3 Factor IX

3 Factor IX FRANCESCO GIANNELLI

Haemophilia was well known in ancient times and a clear reference to it can be found in the Babylonian talmud of the fifth century AO (Rosner, 1969). The pattern of inheritance of this disorder (typical of X-linked recessive disease) was clearly described at the beginning of the nineteenth century, well before the chromosomal basis of inheritance was understood (Otto, 1803). Nevertheless, the existence of two forms of haemophilia, A and B, was not suspected even after the correct description of the function of the antihaemophilic factor (factor VIII) by Patek and Taylor (1937). Possibly the first hint of such an heterogeneity can be found in an investigation of the linkage between haemophilia and colour blindness (another X-linked trait) by Haldane and Smith (1947). Of 17 pedigrees, 16 showed clear linkage between the two traits but one was exceptional and showed apparently independent segregation of the two disorders. The possibility of heterogeneity was briefly entertained but was not pursued further as no more informative families were available at the time. The existence of two forms of haemophilia was clearly demonstrated in 1952 when plasma from patients with haemophilia A was found to induce correct clotting of plasma from patients with haemophilia B who were found to lack a different coagulation factor, now called factor IX (Aggeler et al, 1952; Biggs et al, 1952). This factor was later purified from bovine blood and sequenced (Fujikawa et al, 1973; Katayama et al, 1979). Definition of the sequence of human factor IX was achieved indirectly by the cloning and sequencing of the factor IX gene (Choo et al, 1982; Kurachi and Davie, 1982; Jaye et al, 1983). Cloned factor IX sequences also allowed assignment of the human gene to the long arm of the X chromosome at band Xq27 near to the boundary with Xq26 (Chance et al, 1983; Schwartz et al, 1987). The factor IX gene is, therefore, relatively close, physically, to the genes for factor VIII, colour blindness and glucose 6-phosphatedehydrogenase at band Xq28, but it is only loosely linked to them. Between the genes for factor IX and factor VIII lies the fragile site, a chromosome region that, in individuals with the Martin-Bell syndrome and under appropriate culture conditions, appears extended and poorly stained. Linkage data have shown that in some families with the Martin-Bell syndrome (the most common form of inherited mental deficiency after Down's syndrome) a gene responsible for the syndrome is closely linked to factor IX (Giannelli et al, 1987). Bailli~re's ClinicalHaematology--Vol. 2, No. 4, October1989

821

822

F. GIANNELLI

The cloning and characterization of the factor IX gene has started a new phase in the understanding of factor IX and haemophilia B. I will try to illustrate how this has improved the perception of factor IX structure and function, the genetic advice provided to families with haemophilia B, and the prospects for treatment. For other reviews on factor IX the reader is referred to Brownlee (1987; 1989); McGraw et al, (1985a) and Thompson (1986). THE FUNCTION AND STRUCTURE OF FACTOR IX Function

Factor IX is a plasma serine protease which circulates as a zymogen and is activated by specific proteolytic cleavage. This can be performed either by activated factor XI (Walsh et al, 1984), a member of the intrinsic coagulation pathway, or by the factor VII-tissue factor complex of the extrinsic pathway (Jesty and Morrison, 1983). Thus, factor IX represents a node between the two pathways. Activated factor IX (IXa) cleaves factor X at a specific site and transforms it into an active serine protease (Xa). The study of coagulation in reconstituted solutions indicates that factor IXa has low but measurable factor X activating capacity. Addition of Ca 2+ alone modestly stimulates such an action. The combined addition of Ca z+ and appropriate phospholipids decreases the K,, for factor X substantially but has a modest effect on the reaction velocity. By contrast, the addition of activated factor VIII (VIIIa) to the above complex increases the velocity of the reaction by at least 4000-fold (Griffith et al, 1982). It is believed that factor IXa and factor Villa form a high affinity complex for factor X on phospholipidic surfaces. Studies of the properties of such surfaces indicate that the density and homogeneity of negative charges (Daemen et al, 1965) and the fluidity of the membranes are important (Tans et al, 1979). In vivo phospholipidic membranes are provided by platelets and endothelial cells upon platelet activation and trauma. Endothelial cells have a high affinity surface receptor for factor IX (Rimon et al, 1987) that together with lower affinity but more abundant receptors for factor X assure efficient and localized activation of factor X by the factor IXa-VIIIa complex. In physiological conditions the latter acts as a unit and therefore the control of this factor X-activation complex depends on both the activation and degradation of factor VIII (see Chapter 4) and factor IX. Inactivation of factor IX may occur by binding with antithrombin III or by proteolysis. The factor IXa-antithrombin III complex is cleared by the liver through a receptor that also binds antitrypsin complexes (Fuchs et al, 1984). Proteolytic degradation probably plays a significant role in factor IX inactivation but the details are still largely unknown. Factor IX has a rapid turnover: upon infusion into patients with haemophilia B, it shows a rapid clearance (50 min) as though it were diluted into a volume larger than plasma, and then a slower clearance with a half-life of approximately 24 h (Thompson, 1981). The sketch of the factor IX function presented above indicates that this

~Acro~ Ix

823

protein specifically interacts with many other molecules and therefore factor IX can be expected to have many different structural features endowed with definite functions. These are still mostly uncharacterized but progress in this area is gathering momentum. Structure Factor IX (Figure 1) is synthesized primarily in the liver as a zymogen with a leader peptide of at most 46 residues (numbered from - 4 6 to - 1 ) and is secreted into the circulation as a mature protein of 415 amino acids (+ 1 to 415). The leader peptide has two domains (pre and pro) while circulating RL..E N

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Figure 1. Diagram of the human pre-profactor IX zymogen. The protein domains and the position of the intron/exon boundaries in the factor IX gene are indicated. The latter are marked by the numbers of the codon they interrupt, e.g. intron C (47), or the codons they separate, e.g. intron B (38/39). The cleavages necessary for the processing of the zymogen are indicated by small arrows and those for activation by large arrows. According to usual convention, amino acids in the leader peptide are given negative numbers increasing from the carboxy to the amino end of the peptide and those of the zymogen are given positive numbers increasing from the amino to the carboxy end. Pairs of cysteines thought to form disulphide bridges are united by a hyphen. N • indicates glycosylatable asparagines. Amino acids are represented by the one-letter code: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Ash; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr; -y = -/-carboxylated glutamate, B = 13-hydroxyaspartate. Modified from Foster et al (1985).

