤な神経学的後遺症を残すことになる。 Moyamoya disease is a rare cerebrovascular disease that progressively causes stenosis or occlusion of the end of the internal carotid artery and the main artery that branches from it. When blood vessel occlusion occurs, an abnormal vascular network is formed in a compensatory manner, which is called “moyamo blood vessel”, and the angiographic findings look like “cigarette smoke”. ¹ Moyamoya disease is seen all over the world, but prevalence is high in East Asia, especially in Japan and Korea. ^{2 and 3-year} incidence is 0.35-0.54 / 100,000 person-years in Japan, but in the ^{4, 5} Europe is one of the 10 minutes. ^6,7 Occurrence age shows a bimodal peak. That is, there is a big peak in the group under 10 years old, and a small peak in the 30-40s. ^In the case of childhood onset, transient cerebral ischemia (TIA) or infarction is the most typical symptom, but cerebral hemorrhage is more common in adult onset. Moyamoya disease is an important disease as the most frequent cause of stroke in children, and if left untreated ^8-10 , it will leave irreversible and severe neurological sequelae.

もやもや病は一般に進行する病気であるが、その自然経過は幅があり、ゆっくり経過するものから急速に進行し急激に神経学的レベルの低下をきたすものまである。¹¹ 外科的加療によって脳循環を改善し、脳梗塞の再発を防ぐことができるので、¹² 早期の診断と外科的介入が肝要である。手術前にすでに脳梗塞が見られるもの、発症年齢が若いもの、知的機能の低下やけいれんといった症状が合併しているもの、あるいはPCAにも病変が及んでいるものは予後不良であることが臨床的に知られている。^12-15
もやもや病の遺伝学的関与は臨床的に重要な点である。疫学調査により、約15％の患者に家族歴がみられ、高い家族集積性が知られている。遺伝形式は多因子遺伝もしくは浸透率が低い常染色体優性遺伝が想定されている。^16-18 表現促進現象も家族性もやもや病ではよく観察される事象である。¹⁸ これまで行われた連鎖解析では、5つの異なるもやもや病の遺伝子座が報告されているが、^19-22 いずれの遺伝子座からも疾患責任遺伝子は同定されていない。2010年に、Kamadaらが、GWAS studyにより、初のもやもや病の疾患感受性遺伝子として染色体17q25に位置するRNF213遺伝子を同定した。²³しかし、臨床症状との関連については知られていなかった。 Moyamoya disease is a disease that progresses in general, but its natural history varies, ranging from slowly progressing to rapidly progressing to rapidly reducing neurological levels. ¹¹ Early treatment and improvement of cerebral circulation can prevent recurrence of cerebral infarction. ¹² Early diagnosis and surgical intervention are essential. Prognosis may be poor if cerebral infarction is already seen before surgery, age is young, onset of symptoms such as decline in intellectual function or convulsions, or PCA lesions Clinically known. ^12-15
The genetic involvement of moyamoya disease is a clinically important point. Epidemiological studies show that about 15% of patients have a family history and high family accumulation. Inheritance is assumed to be multifactor inheritance or autosomal dominant inheritance with low penetrance. ^16-18 Expression-promoting phenomenon and familiality are common events in Moyamoya disease. ¹⁸ Linkage analyzes conducted so far have reported five different Moyamoya disease loci, but no disease responsible gene has been identified from any of the ^19-22 loci. In 2010, Kamada et al. Identified the RNF213 gene located on chromosome 17q25 as the first disease susceptibility gene for moyamoya disease using the GWAS study. ²³ However, the association with clinical symptoms was not known.

今回私たちは、もやもや病患者におけるRNF213遺伝子の遺伝学的及び臨床病型に関する包括的な研究を行った。その詳細について以下に述べる。 We conducted a comprehensive study on the genetic and clinical type of RNF213 gene in patients with moyamoya disease. The details are described below.

方法と対象
対象
RNF213遺伝子の変異解析の対象は204人の日本人もやもや病患者で、その血液サンプルを収集した。もやもや病の診断は厚生労働省の診断基準によって行われた。²⁴ これらの患者の臨床症状は、遺伝学的解析の結果を知らされていない臨床医たちによって記載された。204人の患者の概要はtable1に示す。また62組の患者の両親と、13人の母親、4人の父親の血液もしくは唾液サンプルも収集し、変異解析した。RNF213遺伝子で検出された変異について、最大283人の日本人健常コントロールにも見られるかどうか検索した。この研究のプロトコールは横浜市立大学医学部の倫理委員会に承認されており、すべての患者もしくは患者の親に対し、研究参加についての説明が行われ同意が得られた。 Method and target
The subject of mutation analysis of RNF213 gene was 204 Japanese moyamoya disease patients, and blood samples were collected. Moyamoya disease was diagnosed according to the diagnostic standards of the Ministry of Health, Labor and Welfare. ²⁴ clinical symptoms of these patients has been described by clinicians who are not informed of the results of genetic analysis. A summary of 204 patients is shown in table1. Blood or saliva samples from 62 pairs of parents, 13 mothers, and 4 fathers were also collected and analyzed for mutations. We examined whether mutations detected in the RNF213 gene were also found in up to 283 Japanese healthy controls. The protocol for this study was approved by the ethics committee of Yokohama City University School of Medicine, and all patients or their parents were explained and agreed to the study.

