Role of Molecular Genetics in Hemophilia: From Diagnosis to Therapy

Abstract

Despite significant advancements, state-of-the-art care remains inaccessible to patients with hemophilia, especially those from developing countries. Thus, innovative approaches in the management of this condition are needed to improve their quality of life. In this context, genetic studies in hemophilia have contributed to the better understanding of its biology, the detection of carriers, and prenatal diagnosis, and even fostering newer therapeutic strategies. This article reviews the applications of molecular genetics in hemophilia, in general, and how such techniques can be useful for optimizing patient care, in particular.

Keywords

hemophilia - genetic diagnosis - phenotype - variation - inhibitor - gene therapy

• References

• 1 O’Mahoney B. Global haemophilia care challenge and opportunities: World Federation of Hemophilia. 2002
• 2 Gitschier J, Wood WI, Goralka TM , et al. Characterization of the human factor VIII gene. Nature 1984; 312 (5992) 326-330
• 3 Yoshitake S, Schach BG, Foster DC, Davie EW, Kurachi K. Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). Biochemistry 1985; 24 (14) 3736-3750
• 4 Drayna D, White R. The genetic linkage map of the human X chromosome. Science 1985; 230 (4727) 753-758
• 5 Gitschier JWW, Wood WI, Goralka TM , et al. Characterization of the human factor VIII gene. Nature 1984; 312 (5992) 326-330
• 6 Leuer M, Oldenburg J, Lavergne JM , et al. Somatic mosaicism in hemophilia A: a fairly common event. Am J Hum Genet 2001; 69 (1) 75-87
• 7 Peake IR, Lillicrap DP, Boulyjenkov V , et al. Haemophilia: strategies for carrier detection and prenatal diagnosis. Bull World Health Organ 1993; 71 (3-4) 429-458
• 8 Peyvandi F. Carrier detection and prenatal diagnosis of hemophilia in developing countries. Semin Thromb Hemost 2005; 31 (5) 544-554
• 9 Lalloz MR, Schwaab R, McVey JH, Michaelides K, Tuddenham EG. Haemophilia A diagnosis by simultaneous analysis of two variable dinucleotide tandem repeats within the factor VIII gene. Br J Haematol 1994; 86 (4) 804-809
• 10 Jayandharan G, Shaji RV, George B, Chandy M, Srivastava A. Informativeness of linkage analysis for genetic diagnosis of haemophilia A in India. Haemophilia 2004; 10 (5) 553-559
• 11 de Carvalho FM, de Vargas Wolfgramm E, Paneto GG , et al. Analysis of factor VIII polymorphic markers as a means for carrier detection in Brazilian families with haemophilia A. Haemophilia 2007; 13 (4) 409-412
• 12 Ranjan R, Biswas A, Kannan M, Meena A, Deka D, Saxena R. Prenatal diagnosis of haemophilia A by chorionic villus sampling and cordocentesis: all India Institute of Medical Science experience. Vox Sang 2007; 92 (1) 79-84
• 13 Fang Y, Wang XF, Dai J, Wang HL. A rapid multifluorescent polymerase chain reaction for genetic counselling in Chinese haemophilia A families. Haemophilia 2006; 12 (1) 62-67
• 14 Liu Q, Nozari G, Sommer SS. Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in hemophilia A. Blood 1998; 92 (4) 1458-1459
• 15 Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood 2002; 99 (1) 168-174
• 16 Bowen DJ. Haemophilia A and haemophilia B: molecular insights. Mol Pathol 2002; 55 (2) 127-144
