摘要： 目的·对遗传性凝血因子Ⅴ缺乏 （inherited factor Ⅴ deficiency，FⅤD）患者进行临床表型诊断、凝血酶生成试验（thrombin generation assay，TGA）及FⅤ基因（
F5）分析，综合评估患者的临床出血风险，探讨其基因型和表型的关系。方法·选取2020年1月—11月于广州医科大学附属第二医院血液内科门诊的FⅤD患者5例，常规凝血筛查法检测患者的凝血酶原时间（prothrombin time，PT）、活化部分凝血活酶时间（activated partial thromboplastin，APTT）；一期凝固法检测血浆FⅤ活性水平（FⅤ∶C）和血浆FⅧ活性水平（FⅧ∶C）；酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测血浆FⅤ抗原（FⅤ∶Ag）以及游离和总组织因子途径抑制物（tissue factor pathway inhibitor，TFPI）水平。依照出血评分表对患者的出血风险进行评分，结合TGA评估FⅤD患者临床出血风险。用Sanger测序法分析患者
F5的全部外显子及侧翼序列，并对突变位点进行反向测序验证。采用AccuCopy 多重基因拷贝数检测技术对FⅤD患者
F5进行拷贝数变异（copy number variation，CNV）检测。结果·5例先证者均属于Ⅰ型FⅤD，其中有2例为重型FⅤD（FⅤ∶C<1%）；5例先证者的游离和总TFPI水平均有不同程度的降低；TGA结果延迟时间和达峰时间均明显延长，但峰值和凝血酶生成潜力水平在不同患者间有较大差异。
F5突变分析发现8种突变，类型包括错义突变、无义突变、移码突变、CNV，其中错义突变占75%；p.Cys603Ser、p.Leu949Trpfs*、p.Leu1262_Gln1657del为新突变；CNV检测到先证者2的13~14外显子的大片段缺失（c.3784_4971del、p.Leu1262_Gln1657del），mRNA水平进一步分析提示缺失导致患者mRNA剪接异常，产生新的剪切位点，进而表达3种异常转录本（c.3577_4971del、c.3577_4456del、c.3331_4456del）。结论·重型FⅤD患者体内FⅤ∶C水平和出血严重程度无关，TGA和出血评分表可较好地关联患者的出血风险。FⅤD的出血严重程度或与
F5突变类型有关。
关键词: 遗传性凝血因子V缺乏, 出血评分, 凝血酶生成试验, 表型, 基因型 Abstract: Objective·To perform clinical phenotype diagnosis,thrombin generation assay (TGA) and F5 analysis in FⅤD patients, evaluate clinical bleeding risk and discuss the correlation between genotype and phenotype.
Methods·Five patients with FⅤD who visited the Department of Hematology of The Second Affiliated Hospital of Guangzhou Medical University from January to November 2020 were selected. PT and APTT were detected by routine coagulation screening. Plasma FⅤ∶C and plasma FⅧ∶C were detected by one-stage coagulation. Plasma FⅤ∶Ag, free and total TFPI was measured by ELISA. The risk of bleeding was graded according to the bleeding score recommended by ISTH.TGA was used to evaluate the risk of clinical bleeding in the patients with FⅤD. All exons and flanking sequences of F5 were analyzed by Sanger sequencing, and the mutation sites were verified by reverse sequencing. AccuCopy multiple gene copy number detection technique was used to detect F5 copy number variation (CNV) in patients with FⅤD.
Results·All 5 probands belonged to type Ⅰ FⅤD, including 2 cases of severe FⅤD (FⅤ∶C <1%). The levels of free and total TFPI decreased in varying degrees. The lagtime and time to peak of TGA were significantly prolonged, but the levels of peak height and endogenous thrombin potential varied. Mutation analysis revealed 8 kinds of mutations, including missense, nonsense, frameshift mutations and CNV, of which missense mutations accounted for 75% of the total mutations. p.Cys603Ser, p.Leu949Trpfs*, p.Leu1262_Gln1657del were novel mutations. Large fragment deletions in exon 13?14 of proband 2 were detected by AccuCopy (c.3784_4971del, p.Leu1262_Gln1657del). Further analysis of mRNA level showed that the large fragment deletion mutation led to abnormal mRNA splicing, resulting in novel splice site with three kinds of abnormal transcripts (c.3577_4971del, c.3577_4456del, c.3331_4456del).
Conclusion·The level of FⅤ∶C in patients with severe FⅤD is not related to the severity of bleeding, but TGA and bleeding score can well correlate the bleeding risk. The bleeding severity of FⅤD may be related to the type of F5 mutation.
Key words: inherited factor Ⅴ deficiency, bleeding score, thrombin generation assay, phenotype, genotype PDF全文下载地址:
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