824

F. GIANNELLI

factor IX has at least six. These, from the amino end, are as follows: a Gla region containing 12 glutamic acid residues that become ~/-carboxylated in the endoplasmic reticulum by a vitamin K-dependent carboxylase; a short segment called the hydrophobic domain or aromatic amino acid stack; two epidermal growth factor (EGF) regions; the activation peptide domain; and finally the catalytic or serine protease region. The predomain is hydrophobic and shows the characteristics of segments involved in the transport of proteins across cell membranes. A processing enzyme cleaves the bond between cysteine -19 and threonine - 1 8 to separate the prepeptide from the propeptide. The latter shows marked homology to the analogous regions of other Gla proteins (Bentley et al, 1986; Jorgensen et al, 1987) and contains a signal for the ~/-carboxylase that modifies the glutamic acid residues of the Gla region. Recombinant factor IX lacking the propeptide or with substitutions of the very conserved residues at positions - 1 6 and - 1 0 (alanine for phenylalanine and glutamic acid for alanine respectively) are not ~-carboxylated (Jorgensen et al, 1987), while that with arginine - 4 substituted by glutamine shows a 50% reduction in ~-carboxylation (Galeffi and Brownlee, 1987). A natural mutant with serine instead of arginine at position - 1 is also very poorly ~/-carboxylated (Diuguid et al, 1986). The Gla region comprises the first 40 residues of mature factor IX and contributes most of its Ca 2+ binding activity. This is also a conserved region among Gla proteins and, in particular, Price et al (1987) propose that the consensus* Gla-Xaa-Xaa-Xaa-Gla-Xaa-Cys, which in factor IX takes the form Gla-Cys-Met-Gla-Gla-Lys-Cys, is an important landmark for the action of ~/-carboxylase. Crystallographic data have indicated that in the absence of Ca 2+ the Gla region is disorganized, while in its presence it is ordered and at its carboxyl end assumes an a-helical configuration. Harlos et al (1987) suggest that the Gla region tends to form dimers and especially heterodimers with regions of homologous proteins, thus favouring the necessary interactions between members of the coagulation cascade. They also assign to the hydrophobic domain the role of orienting such dimerization in a parallel direction. The hydrophobic domain is a conserved region of Gla proteins and contains the consensus Phe-Trp-Xaa-Xaa-Tyr. The two EGF regions of factor IX consist of approximately 40 amino acids, each including six cysteines that form three disulphide bridges. These domains, showing limited homology to EGF, derive from a module widespread in nature and shared by many proteins, not only of vertebrates but also of lower organisms (reviewed in Rees et al, 1988). Three types of EGF domain have been defined, A, B and C, on the basis of sequence comparisons (Rees et al, 1988). Analysis of the solution structure of EGF suggests a [3-sheet conformation expected to apply also to the EGF domains of factor IX (Cooke et al, 1987). The first of these domains is of type B. This is characterized by the consensus Cys-Xaa-Asp*/Asn*-Xaa-Xaa-Xaa-XaaPhe/Tyr-Xaa-Cys-Xaa-Cys (Stenflo et al, 1987) associated with the *A consensussequenceis a sequencehighlyconservedin groupsofrelatedgenesor proteinsso that its characteristicelementsare presentin the majorityofthe relatedgenesor geneproducts. Xaa standsfor any aminoacid residue.

FACTOR IX

825

[3-hydroxylation of the aspartate or asparagine residue marked by the asterisk. ~-hydroxylation is a post-translational modification observed in 25-40% of human factor IX molecules (Fernlund and Stenflo, 1983; Rabiet et al, 1987), and its function is not yet clear. However, B-type growth factor domains appear associated with a high affinity, Gla region independent, Ca 2+ binding site (Ohlin et al, 1988). It has been suggested that in factor IX the side-chains of the aspartates at positions 47 and 49 plus that of aspartate 64 (which is the target of [3-hydroxylation) form the high affinity Ca 2+ binding site. These side-chains are on the same face of the B-sheet and their binding to Ca 2+ would cause a change in conformation important to factor IX activity (Rees et al, 1988). The second EGF domain of factor IX is of type A. The function of the EGF region is still uncertain but a role in protein and cell membrane interaction seems likely (Nawroth et al, 1986; Rees et al, 1988). The activation peptide domain comprises a segment of 35 residues that is excised during the conversion of factor IX into factor IXa. This is acidic in nature and contains two asparagine residues in the appropriate sequence for N-glycosylation (i.e. Asn-Xaa-Ser/Thr). Sugars account for 17% of the molecular weight of circulating human factor IX and most of this may be in N-linked chains (Balland et al, 1988). The functional role of these sugar chains is uncertain. It has been suggested that they might protect factor IX from activation by non-specific proteases (Mizuochi et al, 1983), and it is also possible that they may play a part in secretion since disruption of N-linked glycosylation affects the secretion of factor VIII and tissue-type •plasminogen activator (Dorner et al, 1987). The activation peptide domain is the less conserved region of factor IX and the only known protein polymorphism is found in this segment at position 148 where either alanine or threonine can exist without any apparent effect on factor IX function (McGraw et al, 1985b). The catalytic domain of factor IX has the typical features of a trypsin-like serine protease. The active site has the characteristic triad of the charge relay system: histidine, aspartate and serine plus an acidic residue at the bottom of the substrate binding pocket which confers specificity for cleavage at basic residues. These key elements of the catalytic site are at positions 221,269, 365 and 359 respectively. Other segments of the catalytic domain are less conserved than the region of the active site and binding pocket (Furie et al, 1982). After activation factor IX consists of a light and a heavy chain held together by a disulphide bridge between cysteine 132 and 289. The first residue of the latter chain (valine 181) is thought to interact with aspartate 364 and cause a conformational change in the active site that increases catalytic activity (Titani and Fujikawa, 1982). THE FACTOR IX GENE AND ITS MUTATIONS The gene Factor IX is coded by a gene of approximately 33.5 kb containing eight exons

826

F. GIANNELLI

(a-h or I-VIII) separated by seven introns (Anson et al, 1984; Yoshitake et al, 1985). The exons range in length from 25 to 1935 nucleotides (exons c and h respectively) and the introns from 188 (intron 2) to 9473 (intron 6) nucleotides. The promoter of the gene is still poorly characterized. The exons correspond fairly closely to the protein domains. The first exon codes for the untranslated 5' leader of the mRNA and the predomain of the leader peptide, the second for the propeptide and the Gla region, the third for the hydrophobic amino acid stack, the fourth and fifth for the two EGF domains, the sixth for the activation peptide and neighbouring regions, the seventh for the segment of the serine protease containing the histidine residue of the charge relay system, and the eighth for the rest of the catalytic region. This exon also codes for the 1390 nucleotides long 3' non-coding tail of the message. This starts with a U A A U G A double stop and terminates 15 residues 3' of the polyadenylation signal A A U A A A . The introns of the gene and the flanking sequences contain repetitive elements: one Alu repeat in the second, three in the sixth intron plus one immediately flanking the 3' end of the gene, two KpnI repeats (a member of a family of long interspersed repeats) in the 5' flanking region, and one in the fourth intron. The exon/intron organization of the factor IX gene is identical to that of factor X, protein C and factor VII although the latter may possess an additional exon at the 5' end of the gene (Foster et al, 1985; Leytus et al, 1986; O'Hara et al, 1987). In addition the organization of the first three exons is also identical in prothrombin (Degen and Davie, 1987). It is likely therefore that factors VII, IX, X and protein C are the product of a recent duplication of a common precursor gene related, but more remotely, to the precursor of the prothrombin gene. The factor IX gene shows sequence homology to the other members of its evolutionary group but only in the coding regions; even here, however, its level of G + C richness is clearly different from that of factor VII, X and protein C (i.e. 41.3 % versus 62.3, 60 and 62% respectively). The mutation rate