• 17 Peyvandi F, Jayandharan G, Chandy M , et al. Genetic diagnosis of haemophilia and other inherited bleeding disorders. Haemophilia 2006; 12 (Suppl 3) 82-89
• 18 Jeffreys AJ, Royle NJ, Wilson V, Wong Z. Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA. Nature 1988; 332 (6161) 278-281
• 19 Williams IJ, Abuzenadah A, Winship PR , et al. Precise carrier diagnosis in families with haemophilia A: use of conformation sensitive gel electrophoresis for mutation screening and polymorphism analysis. Thromb Haemost 1998; 79 (4) 723-726
• 20 Pieneman WC, Deutz-Terlouw PP, Reitsma PH, Briët E. Screening for mutations in haemophilia A patients by multiplex PCR-SSCP, Southern blotting and RNA analysis: the detection of a genetic abnormality in the factor VIII gene in 30 out of 35 patients. Br J Haematol 1995; 90 (2) 442-449
• 21 Kogan S, Gitschier J. Mutations and a polymorphism in the factor VIII gene discovered by denaturing gradient gel electrophoresis. Proc Natl Acad Sci U S A 1990; 87 (6) 2092-2096
• 22 Herbert O, Trossaërt M, Boisseau P, Fressinaud E, Gerson F. Evaluation of denaturing high-performance liquid chromatography (DHPLC) in the screening of mutations in hemophilia B patients. J Thromb Haemost 2004; 2 (12) 2267-2269
• 23 Jayandharan G, Shaji RV, Chandy M, Srivastava A. Identification of factor IX gene defects using a multiplex PCR and CSGE strategy-a first report. J Thromb Haemost 2003; 1 (9) 2051-2054
• 24 Jayandharan G, Shaji RV, Baidya S, Nair SC, Chandy M, Srivastava A. Identification of factor VIII gene mutations in 101 patients with haemophilia A: mutation analysis by inversion screening and multiplex PCR and CSGE and molecular modelling of 10 novel missense substitutions. Haemophilia 2005; 11 (5) 481-491
• 25 Jayandharan G, Nelson EJ, Baidya S, Chandy M, Srivastava A. A new multiplex PCR and conformation-sensitive gel electrophoresis strategy for mutation detection in the platelet glycoprotein alphaIIb and beta3 genes. J Thromb Haemost 2007; 5 (1) 206-209
• 26 Silva Pinto C, Fidalgo T, Salvado R , et al. Molecular diagnosis of haemophilia A at Centro Hospitalar de Coimbra in Portugal: study of 103 families - 15 new mutations. Haemophilia 2012; 18 (1) 129-138
• 27 Oldenburg J, El-Maarri O. New insight into the molecular basis of hemophilia A. Int J Hematol 2006; 83 (2) 96-102
• 28 Goodeve AC. Another step towards understanding hemophilia A molecular pathogenesis. J Thromb Haemost 2010; 8 (12) 2693-2695
• 29 Tizzano EF, Venceslá A, Baena M , et al. First report of two independent point factorVIII mutations in a family with haemophilia a: a word of caution for carrier diagnosis. Thromb Haemost 2005; 94 (3) 675-677
• 30 Ogata K, Selvaraj SR, Miao HZ, Pipe SW. Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe hemophilia A: implications for genotyping. J Thromb Haemost 2011; 9 (6) 1183-1190
• 31 Berber E, Leggo J, Brown C , et al. DNA microarray analysis for the detection of mutations in hemophilia A. J Thromb Haemost 2006; 4 (8) 1756-1762
• 32 Chan K, Sasanakul W, Mellars G , et al. Detection of known haemophilia B mutations and carrier testing by microarray. Thromb Haemost 2005; 94 (4) 872-878
• 33 Tizzano EF, Barceló MJ, Baena M , et al. Rapid identification of female haemophilia A carriers with deletions in the factor VIII gene by quantitative real-time PCR analysis. Thromb Haemost 2005; 94 (3) 661-664
