Significance of mutation analysis in patients with haemophilia A

Haemophilia A represents the most frequent hereditary bleeding disorder in humans. The disease is caused by mutations within the factor VIII gene leading to decreased or absent factor VIII activities with a bleeding tendency depending on the degree of factor VIII deficiency. Nowadays, the causative mutations can be routinely detected and have substantially improved diagnostic and understanding of the pathophysiology of haemophilia A. Identification of the gene defects in haemophilic families have enabled fast and save carrier diagnosis. The correlation of the genetic defects with the clinical course revealed that the type of mutation represents the most important genetic predisposing factor for inhibitor formation, the most severe complication of treatment with factor VIII concentrates. Mitigated clinical courses of haemophilia A were shown to be due to special types of mutations or the presence of concomitant thrombophilic mutations. Molecular models of the factor VIII protein allowed to investigate the effects of specific mutations thus giving new insights in the structure/function relationship of the factor VIII molecule. These findings might promote the development of novel recombinant factor VIII concentrates with higher efficacy, longer half life and reduced immunogenicity.     

血友病A／B的携带者和产前诊断研究

目的：建立一套简易、快速且准确率高的血友病携带者和产前诊断体系。方法：对血友病A（HA）家系采用长距离聚合酶链反应（LD-PCR）及序列特异PCR进行F8基因内含子22及内含子1倒位检测。对有家族史的HA家系可联合F8基因内外的8个多态性位点进行遗传连锁分析．产前诊断加测性别位点（Amelo）。而对散发家系，则通过直接核苷酸测序查找先证者的基因突变，继而对家系女性成员进行携带者与产前诊断。对血友病B（HB）家系，采用直接核苷酸测序法确定其基因突变．结果：100个HA家系中。22例先证者的F8基因内含子22倒位检测结果为阳性；2例患者母亲为F8基因内含子1倒位的携带者：对有家族史的家系，综合应用倒位检测和遗传连锁分析，携带者与产前诊断率均为100％。34个HA散发家系除2个家系外均可找到致病突变：发现可能携带者105例，产前诊断65例，总诊断率为95．9％：23个HB家系中19个家系通过直接测序可找到突变．而联合F9基因外6个STR位点对HB家系进行遗传连锁分析，发现31例可能携带者及需产前诊断者16例，总诊断率为95．7％。结论：F8基因内含子22及1倒位筛检联合F8基因内、外多个位点的遗传连锁分析．可作为HA的携带者检测及产前诊断的方法；直接核苷酸测序可确定F9基因突变．而联合基因外多个多态性位点检测并进行遗传连锁分析．是HB携带者检测及产前诊断的简便、快速方法。     

Genetic analysis of non-severe hemophilia A phenotype with A discrepancy between one-stage and chromogenic factor VIII activity assays

IntroductionThe diagnosis of hemophilia A (HA) is based on the measurement of factor VIII activity (VIII:C). About one-third of non-severe HA patients show a discrepancy of VIII:C measured by one-stage (VIII:C 1st) and chromogenic (VIII:C chr) assays. Different mutations in the F8 gene may cause the discrepancy in results of the FVIII activity assay.The aim of this study was to investigate F8 gene mutations in patients with assay discrepancies and to evaluate their impact on the results of VIII:C assays.MethodsMutation analysis was performed on 41 individuals with a discrepancy in VIII:C 1st and FVIII: C chr assays by direct sequencing. In addition, the effect of the variants on FVIII macromolecule structure was investigated by in silico and bioinformatics tools.ResultsGenetic analysis disclosed 22 different variants, of which 19 were identified for the first time to be involved in the phenotype of VIII:C discrepancy. Most of the variants related to the higher VIII:C 1st were found in A1, A2, A3 domains. The variant related to VIII:C chr > VIII:C 1st was located in the thrombin cleavage site. In silico analysis showed the effect of variants on FVIII macromolecule stability, which may be the possible mechanism causing the discrepancy.ConclusionOur data shed light on the impact of genetic defects on VIII:C assay and provided evidence that the consideration of these mutations may open a new window to the proper diagnosis and treatment monitoring of non-severe HA patients.     

