	The text of this Review was most recently updated in October 2003, after which it will slowly go out of date! However the linked tables are part of the database so are always fully up to date.
	The Molecular Pathology Of Haemophilia A Table of Contents Introduction 1 Structure and function of the factor VIII gene and protein 1.1 Factor VIII Gene 1.2 Structure and Function of Factor VIII 1.3 Expression of Factor VIII 2 Molecular Pathology of Haemophilia A 2.1 Gene Rearrangements 2.2 Single Base Substitutions 2.3 Sequence Deletions 2.4 Sequence Insertions 2.5 Detection of FVIII Mutations 3 Structure-Function Relationships of Altered FVIII:C Molecules in CRM+ve Haemophilia A 4 References to the Review Introduction Over the last decade there has been a dramatic increase in our understanding of the pathology of haemophilia A in molecular terms, at the levels both of nucleic acid sequence and to a much lesser extent, protein structure. By 1983 factor VIII (FVIII), the protein absent or defective in haemophilia A, had been purified to homogeneity (Rotblat et al 1983) and shortly afterwards the gene was successfully cloned (Gitschier et al 1984). Previously, it was possible to carry out laboratory assays both for FVIII activity and for antigenic protein in the plasma of haemophilia A patients, allowing the distinction to be made between those lacking any FVIII protein and those with normal or reduced levels of a dysfunctional protein: however in the absence of molecular genetic studies there was little further analysis to be made of the molecular nature of the defects. The cloning of the FVIII gene and the more recent adoption of the polymerase chain reaction (PCR) (Saiki et al 1988) and other technical innovations to probe for missense mutations, insertion/deletions, and other gene defects has now allowed the definition of the molecular defect in a large majority of patients with clinically significant bleeding due to haemophilia A. An overview of the molecular pathology of haemophilia A, together with some speculations on the structure-function aspects of FVIII revealed by these data, is presented below. (Back to Table of Contents) 1 Structure and function of the factor VIII gene and protein 1.1 Factor VIII Gene The human factor VIII gene was cloned between 1982 and 1984 by Gitschier and colleagues at Genentech Inc. (Gitschier et al 1984): at the time the gene was the largest described (186kb). Mapping positions the FVIII gene in the most distal band (Xq28) of the long arm of the X chromosome (Poustka et al 1991; Freije & Schlessinger 1992). As shown in Figure 1, analysis of the gene reveals 26 exons, 24 of which vary in length from 69 to 262 basepairs (bp): the remaining much larger exons, 14 and 26, contain 3106 and 1958 bp respectively (the large majority of exon 26 is 3' untranslated sequence). The spliced FVIII mRNA is approximately 9kb in length and predicts a precursor protein of 2351 amino acids. Of the introns, 6 are larger than 14 kilobases (kb). Unusually, the intron separating exons 22 and 23 (IVS22) contains a CpG island associated with two additional transcripts, termed F8A (Levinson et al 1990) and F8B (Levinson et al 1992). F8B is a transcript of 2.5kb and is transcribed in the same direction as the FVIII gene, using a private exon plus FVIII exons 23-26. F8A however contains no introns and is transcribed in the opposite direction to the FVIII gene: furthermore, two additional copies of F8A have been found approximately 400kb telomeric to the FVIII gene (Levinson et al 1990): these F8A copies are implicated in almost half of severe haemophilia A via a partial inversion mechanism (see section 2.1 ). The functions if any of the F8A and F8B transcripts and their potential translated products are unknown, although F8A transcripts have been found in a wide variety of tissues. (Back to Table of Contents) 1.2 Structure and Function of Factor VIII Factor VIII circulates in plasma as a large glycoprotein complexed non-covalently to the giant multimeric adhesive protein von Willebrand factor (vWf) which acts as a carrier for factor VIII both during its secretion and in the general circulation. Sequencing of FVIII cDNA predicted a mature secreted protein consisting of 2332 amino acids with a calculated molecular weight of 265kDa (without carbohydrate). Analysis of the sequence showed very clearly a repeating domain structure A1-A2-B-A3-C1-C2 (Vehar et al 1984). In addition close homology was seen (Figure 2) to coagulation factor V (also A1-A2-B-A3-C1-C2, although the B domains are apparently unrelated) and to the plasma protein caeruloplasmin (A1-A2-A3) (Vehar et al 1984; Koschinsky et al 1986; Kane and Davie 1986). Less obvious homology of the C domains has also been noted with milk fat globule membrane protein (Stubbs et al 1990), discoidin I (Vehar et al 1984) and a receptor tyrosine kinase found in breast carcinoma cells (Johnson et al 1993). No significant homology has yet been identified between the B domain of FVIII and any other protein sequence in protein sequence databases. FVIII is highly sensitive to proteolytic processing before and after secretion and only a small fraction of circulating FVIII is in the single-chain form: the majority consists of heavy chains of variable length (consisting of the A1 and A2 domains together with variable lengths of B domain) linked non-covalently to light chains consisting of the A3, C1 and C2 domains (Vehar et al 1984). Expression of active recombinant FVIII lacking the entire length of the B domain has confirmed that this domain is unnecessary for coagulation activity (Eaton et al 1986): a cleavage after R740 (probably by thrombin) during coagulation serves to remove it. The function of FVIII is to act as an essential cofactor for the activation of factor X (FX) by activated factor IX (FIXa) on a suitable phospholipid surface, thus amplifying the clotting stimulus many-fold (van Dieijen et al 1981; Mann et al 1990). Proteolytic processing mediates both the generation and destruction of this activity. Thus, in order to participate in this reaction plasma FVIII must first be proteolytically cleaved at two distinct sites which lie at the interfaces between domains - after R372 (A1/A2 junction) and after R1689 (B/A3 junction) (Pittman & Kaufman 