Haemophilia (2014), 20 (Suppl. 4), 50–53
DOI: 10.1111/hae.12424
REVIEW ARTICLE
Genomics of bleeding disorders A . C . G O O D E V E , * A . P A V L O V A † and J . O L D E N B U R G † *Sheffield Diagnostic Genetics Service, Sheffield Children’s NHS Foundation Trust and Haemostasis Research Group, Department of Cardiovascular Science, University of Sheffield, Sheffield, UK; and †Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany
Summary. Molecular genetic tools are widely applied in inherited bleeding disorders. New genes involved in haemorrhagic disorders have been identified by genome wide linkage analysis on families with a specific phenotype. LMNA1 or MCFD in combined FV/FVIIIdeficiency and VKORC1 in vitamin K coagulation factor deficiency type 2 are two examples. Identification of the causative gene mutation has become standard for most bleeding disorders. Knowledge of the causative mutation allows genetic counselling in affected families and most importantly adds to the pathophysiological
understanding of phenotypes. Haemophilia A represents a model as the F8 gene mutation predicts the risk of developing an inhibitor and more recently also the bleeding phenotype. In this review novel genetic diagnostic strategies for bleeding disorders are outlined and inhibitor formation is presented as an example for clinical relevant phenotype/genotype correlation studies.
Novel genetic diagnostic strategies for bleeding disorder genetic analysis
widely adopted. It has enabled identification of deletions and duplications where standard PCR (and DNA sequencing) cannot detect these exon dosage changes [6,7]. An alternative technique for analysing dosage uses array comparative genomic hybridization with a high probe density. Arrays can be custom-designed for a specific set of genes and probes included for exons and flanking intronic sequence for a panel of haemostatic genes. Array analysis has been used to detect large VWF deletions [8]. As more probes can be used in this technique than the typical single probe set per exon used for MLPA, its resolution for dosage change detection is higher, and deletions down to 12 bp have been detected [9]. Inclusion of probes in intronic regions provides the opportunity to more closely define mutation breakpoints. Next generation DNA sequencing (NGS) is becoming available in diagnostic laboratories and starting to be used for bleeding disorder genetic analysis. The technique enables parallel sequencing of many gene regions at once. It can be undertaken on a number of different scales ranging from single gene analysis, or a defined panel of disorders, e.g. known coagulation factors and platelet bleeding disorders [10]. At the other end of the scale, the whole exome (analysis of all exons of known protein coding genes) or whole genome can be sequenced. These latter analyses may be used where the cause of the disorder in a patient remains unclear from their phenotype and no likely
The inherited bleeding disorders include coagulation factor and platelet bleeding disorders. Genetic analysis for haemophilia A (HA), haemophilia B and von Willebrand disease (VWD) is routine in many diagnostic laboratories, but is less widespread for many of the rarer disorders [1–5]. Genotype–phenotype correlations have been possible by genetic analysis [4–5]. When genetic analysis is undertaken, the strategy is often similar; all exons, closely flanking intronic sequence plus 50 and 30 untranslated regions are PCR amplified and analysed using Sanger DNA sequencing, sometimes following mutation scanning to highlight candidate variants. This process identifies mutations in a good proportion of patients for most disorders. Within recent years, gene dosage analysis using multiplex ligation-dependent probe amplification (MLPA; MRC Holland, Amsterdam, Netherlands) has become available to search for large deletions and duplications within F8, F9 and VWF genes and has been Correspondence: Johannes Oldenburg, MD/PhD, Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Siegmund-Freud-Str. 25, 53105 Bonn, Germany. Tel.: +49 228 28715175; fax: +49 228 28714783; e-mail: [email protected] Accepted 5 March 2014 50
Keywords: bleeding disorders, genetics, haemophilia, inhibitor development
genomics,
© 2014 John Wiley & Sons Ltd
GENOMICS OF BLEEDING DISORDERS
