FⅧ基因内5个SNPs位点多态性对血友病A基因诊断的意义

目的 通过对FⅧ基因内5个单核苷酸多态性(SNP)位点的杂合度进行分析,以寻找新的可用于血友病A(HA)家系基因诊断的多态位点.方法 50例汉族正常女性血样用于5个SNP位点的多态性分析;收集16个无血缘关系的HA家系成员血样,将具有多态性的SNP位点用于家系连锁分析,评价其对HA基因诊断的意义.结果 5个FⅧ基因内SNP位点在所研究的人群中有3个位点具有多态性,即rs1509787、rs3861554和rs1470586,多态信息量分别为0.106 4、0.344 4和0.310 8.利用这3个SNP位点对16个家系中的7个家系可作出诊断,诊断率为43.75％,其中rs3861554对HA的诊断率最高(31.5％).结论 在所选人群中有3个SNP位点具有多态性,对HA家系具有一定的诊断意义;rs3861554可作本地人群HA诊断的常用多态位点.     

基因重组人凝血因子Ⅷ在中国人血友病A患者中使用的研究

目的 探讨第二代rFⅧ（Kogenate FS）在中国人血友病A患者中使用的安全性和有效性。方法 采用第二代rFⅧ（Kogenate FS）治疗14例血友病A志愿者患者，用药前进行FⅧ抗体、FⅧ：C活性、血清病毒学（HBV、HCV和HIV）、血常规、尿常规、大便常规以及肝肾功能检查，于第一次用药后10min和60min进行FⅧ：C测定，治疗结束后进行FⅧ抗体和肝肾功能检测。结果 ①采用KogenateFS治疗的14例血友病A志愿者患者中，KogenateFS用量12．99～25．00u／kg（平均18．74u／kg），总用量3000～11000u（平均6570u），自治疗至出血停止或出血症状改善所需的时间为1．5～5．5d（平均3．29d），除1例FⅧ抗体滴度较高者外，其余13例患者第一次用药后10min和60minFⅧ：C活性均明显升高，用药前后FⅧ：C活性比较差异具有显著性（P〈0．01）。显效13例（占92．86％），好转1例（占7．14％），总有效率为100％。②14例血友病A志愿者患者中，用药前FⅧ抗体阳性2例，其中1例FⅧ抗体〉5Bu，第一次用药后10min和60minFⅧ：C活性无明显升高，治疗结束后FⅧ抗体仍然〉5Bu，但出血症状改善；另1例FⅧ抗体为4Bu，第一次用药后10min和60minFⅧ：C活性显著升高，治疗结束后FⅧ抗体转为阴性。③14例血友病A志愿者患者在应用KogenateFS治疗期间和治疗结束后均表现出良好的耐受性，无不良事件或药物不良反应发生。结论 第二代rFⅧ（KogenateFS）治疗中国人血友病A患者耐受性好，安全有效。     

重组人凝血因子Ⅷ在中国血友病A患者的有效性及抑制物产生的观察

目的探讨重组人凝血因子Ⅷ（Kogenate FS，拜科奇）在中国血友病A患者的有效性、安全性及抑制物产生率。方法采用拜科奇治疗30例血友病A患者的出血事件，观察其临床止血效果及用药后4周FⅧ抑制物的产生情况。其中11例患者在用药前后进行血、尿便常规、肝肾功、血清病毒学（HBV、HCV、HIV）、FⅧ抑制物、FⅧ活性检测，并于第1次用药后10min、60min检测FⅧ活性；同时随访2年检测FⅧ抑制物。结果11例患者在用药后10min、60min较用药前FⅧ活性明显提高，达到或接近预期升高值；所有30例患者用药后出血症状停止，显效率达100％。每次出血事件约86．7％的患者在≤3次输注即可较好的控制症状，提示具有较好的有效性。30例患者共输注重组FⅧ70次，总计51294u，在用药期间无任何不良反应出现。本研究发现1例患者在用药后4周产生FⅧ抑制物，提示短期抑制物产生率为3．3％（1／30），11例2年随访未见抑制物产生增加趋势。结论重组人凝血因子Ⅷ（Kogenate FS，拜科奇）在中国血友病A患者使用中具有较好的有效性、安全性及较低的抑制物产生率。     

获得性血友病A一例

患者,男,35岁,因四肢皮肤出现瘀斑,左大腿内侧出现包块1月余而就诊.患者平素体健,1月前,四肢出现不明原因的片状瘀斑伴肿痛;无血友病家族史、无输血史、无青霉素过敏史,否认免疫相关性疾病.查体:轻度贫血貌,左大腿内侧明显肿胀,能触及约10 cm×10 cm的包块,质较硬,边界不清,不活动,皮肤表面为片状瘀斑,左右上臂及右膝盖下亦见片状瘀斑;浅表淋巴结无肿大,腹软,肝脾肋下未及.     