• 34 Favaloro EJ. Learning from peer assessment: the role of the external quality assurance multilaboratory thrombophilia test process. Semin Thromb Hemost 2005; 31 (1) 85-89
• 35 Perry DJ, Goodeve A, Hill M, Jennings I, Kitchen S, Walker I ; UK NEQAS for Blood Coagulation. The UK National External Quality Assessment Scheme (UK NEQAS) for molecular genetic testing in haemophilia. Thromb Haemost 2006; 96 (5) 597-601
• 36 Preston FE, Kitchen S, Jennings I, Woods TA. A UK National External Quality Assessment scheme (UK Neqas) for molecular genetic testing for the diagnosis of familial thrombophilia. Thromb Haemost 1999; 82 (5) 1556-1557
• 37 Favaloro EJ, Bonar R, Sioufi J , et al; RCPA QAP in Haematology. Multilaboratory testing of thrombophilia: current and past practice in Australasia as assessed through the Royal College of Pathologists of Australasia Quality Assurance Program for Hematology. Semin Thromb Hemost 2005; 31 (1) 49-58
• 38 College of American Pathologists Proficiency Testing Program. http://www.cap.org/apps/cap.portal?_nfpb=true&_pageLabel=home . Accessed on January 2, 2012
• 39 Preston FE, Lippi G, Favaloro EJ, Jayandharan GR, Edison ES, Srivastava A. Quality issues in laboratory haemostasis. Haemophilia 2010; 16 (Suppl 5) 93-99
• 40 Schramm W, Royal S, Kroner B , et al; for the European haemophilia economic study group. Clinical outcomes and resource utilization associated with haemophilia care in Europe. Haemophilia 2002; 8 (1) 33-43
• 41 Molho P, Rolland N, Lebrun T , et al. Epidemiological survey of the orthopaedic status of severe haemophilia A and B patients in France. The French Study Group. [e-mail]. Haemophilia 2000; 6 (1) 23-32
• 42 Aledort LM, Haschmeyer RH, Pettersson H ; The Orthopaedic Outcome Study Group. A longitudinal study of orthopaedic outcomes for severe factor-VIII-deficient haemophiliacs. J Intern Med 1994; 236 (4) 391-399
• 43 Bolton-Maggs PH, Pasi KJ. Haemophilias A and B. Lancet 2003; 361 (9371) 1801-1809
• 44 Pollmann H, Richter H, Ringkamp H, Jürgens H. When are children diagnosed as having severe haemophilia and when do they start to bleed? A 10-year single-centre PUP study. Eur J Pediatr 1999; 158 (Suppl 3) S166-S170
• 45 Ramgren O. Haemophilia in Sweden. III. Symptomatology, with special reference to differences between haemophilia A and B. Acta Med Scand 1962; 171: 237-242
• 46 Rainsford SG, Hall A. A three-year study of adolescent boys suffering from haemophilia and allied disorders. Br J Haematol 1973; 24 (5) 539-551
• 47 Blanchette P, Rivard G, Israels S , et al; Association of Hemophilia Clinic Directors of Canada and Canadian Association of Nurses in Hemophilia Care. A survey of factor prophylaxis in the Canadian haemophilia A population. Haemophilia 2004; 10 (6) 679-683
• 48 Beltrán-Miranda CP, Khan A, Jaloma-Cruz AR, Laffan MA. Thrombin generation and phenotypic correlation in haemophilia A. Haemophilia 2005; 11 (4) 326-334
• 49 Shima M, Matsumoto T, Fukuda K , et al. The utility of activated partial thromboplastin time (aPTT) clot waveform analysis in the investigation of hemophilia A patients with very low levels of factor VIII activity (FVIII:C). Thromb Haemost 2002; 87 (3) 436-441
