Info

III:1 David

11:4 Helen

I:2 Carol

0.88

0.88

Marker

Probe

Enzyme(s)

Alleles kb

Bcl I RFLP Intron 18

p114.12

Bcl I

1.1 + 0.88

Xba I RFLP Intron 22

p482.6

Xba I + Kpn I

6.2+4.8

Bgl II RFLP DXS15

DX13

Bgl II

5.8 + 2.8

Q Unaffected female

^ Hemophilic male

Q Unaffected female

^ Hemophilic male

Q Carrier

I I Unaffected male and the wider adoption of prophylaxis, offer the prospect of an essentially normal life for the younger generation of people with hemophilia.

Amniocentesis was the first technique employed for the antenatal diagnosis of hemophilia and other X-linked disorders, such as muscular dystrophy. Whilst amniocentesis is both technically simple and safe, an important limitation is the fact that it may only be employed in the second trimester of preg-

Fig. 16.10 The use of restriction fragment length polymorphism (RFLP) analysis in a family to determine carrier status

Robin and David both have severe hemophilia A. Victor and Brian are normal. It is clear from the family tree that the disorder is associated with the 1.1/4.8/5.8 haplotype, and this permits the identification of Robin's sister Helen as a carrier. Anne and Carol are not carriers.

nancy, after approximately 15 weeks of gestation. Chorionic villus sampling (CVS) was first applied to the antenatal diagnosis of a number of genetic disorders in the early 1980s, but this technique is now the principal method used for the antenatal diagnosis of hemophilia and several other single-gene disorders. The principal advantage is that the method may be applied during the first trimester, so that if termination of the pregnancy is required this is easier to carry out. Furthermore, the results of the test are often available within only a few days of the procedure, as (in contrast to amniocentesis) there is no need to culture cells before genetic analysis. A sample is obtained either by a transabdominal or a transvaginal route, under ultrasound guidance (Figure 16.11). However, CVS should not be undertaken before 11 weeks of pregnancy in order to minimize the risk of inducing congenital limb abnormalities. A disadvantage of CVS is that the procedure has to be carried out at a time when fetal sexing through ultrasound scanning is not feasible, so that a female fetus is unnecessarily exposed to risk.

Direct fetal blood sampling may be used for the antenatal diagnosis of hemophilia but this method is usually only offered as a last resort, either because it was not possible to carry out DNA-based family studies in time or because such studies were carried out but were not informative. In this technique, fetal blood is taken from fetal umbilical vessels under ultrasound guidance. The procedure requires considerable expertise and will thus not be available in all hospitals. It is usually carried out at a minimum of 18 weeks of gestation. The levels of factors VIII and IX in a normal fetus at around 19 weeks of gestation are significantly lower than those in an adult, at approximately 40 IU/dl and 10 IU/dl, respectively.

In future, it is likely that antenatal diagnosis of hemophilia (and other genetic disorders) will be based on the isolation of fetal cells from the maternal circulation. It is well documented that fetal lymphocytes may be isolated from the maternal circulation during pregnancy, and determination of the karyo-

Uterine wall

Uterine wall

Chorionic Villus Tissue

Fig. 16.11 Chorionic villus sampling (CVS)

A sample of trophoblastic tissue from the placental area is aspirated with a fine needle under general anesthesia. The procedure is usually carried out after 11 weeks of gestation. DNA isolated from the fetal tissue can then be analyzed to determine fetal sex and status with regard to hemophilia.

Fig. 16.11 Chorionic villus sampling (CVS)

A sample of trophoblastic tissue from the placental area is aspirated with a fine needle under general anesthesia. The procedure is usually carried out after 11 weeks of gestation. DNA isolated from the fetal tissue can then be analyzed to determine fetal sex and status with regard to hemophilia.

type in fetal cells has been used to predict fetal sex. However, fetal lymphocytes may persist in the maternal circulation for many years after a pregnancy and this limits use of this method for second or subsequent pregnancies. More recently, fetal normoblasts have been isolated from the maternal circulation with a flow cytometer/cell sorter and fetal DNA extracted from these in order to probe for markers of hereditary genetic disorders. The attraction of this approach is that it is noninvasive and also offers the prospect of very early diagnosis, as early as 7 or 8 weeks of gestation. Preimplantation diagnosis is another technique that has been developed, involving the determination of embryonic sex using dual fluorescence in situ hybridization of blastomere cells with labeled probes specific for the sex chromosomes. The method may be particularly attractive to women who would not be prepared to undergo a conventional termination of a well-established pregnancy.

