Partial F8 gene duplication (Factor VIII Padua) associated with high factor VIII levels and familial thrombophilia

Blood ◽  
2020 ◽  
Author(s):  
Paolo Simioni ◽  
Stefano Cagnin ◽  
Francesca Sartorello ◽  
Gabriele Sales ◽  
Luca Pagani ◽  
...  

High coagulation factor VIII (FVIII) levels are a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in two Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before the age of 50, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defect. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1 and a large part of intron 1), which co-segregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a ≥2-fold up-regulation of the F8 mRNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In conclusion, we report the first thrombophilic defect in the F8 gene (designated "FVIII Padua") associated with markedly elevated FVIII levels and severe thrombophilia in two Italian families.

2010 ◽  
Vol 30 (S 01) ◽  
pp. S150-S152
Author(s):  
G. Jiménez-Cruz ◽  
M. Mendez ◽  
P. Chaverri ◽  
P. Alvarado ◽  
W. Schröder ◽  
...  

SummaryHaemophilia 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.


Author(s):  
Maysoon Mohammed Hassan

The background:One of the prevalent main concerns in the medical world is the identification of Intron22 mutations in the Factor VIII gene carried by Iraqi patient in Wasit town, in Iraq suffering Hemophilia A (classical hemophilia) which is related to a X-chromosome recessive haemorrhage afflictions as the result of a flaw in the coagulation factor VIII (FVIII). It is essentially related with F8 mutations of Intron22 in version which forms the most typical kind of mutations of blood afflictions worldwide involving half the patients suffering from severe Hemophilia A that possesses mutations, in addition to Intron 1 inversion suffered by 5% of severe Hemophilia A patients.All of the inversion mutations are suffered mainly by males,and uncommonly by females due to the intra chromosomal recombination among the homologous areas, in inversion 1 or 22, with extragenic copy posited the telomeric to the Factor VIII gene. Unfortunately, there is an absence in Iraq on researches pertaining blood affliction gene identification in persons who carries the Intron22 mutations exception in the current research.Aims of study:The objectives of the research is to to analyze through the detection mechanisms, the existence of Intron 22 mutations in the Factor VIII gene of 10 Hemophilia A Iraqi carriers cohort families. The hypothesis and anticipated result is that there will be a minimal margin of hazardous possibility for the recurrence. The hereditary F8 mutation is unknown to be present on the maternal side of the patient sufferer due to the possibilty of germline mosaics that exists within the community.


Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4096-4096
Author(s):  
Lizbeth Salazar-Sanchez ◽  
Guillermo Jimenez-Cruz ◽  
Pilar Chaverri ◽  
Winnie Schroeder ◽  
Karin Wulff ◽  
...  

Abstract Hemophilia A (HA) is X-chromosome linked bleeding disorders caused by deficiency of the coagulation factor VIII. The disease is caused by Factor VIII gene intron 22 inversion in approximately 50% of the patients and by intron 1 inversion in 5% of the patients. Both inversions occur as a result of intrachromosomal recombination between homologous region, in intron 1 or 22 and their extragenic copy located telomeric to the FVIII gene. The goal of the present study was to analyze the presence of these mutations in 15 HA severe Costarrican families. Methods: We studied 122 unrelated HA patients and obligate or possible carriers for the presence of intron 22 or intron 1 by Southern blotting and polymerase chain reaction (PCR). Results: We found altered intron 22 restriction profiles by Southern analyses in 14 of the families, 12 cases type 1 (Figure 1) and 2 cases type 2 inversion. During the screening for intron 1 inversion among the HA patient, who were intron 22 inversion negative, we did not identified this mutation. Interpretation and Conclusions: This report is the first in our haemophilia families dealing with mutations in the intron 22 and intron 1. Our data highlight the importance of the analysis of intron 22 inversion for the association with development of inhibitors in the HA patients and we are continuous searching of intron 1 mutation. This will benefit both genetic counselling and the study of the relationship between genotype and inhibitor development. Fig. 1 Southern blot of HA-I showing the intron 22 inversion of the factor VIII gene. Lane 1 and 2 normal Lane 3: heterozygote carrier and lane 4: patient with the altered fragment, (inversion of the intron 22). Fig. 1. Southern blot of HA-I showing the intron 22 inversion of the factor VIII gene. Lane 1 and 2 normal Lane 3: heterozygote carrier and lane 4: patient with the altered fragment, (inversion of the intron 22).