• 50 van Dijk K, van der Bom JG, Lenting PJ , et al. Factor VIII half-life and clinical phenotype of severe hemophilia A. Haematologica 2005; 90 (4) 494-498
• 51 Arbini AA, Mannucci PM, Bauer KA. Low prevalence of the factor V Leiden mutation among “severe” hemophiliacs with a “milder” bleeding diathesis. Thromb Haemost 1995; 74 (5) 1255-1258
• 52 Escuriola Ettingshausen C, Halimeh S, Kurnik K , et al. Symptomatic onset of severe hemophilia A in childhood is dependent on the presence of prothrombotic risk factors. Thromb Haemost 2001; 85 (2) 218-220
• 53 Ghosh K, Shetty S, Mohanty D. Milder clinical presentation of haemophilia A with severe deficiency of factor VIII as measured by one-stage assay. Haemophilia 2001; 7 (1) 9-12
• 54 van Dijk K, van der Bom JG, Fischer K, Grobbee DE, van den Berg HM. Do prothrombotic factors influence clinical phenotype of severe haemophilia? A review of the literature. Thromb Haemost 2004; 92 (2) 305-310
• 55 Jayandharan GR, Srivastava A. The phenotypic heterogeneity of severe hemophilia. Semin Thromb Hemost 2008; 34 (1) 128-141
• 56 Jayandharan GR, Nair SC, Poonnoose PM , et al. Polymorphism in factor VII gene modifies phenotype of severe haemophilia. Haemophilia 2009; 15 (6) 1228-1236
• 57 Jayandharan GR, Chapla A, Nair SC , et al. A polymorphism in Interferon gamma gene impacts the extent of joint damage in patients with severe hemophilia. Blood 2010; 110: A546 (Suppl.) Abstract 546
• 58 Franchini M, Mannucci PM. Inhibitors of propagation of coagulation (factors VIII, IX and XI): a review of current therapeutic practice. Br J Clin Pharmacol 2011; 72 (4) 553-562
• 59 Ehrenforth S, Kreuz W, Scharrer I , et al. Incidence of development of factor VIII and factor IX inhibitors in haemophiliacs. Lancet 1992; 339 (8793) 594-598
• 60 Goodeve AC, Peake IR. The molecular basis of hemophilia A: genotype-phenotype relationships and inhibitor development. Semin Thromb Hemost 2003; 29 (1) 23-30
• 61 High KA. Factor IX: molecular structure, epitopes, and mutations associated with inhibitor formation. Adv Exp Med Biol 1995; 386: 79-86
• 62 Chambost H. Assessing risk factors: prevention of inhibitors in haemophilia. Haemophilia 2010; 16 (Suppl 2) 10-15
• 63 Oldenburg J, Schröder J, Brackmann HH, Müller-Reible C, Schwaab R, Tuddenham E. Environmental and genetic factors influencing inhibitor development. Semin Hematol 2004; ; 41 (1) (Suppl 1) 82-88
• 64 Fakharzadeh SS, Kazazian Jr HH. Correlation between factor VIII genotype and inhibitor development in hemophilia A. Semin Thromb Hemost 2000; 26 (2) 167-171
• 65 Fay PJ, Jenkins PV. Mutating factor VIII: lessons from structure to function. Blood Rev 2005; 19 (1) 15-27
• 66 Graw J, Brackmann HH, Oldenburg J, Schneppenheim R, Spannagl M, Schwaab R. Haemophilia A: from mutation analysis to new therapies. Nat Rev Genet 2005; 6 (6) 488-501
• 67 Green PM, Montandon AJ, Bentley DR, Giannelli F. Genetics and molecular biology of haemophilias A and B. Blood Coagul Fibrinolysis 1991; 2 (4) 539-565
• 68 Astermark J, Berntorp E, White GC, Kroner BL ; MIBS Study Group. The Malmö International Brother Study (MIBS): further support for genetic predisposition to inhibitor development in hemophilia patients. Haemophilia 2001; 7 (3) 267-272