Recombinant blood products

The development of plasma-derived coagulation factor concentrates in the early 1970s dramatically improved both the longevity and quality of life of patients with hemophilia, and the demand for factors VIII and IX has risen steadily. The burgeoning global demand for factor VIII can no longer be met by products derived from volunteer blood donors. The manufacture of recombinant coagulation factor proteins offers the promise of unlimited supplies, albeit at increased cost. However, the most important advantage of recombinant products is safety with regard to the transmission of human pathogens. Many patients with hemophilia were infected with either HIV and/or hepatitis C before the introduction of physical methods of viral inactivation of plasma-derived coagulation factor concentrates in 1985. More recently, there has been concern about the possibility of transmission of variant Creutzfeldt-Jakob (vCJD) disease via blood products, as the prions believed to be the cause of this neurological disorder are extremely resistant to the usual viral inactiva-tion procedures, such as heat treatment and exposure to a solvent/detergent mixture. Recombinant coagulation factor products offer the best possible protection from transmission of human blood-borne viruses and are regarded as the treatment of choice for all patients with hemophilia. However, the increased cost compared with conventional plasma-derived products has limited the availability of these products.

Recombinant coagulation factor concentrates are manufactured by insertion of the human gene into mammalian cell lines (such as Chinese hamster ovary cells or baby hamster kidney cells), which are then grown in culture on an industrial scale. Factor VIII (or IX) is then secreted into the growth medium, from which it is subsequently extracted by monoclonal or other immunoaffinity chromatography (Figure 16.12).

(Circle of bacterial DNA that replicates) (a) Plasmid vector

FVIII

(Circle of bacterial DNA that replicates) (a) Plasmid vector

FVIII

Nucleus

FVIII

Nucleus

Insert recombinant plasmid into host cell

(c) Chinese hamster ovary (CHO) cell vWF

FVIII

(b) Isolate human gene sequence and splice into a suitable vector vWF

(b) Isolate human gene sequence and splice into a suitable vector c-^Hf-*

Insert recombinant plasmid into host cell

(c) Chinese hamster ovary (CHO) cell

(d) Bioreactor

Cells in nutrient media

Vial of cells is 'cultured' in media, cells multiply into millions of cells, each expressing FVIII into the media

FVIII

Impurities

Impurities pass through column and rFVIII binds to antibodies

-<

-<

>-

-<

-<

>-

-<

Column is washed

-<

>-

-< -< -<

and rFVIII protein is released

-< -< -<

>->->-

-<

-<

>-

-<

-<

>-

(e) FVIII is purified from media using a monoclonal Ab column and ion-exchange columns rFVIII Protein

Fig. 16.12 Manufacture of recombinant factor VIII

The gene for human factor VIII is incorporated into a bacterial vector (a, b). Inclusion of the von Willebrand factor gene also enhances production of factor VIII. The vector is then inserted into a mammalian host cell (c). The cells grow and multiply in a nutrient medium, and then secrete factor VIII (d). Factor VIII can be extracted and purified by immunoaffinity chromatography (e). The final product contains no von Willebrand factor.

The original recombinant factor VIII products all contained added human albumin as a stabilizer. However, second- and third-generation products are now available in which alternative stabilizers are used and bovine proteins have also been eliminated from the culture media. These measures will further increase the margin of safety as regards viral and other infections. Recombinant factor VIII has an essentially identical structure [with the exception of one brand, Refacto (Wyeth), which has no B-domain] and glycosylation profile to natural plasma factor VIII. The pharmacokinetic profile and postinfusion recovery are also identical to that observed in plasma-derived concentrates.

Initial concerns about a potential increase in the incidence of inhibitors among people with hemophilia receiving recombinant products have proved unfounded. Although randomized trials comparing the incidence in previously untreated patients receiving recombin ant products with those receiving plasma-derived products have never been conducted, it would be fair to say that the current consensus is that the incidence of inhibitor development is very similar for the two types of product.

Recombinant factor IX is also available. Recombinant factor IX is identical in amino acid sequence to the Ala148 (as opposed to the less common Thr148) human polymorphic variant. Plasma factor IX is synthesized in the liver and undergoes post-translation glycosylation of a number of glutamic acid residues. Vitamin K is a vital cofactor for this process and is essential for its activity, but recombinant factor IX is not as effectively carboxylated. The postinfusion recovery does appear to be reduced when compared with plasma-derived products, although the plasma half-life is identical. It is a smaller molecule than factor VIII and requires no albumin or other material to be added to the final product as a stabilizer. The cell line is grown in media that contain no animal or human-derived proteins but the product is subjected to nano-filtration to enhance its safety profile. There is no suggestion of an increased risk of inhibitor development associated with the use of recombinant factor IX.