‘candidate genes’ can be suggested. Either PCR amplification or sequence capture using hybridization can be used to prepare the NGS target sequence. Analysis of F8 and VWF has been reported using NGS. For VWF, individual exons were amplified and then sequenced [11], whereas for F8, all exons together with both inversions were analysed using molecular inversion probe sequence capture [12] and the entire gene locus has been amplified and analysed using PCR [13]. A panel may include 50–100 specific genes. For many patients with inherited bleeding disorders, the diagnosis would indicate only one or two genes relevant to investigate and the computer software enables interrogation of only those genes relevant to the symptoms and phenotype in that patient. However, having a single sequencing workflow for many genes followed by selective analysis of the relevant gene(s) can greatly streamline laboratory process. This has particularly utility where more than one gene is associated with a disorder, e.g. in Glanzmann thrombasthenia and FXIII deficiency, where two different genes require analysis per disorder. It is also useful where there is phenotypic overlap between disorders; for example, a patient presenting with ‘mild HA’ with no previous family history may be analysed for mutations in F8, but when none are found, VWF data could then be interrogated, enabling mutations resulting in 2N VWD to be identified without undertaking any further laboratory work. The technology has particular potential where several different genes may cause the same disorder, e.g. in Hermansky–Pudlack syndrome where nine different currently known genes may be responsible [14].
The genetic predictors of inhibitors In haemophilia patients, in whom the endogenous FVIII/FIX is either absent or functionally inactive, the allo-antibodies (inhibitors) are produced as part of the individual’s immune response to a foreign antigen following replacement therapy and cause neutralization of the coagulant activity of factor FVIIIFIX. Although the aetiology of inhibitor development is increasingly more figured out, still the question why inhibitors develop in only 25–30% of patients rather than in all patients with severe haemophilia is poorly understood. Identifying factors favouring inhibitor development would allow stratifying patients’s therapy by inhibitor risk and have a major clinical and economical impact. Certain genetic factors have been shown to play an important role in this complex process. The most widely acknowledged risk factor is the type of haemophilia-causing mutation. The risk is associated with the severity of the disease, and the highest incidence (25–30% FVIII and 3–5% FIX) occurs in those patients with the severe form. Those mutations that result in the absence or severe © 2014 John Wiley & Sons Ltd
51
truncation of circulating proteins (null mutations) are associated with the highest risk. Although the reported absolute and relative risk of different mutation types vary between the studies it is well proved that the mutations with the highest inhibitor incidence are the large deletion, with prevalence ranges between 42% and 74%. These patients are not only at the highest risk of developing inhibitors (OR 3.57) but furthermore most of inhibitors are high-titter (OR 5.16) [15]. In all other null mutations (intron 22/1 inversions, nonsense and splice site mutations, small deletions and insertions outside sequences of adenine repeats (A-runs) the inhibitor incidence spread in a window between 14% and 36% [16,17]. Missense mutations, small insertions/deletions within A-runs and non-conserved splice site mutations are considered to be lowrisk mutations with an average frequency of inhibitors below 5% [18]. Inhibitor development is less frequently observed in patients with non-severe HA, generally caused by missense mutations. Nineteen missense mutations associated with inhibitor development were identified, suggesting that these single amino acid variants exhibit a higher immunogenicity. Position, type of substitution, physicochemical class of the affected amino acid may influence the inhibitor risk. The mutations associated with inhibitors are mainly located within the regions encoding for the light chain and the A2 domain of the F8 [19,20]. Several studies indicate that the immune response triggered by the presence of exogenous FVIII is a T helper cell-mediated event. The start of the antibody production process involves the processing of proteins by antigen-presenting cells and subsequent association of these peptides to HLA molecules [21,22]. The majority of associations with inhibitor production are related to HLA class II alleles: HLA-DRB1*14, DRB1*15, HLA-DQB1*06:02, DQB1*06:03. A positive association of the DRB1*15:01/DQB1*06:02 haplotype and inhibitor prevalence was reported in severe haemophilia patients. On the contrary DRB1*16 and DQB1*05:02 alleles were found to lower inhibitor risk [23–26]. The weak association of HLA types with inhibitor development suggests that the ability of a patient’s MHC class II to present one or more FVIIIderived peptides is a necessary but not sufficient condition to stimulate helper T cells and produce neutralizing antibodies. In attempts to find new markers allowing a stratification of the risk patients to develop inhibitors, singlenucleotide polymorphisms (SNPs) in the regulatory regions of cytokine genes have been studied. Certain polymorphisms, mainly localized in the promoter regions, in the exons or in microsatellites of intron regions can affect the transcription and influence the production of cytokines and subsequently modify the profile of the immune response. Genetic polymorphisms in immune-response associated genes, i.e. IL1b, IL4, Haemophilia (2014), 20 (Suppl. 4), 50--53
