中华血液学杂志
中華血液學雜誌
중화혈액학잡지
Chinese Journal of Hematology
2011年
9期
587-591
,共5页
秦欢欢%王学锋%丁秋兰%陆晔玲%戴菁%奚晓东%王鸿利
秦歡歡%王學鋒%丁鞦蘭%陸曄玲%戴菁%奚曉東%王鴻利
진환환%왕학봉%정추란%륙엽령%대정%해효동%왕홍리
血友病A%因子Ⅷ%DNA突变分析%分子发病机制
血友病A%因子Ⅷ%DNA突變分析%分子髮病機製
혈우병A%인자Ⅷ%DNA돌변분석%분자발병궤제
Hemophilia A%Coagulation factor Ⅷ%Gene mutation%Molecular mechanism
目的 探讨1例临床出血表现与凝血因子Ⅷ活性(FⅧ:C)检测结果明显不符的血友病A患者表型、基因型诊断及其分子发病机制。方法 凝血、抗凝及纤溶相关指标检测，一步法及发色底物法检测FⅧ:C，ELISA法检测FⅧ的抗原(FⅧ:Ag)。活化部分凝血活酶时间(APTT)纠正实验筛查FⅧ抑制物;PCR扩增先证者F8基因的所有外显子及其侧翼序列，利用直接核苷酸测序法进行基因序列分析。针对先证者的基因突变位点，对其家系成员进行相应外显子基因检测;定点突变法构建B亚基缺失(760aa～1639aa)的人FⅧHis99Arg和His99Ala两种突变的表达质粒(pRC/RS Ⅴ-BDhFⅦcDNA)，两种突变质粒分别瞬时转染HEK293T细胞，测定细胞上清液中的FⅧ:C和细胞上清液及裂解液中的FⅧ:Ag。结果 先证者APTT 延长，一步法及发色底物法检测的F Ⅷ:C均低于1.0％，血浆FⅧ:Ag为120.0％。APTT纠正试验阴性，其他凝血、抗凝及纤溶相关检测均未见异常。诊断为交叉反应阳性(CRM+)的血友病A。患者F8基因直接核苷酸测序发现3号外显子存在28828A→G突变，导致His99Arg氨基酸改变，其母亲该位点为杂合突变。体外表达研究结果显示细胞上清液中His99Arg突变蛋白FⅧ:Ag及FⅧ:C分别为野生型的180.0％及5.8％，His99Ala突变蛋白FⅧ:Ag及F Ⅷ:C分别为野生型的45.0％和20.0％。Western blot显示，His99Arg突变质粒表达上清液中FⅧ蛋白含量较野生型明显增高，而His99Ala突变质粒表达上清液FⅧ蛋白含量较野生型减少。提示His99Arg为CRM+血友病A突变而His99Ala为CRM-血友病A突变。结论 体外检测发现His99Arg和His99Ala两种FⅧ能够表达，但常规凝血检测Hi99Arg突变FⅧ基本无功能，而His99Ala凝血功能也降低。
目的 探討1例臨床齣血錶現與凝血因子Ⅷ活性(FⅧ:C)檢測結果明顯不符的血友病A患者錶型、基因型診斷及其分子髮病機製。方法 凝血、抗凝及纖溶相關指標檢測，一步法及髮色底物法檢測FⅧ:C，ELISA法檢測FⅧ的抗原(FⅧ:Ag)。活化部分凝血活酶時間(APTT)糾正實驗篩查FⅧ抑製物;PCR擴增先證者F8基因的所有外顯子及其側翼序列，利用直接覈苷痠測序法進行基因序列分析。針對先證者的基因突變位點，對其傢繫成員進行相應外顯子基因檢測;定點突變法構建B亞基缺失(760aa～1639aa)的人FⅧHis99Arg和His99Ala兩種突變的錶達質粒(pRC/RS Ⅴ-BDhFⅦcDNA)，兩種突變質粒分彆瞬時轉染HEK293T細胞，測定細胞上清液中的FⅧ:C和細胞上清液及裂解液中的FⅧ:Ag。結果 先證者APTT 延長，一步法及髮色底物法檢測的F Ⅷ:C均低于1.0％，血漿FⅧ:Ag為120.0％。APTT糾正試驗陰性，其他凝血、抗凝及纖溶相關檢測均未見異常。診斷為交扠反應暘性(CRM+)的血友病A。患者F8基因直接覈苷痠測序髮現3號外顯子存在28828A→G突變，導緻His99Arg氨基痠改變，其母親該位點為雜閤突變。體外錶達研究結果顯示細胞上清液中His99Arg突變蛋白FⅧ:Ag及FⅧ:C分彆為野生型的180.0％及5.8％，His99Ala突變蛋白FⅧ:Ag及F Ⅷ:C分彆為野生型的45.0％和20.0％。Western blot顯示，His99Arg突變質粒錶達上清液中FⅧ蛋白含量較野生型明顯增高，而His99Ala突變質粒錶達上清液FⅧ蛋白含量較野生型減少。提示His99Arg為CRM+血友病A突變而His99Ala為CRM-血友病A突變。結論 體外檢測髮現His99Arg和His99Ala兩種FⅧ能夠錶達，但常規凝血檢測Hi99Arg突變FⅧ基本無功能，而His99Ala凝血功能也降低。