中国人凝血因子Ⅷ基因多态性和血友病A的产前诊断

本文应用克隆化的凝血因子Ⅷ(下称FⅧ)基因cDNA及其旁侧的DNA片段作为探针,分析了123名中国正常人和22例血友病A患者的FⅧ基因及其旁侧的多态性位点Bcl Ⅰ/18e,Xbal/221,MspI/St14和BgI I/DX13。获得了中国人上述4个位点的RFLP资料,并应用RFLP连锁分析对2例血友病A高危妊娠进行了产前诊断。     

获得性血友病A1例

非血友病患者产生抗因子Ⅷ抑制物,临床又名获得性血友病。此病罕见,国内仅有个例报道,国外文献报道已逾300例。本院曾收治1例,现报道如下：     

血友病A家系因子Ⅷ基因内含子重复序列多态性的检测及分析

目的探索一种更简便和更特异的方法，用于血友病A的基因诊断及其家系遗传咨询。方法提取两个血友病A家系中成员外周血DNA，进行因子Ⅷ基因中内含子13（CA）n、内含子22（GT）n和（AG）n二核苷酸重复序列多态性分析。结果通过对内含子13和内含子22的两个STR位点的检测，两个家系均检出携带者。结论凝血因子Ⅷ基因中内含子重复序列是血友病A高多态性遗传标志之一，用于血友病A家系分析有较大的实用价值。     

女性A型血友病FⅧ基因突变分析

报道罕见的女性A型血友病1例,并对其凝血因子Ⅶ进行基因突变分析。患者,女,65岁,因为跌倒后右胸痛2d入院,院内查体提示胸壁皮下血肿,双下肢等长,髋屈曲及内旋受限。测定凝血指标提示APTT61．3s,正常血浆纠正后为41．3s,而PT、FIB、TT均正常。有既往出血史。FⅧ活性为2％,FⅨ活性为200％,vWF：Ag为120％,vWF：RCof100％,vWF：CBAl28％,FⅧ结合分析正常;髋关节X片提示;双侧髋臼发育不良,髋关节骨关节炎。临床诊断为血友病A型。提取该患者外周血DNA,根据NM_000132之凝血因子FⅦ基因序列设计合成了其第14外显子特异的引物,行聚合酶链反应扩增,并对扩增产物进行测序分析,测序结果与标准序列进行比较,发现该患者出现4111A→C杂合突变,使1314位氨基酸由苏氨酸变为脯氨酸,产生一错意突变,该突变未见其它文献报道．     

获得性血友病A的治疗进展

获得性血友病A是一种由于多种原因导致机体产生凝血因子Ⅷ抗体引起的凝血缺陷性疾病, 该病发病率极低, 且病死率较高, 因该病少见, 故常误诊, 以致延误治疗, 故早期诊断及及时治疗对减少该病的死亡率至关重要.对于该病的治疗原则主要为控制急性出血、根除抑制物、治疗原发病, 故合理及个性化选择治疗方案也很关键.     

中国血友病A患者因子Ⅷ抑制物形成特征及随访研究

目的 随访研究中国血友病A患者因子Ⅷ(FⅧ)抑制物发生率和特征.方法 215例血友病A患者在24月(2007年6月至2009年6月)的连续随访中,监测FⅧ抑制物发生、变化及转归,并观察患者临床特征.结果 215例血友病A患者随访24月FⅧ抑制物累积发病率为11.6%(25/215);其中低滴度者占72%(18/25),高滴度者占28% (7/25) ; FⅧ抑制物阳性发生时的中位年龄为25岁(6-59岁)、累积中位暴露日为150日;15/25(60%)低滴度阳性者(中位滴度1.25BU/ml)在自然情况下于6-15月(中位10月)转为阴性,5/25(20%)高滴度抑制物者(中位滴度100 BU/ml)则随访24月持续阳性,另外5/25 (20%)FⅧ抑制物阳性无变化;25例FⅧ抑制物阳性者出血频率较其阴性时显著增加(P=0.025);18/25例继续应用FVl者,FⅧ产品用量(IU/kg.月)较前显著增加(P=0.015),但靶关节数目在24月随访期间并无增加(P=0.329).结论 我国血友病A患者FⅧ抑制物发病率和特征与欧美等国家存在差异.     