It can be estimated that the factor IX gene may suffer mutations so detrimental as to cause haemophilia B at a rate of about 5 × 10.6 per gene per gamete per generation. This estimate is based on the knowledge that patients with haemophilia, at least until recently, had a much lower chance of reproducing than normal individuals (Ferrari and Rizza, 1986). Loss of haemophilia B alleles, through patients' reproductive failure, must be compensated by the generation of new mutant genes if the disease is to persist in the population. For this reason equilibrium is usually assumed between loss and gain of detrimental genes found in the population. On the strength of this assumption and the knowledge that one third of all X-linked genes in the population are in males, the mutation rate for X-linked recessive genes is estimated by the formula p. = 1/3sI, where p. is the mutation rate, s the selection coefficient or, in practice, the relative fertility of haemophilia B patients, and I the incidence of the disease. Since s was probably equal to 0.5 before the introduction of factor IX concentrates into therapy (Ferrari

FACTOR IX

827

and Rizza, 1986), the above formula indicates that p. = 1~61andhence that ¼ of the haemophilia B alleles was replaced at each generation. The disease should therefore be heterogeneous and overtly unrelated patients should carry different mutations. Now this prediction can be tested, frequent sites of mutations can be identified and correlations can be established between structural defects and clinical features. Point mutations

Ninety-eight per cent of the factor IX mutations are small changes involving one or a few base pairs. Several of these mutations have been analysed in detail (see Appendix I) and have offered insights into the molecular pathology of haemophilia B. For example, in patients with the haemophilia B Leyden phenotype--a variant that manifests with low factor IX levels in children but gradually improves after puberty, so that the patients may become asymptomatic--partial sequencing of the gene has revealed an A to T transversion in the promoter region at position - 2 0 (i.e. 20 residues upstream of the start of R N A transcription, designated + 1: see Yoshitake et al, 1985 for numbering of the factor IX gene) in one patient and defects at nucleotide +13 in three: deletion of the nucleotide in one and A to G transition in the others (Crossley et al, 1989; Reitsma et al, 1989). This suggests that alterations of residue + 13 and possibly also - 2 0 of the factor IX gene may cause misregulation of factor IX expression. Five mutants have been reported with substitution of arginine - 4 by glutamine (factor IX Oxford 3, San Dimas, Troed-y-Rhiw, London 3 and London 4). These patients have in circulation a factor IX of abnormally high molecular weight that retains residues - 1 8 to - 1 (Bentley et al, 1986; Ware et al, 1986; Liddell et al, 1988; Green et al, 1989). This proves that arginine - 4 is important for the cleavage of the propeptide from mature factor IX. Arginine - 4 is substituted by tryptophan in factor IXMalmo6 (Green et al, 1989). Substitution of a second arginine (Arg - 1 by Ser) in factor IXCambridge 1 also causes failure of propeptide cleavage. Furthermore this factor IX is clearly defective in ~-carboxylation (Diuguid et al, 1986). Haemophilia B London 10 with a three base pair deletion causing the loss of arginine 37 demonstrates the importance of the carboxyl end of the Gla region. This together with the hydrophobic domain forms an oL-helix where hydrophilic and hydrophobic residues are sharply segregated to opposite faces of the helix. In factor IXLonaon 10the loss of arginine-37 grossly disturbs sucha segregation and the amphipathic nature of the oL-helixis lost (Green et al, 1989). Different mutations have been detected in the first growth factor domain (type B). In factor IXAlabama and factor IXLondon6 glycine substitutes aspartate-47 and 64 respectively (Davis et al, 1987; Green et al, 1989). These mutations, and data on recombinant factor IX mutated at aspartate-47, 49 and 64 (Rees et al, 1988), support the idea that these three acidic residues form a functionally important feature of factor IX (see above). Further mutations confirm the important functional role of this domain. Factor IXLonaon 7, which causes moderate to mild haemophilia, has alanine instead

828

F. GIANNELLI

of proline at position 55. A glycine-60 to serine substitution has been reported in two patients with mild haemophilia who were not known to be related. The factor IX of these two patients reacted poorly to a monoclonal antibody directed against the growth factor region and only exons d and e were sequenced (Denton et al, 1988). So far only one mutation has been localized to the second growth factor domain (Green et al, 1989). This is the substitution of asparagine-120 by tyrosine observed in a patient with severe haemophilia B (London 9). Factor IXChapel Hill and factor [XHilo (Noyes et al, 1983; Huang et al, 1989) demonstrate the importance of the residues that form the dipeptides cleaved during the activation of factor IX, since substitution of arginine-145 by histidine in the former and arginine-180 by glutamine in the latter prevent the release of the activation peptide. Various mutations have been localized to the catalytic region. Factor IXLonaon2 and factor IXLonaon5 show independent mutations causing the substitution of arginine-333 by glutamine (Tsang et al, 1988; Green et al, 1989), and a third mutant has been found in a patient from a different ethnic group (P. M. Green, I. M. Nilsson and F. Giannelli, unpublished data). The positively charged amino acid, arginine-333, and a negatively charged amino acid corresponding to position 332 in factor IX, are absolutely conserved in factors IX, X and prothrombin. These residues appear to belong to a surface loop of the catalytic region where they constitute an important electrostatic feature presumably concerned with substrate selection, cofactor binding or both. The presence of glutamine in the mutants causes loss of a positive charge and disrupts this newly recognized feature. Haemophilia B London 8 shows substitution of 6ysteine-336 by arginine. Other mutations affect isoleucine-397 (substituted by threonine) and alanine-390 (substituted by valine). The first has been observed in a number of patients with moderate haemophilia (see Appendix I). Geddes et al (1989) suggest that hydrogen bonding between the O H group of threonine and tryptophan-385 may impair the binding of factor X 'in a configuration favouring catalysis. The second (Spitzer et al, 1988; Sugimoto et al, 1988) has a fairly conservative amino acid substitution, but nevertheless the patients have very low coagulant activity (see Appendix I). A number of patients show mutations that cause premature termination of factor IX synthesis. These are frameshifts due to 1, 2 and 8 base pair delections, i.e. haemophilia B London 12, London 11 and Malmo 1 respectively; or single base pair substitutions generating stop codons, i.e. haemophilia B Malmo 3, 4 and 5 and some detected by Taq I restriction analysis and partial sequencing. Two splice site mutations have also been reported, i.e. haemophilia B Oxford 1 and 2 (see Appendix I). Gross mutations

A small proportion of factor IX mutations, approximately 2%, is due to gross rearrangements and even complete loss of the gene (Appendix II). These were first observed in UK patients who had developed inhibiting antibodies to administered factor IX (inhibitors) and it was proposed,

FACTOR IX

829

therefore, that mutations not allowing the development of immune tolerance to factor IX are important in predisposing to the inhibitor complication (Giannelli et al, 1983; Giannelli and Brownlee, 1986). So far, 22 unrelated patients with such mutations have been reported and 18 had inhibiting antibodies against factor IX (Appendix II). Two patients had no inhibitors but produced significant amounts of factor IX protein. One of these (Strasbourg 1) had significant amounts of factor IX antigen in circulation. His deletion removed only exon d and caused the loss of the first growth factor domain without changing the reading frame of the m R N A (Vidaud et al, 1986). The other (Seattle 1) showed significant amounts of factor IX antigen only in the urine (Bray and Thompson, 1986). The mutation removed exons e and f, which code for the second growth factor and activation domain. The gene product had the molecular weight expected if the two domains had been lost without alteration of the remaining segment of factor IX. However, since the loss of exons e and f should cause a frameshift in exons g and h, the interpretation of the functional consequences of this mutation awaits more detailed characterization of the gene defect. Complete deletion of the factor IX gene, not associated with the inhibitor complication, has been observed in three related patients (Wadelius et al, 1988) and a similar deletion has been found in two relatives: one free of inhibitors and the other with a transient inhibitor response (Taylor et al, 1988). In one deletion (London 1) where the breakpoints and deletion junction were precisely defined, it was shown that the mutation had occurred by non-homologous (or illegitimate) recombination (Green et al, 1987). It is interesting that at least four deletions, Manchester 1 and 2, Jersey and Boston 1, are so large as to include an adjacent gene, rncf2 (Anson et al, 1988). This belongs to the broad and heterogeneous group of transforming genes (oncogenes), but its loss in the patients carrying the above mutations causes no obvious clinical signs. Functional interpretation of the factor IX mutations and correlations between genotype and phenotype

