CAJ | 학술논문

目的探讨一个遗传性无纤维蛋白原血症家系的分子发病机制.方法应用Clauss法测定血浆纤维蛋白原活性,应用免疫比浊法测定纤维蛋白原抗原.提取先证者及其家系成员外周血DNA,PCR扩增纤维蛋白原FGA、FGB和FGG基因所有外显子和侧翼序列,构建纤维蛋白原野生型和突变型表达载体.在先证者血浆中加入凝血酶进行纤维蛋白聚集曲线测定；应用Western blot分析血浆纤维蛋白原,用野生型或FGB突变型质粒转染COS-7细胞,用Western blot和ELISA法检测转染后的细胞裂解液及细胞培养上清中纤维蛋白原的表达.结果先证者APTT、PT、凝血酶时间明显延长；血浆纤维蛋白原活性及纤维蛋白原抗原检测结果均为0；先证者FGB基因2号外显子核苷酸2833～2834之间插入GTTT(纯合突变),先证者父亲、母亲、胞弟和儿子为杂合突变；凝血酶诱导的血浆纤维蛋白聚集曲线显示患者血浆无纤维蛋白聚集；Western blot分析显示,非还原条件下先证者血浆缺乏完整的纤维蛋白原分子和纤维蛋白原半分子,在还原条件下未检出截短的Bβ链.在转染突变型质粒的COS-7细胞裂解液中检出异常纤维蛋白原分子(相对分子质量＞340 000),细胞培养上清中未检出异常纤维蛋白原.野生型和突变型质粒转染的COS-7细胞裂解液中纤维蛋白原含量差异无统计学意义[(2.47±0.30)μg/ml对(2.65±0.60)μg/ml,P=0.0889];转染突变型质粒的COS-7细胞培养上清中纤维蛋白原含量低于转染野生型质粒的COS-7细胞,差异有统计学意义[(0.01±0.01)tg/ml对(3.80±0.80)μg/ml,P=0.0001].结论纤维蛋白原FGB基因插入突变是该家系遗传性无纤维蛋白原血症的分子发病机制；该突变导致纤维蛋白原分子合成异常、组装及分泌障碍.
목적 탐토일개유전성무섬유단백원혈증가계적분자발병궤제.방법 응용Clauss법측정혈장섬유단백원활성,응용면역비탁법측정섬유단백원항원.제취선증자급기가계성원외주혈DNA,PCR확증섬유단백원FGA、FGB화FGG기인소유외현자화측익서렬,구건섬유단백원야생형화돌변형표체재체.재선증자혈장중가입응혈매진행섬유단백취집곡선측정；응용Western blot분석혈장섬유단백원,용야생형혹FGB돌변형질립전염COS-7세포,용Western blot화ELISA법검측전염후적세포렬해액급세포배양상청중섬유단백원적표체.결과 선증자APTT、PT、응혈매시간명현연장；혈장섬유단백원활성급섬유단백원항원검측결과균위0；선증자FGB기인2호외현자핵감산2833～2834지간삽입GTTT(순합돌변),선증자부친、모친、포제화인자위잡합돌변；응혈매유도적혈장섬유단백취집곡선현시환자혈장무섬유단백취집；Western blot분석현시,비환원조건하선증자혈장결핍완정적섬유단백원분자화섬유단백원반분자,재환원조건하미검출절단적Bβ련.재전염돌변형질립적COS-7세포렬해액중검출이상섬유단백원분자(상대분자질량＞340 000),세포배양상청중미검출이상섬유단백원.야생형화돌변형질립전염적COS-7세포렬해액중섬유단백원함량차이무통계학의의[(2.47±0.30)μg/ml대(2.65±0.60)μg/ml,P=0.0889];전염돌변형질립적COS-7세포배양상청중섬유단백원함량저우전염야생형질립적COS-7세포,차이유통계학의의[(0.01±0.01)tg/ml대(3.80±0.80)μg/ml,P=0.0001].결론 섬유단백원FGB기인삽입돌변시해가계유전성무섬유단백원혈증적분자발병궤제；해돌변도치섬유단백원분자합성이상、조장급분비장애.
Objective To investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia.Methods The plasma fibrinogen activity and antigen of the patient was determined using the Clauss method and immuno-nephelometric assay,respectively.Genomic DNA was isolated from peripheral blood of the proband and his related family members.All exons and exon-intron boundaries of the three fibrinogen genes (FGA,FGB,FGG) were amplified by PCR followed by direct sequencing.Thrombin fibrin aggregation curve were detected in the plasma of the patient.Wild-type and mutation type fibrinogen vectors were constructed,and then transfected into COS-7 cells.The wild-type and mutant proteins from the culture media and cell lysates were tested by Western blot and ELISA.Results APTT,PT,TT were significantly longer in the proband.Plasma fibrinogen activity and antigen of the patient could not be detected using the Clauss method and immuno-nephelometry,respectively.Gene analysis revealed that a novel homozygous GTTT insertion between nucleotides 2833 and 2834 in FGB exon 2 in the proband.The proband' s father,mother,brother and son were heterozygous.The polymerization curves of the patient did not show a lag phase or final turbidity,compared with the normal controls.Western blot analysis showed the lack of complete half-molecules of the fibrinogen molecule and fibrinogen in patient's plasma under non-reducing conditions.It also could not detect the truncated Bβ chain under reducing conditions.Abnormal fibrinogen molecule (molecule weight ＞340 000) were found in transfected COS-7 cells by Western blot,which indicated that the mutation caused the abnormal intracellular fibrinogen molecule assembly.The fibrinogen band was absent in culture media transfected by the mutation.Fibrinogen levels of mutant fibrinogen were no significant different from those of wild-type fibrinogen in cell lysates by ELISA analysis [(2.47+0.30) μg/ml for (2.65+0.60) μg/ml,P =0.0889] ;However,the levels of the mutant fibrinogen were statistically significant lower than those of wild type fibrinogen in culture media [(0.01 ±0.01) μg/ml for (3.80±0.80) μg/ml,P=0.0001].Conclusion Congenital afibrinogenemia was caused by this frameshift mutation in exon 2 of FGB.This novel mutation impaired fibrinogen assembly and secretion.