1988; Hill-Eubanks et al 1989). The first cleavage may serve to allow the short (~30 residue) acidic linker a1 between the A1 and A2 domains to function as a binding-site for A2 in the proposed heterotrimeric active FVIIIa species (Fay et al 1993), while the second allows the dissociation of FVIII from vWf by removing the 40-residue acidic peptide a2 bearing a vWf binding-site (Leyte et al 1991): it is thought that FVIIIa is then able to interact with a phospholipid surface via its C2 domain and form a macromolecular complex with membrane- bound factors IXa and X. Further proteolytic cleavage by activated protein C (APC), thrombin, FIXa or FXa may specifically inactivate FVIIIa by cleavage after R336 (all four enzymes) after 1719 (FIXa only), or after R562 (APC only) (Vehar et al 1984; Walker et al 1987; O'Brien et al 1992): this latter may be the most important in the downregulation of FVIII activity following coagulation. Additionally, the A2 subunit of highly-purified heterotrimeric FVIIIa has been shown to dissociate spontaneously from the complex in vitro with complete loss of activity, although the active complex may be stabilised against this dissociation when complexed with FIXa and phospholipid (Curtis et al, 1994). Figure 3 shows in diagrammatic form a scheme for the activation and inactivation of FVIII. (Back to Table of Contents) 1.3 Expression of Factor VIII The results of immunohistology with a monoclonal antibody against FVIII antigen (Zelechowska et al 1985), the presence of FVIII mRNA in hepatocytes (Wion et al, 1985) and liver transplantation studies in haemophiliacs (Bontempo et al 1984) suggest that in humans the primary site of production of FVIII is the liver, although other tissues do contain detectable FVIII mRNA (Wion et al 1985). Analysis of in vitro expression using cloned FVIII cDNA transfected into mammalian cells in tissue culture has shown that mRNA accumulation is grossly reduced by a dominant inhibitor of transcriptional elongation found in a 1.2kb portion of the cDNA (Koeberl et al 1995). Following synthesis the 19 amino acid leader peptide is removed on translocation into the endoplasmic reticulum (ER) (Kaufman et al 1988). The precise fate of FVIII from this stage is unclear in that, in addition to glycosylations being added to asparagine residues, a proportion of the molecules associate tightly with an ER protein called BiP or GRP78 (Dorner et al 1987; Munro & Pelham 1986) and may not be successfully expressed. This binding to BiP appears to be mediated via the C-terminal portion of the A1 domain (Marquette et al 1995). In the Golgi apparatus, successfully exported molecules have further O-linked glycosylations carried out, six tyrosine residues are sulphated and a range of proteolytic cleavages made in the B domain (Kaufman et al 1988; Pittman et al 1992). The resulting two-chain molecule consists of a C-terminal 80kDa light chain and heavy chains ranging in size from 90 to 200kDa, associated via metal ion interaction (Vehar et al 1984). (Back to Table of Contents) 2 Molecular Pathology of Haemophilia A The results from genetic analysis of nearly 2000 individual DNA samples from haemophilia A patients are summarised in a mutation database first published in 1991, updated in 1994 (Tuddenham et al 1994) and 1996, and now available online at this World Wide Web site. The results presented below constitute a very selective summary, and for full details readers should consult the database. Defined FVIII gene defects may be broadly split into several categories: (i) gross gene rearrangements of DNA sequence involving the FVIII gene (ii) single DNA base substitutions leading to either amino-acid replacement ('missense'), premature peptide chain termination ('nonsense' or stop mutations) or mRNA splicing defects (iii) deletions of genetic sequence of a size varying from one base-pair up to the entire gene (iv) insertions of DNA of varying size. A summary of mutations in the above categories is given in Table 1. (Back to Table of Contents) 2.1 Gene Rearrangements Gross gene rearrangements reported consist almost entirely of a unique inversion elaborated quite recently, yet now known to be responsible for more than 40% of all cases of severe haemophilia A. During intensive attempts to define the causative mutations in a population of severe patients by PCR amplification of all 26 exons, mutations were found in only about 50% of cases (Higuchi et al 1991): in the rest, all the exonic sequences appeared normal. However, on RT-PCR of FVIII mRNA from these cases it was found that no amplification was possible between exons 22 and 23 (Naylor et al 1992; Naylor et al 1993) . It is now known that in all these patients there is a large inversion and translocation of exons 1-22 (together with introns) away from exons 23-26, the mechanism of which is homologous recombination between the F8A gene in intron 22 (Figure 1) and one of the extragenic F8A copies 400Kb 5' to the FVIII gene (Lakich et al 1993; Naylor et al 1993). Figure 4 shows how a simple crossover event during the meiotic division of spermatogenesis can lead to fragmentation of the gene with subsequent severe disease. Indeed, family studies show that the origin of such inversions is almost exclusively a gamete supplied by a normal male (Rossiter et al 1994). A recent study (Antonarakis et al, 1995b) determined that of the two common types of inversion, the distal F8A copy is responsible for 35% of severe haemophilia A cases, while crossover with the proximal copy results in a further 7% of cases. In addition to the Intron 22 inversion, recently an inversion resulting from recombinantion of an intron 1 sequence with a 5' extragenic sequence has been reported, probably responsible for 2-5% of severe cases. (Back to Table of Contents) 2.2 Single Base Substitutions To date (October 2003) there have been 615 different single base substitutions described, of which 462 (75%) predict a single amino-acid change from the wild-type sequence (missense). A further 100 lead to creation of preliminary peptide chain termination or STOP codons, while 62 may give rise to altered or absent splicing of the FVIII mRNA: of these latter, some also predict an amino-acid substitution. Table 2 lists the different substitutions together with their predicted effect, the number of independent reports of the change in the database, any data available on FVIII activity or antigen levels, plus an indication of clinical severity and anti-FVIII inhibitor status (if known). As will be seen, many defects (e.g. Val162 to Met) occur in multiple reports: for the purposes of this Table any available data has been pooled from such reports to provide a summary entry: click here to consult the entire database (with all multiple occurrences) for details of individual cases, together with appropriate references. (Back to Table of Contents) 2.3 Sequence Deletions FVIII gene deletions have been divided arbitrarily into large (>50bp) and small (<50bp), and are listed in Table 3A and Table 3B respectively. 