血友病A患者凝血因子Ⅷ基因内含子1及22倒位分析

目的 对血友病A(HA)患者及其家系携带者进行凝血因子Ⅷ(FⅧ)基因内含子1及22倒位分析,并探讨其与FⅧ抗体产生的相关性.方法 对157例HA患者进行FⅧ活性(FⅧ:C)及FⅧ抗体检测;同时采用双管多重PCR及长距离PCR(LD-PCR)技术对其中81例HA患者进行FⅧ内含子1及内含子22倒位分析;并对其中6例患者进行家系调查.结果 81例HA患者中3例为内含子1倒位,均为重型,占重型HA的4.6%(65例中3例);内含子1倒位患者中1例为FⅧ抗体阳性.用该基因信息对1个内含子1倒位患者家系的2名女性进行了检测,1例为携带者,1例为非携带者.81例HA患者中25例为内含子22倒位,均为重型,占重型HA的38.5%(65例中25例),内含子22倒位患者中发现1例为FⅧ抗体阳性.用该基因信息对5个内含子22倒位患者家系的9名女性进行了检测,7例为携带者,2例为非携带者.结论 内含子1倒位是继内含子22倒位外导致重型HA的另一重要分子发病机制.FⅧ内含子倒位检测不仅可用于血友病直接基因诊断、进行家系调查;同时还可纠正表型分型的偏差.FⅧ内含子1和内含子22倒位可能是抗体产生的高危因素之一.     

Detection of factor VIII gene mutations by high-resolution melting analysis

BACKGROUND: Single base-pair substitution mutations in the gene for coagulation factor VIII, procoagulant component (hemophilia A) (F8) account for approximately 50% of severe cases of hemophilia A (HA), and almost all moderate or mild cases. Because F8 is a large gene, mutation screening using denaturing HPLC or DNA sequencing is time-consuming and expensive. METHODS: We evaluated high-resolution melting analysis as an option for screening for F8 gene mutations. The melting curves of amplicons heterozygous for known F8 gene mutations were compared with melting curves of the corresponding normal amplicons to assess whether melting analysis could detect these variants. We examined 2 platforms, the Roche LightCycler 480 (LC480) and the Idaho Technology LightScanner. RESULTS: On both instruments, 18 (90%) of the 20 F8 gene variants we examined were resolved by melting analysis. For the other 2 mutations, the melting curves of the heterozygous amplicons were similar to the corresponding normal amplicons, suggesting these variants may not be detected by this approach in a mutation-scanning screen. CONCLUSION: High-resolution melting analysis is an appealing technology for F8 gene screening. It is rapid and quickly identifies mutations in the majority of HA patients; samples in which no mutation is detected require further testing by DNA sequencing. The LC480 and LightScanner platforms performed similarly.     

Unusual genomic rearrangements in introns 1 and 22 of the F8 gene

Intron 1 and intron 22 inversions, two large rearrangements of the factor VIII gene, are generally associated with a severe phenotype of haemophilia A and a high risk of inhibitor formation. In several haemophiliacs, diagnostic analyses for detection of these inversions revealed unusual band patterns. Upon further examination, different copy number variations were detected in the factor VIII gene of these patients by multiplex ligation-dependent probe amplification (MLPA). Since these duplications or deletions alone could not sufficiently explain the abnormal band patterns of the first analyses, we assumed a combination of intron 1 or intron 22 inversions together with a copy number variation. Result: We could confirm this hypothesis by specific long range PCRs but a detailed characterization of the breakpoints and the mechanisms for these complex rearrangements have yet to be elucidated.     

Haemophilia B: from molecular diagnosis to gene therapy.

Thanks to its typical expression, haemophilia can be identified in writings from the second century AD. Haemophilia B, an X-linked recessive bleeding disorder due to factor IX (FIX) deficiency, has an incidence of about 1:30,000 live male births. The factor 9 (F9) gene was mapped in 1984 on Xq27.1. Haemophilia is diagnosed from prothrombin time, activated partial thromboplastin time, and FIX levels. Carrier females are usually asymptomatic and must be identified only with molecular analysis. Linkage analysis of F9 polymorphisms is rapid and inexpensive but limited by non-informative families, recombinant events, and the high incidence of germline mutations; thus, various procedures have been used for the direct scan of F9 mutations. We set up a novel denaturing high performance liquid chromatographic procedure to scan the F9 gene. This rapid, reproducible procedure detected F9 mutations in 100% of a preliminary cohort of 18 haemophilia B patients. Parallel to the development of more efficient diagnostic tools, the life expectancy and reproductive fitness of haemophilic patients have greatly improved and will continue to improve thanks to the use of less immunogenic recombinant FIX. Hopefully, new approaches based on gene therapy now being evaluated in clinical trials will revolutionise haemophilia B treatment.     