获得性血友病A确诊前病程分析

目的分析获得性血友病A（AHA）诊治的临床特点,探讨国内外治疗该病的差异。方法整理分析2007—2012年本院收治的9例AHA患者资料．对比近5年国内AHA相关文献及欧洲获得性血友病登记管理组织（EACH2）的研究结果,分析国内外AHA确诊前病程与完全缓解率（CR）的差异。结果本组9例及国内文献100例AHA患者确诊前病程均延迟于EACH2的研究报道,CR率也均低于后者,差异具有统计学意义（P〈0．05）。结论我国AHA患者治疗状况与欧洲国家存在差异,加大知识宣传,降低漏诊、误诊率可能有助于缩短差距。     

血友病A家系的单体型基因连锁分析

目的：该研究开展温州地区血友病A（HA）家系的可变数目串联重复序列（VNTR）多态性、短串联重复序列（STR）多态性和限制性片段长度多态性（RFLP）单体型基因连锁分析,为HA遗传咨询和生育指导提供症状前、携带者基因诊断方面的依据。方法：针对HA先证者及其有关家系成员,进行单体型基因连锁分析。采用PCR检测凝血因子VIII（FVIII）基因外的DXS52（St14）位点的VNTR多态性,检测FVIII基因外的DXS15（CA）n、DXS9901（GT）n、DXS1073（GT）n位点和内含子1（GT）n、13（CA）n、22（GT）n（AG）n、24（GT）n位点的STR多态性并经毛细管电泳确证。另外采用PCR产物限制酶切检测FⅧ基因的内含子18、19、22位点的RFLP。结果：以2个HA家系为例报道研究结果。家系一先证者年幼弟弟肯定为正常人,先证者母亲、外祖母为携带者,先证者小姨肯定为携带者而其年幼儿子肯定为正常人。家系二先证者外祖母肯定不是携带者,先证者的X染色体来自外祖父,但已知外祖父不是患者,那么按照最大风险估计,母亲的那条来自外祖父的X染色体在外祖父生殖细胞中FVIII基因发生了突变,因此母亲是携带者。家系二先证者年幼妹妹是携带者,将来有生育患儿的风险,但先证者大姨及其年幼女儿不是携带者。结论：该研究的HA家系的VNTR-PCR、STR-PCR和PCR/RFLP单体型基因连锁分析,特别是对症状前男孩的诊断、对未曾有患病后代的女性携带者的检出,具有非常重要的实际意义,可以为遗传咨询和生育指导提供可靠依据。     

Identification of seven novel mutations in the factor VIII gene in 18 unrelated Chinese patients with hemophilia A

Background  Hemophilia A (HA) is an X-linked inherited bleeding disorder caused by decreased activity of factor VIII (FVIII) due to heterogenous mutations in the FVIII coding gene (F8). The type of mutation plays an important role in the FVIII inhibitor formation. To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we reported the distribution of the F8 gene mutations in 18 unrelated Chinese patients with HA.
Methods  Intron 22 and intron 1 inversions in the F8 gene were screened in 158 unrelated patients with HA using a long-distance PCR and multiplex PCR method. Direct sequencing of the coding region of the F8 gene was used to identify the mutations responsible for HA in 18 unrelated Chinese HA patients who were negative for intron 22 and intron 1 inversions; sequences were compared with the HAMSTeRS database. A clotting method was used to assay the FVIII activity level and the Bethesda assay was used to detect the FVIII inhibitor.
Results  A total of 18 different HA F8 mutations were identified, seven of which were described for the first time. These novel mutations included five small deletions, one point mutation and one small insertion. One novel mutation (4382-3 AC deletion) was associated with inhibitor development.
Conclusion  These data extend our insight into the mechanisms by which novel amino acid mutations may lead to HA and how the HA patient genotypes influence the risk of FVIII inhibitor.

     