2.3.1 Large Deletions There are 120 unique large deletions reported in the database (Table 3A), from less than 1kb up to more than 210kb deleting the entire gene: the mechanism in most of these is probably non-homologous recombination (Woods-Samuels et al 1991), and is responsible for about 5% of severe haemophilia A cases. As might be expected, large deletions in the FVIII gene almost invariably give rise to clinically severe disease with no FVIII activity measurable in plasma samples and no antigen detected (where assays have been performed): truncated proteins, if produced, are likely to be poorly expressed, inactive and/or rapidly cleared from the circulation. There are few reports of clinically merely moderate disease associated with deletions involving C-terminal exons e.g.22 (Youssoufian et al 1987) and exons 23-24 (Wehnert et al 1989; Lavergne et al 1992) and these may result from in-frame splicing of mRNA to delete exon 22 or both 23 and 24 with subsequent secretion of hypoactive FVIII lacking short stretches of amino-acids. 2.3.2 Small Deletions There are reports of 152 unique small (<50bp) deletions in the database (Table 3B), varying in size from 1bp to 86bp: they are distributed fairly evenly through the exons and are almost all associated with severe disease. Most of these small deletions produce frameshifts and consequent abolition of FVIII expression, but there are a small number of interesting in-frame deletions. There are "hotspots" of single A deletions e.g. in a string of 9 A bases at codons 1191-1194, where single A insertions are also seen. (Back to Table of Contents) 2.4 Sequence Insertions The haemophilia A database lists 57 different insertions (Table 4) varying in size from 1bp up to 2.1Kb and 3.8Kb LINE elements (retrotransposon sequences found distributed throughout the genome in approximately 105 copies) (Kazazian et al 1988). Most insertions are of 1bp, often an A in a stretch of A residues. There are now several cases reported of a single A insertion in such a stretch of 8 As (e.g.Higuchi et al 1991; David et al 1994: Pieneman et al, 1995) where a single A deletion has also been described (codons 1439-1441) (e.g. Antonarakis et al, personal communication). All insertions are associated with severe disease and are either gross insertions or predicted frameshifts. (Back to Table of Contents) 2.5 Detection of FVIII Mutations The introduction of the polymerase chain reaction (PCR (Saiki et al 1988) for the specific amplification in vitro of short stretches of DNA has revolutionised analysis of patient DNA for mutations. Genomic DNA may be derived from a haemophilic subject, for example by extraction from white blood cells, then all the exons including splice junctions may be amplified by use of sequence-specific DNA primers: the amplified stretches can then be directly sequenced to indicate the presence of any of the defects described above. There are however two problems with this approach when analysing the FVIII gene: (i) the large size of the coding region - 26 exons with over 9kb of coding sequence to be searched (ii) this strategy may not detect gene rearrangements where the exonic sequence is unaltered (see Section 2.1 above). These and other considerations have let to the adoption of other strategies which, while still dependent on PCR, give faster and easier identification of the defects. For missense/polymorphism detection, a variety of gel-based presecreening methods have been used to target a particular exon for sequencing (for a detailed review see Michaelides et al 1994), while non-deletional rearrangements may be detected by Southern blotting using a probe corresponding to the F8A region of intron 22 (Lakich et al 1993). Alternatively, a "long PCR" method may be employed. (Back to Table of Contents) 3 Structure-Function Relationships of Altered FVIII:C Molecules in CRM+ve Haemophilia A In the case of predicted single amino-acid substitutions two broad categories of phenotype are found: (i) cases (CRM-reduced or CRM-ve) in which plasma FVIII antigen is reduced concomitantly with activity, presumably as a result of a coagulation-normal protein being either poorly expressed or more rapidly cleared from the circulation than normal: the ways in which the substitutions produce this phenotype are unknown in all cases (ii) approximately normal circulating levels of FVIII antigen with reduced or absent FVIII:C activity (CRM+ve). From the standpoint of an understanding of the structure-function relationships of the molecule in coagulation it is primarily this latter CRM+ve group that is of interest: unfortunately the CRM status is only reported in a minority of the single amino-acid substitutions in the database, and of this minority, most are found to be CRM-ve. Of the remainder, the defect is broadly understood in a small number only. Mutations at or after critical Arg residues at thrombin cleavage sites (residues 372/373 and 1689/1690) have been shown to render the molecule resistant to thrombin activation resulting in reduced coagulant activity (Shima et al 1989; Gitschier et al 1988; Pattinson et al 1990; Higuchi et al 1990; Schwaab et al 1991; Arai et al 1989; O'Brien et al 1990; Arai et al 1990; O'Brien et al 1989; Johnson et al 1994); mutation of a Tyr residue at 1680 (to Phe) which when sulphated forms part of a binding-site for carrier von Willebrand factor is a frequently-reported defect resulting in low plasma FVIII antigen levels with even lower activity (e.g. Higuchi et al 1991); and there are two predicted new