Haematologica 2000;85:supplement to no. 10 - Supplements ...

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Immune Tolerance and the Treatment of Hemophilacs with an Inhibitor 9 Table 1. Mutation type and inhibitor prevalence in severe hemophilia A according to Schwaab et al. 3 The proportion of inhibitor patients is given in absolute numbers in brackets and as relative percentage number (data from the hemophilia center Bonn and the hemophilia A mutation databases. 9 Point mutations Deletions Intron 22 Stop Missense Large Small inversions Pts. from Bonn 20 (7) 30 (2) 13 (3) 11 (1) 59 (25) 35.0 % 6.7% 23.1% 9.1% 42.4% IMD 53 (21) 39 (1) 57 (22) 16 (1) 66 (18) 39.6 % 2.6% 38.6% 6.3% 27.3% Total 73 (28) 69 (3) 70 (25) 27 (2) 125 (43) 38.4 % 4.3% 35.7% 7.4% 34.4% IMD: International mutation database. Table 3. Coagulation parameters of patients with small deletion/insertion mutations in exon 14 (upper part) and age-matched patients with standard (stop codon, intron 22 inversion) mutations according to Oldenburg et al. 12 Patients & mutation FVIII:C Thrombelastogram (U/mL) r (min) r+k (min) Group A HD25, Exon 14 DelACAC, Codon 1187/88 < 0.01 29 36 HD26, Exon 14, DelA, Codon 1192 < 0.01 26 39 HD26, Exon 14, DelA, Codon 1192 < 0.01 48 71 HD26, Exon 14, DelA, Codon 1192 < 0.01 26 35 Group B HP141, Exon 14, CGC(Arg)1689TGC(Stop) < 0.01 158 199 HP131, Exon 13, TAC(Tyr)636TAG(Stop) < 0.01 134 202 HP127, Exon 12, CGA(Arg)583TAG(Stop) < 0.01 175 224 HI36, Proximal Intron 22 inversion < 0.01 178 259 HI40, Distal Intron 22 inversion < 0.01 159 219 Table 2. Mutation type in patients with severe hemophilia A according to Becker et al. 2 Type of mutation Patients n=147 Intron 22 inversion 55 (37.4%) Point mutation 47 (32.0%) Stop codon 20 (13.6%) Missense 27 (18.4%) Large deletion 8 (5.4%) Small deletion 14 (9.5%) Insertion 2 (1.4%) Mutation not characterized 4 (2.7%) Mutation not identified 17 (11.6%) HIGH RISK 75% multi domain 0% Large deletions light chain Nonsense mutations single domain heavy chain Intron 22 inversions LOW RISK Non A-run C1-C2-junction Small Missense deletions mutations Non C1-C2-junction Splice site A-run mutations Figure 1. Mutation types and risks of inhibitor development. Main types of mutations are indicated by a grey background. Most of them show subgroups of mutations with different risks of inhibitor formation. investigated the phenotype of 5 hemophilia A patients in whom we had found a small deletion or insertion within or near another run of 9 adenines of the B-domain (codons 1191- 1194). 12 In all five patients the mutations were associated with a frame shift resulting in a nonsense codon shortly after the mutation. However, although FVIII activity was found to be below 1% – thus fulfilling the criteria of severe hemophilia A – the thrombelastogram parameters r- and r+k-time revealed significant residual clotting activity compared to that in patients with a nonsense mutation or intron 22 inversion (Table 3). These findings clearly point to the synthesis of some functional residual FVIII protein that mitigates the severe hemophilia A phenotype and may cause an almost complete protection against inhibitor development. Considering that the run of adenines at codons 1191-1194 represents a mutation hotspot responsible for one third of all deletion/insertion mutations within the FVIII gene, 8 our findings might explain the low risk of inhibitor formation found in association with the mutation type of small deletions/insertions. However, it should be noted that those small deletions/insertions that do not affect a series of adenines may produce a significantly higher risk for the development of inhibitors. Inhibitors in mild hemophilia A Recently, some very interesting results from studies of inhibitor patients with non-severe hemophilia A have been published. Fijnvandraat et al. 13 described a mild hemophilia A patient in whom FVIII antibodies were directed against sub- Haematologica vol. 85(supplement to n. 10):October 2000
10 J. Oldenburg et al. Table 4. Mutation type and relative risk of inhibitor formation. Mutation type Patients with Patients without Relative inhibitor n=28 inhibitor n=67 Risk Intron 22 inverson 18 (64.3%) 34 (50.7%) 1.7 Nonsense mutation 6 (21.4%) 7 (10.4%) 2.3* Missense mutation 1 (3.6%) 18 (26.9%) 0.1° Splice-mutation 0 (0.0%) 3 (4.5%) n.d. Small deletion 1 (3.6%) 4 (6.0%) 0.6 Large deletion 2 (7.1%) 1 (1.5%) 5.1 Not detected 5 (15.2%) 7 (9.5%) 1.7 *p < 0.05, ° p < 0.01, n.d. = not determined. stituted FVIII but not against the mutated endogenous FVIII protein. This led to the interesting phenomenon that DDAVP was able to induce a considerable increase of the FVIII activity while substituted FVIII did not. Investigating 26 patients with non-severe hemophilia A and inhibitor development, Hay et al. 14 found that 41% of the patients had