甲型血友病的基因诊断研究

目的 建立甲型血友病的基因诊断方法.方法 对33例甲型血友病患者以一期法检测血浆凝血因子FⅧ(F8)活性.采用长距离聚合酶链式反应扩增法(LD-PCR)进行FⅧ内含子22倒位检测,并对反应体系进行优化;聚合酶链式反应(PCR)技术检测内含子1倒位,凝胶成像技术分析扩增产物.捕获测序技术检测是否存在其他突变类型.结果 33例患者的FⅧ活性检测结果均小于1%,为重型;LD-PCR技术检测出13例患者存在Int22倒位,倒位发生率为39.4%;Int1倒位患者1人,发生率3%;对13例内含子 22倒位患者进行测序比对,未发现其他基因突变.结论 LD-PCR技术和多重PCR技术可以有效的检测重型甲型血友病FⅧ的基因倒位.     

Molecular analysis of FVIII gene in severe HA patients of Costa Rica

Haemophilia A (HA) is X-chromosome linked bleeding disorders caused by deficiency of the coagulation factor VIII (FVIII). It is caused by FVIII gene intron 22 inversion (Inv22) in approximately 45% and by intron 1 inversion (Inv1) in 5% of the patients. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22 and their extragenic copy located telomeric to the FVIII gene. The aim of this study was to analyze the presence of these mutations in 25 HA Costa Rican families. Patients, methods: We studied 34 HA patients and 110 unrelated obligate members and possible carriers for the presence of Inv22or Inv1. Standard analyses of the factor VIII gene were used incl. Southern blot and long-range polymerase chain reaction for inversion analysis. Results: We found altered Inv22 restriction profiles in 21 patients and 37 carriers. It was found type 1 and type 2 of the inversion of Inv22. During the screening for Inv1 among the HA patient, who were Inv22 negative, we did not found this mutation. Discussion: Our data highlight the importance of the analysis of Inv22 for their association with development of inhibitors in the HA patients and we are continuous searching of Inv1 mutation. This knowledge represents a step for genetic counseling and prevention of the inhibitor development.     

Thirty-four novel mutations detected in factor VIII gene by multiplex CSGE: modeling of 13 novel amino acid substitutions

Summary.  Detection of causal mutations is required for genetic counseling. Molecular modeling combined with patients' phenotype provides significant insight into structure–function relationship of factor (F)VIII molecule. Our objective was to identify defects in the gene of FVIII by a sensitive and simple scanning technique with high throughput in order to study molecular mechanisms by which novel amino acid substitutions may lead to hemophilia A. A cohort of 81 families with mild, moderate and severe hemophilia A negative in intron 22 inversion was studied. For detection of mutations in the FVIII gene a conformation sensitive gel electrophoresis (CSGE) was modified by multiplexing. Thirteen novel amino acid substitutions were studied by molecular modeling and a correlation with the cross-reactive material (CRM) phenotype was performed. In 74 families, 59 different mutations were detected. Six different mutations were recurrent in 21 unrelated families. Thirty-four novel mutations included 19 point mutations, four small insertions, nine small deletions and two complex mutations. Thirteen novel amino acid substitutions occurred at residues conserved in FVIII orthologs. Five of them were associated with a discrepancy between FVIII activity and antigen; another five with CRM reduced phenotype and one with undetectable FVIII antigen. Multiplexing of the CSGE significantly increased its throughput without substantial loss of sensitivity. Molecular modeling suggested mechanisms by which substitutions at residues 382 and 569, located outside the proposed FIXa-binding region, may influence FVIII/FIXa interaction. His2155 was predicted to participate in FVIII/VFW binding.     