N-glycosylation sites created by mutations I566T and M1772T which result in normal circulating antigen levels with grossly-reduced or absent activity (Aly et al 1992; Tuddenham et al, personal communication): deglycosylation of the plasma FVIII restored some coagulant activity. Detailed understanding on a molecular level of how substitutions result in dysfunctional FVIII will have to remain in abeyance until a three-dimensional crystal structure is available: it would be expected that in some cases defective interaction with one of the ligands of FVIII (factor IXa, phospholipid membrane, divalent cations) will be responsible for reduced functional activity. In other cases, mutations impacting the stability of the heterotrimeric form of FVIIIa might give rise to haemophilic consequences. Currently these possibilities are being studied in a handful of laboratories. In tandem, molecular models of FVIII domains have been made by a combination of electron crystallography and homology modelling. There is now also a published crystal structure for the FVIII C2 domain (Pratt K. et al 1999).In some cases, it is possible to reconcile the phenotypic presentation in a haemophiliac with a predicted molecular consequence derived from the model, and there are now a number of reports in the literature which attempt this. (Back to Table of Contents) 4 References to the Review Aly, A.M., Higuchi, M., Kasper, C.K., Kazazian, H.H., Jr., Antonarakis, S.E. and Hoyer, L.W. (1992) Hemophilia A due to mutations that create new N-glycosylation sites. Proc. Natl. Acad. Sci. USA 89, 4933-4937. Antonarakis, S.E., Kazazian, H.H., Jr. and Tuddenham, E.G.D. (1995a) Molecular etiology of factor VIII deficiency in hemophilia A. Hum. Mutat. 5, 1-22. Antonarakis, S.E. and a consortium of more than 50 international authors (1995b) Factor VIII gene inversions in severe haemophilia A: results of an international consortium study. Blood 86, 2206-2212. 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Pattinson, J.K., Millar, D.S., McVey, J.H., Grundy, C.B., Wieland, K., Mibashan, R.S., Martinowitz, U., Tan Un, K., Vidaud, M., Goossens, M., Sampietro, M., Mannucci, P.M., Krawczak, M., Reiss, J., Zoll, B., Whitmore, D., Bowcock, S., Wensley, R., Ajani, A., Mitchell, V., Rizza, C., Maia, R., Winter, P., Mayne, E.E., Schwartz, M., Green, P.J., Kakkar, V.V., Tuddenham, E.G.D. and Cooper, D.N. (1990) The molecular genetic analysis of hemophilia A: a directed search strategy for the detection of point mutations in the human factor VIII gene. Blood 76, 2242-2248. Pan, Y., DeFay, T, Gitschier, J. and Cohen, F. (1995) Proposed structure of the A domains of FVIII by homology modelling. Nature Structural Biology 2, 740-743. Pittman, D.D., Wang, J.H. and Kaufman, R.J. (1992) Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII. Biochemistry 31, 3315-3325. Pittman, D.D. and Kaufman, R.J. (1988) Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII). Proc. Natl. Acad. Sci. U. S. A. 85, 2429-2433. Poustka, A., Dietrich, A., Langenstein, G., Toniolo, D., Warren, S.T. and Lehrach, H. (1991) Physical map of human Xq27-qter: localizing the region of the fragile X mutation. Proc. Natl. Acad. Sci. U. S. A. 88, 8302-8306. Rossiter, J.P., Young, M., Kimberland, M.L., Hutter, P., Ketterling, R.P., Gitschier, J., Horst, J., Morris, M.A., Schaid, D.J., de Moerloose, P., Sommer, S.S., Kazazian, H.H. and Antonarakis, S.E. (1994) Factor VIII gene inversions causing severe hemophilia A originate almost exclusively in male germ cells. Hum. Mol. Genet. 3, 1035-1039. Rotblat, F., Goodall, A.H., O'Brien, D.P., Rawlings, E., Middleton, S. and Tuddenham, E.G.D. (1983) Monoclonal antibodies to human procoagulant factor VIII. J. Lab. Clin. Med. 101, 736-746. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Schwaab, R., Ludwig, M., Kochhan, L., Oldenburg, J., McVey, J.H., Egli, H., Brackmann, H.H. and Olek, K. (1991) Detection and characterisation of two missense mutations at a cleavage site in the factor VIII light chain. Thromb. Res. 61, 225-234. Shima, M., Ware, J., Yoshioka, A., Fukui, H. and Fulcher, C.A. (1989) An arginine to cysteine amino acid substitution at a critical thrombin cleavage site in a dysfunctional factor VIII molecule. Blood 74, 1612-1617. Stubbs, J.D., Lekutis, C., Singer, K.L., Bui, A., Yuzuki, D., Srinivasan, U. and Parry, G. (1990) cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences. Proc. Natl. Acad. Sci. U. S. A. 87, 8417-8421. Tuddenham, E.G., Schwaab, R., Seehafer, J., Millar, D.S., Gitschier, J., Higuchi, M., Bidichandani, S., Connor, J.M., Hoyer, L.W., Yoshioka, A., Peake, I.R., Olek, K., Kazazian, H.H., Lavergne, J.-M., Giannelli, F., Antonarakis, S.E. and Cooper, D.N. (1994) Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition. Nucleic. Acids. Res. 22, 4851-4868. van Dieijen, G., Tans, G., Rosing, J. and Hemker, H.C. (1981) The role of phospholipid and factor VIIIa in the activation of bovine factor X. J. Biol. Chem. 256, 3433-3442. Vehar, G.A., Keyt, B., Eaton, D., Rodriguez, H., O'Brien, D.P., Rotblat, F., Oppermann, H., Keck, R., Wood, W.I., Harkins, R.N., Tuddenham, E.G.D., Lawn, R.-M. and Capon, D.J. (1984) Structure of human factor VIII. Nature 312, 337-342. Walker, F.J., Chavin, S.I. and Fay, P.J. (1987) Inactivation of factor VIII by activated protein C and protein S. Arch. Biochem. Biophys. 252, 322-328. Wehnert, M., Herrmann, F.H. and Wulff, K. (1989) Partial deletions of factor VIII gene as molecular diagnostic markers in haemophilia A. Dis. Markers 7, 113-117. Wion, K.L., Kelly, D., Summerfield, J.A., Tuddenham, E.G.D. and Lawn, R.M. (1985) Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature 317, 726-729. Woods-Samuels, P., Kazazian, H.H., Jr. and Antonarakis, S.E. (1991) Nonhomologous recombination in the human genome: deletions in the human factor VIII gene. Genomics 10, 94-101. Youssoufian, H., Antonarakis, S.E., Aronis, S., Tsiftis, G., Phillips, D.G. and Kazazian, H.H. (1987) Characterization of five partial deletions of the factor VIII gene. Proc. Natl. Acad. Sci. U. S. A. 84, 3772-3776. Zelechowska, M.G., van Mourik, J.A. and Brodniewicz-Proba, T. (1985) Ultrastructural localization of factor VIII procoagulant antigen in human liver hepatocytes. Nature 317, 729-731. (Back to Table of Contents)