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1223-1223
Author(s):  
Hiroshi Inaba ◽  
Keiko Shinozawa ◽  
Takeshi Hagiwara ◽  
Kagehiro Amano ◽  
Katsuyuki Fukutake

Abstract Abstract 1223 Introduction/Background: Hemophilia A is a congenital X-linked bleeding disorder caused by various mutations in the coagulation factor VIII gene (F8). However, recent studies have described that no genetic mutation could be found in the F8 of about 2% of hemophilia A patients, even after nucleotide sequencing including the entire coding region, exon/intron boundaries, and the 5'- and 3'-untranslated region (Vidal et al, 2001; Klopp et al, 2002). Factor VIII deficient mechanisms underlying this phenomenon remain unexplained. To further elucidate the mechanisms causing hemophilia A in these patients, we performed a detailed analysis of F8 mRNA. Materials and methods: F8 mRNA from a Japanese hemophilia A patient with undetectable mutations was analyzed. Total RNA was isolated from peripheral blood cells using a QIAamp® RNA Blood Mini Kit (Qiagen) or PAXgene® Blood RNA Kit (Qiagen). Both preparations were performed following the manufacturer's instructions. In order to analyze the F8 mRNA, we performed the cDNA-amplification in two rounds of PCR using the nested approach reported by El-Maarri et al (2005). The nucleotide sequences of primer used followed those of their report. OneStep RT-PCR Kit (Qiagen) and TaKaRa LA Taq ™ (TaKaRa) were used for first and second round PCR amplification, respectively. Ectopic F8 mRNA expression level was relatively quantified by a real-time PCR technique using 4 TaqMan gene expression assays (Hs00240767_m1 amplify exon 1–2 boundary, Hs01109548_m1 amplify exon 6–7 boundary, Hs01109541_m1 amplify exon 14–15 boundary, Hs01109543_m1 amplify exon 20–21 boundary; Applied Biosystems). Results: Because the size of the F8 mRNA is very large ∼9kb, the entire F8 cDNA was divided into four different regions: exons 1–8 (region A); exons 8–14 (region B); exons 14–22 (region C); and exons 19–26 (region D) and amplified in the first round. Then, each of four regions were further divided into two different regions (a total of 8 overlapping regions; region 1–8), and amplified in the second round. An abnormality was observed in the amplification. Although the PCR products of regions 1 and 2, (region A), were obtained, the products remaining in all later regions (regions 3–8) were not. A similar phenomenon was also confirmed in the semi-quantification of the mRNA. Though we were able to quantify the mRNA by using both exon 1–2 and 5–6 boundary amplifications, we were not able to quantify the mRNA using the 14–15 and 20–21 boundaries. These results suggested that the quantity of the mRNA decrease remarkably in the vicinity of exon 8 as a boundary. Further analysis of the mRNA showed that quantity of the mRNA is normal from exon 1 through 9. Nucleotide sequencing of intron 9 revealed a single nucleotide substitution, adenine to guanine transition, at 602bp downstream from the 3' end of exon 9. This transition has not been registered in any international database as a mutation or a polymorphism and was not found in the F8 from 124 Japanese. These results strongly suggest that the transition is very rare and may be involved in factor VIII deficiency in these patients. Analysis of the nucleotide sequence of the substitution by splicing site prediction software predicted the formation of a new acceptor splice site. This result suggested the existence of splice abnormality. However, further characterization is needed to elucidate the mechanism that causes the decrease in mRNA in the middle of the gene. Conclusion: The mechanism behind factor VIII deficiency in hemophilia A patients with undetectable mutations is very interesting and various possibilities are conceivable. This study provides the possibility that some causative genetic abnormality remains in a further unanalyzed F8 region, most likely deep inside the intron, of these patients. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4924-4924
Author(s):  
Min Zhou ◽  
Xiaobo Luo ◽  
Qing Zhang ◽  
Xiaolan Yang ◽  
Xiaojing Li ◽  
...  