系统性红斑狼疮合并获得性血友病A1例

Acquired hemophilia A (AHA) is anunusual disease resulting from autoantibodies (inhibitors) against coagulation factor Ⅷ (FⅧ) and clinically manifests as bleeding, which sometimes can cause potentially limb-threatening or life-threatening situations. AHA is associated with cancers, auto-immune disorders, infections, dermatologic conditions and certain medications, among which it is commonly secondary to multiple rheumatologic conditions,such as rheumatoid arthritis, systemic lupus erythematosus (SLE), pollymyositis, autoimmune hemolytic anemia and undifferentiated connective tissue disease. In autoimmune diseases, it may be the result of autoantibody producing against FⅧ, and some cases of AHA may act as the first manifestation of SLE. AHA should be suspected in patients who have spon-taneously hemorrhagic events with an isolated prolonged activated partial thromboplastin time (APTT), reduced FⅧ activity and a negative lupus anticoagulant (LA). When FⅧ inhibitor is found, it can be diagnosed. The management of AHA focuses on the following goals: (1)controlling and preventing blee-ding,(2)eradication of the inhibitor,(3)treatment of the underlying disease. Here, a case of AHA in a patient with lupus is reported. A 53-year-old man with a 4-year history of SLE developed arthralgia and ecchymotic skin lesions after arthrocentesis of knee joint. Ultrasound confirmed the presence of an intramuscular hematoma. Coagulation tests revealed that FⅧ activity reduced to 1% and a prolonged APTT (92.2 s), FⅧ inhibitors were found to be as high as 60.0 Bethesda Units. Initial treatments with me-thylprednisolone 200 mg/d were started but new hemorrhagic manifestation occurred and hisbiological indexes were not good. Then the patient was treated with intravenous pulse corticosteroids (methylprednisolone 500 mg/d),intravenous cyclophosphamide, and also plasma and prothrombin complex infusion. Sub-sequently, FⅧ activity returned within normal ranges, FⅧ inhibitors decreased and clinical improvement was significantly obtained. The patient’s condition kept stable till now.Hemorrhagic events due to produc-tion of antibodies directed against coagulation factors were rarely observed in SLE and attentions should be paid to the association between SLE and AHA.Bypass treatment was considered as the immediate antihemorrhagic treatment. Corticosteroid combined with immunosuppressor was recommended as the main therapy to eradicate the inhibitors. However we still lack the therapeutic guide-lines and standar-dized treatment in patients of AHA with SLE at present.     

中国重型血友病A成人患者低中剂量三级预防治疗突破性出血特点及影响因素

目的 研究中国重型血友病A成人患者低中剂量三级预防治疗的突破性出血特点及其影响因素.方法49例患者(31.53± 7.33岁)按照预防剂量分为低剂量组和中剂量组;评估临床出血表型(Pre-AJBR)、72 hFⅧ谷活性、活动能力(FISH评分),利用"血友管家"APP前瞻性记录出血与治疗情况,中位随访6月.结果 低剂量组15例;中剂量组34例;低剂量组和中剂量组关节突破性出血(AJBR)为18.79±13.03次/年和9.28±7.02次/年(P=0.016),自发性出血比例为75.0%和47.7%,存在靶关节患者比例为80%和44%,靶关节出血占比为59%和41%,中位突破性出血出现时间为预防注射后40.08 h和46.08 h(P=0.008),预防注射后0~12 h突破性出血发生率为4.86%和5.18%,72 hFⅧ谷活性<1%比例为44.4%和34.8%;AJBR与预防消耗因子量负相关(r=-0.57,P=0.000,n=49);两组AJBR均与FISH评分负相关,与Pre-AJBR正相关(P<0.05).结论 低中剂量三级预防治疗尚无法使多数中国重型血友病A成人患者获得阻止关节病变进展的目标;因子剂量虽是预防疗效的最主要影响因素,但通过非因子途径也可能改善疗效.     

DNA多态性连锁分析在血友病A家系产前诊断中的应用

目的探讨提高运用DNA多态性连锁分析对血友病A携带者进行筛查及产前诊断检出率的途径。方法对3个血友病A家系的成员进行BclⅠ、St14VNTR、Intro13(CA)n和DXS15 4个位点的连锁分析。结果家系a中II7的BclⅠ位点和Intro13(CA)n位点均呈纯合状态,无信息;St14VNTR和DXS15的连锁分析显示III6为携带者,通过对BclⅠ的进一步分析显示胎儿为患儿的可能性大。家系b中II1的BclⅠ位点和St14VNTR多态位点为纯合状态,无信息;联合应用Intro13(CA)n和DXS15进行连锁分析显示II1为携带者,胎儿为正常女性。家系c中St14VNTR、Intro13(CA)n以及DXS15位点在II4均为纯合状态,BclⅠ位点为杂合状态,连锁分析显示胎儿为正常男性可能性较大。结论联合应用多个遗传多态性位点可以提高血友病A的诊断率和携带者分析的正确率。     

Osteopontin gene polymorphism in association with systemic lupus erythematosus in Chinese patients

