一例凝血因子ⅧB區(qū)錯(cuò)義突變導(dǎo)致重型血友病A

1、    一例凝血因子B區(qū)錯(cuò)義突變導(dǎo)致重型血友病A        摘要目的：檢測一例重型血友病A患者(SH9)的基因突變。方法：用PCR、變性梯度凝膠電泳(DGGE)和DNA測序檢測因子基因突變。先用Southern blotting排除內(nèi)含子22倒位，然后應(yīng)用PCR對(duì)凝血因子基因進(jìn)行擴(kuò)增。擴(kuò)增范圍包括所有外顯子及其側(cè)翼內(nèi)含子序列。結(jié)果：片段14-2在DGGE中泳動(dòng)異常。DNA測序證實(shí)C2535A導(dǎo)致B區(qū)錯(cuò)義突變826Asp(GAC)Glu(GAA)。結(jié)論：該突變可能是導(dǎo)致重型

2、血友病A的原因，但有待進(jìn)一步研究證實(shí)。關(guān)鍵詞變性梯度凝膠電泳DNA測序錯(cuò)義突變凝血因子血友病A A novel missense mutation in the B domain of Factor causes severe hemophilia ALiu Jianxiang, Zhang Yuzhou, Wang Hongli, et al. Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai200025AbstractObjective:To ide

3、ntify the genetic defect of a patient with severe hemophilia A (SH9). Methods: PCR, denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to screen mutations in the factor gene. Intron 22 inversion was excluded previously by Southern blotting with F8A probe. PCR primers were de

4、signed to cover all the coding regions and flanking intron sequences. Amplified products were analysed with DGGE, and bands of abnormal mobility were directly sequenced. Results: PCR fragment 14-2 showed slower mobility than normal. A single nucleotide substitution C2535A causing a missense mutation

5、 in the B domain, 826Asp(GAC)Glu(GAA) was identified by DNA sequencing. Conclusion: This nucleotide substitution may be the molecular etiology of SH9. The possible molecular mechanisms underlying this candidate mutation is yet to be clarified.Key wordsDenaturing gradient gel electrophoresisDNA seque

6、ncingMissense mutationCoagulation factor Hemophilia A血友病A為最常見的遺傳性出血性疾病，發(fā)病率為1/(500010000)(男性)，是由血漿凝血因子(F)缺陷所致。編碼F的基因位于Xq28，全長186kb，含26個(gè)外顯子。成熟的 F蛋白由2332個(gè)氨酸酸殘基組成，可分為6個(gè)結(jié)構(gòu)域，即3個(gè)A區(qū)(A1、A2、A3)，2個(gè)C區(qū)(C1、C2)和1個(gè)B區(qū)，排列成A1-A2-B-A3-C1-C2。經(jīng)凝血酶作用后B區(qū)被切除，形成由A1/A2/A3-C1-C2組成的活性形式1。缺失B區(qū)的重組F促凝活性不變。目前還不清楚B區(qū)的具體生物學(xué)功能2。病例和方法1病

7、例患者SH9經(jīng)本院測定血漿FC(一期法)為0.7%、vWFAg(免疫火箭電泳法)為108.8%，診斷為重型血友病A。44例無親緣關(guān)系的血友病A患者亦用同法診斷。110名正常人DNA標(biāo)本來源于體檢學(xué)生和健康獻(xiàn)血者。采ACD抗凝全血，按常規(guī)方法抽提白細(xì)胞高分子量DNA。2聚合酶鏈反應(yīng)(PCR)應(yīng)用Bio-Rad公司軟件MacMelt對(duì)待測DNA序列進(jìn)行分析，根據(jù)解鏈溫度曲線確定PCR引物位置3。先設(shè)計(jì)25對(duì)引物擴(kuò)增除外顯子14的所有外顯子及其側(cè)翼內(nèi)含子序列4。14號(hào)外顯子另設(shè)計(jì)8對(duì)引物(見附表)分別擴(kuò)增。在每對(duì)引物中的一個(gè)引物的5端加一“GC-clamp”，使PCR產(chǎn)物一端形成Tm較高、不易解鏈的