52
A. C. GOODEVE et al.
IL10, TNF-a and CTLA4, have been analysed. The association between the 308A/A genotype in TNF-a gene and the formation of inhibitors was evident in several studies. For the cytogene IL10, the 1082G allele and 134 bp allele of a ‘CA’ dinucleotide repeat microsatellite in the promoter region of the IL10 gene were found to be more common in patients with inhibitors patients. A clear predominance of the high-producer GCC haplotype (0.55 vs. 0.32) and a lower frequency of the low-producer ACC haplotype (0.20 vs. 0.32; P = 0.002) was observed in patients with inhibitors [26–28]. Furthermore, several new candidates as potentially predictors for inhibitor development (CD44, CSF1R, DOCK2, MAPK9 and IQGAP2) have been identified in Haemophilia Inhibitor Genetic Study [29]. Ethnicity and family history have been shown to predispose for the development of FVIII inhibitors. The incidence of inhibitors is high in the subgroup of patients of African descent when compared with Caucasians (55.6% vs. 27.4%). As the F8 mutation spectrum does not differ between races this difference might be based on ethnic-specific genetic variants in immune response determinants. Another hypothesis is related to ethnic-specific F8 gene variants. Four common, non-synonymous SNPs within the F8 have been identified, which occur as six haplotypes in the human population (H1–H6). Three of these haplotypes (H3, H4 and H5) have been associated with an increased risk of inhibitor development and were detected mainly in black people [30]. The risk for the formation of inhibitors increases significantly in patients with a family history of inhibitors, where the absolute
References 1 Nichols WC, Seligsohn U, Zivelin A et al. Mutations in the ER-Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII. Cell 1998; 93: 61–70. 2 Zhang B, McGee B, Yamaoka JS et al. Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2. Blood 2006; 107: 1903–7. 3 Rost S, Fregin A, Ivaskevicius V et al. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 2004; 427: 537–41. 4 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: 737–43. 5 Carcao MD, van den Berg HM, Ljung R, Mancuso ME; PedNet and the Rodin Study Group. Correlation between phenotype and genotype in a large unselected cohort of children with severe hemophilia A. Blood 2013; 121: 3946–52. 6 Rafati M, Ravanbod S, Hoseini A et al. Identification of ten large deletions and one duplication in the F8 gene of eleven
Haemophilia (2014), 20 (Suppl. 4), 50--53
7
8
9
10
11
risk in such patients is determined to be 48%, whereas the risk in patients with no family history only 15%. As inhibitors affect the treatment of haemophilia patients, increase the cost, mortality and morbidity of patients, it is of importance to identify those patients who exhibit a high risk to develop inhibitors. This would allow adjusting the treatment on an individual basis with the intention to reduce the risk of inhibitor formation. Potential strategies that have been identified are early low-dose prophylaxis and avoidance of intensified treatment periods [31]. There is general agreement that a major gene defect, a positive family history for inhibitor development and early intensive treatment are associated with a greater risk of inhibitor development. However, although a growing number of factors have been identified none of them alone is able to assess the risk for an individual patient. In conclusion, identifying the genetic markers as predictors for inhibitor may be used to assess clinical prediction scores and models for inhibitor development, which may be subjected to individualized treatment regimens that lower the risk of inhibitor formation.