Another useful recombinant product is recombinant factor VIIa (Novo Seven; Novo Nordisk). It is now recognized that factor VII plays a key role in the initiation of the coagulation cascade through contact with tissue factor released from damaged tissues, to form activated factor VII (VIIa) (see also Chapter 15). Recombinant factor VIIa is very useful in the clinical management of patients with hemophilia A or B and inhibitory antibodies, as well as patients with acquired hemophilia.

Looking to the future, it is likely that the direction of future research in genetic engineering will increasingly be applied to the production of modified molecules with more favorable properties. For example, it would obviously be useful to produce factor VIII molecules with a longer plasma half-life or reduced propensity to stimulate inhibitor development. Hybrid factor VIII molecules have been developed in which the A2 and C2 domains have been replaced by porcine equivalents. Since almost 90% of inhibitory antibodies bind to these two domains of the human factor VIII molecule, it is hoped that these new constructs may be of clinical use in the treatment of people with inhibitory antibodies. It has recently been discovered that factor VIII catabolism is mediated by low-density lipoprotein receptor-related protein (LRP), a hepatic clearance receptor with broad ligand specificity. Pharmacological blockade of these catabolic receptors is another potential target for prolonging the plasma half-life of infused factor VIII in patients with hemophilia. A further development has been the generation of transgenic livestock such as sheep, pigs and goats for the production of human coagulation proteins. Transgenic animals that secrete antithrombin, factor VIII or factor IX into their milk have been produced, and this approach is being explored with a view to the production of relatively cheap and unlimited supplies of biologically active products free of the risk of transmission of human pathogens (Figure 16.13). More recently, this work has been extended by the successful cloning of sheep. The production of transgenic animals by nuclear transfer may permit the establishment of large breeding colonies of livestock more quickly and efficiently than would be possible through the production of individual transgenic sheep by pronuclear microinjection.

(See also Chapter 23.) Gene therapy offers the prospect of a cure for hemophilia in the long term, but it must be empha sized that there is no prospect of large-scale application for some years. The results of five recent clinical trials in the USA of gene therapy for hemophilia have yielded encouraging results, with some evidence of increased circulating levels of factor VIII or IX. However, all these studies have been Phase I studies, primarily designed to test safety rather than efficacy. There are two basic approaches to gene delivery into cells. The first technique involves the direct injection of transducing vector into the bloodstream or target tissue, with subsequent in vivo transformation of the cells that take up the gene. Alternatively, target cells may be modified by removal of cells from a patient, with subsequent modification ex vivo of these cells followed by reinfusion. Of five clinical studies initiated so far, three have targeted the liver (the natural site of factor VIII and IX synthesis), whilst two other groups have focussed on skeletal muscle cells and dermal fibroblasts.

Retroviruses and adenoviruses have been used extensively as vectors (Table 16.3). The principal advantage of using retroviruses as vectors is that the genetic material is actually integrated into the genome of the target cell, so expression of the transfected gene is permanent. However, integration is random, introducing the potential for oncogenesis through the disruption of oncogenes. A further problem with the use of retroviruses as vectors is that there is a physical limit of approximately 8 kb in the size of cassette that can be accommodated within the virus. The factor IX gene may be accommodated, but the full-length factor VIII gene cannot. Adenoviruses permit the transfer of larger genes and can transfect non-dividing cells but transferred DNA does not integrate permanently, so expression of the transfected gene is transient. A further limitation is that the immune response to adenoviral proteins, which are commonly encountered in everyday life, may limit the efficiency of transfer.

As there are a number of legitimate concerns relating to the use of viruses for gene transfer, other modes of gene transfer have been sought. For example, liposomes have a number of other advantages, in that there is no limit to the size or conformation of the DNA that may be incorporated and the liposomes themselves are composed of non-toxic materials that are easily degraded, thus facilitating repeated treatment. In another study, dermal fibroblasts were transfected with a plasmid containing sequences of the factor VIII gene. Cloned cells which were selected for their capacity to produce factor VIII were harvested and implanted into the omentum by laparoscopy. In four of six patients, plasma levels of factor VIII rose above baseline levels and were sustained for up to 10 months.