Preimplantation genetic diagnosis of hemophilia A

Preimplantation genetic diagnosis (PGD) is a powerful tool to tackle the transmission of monogenic inherited disorders in families carrying the diseases from generation to generation. It currently remains a challenging task, despite PGD having been developed over 25 years ago. The major difficulty is it does not have an easy and general formula for all mutations. Different gene locus needs individualized, customized design to make the diagnosis accurate enough to be applied on PGD, in which the quantity of DNA is scanty, whereas timely laboratory diagnosis is mandatory if fresh embryo transfer is desired occasionally. Indicators for outcome assessment of a successful PGD program include the successful diagnosis rate on blastomeres (Day 3 cleavage-stage embryo biopsy) or trophectoderm cells (Day 5/6 blastocyst biopsy), the implantation rate per embryo transferred, and the livebirth rate per oocyte retrieval cycle. Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by various types of pathological defects in the factor VIII gene (F8). The mutation spectrum of the F8 is complex, according to our previous report, including large segmental intra-gene inversions, large segmental deletions spanning a few exons, point mutations, and total deletion caused by chromosomal structural rearrangements. In this review, the molecular methodologies used to tackle different mutants of the F8 in the PGD of HA are to be explained, and the experiences of successful use of amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR) and linkage analysis for PGD of HA in our laboratory are also provided.     

血友病携带者与产前基因诊断

目的建立1种简便、快速的血友病携带者检测与产前诊断体系。方法对38个血友病A家系,用长链PCR及序列特异PCR作F8基因内含子22及1倒位检测;有家族史家系可联合F8基因内外的8个多态性位点进行遗传连锁分析,DXS 52(ST14)位点多态性以PCR产物凝胶电泳法检测,7个STR位点(F8C ivs13、CA22、DXS15、DXS9901、G6PD、DXS1073、DXS1108)的多态性以多重荧光PCR法检测,产前诊断加用性别位点(Amelo);对于散发家系,通过直接核苷酸测序查找先证者的基因突变,继而对家系女性成员作携带者与产前诊断。对12个血友病B家系采用直接核苷酸测序法确定基因突变,多重荧光PCR法检测F9基因外6个STR位点(DXS1192、DXS1211、DXS8094、DXS8013、DXS1227、DXS102)的多态性。结果38个血友病A家系中,10名先证者的F8基因内含子22倒位检测为阳性,1例先证者为F8基因内含子1倒位阳性,对于有家族史的家系,综合应用倒位检测和遗传连锁分析,携带者与产前诊断率均为100%;4个散发家系均可找到致病突变;38个血友病A家系的携带者及产前诊断总诊断率为94.81%。12个血友病B家系中有10个家系通过直接测序可找到突变,联合F9基因外6个STR位点对血友病B家系的遗传连锁分析的可诊断率为96.88%。结论F8基因内含子22及1倒位筛检联合F8基因内、外多个位点的遗传连锁分析可以进行血友病A的携带者及产前诊断;直接核苷酸测序可确定F9基因突变,而联合基因外多个多态性位点检测并进行遗传连锁分析,是血友病B携带者检测与产前诊断的简便、快速的方法。     

一例凝血因子Ⅷ B区错义突变导致重型血友病A

目的：检测一例重型血友病Ａ患者（ＳＨ９）的基因突变。方法：用ＰＣＲ、变性梯度凝胶电泳（ＤＧＧＥ）和ＤＮＡ测序检测因子Ⅷ基因突变。先用Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ排除内含子２２倒位，然后应用ＰＣＲ对凝血因子Ⅷ基因进行扩增。扩增范围包括所有外显子及其侧翼内含子序列。结果：片段１４２在ＤＧＧＥ中泳动异常。ＤＮＡ测序证实Ｃ２５３５Ａ导致Ｂ区错义突变８２６Ａｓｐ（ＧＡＣ）→Ｇｌｕ（ＧＡＡ）。结论：该突变可能是导致重型血友病Ａ的原因，但有待进一步研究证实。     

