筑波大学技術報告24: 76-80, 2004‫ݏ‬ਬ൉ൟ‫੾ڠ‬ƳǐǓ३‫ࡏډ‬ՔưƥƶЋஞࠃӕ৖ॠ໏ࢆ೵ࠃ૿ౡપԙॶյ੒‫ݜ‬௖ؐА‫ࠊףڽ‬ѬࡋáЌԙ‫ڈ‬âĞ(%*"-*,* Иश‫ۀ‬ƬƗƸࠅஔ҉઩ &"&"&Ӷ຤ō¼ධ඲ư‫ڤ‬ү¸ф௫ȆȠÓȨȮƔਏવஇƳ඄঴ơǓ‫ص‬Їࢹ঴ܺҠऔáALSâƳનഒƝǔǓ‫ۍ‬Пഩฯƶ३‫ډ‬బഗƶଇƳƷ¹ƊǓࡾƶ‫ݏ‬ਬЂऺƯ་ࠨƶऔीǚ֬ƛơǊƶƔ
ƊǓºƛǔǑƶഗ֡Ʒऺ৾ॐઑ༟঴ЋஞƯ¹঵ॶ༎ƯƷ೪ஒ‫ٸ‬இƲ‫ډ‬ӄǚƧƱǓǊƶƔંƌº३‫ډ‬ఝҭƯƷƛǔǑƶഗ֡ƶ॰૨ƶГղưƟƮ¹ಋ‫עڥ‬ȤǟǸǹÓțƳƒƙǓőࡾƶ‫ݏ‬ਬԩ঴ƶ੯୩ǚ݉ƫƮƌǓº‫ݶ‬ӖƷɋ-ȓǨǸǰȚȆǻÓǷԩ঴ƳЂऺƶƊƫƧՔࡣƶ༎ǚದഒơǓº‫ݏ‬ਬԩ঴Ʒ়ऺƳƗǑǁŌŋĊ୷ஶƯ¹ɋ-ȓǨǸǰȚȆǻÓǷƶ A ԩ঴ư
B ԩ঴ƶ໩ࡣƶୢҟƓǑ Sandhoff ഗƔּǗǔƧº2.1 ‫ݏ‬ਬԩ঴Ō¼ƷƠǉƳ¸ɋ-ȓǨǸǰȚȆǻÓǷ(HEX)Ʒ GM2-ǧȮǫȥǥǲȄ(GM2)ƶҦ঎ൟӕ‫ݏ‬ਬƯ¹ƥƶԩ঴ୢҟƳǐǒȤǟǸǹÓțఝƳ GM2 Ɣ଀৘Ɵެභౖ֞ƔमӳƝǔǓºHEX Ʒƥƶൖ‫ݜ‬ઑ‫ٺ‬঵ƶࡾ་ƳǐƫƮ A ൟӋư B ൟӋƶōƬƳൟƓǔǓºHEXA ƷɊ‫(ޒ‬HEXɊ)ưɋ‫(ޒ‬HEXɋ)ƶȓȁȨǻǟșÓ¹HEXB Ʒ HEXɋƶȖȝǻǟșÓưƟƮ‫ٺ‬঵ƝǔǓºɊ‫ޒ‬ǚǮÓȄơǓ HEXɊЋஞࠃƶЂऺƳǐǓǊƶƷ A ൟӋƶԩ঴ƶǇୢҟƟ Tay-Sachs ഗư‫۝‬Ƹǔ¹ɋ‫ޒ‬ǚǮÓȄơǓ HEXɋЋஞࠃƶЂऺƳǐǓǊƶƷ A¹B ໩ൟӋƶԩ঴ƔୢҟƟ Sandhoff ഗư‫۝‬ƸǔǓº¸ՔࡣƷ 15 ‫࠯ޣ‬ǐǒҟ࠙‫ص‬໺ୢҟǚ࠺ԑ¹23 ‫࠯ޣ‬ӭ૫अ‫ݷݔ‬బƳƮઁЦࢄ॰º‫ص‬஢ইƥƶઁǐǒ¹ॳ݉ƶૺƌ ALS ư॰૨ƝǔƧº35 ‫֔ލু࠯ޣ‬ාƯ෭Цǚࢄ॰ƟƧº¸ǆƢՔࡣƶೠ഻ਅЄӍެභǚԵҠƟƧº‫ࡣऺګ‬ƒ
ǐƻ Tay-Sachs ഗƶਅЄӍެභƷ໊‫ڽ‬ƶȋȮǪǐǒ୲‫׼‬ǚࢄƙƧºൿƳ‫ࡣऺګ‬¹Քࡣ¹໩९ƶงऊ‫ڥ‬ǐǒಋ‫עڥ‬ǚൟ໐Ɵ¹ԵҠƟƧਅЄӍެභưưǊƳ‫ݏ‬ਬԩ঴ǚ੯୩ƟƧºԩ঴੯୩ƳƷȓǨǸǰȚȆǻÓǷ ƶ ॶ ‫ ࡐ ֏ ܞ‬Ư Ɗ Ǔ 4-methylumbelliferyl-2acetamido-2-deoxy-ɋ-D-glucopyranoside (4MUG) ǚພƌƮ੯୩ƟƧºƝǑƳేഩԩ঴඲Ƴǐǒ A ԩ঴¹Bԩ঴ǚൟൿ੯୩ƟƧáഒ 1âº‫ڤ‬ү¹੒ Hex ૮Ʒ‫ګ‬ऺࡣƶ๢ 12ĊƳ¹HexA Ʒ๢ 15Ċ¹HexB Ʒ๢ŒĊƳୢҟƟƮƌƧºǆƧ¹ȓǨǸǰȚȆǻÓǷ A Ƴਏવ঴ƶƊǓॶ‫ࡐ֏ܞ‬ƯƊǓ 4-methylumbelliferyl-7-(6sulfo-2-acetamido-2-deoxy-β-D-glucopyranoside)(4MUGS) ǚǊƩƌƮՔࡣೠ഻ਅЄӍެභƶȓǨǸǰȚȆǻÓǷ A ԩ঴ǚ੯୩Ɵ¹‫ࡣऺګ‬ư Tay-Sachsഗƶೠ഻ਅЄӍެභƯƶȓǨǸǰȚȆǻÓǷ A ԩ঴ưೝԔƟƧº‫ڤ‬үƷ‫ࡣऺګ‬ƶ๢ 20Ċƶԩ঴ƯƊƫƧºƝǑƳ 4-MUG ǚພƌƮ໩९ƶȓǨǸǰȚȆǻÓǷԩ঴ǚ੯୩ƟƧºസ९ƳƒƌƮƷ੒ԩ঴૮Ʒ‫ࡣऺګ‬ư௬ȧȔȦƯƊǓƶƳઓƟƮ B ԩ঴ƶ੒ԩ঴ƳઓơǓԥ‫ݜ‬Ɣ 19ĊƯƊǒ¹‫ࡣऺګ‬Ưƶ 35õ6ĊƳೝƟୢ૮ƯƊƫƧºǆƧඟ९ƯƷ੒ԩ঴¹A¹B ԩ঴૮ƌƢǔǊ‫ࡣऺګ‬ƶ๢ 1/3 ƯƊƫƧº4-MUGS ǚພƌƧA ԩ঴ƶ੯୩ƯǊ¹സ९ƶԩ঴Ɣ‫ࡣऺګ‬ư௬ȧȔȦƯƊƫƧƶƳઓƟ¹ඟ९ƯƷ๢ವൟƯƊƫƧºƛǔǑƶ‫ڤ‬үƓǑ¹ඐПࡣư݂ƐǑǔǓസ९ưඟ९ƳƒƙǓȓǨǸǰȚȆǻÓǷԩ঴ƶЂऺȌǺÓȮƔЂƲǓƛưǚ࠷ƟƮƌǓư݂ƐǑǔƧºഒ 1¸ॶ‫ࡐ֏ܞ‬ǚພƌƧɋ-ǫȦǮǰȚȆǻÓǷԩ঴੯୩-76-