Disclosures JO received reimbursement for attending symposia/congresses and/or honoraria for speaking and/or honoraria for consulting, and/or funds for research from Baxter, Bayer, Biogen Idec, Biotest, CSL Behring, Grifols, Novo Nordisk, Octapharma, Swedish Orphan Biovitrum and Pfizer. AP received reimbursement for attending symposia/congresses from Bayer, Octapharma, Novo Nordisk and honoraria for speaking from Octapharma and Novo Nordisk. AG has no relevant disclosures.
unrelated Iranian severe haemophilia A families using the multiplex ligation-dependent probe amplification technique. Haemophilia 2011; 17: 705–7. Yadegari H, Driesen J, Hass M, Budde U, Pavlova A, Oldenburg J. Large deletions identified in patients with von Willebrand disease using multiple ligation-dependent probe amplification. J Thromb Haemost 2011; 9: 1083–6. Bellissimo DB, Chin E, Hegde M, Christopherson PA, Haberichter SL, Montgomery RR. Detection of VWF copy number variations by microarray analysis in von Willebrand disease type 3 index cases. Blood (ASH Annual Meeting Abstracts) 2011; 118: 380. Askree SH, Chen ELH, Bean LH, Coffee B, Tanner T, Hedge M. Detection limit of intragenic deletions with targeted array comparative genomic hybridization. BMC Genet 2013; 14: 116. Watson SP, Lowe GC, Lordkipanidze M, Morgan NV; GAPP consortium. Genotyping and phenotyping of platelet function disorders. J Thromb Haemost 2013; 11 (Suppl. 1): 351–63. Corrales I, Catarino S, Ayats J et al. Highthroughput molecular diagnosis of von Willebrand disease by next generation
12
13
14
15
16
17
sequencing methods. Haematologica 2012; 97: 1003–7. Josephson NC, Martin B, Nakaya S et al. A next generation sequencing approach for genotyping patients with hemophilia. J Thromb Haemost 2013; 11: 85–289. Pezeshkpoor B, Zimmer N, Marquardt N et al. Deep intronic ‘mutations’ cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA. J Thromb Haemost 2013; 11: 1679–87. Dotta L, Parolini S, Prandini A et al. Clinical, laboratory and molecular signs of immunodeficiency in patients with partial oculo-cutaneous albinism. Orphanet J Rare Dis 2013; 8: 168. Gouw SC, van den Berg HM, Oldenburg J, et al. F8 gene mutation type and inhibitor development in patients with severe hemophilia A: systematic review and meta-analysis. Blood 2012; 22: 2922–34. Schwaab R, Brackmann HH, Meyer C et al. Haemophilia A: mutation type determines risk of inhibitor formation. Thromb Haemost 1995; 74: 1402–6. Oldenburg J, Pavlova A. Genetic risk factors for inhibitors to factor VIII and IX. Haemophilia 2006; 12(Suppl. 6): 15–22.