8、“夾子”。PCR反應(yīng)體系同前4。片段14-2的PCR反應(yīng)條件為：94 1分鐘，56.5 1分鐘，72 1分鐘，35個(gè)循環(huán)，72延伸7分鐘。3變性梯度凝膠電泳(DGGE)分析應(yīng)用D GENETM System(Bio-Rad公司)，片段14-2的DGGE條件為：聚丙烯酰胺凝膠濃度為7.5%，變性劑濃度梯度范圍30%60%(40%甲酰胺加7%尿素為100%變性劑濃度)，電泳溫度58，電壓150V，電泳時(shí)間810小時(shí)。4DNA測序在DGGE電泳中發(fā)現(xiàn)異常條帶，則將條帶割下，溶于雙蒸水中，用相同引物再進(jìn)行PCR反應(yīng)，用Taq DyeDeoxTM Terminator Cycle Sequencing

9、kit(Applied Biosystems)對(duì)PCR產(chǎn)物用熒光標(biāo)記后，在ABI377測序儀上進(jìn)行直接測序。正鏈和反鏈序列均同時(shí)檢測。結(jié)果患者SH9擴(kuò)增片段14-2在DGGE電泳中泳動(dòng)比正常明顯減慢(1)。DNA測序結(jié)果表明在826號(hào)密碼子上發(fā)生了一個(gè)堿基置換C2535A，導(dǎo)致B區(qū)錯(cuò)義突變826Asp(GAC)Glu(GAA)(2)。經(jīng)查詢最新文獻(xiàn)及F基因突變數(shù)據(jù)庫，這一突變在國內(nèi)外尚未見報(bào)道。另對(duì)44例無親緣關(guān)系的血友病A患者及110名正常人所做的相同檢測，未發(fā)現(xiàn)這一堿基置換。    15為正常人對(duì)照；610為血友病A患者；患者SH9的14-2條帶泳動(dòng)

10、明顯減慢1片段14-2的變性梯度凝膠電泳結(jié)果附表14號(hào)外顯子擴(kuò)增引物外顯子5端引物3端引物擴(kuò)增片 段長度位置14-1GTAACCAGAGTCTTATTCTTCCT(GC)-TTCTGGAATTGTGGTGGCATTA265bp-234614-2TAGCACTAGGCAAAAGCA(GC)-TGATGGAATTGTTGAAAT429bp2302-273014-3AACAGAGTTGAAGAAACTTGA(GC)-CCAGACTGATGGACTATTCTCA684bp2462-314514-4GAAAGACTCACATTGATGGCC(GC)-GCTTGGAAAAACCATCTC479bp3090

11、-356814-5(GC)-AAGGGTGAATTTACAAAGGACTGTTTCTTCTAGTGGGAG517bp3517-403314-6CAGAATTTTGTCACGCAACGT(GC)-AGTCATCTCCAAGGTTAGAAT450bp3967-441614-7(GC)-GTCCAAGAAAGCAGTCATTCTGATTGCTTTCACAAGCGTTCAGG542bp4340-488114-8TGGAAATCCCAAGAGAAGTCACCA(GC)-AGCAGAGCAAAGGAATAACCA357bp4796-       

12、  在F基因的2535位核苷酸發(fā)生了CA堿基置換，導(dǎo)致826Asp(GAC)Glu(GAA)錯(cuò)義突變A為正常對(duì)照；B為血友病A患者SH92患者SH9片段14-2的DNA測序結(jié)果討論缺失B區(qū)的F促凝活性與野生型相當(dāng)，說明B區(qū)并非F功能活性所必需5。我們先用F8A探針排除了內(nèi)含子22倒位6，然后篩查除14號(hào)外顯子以外的所有編碼序列及側(cè)翼內(nèi)含子序列，未發(fā)現(xiàn)異常4。為此我們又設(shè)計(jì)了8對(duì)引物擴(kuò)增14號(hào)外顯子，發(fā)現(xiàn)14-2片段在DGGE電泳中泳動(dòng)異常，經(jīng)3次重復(fù)測序證實(shí)826Asp(GAC)Glu(GAA)突變。為排除多態(tài)性的可能，我們對(duì)110名正常人和44例無親緣關(guān)系的血友病A患者同