2.2 HEXB Ћஞࠃ¸ǟȮȐǤÓțȄǮȮǶȮȃǚ௻Ƨ‫ۼ‬¹Քࡣư໩९
ƶ DNA ǚಋ‫עڥ‬ǐǒଋࢽ¹HEXB ЋஞࠃƶơǁƮƶ exon ư exon-intron junction ǚ PCR Ư੢൒ƟƧƶƩুৄƟ¹ABI377 ǲÓǪǣȮǰÓƳǐǒǲÓǪǣȮǴƟƧáইŌâº‫ڤ‬ү¹ՔࡣƳƷōƬƶॠƟƌ඄ЂƔ੻޲ƟƧºГƬƷඟ९຀ີƯ Intron 2/exon 3junction ƳƒƙǓ GğA ૷՘(IVS2-1G>A)¹ઁƷസ९຀ີƯ exon¸13 ƳƒƙǓ GğA ૷՘(R533H)ƯƊƫ
ƧºƛǔƳಳƌ 533 ್๋ƶǝȦǩȆȮƔȍǴǼǳȮƳ඄՘ƝǔǓºǲÓǪǣȮǴƶ‫ڤ‬үǐǒ¹ՔࡣƷƌƢǔǊƛǔǆƯƳඦ‫ݥ‬ƶƲƌ 2 ƬƶॠƟƌ඄ЂƳǐ
Ǔ compound heterozygote ưथฯƝǔƧºই 1¸ॠƟƌమƬƶ඄Ђǚ࠮Ƭ HEXB ЋஞࠃƶǲÓǪǣȮǴ