変異解析
ゲノムＤＮＡは、末梢血白血球からQuickGene 610-L (FUJIFILM) を用いて抽出するか、唾液サンプルからOragene（登録商標）・DNA (DNA genotek)キットを用いて抽出した。変異解析のためにＤＮＡをGenomiphi version 2 (GE healthcare)を用いて増幅した。エキソン61にはc.14576G>A変異が存在するが、このエキソンを除く全コーディングエキソンとエキソンーイントロン境界領域について、LightCycler（登録商標）480 System II (Roche Diagnostics).を用いてhigh resolution melting (HRM) 解析を行った。プライマー配列、PCRやHRMの条件は、リクエストに応じて提供可能である。ホモ接合性変異を検出するために、HRMはspike-in法を併用した。²⁵ HRMにおいて、他と異なるメルティング波形を示すサンプルがあれば、ダイレクトシークエンス法にて塩基配列を決定した。使用機器はABI Genetic Analyzer 3100 もしくは3500xL (Applied Biosystems) で、解析ソフトはsequence analysis software version 5.1.1 (Applied Biosystems) およびSequencher
4.10-build 5828 (GeneCodes Corporation)である。エキソン61については、全てのもやもや病患者とその親の検体について、ダイレクトシークエンス法で塩基配列を決定した。以上の変異解析で、検出された変異について、最大283人の健常日本人のサンプルを用いて、HRM解析を行い、これらのサンプルの中に同じ変異が見られるかどうか確認した。全ての変異／多型は、ゲノムDNAもしくは、再度Genomiphiを用いてDNA増幅したものを使って再度PCR及びダイレクトシークエンスを行い、その再現性を確認した。 Mutation analysis genomic DNA was extracted from peripheral blood leukocytes using QuickGene 610-L (FUJIFILM), or from saliva samples using Oragene® DNA (DNA genotek) kit. DNA was amplified using Genomiphi version 2 (GE healthcare) for mutation analysis. Exon 61 has a c.14576G> A mutation. Except for this exon, all coding exons and exon-intron boundary regions were subjected to high resolution melting using LightCycler® 480 System II (Roche Diagnostics). HRM) analysis was performed. Primer sequences, PCR and HRM conditions can be provided upon request. To detect homozygous mutations, HRM used the spike-in method. ^{In 25} HRM, if there was a sample showing a melting waveform different from the others, the base sequence was determined by the direct sequencing method. The equipment used is ABI Genetic Analyzer 3100 or 3500xL (Applied Biosystems), and the analysis software is sequence analysis software version 5.1.1 (Applied Biosystems) and Sequencher
4.10-build 5828 (GeneCodes Corporation). The base sequence of exon 61 was determined for all moyamoya disease patients and their parents by direct sequencing. In the above mutation analysis, HRM analysis was performed on the detected mutations using samples of up to 283 healthy Japanese people, and it was confirmed whether the same mutations were found in these samples. All mutations / polymorphisms were confirmed by PCR and direct sequencing again using genomic DNA or DNA amplified again using Genomiphi.

統計解析
各々の臨床項目について、その情報がない患者は、各々の統計解析から除外した。（Table 1 並びに supplemental tables 2 及び4に各々の項目について除外した患者数を示している）、全ての統計解析はSPSS Statistics 19 (IBM)を用いて行った。カイ二乗検定は、初発症状、家族歴の有無、知的機能低下やてんかんの合併の有無、もやもや病の分布が両側性か片側性か、といった項目について行った。発症年齢、障害されているPCAの本数といった正規分布に従わない連続変数は、Mann-WhitneyのU 検定やKruskal-Wallis 検定を用いて解析した。P値が<0.05である時、統計学的に有意と判定した。Kaplan-Meier曲線を用いて累積発症率を算出し、log-rank検定で統計学的有意性を確認した。Cox回帰モデルを用いて、発症年齢に関連する因子を検出した。もやもや病の発症確率の正確検定の95％信頼区間は二項分布を用いて算出した。親子発症例や同胞発症例での臨床項目の比較はWilcoxinの符号順位検定もしくはMcNemar検定を用いた。 Statistical analysis Patients with no information about each clinical item were excluded from each statistical analysis. (Table 1 and supplemental tables 2 and 4 show the number of patients excluded for each item). All statistical analyzes were performed using SPSS Statistics 19 (IBM). The chi-square test was performed on items such as initial symptoms, presence of family history, presence of intellectual function decline and epilepsy, and whether Moyamoya disease was bilateral or unilateral. Continuous variables that do not follow the normal distribution, such as age at onset and the number of PCAs injured, were analyzed using the Mann-Whitney U test and Kruskal-Wallis test. A P value of <0.05 was considered statistically significant. Cumulative incidence was calculated using Kaplan-Meier curve, and statistical significance was confirmed by log-rank test. Cox regression model was used to detect factors related to age at onset. The 95% confidence interval of the exact test for the probability of Moyamoya disease was calculated using the binomial distribution. Wilcoxin's sign rank test or McNemar's test was used to compare clinical items in patients with onset of parents and siblings.