© 2014 John Wiley & Sons Ltd
GENOMICS OF BLEEDING DISORDERS 18 Oldenburg J, Schroder J, Schmitt C, Brackmann HH, Schwaab R. Small deletion/ insertion mutations within poly-A runs of the factor VIII gene mitigate the severe haemophilia A phenotype. Thromb Haemost 1998; 79: 452–3. 19 Eckhardt CL, van Velzen AS, Peters M et al. Factor VIII gene (F8) mutation and risk of inhibitor development in nonsevere hemophilia A. Blood 2013; 122: 1954–62. 20 Schwaab R, Pavlova A, Albert T, Caspers M, Oldenburg J. Significance of F8 missense mutations with respect to inhibitor formation. Thromb Haemost 2013; 109: 464–70. 21 van Helden PM, Kaijen PH, Fijnvandraat K, van den Berg M, Voorberg J. Factor VIII-specific memory B cells in patients with hemophilia A. J Thromb Haemost 2007; 5: 2306–8. 22 Reipert BM, Allacher P, Hausl C et al. Modulation of factor VIII-specific memory B cells. Haemophilia 2010; 16: 25–30. 23 Hay CR, Ollier W, Pepper L et al. HLA class II profile: a weak determinant of factor
© 2014 John Wiley & Sons Ltd
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25
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VIII inhibitor development in severe haemophilia A. UKHCDO Inhibitor Working Party. Thromb Haemost 1997; 77: 234–7. Oldenburg J, Picard JK, Schwaab R, Brackmann HH, Tuddenham EG, Simpson E. HLA genotype of patients with severe haemophilia A due to intron 22 inversion with and without inhibitors of factor VIII. Thromb Haemost 1997; 77: 238–42. Hay CR. The epidemiology of factor VIII inhibitors. Haemophilia 2006; 12(Suppl. 6): 23–8. Pavlova A, Delev D, Lacroix-Desmazes S et al. Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10, tumor necrosis factor-alpha and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe hemophilia A. J Thromb Haemost 2009; 7: 2006–15. Astermark J, Oldenburg J, Pavlova A, Berntorp E, Lefvert AK and MIBS Study Group. Polymorphisms in the IL10 but not in the IL1 beta and IL4 genes are associated with
28
29
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31
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inhibitor development in patients with hemophilia A. Blood 2006; 107: 3167–72. 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: 3739–45. Astermark J, Donfield SM, Gomperts ED et al. Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort. The polygenic nature of inhibitors in hemophilia A: results from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort. Blood 2013; 121: 1446–54. Viel KR, Ameri A, Abshire TC et al. Inhibitors of factor VIII in black patients with hemophilia. N Engl J Med 2009; 360: 1618–27. Kurnik K, Bidlingmaier C, Engl W, Chehadeh H, Reipert B, Auerswald G. New early prophylaxis regimen that avoids immunological danger signals can reduce FVIII inhibitor development. Haemophilia 2010; 16: 256–62.
Haemophilia (2014), 20 (Suppl. 4), 50--53
DOI: 10.1111/hae.12424
REVIEW ARTICLE
Genomics of bleeding disorders A . C . G O O D E V E , * A . P A V L O V A † and J . O L D E N B U R G † *Sheffield Diagnostic Genetics Service, Sheffield Children’s NHS Foundation Trust and Haemostasis Research Group, Department of Cardiovascular Science, University of Sheffield, Sheffield, UK; and †Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany
Summary. Molecular genetic tools are widely applied in inherited bleeding disorders. New genes involved in haemorrhagic disorders have been identified by genome wide linkage analysis on families with a specific phenotype. LMNA1 or MCFD in combined FV/FVIIIdeficiency and VKORC1 in vitamin K coagulation factor deficiency type 2 are two examples. Identification of the causative gene mutation has become standard for most bleeding disorders. Knowledge of the causative mutation allows genetic counselling in affected families and most importantly adds to the pathophysiological
understanding of phenotypes. Haemophilia A represents a model as the F8 gene mutation predicts the risk of developing an inhibitor and more recently also the bleeding phenotype. In this review novel genetic diagnostic strategies for bleeding disorders are outlined and inhibitor formation is presented as an example for clinical relevant phenotype/genotype correlation studies.