Our knowledge of the structure and function of factor IX is too incomplete to allow prediction of the functional consequences of every sequence change detected in the factor IX gene (see Appendix I). The latter is possible when the sequence change can be expected seriously to disrupt protein synthesis, or important recognized features of factor IX, or conserved elements of protein domains common to factor IX and its homologues. It is also reasonable to expect a correlation between the clinical features of patients and the defect in their factor IX gene. Thus patients with levels of factor IX antigen within the normal range but deficient factor IX activity, called crm +, can be expected to carry mutations that alter functionally important regions of the factor IX protein so as to produce an enzyme of low specific activity. Good examples of this are: (a) the mutants with substitution of arginine - 4 and - 1 as they cause a well documented and gross alteration to the structure of circulating factor IX (propeptide retention); (b) factor IXChapel Hill and

830

F. GIANNELLI

factor IXHilo which show modification of recognized important features of factor IX (the dipeptides cleaved during activation); (c) factor lXAlabamaand factor IXLondon6 that alter conserved features of the first epidermal growth factor domain expected to contribute a Ca 2+ binding site; and (d) the substitution of arginine-333 by glutamine. This alters a conserved feature of the catalytic domain of factors IX, X and prothrombin in three independent mutants that show identical phenotypes (normal factor IX antigen levels but extremely low activity). Therefore the substitution of arginine-333 by glutamine clearly disrupts an essential feature of factor IX that had not been previously recognized. Patients with reduced factor IX antigen and greater, disproportionate reduction of factor IX activity, called crm R, can be expected to carry mutations that either alter both the stability and function of factor IX or cause reduced production of a factor IX of low specific activity. A good example of the former is factor IXLondon 10where loss of arginine-37 disrupts the amphipathic nature of the c~-helix formed by the hydrophobic domain and the carboxyl end of the Gla region. This can be expected to alter the tertiary structure of factor IX so as to reduce both stability and specific activity. Patients with a proportionate reduction in factor IX activity and antigen level, called c r m - , may carry mutations that impair protein synthesis or protein stability. Classical examples of the first kind are splice site mutations, such as Oxford 1 and 2, that interfere with the maturation of m R N A and hence with the synthesis of factor IX. A good example of the second kind is patient London 8 because in his factor IX the substitution of cysteine-336 by arginine causes loss of a conserved residue contributing to a disulphide bridge. Such a structure is known to stabilize protein conformation. Furthermore, the acquisition of a positive charge and the presence of an unpaired cysteine (residue 350) that could compete with similar residues and form abnormal disulphide bridges might add to the instability of this factor IX. Mutations interrupting the reading frame, such as Seattle 2 and London 11, are also consistent with a c r m - phenotype because they lead to premature termination of protein synthesis and may result in nonfunctional products that are unstable, or react poorly with antibodies against normal factor IX. Furthermore, this class of mutants in the globin genes has sometimes shown instability of the m R N A (Kazazian and Boehm, 1989).

Factor IX mutations and the inhibitor complication

Some haemophilia B patients develop antibodies to therapeutic factor IX that severely complicate treatment and worsen prognosis. Mutations causing major loss of antigenic determinants (epitopes) of factor IX are very frequent in these patients; so far, at least half (18/36) of these patients have shown gross deletions of the factor IX gene (Giannelli, 1987a). The remaining patients have point mutations but recently we have examined five of these patients (London 12, Malmo 1, 3, 4 and 5) and found that their genes have suffered functional loss of protein-coding information because of

FACTOR IX

831

frameshift or nonsense mutations (see Green et al (1989) and Appendix I). This strongly supports the idea that the nature of the factor IX mutation is important in predisposing to the inhibitor complication. A few individuals have been reported with mutations causing loss of protein information but clearly no inhibitors (Schach et al, 1987; Taylor et al, 1988; Wadelius et al, 1988; Green et al, 1989: haemophilia B London 11) and a number of factors could account for this, for example the immunogenetic background, the immune physiopathology of the patient, and possibly variations in environmental factors, including the leakage of factor IX from the maternal to the fetal circulation. There is also the possibility that some individuals may develop 'cross-tolerance' to absent epitopes of factor IX because of their similarity to those of homologous proteins such as factors VII, X, protein C and prothrombin. This possibility is consistent with the isolation of monoclonal antibodies that cross-react with many vitamin Kdependent proteins (Church et al, 1988). Individual variations are observed in the strength of the immune response in inhibitor patients. Some may, at least for some time, have antibodies that are hidden in antibody-antigen complexes (Goodnight et al, 1979; Miller et al, 1985); others have detectable persistent inhibitors of either low or high titre and others, rarely, may show only a transient inhibitor status (Taylor et al, 1988). Successful persistent reversion of the inhibitor complication has been obtained in two Swedish patients with high antibody titres after combined treatment with cyclophosphamide, high intravenous doses of immunoglobulin G and factor IX. In a third patient, with a low antibody titre, persistent reversion was obtained simply by treatment with cyclophosphamide and factor IX (Nilsson et al, 1986; I. M. Nilsson, personal communication).

Heterogeneity of haemophilia B and mutational hotspots The data presented above clearly indicate that haemophilia B is genetically very heterogeneous. The different size, and in some cases the recent origin of, the deletions considered above (Matthews et al, 1987; Brownlee, 1989) indicate that they represent independent mutational events. The same applies to the point mutations that have been characterized so far, as most patients carry different defects. There are, however, some mutations that are repeatedly observed. As mentioned above, five G to A transitions causing substitution of arginine - 4 by glutamine have been reported in individuals who are not known to be related (Bentley et al, 1986; Ware et al, 1986; Liddell et al, 1988; Green et al, 1989). The abnormally large protein of these patients can be identified by western blot analysis, and at least two patients were selected on this ground (Bentley et al, 1986; Liddell et al, 1988). However, patients London 3 and 4 were not selected by this criterion and they could be shown by DNA analysis not to be related to each other or to patient Oxford 3 (Green et al, 1989; Montandon et al, 1989; P . R . Winship, personal communication). A sixth patient, Malmo 6, has arginine