• 69 Astermark J, Oldenburg J, Pavlova A, Berntorp E, Lefvert AK ; MIBS Study Group. Polymorphisms in the IL10 but not in the IL1beta and IL4 genes are associated with inhibitor development in patients with hemophilia A. Blood 2006; 107 (8) 3167-3172
• 70 Viel KR, Ameri A, Abshire TC , et al. Inhibitors of factor VIII in black patients with hemophilia. N Engl J Med 2009; 360 (16) 1618-1627
• 71 Astermark J, Wang X, Oldenburg J, Berntorp E, Lefvert AK ; MIBS Study Group. Polymorphisms in the CTLA-4 gene and inhibitor development in patients with severe hemophilia A. J Thromb Haemost 2007; 5 (2) 263-265
• 72 Astermark J, Oldenburg J, Carlson J , et al. Polymorphisms in the TNFA gene and the risk of inhibitor development in patients with hemophilia A. Blood 2006; 108 (12) 3739-3745
• 73 White II GC, Kempton CL, Grimsley A, Nielsen B, Roberts HR. Cellular immune responses in hemophilia: why do inhibitors develop in some, but not all hemophiliacs?. J Thromb Haemost 2005; 3 (8) 1676-1681
• 74 Key NS. Inhibitors in congenital coagulation disorders. Br J Haematol 2004; 127 (4) 379-391
• 75 Goodeve A. The incidence of inhibitor development according to specific mutations—and treatment?. Blood Coagul Fibrinolysis 2003; 14 (Suppl 1) S17-S21
• 76 High KA. The Jeremiah Metzger Lecture: gene therapy for inherited disorders: from Christmas disease to Leber’s amaurosis. Trans Am Clin Climatol Assoc 2009; 120: 331-359
• 77 Donahue RE, Kessler SW, Bodine D , et al. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med 1992; 176 (4) 1125-1135
• 78 Hacein-Bey-Abina S, Von Kalle C, Schmidt M , et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302 (5644) 415-419
• 79 Kohn DB, Sadelain M, Dunbar C , et al; American Society of Gene Therapy (ASGT). American Society of Gene Therapy (ASGT) ad hoc subcommittee on retroviral-mediated gene transfer to hematopoietic stem cells. Mol Ther 2003; 8 (2) 180-187
• 80 Blacklow N. Parvoviruses and Human Disease. Boca Raton: : CRC Press;; 1988
• 81 Daya S, Berns KI. Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev 2008; 21 (4) 583-593
• 82 Flotte TR. Recent developments in recombinant AAV-mediated gene therapy for lung diseases. Curr Gene Ther 2005; 5 (3) 361-366
• 83 Kaplitt MG, Feigin A, Tang C , et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet 2007; 369 (9579) 2097-2105
• 84 Bainbridge JW, Smith AJ, Barker SS , et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med 2008; 358 (21) 2231-2239
• 85 Maguire AM, Simonelli F, Pierce EA , et al. Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med 2008; 358 (21) 2240-2248
• 86 Wang L, Herzog RW. AAV-mediated gene transfer for treatment of hemophilia. Curr Gene Ther 2005; 5 (3) 349-360
• 87 Gnatenko DV, Saenko EL, Jesty J, Cao LX, Hearing P, Bahou WF. Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy. Br J Haematol 1999; 104 (1) 27-36
• 88 Sarkar R, Xiao W, Kazazian Jr HH. A single adeno-associated virus (AAV)-murine factor VIII vector partially corrects the hemophilia A phenotype. J Thromb Haemost 2003; 1 (2) 220-226
• 89 Walsh CE, Chao H. Parvovirus-mediated gene transfer for the haemophilias. Haemophilia 2002; 8 (Suppl 2) 60-67
• 90 Sarkar R, Tetreault R, Gao G , et al. Total correction of hemophilia A mice with canine FVIII using an AAV 8 serotype. Blood 2004; 103 (4) 1253-1260