FⅧ基因内含子22倒位及X染色体非随机灭活导致的女性血友病A

目的 对1例女性血友病A(HA)患者进行产前诊断,探讨其分子发病机制.方法 利用测定FⅧ活性(FⅧ:C)、出血时间(BT)、血管性血友病因子(VWF)等指标进行HA表型诊断;用长链PCR及序列特异PCR进行FⅧ基凶内含子22及1倒位检测,对FⅧ基因的所有外显子及其侧翼序列进行测序.联合FⅧ基因内外的8个多态性位点进行遗传连锁分析,DXS 52(ST14)位点多态性以PCR产物凝胶电泳法榆测,7个微卫星位点(F8Civs13、CA22、DXS15、DXS9901、G6PD、DXS1073、DXS1108)的多态性以多重荧光PCR法检测,产前诊断加用性别位点(Amelo).利用甲基化敏感的限制性内切酶HpaⅡ对基因组DNA进行酶切,荧光PCR法对HpaⅡ酶切前后的基因组DNA进行人类雄激素受体基丙(HUMARA)中CAG重复序列的扩增,荧光扫描法对扩增产物进行检测,根据酶切前后PCR产物的峰值变化来分析判断X染色体的随机灭活模式.结果 先证者的FⅧ:C为2.1%,其余表型检测结果均正常,其家系成员表型检测结果均正常.FⅧ基因内含子22倒位检测示先证者为22倒位阳性携带者,家系其他成员及胎儿均为22倒位阴性;先证者FⅧ基因内含子1倒位检测结果为阴性,其FⅧ基因测序未发现突变.遗传连锁分析发现先证者的X染色体分别遗传自其父亲、母亲;其胎儿为女性且遗传了先证者来源于其母亲的1条X染色体.先证者为X染色体非随机灭活,其来自母亲的x染色体被大部分灭活,而来自父亲的x染色体大部分被保留活性.结论 FⅧ基冈内含子22倒位及X染色体非随机灭活导致了该例女性血发病A的发生,其胎儿为正常女性.     

两种新的F8内含子突变导致剪接异常的机制研究

目的：分析从2个血友病A家系中检出的2个未报道的内含子突变对剪接的影响,明确这2种突变的致病性,为遗传咨询提供依据。方法：通过检测相关的凝血指标,明确血友病A的诊断。进行F8相关检测,包括PCR扩增及测序分析F8的26个外显子及其侧翼序列、拷贝数检测、内含子1和内含子22倒位检测,明确致病突变。采用巢式PCR扩增外周血中的mRNA,从异位转录水平分析内含子突变对剪接的影响。针对6个短串联重复序列（short tandem repeats, STR）位点F8Up226、F8Up146、F8Int13、F8Int25、F8Down48和DXS1073进行家系遗传连锁分析,用SNaPshot SNP分型技术检测外周血、口腔黏膜细胞和毛囊细胞嵌合情况,分析突变来源。结果：家系1中血友病A先证者1的 FⅧ：C为0.9%,检测到F8的9号内含子c.1444-2dupA突变,mRNA分析显示该突变导致F8的10号和11号外显子缺失;家系2中血友病A先证者2的 FⅧ：C为5.1%,检测到F8的18号内含子c.5999-29T>G突变,mRNA分析显示该突变产生2种转录本,即缺失19号外显子的异常转录本和少量正常转录本。家系1无血友病A家族史,遗传分析显示突变来源于先证者的外公,但外公的血液、毛发和口腔样本嵌合检测均未检出突变。结论：c.1444-2dupA和c.5999-29T>G突变均为国际首次报道,突变导致了不同程度的剪接异常,分别引起重型和轻型血友病A。家系1的c.1444-2dupA为新发突变,可能是在先证者外公的精子形成过程中发生。     

Prediction of factor VIII inhibitor development in the SIPPET cohort by mutational analysis and factor VIII antigen measurement

Essentials

• A residual factor VIII synthesis is likely to be protective towards inhibitor (INH) development.
• Mutation type‐inhibitor risk association was explored in 231 patients with severe hemophilia A.
• A 2‐fold increase in INH development for in silico null vs. non‐null mutations was found.
• A 3.5‐fold increase in INH risk for antigen negative vs. antigen positive mutations was found.

Summary

Background

The type of F8 mutation is the main predictor of inhibitor development in patients with severe hemophilia A. Mutations expected to allow residual synthesis of factor VIII are likely to play a protective role against alloantibody development by inducing immune tolerance. According to the expected full or partial impairment of FVIII synthesis, F8 variants are commonly classified as null and non‐null.

Objectives

To explore the mutation type–inhibitor risk association in a cohort of 231 patients with severe hemophilia A enrolled in the Survey of Inhibitors in Plasma‐Product Exposed Toddlers (SIPPET) randomized trial.