832

F. GIANNELLI

- 4 replaced by tryptophan. This clearly indicates a relatively high rate of mutation at this site. Arginine-333 is substituted by glutamine in patients London 2 and 5 who, on the basis of D N A analysis, are not related (Green et al, 1989), and also in a third presumably unrelated patient (P. M. Green, F. Giannelli and I. M. Nilsson, unpublished data). Probably unrelated also are the two patients with serine instead of glycine at residue 60 (Denton et al, 1988); those with valine instead of alanine at residue 390 (Spitzer et al, 1988; Sugimoto et al, 1988) or isoleucine-397 substituted by threonine (Ware et al, 1988; Attree et al, 1989; Geddes et al, 1989). The substitution of arginine - 4 by glutamine may be due to a 5'CpG3' to 5'TpG3' transition on the non-coding strand and that of arginine - 4 by tryptophan to the same type of change on the coding strand. Similar CpG to TpG changes probably explain also the two reported substitutions of glycine-60 by serine and those of arginine-333 by glutamine as well as a number of mutations identified by the analysis of Taq I restriction sites (e.g. patient Portland 2 (Chen et al, 1989)). In fact, even if one excludes mutations identified by Taq I restriction analysis, 44% of the single base substitutions reported so far are of this type (Appendix I and P. M. Green, personal communication). High frequency of mutations at CpG sites has also been observed in the factor VIII (Youssoufian et al, 1988) and glucose-6-phosphate dehydrogenase gene (Vulliamy et al, 1988). CpG to TpG transitions are thought to account for the relative rarity of the CpG dinucleotide in eukaryotic D N A (Bird, 1980), and the high frequency of mutations at restriction sites containing the CpG dinucleotides such as Taq I and Msp I (Barker et al, 1984). It is believed that the unusual frequency of CpG to TpG transitions is due to methylation of C followed by degradative deamination and convertion into T: a normal component of DNA. Such a T, however, is incorrectly paired to a G and forms a premutational lesion susceptible of repair. Recent data in mammalian cells suggest that correction of G:T mismatches may occur with restoration of the orginal G:C pair in 96% of cases (Brown and Jiricny, 1988). This presumably is still not enough to conserve CpG sequences to the same degree as others. The factor IX gene has far fewer C + G bases in its coding regions than its homologues, factors VII, X and protein C. This may be due to the accelerated conversion of CpG into TpG arising from the hypermethylation of the inactive X chromosome in females (Cullen et al, 1986). If this were so, one might suspect that the proportion of CpG dinucleotides at important functional sites would be higher in factor IX as such CpGs should be fixed in the gene by natural selection. In agreement with this hypothesis, transition from CpG to TpG in the coding region of factor IX results in codons blocking protein synthesis in 15% of the cases, while the same occurs in only 0, 1.3 and 1.5% of the cases in factors X, VII and protein C respectively. Selective retention of functionally important CpG dinucleotides during the evolution of factor IX xftay in part contribute to the excess of haemophilia B alleles with CpG to TpG transitions reported so far (44% of all single base substitutions), when the ratio of CpG to other dinucleotide targets in the coding sequence and splice sites of factor IX is 40/1411.

FACTOR IX

833

GENETIC COUNSELLING OF HAEMOPHILIA B The problem Haemophilia B is an X-linked, relatively rare, recessive disease, and therefore affected males generally produce only normal sons and carrier daughters. At each pregnancy, carrier females have a one in two chance of producing affected sons or carrier daughters. Affected females are very rare, and some have clearly been shown to be expressing heterozygotes (Holmberg et al, 1978; Nisen et al, 1986). A well-documented explanation for this phenomenon is abrogation of the random pattern of X chromosome inactivation in early female embryos so that systematic inactivation of either the maternally or paternally derived X chromosome occurs. This usually is brought about by structural aberrations of one X chromosome. Less extreme bias in the X chromosome inactivation of factor IX synthesizing cells occurs by chance. This causes a very broad spectrum of factor IX levels in heterozygotes. Consequently, haematological data are equivocal about the carrier status except when the factor IX assays give very low values or when, in members of families segregating for c r m + mutations, the coagulant activity is much more reduced than the factor IX antigen. One in 15 000 females carries an haemophilia B gene, and identification of such individuals is crucial to genetic counselling in haemophilia B. However, it is practical to test for carrier status only individuals at a high risk. Each patient may be expected to have five or six female relatives at such a risk, leading to a total of at least 5000 individuals in the UK. Prenatal diagnosis is also essential to the genetic counselling and prevention of haemophilia B. Factor IX assays on fetal blood samples provide reliable diagnoses, but these have to wait until the 18th week of pregnancy, can be performed in only one or two practised centres, and carry a risk, although small, to the fetus (Mibashan et al, 1986). The partial solution: indirect diagnoses based on analysis of DNA markers The cloning of the factor IX gene has transformed the genetic counselling of haemophilia B. This transformation has occurred in two phases that are described in this and the next section. Up to 1989, the individuality of the haemophilia B genes, illustrated above, complicated carrier and prenatal diagnoses because it did not allow the direct identification of mutations by a few mutation-specific probes, as is done in the thalassaemias, where a small number of mutations accounts for all affected individuals in discrete populations. Therefore, an indirect procedure was used to identify defective factor IX genes and to perform carrier or prenatal diagnoses in haemophilia B. Functionally irrelevant sequence variations in the factor IX gene were used to mark the defective alleles of individual families and to follow their transmission. So far (Figure 2), seven high frequency, or polymorphic, sequence variations in and around the factor IX gene are known to exist in Caucasians (Giannelli, 1987b; Winship

834

I

F. GIANNELLI

B

(B)

X

D D i (s')l H H ~(X)

,

II

I

I I11[]

.

T

I

(5~o) ~ ~

(T)TM

II d

'

I11

X i

(M) ,

i

M

Act GCT

B

I

/

,

i

l I

•

o

h

vm

OLIGOPROBE

IKb

Figure 2. Scale diagram of the factor IX gene. Solid regions (a-h) are exons (NB: exon C is only 25 base pairs long and is not drawn to scale). Single capital letters are restriction sites, and those in brackets are polymorphic or facultative sites: B, Bam HI, D, Dde I; H, Hinf I; M, Msp I; T, Taq I; X, Xmn I; (B*) = facultative Bam HI site so far observed only in American blacks (Driscoll et al, 1988b). ACT and GCT are alternative codons specifying the polymorphic residue (148) in the activation peptide. Horizontal lines labelled II, VIII, XII and XIII are probes that can be used to detect the restriction fragment length polymorphisms. Sequence specific oligonucleotides have been used to detect the ACT/GCT polymorphism but this can also be detected by Mnl I cleavage of amplified DNA (Tsang et al, 1989). Note that a further polymorphism, affecting a Hha I site, exists 8 kb 3' of exon h (Winship et al, 1989). Modified from Giannelli (1987b). et al, 1989). These modify the pattern of D N A digestion when sequencespecific enzymes (restriction endonucleases) are used, and are called restriction fragment length polymorphisms (RFLPs). In order to use such R F L P s for diagnostic purposes, the following must apply: (a) (b)

(c)

(d)

(e)

The family must be known to segregate for the disease. T h e r e must be genetic information sufficient to define the m a r k e r s of the defective gene segregating in the family. In the simplest and most c o m m o n case this entails the determination of the markers of the factor I X gene in one affected m e m b e r of the family. Individuals who m a y have transmitted the defective gene to the proband must be informative (i.e. heterozygous) for at least one R F L P so that the defective gene m a y be unequivocally defined by its polymorphic marker. Co-operation must be obtained from all the family m e m b e r s needed to trace the segregation of the defective gene. These usually are: one affected blood relative, the mother of the proband, and, often, the father of the p r o b a n d (Figure 3). Confirmation of paternity is needed whenever diagnosis requires samples f r o m the p r o b a n d ' s father.