• 91 Jiang H, Lillicrap D, Patarroyo-White S , et al. Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs. Blood 2006; 108 (1) 107-115
• 92 Jayandharan GR, Zhong L, Li B, Kachniarz B, Srivastava A. Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo. Gene Ther 2008; 15 (18) 1287-1293
• 93 Jayandharan GR, Zhong L, Sack BK , et al. Optimized adeno-associated virus (AAV)-protein phosphatase-5 helper viruses for efficient liver transduction by single-stranded AAV vectors: therapeutic expression of factor IX at reduced vector doses. Hum Gene Ther 2010; 21 (3) 271-283
• 94 Monahan PE, Lothrop CD, Sun J , et al. Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application. Mol Ther 2010; 18 (11) 1907-1916
• 95 Sabatino DE, Lange AM, Altynova ES , et al. Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors. Mol Ther 2011; 19 (3) 442-449
• 96 Mingozzi F, High KA. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet 2011; 12 (5) 341-355
• 97 Herzog RW, Hagstrom JN, Kung SH , et al. Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Natl Acad Sci U S A 1997; 94 (11) 5804-5809
• 98 Snyder RO, Miao CH, Patijn GA , et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat Genet 1997; 16 (3) 270-276
• 99 Nathwani AC, Gray JT, McIntosh J , et al. Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates. Blood 2007; 109 (4) 1414-1421
• 100 Nathwani AC, Gray JT, Ng CY , et al. Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood 2006; 107 (7) 2653-2661
• 101 Wang L, Takabe K, Bidlingmaier SM, Ill CR, Verma IM. Sustained correction of bleeding disorder in hemophilia B mice by gene therapy. Proc Natl Acad Sci U S A 1999; 96 (7) 3906-3910
• 102 Monahan PE, Samulski RJ, Tazelaar J , et al. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia. Gene Ther 1998; 5 (1) 40-49
• 103 Niemeyer GP, Herzog RW, Mount J , et al. Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy. Blood 2009; 113 (4) 797-806
• 104 Snyder RO, Miao C, Meuse L , et al. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat Med 1999; 5 (1) 64-70
• 105 Manno CS, Chew AJ, Hutchison S , et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 2003; 101 (8) 2963-2972
• 106 Kay MA, Manno CS, Ragni MV , et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 2000; 24 (3) 257-261
• 107 Manno CS, Pierce GF, Arruda VR , et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006; 12 (3) 342-347
• 108 Mingozzi F, Maus MV, Hui DJ , et al. CD8( + ) T-cell responses to adeno-associated virus capsid in humans. Nat Med 2007; 13 (4) 419-422
• 109 Mingozzi F, High KA. Immune responses to AAV in clinical trials. Curr Gene Ther 2007; 7 (5) 316-324
• 110 Zaiss AK, Muruve DA. Immunity to adeno-associated virus vectors in animals and humans: a continued challenge. Gene Ther 2008; 15 (11) 808-816
• 111 Basner-Tschakarjan E, Mingozzi F, Chen Y, Nathwani AC, Tuddenham EGD, Rosales C, McIntosh MF, Riddell A, Rustagi P, Glader B, Kay MA, Allay J, Coleman J, Sleep S, Gray J, Reiss U, Nienhuis AW, Davidoff AM, High KA. Dose-dependent activation of capsid-specific T cells after AAV serotype 8 vector adminstration in a clinical study for hemophilia B. Molecular Therapy 2011; ;19, Suppl 1:Abstract 602.