The text of this Review was most recently updated in October 2003, after which it will slowly go out of date! However the linked tables are part of the database so are always fully up to date.

The Molecular Pathology Of Haemophilia A

Introduction
1 Structure and function of the factor VIII gene and protein
2 Molecular Pathology of Haemophilia A
3 Structure-Function Relationships of Altered FVIII:C Molecules in CRM+ve Haemophilia A
4 References to the Review

Introduction

Over the last decade there has been a dramatic increase in our understanding of the pathology of haemophilia A in molecular terms, at the levels both of nucleic acid sequence and to a much lesser extent, protein structure. By 1983 factor VIII (FVIII), the protein absent or defective in haemophilia A, had been purified to homogeneity (Rotblat et al 1983) and shortly afterwards the gene was successfully cloned (Gitschier et al 1984). Previously, it was possible to carry out laboratory assays both for FVIII activity and for antigenic protein in the plasma of haemophilia A patients, allowing the distinction to be made between those lacking any FVIII protein and those with normal or reduced levels of a dysfunctional protein: however in the absence of molecular genetic studies there was little further analysis to be made of the molecular nature of the defects.

The cloning of the FVIII gene and the more recent adoption of the polymerase chain reaction (PCR) (Saiki et al 1988) and other technical innovations to probe for missense mutations, insertion/deletions, and other gene defects has now allowed the definition of the molecular defect in a large majority of patients with clinically significant bleeding due to haemophilia A. An overview of the molecular pathology of haemophilia A, together with some speculations on the structure-function aspects of FVIII revealed by these data, is presented below.

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1 Structure and function of the factor VIII gene and protein

1.1 Factor VIII Gene

The human factor VIII gene was cloned between 1982 and 1984 by Gitschier and colleagues at Genentech Inc. (Gitschier et al 1984): at the time the gene was the largest described (186kb). Mapping positions the FVIII gene in the most distal band (Xq28) of the long arm of the X chromosome (Poustka et al 1991; Freije & Schlessinger 1992). As shown in Figure 1, analysis of the gene reveals 26 exons, 24 of which vary in length from 69 to 262 basepairs (bp): the remaining much larger exons, 14 and 26, contain 3106 and 1958 bp respectively (the large majority of exon 26 is 3' untranslated sequence). The spliced FVIII mRNA is approximately 9kb in length and predicts a precursor protein of 2351 amino acids. Of the introns, 6 are larger than 14 kilobases (kb). Unusually, the intron separating exons 22 and 23 (IVS22) contains a CpG island associated with two additional transcripts, termed F8A (Levinson et al 1990) and F8B (Levinson et al 1992). F8B is a transcript of 2.5kb and is transcribed in the same direction as the FVIII gene, using a private exon plus FVIII exons 23-26. F8A however contains no introns and is transcribed in the opposite direction to the FVIII gene: furthermore, two additional copies of F8A have been found approximately 400kb telomeric to the FVIII gene (Levinson et al 1990): these F8A copies are implicated in almost half of severe haemophilia A via a partial inversion mechanism (see section 2.1 ). The functions if any of the F8A and F8B transcripts and their potential translated products are unknown, although F8A transcripts have been found in a wide variety of tissues.

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1.2 Structure and Function of Factor VIII

Factor VIII circulates in plasma as a large glycoprotein complexed non-covalently to the giant multimeric adhesive protein von Willebrand factor (vWf) which acts as a carrier for factor VIII both during its secretion and in the general circulation. Sequencing of FVIII cDNA predicted a mature secreted protein consisting of 2332 amino acids with a calculated molecular weight of 265kDa (without carbohydrate). Analysis of the sequence showed very clearly a repeating domain structure A1-A2-B-A3-C1-C2 (Vehar et al 1984). In addition close homology was seen (Figure 2) to coagulation factor V (also A1-A2-B-A3-C1-C2, although the B domains are apparently unrelated) and to the plasma protein caeruloplasmin (A1-A2-A3) (Vehar et al 1984; Koschinsky et al 1986; Kane and Davie 1986). Less obvious homology of the C domains has also been noted with milk fat globule membrane protein (Stubbs et al 1990), discoidin I (Vehar et al 1984) and a receptor tyrosine kinase found in breast carcinoma cells (Johnson et al 1993). No significant homology has yet been identified between the B domain of FVIII and any other protein sequence in protein sequence databases.

FVIII is highly sensitive to proteolytic processing before and after secretion and only a small fraction of circulating FVIII is in the single-chain form: the majority consists of heavy chains of variable length (consisting of the A1 and A2 domains together with variable lengths of B domain) linked non-covalently to light chains consisting of the A3, C1 and C2 domains (Vehar et al 1984). Expression of active recombinant FVIII lacking the entire length of the B domain has confirmed that this domain is unnecessary for coagulation activity (Eaton et al 1986): a cleavage after R740 (probably by thrombin) during coagulation serves to remove it.