A-C ƷǝȮǼǶȮǴ੩ƶ intron 2/exon 3 junction ƶǲÓǪǣȮǴ¹D-F ƷǶȮǴ੩ƶ exon 13 ǚ࠷ơº¸A¹D:Քࡣƶ HEXB Ћஞࠃ¸B¹E:സ९ƶ HEXB Ћஞࠃ¸C¹F:ඟ९ƶ HEXB Ћஞࠃ
A ƷՔࡣƶǝȮǼǶȮǴ੩Ư CğT ૷՘ƟƧ intron 2/exon 3 junction Ʒ HEXB ЋஞࠃƶǶȮǴƯ intron 2 ƶ 3’-splice
acceptor site ƶ GğA ૷՘Ƴ੏௓ơǓ (IVS2-1G>A)ºA ư௬Ơ඄ЂƔඟ९áCâƳǊԏీƝǔǓƔ¹സ९áBâƳ(A) RT-PCR Ơƽǀ IVS2-1G>A mRNA ӂৃƷƲƌºՔࡣƶ HEXB ЋஞࠃáDâƶ exon 13 Ưƶ c.1598G>A ૷՘ƶ‫ڤ‬үƒƛǓ R533H ඄ЂƷസ९áEâƳƷƊǓƔඟ९áFâƳƷƲƌºƛƶȂÓǺƳǐǒՔࡣƷమƬƶॠƟƌ඄Ђǚ࠮Ƭ compound heterozygote ƯƊǓƛưƔ
಴ฯƟƧº-77-

2.3 RT-PCR ƳǐǓ IVS2-1G>A mRNA ӕ৖¸ඟ९຀ີƶ඄Ђ‫ݏ‬ਬƶ mRNA ƶದ‫۔‬ǚ‫ۂ‬ǓƧǉ
Ƴ¹ՔࡣǐǒԵҠƟƧਅЄӍެභǐǒ RNA ǚଋࢽƟ RT-PCR ǚ݉ƌ exon2 ƓǑ 5 ǚ੢൒ƟƮӕ৖ƟƧº‫ࡣऺګ‬ƶ cDNA ƯƷ HEXɋ‫ޒ‬຀ີƶ 225bp ƶȋȮȄǚԏీƟƧºՔࡣƶ cDNA ƓǑƷమ෭ƶȋȮȄƔ
ԏీƝǔƧºГƬƷ়ऺƯǇǑǔǓȋȮȄư௬Ơǰ
ǟǵƯ¹ǊƎГƬƷƥǔǐǒ૜ƌȋȮȄƨƫƧáই
2AâºƝǑƳƥǔǑƶȐȤǫȜȮȃǚ pCR-Script Amp