Background: The concept of developmental hemostasis has been universally accepted. Plasma concentrations of many coagulation factors in childhood are significantly different from adults for both mean values and ranges of normal. Thus, an understanding of developmental hemostasis and the development of appropriate age-dependent reference ranges are pivotal for prevention, diagnosis, and treatment of hemostatic problems during childhood. However, no data of developmental hemostasis are available in healthy Chinese children. Methods: Coagulation data from children aged 0-18 years old receiving minor elective surgery in Chengdu Women and Children's Central Hospital, from Sep. 2017 to Feb. 2019 was collected, and patient medical records were reviewed. Ethical approval was obtained from the parents of all children and the study was approved by the Ethics Review Committee at Chengdu Women and Children's Central Hospital. To qualify for the study, enrolled subjects had to meet the following criteria: (1) Children aged 0-18 years receiving minor elective surgery.(2) No history of bleeding problems.(3)No family history of bleeding nor thrombosis .(4) No history of heart, lung, liver or kidney diseases, normal physical examination. (5) Normal blood routine test. Normal liver function and kidney function, (6)No history of medication use for at least two weeks prior to specimen collection.(7) The operation gone successfully, and postoperative recovery was well. Blood samples (3 mL) were obtained by venipuncture into plastic tube containing 3.2% (109 mmol/L) buffered sodium citrate (1 part citrate:9 parts blood). The specimens were centrifuged immediately at 2500 g for 15 min at room temperature to prepare platelet-poor plasma (PPP) for the preoperative coagulation screening tests, consisting of activated partial thromboplastin time (APTT), prothrombin time (PT)), international normalized ratio (INR), thrombin time (TT) and fibrinogen. Activity of coagulation factors VIII, IX, XI, XII,Ⅱ,Ⅴ,Ⅶ,Ⅹ were determined using one-stage clotting methods with respective factor-deficient plasma. Results: A total of 82 samples were collected, while only 67 met the enrollment criteria. Specimens with inappropriate ratio of blood to anticoagulant, hemolysis or abnormal values of coagulation tests (APTT, PT, TT, fibrinogen) were excluded from analysis. The total of 67 children (40 Male 27 female), with a median age of 2.0 years (range: 1month -14 years) were divided into three groups according to the age: <1y group 15 cases, 1-5y group 45 cases, and >6y group 7cases. (1) The values of APTT, PT, TT, and fibrinogen were (36.1±5.2) seconds, (11.1±0.85) seconds, (20.3±1.6) seconds, (2.4±0.89) g/L respectively. No significant differences were found between groups. (2) In all children ,the activities of factor VIII、IX、XI、XII、vWF、II、V、VII、X were(123.6±48.3)%、(75.9±16.9)%、(95.9±24.3)%、(43.7±16.3)%、(111.3±50.4)%、(90.2±14.0)%、(104.7±21.1)%、(81.6±19.1)%、(93.0±21.8)% respectively. Factor VIII and vWF activities were significantly higher than other factors, while factor XII activities were significantly lower than others. (3)Mean values of FII:C, FVIII:C, FIX:C, FXI:C were significantly lower in children below <1year old group than those in 1-5y group. No significant difference were seen in FVII:C, FV:C, FX:C, FXII:C, vWF:C among three groups. Conclusions: Coagulation test is just a simple and easy screening test. Coagulation factor activities changed dynamically with age during childhood, especially the FII:C, FVIII:C, FIX:C and FXI:C. Physiological reference ranges for coagulation factor activities in Chinese children of different ages should be established in order to evaluate the children with congenital or acquired bleeding diseases correctively. Table Disclosures No relevant conflicts of interest to declare.


2018 ◽  
Vol 2018 ◽  
pp. 1-3
Author(s):  
Manori Gamage ◽  
Sadeepa Weerasinghe ◽  
Mohamed Nasoor ◽  
A. M. P. W. Karunarathne ◽  
Sashi Praba Abeyrathne

Acquired hemophilia A (AHA) is a rare bleeding disorder due to acquired antibodies against coagulation factor VIII (FVIII). It is rare in children less than 16 years old, and the incidence is 0.45/million/year. An otherwise healthy, 12-year-old boy was admitted to the ward with a history of swelling of the right and left forearms, for 1 day duration. He did not have any history of trauma or bleeding disorder. He had prolonged APPTT level with very high antibody titer against factor VIII. His gene expression for factor VIII was found to be normal. He was managed with FEIBA and recombinant FVII activated complexes and prednisolone 1 m/kg/day regime to control bleeding. AHA is associated with several underlying pathologies such as pregnancy, autoimmune diseases, malignancy, medications and infections; however, up to 50% of reported cases are idiopathic. In contrast to congenital haemophilia A, in which haemarthrosis is the hallmark clinical presentation, patients with AHA mainly bleed in to the skin, muscles, and soft tissues. High mortality rate of more than 20% is either to retroperitoneal or intracranial bleeds. Diagnosis is confirmed on isolated prolongation of activated partial thromboplastin time which does not normalize after addition of normal plasma, reducing the factor VIII levels with evidence of FVIII inhibitor activity. They have normal prothrombin time and platelet functions. Management of AHA involves two aspects, namely, eradication of antibodies and maintaining effective haemostasis during a bleeding episode.