It is likely that gene therapy for hemophilia B will be achieved earlier than gene therapy for classical hemophilia A since the smaller size of the factor IX gene compared with the factor VIII gene permits the use of retroviral vectors; furthermore, factor IX (in contrast to factor VIII) may be absorbed

Gene therapy for hemophilia

Human coding sequence for protein of interest

DNA fragment containing new hybrid gene

Human coding sequence for protein of interest

Mouse coding sequence for promotion of a milk protein

Collection of pig embryos

Collection of pig embryos

DNA fragment containing new hybrid gene

Male pronucleus

Female pronucleus

Holding pipette

Mouse coding sequence for promotion of a milk protein

Male pronucleus

Female pronucleus

Holding pipette

Fig. 16.13 Production of recombinant proteins using transgenic livestock ('pharming')

from subcutaneous tissues after local injection. Although the liver is the site of synthesis of factor IX, a number of other cells can produce factor IX very effectively after transfection with the human factor IX gene, even in the absence of vitamin K. Both human fibroblasts and keratinocytes can produce factor IX, but keratinocytes are particularly attractive cells for gene therapy as they are very accessible, grow well in culture and can be grafted with ease.

Gene therapy poses a number of ethical problems, particularly since effective and safe treatment with recombinant coagulation factors is now/a available for patients with either hemophilia A or hemophilia B. The use of viral vectors introduces risks such as oncogenesis and infection, or even modification of patient germlines. Patients will also need to be enrolled in clinical trials of very long duration, so that close follow-up will identify problems. Finally, in contrast to the usual form of clinical study, it is likely that children will have to be used in the initial clinical studies in preference to adults because the limited yield of coagulation proteins from current cell culture systems would not suffice for larger subjects.

Table 16.3 Comparison of adenoviruses and retroviruses as vectors.

Retroviruses

Adenoviruses

Physical limit to gene cassette (8 kb) Relatively low copy number Can only infect dividing cells

Permanent integration: sustained expression of transduced gene and potential for oncogenesis Poor immune response of host

Accommodate much larger genes More efficient transfer Infect non-dividing cells

Not integrated after infection: transient gene expression only Stimulate immune responses of host

Conclusions Further reading

Hemophilia is an inherited disorder of coagulation, associated with congenital deficiency of factor VIII (or IX). It is inherited in a sex-linked fashion, so that only males are affected. Approximately one-third of cases arise in families with no previous family history, and represent new mutations. The typical features of severe hemophilia include spontaneous bleeding into the joints, but in the absence of treatment more serious complications (such as intracranial hemorrhage) will lead to early death.

The first products used for the treatment of hemophilia were derived from human plasma, but unfortunately the use of pooled plasma products before 1985 resulted in the transmission of serious viral infections to many patients, such as HIV and hepatitis. In recent years, the development of recombinant blood products has eliminated the risk of transmission of these infections, and also offers the prospect of unlimited supplies. The life expectancy of the younger generation of hemophiliacs now approaches that of the normal population.

The commonest molecular defect in hemophilia A is an inversion in intron 22 of the factor VIII gene on the X chromosome, which accounts for approximately half of all cases. Genetic testing is now readily available in many centers to document the genetic defect in each family and to identify carriers within families. Antenatal diagnosis is now easily available, facilitating early termination of the pregnancy if hemophilia is identified. Recent trials of gene therapy for hemophilia have yielded encouraging results.

Factor VIII (and VII) mutation database: europium.csc.mrc. ac.uk

Factor IX mutation database: www.kcl.ac.uk/ip/petergreen/

haemBdatabase.html World Federation of Haemophilia: www.wfh.org National Hemophilia Foundation (USA): www.hemophilia.org Oxford Haemophilia Centre: www.medicine.ox.ac.uk/ohc/

Introduction: clinical features of hemophilia

Rizza C, Lowe G (eds). (1997) Haemophilia and Other Inherited Bleeding Disorders. Eastbourne: W.B. Saunders.

Zeepvat C. (1998) Prince Leopold: The Untold Story of Queen Victoria's Youngest Son. Stroud, UK: Sutton Publishing.

Hemophilia A

Antonarakis SE, Rossiter JP, Young M etal. (1995) Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood, 86, 2206-2212.

Bagnall RD, Waseem N, Green PM et al. (2002) Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood, 99, 168-174.