Novel genetic diagnostic strategies for bleeding disorder genetic analysis
widely adopted. It has enabled identification of deletions and duplications where standard PCR (and DNA sequencing) cannot detect these exon dosage changes [6,7]. An alternative technique for analysing dosage uses array comparative genomic hybridization with a high probe density. Arrays can be custom-designed for a specific set of genes and probes included for exons and flanking intronic sequence for a panel of haemostatic genes. Array analysis has been used to detect large VWF deletions [8]. As more probes can be used in this technique than the typical single probe set per exon used for MLPA, its resolution for dosage change detection is higher, and deletions down to 12 bp have been detected [9]. Inclusion of probes in intronic regions provides the opportunity to more closely define mutation breakpoints. Next generation DNA sequencing (NGS) is becoming available in diagnostic laboratories and starting to be used for bleeding disorder genetic analysis. The technique enables parallel sequencing of many gene regions at once. It can be undertaken on a number of different scales ranging from single gene analysis, or a defined panel of disorders, e.g. known coagulation factors and platelet bleeding disorders [10]. At the other end of the scale, the whole exome (analysis of all exons of known protein coding genes) or whole genome can be sequenced. These latter analyses may be used where the cause of the disorder in a patient remains unclear from their phenotype and no likely
The inherited bleeding disorders include coagulation factor and platelet bleeding disorders. Genetic analysis for haemophilia A (HA), haemophilia B and von Willebrand disease (VWD) is routine in many diagnostic laboratories, but is less widespread for many of the rarer disorders [1–5]. Genotype–phenotype correlations have been possible by genetic analysis [4–5]. When genetic analysis is undertaken, the strategy is often similar; all exons, closely flanking intronic sequence plus 50 and 30 untranslated regions are PCR amplified and analysed using Sanger DNA sequencing, sometimes following mutation scanning to highlight candidate variants. This process identifies mutations in a good proportion of patients for most disorders. Within recent years, gene dosage analysis using multiplex ligation-dependent probe amplification (MLPA; MRC Holland, Amsterdam, Netherlands) has become available to search for large deletions and duplications within F8, F9 and VWF genes and has been Correspondence: Johannes Oldenburg, MD/PhD, Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Siegmund-Freud-Str. 25, 53105 Bonn, Germany. Tel.: +49 228 28715175; fax: +49 228 28714783; e-mail: [email protected] Accepted 5 March 2014 50
Keywords: bleeding disorders, genetics, haemophilia, inhibitor development
genomics,
© 2014 John Wiley & Sons Ltd
GENOMICS OF BLEEDING DISORDERS
‘candidate genes’ can be suggested. Either PCR amplification or sequence capture using hybridization can be used to prepare the NGS target sequence. Analysis of F8 and VWF has been reported using NGS. For VWF, individual exons were amplified and then sequenced [11], whereas for F8, all exons together with both inversions were analysed using molecular inversion probe sequence capture [12] and the entire gene locus has been amplified and analysed using PCR [13]. A panel may include 50–100 specific genes. For many patients with inherited bleeding disorders, the diagnosis would indicate only one or two genes relevant to investigate and the computer software enables interrogation of only those genes relevant to the symptoms and phenotype in that patient. However, having a single sequencing workflow for many genes followed by selective analysis of the relevant gene(s) can greatly streamline laboratory process. This has particularly utility where more than one gene is associated with a disorder, e.g. in Glanzmann thrombasthenia and FXIII deficiency, where two different genes require analysis per disorder. It is also useful where there is phenotypic overlap between disorders; for example, a patient presenting with ‘mild HA’ with no previous family history may be analysed for mutations in F8, but when none are found, VWF data could then be interrogated, enabling mutations resulting in 2N VWD to be identified without undertaking any further laboratory work. The technology has particular potential where several different genes may cause the same disorder, e.g. in Hermansky–Pudlack syndrome where nine different currently known genes may be responsible [14].