The function of FVIII is to act as an essential cofactor for the activation of factor X (FX) by activated factor IX (FIXa) on a suitable phospholipid surface, thus amplifying the clotting stimulus many-fold (van Dieijen et al 1981; Mann et al 1990). Proteolytic processing mediates both the generation and destruction of this activity. Thus, in order to participate in this reaction plasma FVIII must first be proteolytically cleaved at two distinct sites which lie at the interfaces between domains - after R372 (A1/A2 junction) and after R1689 (B/A3 junction) (Pittman & Kaufman 1988; Hill-Eubanks et al 1989). The first cleavage may serve to allow the short (~30 residue) acidic linker a1 between the A1 and A2 domains to function as a binding-site for A2 in the proposed heterotrimeric active FVIIIa species (Fay et al 1993), while the second allows the dissociation of FVIII from vWf by removing the 40-residue acidic peptide a2 bearing a vWf binding-site (Leyte et al 1991): it is thought that FVIIIa is then able to interact with a phospholipid surface via its C2 domain and form a macromolecular complex with membrane- bound factors IXa and X. Further proteolytic cleavage by activated protein C (APC), thrombin, FIXa or FXa may specifically inactivate FVIIIa by cleavage after R336 (all four enzymes) after 1719 (FIXa only), or after R562 (APC only) (Vehar et al 1984; Walker et al 1987; O'Brien et al 1992): this latter may be the most important in the downregulation of FVIII activity following coagulation. Additionally, the A2 subunit of highly-purified heterotrimeric FVIIIa has been shown to dissociate spontaneously from the complex in vitro with complete loss of activity, although the active complex may be stabilised against this dissociation when complexed with FIXa and phospholipid (Curtis et al, 1994). Figure 3 shows in diagrammatic form a scheme for the activation and inactivation of FVIII.

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1.3 Expression of Factor VIII

The results of immunohistology with a monoclonal antibody against FVIII antigen (Zelechowska et al 1985), the presence of FVIII mRNA in hepatocytes (Wion et al, 1985) and liver transplantation studies in haemophiliacs (Bontempo et al 1984) suggest that in humans the primary site of production of FVIII is the liver, although other tissues do contain detectable FVIII mRNA (Wion et al 1985). Analysis of in vitro expression using cloned FVIII cDNA transfected into mammalian cells in tissue culture has shown that mRNA accumulation is grossly reduced by a dominant inhibitor of transcriptional elongation found in a 1.2kb portion of the cDNA (Koeberl et al 1995). Following synthesis the 19 amino acid leader peptide is removed on translocation into the endoplasmic reticulum (ER) (Kaufman et al 1988). The precise fate of FVIII from this stage is unclear in that, in addition to glycosylations being added to asparagine residues, a proportion of the molecules associate tightly with an ER protein called BiP or GRP78 (Dorner et al 1987; Munro & Pelham 1986) and may not be successfully expressed. This binding to BiP appears to be mediated via the C-terminal portion of the A1 domain (Marquette et al 1995). In the Golgi apparatus, successfully exported molecules have further O-linked glycosylations carried out, six tyrosine residues are sulphated and a range of proteolytic cleavages made in the B domain (Kaufman et al 1988; Pittman et al 1992). The resulting two-chain molecule consists of a C-terminal 80kDa light chain and heavy chains ranging in size from 90 to 200kDa, associated via metal ion interaction (Vehar et al 1984).

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2 Molecular Pathology of Haemophilia A

The results from genetic analysis of nearly 2000 individual DNA samples from haemophilia A patients are summarised in a mutation database first published in 1991, updated in 1994 (Tuddenham et al 1994) and 1996, and now available online at this World Wide Web site. The results presented below constitute a very selective summary, and for full details readers should consult the database. Defined FVIII gene defects may be broadly split into several categories: (i) gross gene rearrangements of DNA sequence involving the FVIII gene (ii) single DNA base substitutions leading to either amino-acid replacement ('missense'), premature peptide chain termination ('nonsense' or stop mutations) or mRNA splicing defects (iii) deletions of genetic sequence of a size varying from one base-pair up to the entire gene (iv) insertions of DNA of varying size. A summary of mutations in the above categories is given in Table 1.

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2.1 Gene Rearrangements

Gross gene rearrangements reported consist almost entirely of a unique inversion elaborated quite recently, yet now known to be responsible for more than 40% of all cases of severe haemophilia A. During intensive attempts to define the causative mutations in a population of severe patients by PCR amplification of all 26 exons, mutations were found in only about 50% of cases (Higuchi et al 1991): in the rest, all the exonic sequences appeared normal. However, on RT-PCR of FVIII mRNA from these cases it was found that no amplification was possible between exons 22 and 23 (Naylor et al 1992; Naylor et al 1993) . It is now known that in all these patients there is a large inversion and translocation of exons 1-22 (together with introns) away from exons 23-26, the mechanism of which is homologous recombination between the F8A gene in intron 22 (Figure 1) and one of the extragenic F8A copies 400Kb 5' to the FVIII gene (Lakich et al 1993; Naylor et al 1993). Figure 4 shows how a simple crossover event during the meiotic division of spermatogenesis can lead to fragmentation of the gene with subsequent severe disease. Indeed, family studies show that the origin of such inversions is almost exclusively a gamete supplied by a normal male (Rossiter et al 1994). A recent study (Antonarakis et al, 1995b) determined that of the two common types of inversion, the distal F8A copy is responsible for 35% of severe haemophilia A cases, while crossover with the proximal copy results in a further 7% of cases.

In addition to the Intron 22 inversion, recently an inversion resulting from recombinantion of an intron 1 sequence with a 5' extragenic sequence has been reported, probably responsible for 2-5% of severe cases.

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2.2 Single Base Substitutions

To date (October 2003) there have been 615 different single base substitutions described, of which 462 (75%) predict a single amino-acid change from the wild-type sequence (missense). A further 100 lead to creation of preliminary peptide chain termination or STOP codons, while 62 may give rise to altered or absent splicing of the FVIII mRNA: of these latter, some also predict an amino-acid substitution. Table 2 lists the different substitutions together with their predicted effect, the number of independent reports of the change in the database, any data available on FVIII activity or antigen levels, plus an indication of clinical severity and anti-FVIII inhibitor status (if known). As will be seen, many defects (e.g. Val162 to Met) occur in multiple reports: for the purposes of this Table any available data has been pooled from such reports to provide a summary entry: click here to consult the entire database (with all multiple occurrences) for details of individual cases, together with appropriate references.