2018 ◽  
Vol 2018 ◽  
pp. 1-3 ◽  
Author(s):  
Tomohisa Kitamura ◽  
Tsuyoshi Sato ◽  
Eiji Ikami ◽  
Yosuke Fukushima ◽  
Tetsuya Yoda

Background. Acquired hemophilia A (AHA) is a rare disorder which results from the presence of autoantibodies against blood coagulation factor VIII. The initial diagnosis is based on the detection of an isolated prolongation of the activated partial thromboplastin time (aPTT) with negative personal and family history of bleeding disorder. Definitive diagnosis is the identification of reduced FVIII levels with evidence of FVIII neutralizing activity. Case report. We report a case of a 93-year-old female who was diagnosed as AHA after tooth extraction at her home clinic. Prolongation of aPTT and a reduction in factor VIII activity levels were observed with the presence of factor VIII inhibitor. AHA condition is mild. However, acute subdural hematoma of this patient occurred due to an unexpected accident in our hospital. Hematoma was gradually increased and the patient died 13 days after admission. Discussion. Although AHA is mild, intracranial bleeding is a life-threatening condition. We also should pay attention to the presence of AHA patients when we extract teeth.


Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 168-174 ◽  
Author(s):  
Richard D. Bagnall ◽  
Naushin Waseem ◽  
Peter M. Green ◽  
Francesco Giannelli

The messenger RNA (mRNA) from 5 of 69 patients with severe hemophilia A did not support amplification of complementary DNA containing the first few exons of the factor VIII (F8) gene but supported amplification of mRNA containing exon 1 ofF8 plus exons of the VBP1 gene. This chimeric mRNA signals an inversion breaking intron 1 of the F8 gene. Using an inversion patient, one deleted for F8 exons 1 to 6, and cosmids mapped 70 to 100 kb telomeric of the F8gene, this study shows that this break strictly affects a sequence (int1h-1) repeated (int1h-2) about 140 kb more telomerically, between the C6.1A andVBP1 genes. The 1041-base pair repeats differ at a single nucleotide (although int1h-2 also showed one polymorphism) and are in opposite orientation. The results demonstrate that they cause inversions by intrachromosome or intrachromatid homologous recombination. The genomic structure of the inversion region shows that transcription traverses intergenic spaces to produce the 2 chimeric mRNAs containing the F8 sequences and characteristic of the inversion. This observation prompts the suggestion that nature may use such extended transcription to test whether the addition of novel domains from neighboring genes creates desirable new genes. A rapid polymerase chain reaction test was developed for the inversion in both patients and carriers. This has identified 10 inversions, affectingF8 genes with 5 different haplotypes for the BclI, introns 13 and 22 VNTR polymorphism, among 209 unrelated families with severe hemophilia A. This indicates a prevalence of 4.8% and frequent recurrence of the inversion. This should result in absence ofF8, and one inversion patient is known to have inhibitors.


Author(s):  
Sanchuan Luo ◽  
Zhongxiang Li ◽  
Xin Dai ◽  
Rui Zhang ◽  
Zhibing Liang ◽  
...  

Hemophilia A (HA), a common bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII), has long been considered an attractive target for gene therapy studies. However, full-length F8 cDNA cannot be packaged efficiently by adeno-associated virus (AAV) vectors. As the second most prevalent mutation causing severe HA, F8 intron 1 inversion (Inv1) is caused by an intrachromosomal recombination, leaving the majority of F8 (exons 2–26) untranscribed. In theory, the truncated gene could be rescued by integrating a promoter and the coding sequence of exon 1. To test this strategy in vivo, we generated an HA mouse model by deleting the promoter region and exon 1 of F8. Donor DNA and CRISPR/SaCas9 were packaged into AAV vectors and injected into HA mice intravenously. After treatment, F8 expression was restored and activated partial thromboplastin time (aPTT) was shortened. We also compared two liver-specific promoters and two types of integrating donor vectors. When an active promoter was used, all of the treated mice survived the tail-clip challenge. This is the first report of an in vivo gene repair strategy with the potential to treat a recurrent mutation in HA patients.


2015 ◽  
Vol 5 (2S) ◽  
pp. 15-19
Author(s):  
Irene Ricca ◽  
Marisa Coggiola ◽  
Silvia Destefanis ◽  
Claudio Pascale

Acquired haemophilia A (AHA) is a rare disorder with a high mortality rate. It occurs due to autoantibodies against coagulation factor VIII (FVIII) which neutralise its procoagulant function resulting in severe bleeding. This disease may be associated with autoimmune diseases, malignancies, infections or medications and occurs most commonly in the elderly. Diagnosis is based on the isolated prolongation of aPTT which does not normalise after the addition of normal plasma along with reduced FVIII levels. Treatment involves eradication of antibodies and maintaining effective haemostasis during bleeding. We report a case of a 76-year-old patient with a history of haemorrhage with severe anaemia. The article describes difficulties and complexities of clinical and therapeutic management of the patient.


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