The genetic predictors of inhibitors In haemophilia patients, in whom the endogenous FVIII/FIX is either absent or functionally inactive, the allo-antibodies (inhibitors) are produced as part of the individual’s immune response to a foreign antigen following replacement therapy and cause neutralization of the coagulant activity of factor FVIIIFIX. Although the aetiology of inhibitor development is increasingly more figured out, still the question why inhibitors develop in only 25–30% of patients rather than in all patients with severe haemophilia is poorly understood. Identifying factors favouring inhibitor development would allow stratifying patients’s therapy by inhibitor risk and have a major clinical and economical impact. Certain genetic factors have been shown to play an important role in this complex process. The most widely acknowledged risk factor is the type of haemophilia-causing mutation. The risk is associated with the severity of the disease, and the highest incidence (25–30% FVIII and 3–5% FIX) occurs in those patients with the severe form. Those mutations that result in the absence or severe © 2014 John Wiley & Sons Ltd
51
truncation of circulating proteins (null mutations) are associated with the highest risk. Although the reported absolute and relative risk of different mutation types vary between the studies it is well proved that the mutations with the highest inhibitor incidence are the large deletion, with prevalence ranges between 42% and 74%. These patients are not only at the highest risk of developing inhibitors (OR 3.57) but furthermore most of inhibitors are high-titter (OR 5.16) [15]. In all other null mutations (intron 22/1 inversions, nonsense and splice site mutations, small deletions and insertions outside sequences of adenine repeats (A-runs) the inhibitor incidence spread in a window between 14% and 36% [16,17]. Missense mutations, small insertions/deletions within A-runs and non-conserved splice site mutations are considered to be lowrisk mutations with an average frequency of inhibitors below 5% [18]. Inhibitor development is less frequently observed in patients with non-severe HA, generally caused by missense mutations. Nineteen missense mutations associated with inhibitor development were identified, suggesting that these single amino acid variants exhibit a higher immunogenicity. Position, type of substitution, physicochemical class of the affected amino acid may influence the inhibitor risk. The mutations associated with inhibitors are mainly located within the regions encoding for the light chain and the A2 domain of the F8 [19,20]. Several studies indicate that the immune response triggered by the presence of exogenous FVIII is a T helper cell-mediated event. The start of the antibody production process involves the processing of proteins by antigen-presenting cells and subsequent association of these peptides to HLA molecules [21,22]. The majority of associations with inhibitor production are related to HLA class II alleles: HLA-DRB1*14, DRB1*15, HLA-DQB1*06:02, DQB1*06:03. A positive association of the DRB1*15:01/DQB1*06:02 haplotype and inhibitor prevalence was reported in severe haemophilia patients. On the contrary DRB1*16 and DQB1*05:02 alleles were found to lower inhibitor risk [23–26]. The weak association of HLA types with inhibitor development suggests that the ability of a patient’s MHC class II to present one or more FVIIIderived peptides is a necessary but not sufficient condition to stimulate helper T cells and produce neutralizing antibodies. In attempts to find new markers allowing a stratification of the risk patients to develop inhibitors, singlenucleotide polymorphisms (SNPs) in the regulatory regions of cytokine genes have been studied. Certain polymorphisms, mainly localized in the promoter regions, in the exons or in microsatellites of intron regions can affect the transcription and influence the production of cytokines and subsequently modify the profile of the immune response. Genetic polymorphisms in immune-response associated genes, i.e. IL1b, IL4, Haemophilia (2014), 20 (Suppl. 4), 50--53
