許小菁 張月華
SCN1A基因定位于染色體2q24.3,長81 kb,有26個外顯子,編碼Na+通道α1亞單位。SCN1A基因屬電壓門控Na+通道編碼基因家族中的一員,該家族還包括SCN2A、SCN3A、SCN7A和SCN9A基因[3]。
α1亞單位作為主體形成通道孔,由2 000個氨基酸組成,具有典型的4×6結構,由4個高度同源的結構域(D1~D4)組成,每個結構域含有6個跨膜區(qū)域(S1~S6,從左至右),N端和C端直接朝向細胞內部[4](圖1)。
圖1 Na+通道α1亞單位結構模式圖[4]
S4區(qū)含5~8個帶正電荷的氨基酸殘基,稱為“電壓感受區(qū)”;S5和S6間的結構形成反向平行的β折疊襯于孔道內壁,稱為“門孔區(qū)”,Na+經(jīng)此區(qū)而由細胞膜外流入膜內;D3和D4間的細胞內連接環(huán)為“失活門”。
電壓門控Na+通道存在于大多數(shù)可興奮組織細胞膜上的跨膜大分子糖蛋白,是一種電壓敏感性通道,主要具有觸發(fā)動作電位作用。通道內部傳感結構能夠感知細胞膜上的微小電位變化,同時也能被S4跨膜區(qū)域上的正電荷殘基感知,產生去極化,使S4區(qū)域的堿性殘基朝向細胞外側。這些變化反過來可引發(fā)“活化門”的構象重排,使離子孔傳導時間縮短,僅為數(shù)毫秒,離子孔即關閉成為“失活門”。任何細微的電壓變化對于門控通道的影響都是重大的,進而影響到細胞的興奮性[2]。
Escayg等[14]首次報道在GEFS+家系中發(fā)現(xiàn)SCN1A基因突變。5%~10%的GEFS+家系存在SCN1A基因突變[15,16]。迄今為止,在GEFS+家系中已發(fā)現(xiàn)42種SCN1A基因突變類型,且均為錯義突變(http://www.molgen.ua.ac.be/SCN1AMutations/ Mutations/ Default.cf-m)。
GEFS+家系具有遺傳異質性,雖然SCN1A基因是GEFS+最常見的致病基因;但SCN1B和氨基丁酸受體γ2亞單位基因(GABRG2)基因突變也與GEFS+家系發(fā)病有關[12,16,17];SCN2A和GABRD基因突變也分別見諸于一個GEFS+家系報道[18,19]。
表1 20個SCN1A 基因突變GEFS+家系表型分析[14~16,20~33]
近年的研究結果顯示,約80%的Dravet 綜合征患兒具有SCN1A基因突變,基因突變類型也達300余種,包括截斷突變、錯義、無義、堿基缺失及重復等,其中超過一半的患者因無義和框移突變導致蛋白質的截斷[34]。這些突變廣泛分布于整個亞單位蛋白的C端至N端,包括形成Na+通道孔的重要功能區(qū)(S5~6)[36]。SMEB和SMEI患兒的SCN1A基因突變率無明顯區(qū)別,從分子遺傳學的水平也證實兩者無本質區(qū)別,故兩者均稱為Dravet綜合征。Fujiwara等[37]在10例ICEGTC患兒中發(fā)現(xiàn)7例均為SCN1A基因錯義突變,與SMEI、SMEB中SCN1A基因突變率接近。Harkin等[38]在2例ICEGTC患兒中發(fā)現(xiàn)了1例缺失突變(Q1914fsX1943)。
PCR-DNA測序未發(fā)現(xiàn)SCN1A基因突變的病例,采用多重連接依賴的探針擴增技術(MLPA) 已發(fā)現(xiàn)SCN1A基因的片段缺失或重復,從單一外顯子至整個基因的缺失,在Dravet綜合征中的陽性率為10%~15%[39~42]。采用MLPA的方法還可發(fā)現(xiàn)染色體的微缺失,再通過比較基因組雜交和熒光原位雜交的方法驗證缺失位置和大小,并且能發(fā)現(xiàn)SCN1A鄰近基因的缺失。
有研究[46]發(fā)現(xiàn),8%的Dravet綜合征病例有SCN9A基因突變, 9例SCN9A基因突變中有6例同時存在SCN1A基因突變,分析認為SCN9A可能是SCN1A的修飾調節(jié)基因。 Depienne等[47]發(fā)現(xiàn)SCN1A基因突變陰性的SEMI患兒中,15%的女性患兒有原鈣黏蛋白基因PCDH19 突變, 該基因定位于染色體Xq22。提示PCDH19 基因與Dravet綜合征女性患者致病高度相關,此外有1例男性SMEI患者為PCDH19 基因突變嵌合體。
SIMFE臨床特點:SIMFE可被歸入CFE。SIMFE臨床表現(xiàn)為1歲以內早發(fā)的多變的部分性發(fā)作;EEG以大量多灶性放電為主,不伴全面性或雙側同步化放電;隨病情發(fā)展出現(xiàn)不同程度的智力發(fā)育落后。已在5例患者中發(fā)現(xiàn)3種SCN1A基因突變(F575fsSX48、F1543S和F1543S)[38]。
CGE臨床特點:可表現(xiàn)為多種發(fā)作形式,智力倒退,EEG有全導棘慢波。已發(fā)現(xiàn)6例CGE患兒SCN1A基因的錯義突變(T226M、A395P、V422E、S626G、M973V和IVS15+1G→T)[38]。
圖2SCN1A基因突變位點分布(不包括大片段缺失和重復)[2]
電壓門控Na+通道負責控制神經(jīng)元和其他可興奮細胞動作電位的上升支[4]。Na+通道α亞單位蛋白編碼基因SCN1A的突變可導致Na+通道的功能獲得,如持續(xù)的Na+內流,也可導致Na+通道的功能缺失,如Na+通道密度減少,激活與失活的電壓依賴性改變[62]。
在SCN1A基因突變功能研究中,GEFS+中SCN1A基因突變常導致Na+通道失活[65]。Sugawara等[69]將Dravet綜合征SCN1A基因的錯義突變和無義突變(G979R、N985I、F1831S、R712X,R1407X、R1892X)轉入HEK293細胞,通過膜片鉗功能研究發(fā)現(xiàn),細胞Na+電流明顯減少,通道功能缺失;其中截斷突變R712X、R1407X和R1892X可引起單倍體功能不足,通道表達量下降,導致通道活性的完全缺失。Dravet綜合征相關的SCN1A基因突變R1648C與F1661S在體外突變模型中功能獲得和缺失同時存在[68]。
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