SK(ó)vector ƳǪȨÓȆȮǫƟƮǲÓǪǣȮǴǚԏీƟƧºՔࡣƶସƌȋȮȄƷ‫ࡣऺګ‬ƶ 225bp ƶȋȮȄƶǲÓǪǣȮǴư௬ƠǊƶƨƫƧº૜ƌȋȮȄƷ
159bp ƶǰǟǵƯ¹ƛǔƷ exon 3 Ɣ‫ࡉڠ‬ƟƧǊƶƨ
ƫƧáই 2Bâºই 2C Ʒ HEXB Ћஞࠃƶ exon 3 പ‫ؼ‬ƶܲ੧ǚഒơºGğA ૷՘ƳǐƫƮ intron 2 ƶ 3’ǴȒȤǟǴ൉ϳƯЂऺƔ֬ƕ¹‫ڤ‬ү mRNA Ʒ exon 3ǚ‫ࡉڠ‬ơǓഩՏਙƯ૜ƌɋ-‫ޒ‬ǚ߫ࢽơǓưࠈǗǔǓºই 2¸RT-PCR ƳǐǓՔࡣưǮȮȃȨÓȦƶೠ഻ਅЄӍެභƶ HEXB ƶ mRNA ƶӕ৖(A) PCR Ư exon 2 ƓǑ exon 5 ǚ੢൒º‫ࡣऺګ‬ƶೠ഻ਅЄӍެභƶ cDNA ƓǑƷ 225bp ƶǇ੢൒ƝǔǓºГධՔࡣƶ cDNA
ƓǑƷ 225¹159bp Ɣ੢൒ƝǔǓº(B) Քࡣƶ RT-PCR ƓǑ੢൒ƝǔƧమࡾ་ƶ DNA ૨අº159bp ƶ DNA ૨අƷ exon 3 Ɣ‫੾ڠ‬ƟƮƌƧº(C) exon 3 പ‫ؼ‬ƶ HEXB Ћஞࠃƶ౴༞G ƓǑ A ƶ૷՘ǚ๟МƯ࠷ơºƛƶ඄ЂƷ intron 2 ƶ 3’-splice acceptor site ǚฤܑƳơǓºƥƶ‫ڤ‬ү cDNA Ʒ exon 3ǚࡉƎº-78-2.4 RFLP ƳǐǓ R533H ƶӕ৖¸Քࡣư໩९¹ష෭ॶǮȮȃȨÓȦ 100 ॶƶ exon 13ǚ PCR ƳǐƫƮ੢൒Ɵরۙ‫ݏ‬ਬ Fnu4H˿Ưৡ૨Ɵ
RFLP ӕ৖ǚ݉ƫƧº๝িԵƶ 200 ǝȧȦƯƷơǁƮরۙ‫ݏ‬ਬ Fnu4H˿ƯՏਙƳৡ૨Ɲǔ¹225bp¹181bp
ƶȋȮȄƔԏీƝǔƧºՔࡣưസ९ƶ඄ЂԵǝȧȦ
ƯƷ 225bp¹181bp ƶȋȮȄƶઁƳ 406bp ƶȋȮȄAǊԏీƝǔƧáইŎBâºƛǔƷ c.1598G>A ƶ඄ЂƳǐƫƮ Fnu4H˿ৡ૨൉ϳƔ‫ࡉڠ‬ƟƧƛưǚ࠷ơºRFLP ӕ৖ƷՔࡣưസ९ƶ඄ЂǝȧȦƔ়ऺƲં‫ٸ‬ƯƷƲƗ¹‫ئ‬ǉƮ֦Ʋ඄ЂƨưƌƎƛưǚ࠷ƟƮƌ
ǓºǲÓǪǣȮǴƶ‫ڤ‬үƓǑǊ c.1598G>A ƶ඄ЂƯ
Fnu4H˿ৡ૨൉ϳƶ‫੾ڠ‬ƔԏీƝǔƧáইŎAâº406 bp181 bp

Wild type 5'