結果
RNF213遺伝子の19種類の多型が同定された。(Fig.1及びsupplemental table1に詳細を示す。) そのうち16個は新規の変異であった。C.14576G>Aは41例の家族歴のあるもやもや病患者のうち39例に（95.1%）、163例の孤発性の患者のうち129例(79.2%)に、そして283人の健常日本人のうち5人に(1.8%)検出された。62組の患者の両親についてもこの多型の有無を調べたところ、この多型は全例で、一方もしくは両方の親からの遺伝であった。C.14576G>A多型をもつ168例のうち、15例でホモ接合性変異が検出されたが、健常コントロールと非罹患の患者の親では1例も検出されなかった。以上からヘテロ接合性のc.4576G>A多型はもやもや病の発症リスクを高めそのOdds比は236、95%信頼区間は91-615、p<0.001であった。ホモ接合性変異がコントロールや非罹患の親たちに見られなかったことから、ホモ接合性多型の発症リスクはOdds比が計算上無限大となって算出はできなかった。しかしかなり強い効果をもつものと想定された。ホモ接合性変異を有する場合のもやもや病の発症確率は、非常に高く、95%信頼区間でいうと、78-100%であった。その他に見つかった変異／多型に関しては、もやもや病発症のリスクに関するOdds比は小さく、どれも有意差は見られなかった。(Supplemental table 1)
31例が、その他の多型を有していた。（table.2）そのうち15人は、c.14576G>Aをヘテロ接合性に有しており、その中で両親の検体が解析可能だった5例中4例が、これら2つの多型を複合ヘテロ接合性に有していた。（つまり1つの多型が父親から、もうひとつが母親から由来していた）。それ以外の16例はc.14576G>A多型を持っていなかった。そのうち1人はc.13342G>A多型をホモ接合性に有しており、他の２人は２つの別の多型であるc.13342G>Aとc.14053G>Aを複合ヘテロ接合性に有していた。16個の新規の多型のうち、11個は188人の健常日本人には1例も見られず、これらは全てプライベートな変異であった。（つまり各々が1つの家系にしか見られなかった）
私たちは、c.14576G>A多型のパターンによって、患者を3つのグループに分けて、各グループでの臨床項目を比較した。3つのグループとは、多型をもたない野生型(遺伝型:
GG, これをGGグループとする)、ヘテロ接合型(遺伝型: GA,GAグループ),ホモ接合型(遺伝型: AA, AAグループ) である。発症年齢は、GA, GGクループに比べてAAグループで有意に若かった。(AA vs. GA: p=0.002, AA vs. GG: p=0.007) (Fig. 2A 及びsupplemental table 2参照) 。発症年齢の中央値はそれぞれ、AAグループで3歳、GAグループで7歳、GGグループで8歳だった。加齢に伴う二次性の血管病変の影響を無視でき、そのため遺伝学的効果が純粋に観察できると思われる15歳以下の患者に限ってみても、同様の結果が再現された。(AA vs. GA: p=0.001, AA vs. GG: p=0.007) (Supplemental table 2) 。小児発症と成人発症では臨床経過が異なるが、この2群間にこの多型を持つ患者の割合の有意差はなく、それぞれ83.2%と79.6%であった。これは異なる遺伝子によって規定される別の疾患というより、臨床の多様性を示唆するものと思われた。単変量のCox回帰分析では、発症年齢に関連するのはc.14576G>A遺伝型のみであった。(Supplemental table 3) 。累積発症頻度はAAグループで有意に高く、この傾向はほとんどどの年代にも見られた。(Log Rank検定: p=0.03) (Fig.2B) しかし、この傾向は10歳前の年代で特に顕著であった。AAグル―プの60％もの患者が4歳未満で発症しているのに対し、GAグループでは15％、GGグループでは14.3％が4歳未満で発症していて、早期発症例の頻度は有意差があった。(AA vs. GA vs. GG: p<0.001) (Fig. 3A)。これらのAAグループの早期発症例では全例脳梗塞で発症していた。 result
Nineteen polymorphisms of the RNF213 gene were identified. (Details are shown in Fig. 1 and supplemental table 1.) 16 of them were novel mutations. C.14576G> A was found in 39 of moyamoya disease patients with a family history of 41 (95.1%), 129 of 163 sporadic patients (79.2%), and 283 healthy Japanese patients It was detected in 5 of the people (1.8%). The parents of the 62 pairs of patients were also examined for the presence or absence of this polymorphism, all of which were inherited from one or both parents. Of the 168 cases with the C.14576G> A polymorphism, homozygous mutations were detected in 15 cases, but none were found in the parents of healthy controls and unaffected patients. Based on the above, the heterozygous c.4576G> A polymorphism increased the risk of Moyamoya disease and its Odds ratio was 236, with a 95% confidence interval of 91-615 and p <0.001. Since no homozygous mutations were found in controls or unaffected parents, the risk of developing homozygous polymorphisms could not be calculated because the Odds ratio was infinite. However, it was assumed to have a fairly strong effect. The incidence of moyamoya disease in the case of having a homozygous mutation was very high, 78-100% in the 95% confidence interval. For other mutations / polymorphisms found, the odds ratio for the risk of developing moyamoya disease was small, and no significant difference was observed. (Supplemental table 1)
31 cases had other polymorphisms. (Table.2) 15 of them have heterozygosity of c.14576G> A, of which 4 out of 5 cases where parents' samples could be analyzed combined these 2 polymorphisms It had heterozygosity. (That is, one polymorph was from the father and the other was from the mother.) The other 16 cases did not have the c.14576G> A polymorphism. One of them is homozygous for the c.13342G> A polymorphism and the other two are heterozygous for two other polymorphisms, c.13342G> A and c.14053G> A. Had. Of the 16 new polymorphisms, 11 were not seen in 188 healthy Japanese, all of which were private mutations. (In other words, each was found in only one family)
We divided patients into three groups according to the c.14576G> A polymorphism pattern and compared clinical items in each group. The three groups are the wild type without the polymorphism (genotype:
GG, which is a GG group), heterozygous (genotype: GA, GA group), and homozygous (genotype: AA, AA group). The age of onset was significantly younger in the AA group than in the GA and GG groups. (AA vs. GA: p = 0.002, AA vs. GG: p = 0.007) (See Fig. 2A and supplemental table 2). The median age of onset was 3 years in the AA group, 7 years in the GA group, and 8 years in the GG group, respectively. Similar effects were replicated in patients under 15 years of age who could ignore the effects of secondary vascular lesions associated with aging, so that the genetic effect would be purely observable. (AA vs. GA: p = 0.001, AA vs. GG: p = 0.007) (Supplemental table 2). Although the clinical course differs between childhood onset and adult onset, there was no significant difference in the proportion of patients with this polymorphism between the two groups, 83.2% and 79.6%, respectively. This seemed to suggest clinical diversity rather than another disease defined by different genes. In the univariate Cox regression analysis, only the c.14576G> A genotype was associated with age at onset. (Supplemental table 3). Cumulative incidence was significantly higher in the AA group, and this trend was seen in almost every age. (Log Rank test: p = 0.03) (Fig.2B) However, this tendency was particularly prominent in the age before 10 years. As many as 60% of patients in the AA group develop before the age of 4 years, whereas 15% of the GA group and 14.3% of the GG group develop before the age of 4 years. There was a difference. (AA vs. GA vs. GG: p <0.001) (Fig. 3A). All of these AA groups had early onset cases with cerebral infarction.