Since the m o t h e r of the individual examined must be heterozygous for the m a r k e r , it follows that the m a x i m u m diagnostic efficiency of a m a r k e r cannot exceed the frequency of haemophilia B carriers heterozygous for the m a r k e r or, in practice, the frequency of individuals heterozygous for the m a r k e r in the general population. For each dimorphic R F L P , such as those detected in and around the factor I X gene, the m a x i m u m possible frequency of heterozygotes is 50%. This is often approached but seldom achieved. F u r t h e r m o r e , the efficiency of the different R F L P s is not additive, because each m a r k e r wastes some of its power confirming the results obtained by the others, a p h e n o m e n o n aggravated by linkage disequilibrium or, in other words, the n o n - r a n d o m association of the different forms of the markers.

FACTOR IX

835

,

,-y rl.aJl.3

.

T1.6/I.3

T1 8 [ 1 , 3

T18/13

TL8/1.8

rla/~a

T18

I T 1 . 8 l 1.0

Tl.O

T1.0/1.0

Figure 3. Pedigree of a large family segregating for haemophilia B and tested for the Taq I polymorphism (T1.8 or T1.3). ~ ~ , deceased male and female; m, affected male; ~ , obligatory carder; ~ , carrier diagnosed by Taq I marker analysis; ~ , female diagnosed as normal by Taq I analysis. Note that all affected individuals have the same Taq I marker (TI.8) because this is the marker of the haemophilia B allele segregating through this family, but, of course, normal genes of individuals marrying into the family (e.g. III1, 1116) may also carry the 1.8 Taq I marker and hence heterozygotes for the factor IX mutation may be homozygous for the 1.8 Taq I marker (e.g. IV2). Two females are diagnosed as carriers because they have inherited the abnormal maternal X chromosome with the 1.8 marker, while four could be reassured as they have inherited the normal maternal gene with the Taq 1.3 marker. The carrier status of two females: V1 and V3 cannot be determined with the Taq I polymorphism because their mother (IV2) is homozygous for the marker.

Table 1. Intragenic polymorphisms of factor IX*

Polymorphism

Allele

Taq I

1.8 1.3 11.5 6.5 1.75 1.7 6.3 2.3 25 23 Threonine Alanine 230 150 + 80

Xmn I Dde I Msp I Bam HI Residue 148 Hha I

Allele frequency (in Caucasians) 0.65 0.35 0.71 0.29 0.24 0.76 0.2 0.8 0.94 0.06 0.67 0.33 0.39 0.61

Maximum increment in Heterozygote diagnostic efficiency? frequency % 0.45

45.0

0.41

5.0

0.36

20.0

0.32

1.5

0.11

3.0

0.44

3.5

0.48 Total

* From Giannelli (1987b) and Winship et al (1989). t if markers are used in the order in which they appear in the table.

11.0 89.0

836

F. GIANNELLI

This is often observed when markers are physically close. The seven markers available in Caucasians allow successful diagnoses in, at most, 89% of families segregating for haemophilia B (Table 1), but in other ethnic groups (e.g. Orientals), the success is lower because the frequency of the known polymorphic markers is less favourable (Brownlee, 1989). A significant proportion of haemophilia B patients (20 %) are isolated cases and the segregation of the defective gene within the family cannot be clearly established. In this situation the RFLP markers can provide only negative diagnoses: that is, carrier and disease exclusion diagnoses in a small proportion of the patient's female and male relatives, respectively (i.e. 44% of the patient's sisters, 14% of aunts, and even fewer female first cousins; 29%. of nephews and 38% of male first cousins). When successful, this indirect approach has provided definite carrier diagnoses unaffected by the pattern of X chromosome inactivation, and prenatal diagnoses based on chorion biopsy at the 9-10th week of pregnancy or on amniotic cell samples at the 16th week. The definitive solution: direct identification of factor IX mutations

The indirect detection of gene mutations outlined above has several drawbacks. It fails in approximately 30% of the families because in at least 11% of the multiply affected Caucasian families the markers are not informative, and in the families of isolated patients (20% of total) it does not indicate who has a defective gene among the patient's sisters, collateral and ascendant relatives. Each individual diagnosis requires the analysis of complete family groups. Several polymorphisms must be tested to obtain maximum diagnostic success. Confirmation of paternity is necessary. In addition such a diagnostic activity does not advance understanding of the molecular pathology of haemophilia B. Carrier and prenatal diagnoses based on the direct detection of gene defects were, therefore, advocated (Giannelli, 1987b) and attained by two independent means: the direct sequencing of genomic D N A and the detection of mismatches in hybrid molecules formed by test and wild-type (probe) D N A strands (Green et al, 1989; Montandon et al, 1989).

Direct sequencing of genornic DNA Direct sequencing (Figure 4) became practical when, after the development of the polymerase chain reaction, the thermoresistant Taq polymerase was purified and used in this reaction (Saiki et al, 1988). The polymerase chain reaction proceeds as follows. Two oligonucleotides, one complementary to a sequence on the coding strand of the target DNA, and the other complementary to a sequence on the non-coding strand and separated from the former by up to a few thousand nucleotides, are annealed to the denatured genomic DNA, The primers are then extended by two D N A polymerase molecules which proceed in antiparallel directions to produce two new D N A strands, each one starting at one of the primers and extending to and beyond the region of the other primer. Then the D N A is denatured and reannealed

FACTOR IX

837

Figure 4. Carrier and prenatal diagnoses based on the direct sequencing of a factor IX mutation. The sequencing gel shows a segment of exon b and part of the pedigree of the family of patient Malmo 6. The C tracks show that the patient (I1) and his sister's fetus (II1) lack a C residue (left arrow) substituted by a T (right arrow). The patient's sister (12) shows both a C and T residue at this position of the factor IX gene. This indicates that she is a carrier with one mutant and one normal gene, revealed by the arrowed T and C bands respectively. N, normal control sequence. From Green et al (1989).

to the primers that are extended by D N A polymerase, thus repeating the replication or amplification cycle. Now, however, the strands synthesized during the first cycle have one end defined by the primers. When these strands act as templates during the second cycle, DNA synthesis will start from the primers and terminate at the defined end of the template, thus yielding strands with both ends delimited by the primers and their complementary sequences. After 20 to 30 cycles of logarithmic amplification by the polymerase chain reaction, large amounts of perfectly defined sequences can be obtained. The Taq D N A polymerase allows the use of high temperatures in the reaction and this assures the very specific annealing of primers to their complementary sequences and the isolation of very pure amplified DNA. This can be directly sequenced using the dideoxy procedure as described by Green et al (1989). Using this approach all the essential regions of the factor IX gene (promoter, eight coding regions plus extensive sections of introns at the intron/exon boundaries, and the 3' region of the gene which contains sequences important for the cleavage and maturation of the primary transcript) can be sequenced in 3-4 days. Mismatch detection