52
A. C. GOODEVE et al.
IL10, TNF-a and CTLA4, have been analysed. The association between the 308A/A genotype in TNF-a gene and the formation of inhibitors was evident in several studies. For the cytogene IL10, the 1082G allele and 134 bp allele of a ‘CA’ dinucleotide repeat microsatellite in the promoter region of the IL10 gene were found to be more common in patients with inhibitors patients. A clear predominance of the high-producer GCC haplotype (0.55 vs. 0.32) and a lower frequency of the low-producer ACC haplotype (0.20 vs. 0.32; P = 0.002) was observed in patients with inhibitors [26–28]. Furthermore, several new candidates as potentially predictors for inhibitor development (CD44, CSF1R, DOCK2, MAPK9 and IQGAP2) have been identified in Haemophilia Inhibitor Genetic Study [29]. Ethnicity and family history have been shown to predispose for the development of FVIII inhibitors. The incidence of inhibitors is high in the subgroup of patients of African descent when compared with Caucasians (55.6% vs. 27.4%). As the F8 mutation spectrum does not differ between races this difference might be based on ethnic-specific genetic variants in immune response determinants. Another hypothesis is related to ethnic-specific F8 gene variants. Four common, non-synonymous SNPs within the F8 have been identified, which occur as six haplotypes in the human population (H1–H6). Three of these haplotypes (H3, H4 and H5) have been associated with an increased risk of inhibitor development and were detected mainly in black people [30]. The risk for the formation of inhibitors increases significantly in patients with a family history of inhibitors, where the absolute
References 1 Nichols WC, Seligsohn U, Zivelin A et al. Mutations in the ER-Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII. Cell 1998; 93: 61–70. 2 Zhang B, McGee B, Yamaoka JS et al. Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2. Blood 2006; 107: 1903–7. 3 Rost S, Fregin A, Ivaskevicius V et al. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 2004; 427: 537–41. 4 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: 737–43. 5 Carcao MD, van den Berg HM, Ljung R, Mancuso ME; PedNet and the Rodin Study Group. Correlation between phenotype and genotype in a large unselected cohort of children with severe hemophilia A. Blood 2013; 121: 3946–52. 6 Rafati M, Ravanbod S, Hoseini A et al. Identification of ten large deletions and one duplication in the F8 gene of eleven
Haemophilia (2014), 20 (Suppl. 4), 50--53
7
8
9
10
11
risk in such patients is determined to be 48%, whereas the risk in patients with no family history only 15%. As inhibitors affect the treatment of haemophilia patients, increase the cost, mortality and morbidity of patients, it is of importance to identify those patients who exhibit a high risk to develop inhibitors. This would allow adjusting the treatment on an individual basis with the intention to reduce the risk of inhibitor formation. Potential strategies that have been identified are early low-dose prophylaxis and avoidance of intensified treatment periods [31]. There is general agreement that a major gene defect, a positive family history for inhibitor development and early intensive treatment are associated with a greater risk of inhibitor development. However, although a growing number of factors have been identified none of them alone is able to assess the risk for an individual patient. In conclusion, identifying the genetic markers as predictors for inhibitor may be used to assess clinical prediction scores and models for inhibitor development, which may be subjected to individualized treatment regimens that lower the risk of inhibitor formation.
Disclosures JO received reimbursement for attending symposia/congresses and/or honoraria for speaking and/or honoraria for consulting, and/or funds for research from Baxter, Bayer, Biogen Idec, Biotest, CSL Behring, Grifols, Novo Nordisk, Octapharma, Swedish Orphan Biovitrum and Pfizer. AP received reimbursement for attending symposia/congresses from Bayer, Octapharma, Novo Nordisk and honoraria for speaking from Octapharma and Novo Nordisk. AG has no relevant disclosures.
unrelated Iranian severe haemophilia A families using the multiplex ligation-dependent probe amplification technique. Haemophilia 2011; 17: 705–7. Yadegari H, Driesen J, Hass M, Budde U, Pavlova A, Oldenburg J. Large deletions identified in patients with von Willebrand disease using multiple ligation-dependent probe amplification. J Thromb Haemost 2011; 9: 1083–6. Bellissimo DB, Chin E, Hegde M, Christopherson PA, Haberichter SL, Montgomery RR. Detection of VWF copy number variations by microarray analysis in von Willebrand disease type 3 index cases. Blood (ASH Annual Meeting Abstracts) 2011; 118: 380. Askree SH, Chen ELH, Bean LH, Coffee B, Tanner T, Hedge M. Detection limit of intragenic deletions with targeted array comparative genomic hybridization. BMC Genet 2013; 14: 116. Watson SP, Lowe GC, Lordkipanidze M, Morgan NV; GAPP consortium. Genotyping and phenotyping of platelet function disorders. J Thromb Haemost 2013; 11 (Suppl. 1): 351–63. Corrales I, Catarino S, Ayats J et al. Highthroughput molecular diagnosis of von Willebrand disease by next generation
12
13
14
15
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