13、時(shí)進(jìn)行PCR-DGGE檢測，均未發(fā)現(xiàn)這一堿基置換?，F(xiàn)已知F基因點(diǎn)突變中B區(qū)點(diǎn)突變共13種，其中6種為無義突變，其余7種為錯(cuò)義突變7。其中一例1242Asp(GAC)Glu(GAG)突變導(dǎo)致重型血友病A，與SH9相似。Kamisue等8在狗血友病A模型中發(fā)現(xiàn)B區(qū)909SerGlu突變，也導(dǎo)致重型血友病A。錯(cuò)義突變導(dǎo)致重型血友病A的具體分子機(jī)制還不清楚。最近，Liu等9首次報(bào)道一例馬凡氏綜合征患者FBN1基因外顯子51中一個(gè)靜止突變(氨基酸未改變)C6354T導(dǎo)致mRNA剪接錯(cuò)誤，使整個(gè)外顯子51丟失(exon skipping)，其發(fā)生機(jī)制不明。剪接錯(cuò)誤一般由于剪接位點(diǎn)的絕對(duì)保守序列或套索分支點(diǎn)

14、(lariat branch point)區(qū)域的突變影響剪接體的組裝所致。堿基置換產(chǎn)生新的剪接位點(diǎn)(cryptic splice site)也可使mRNA前體的拼接發(fā)生錯(cuò)誤。此外，剪接位點(diǎn)鄰近的核苷酸序列也有一定的保守性，對(duì)正確的剪接也有作用10。編碼區(qū)點(diǎn)突變使整個(gè)外顯子丟失大多見于無義突變，說明保持完整讀框在剪接過程中也發(fā)揮一定作用9。826Asp(GAC)Glu(GAA)突變距兩端剪接位點(diǎn)有相當(dāng)距離，不可能影響剪接體的結(jié)合組裝。對(duì)兩側(cè)翼序列分析表明，這一堿基置換并沒有產(chǎn)生新的剪接位點(diǎn)。這一突變是否確實(shí)影響mRNA剪接還有待進(jìn)一步研究。目前還不能確定在未被檢測的內(nèi)含子、F基因的調(diào)控序列或其他

15、位點(diǎn)中是否還有突變。志謝：對(duì)提供最新F基因點(diǎn)突變數(shù)據(jù)庫的EGD Tuddenham教授、ST Antonarakis教授及G Kemball-Cook博士表示感謝參 考 文 獻(xiàn)1Gitchier J, Wood WI, Goralka TM, et al. Characterization of the human factor gene. Nature, 1984, 312:326-330.2Antonarakis ST. Molecular genetics of coagulation factor gene and haemophilia A. Thromb Haemost, 1995

16、, 74:322-328.3Myers R, Maniatis T, Lerman M. Detection and localization of single base changes by denaturing gradien gel electrophoresis. Meth Enzymol, 1987, 88:501-527.4劉建湘，張宇舟，王鴻利，等. 應(yīng)用DGGE檢測中國人血友病甲基因突變. 中華血液學(xué)雜志，1997， 18：464-467.5Erik B. Second generation,B domain deleted recombinant factor . Thro

17、mb Haemost, 1997, 78:256-260.6張宇舟，邵慧珍，王鴻利，等. 重型血友病甲凝血因子基因內(nèi)含子22倒位的研究. 中華血液學(xué)雜志，1995，16：454-456.7Kemball-Cook G, Tuddenham EGD. The factor mutation database on the world wide web: the heamophilia A mutation, search, test and resource site. HAMSTeRS update (version 3.0). Nucleic Acids Res, 1997, 25:128-132.8Kamisue S, Cameron C, Notley C, et al. A candidate mutation in the factor B domain as a cause of severe canine ha