Mismatch detection (Figure 5) offers an even faster procedure, which avoids extensive sequencing. If a segment of a proband's factor IX gene contains a mutation, this D N A will not form a perfect double helix upon annealing to the normal complementary sequence. To date a number of methods have been proposed for the detection of such imperfect or mismatch-containing DNA. These, however, have lacked either the ability to detect all possible mutations or the sensitivity to analyse human genomic DNA. RNase cleav-

838

r. GIANNELLI

Figure 5. Direct carrier diagnosis by amplification and mismatch detection. The pedigrees show patients London 3 (a) and 4 (b), both with arg - 4 substituted by Gin, and their immediate relatives. The lanes of the acrylamide gel electrophoresis show the results of mismatch tests on the exon b of the factor IX genes of such individuals. An amplified segment of 551 base pairs containing exon b is hybridized to a normal probe. A mismatch in the hybrids containing DNA from patient 3, patient 4 and his mother is revealed by the 152 base pairs band (a, lane 1; b, lanes 1 and 2). The sister and the mother of patient London 3 are not carriers. The mutation must, therefore, have occurred in the germ cells of the mother. The mother of patient London 4 is a carrier but her daughter is not. This analysis clearly demonstrates that the mutations of patients London 3 and 4, ahhough identical, are of independent origin. From Montandon et al (1989).

age of m i s m a t c h e s in hybrids f o r m e d by test D N A and R N A p r o b e s detects 6 0 - 7 0 % o f all possible point mutations (Myers et al, 1985). Gel electrophoresis in d e n a t u r i n g gradients m a y detect all mismatches w h e n special p r o c e d u r e s are used (Sheffield et al, 1989), but it does not indicate the exact position o f the mismatch. This is also the case w h e n mismatches are d e t e c t e d by the binding o f carbodiimide to mispaired t h y m i n e a n d guanine residues ( N o v a c k et al, 1986). H y d r o x y l a m i n e and o s m i u m tetroxide have b e e n used to detect and accurately localize all possible mismatches by cleaving, with piperidine, the D N A modified at mispaired cytosines a n d thymines, but this p r o c e d u r e was n o t directly applied to g e n o m i c D N A ( C o t t o n et al, 1988). W e have d e v e l o p e d an amplification and m i s m a t c h detection ( A M D )

FACTOR IX

839

procedure with hydroxylamine and osmium tetroxide that is capable of identifying all sequence variations and have applied it to the detection of factor IX mutations (Montandon et al, 1989). The two strands of an amplified or cloned segment of the factor IX gene are labelled at the 3' end, denatured and annealed to the corresponding segment amplified from a proband!s DNA. Mismatched C or T residues in the heterodiaplexes formed during the previous step are reacted with hydroxylamine and osmium tetroxide, respectively, and then cleaved by piperidine. Such a cleavage shortens the end labelled probes and indicates the sites of sequence changes. Since any point mutation must result in either a T or C mismatch in heteroduplexes formed by mutant and wild-type DNA, AMD can detect virtually all point mutations with two chemical modification reactions. Single base or small insertions in the proband's D N A may allow adequate pairing of the probe to the template with extrusion of a single-stranded loop in the latter. There is evidence, however, that both A : T and G : C pairs adjacent to a mismatch are sufficiently disturbed to increase the chemical reactivity of the T and C residues enough to detect even the insertions considered above (Montandon et al, 1989; Cotton and Campbell, 1989). AMD, faster and more convenient than direct sequencing, is ideal for rapid diagnosis and mutation screening but does not determine the exact nature of the mutations, because no mismatch method can easily and clearly distinguish between single base-pair substitutions and other point mutations or between single and multiple base-pair insertions or deletions. However, by combining AMD with direct sequencing one can characterize the haemophilia B mutations of large populations.

Direct detection of factor I X mutations Direct detection (Figures 4 and 5) provides important information on the molecular pathology of haemophilia B and will completely replace the earlier indirect diagnostic procedures, based on the analysis of polymorphic markers, for the following reasons. It guarantees successful diagnoses in virtually every family. It does not require collaboration from the proband's relatives. It needs no verification of paternity. Detection of the mutation in an individual reduces the cost and time required for precise diagnoses in all his relatives for generation after generation, because it allows diagnostic tests to focus only on the region of the gene defective in the index case. This has prompted us to propose the creation of a national database of haemophilia B mutations that will enable any individual to obtain carrier or prenatal tests, in only a few days, simply by offering a sample of cells and information to identify their affected relative in the database. FUTURE PERSPECTIVES FOR TREATMENT

The recent advances in the molecular biology of factor IX have not yet produced palpable improvements in treatment but promise benefits in the future. Current blood products carry the risk of serious viral infection, and it

Haemophilia B Leyden (Dutch)* Haemophilia B Leyden (Greek)* Haemophilia B Leyden (US)* Haemophilia B Norwich* Factor IX Malm6 6 Factor IX Oxford 3 Factor IX San Dimas Factor IX Troed-y-Rhiw* Factor IX London 3 Factor IX London 4 Factor IX Cambridget Unnamed (HB2) Unnamed (HB9):~ Factor IX London 10 Factor IX Alabama* Factor IX London 7 Factor IX Durham§ Factor IX London 6 Haemophilia B L o n d o n 9 Factor IX Chapel-Hillt Factor IX Hilo Factor IX London 13" Factor IX London 2 Factor IX London 5

Factor IX point mutations

APPENDIX I

Nucleotide -20: T---~A Nucleotide +13: deletion Nucleotide + 13: A---~G Nucleotide + 13: A---~G Arg---~Trp ( - 4 ) Arg---~Gln ( - 4 ) Arg--~Gln ( - 4 ) Arg---~Gln ( - 4 ) Arg---~Gln ( - 4 ) Arg-->Gln ( - 4 ) Arg--->Ser ( - 1) Arg---~Gln (29) Gla--->Asp (33) Arg-37 lost Asp~Gly (47) Pro--~Ala (55) Gly---~Ser (60) Asp~Gly (64) Asn---~Tyr (120) Arg--~His (145) Arg--~Gln (180) Ala--~Thr (291) Arg--*Gln (333) Arg---~Gln (333)

Defect + + + + + + + + + + + NK NK R + R + + + + NK + +

Variable Variable Variable Variable Severe Severe Severe Severe Severe Severe Severe Mild Moderate Severe Mild Mild Mild Moderate Severe Mild Severe Moderate Severe Severe

ttaematological type crm Severity

Reitsma et al (1989) Reitsma et al (1989) Reitsma et al (1989) Crossley et al (1989) Green et al (1989) Bentley et al (1986) Ware et al (1986) Liddell et al (1988) Green et al (1989) Green et al (1989) Diuguid et al (1986) Koeberl et al (1988) Koeberl et al (1988) Green et al (1989) Davis et al (1987) Green et al (1989) Denton et al (1988) Green et al (1989) Green et al (1989) Noyes et al (1983) Huang et al (1989) Montandon et al (1989) Tsang et al (1988) Green et al (1989)

Reference

Cys---~Arg (336) Ala---~Val (390) Ala---}Val (390) Arg---~Gly (396) Ile---~Thr (397) Ile---~Thr (397) Ile---~Thr (397) Ile---~Thr (397) Ile---~Thr (397) GT---~GG donor splice exon c GT---~TF donor splice exon f Frameshift at Leu-6 Arg---~Stop (29) Arg---~Stop (29) Frameshift at Asp-85 Trlr~Sto p (194) Arg---}Stop (248) Arg---~Stop (252) Arg---}Stop (252) Frameshift at Asp-276 Frameshift at Val-313 Arg---~Stop (338) Lys---~Stop (411)

* Data obtained by sequencing only some of the factor 1x essential regions. t Data obtained by protein sequencing. :~Also has Ile---~Thr (397) substitution. § Two probably unrelated patients; gene partially sequenced. IIMutation detected by restriction analysis (Taq I) and verified by partial sequencing. N K = factor IX antigen level unknown; R = c r m reduced.