목적 탐토1례림상출혈표현여응혈인자Ⅷ활성(FⅧ:C)검측결과명현불부적혈우병A환자표형、기인형진단급기분자발병궤제。방법 응혈、항응급섬용상관지표검측，일보법급발색저물법검측FⅧ:C，ELISA법검측FⅧ적항원(FⅧ:Ag)。활화부분응혈활매시간(APTT)규정실험사사FⅧ억제물;PCR확증선증자F8기인적소유외현자급기측익서렬，이용직접핵감산측서법진행기인서렬분석。침대선증자적기인돌변위점，대기가계성원진행상응외현자기인검측;정점돌변법구건B아기결실(760aa～1639aa)적인FⅧHis99Arg화His99Ala량충돌변적표체질립(pRC/RS Ⅴ-BDhFⅦcDNA)，량충돌변질립분별순시전염HEK293T세포，측정세포상청액중적FⅧ:C화세포상청액급렬해액중적FⅧ:Ag。결과 선증자APTT 연장，일보법급발색저물법검측적F Ⅷ:C균저우1.0％，혈장FⅧ:Ag위120.0％。APTT규정시험음성，기타응혈、항응급섬용상관검측균미견이상。진단위교차반응양성(CRM+)적혈우병A。환자F8기인직접핵감산측서발현3호외현자존재28828A→G돌변，도치His99Arg안기산개변，기모친해위점위잡합돌변。체외표체연구결과현시세포상청액중His99Arg돌변단백FⅧ:Ag급FⅧ:C분별위야생형적180.0％급5.8％，His99Ala돌변단백FⅧ:Ag급F Ⅷ:C분별위야생형적45.0％화20.0％。Western blot현시，His99Arg돌변질립표체상청액중FⅧ단백함량교야생형명현증고，이His99Ala돌변질립표체상청액FⅧ단백함량교야생형감소。제시His99Arg위CRM+혈우병A돌변이His99Ala위CRM-혈우병A돌변。결론 체외검측발현His99Arg화His99Ala량충FⅧ능구표체，단상규응혈검측Hi99Arg돌변FⅧ기본무공능，이His99Ala응혈공능야강저。
Objective To investigate the molecular mechanism of a Chinese hemophilia A patient in whom there was a discrepancy between the clinical bleeding symptoms and laboratory assay of FⅧ activity (FⅧ: C). Methods FⅧ: C was detected by chromogenic and one-stage methods, and FⅧ: Ag by ELISA.The APTT corrected test was used to screen the FⅧ inhibitor and PCR amplification to analyze all the exons and flanking sequences of F8 gene of the proband, PCR products were purified and sequenced directly. The corresponding gene sites of family members were detected according to the gene mutation sites. Two B domain deleted human FⅧ mutant expression plasmids His99Arg and His99Ala( pRC/RS Ⅴ -BDhFⅧcDNA) were constructed and transfected into HEK293T transiently. FⅧ: Ag and FⅧ: C of the expression products were assayed. Results The proband APTT was prolonged, FⅧ: Ag was 120％ but FⅧ: C ＜ 1％ and no FⅧ inhibitor in plasma. The results of anticoagulation and fibrinolytic functions were normal. The cross reacting material positive ( CRM + ) hemophlia A was diagnosed. Gene analysis revealed a A28828G substitution in exon 3 resulted in a H(His) to R(Arg) missense mutation and the same heterozygous was identified in his mother.In vitro expression of FⅧ: Ag and FⅧ: C of His99Arg were 180.0％ and 5.8％, respectively, while FⅧ: Ag and FⅧ: C of His99Ala were 45.0％ and 20.0％ of that of wild type, respectively. His99Arg and His99Ala were diagnosed as CRM+ and CRM- mutations, respectively. Conclusion Both the two FⅧ mutations could express FⅧ protein. However, CRM + His99Arg mutant protein has little FⅧ procoagulant activity and His99Ala has reduced FⅧ function by routine methods.