hemophilic relatives with inhibitors. In 9 of 11 families in which the FVIII gene mutation was known, the mutation affects the C1/C2 junction (amino acids 2009 to 2229). Moreover, five patients out of three families with an Arg2229Cys mutation developed anti-FVIII antibodies. Suzuki et al. 15 found that a mutation in this region at amino acid 2153 modifies the antigenicity of the C2 domain. Jacquemin et al. 16 and Peerlinck et al. 17 described that the mutations Ile2098Ser, Asn2129Ser, Trp2229Cys, Gln2246Arg 16 and Arg2150His 17 are associated with normal function of the FVIII molecule except for reduced FVIII binding to vWF that causes the decrease of FVIII:C. These findings point to a critical role of this region in the pathogenesis of inhibitor formation. The recently discovered crystal structure of the C-domains of the FVIII molecule 18,19 will form the base for efficient evaluation of this important aspect. Table 5. Common (upper part) and rare (lower part) MHC class I and II alleles in severe hemophilia A patients with intron 22 inversion and inhibitor formation according to Oldenburg et al. 4 For the MHC class I loci A, B, C the number of individuals and for the MHC class II loci DR, DQA, DQB the number of chromosomes is given. Allele Patients with inhibitor Patients without inhibitor Relative No. % No. % risk Common MHC-alleles in patients with inhibitor A3 11 37.9 9 21.4 2.2 B7 14 48.3 8 19.1 4.0 C7 17 58.6 16 38.1 2.3 DQA0102 20 34.5 16 19.1 2.2 DQB0602 18 31.0 12 14.3 2.7 DR15 19 32.8 17 20.2 1.9 Rare MHC-alleles in patients with inhibitor C2 1 3.4 6 14.3 0.2 DQA0102 1 1.7 12 14.3 0.1 DQB0602 0 0 6 7.1 0.1 DR15 1 1.7 9 10.7 0.1 Mutation type and relative risk of inhibitor development At present only retrospective data and data from the hemophilia A mutation register (HAM- STeRS) 9 are available to correlate the inhibitor risk to the mutation type. These data may be biased to some extent. In the mutation register no information about the cumulative exposure days is given, thus some of these patients might still be at risk of inhibitor development. Moreover the mutation register provides no information about the characteristics of the inhibitor (permanent vs. transient, high titer vs low titer). Another problem is that in the mutation register the same mutation is associated with different phenotypes ranging from severe to mild hemophilia A. It is also possible that inhibitor patients have been analyzed with higher priority and therefore may be overrepresented in the mutation register. For these reasons we initiated a further study in a well defined and characterized cohort of patients. The criteria for entering the study were that the patient had severe hemophilia A, was registered and treated at the Bonn hemophilia center and had been born after the 1.1.1978 (because from this time onwards, computerassisted patient documentation allowed for complete registration of all patients). Genetic analysis has been completed in 107 patients. The mutation within the FVIII gene could be identified in 95 patients, comprising 28 patients with an inhibitor and 67 patients without an inhibitor. In 12 patients we were not able to find the mutation. Interestingly, 5 of these 12 patients had developed an inhibitor, thus pointing to the fact that mutations not detected by our screening strategy may represent genetic defects associated with a higher risk of inhibitor formation. The strong selection criteria of the patients allowed the relative risks of inhibitor formation for certain mutation types to be calculated for the first time (Table 4). Intron 22 inversions showed a relative risk of 1.7 in favor of inhibitor development, nonsense mutations of 2.3 and Haematologica vol. 85(supplement to n. 10):October 2000
Page 1 and 2: Haematologica established in 1920 e
Page 3 and 4: Acknowledgments We wish to thank th
Page 5 and 6: Haematologica 2000; 85(suppl. to n.
Page 7 and 8: 4 J.M. Lusher (range 1-34) for pati
Page 9 and 10: 6 J.M. Lusher ALEDORT: I’d just l
Page 11: 8 J. Oldenburg et al. Tuddenham et
Page 15 and 16: 12 J. Oldenburg et al. reactions ac
Page 17 and 18: 14 J. Oldenburg et al. Dr. Schwaab
Page 19 and 20: 16 J. Ingerslev be established more
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Page 35 and 36: 32 Immune Tolerance and the Treatme
Page 37 and 38: 34 Immune Tolerance and the Treatme
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60 C.M. Kessler in eradicating allo
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62 C.M. Kessler ously. Do you see d
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66 L. Nemes et al. Figure 1. A gian
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68 L. Nemes et al. trast to the ear
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II Immune Tolerance and the Treatme
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