Haemophilia B London 8 Factor IX Lake Elsinore Factor IX Niigatat Factor IX Angers* Factor IX Vancouver Factor IX 'Vancouver'* Factor IX 'Vancouver'* Factor IX 'Vancouver'* Factor IX Long Beach Haemophilia B Oxford 2 Haemophilia B Oxford 1 Haemophilia B London 12 Haemophilia B Maim6 4 Haemophilia B London 14" Haemophilia B Seattle 2 Haemophilia B Maim6 5 Haemophilia B Maim6 3 Portlandll Unnamedll Haemophilia B Malm6 1 Haemophilia B London 11 Unnamedl[ Haemophilia B Bordeaux* Inhibitor

Inhibitor Inhibitor

Inhibitor Inhibitor

+ + + + + + + +

Severe Severe Severe Severe Moderate Severe Moderate Severe Moderate Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe

Green et al (1989) Spitzer et al (1988) Sugimoto et al (1988) Attree et al (1989) Geddes et al (1989) Attree et al (1989) Attree et al (1989) Attree et al (1989) Ware et al (1988) Winship (1986) Rees et al (1985) Green et al (1989) Green et al (1989) Montandon et al (1989) Schach et al (1987) Green et al (1989) Green et al (1989) Chen et al (1989) Siguret et al (1988) Green et al (1989) Green et al (1989) Driscoll et al (1988a) Attree et al (1989)

o~

842

F. GIANNELLI

is hoped that factor IX synthesized from recombinant genes may eventually represent a safer therapeutic agent. However, laboratory production of factor IX is complicated by the post-translational modifications necessary to produce functional factor IX. These do not occur in bacterial cells, and none of the transformed mammalian cell lines containing recombinant factor IX genes has produced more than 1 mg of active factor IX per litre of tissue culture medium (Brownlee, 1987; Balland et al, 1988). One successful attempt at expressing human factor IX in transgenic mice lends hope to the production of factor IX in transgenic mammals (Choo et al, 1987). However, the ultimate therapeutic goal is the correction of the gene defect by providing a permanent endogenous source of factor IX. This is conceivable because factor IX is secreted and does not act exclusively at the site of production. Furthermore, one fifth to one tenth of the normal amount of factor IX is sufficient in ordinary circumstances. Schemes have been considered where the correction of the gene defect in some cells of the patient by transfection with functional, recombinant factor IX genes provides a pool of immune-compatible, factor IX-producing cells.These could then be transplanted back into the patient. Many obstacles have to be overcome before achieving this goal (see Brownlee (1989) for detailed discussion) but experiments of this type have begun in mice. St Louis and Verma (1988), using improved constructs of retroviral vectors with human factor IX sequences, have infected mouse fibroblasts and obtained cells capable of producing human factor IX. When transplanted subcutaneously into syngeneic mice, such fibroblasts produced biologically active human factor IX detectable in the general circulation. Further experiments in haemophilic animals are now necessary to test the life-span of the transplanted cells and the safety of the recombinant DNA constructs. CONCLUSION The cloning of the factor IX gene has allowed the characterization of haemophilia B mutants, some producing abnormal factor IX, and others no factor IX at all. Rapid methods of gene analysis, such as the direct sequencing of DNA amplified by the polymerase chain reaction and the amplification and mismatch detection, promise rapid accumulation of information on the molecular pathology of haemophilia B and elucidation of the structure-function relationships in factor IX. Carrier and prenatal diagnoses can now be based on the direct detection of sequence changes. This ensures success in virtually every case. One can also hope that, in the future, new and safe therapeutic strategies will be implemented.

Acknowledgements I am gratefulto mycollaboratorsDr D. R. Bentley,Dr P. M. Greenand Mrs A. J. Montandon for usefuldiscussions,to ProfessorM. Bobrow for reading the manuscriptand to Adrienne Knightfor her secretarial help. Thisworkwas supported by MedicalResearch Councilgrant no. G8611750CB, ActionResearchfor the CrippledChildgrant no. AJ8/1672,and alsoby the Spastics Societyand the GenerationTrust.

843

FACTOR IX APPENDIX II Factor IX deletions

Manchester 1 Manchester 2 London 1 Jersey 1 Pisa 1 Boston 1 Chicago 1 Malmo 2 Bad 1 Ludwig et al (1987) Casarino et al (1986) Mikami et al (1987) 1 Mikami et al (1987) 2 McGraw et al (1985a) Tanimoto et al (1988) 1 Tanimoto et al (1988) 2 Tanimoto et al (1988) 3 Taylor et al (1988) Seattle 1 Strasbourg 1 Wadelius et al (1988)

Defect

Haematological group

Complete Complete Exons f,g,h Complete Complete Complete Exons e,g,h Complete Exons a to h Exons a,b,c At least exon d Complete Complete At least exon h Complete Complete Complete Complete Exons e,f Exon d Complete

Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor + / - inhibitor* no inhibitor c r m + / no inhibitor crm + no inhibitor

Reference (a) (a) (b) (c) (d) (c) (c) (e) (d)

(f) (g)

(a) Giannelli et al (1983) and Anson et al (1988). (b) Giannelli et al (1983) and Green et al (1987). (c) Matthews et al (1987). (d) Quoted in Giannelli (1987a). (e) C. Tsang, I-M. Nilsson and F. Giannelli, unpublished data. (f) Bray and Thomson (1986). (g) Vidaud et al (1986). * One relative with transient inhibitor antibody response and one with no inhibitor.

REFERENCES

Aggeler PM, White SG, Glendening MB et al (1952) Plasma thromboplastin component (PTC) deficiency: a new disease resembling hemophilia. Proceedings o f the Society for Experimental Biology and Medicine 79: 692--694. Anson DS, Choo KH, Rees DJG et al (1984) The gene structure of human anti-haemophilic factor IX. E M B O Journal 3: 1053-1060. Anson DS, Blake D J, Winship PR, Birnbaum D & Brownlee GG (1988) Nullisomic deletion of the mcI2 transforming gene in two haemophilia B patients. E M B O Journal 7: 2795-2799. Attree O, Vidaud D, Vidaud M et al (1989) Mutations in the catalytic domain of human coagulation factor IX: rapid characterisation by direct genomic sequencing of DNA fragments displaying an altered melting behaviour. Genomics 4: 266-272. Balland A, Faure T, Carvallo D et al (1988) Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector. European Journal o f Biochemistry 172: 565--572. Barker D, Schafer M & White R (1984) Restriction sites containing CpG show a higher frequency of polymorphism in human DNA. Cell 36: 131-138. Bentley AK, Rees DJG, Rizza C & Brownlee GG (1986) Defective propeptide processing of

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