• 112 Mingozzi F, Kleefstra A, Meulenberg J , et al. Modulation of T cell response to the AAV capsid in subjects undergoing intramuscular gene transfer for lipoprotein lipase deficiency. Hum Gene Ther 2008; 19 (10) 1090
• 113 Zhong L, Li B, Mah CS , et al. Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci U S A 2008; 105 (22) 7827-7832
• 114 Markusic DM, Herzog RW, Aslanidi GV , et al. High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines. Mol Ther 2010; 18 (12) 2048-2056
• 115 Jayandharan GR, Aslanidi G, Martino AT , et al. Activation of the NF-κB pathway by AAV vectors and its implications in immune response and gene therapy. Proc Natl Acad Sci U S A 2011; 108 (9) 3743-3748
• 116 Nathwani AC, Cochrane M, McIntosh J , et al. Enhancing transduction of the liver by adeno-associated viral vectors. Gene Ther 2009; 16 (1) 60-69
• 117 Martino AT, Suzuki M, Markusic DM , et al. The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver. Blood 2011; 117 (24) 6459-6468
• 118 Ahmed R, Kannan M, Choudhry VP, Saxena R. Does the MTHFR 677T allele alter the clinical phenotype in severe haemophilia A?. Thromb Res 2003; 109 (1) 71-72
• 119 Barnes C, Blanchette V, Lillicrap D , et al. Different clinical phenotype in triplets with haemophilia A. Haemophilia 2007; 13 (2) 202-205
• 120 Biguzzi E, Mancuso P, Franchi F , et al. Circulating endothelial cells (CECs) and progenitors (CEPs) in severe haemophiliacs with different clinical phenotype. Br J Haematol 2009; 144 (5) 803-805
• 121 Cruz E, Porto G, Morais S, Campos M, de Sousa M. HFE mutations in the pathobiology of hemophilic arthropathy. Blood 2005; 105 (8) 3381-3382
• 122 Grünewald M, Siegemund A, Grünewald A, Konegan A, Koksch M, Griesshammer M. Paradoxical hyperfibrinolysis is associated with a more intensely haemorrhagic phenotype in severe congenital haemophilia. Haemophilia 2002; 8 (6) 768-775
• 123 Klintman J, Berntorp E, Astermark J ; MIBS Study Group. Thrombin generation in vitro in the presence of by-passing agents in siblings with severe haemophilia A. Haemophilia 2010; 16 (1) e210-e215
• 124 Lee DH, Walker IR, Teitel J , et al. Effect of the factor V Leiden mutation on the clinical expression of severe hemophilia A. Thromb Haemost 2000; 83 (3) 387-391
• 125 Nowak-Göttl U, Escuriola C, Kurnik K , et al. Haemophilia and thrombophilia. What do we learn about combined inheritance of both genetic variations?. Hamostaseologie 2003; 23 (1) 36-40
• 126 Ninivaggi M, Dargaud Y, van Oerle R, de Laat B, Hemker HC, Lindhout T. Thrombin generation assay using factor IXa as a trigger to quantify accurately factor VIII levels in haemophilia A. J Thromb Haemost 2011; 9 (8) 1549-1555
• 127 Petkova R, Chakarov S, Horvath A, Ganev V, Kremensky I. Coexistence of a common prothrombotic risk factor and hemophilia in the Bulgarian hemophilic population:genotype/phenotype correlations. Balkan J Med Genet 2001; 4 (3–4) 37-39
• 128 Santagostino E, Mancuso ME, Tripodi A , et al. Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile. J Thromb Haemost 2010; 8 (4) 737-743
• 129 Schulman S, Eelde A, Holmström M, Ståhlberg G, Odeberg J, Blombäck M. Validation of a composite score for clinical severity of hemophilia. J Thromb Haemost 2008; 6 (7) 1113-1121
• 130 Shetty S, Vora S, Kulkarni B , et al. Contribution of natural anticoagulant and fibrinolytic factors in modulating the clinical severity of haemophilia patients. Br J Haematol 2007; 138 (4) 541-544
• 131 Yee DL. Platelets as modifiers of clinical phenotype in hemophilia. Scientific World J 2006; 6: 661-668
• 132 van Dijk K, Fischer K, van der Bom JG, Grobbee DE, van den Berg HM. Variability in clinical phenotype of severe haemophilia: the role of the first joint bleed. Haemophilia 2005; 11 (5) 438-443
• 133 van Dijk K, van der Bom JG, Fischer K, de Groot PG, van den Berg HM. Phenotype of severe hemophilia A and plasma levels of risk factors for thrombosis. J Thromb Haemost 2007; 5 (5) 1062-1064
• 134 Nathwani AC, Tuddenham EG, Rangarajan S , et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 2011; 365 (25) 2357-2365