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2.3 Sequence Deletions

FVIII gene deletions have been divided arbitrarily into large (>50bp) and small (<50bp), and are listed in Table 3A and Table 3B respectively.

2.3.1 Large Deletions

There are 120 unique large deletions reported in the database (Table 3A), from less than 1kb up to more than 210kb deleting the entire gene: the mechanism in most of these is probably non-homologous recombination (Woods-Samuels et al 1991), and is responsible for about 5% of severe haemophilia A cases. As might be expected, large deletions in the FVIII gene almost invariably give rise to clinically severe disease with no FVIII activity measurable in plasma samples and no antigen detected (where assays have been performed): truncated proteins, if produced, are likely to be poorly expressed, inactive and/or rapidly cleared from the circulation. There are few reports of clinically merely moderate disease associated with deletions involving C-terminal exons e.g.22 (Youssoufian et al 1987) and exons 23-24 (Wehnert et al 1989; Lavergne et al 1992) and these may result from in-frame splicing of mRNA to delete exon 22 or both 23 and 24 with subsequent secretion of hypoactive FVIII lacking short stretches of amino-acids.

2.3.2 Small Deletions

There are reports of 152 unique small (<50bp) deletions in the database (Table 3B), varying in size from 1bp to 86bp: they are distributed fairly evenly through the exons and are almost all associated with severe disease.

Most of these small deletions produce frameshifts and consequent abolition of FVIII expression, but there are a small number of interesting in-frame deletions. There are "hotspots" of single A deletions e.g. in a string of 9 A bases at codons 1191-1194, where single A insertions are also seen.

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2.4 Sequence Insertions

The haemophilia A database lists 57 different insertions (Table 4) varying in size from 1bp up to 2.1Kb and 3.8Kb LINE elements (retrotransposon sequences found distributed throughout the genome in approximately 105 copies) (Kazazian et al 1988). Most insertions are of 1bp, often an A in a stretch of A residues. There are now several cases reported of a single A insertion in such a stretch of 8 As (e.g.Higuchi et al 1991; David et al 1994: Pieneman et al, 1995) where a single A deletion has also been described (codons 1439-1441) (e.g. Antonarakis et al, personal communication). All insertions are associated with severe disease and are either gross insertions or predicted frameshifts.

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2.5 Detection of FVIII Mutations

The introduction of the polymerase chain reaction (PCR (Saiki et al 1988) for the specific amplification in vitro of short stretches of DNA has revolutionised analysis of patient DNA for mutations. Genomic DNA may be derived from a haemophilic subject, for example by extraction from white blood cells, then all the exons including splice junctions may be amplified by use of sequence-specific DNA primers: the amplified stretches can then be directly sequenced to indicate the presence of any of the defects described above. There are however two problems with this approach when analysing the FVIII gene: (i) the large size of the coding region - 26 exons with over 9kb of coding sequence to be searched (ii) this strategy may not detect gene rearrangements where the exonic sequence is unaltered (see Section 2.1 above). These and other considerations have let to the adoption of other strategies which, while still dependent on PCR, give faster and easier identification of the defects. For missense/polymorphism detection, a variety of gel-based presecreening methods have been used to target a particular exon for sequencing (for a detailed review see Michaelides et al 1994), while non-deletional rearrangements may be detected by Southern blotting using a probe corresponding to the F8A region of intron 22 (Lakich et al 1993). Alternatively, a "long PCR" method may be employed.

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3 Structure-Function Relationships of Altered FVIII:C Molecules in CRM+ve Haemophilia A

In the case of predicted single amino-acid substitutions two broad categories of phenotype are found: (i) cases (CRM-reduced or CRM-ve) in which plasma FVIII antigen is reduced concomitantly with activity, presumably as a result of a coagulation-normal protein being either poorly expressed or more rapidly cleared from the circulation than normal: the ways in which the substitutions produce this phenotype are unknown in all cases (ii) approximately normal circulating levels of FVIII antigen with reduced or absent FVIII:C activity (CRM+ve). From the standpoint of an understanding of the structure-function relationships of the molecule in coagulation it is primarily this latter CRM+ve group that is of interest: unfortunately the CRM status is only reported in a minority of the single amino-acid substitutions in the database, and of this minority, most are found to be CRM-ve. Of the remainder, the defect is broadly understood in a small number only. Mutations at or after critical Arg residues at thrombin cleavage sites (residues 372/373 and 1689/1690) have been shown to render the molecule resistant to thrombin activation resulting in reduced coagulant activity (Shima et al 1989; Gitschier et al 1988; Pattinson et al 1990; Higuchi et al 1990; Schwaab et al 1991; Arai et al 1989; O'Brien et al 1990; Arai et al 1990; O'Brien et al 1989; Johnson et al 1994); mutation of a Tyr residue at 1680 (to Phe) which when sulphated forms part of a binding-site for carrier von Willebrand factor is a frequently-reported defect resulting in low plasma FVIII antigen levels with even lower activity (e.g. Higuchi et al 1991); and there are two predicted new N-glycosylation sites created by mutations I566T and M1772T which result in normal circulating antigen levels with grossly-reduced or absent activity (Aly et al 1992; Tuddenham et al, personal communication): deglycosylation of the plasma FVIII restored some coagulant activity.

Detailed understanding on a molecular level of how substitutions result in dysfunctional FVIII will have to remain in abeyance until a three-dimensional crystal structure is available: it would be expected that in some cases defective interaction with one of the ligands of FVIII (factor IXa, phospholipid membrane, divalent cations) will be responsible for reduced functional activity. In other cases, mutations impacting the stability of the heterotrimeric form of FVIIIa might give rise to haemophilic consequences. Currently these possibilities are being studied in a handful of laboratories. In tandem, molecular models of FVIII domains have been made by a combination of electron crystallography and homology modelling. There is now also a published crystal structure for the FVIII C2 domain (Pratt K. et al 1999).In some cases, it is possible to reconcile the phenotypic presentation in a haemophiliac with a predicted molecular consequence derived from the model, and there are now a number of reports in the literature which attempt this.