Background Osteopontin (OPN) is one kind of cytokine which can play a number of roles in promoting activation of T lymphocyte, regulating balance between Th1 and Th2, participating in cell-induced immunologic response and stimulating B lymphocyte to express multi-clone antibodies. Some researches have showed that OPN may be involved in the pathogenesis of systemic lupus erythematosus (SLE). The aim of this study was to investigate possible association of a single nucleotide polymorphism（SNP）at position 9250 in exon 7 of the OPN gene (OPN gene 9250) with SLE in Chinese patients.Methods Totally 158 patients (18 males and 140 females) fulfilled the revised criteria for SLE by the American College of Rheumatology in 1982 and 180 healthy volunteer controls (34 males and 146 females), all from the south of China, consented to participate in the study. OPN gene 9250 polymorphism was detected by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP).Results The frequency of TT genotype of the OPN gene 9250 was significantly lower (52.5% vs 70%, P&lt;0.05) and the frequency of TC genotype of the OPN gene 9250 was significantly higher (43.7% vs 29.4%, P&lt;0.05) in SLE patients than in controls. There were significant differences in OPN gene 9250 allele and phenotype frequencies between the SLE patients and controls (P&lt;0.05). When the SLE patients and controls were separated into men and women, significant differences of frequencies were noted in TT genotype, TC genotype and allele of the OPN gene 9250 in women (P&lt;0.05) but not in men (P&gt;0.05). Conclusions OPN gene 9250 polymorphism appears to be associated with susceptibility to SLE in Chinese Han ethnic population.     

FⅧ剂量对青少年重型血友病A的关节结构与功能的影响:43例前瞻性队列研究中期随访报告

目的 运用综合评估手段比较分析不同剂量FⅧ治疗下青少年重型血友病A患者的临床表型、关节结构与功能变化规律。方法 43例青少年（4~18岁）重型血友病A患者,分为按需组（n=7）,低剂量预防组[FⅧ 10~15 U/kg,2~3次/周,且≤30 U/（kg·周）,n=17],中剂量预防组[FⅧ 15~20 U/kg,2~3次/周,且45~60 U/（kg·周）,n=19]。比较分析其24月间临床出血表型[年出血率/年关节出血率/最严重单个关节年出血率]、关节影像评分（超声HEAD-US评分,IPSG MRI评分）、血友病关节健康评分量表（HJHS）、血友病功能独立性评分（FISH）及变化规律。使用Kruskal-Wallis检验、Wilcoxon和重复测量的方差分析进行统计检验。结果 低中剂量组的年出血率较按需组明显减少（P1=0.004,P2=0.000）,中剂量较低剂量组减少32.87%。按需组、低剂量组和中剂量组的年关节出血率差异有统计学意义（P1=0.001,P2=0.000,P3=0.001）。按需组、低剂量组和中剂量组的最严重单个关节年出血差异有统计学意义（P1=0.016,P2=0.000,P3=0.005）,按需组出血次数渐快增加,低剂量组维持不变,中剂量组则有减缓趋势。3组患者最严重单个关节影像学评分均有进展。低中剂量组的超声评分变化值进展较按需组延缓（P1=0.002,P2=0.000）,按需组评分按原速率增长,低、中剂量组均有减缓趋势。低中剂量组的MRI评分变化值进展较按需组均有延缓（P1=0.041,P2= 0.000）,按需、低剂量组评分增长加快,中剂量组速率保持不变。按需组的HJHS评分变化值较低中剂量组增加（P1=0.003,P2= 0.000）;按需组保持原有增长速度,低、中剂量组评分均出现负值趋势。按需组FISH评分降低0.29±3.09分,低中剂量组均回升,与按需组差异有统计学意义（P1=0.000,P2=0.000）。结论 两年中期随访结果显示,中国青少年低中剂量FⅧ的预防治疗在保护关节结构和功能方面明显优于按需治疗,中剂量可能稍优于低剂量预防。     

非凝血因子Ⅷ基因内含子22倒位型血友病A家系11例产前诊断

目的:探讨用多态性位点分析进行非凝血因子Ⅷ基因内含子22倒位型血友病A产前诊断.方法:用PCR-琼脂糖电泳、PCR-聚丙烯酰胺凝胶电泳、PCR-限制性酶切片段长度多态性等方法,选择FⅧ基因内BclⅠ位点、XbaⅠ位点、CA-13、CA-22,FⅧ基因旁侧DXS52(ST14)和DXS15位点,对11例非凝血因子Ⅷ基因内含子22倒位型血友病A家系进行产前诊断.结果:11例非凝血因子Ⅷ基因内含子22倒位型血友病A家系均可通过使用6个多态位点进行产前诊断,其中4例家系胎儿为血友病患者的可能性大,选择了引产,另7例家系胎儿为健康个体的可能性大,出生后经诊断均健康.结论:检测FⅧ基因内、外多个多态性位点可对非凝血因子Ⅷ基因内含子22倒位型血友病A家系进行高效、快速的产前诊断.