その他のもやもや病の臨床症状の項目も、3つのグループ間で比較した。(Fig.3A及び supplemental table 2)。初発症状としては、AAグループでは脳梗塞の頻度が他のグループより有意に高かった(AA vs. GA: 80% vs. 43.1%; p=0.01; OR 5.3; 95%CI 1.43-19.56, AA vs. GG: 80.0% vs. 38.2%; p=0.01; OR 6.5; 95%CI 1.53-27.32) 。TIAは逆にAAでより頻度が少なかった。(AA vs. GA: 13.3% vs. 43.1%; p=0.03; OR 0.2; 95%CI 0.04-0.94) 。脳出血／脳室内出血の頻度は3つのグループ間で有意差はなかった。(AA vs. GA vs. GG: 6.7% vs. 6.9% vs. 17.6%; p=0.13) 両側性のもやもや病はGGグループよりもAAとGAグループでより有意に頻度が高かった。(AA+GA vs. GG: 98.4% vs. 84.6%; p=0.008; OR 11; 95%CI 1.98-66.36) 家族性の頻度はGGグループよりもAAとGAグループで有意に高かった。(AA+GA vs. GG: 23.2% vs. 5.6%; p=0.02; OR5.1; 95%CI 1.18-22.36) 狭窄もしくは閉塞したPCAの本数はGAグループよりもAAグループで有意に多かった。(AA: 18/ 26 PCAs in 13 AA homozygotes; GA: 82/ 230 PCAs in 115 GA heterozygotes;
p=0.01 by Mann-Whitney U test) PCA病変の有無について情報のある152症例中74例(48.6%)にPCA病変が見られ、PCA病変を合併する群ではそうでない群に比べて初発症状に脳梗塞が起こる頻度と知的機能障害を合併する頻度が有意に高かった。(Infarctions: 68.9% vs. 30.4%, p<0.001; intellectual impairment: 26.8% vs. 5.2%, p<0.001) このことは、以前にYamadaらが、報告したデータ（43％の症例にPCA病変が合併し、こうした症例ではより高頻度に脳梗塞や大脳萎縮がみられた）に一致していた。知的機能低下の合併は、GAグループよりもAAグループでより多い傾向がみられたが、有意差はなかった。(AA vs. GA: 33.3% vs. 13.8%; p=0.06, OR 3.1; 95%CI 0.97-10.17) てんかん合併の頻度はグループ間で違いはなかった。(AA vs. GA vs. GG: 26.7% vs. 15.8% vs. 20.6%; p=0.51) 15歳未満発症の小児例においても、c.14576G>Aの多型のタイプと臨床病型の相関は類似していた。ただし小児例では全例が両側性の血管病変を持つことが異なっていた。(Fig.3B)
c.14576G>A多型以外の多型ともやもや病の臨床症状との相関についても検討した。(Supplemental table 4) もやもや病の患者を次の4つのグループに分けた、すなわちc.14576G>A多型を持たず、それ以外の多型を1つ以上持つ群(GG1グループ)、c.14576G>A多型を持たず、それ以外の多型も持たない群(GG0グループ)、ヘテロ接合性にc.14576G>A多型を持ち、それ以外の多型を1つ以上持つ群(GA1グループ)、ヘテロ接合性にc.14576G>A多型を持ち、それ以外の多型を持たない群(GA0グループ)にわけた。GG1とGG0の間には発症年齢に有意差はなかったが(GG1 vs. GG0: p=0.48)、GA0はGA1より発症年齢は有意に低かった(GA1 vs. GA0: p=0.03)。発症年齢の中央値はGA0で7歳、GA1で12歳であった。GA1では初発時に脳梗塞を起こす頻度は有意に低く、脳出血を起こす頻度は有意に高かった(infarctions:
GA1 vs. GA0:14.3% vs. 46.2%; p=0.02; OR 0.19; 95%CI 0.04-0.90, ICH/IV: GA1 vs.
GA0: 28.6% vs. 4.6%; p=0.009; OR 8.3; 95%CI 2.00-35.20) 。しかしpolyphen2²⁶やSIFT²⁷などの変異の病原性を予想するアルゴリズムで病的と判定された変異に限って同様の比較を行うと、上記の結果はコンスタントには見られなかったのでこれらのその他の多型の意義は不明である。多数例でのさらなる解析が、その他の変異の遺伝学的効果の検証を行うためには必要である。 Other clinical symptoms of Moyamoya disease were also compared among the three groups. (Fig.3A and supplemental table 2). As initial symptoms, the AA group had a significantly higher frequency of cerebral infarction than the other groups (AA vs. GA: 80% vs. 43.1%; p = 0.01; OR 5.3; 95% CI 1.43-19.56, AA vs. GG: 80.0% vs. 38.2%; p = 0.01; OR 6.5; 95% CI 1.53-27.32). Conversely, TIA was less frequent with AA. (AA vs. GA: 13.3% vs. 43.1%; p = 0.03; OR 0.2; 95% CI 0.04-0.94). The frequency of cerebral hemorrhage / intraventricular hemorrhage was not significantly different among the three groups. (AA vs. GA vs. GG: 6.7% vs. 6.9% vs. 17.6%; p = 0.13) Bilateral moyamoya disease was significantly more frequent in the AA and GA groups than in the GG group. (AA + GA vs. GG: 98.4% vs. 84.6%; p = 0.008; OR 11; 95% CI 1.98-66.36) The frequency of familiality was significantly higher in the AA and GA groups than in the GG group. (AA + GA vs. GG: 23.2% vs. 5.6%; p = 0.02; OR5.1; 95% CI 1.18-22.36) The number of constricted or occluded PCA was significantly higher in the AA group than in the GA group. (AA: 18/26 PCAs in 13 AA homozygotes; GA: 82/230 PCAs in 115 GA heterozygotes;
p = 0.01 by Mann-Whitney U test) 74 out of 152 cases (48.6%) with information on the presence or absence of PCA lesions, PCA lesions were seen in the group with PCA lesions compared to the other group with the first symptoms The frequency of cerebral infarction and the frequency of intellectual impairment were significantly higher. (Infarctions: 68.9% vs. 30.4%, p <0.001; intellectual impairment: 26.8% vs. 5.2%, p <0.001) This was previously reported by Yamada et al. (43% of cases had PCA lesions) In these cases, these cases coincided with cerebral infarction and cerebral atrophy more frequently). There was a trend toward more intellectual declines in the AA group than in the GA group, but there was no significant difference. (AA vs. GA: 33.3% vs. 13.8%; p = 0.06, OR 3.1; 95% CI 0.97-10.17) The frequency of epilepsy mergers did not differ between groups. (AA vs. GA vs. GG: 26.7% vs. 15.8% vs. 20.6%; p = 0.51) Correlation between c.14576G> A polymorphism and clinical pathology even in children under 15 years of age Were similar. However, all the pediatric cases differed from having bilateral vascular lesions. (Fig.3B)
We also examined the correlation between polymorphisms other than c.14576G> A polymorphism and clinical symptoms of Moyamoya disease. (Supplemental table 4) Moyamoya disease patients were divided into the following 4 groups: c.14576G> A polymorphism, group with more than 1 polymorphism (GG1 group), c.14576G > A polymorphism and no other polymorphism (GG0 group), heterozygous c.14576G> A polymorphism and one or more other polymorphism (GA1 group) ), Heterozygous c.14576G> A polymorphism and other polymorphisms (GA0 group). There was no significant difference in onset age between GG1 and GG0 (GG1 vs. GG0: p = 0.48), but GA0 was significantly lower in onset age than GA1 (GA1 vs. GA0: p = 0.03). The median age of onset was 7 years for GA0 and 12 years for GA1. In GA1, the frequency of cerebral infarction at the first onset was significantly lower and the frequency of cerebral hemorrhage was significantly higher (infarctions
GA1 vs. GA0: 14.3% vs. 46.2%; p = 0.02; OR 0.19; 95% CI 0.04-0.90, ICH / IV: GA1 vs.
GA0: 28.6% vs. 4.6%; p = 0.009; OR 8.3; 95% CI 2.00-35.20). However, when similar comparisons were made only for mutations that were determined to be pathogenic by algorithms that predict pathogenicity of mutations such as polyphen2 ²⁶ and SIFT ²⁷ , the above results were not seen consistently. The significance of the polymorphism is unknown. Further analysis in a number of cases is necessary to verify the genetic effects of other mutations.