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4 References to the Review

Aly, A.M., Higuchi, M., Kasper, C.K., Kazazian, H.H., Jr., Antonarakis, S.E. and Hoyer, L.W. (1992) Hemophilia A due to mutations that create new N-glycosylation sites. Proc. Natl. Acad. Sci. USA 89, 4933-4937.

Antonarakis, S.E., Kazazian, H.H., Jr. and Tuddenham, E.G.D. (1995a) Molecular etiology of factor VIII deficiency in hemophilia A. Hum. Mutat. 5, 1-22.

Antonarakis, S.E. and a consortium of more than 50 international authors (1995b) Factor VIII gene inversions in severe haemophilia A: results of an international consortium study. Blood 86, 2206-2212.

Arai, M., Inaba, H., Higuchi, M., Antonarakis, S.E., Kazazian, H.H., Jr., Fujimaki, M. and Hoyer, L.W. (1989) Direct characterization of factor VIII in plasma: detection of a mutation altering a thrombin cleavage site (arginine-372®histidine). Proc. Natl. Acad. Sci. U. S. A. 86, 4277-4281.

Arai, M., Higuchi, M., Antonarakis, S.E., Kazazian, H.H., Jr., Phillips, J.A., Janco, R.L. and Hoyer, L.W. (1990) Characterization of a thrombin cleavage site mutation (Arg 1689 to Cys) in the factor VIII gene of two unrelated patients with cross-reacting material-positive hemophilia A. Blood 75, 384-389.

Bontempo, F.A., Lewis, J.H., Gorenc, T.J., Spero, J.A., Ragni, M.V., Scott, J.P. and Starzl, T.E. (1987) Liver transplantation in hemophilia A. Blood 69, 1721-1724.

Curtis, J.E., Helgerson, S.L., Parker, E.T. and Lollar, P. (1994) Isolation and characterization of thrombin-activated human factor VIII. J. Biol. Chem. 269, 6246-6251.

David, D., Moreira, I., Lalloz, M.R., Rosa, H.A., Schwaab, R., Morais, S., Diniz, M.J., de Deus, G., Campos, M., Lavinha, J., Johnson, D. and Tuddenham, E.G.D. (1994) Analysis of the essential sequences of the factor VIII gene in twelve haemophilia A patients by single-stranded conformation polymorphism. Blood Coagul. Fibrinolysis 5, 257-264.

Dorner, A.J., Bole, D.G. and Kaufman, R.J. (1987) The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J. Cell Biol. 105, 2665-2674.

Eaton, D., Rodriguez, H. and Vehar, G.A. (1986) Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 25, 505-512.

Economou, E.P., Kazazian, H.H., Jr. and Antonarakis, S.E. (1992) Detection of mutations in the factor VIII gene using single-stranded conformational polymorphism (SSCP). Genomics 13, 909-911.

Fay, P.J., Haidaris, P.J. and Huggins, C.F. (1993) Role of the COOH-terminal acidic region of A1 subunit in A2 subunit retention in human factor VIIIa. J. Biol. Chem. 268, 17861-17866.

Freije, D. and Schlessinger, D. (1992) A 1.6-Mb contig of yeast artificial chromosomes around the human factor VIII gene reveals three regions homologous to probes for the DXS115 locus and two for the DXYS64 locus. Am. J. Hum. Genet. 51, 66-80.

Gitschier, J., Wood, W.I., Goralka, T.M., Wion, K.L., Chen, E.Y., Eaton, D.H., Vehar, G.A., Capon, D.J. and Lawn, R.M. (1984) Characterization of the human factor VIII gene. Nature 312, 326-330.

Gitschier, J., Kogan, S., Levinson, B. and Tuddenham, E.G. (1988) Mutations of factor VIII cleavage sites in hemophilia A. Blood 72, 1022-1028.

Higuchi, M., Wong, C., Kochhan, L., Olek, K., Aronis, S., Kasper, C.K., Kazazian, H.H., Jr. and Antonarakis, S.E. (1990) Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA. Genomics 6, 65-71.

Higuchi, M., Kazazian, H.H., Jr., Kasch, L., Warren, T.C., McGinniss, M.J., Phillips, J.A., Kasper, C., Janco, R. and Antonarakis, S.E. (1991) Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene. Proc. Natl. Acad. Sci. U. S. A. 88, 7405-7409.

Hill-Eubanks, D.C., Parker, C.G. and Lollar, P. (1989) Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin. Proc. Natl. Acad. Sci. U. S. A. 86, 6508-6512.

Johnson, D.J., Pemberton, S., Acquila, M., Mori, P.G., Tuddenham, E.G. and O'Brien, D.P. (1994) Factor VIII S373L: mutation at P1' site confers thrombin cleavage resistance, causing mild haemophilia A. Thromb. Haemost. 71, 428-433.

Johnson, J.D., Edman, J.C. and Rutter, W.J. (1993) A receptor tyrosine kinase found in breast carcinoma cells has an extracellular discoidin I-like domain [published erratum appears in Proc Natl Acad Sci U S A 1993 Nov 15;90(22):10891]. Proc. Natl. Acad. Sci. U. S. A. 90, 5677-5681.

Kane, W.H. and Davie, E.W. (1986) Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin. Proc. Natl. Acad. Sci. U. S. A. 83, 6800-6804.

Kaufman, R.J., Wasley, L.C. and Dorner, A.J. (1988) Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J. Biol. Chem. 263, 6352-6362.

Kazazian, H.H., Jr., Wong, C., Youssoufian, H., Scott, A.F., Phillips, D.G. and Antonarakis, S.E. (1988) Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature 332, 164-166.

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