ACA AGG CAC CGC TGC AGG ATG GTCThrR533H5'B225 bp

Fnu4HI

Arg

HisArgCys Arg Met Val

ACA AGG CAC CAC TGC AGG ATG GTC

1Thr Arg

HisHis2573 4

63'Cys Arg
89 10 Pt

Met3'ValF M λ/φ603
310281/271

406 bp225 bp234

181 bp194

ই 3¸PCR-RFLP ƳǐǓ R533H ඄ЂǚǊƧǑƟƧ c.1598G>A ૷՘ƶӕ৖(A) 406bp ƶ DNA ૨අƷ¹PCR ƳǐƫƮ Fnu4H˿রۙ‫ݏ‬ਬ൉ϳǚ࠮Ƭ wild type ǐǒ੢൒ƝǔǓºc.1598G>A ૷՘
ƳǐǓ R533H ඄ЂƷ Fnu4H˿൉ϳƔࡉǗǔǓº(B) ǮȮȃȨÓȦ¹Քࡣ¹໩९ƶ PCR ૨අǚ Fnu4H˿Ưৡ૨Ɵӕ৖ƟƧºՔࡣưസ९ƶ ੢൒ƝǔƧ DNA ƶГǬࣗƔ Fnu4H˿Ưৡ૨Ɲǔ¹c.1598G>A ૷՘ƶ heterozygous ưԏీƝǔƧºǮȮȃȨÓȦƶ PCR ૨අƷ Fnu4H˿ƯՏਙƳৡ૨Ɲǔ¹c.1598G>A ૷՘Ʒష෭ॶƶଇƯ‫؁‬ୋƳ‫ۂ‬ǑǔǓǐƎƲં‫ٸ‬ƯƷƲƌƛưƔ࠷‫މ‬ƝǔǓº-79-Ŏ¼݂ߒࡤ࠼¸‫ݶ‬ӖƷӄ‫׫‬ƳઁЦƯ ALS ư॰૨ƝǔƧǬÓǴǚিҠԙஇƲ‫ލڸ‬Ư Sandhoff ഗư়ƟƗ॰૨ơǓƛưƔƯƕƧऔ༎ƯƊǓºǆƧƝǑƳЋஞࠃӕ৖Ƴǐƫ
Ʈ ¹ ࢫ ີ ඦ ‫ ݥ‬ƶ Ʋ ƌ ॠ Ɵ ƌ మ Ƭ ƶ compound
heterozygote ƶ඄ЂǊथฯơǓƛưƔƯƕƧºඟ९຀ີƶ intron 2 ƶ 3’-splice acceptor site ƳƒƙǓ GğA ૷՘ƶ඄ЂƷ exon 3 ǚ‫ࡉڠ‬Ɵ¹22 ǝȚȉ߱Ɣ‫ڠ‬ƙƧ૜ƌɋ‫ޒ‬ǚ߁Ǔº4-MUG¹4-MUGS Ưƶ‫ݏ‬ਬԩ঴ƶ੯୩Ɣ࠷ơǐƎƳ¹ƛƶഩՏਙƲɋ‫ޒ‬Ʒ‫ݏ‬ਬԩ঴ƔࡉǗǔƮƌǓư݂ƐǑǔǓºസ९຀ີƶ exon 13Ưƶ GğA ૷՘ƶ඄ЂƷ 533 ್ƶǝȚȉ߱ǚǝȦǩȆȮƓǑȍǴǼǳȮƳƓƐǓºസ९ƶ੒ HEX ԩ঴Ʒ়ऺȧȔȦƨƔ HEXB ƶԥ‫ݜ‬ƔୢҟƟƮƌƧºസ९ƶ‫ݏ‬ਬԩ঴Ɣॶ‫ࡐ֏ܞ‬ƳƧƌƟƮƷ়ऺƯǊ¹িઑఝ֏ࡐƯƊǓ GM2 ǧȮǫȥǥǲȄƳઓƟƮƷୢҟƟƮƌǓҧౖ঴ǌ¹ƛƶ R533H ƶ඄ЂƷ HEXBƶేઔ঴ƳշຌƟƮƌǓҧౖ঴Ǌ݂ƐǑǔǓº¸‫޲۔‬ӉÐƔ੯୩ƟƮƌǓȤǟǸǹÓț‫ݏ‬ਬƶЂऺऔƯƷ¹൹‫ݦ‬ƳƒƌƮǯÓǲǢഗưȐǜȑȥÓഗƯ‫੾ڠ‬ƟƧ‫ݏ‬ਬƥƶǊƶǚ඗ࢩơǓƛưƳǐǒ࠲໰ơǓ‫ݏ‬ਬ඗ࢩ໰඲ƔҧౖưƲƫƮƒǒ¹ష෭ǀƶ௮హ
Ǌյ‫ؼ‬ƯƊǓºSandhoff ഗƯƷ‫ݏ‬ਬ඗ࢩ໰඲ƶ༂ࣽ҃ພƳƷณƨ௛ƫƮƌƲƌƔ¹‫ݶ‬ӖƶǐƎƲ॰૨Ʒ‫࠲ۼݶ‬໰඲Ɣ҃ພҧౖƳƲƫƧ࠯஝ƯơƘ࠲໰ǀƶࠟǇƳƬƲƔǓҧౖ঴Ɣ‫ݗ‬ƌǊƶư֛ગƟƮƌǓº෭‫ףڽ‬ƳƒƌƮ༂ࣽЌԙ‫ڈ‬३‫ډ‬ఝҭƶ‫ׅ‬ᓵ໅‫ࠆ݋ܥ‬ƶ‫ࠉ۽‬௮Ƴǐǒ¹‫ݏ‬ਬԩ঴੯୩ƒǐƻЋஞࠃӕ৖ƶГ൉ǚ૕௓૸ƟǆƟƧº॥ࡤ૸Ɵǆơºߣ݂൫‫ڼ‬[1]

T. Yoshizawa, Y. Kohno, S. Nissato, S.Shoji, Compound
heterozygosity with two novel mutations in the HEXB
gene produces adult Sandhoff disease presenting as a motor
neuron disease phenotype, J. Neurol. Sci. 159 (2002) 129138.
[2] Ҳ๝඾, ‫ׅ‬ᓵ໅‫ܥ‬, પѠؐബ, ‫ا‬ҏ‫ܭ‬, ࣼ߻ॳГ. ժࣦƳॳ݉ơǓф௫ȆȠÓȨȮࡏՔບऔीǚ୧ƟƧ঵ॶ‫ ٸ‬Sandhoff ഗƶ 1 ༎, ༂ࣽ३‫ډ‬ԙ¸41 (2000) 36-39.

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