さらに、５つの親子例と６つの同胞例でRNF213の遺伝型が一致しているペアについて、臨床症状項目を比較した。親子例では、発症年齢は子供で有意に若く(p=0.04)、発症年齢の中央値は子供が5歳、親が37歳であった。このことは従来いわれているもやもや病の表現促進現象¹⁸に矛盾しない。一方同胞例では、発症年齢は同胞間で有意差はなかった(p=0.67)。発症年齢の中央値は年上の同胞で8歳、年下の同胞で12.5歳であった。その他の臨床症状について、親子間、同胞間で比較すると有意な違いはみられなかった。 Furthermore, the clinical symptom items were compared for the pairs in which the genotype of RNF213 was the same in 5 parent-child cases and 6 siblings. In the parent-child case, the onset age was significantly younger in children (p = 0.04), and the median age of onset was 5 years for children and 37 years for parents. This is not inconsistent with the anticipation phenomenon ¹⁸ of moyamoya disease, which is said conventional. On the other hand, in siblings, the age of onset was not significantly different between siblings (p = 0.67). The median age of onset was 8 years for older siblings and 12.5 years for younger siblings. Regarding other clinical symptoms, no significant difference was found between parent and child and siblings.

考察
204例のもやもや病患者について、RNF213遺伝子の包括的な解析及び臨床症状の評価を行った。C.14576G>A多型ともやもや病発症には強い相関があることを確定した。この多型をヘテロ接合性に持っている場合、オッズ比は236, p<0.001、ホモ接合性に持っているときは、その効果が計算上無限大となるほど大きかった。仮にヘテロ接合性多型とホモ接合性多型のもやもや病発症に対する効果がほぼ同等とすると、ホモ接合性多型を持つことで、もやもや病の発症リスクは少なくともオッズ比259, 95%信頼区間 100-674, p<0.001と計算された。しかしホモ接合性多型の効果は、もっと非常に大きいと思われた。何故なら私たちの研究で283人の健常コントロールと132人の非罹患の両親、および先行研究²³で429人の健常人コントロールと28人の非罹患の家族構成員にこの変異が見つからなかったからである。ホモ接合性多型を持つことによる、もやもや病の発症確率は78％以上と計算された。非常にオッズ比は高いが、この多型は純粋なメンデル遺伝には合致しない。何故なら正常集団にもある程度の頻度でこの多型がみつかるからである。この比較的まれな多型は、多因子複合疾患における、いわゆる ‘missing heritability’ （小さな遺伝学的効果を持つ高頻度の多型では説明しえない遺伝学的背景）を説明する１つの好例であると考える。^28,29 RNF213 はAAA (ATPases associated with variety of a cellular activities) ドメインをもつRINGフィンガータンパクであり、E3 ユビキチンリガーゼ活性とエネルギー依存性アンフォルダーゼ活性を有する。³⁰このタンパクのもやもや病発症への関与については更なる研究が必要である。 Consideration
A comprehensive analysis of RNF213 gene and evaluation of clinical symptoms were performed on 204 moyamoya patients. The C.14576G> A polymorphism was confirmed to have a strong correlation with the onset of Moyamoya disease. When this polymorphism was heterozygous, the odds ratio was 236, p <0.001, and when it was homozygous, the effect was so large that it became infinite in the calculation. If the heterozygous polymorphism and the homozygous polymorphism have almost the same effect on the development of moyamoya disease, the risk of developing moyamoya disease is at least an odds ratio of 259, 95% with a confidence interval of 100. -674, p <0.001. However, the effect of the homozygous polymorphism seemed much greater. Because our study did not find this mutation in 283 healthy controls and 132 unaffected parents, and in previous study ²³ 429 healthy controls and 28 unaffected family members is there. The probability of Moyamoya disease due to having a homozygous polymorphism was calculated to be over 78%. Although the odds ratio is very high, this polymorphism does not match pure Mendelian inheritance. This is because the polymorphism is found in the normal population at a certain frequency. This relatively rare polymorphism is a good example of the so-called 'missing heritability' (genetic background that cannot be explained by high-frequency polymorphisms with small genetic effects) in multifactorial complex diseases. I think there is. ^28,29 RNF213 is a RING finger protein with AAA (ATPases associated with a variety of cellular activities) domain, and has E3 ubiquitin ligase activity and energy-dependent unfoldase activity. ³⁰ Further research is needed on the involvement of this protein in the development of moyamoya disease.

ホモ接合性のc.1576G>A多型を持つ患者は、有意に早期発症で初発症状に脳梗塞が多く、またPCAも障害されていることが多かった。これらはすべて予後不良因子とされている。^12,15 これらのことから、ホモ接合性c.14576G>A多型を持つと、より重症で広範な脳血管病変を有することが示唆される。その他予後不良因子として知られている、知的機能低下やけいれん合併者は、ホモ接合性多型を持つ患者に多い傾向があったが有意差は見られなかった。これらの病態は早期診断、早期治療によって修飾あるいは予防されると考えられる。 Patients with a homozygous c.1576G> A polymorphism were significantly early onset, often had cerebral infarction as the first symptom, and PCA was also often impaired. These are all regarded as poor prognostic factors. ^12,15 These suggest that having a homozygous c.14576G> A polymorphism has more severe and widespread cerebrovascular lesions. Other patients with poor intellectual function and convulsions, known as poor prognostic factors, tended to be more common among patients with homozygous polymorphism, but there was no significant difference. These pathological conditions are considered to be modified or prevented by early diagnosis and early treatment.