Kemball-Cook G, Tuddenham EGD, Wacey AI. (1998) The factor VIII structure and function resource site: HAMSTeRS version 4. Nucleic Acids Research, 26, 216-219.

Lakich D, Kazazian HH, Antonarakis SE et al. (1993) Inversions disrupting the factor VIII gene as a common cause of severe haemophilia A. Nature Genetics, 5, 236-241.

Rossiter J, Young M, Kimberland ML et al. (1994) Factor VIII gene inversions causing severe haemophilia A originate almost exclusively in male germ cells. Human Molecular Genetics, 3, 1035-1039.

Hemophilia B

Briet E, Bertina RM, Van Tilburg NH et al. (1982) Hemophilia B Leyden: a sex-linked hereditary disorder that improves after puberty. New England Journal of Medicine, 306, 788-790.

Crossley M, Ludwig M, Stowell KM et al. (1992) Recovery from hemophilia B Leyden: an androgen-responsive element in the factor IX promoter. Science, 257, 377-379.

Giannelli F, Green PM, Sommer SS et al. (1998) Haemophilia B: database of point mutations and short additions and deletion. Nucleic Acids Research, 26, 265-268.

Hemophilia resources on the Internet

Inhibitors

Giannelli F, Choo KH, Rees DJG et al. (1983) Gene deletions in patients with haemophilia B and factor IX antibodies. Nature, 303, 181-182.

Hay CR, Baglin TP, Collins PW et al. (2000) The diagnosis and management of factor VIII and IX inhibitors: a guideline from the United Kingdom Haemophilia Centre Doctors' Organisation (UKHCDO). British Journal of Haematology, 111, 78-90.

Lacroix-Desmazes S, Bayry J, Misra N et al. (2002) The prevalence ofpro-teolytic antibodies against factor VIII in hemophilia A. New England Journal of Medicine, 346, 662-667.

Oldenburg J, El-Maari O, Schwaab R. (2002) Inhibitor development in correlation to factor VIII genotypes. Haemophilia, 8 (Supplement 2), 23-29.

Saenko EL, Ananyeva NM, Kouiavskala DV et al. (2002) Haemophilia A: effect of inhibitory antibodies on factor VIII functional interactions and approaches to prevent their action. Haemophilia, 8, 1-11.

Carrier testing and antenatal diagnosis

Clarke A and the Working Party of the Clinical Genetics Society (UK). (1994) The genetic testing of children. Journal of Medical Genetics, 31, 785-797.

Giangrande PLF. (2002) Pregnancy in women with inherited bleeding disorders. In: Brenner B, Marder V, Conard J (eds). Women's Issues in Thrombosis and Haemostasis. London: Martin Dunitz.

Goodeve AC. (1998) Advances in carrier detection in haemophilia. Haemophilia, 4, 358-364.

Tedgard U, Ljung R, McNeil TF. (1999) Reproductive choices of haemophilia carriers. British Journal of Haematology, 106, 421-426.

Recombinant blood products

Brownlee GG, Giangrande PLF. (2001) Clotting factors VIII and IX. In: Buckel P. (ed.) Recombinant Protein Drugs (Milestones in Drug Therapy series). Basel: Birkhäuser, pp. 67-88.

Giangrande PLF. (2003) Treatment of hemophilia: recombinant products only? Yes. Journal of Thrombosis and Haemostasis, 1, 214-215.

Lusher J, Ingerslev J, Roberts H, Hedner U. (1998) Clinical experience with recombinant factor VIIa. Blood Coagulation and Fibrinolysis, 9, 119-128.

Mannucci PM. (2003) Treatment of hemophilia: recombinant products only? No. Journal of Thrombosis and Haemostasis, 1, 216-217.

Soukharev S, Hammond D, Ananyeva NM et al. (2002) Expression of factor VIII in recombinant and transgenic systems. Blood Cells, Molecules and Diseases, 28, 234-248.

Gene therapy

Kelley K, Verma I, Pierce GF. (2002) Gene therapy: reality or myth for the global bleeding disorders community? Haemophilia, 8, 261-267.

Manno CS. (2002) Gene therapy for bleeding disorders. Current Opinion in Hematology, 9, 511-515.

Roth DA, Tawa NE, O'Brien JM et al. (2001) Nonviral transfer of the gene encoding coagulation factors VIII in patients with severe hemophilia A. New England Journal of Medicine, 344, 1735-1742.

Velander WH, Lubon H, Drohan WN. (1997) Transgenic livestock as drug factories. Scientific American, 276, 70-74.

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