Methods

The genetic defects in these patients, consisting of inversions of intron 22 (n = 110) and intron 1 (n = 6), large deletions (n = 16), and nonsense (n = 38), frameshift (n = 28), missense (n = 19) and splicing (n = 14) variants, of which 34 have been previously unreported, were reclassified according to two additional criteria: the functional effects of missense and splicing alterations as predicted by multiple in silico analyses, and the levels of FVIII antigen in patient plasma.

Results

A two‐fold increase in inhibitor development for in silico null mutations as compared with in silico non‐null mutations (hazard ratio [HR] 2.08, 95% confidence interval [CI] 0.84–5.17) and a 3.5‐fold increase in inhibitor development for antigen‐negative mutations as compared with antigen‐positive mutations (HR 3.61, 95% CI 0.89–14.74] were found.

Conclusions

Our findings confirm an association between the synthesis of minute amounts of FVIII and inhibitor protection, and underline the importance of investigating the residual FVIII antigen levels associated with causative variants in order to understand their clinical relevance.

     

A possible mechanism for Inv22‐related F8 large deletions in severe hemophilia A patients with high responding factor VIII inhibitors

Summary. Background: Intron 22 inversion (Inv22) of the coagulation factor (F)VIII gene (F8) is a frequent cause of severe hemophilia A. In addition to Inv22, a variety of F8 mutations (1492 unique mutations) causing hemophilia A have been reported, of which 171 involve deletions of over 50 bp (HAMSTeRs database; http://hadb.org.uk/ ). However, only 10% of these large deletions have been fully characterized at the nucleotide level. Patients and methods: We investigated gene abnormalities in three unrelated severe hemophilia A patients with high titer FVIII inhibitors. They had previously been shown to carry large deletions of the F8, but the precise gene abnormalities remain to be elucidated. Results: Inverse shifting‐PCR (IS‐PCR) Inv22 diagnostic tests revealed that these patients carried either type I or II Inv22. However, they showed a wild‐type (WT) pattern in the IS‐PCR Inv22 complementary tests. We further analyzed their X chromosomes to account for the puzzling results, and found that they had different centromeric breakpoints in the Inv22 X chromosomes, adjacent to the palindromic regions containing int22h‐2 or ‐3, and their spacer region, respectively. The connections appeared to be shifted towards the telomere of the WT F8 Xq28, resulting in a new telomere with an additional intact int22h copy. Conclusions: These gene rearrangements might result from double‐strand breaks in the most distal regions of the long arms of the Inv22 X chromosomes, followed by DNA restorations using the WT F8 Xq28 by non‐homologous end joining or break‐induced replication; thus leading to large F8 deletions in severe hemophilia A patients.     

Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype

Severity of bleeding phenotype in hemophilia A (HA) depends on the underlying mutation in the F8 gene and, ultimately, on the concentration and functional integrity of the factor VIII (FVIII) protein in circulating plasma. Initial diagnosis for HA and monitoring of treatment is typically performed by measuring of FVIII activity by either one-stage assay or chromogenic assay. We review evidence for why both types of assay do not give comparable results in a significant proportion of patients with non-severe haemophilia A and why the discrepancy in results between both methods segregates with distinct subclasses of known missense mutations causing haemophilia A. The current understanding of the mechanistic basis for how FVIII:C assay discrepancies arise are discussed. Conclusion: We propose that both methods should be used in initial patient diagnosis along with follow-up genetic analysis to avoid potential misdiagnosis and to optimize treatment monitoring of patients with HA phenotypes.     

长距离PCR检测重型血友病A病人凝血因子Ⅷ基因倒位

为了筛查山东省重型血友病A(hemophiliaA，HA)凝血因子Ⅶ基因倒位的患并检出携带，采用长距离DNA扩增(LD-PCR)方法，以0．6％琼脂糖凝胶电泳技术，检测临床上确诊的55例重型HA患及其家系成员中是否存在凝血因子Ⅷ(FⅧ)基因倒位。电泳出现11kb带，示凝血因子Ⅷ基因倒位；12kb带，示非倒位；这两条带同时出现为凝血因子Ⅷ基因倒位携带。结果表明：55例无亲缘关系的重型HA患中，发现22例患(或家系成员)有凝血因子Ⅷ基因倒位，占重型HA患的40%；15个家系中查出基因倒位携带5名。结论：运用LD-PCR技术可以准确、简便、快速地检测重型血友病A患是否存在凝血因子Ⅷ基因倒位。