早期発症もやもや病患者(発症年齢<3-4歳) は、通常より重症になるため、早期の外科的治療が勧められている。早期発症例の約80％は初発症状に脳梗塞を起こし、その後の脳梗塞の再発も多く、再発性脳梗塞の頻度は、より高い年齢で発症した患者群より高い。^31,32 私たちの研究でも、4歳未満で発症した患者の77.1%の症例が初発時脳梗塞を起こしているのに対し、4歳以降に発症した患者の38％が初発時脳梗塞を起こしており(p<0.001)、従来のデータに矛盾しなかった。しかしScottらによると、2歳以下発症の患者のすべてが予後不良であるわけではなく、予後を最も左右するのは、脳梗塞によっておこった神経学的脱落症状が手術する時点でどれくらいみられるかである。^11,33最近、非可逆性の脳梗塞が、最大の予後不良因子であることが多変量解析で明らかになった。¹² 発症年齢は単変量解析では予後と相関があったが、最も特異的な予後不良マーカーではなかった。¹² 脳梗塞と強く相関する特異的なバイオマーカーがあれば、手術時期を決定するのに（手術を急いだ方がよいのかどうか検討するのに）臨床上とても有用である。今回の研究ではホモ接合性多型を持つ患者の60％が4歳までに発症していたが、その全員は初発症状が脳梗塞であった。ホモ接合性のこの多型は、若年発症のもやもや病患者の中で、予後不良群と予後良好群を見分けるためのより特異的な予測因子になり得る。 Early surgical treatment is recommended for patients with early-onset moyamoya disease (onset age <3-4 years) because they are more severe than usual. Approximately 80% of early-onset cases have cerebral infarction as the first symptom, and there are many recurrences of subsequent cerebral infarction, and the frequency of recurrent cerebral infarction is higher than the group of patients who developed at an older age. ^{31,32 In} our study, 77.1% of patients who developed before the age of 4 years had the first stroke, whereas 38% of patients who developed after the age of 4 had the first stroke It was awake (p <0.001) and was consistent with previous data. However, according to Scott et al., Not all patients who develop 2 years of age or younger have a poor prognosis, and the prognosis is most affected by how much neurological dropout caused by cerebral infarction is seen at the time of surgery. It is. ^11,33 Recently, multivariate analysis revealed that irreversible cerebral infarction is the biggest poor prognostic factor. ¹² Age at onset correlated with prognosis in univariate analysis, but was not the most specific poor prognostic marker. ¹² Specific biomarkers that strongly correlate with cerebral infarction are very useful clinically to determine the timing of surgery (whether it is better to rush surgery). In this study, 60% of patients with homozygous polymorphism had developed by the age of 4 years, all of whom had cerebral infarction as the first symptom. This homozygous polymorphism can be a more specific predictor for distinguishing between a poor prognosis group and a good prognosis group in young patients with Moyamoya disease.

私たちは、今回の研究で、c.14576G>A の遺伝型が予後不良の重症型もやもや病を予測するための有用なマーカーになり得ることを提案する。この遺伝型を知ることで、重症の初発脳梗塞を起こす実際のリスクについても知ることができるので、このようなハイリスク保因者では、注意深いフォローアップを行うことで、脳梗塞を起こす前に早期の外科的療法を行い、非可逆的な神経学的後遺症を残すことを防ぐことも可能にするかもしれない。この遺伝型についての情報は、正確な遺伝カウンセリングにも有用であろう。 In this study, we propose that the genotype c.14576G> A could be a useful marker for predicting severe moyamoya disease with poor prognosis. By knowing this genotype, you can also know the actual risk of developing a severe initial cerebral infarction, so for such high-risk carriers, careful follow-up can be done before cerebral infarction occurs. It may also be possible to prevent early surgical treatment and leave behind irreversible neurological sequelae. Information about this genotype may also be useful for accurate genetic counseling.

文献
1. Suzuki J, Takaku A. Cerebrovascular "moyamoya" disease. Disease showingabnormal net-like vessels in base of brain. Arch Neurol 1969;20:288-99.
2. Goto Y, Yonekawa Y. Worldwide distribution of moyamoya disease. Neurol Med Chir (Tokyo) 1992;32:883-6.
3. Suzuki J, Kodama N. Moyamoya disease--a review. Stroke 1983;14:104-9.
4. Wakai K, Tamakoshi A, Ikezaki K, et al. Epidemiological features of moyamoya disease in Japan: findings from a nationwide survey. Clin Neurol Neurosurg 1997;99 Suppl 2:S1-5.
5. Kuriyama S, Kusaka Y, Fujimura M, et al. Prevalence and clinicoepidemiological features of moyamoya disease in Japan: findings from a nationwide epidemiological survey. Stroke 2008;39:42-7.
6. Yonekawa Y, Ogata N, Kaku Y, Taub E, Imhof HG. Moyamoya disease in Europe, past and present status. Clin Neurol Neurosurg 1997;99 Suppl 2:S58-60.
7. Kuroda S, Houkin K. Moyamoya disease: current concepts and future perspectives. Lancet Neurol 2008;7:1056-66.
8. Kim SK, Wang KC, Kim DG, et al. Clinical feature and outcome of pediatric cerebrovascular disease: a neurosurgical series. Childs Nerv Syst 2000;16:421-8.
9. Soriano SG, Sethna NF, Scott RM. Anesthetic management of children with moyamoya syndrome. Anesth Analg 1993;77:1066-70.
10. Nagaraja D, Verma A, Taly AB, Kumar MV, Jayakumar PN. Cerebrovascular disease in children. Acta Neurol Scand 1994;90:251-5.
11. Scott RM, Smith ER. Moyamoya disease and moyamoya syndrome. N Engl J Med
2009;360:1226-37.
12. Kim SK, Cho BK, Phi JH, et al. Pediatric moyamoya disease: An analysis of 410 consecutive cases. Ann Neurol 2010;68:92-101.
13. Karasawa J, Touho H, Ohnishi H, Miyamoto S, Kikuchi H. Long-term follow-up study after extracranial-intracranial bypass surgery for anterior circulation ischemia in childhood moyamoya disease. J Neurosurg 1992;77:84-9.
14. Kurokawa T, Tomita S, Ueda K, et al. Prognosis of occlusive disease of the circle of Willis (moyamoya disease) in children. Pediatr Neurol 1985;1:274-7.
15. Yamada I, Himeno Y, Suzuki S, Matsushima Y. Posterior circulation in moyamoya disease: angiographic study. Radiology 1995;197:239-46.
16. Mineharu Y, Takenaka K, Yamakawa H, et al. Inheritance pattern of familial moyamoya disease: autosomal dominant mode and genomic imprinting. J Neurol Neurosurg Psychiatry 2006;77:1025-9.
17. Fukui M, Kono S, Sueishi K, Ikezaki K. Moyamoya disease. Neuropathology 2000;20 Suppl:S61-4.
18. Nanba R, Kuroda S, Tada M, Ishikawa T, Houkin K, Iwasaki Y. Clinical features of familial moyamoya disease. Childs Nerv Syst 2006;22:258-62.
19. Ikeda H, Sasaki T, Yoshimoto T, Fukui M, Arinami T. Mapping of a familial moyamoya disease gene to chromosome 3p24.2-p26. Am J Hum Genet 1999;64:533-7.
20. Sakurai K, Horiuchi Y, Ikeda H, et al. A novel susceptibility locus for moyamoya disease on chromosome 8q23. J Hum Genet 2004;49:278-81.
21. Mineharu Y, Liu W, Inoue K, et al. Autosomal dominant moyamoya disease maps to chromosome 17q25.3. Neurology 2008;70:2357-63.
22. Inoue TK, Ikezaki K, Sasazuki T, Matsushima T, Fukui M. Linkage analysis of moyamoya disease on chromosome 6. J Child Neurol 2000;15:179-82.
23. Kamada F, Aoki Y, Narisawa A, et al. A genome-wide association study identifies RNF213 as the first Moyamoya disease gene. J Hum Genet 2011;56:34-40.
24. Fukui M. Guidelines for the diagnosis and treatment of spontaneous occlusion of the circle of Willis ('moyamoya' disease). Research Committee on Spontaneous Occlusion of the Circle of Willis (Moyamoya Disease) of the Ministry of Health and Welfare, Japan. Clin Neurol Neurosurg 1997;99 Suppl 2:S238-40.
25. Garritano S, Gemignani F, Voegele C, et al. Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus. BMC Genet 2009;10:5.
26. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods 2010;7:248-9.
27. Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 2003;31:3812-4.
28. Eichler EE, Flint J, Gibson G, et al. Missing heritability and strategies for finding the underlying causes of complex disease. Nat Rev Genet 2010;11:446-50.
29. Manolio TA, Collins FS, Cox NJ, et al. Finding the missing heritability of complex diseases. Nature 2009;461:747-53.
30. Lupas AN, Martin J. AAA proteins. Curr Opin Struct Biol 2002;12:746-53.
31. Kim SK, Seol HJ, Cho BK, Hwang YS, Lee DS, Wang KC. Moyamoya disease among young patients: its aggressive clinical course and the role of active surgical treatment. Neurosurgery 2004;54:840-4; discussion 4-6.
32. Mugikura S, Higano S, Shirane R, Fujimura M, Shimanuki Y, Takahashi S. Posterior circulation and high prevalence of ischemic stroke among young pediatric patients with Moyamoya disease: evidence of angiography-based differences by age at diagnosis. AJNR Am J Neuroradiol 2011;32:192-8.
33. Scott RM, Smith JL, Robertson RL, Madsen JR, Soriano SG, Rockoff MA. Long-term outcome in children with moyamoya syndrome after cranial revascularization by pial synangiosis. J Neurosurg 2004;100:142-9. Literature
1. Suzuki J, Takaku A. Cerebrovascular "moyamoya" disease. Disease showing abnormal net-like vessels in base of brain. Arch Neurol 1969; 20: 288-99.
2. Goto Y, Yonekawa Y. Worldwide distribution of moyamoya disease. Neurol Med Chir (Tokyo) 1992; 32: 883-6.
3. Suzuki J, Kodama N. Moyamoya disease--a review.Stroke 1983; 14: 104-9.
4.Wakai K, Tamakoshi A, Ikezaki K, et al. Epidemiological features of moyamoya disease in Japan: findings from a nationwide survey.Clin Neurol Neurosurg 1997; 99 Suppl 2: S1-5.
5. Kuriyama S, Kusaka Y, Fujimura M, et al. Prevalence and clinicoepidemiological features of moyamoya disease in Japan: findings from a nationwide epidemiological survey.Stroke 2008; 39: 42-7.
6. Yonekawa Y, Ogata N, Kaku Y, Taub E, Imhof HG.Moyamoya disease in Europe, past and present status.Clin Neurol Neurosurg 1997; 99 Suppl 2: S58-60.
7. Kuroda S, Houkin K. Moyamoya disease: current concepts and future perspectives. Lancet Neurol 2008; 7: 1056-66.
8. Kim SK, Wang KC, Kim DG, et al. Clinical feature and outcome of pediatric cerebrovascular disease: a neurosurgical series.Childs Nerv Syst 2000; 16: 421-8.
9. Soriano SG, Sethna NF, Scott RM. Anesthetic management of children with moyamoya syndrome. Anesth Analg 1993; 77: 1066-70.
10. Nagaraja D, Verma A, Taly AB, Kumar MV, Jayakumar PN.Cerebrovascular disease in children. Acta Neurol Scand 1994; 90: 251-5.
11. Scott RM, Smith ER. Moyamoya disease and moyamoya syndrome. N Engl J Med
2009; 360: 1226-37.
12. Kim SK, Cho BK, Phi JH, et al. Pediatric moyamoya disease: An analysis of 410 consecutive cases. Ann Neurol 2010; 68: 92-101.
13. Karasawa J, Touho H, Ohnishi H, Miyamoto S, Kikuchi H. Long-term follow-up study after extracranial-intracranial bypass surgery for anterior circulation ischemia in childhood moyamoya disease.J Neurosurg 1992; 77: 84-9.
14. Kurokawa T, Tomita S, Ueda K, et al. Prognosis of occlusive disease of the circle of Willis (moyamoya disease) in children.Pediatr Neurol 1985; 1: 274-7.
15. Yamada I, Himeno Y, Suzuki S, Matsushima Y. Posterior circulation in moyamoya disease: angiographic study. Radiology 1995; 197: 239-46.
16. Mineharu Y, Takenaka K, Yamakawa H, et al. Inheritance pattern of familial moyamoya disease: autosomal dominant mode and genomic imprinting. J Neurol Neurosurg Psychiatry 2006; 77: 1025-9.
17. Fukui M, Kono S, Sueishi K, Ikezaki K. Moyamoya disease. Neuropathology 2000; 20 Suppl: S61-4.
18. Nanba R, Kuroda S, Tada M, Ishikawa T, Houkin K, Iwasaki Y. Clinical features of familial moyamoya disease.Childs Nerv Syst 2006; 22: 258-62.
19. Ikeda H, Sasaki T, Yoshimoto T, Fukui M, Arinami T. Mapping of a familial moyamoya disease gene to chromosome 3p24.2-p26. Am J Hum Genet 1999; 64: 533-7.
20. Sakurai K, Horiuchi Y, Ikeda H, et al. A novel susceptibility locus for moyamoya disease on chromosome 8q23. J Hum Genet 2004; 49: 278-81.
21. Mineharu Y, Liu W, Inoue K, et al. Autosomal dominant moyamoya disease maps to chromosome 17q25.3. Neurology 2008; 70: 2357-63.
22. Inoue TK, Ikezaki K, Sasazuki T, Matsushima T, Fukui M. Linkage analysis of moyamoya disease on chromosome 6.J Child Neurol 2000; 15: 179-82.
23. Kamada F, Aoki Y, Narisawa A, et al. A genome-wide association study identifies RNF213 as the first Moyamoya disease gene. J Hum Genet 2011; 56: 34-40.
24. Fukui M. Guidelines for the diagnosis and treatment of spontaneous occlusion of the circle of Willis ('moyamoya' disease) .Research Committee on Spontaneous Occlusion of the Circle of Willis (Moyamoya Disease) of the Ministry of Health and Welfare, Japan. Clin Neurol Neurosurg 1997; 99 Suppl 2: S238-40.
25. Garritano S, Gemignani F, Voegele C, et al. Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus. BMC Genet 2009; 10: 5.
26. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods 2010; 7: 248-9.
27. Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function.Nucleic Acids Res 2003; 31: 3812-4.
28. Eichler EE, Flint J, Gibson G, et al. Missing heritability and strategies for finding the underlying causes of complex disease.Nat Rev Genet 2010; 11: 446-50.
29. Manolio TA, Collins FS, Cox NJ, et al. Finding the missing heritability of complex diseases. Nature 2009; 461: 747-53.
30. Lupas AN, Martin J. AAA proteins. Curr Opin Struct Biol 2002; 12: 746-53.
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本発明は、もやもや病の詳細な発症リスクの検出と重症度の予測を行うものであり、医療現場において、早急な手術適応となる症例の抽出や、発症前ハイリスク症例の脳血管イベントの予防のための手段を提供する。本発明が提供するホモ接合例は重症度が高く手術適応をはじめとする臨床方針決定の参考になりうる。 The present invention detects detailed risk of moyamoya disease and predicts its severity, and in the medical field, extracts cases that are promptly indicated for surgery and prevents cerebrovascular events in high-risk cases before onset. Provides a means for The homozygous example provided by the present invention has high severity and can be used as a reference for clinical policy determination including surgical indication.

＜配列番号１＞
配列番号１は、ヒトRNF213遺伝子(isoform1)のゲノム配列を示す。
＜配列番号２＞
配列番号２は、ヒトRNF213遺伝子(isoform1)のmRNA配列を示す。
＜配列番号３＞
配列番号３は、ヒトRNF213遺伝子(isoform1)のmRNAがコードするRNF213蛋白のアミノ酸配列を示す。
＜配列番号４＞
配列番号４は、ヒトRNF213遺伝子(isoform2)のゲノム配列を示す。
＜配列番号５＞
配列番号５は、ヒトRNF213遺伝子(isoform2)のmRNA配列を示す。
＜配列番号６＞
配列番号６は、ヒトRNF213遺伝子(isoform2)のmRNAがコードするRNF213蛋白のアミノ酸配列を示す。
＜配列番号７＞
配列番号７は、RNF213遺伝子の変異解析に使用できるプライマー（フォワードプライマー）の配列を示す。
＜配列番号８＞
配列番号８は、RNF213遺伝子の変異解析に使用できるプライマー（リバースプライマー）の配列を示す。 <SEQ ID NO: 1>
SEQ ID NO: 1 shows the genomic sequence of the human RNF213 gene (isoform 1).
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the mRNA sequence of human RNF213 gene (isoform 1).
<SEQ ID NO: 3>
SEQ ID NO: 3 shows the amino acid sequence of the RNF213 protein encoded by the mRNA of the human RNF213 gene (isoform 1).
<SEQ ID NO: 4>
SEQ ID NO: 4 shows the genomic sequence of human RNF213 gene (isoform2).
<SEQ ID NO: 5>
SEQ ID NO: 5 shows the mRNA sequence of human RNF213 gene (isoform 2).
<SEQ ID NO: 6>
SEQ ID NO: 6 shows the amino acid sequence of the RNF213 protein encoded by the mRNA of the human RNF213 gene (isoform 2).
<SEQ ID NO: 7>
SEQ ID NO: 7 shows the sequence of a primer (forward primer) that can be used for mutation analysis of the RNF213 gene.
<SEQ ID NO: 8>
SEQ ID NO: 8 shows the sequence of a primer (reverse primer) that can be used for mutation analysis of the RNF213 gene.

Claims

被験者のRNF213遺伝子におけるc.14576G>A多型を検出し、ホモ接合性の多型A/Aを持つ場合に、予後不良の重症型である、あるいは発症リスクが高いと判定する、もやもや病の検査方法。 When a c.14576G> A polymorphism in the RNF213 gene of a subject is detected and it has a homozygous polymorphism A / A, it is determined that the prognosis is severe or has a high risk of developing Moyamoya disease. Inspection method.

c.14576G>A多型をゲノムDNAで解析して検出する請求項１記載の検査方法。 The test method according to claim 1, wherein the c.14576G> A polymorphism is analyzed by genomic DNA.

c.14576G>A多型をRNAレベルで解析して検出する請求項１記載の検査方法。 The test method according to claim 1, wherein the c.14576G> A polymorphism is analyzed and detected at the RNA level.

c.14576G>A多型をタンパク質レベルで解析して検出する請求項１記載の検査方法。 The test method according to claim 1, wherein c.14576G> A polymorphism is analyzed and detected at the protein level.