J . Cell Sci. Suppl. 4, 445-458 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
445
THE M O LE CU LAR GENETICS OF HAEMOPHILIA
A AN D B
G. G. BROWNLEE
S ir William D unn S chool o f P athology, U niversity o f Oxford, South Parks Road, Oxford
0X1 3RE, UK
INTRODUCTION
Haemophilia has been known since biblical times as an inherited bleeding
condition, as boys born into families known to have the disease were excluded from
ritual circumcision in the third century a d (quoted by McKee, 1983). The pattern of
inheritance whereby males were affected, whereas females were not (although they
could transmit the disease to future generations), intrigued the 19th century
biologists and was not adequately explained until the genetic basis of sex was
understood. The clinical symptoms of the disease can be very serious and were lifethreatening before replacement therapy was available. Internal bleeding occurred
into muscles and joints, often without obviously following any trauma and patients
could die of massive internal haemorrhage. Haemophilia A and B pose identical
clinical pictures and both show a similar X-linked pattern of inheritance. Their
differential diagnosis depends on laboratory clotting tests and they were first clearly
distinguished in 1952. Subsequent work showed that the former condition involved
defects in the protein factor VIIIC and the latter in factor IX. Both proteins function
in the middle phase of the intrinsic clotting cascade (Fig. 1). Haemophilia A is the
commoner disease, occurring in Caucasians in approximately one in 5000 males, and
is also referred to as classical haemophilia. Haemophilia B occurs in approximately 1
in 30000 males and is also known as Christmas disease, after the name of a patient,
Stephen Christmas, in whom the disease was characterized early on.
Between 1960 and 1980 the methods of analysing the sequence of amino acids in
proteins that had been pioneered by Sanger and his colleagues on insulin were
applied to the proteins of the clotting cascade, including the factor IX protein. By
1979 the entire amino acid sequence of the bovine factor IX was known (Katayama
et al. 1979) and there was evidence suggesting that a rather similar sequence might be
present in humans. This was a considerable feat of work because of the low yield
(about 5 jUgml-1 of the protein) in plasma. But this basic knowledge of protein
structure was not as easily obtained for factor VIIIC, partly because of its minute
yield in plasma, but also because its high molecular weight and instability made
it particularly difficult to characterize. In fact, factor VIIIC exists as a minor
component of the high molecular weight complex referred to as factor VIII, and the
bulk of this complex is composed of another protein, the von Willebrand factor.
G. G. Brownlee
446
In this short review, I will briefly describe and discuss the advances made since
1980 that have enabled the genes for factor VIIIC and IX to be isolated by
recombinant DNA methods. I will then describe progress in the uses of such gene
probes in clinical diagnosis of carriers, as well as their use for studying the molecular
pathology of the disease and the production of genetically engineered protein.
C LONI NG A N D C HA RA CT ERI Z A TI O N OF THE GENES FOR F ACTORS VIIIC A N D IX
In the period between 1970 and 1980, considerable advances were made in the
purification of messenger RNA from highly specialized cells, such as the a and ¡3
globin mRNA from reticulocytes. It became possible to clone these highly enriched
mRNA preparations, first by making complementary DNA, or cDNA, copies of the
mRNA, and then subsequently converting this material to double-stranded DNA
in vitro and cloning in plasmid or in phage A vectors propagated in the bacterium,
Escherichia coli. Subsequently, these cDNA clones could be characterized and used
to isolate and study the gene organization. For clotting factor IX, a different
approach was necessary as its messenger RNA was present in extremely low
concentrations in liver. Nevertheless, using the approach of synthesizing short
oligonucleotide probes based on a knowledge of the amino acid sequence of portions
of the protein, the problem became soluble. We synthesized oligonucleotide probe
mixtures for several regions of the bovine factor IX sequence to enable the isolation
Surface contact
XII
I
Xlla
-Xla
XI-
Ca
IXa
IX-
Ca • VIII
Phospholipid
X-
-»Xa*Ca1
II-
tissue extract
y j[ c a ++
-X
Phospholipid
■Ha
I
-Fibrin
Fig. I. The clotting cascade. The intrinsic pathway is shown from top left to bottom
right. The extrinsic pathway (activation of X by activated VII) is also shown. This is a
simplified version showing the main features, but it omits feedback loops and a step, e.g.
factor V ila activation of IX, interconnecting the two pathways (from Austen & Rhymes,
1975, with permission).
Haemophilia A and B
447
of an initial short cDNA probe from bovine liver (Choo, Gould, Rees & Brownlee,
1982). Others used essentially the same principles, although the details differed
slightly (Kurachi & Davie, 1982; Jaye et al. 1983). These initial cDNA clones were
then used as probes in order to isolate both human genomic and cDNA clones
establishing the nature of the gene structure as about a 34x 103 base long region of
DNA split into eight coding regions or exons of varying length, which are inter­
rupted by seven non-coding regions or introns (Anson et al. 1984). Subsequently,
the entire gene sequence has been established by Davie and co-workers (Yoshitake
et al. 1985).
For factor VIIIC a variation on the method of using oligonucleotide probes was
used. Because there was some uncertainty as to which tissue to use as a source of
mRNA in the preparation of factor VUIC-containing cDNA libraries of clones, both
groups who successfully cloned and characterized the factor VIIIC gene chose to
isolate the initial clone from a short section of the factor VIIIC gene. It turned out to
be extremely long and complicated and is about 186X 103 bases in length, and boasts
26 introns. The mRNA codes for a mature glycoprotein of 2332 amino acids in
length. This gene is the largest known to date and occupies approaching 0-1 % of the
total length of the X chromosome (Gitschier et al. 1984; Toole et al. 1984).
D I A G N O S I S OF C A R R I E R S A N D P R E N A T A L D I A G N O S I S OF A F F E C T E D
INDIVIDUALS
When girls in known haemophiliac families reach child-bearing age, they naturally
wish to know the risk of having an affected son. Girls have, on average, a 50 % chance
of carrying the defective gene, assuming they inherit this from their mother.
Traditional methods of carrier detection in such girls rely on the measurement of the
concentration of factors VIIIC and IX in plasma. On average, the value in carriers is
half that in normal individuals, but unfortunately the diagnostic value of such
measurements is limited because of the wide spread of actual values among both
normal individuals and carriers. The range of values in carriers is explained by
lyonization, i.e. the random inactivation of one X chromosome early in embryonic
life, resulting in the fact that the factor-VIII- or IX-producing cells in an individual
carrier may be strongly biased in favour of one or other of the paternal or maternal
chromosomes.
Although of some value, these classical methods of diagnosis of carriers are
thought to have an error-rate of, on average, 17 % in haemophilia A and a somewhat
similar rate in haemophilia B (see review by Giannelli, 1986; Barrow, Miller, Reisner
& Graham, 1982; Graham, Flyer, Elston & Kasper, 1979). Prenatal diagnosis of
affected males, in contrast, is reliable so long as a foetal blood sample can be obtained
from the foetal circulation (umbilical vein) uncontaminated with maternal blood
(see Mibashan, Giannelli, Pembrey & Rodeck, 1986). A simple clotting assay clearly
distinguishes affected from normal individuals. The disadvantage of this procedure
is that it requires a skilled gynaecologist to carry out this operation. Furthermore,
foetal blood sampling cannot easily be carried out before the 19th or 20th week in
448
G. G. Brownlee
pregnancy, when a termination, if desired, is a reasonably unpleasant experience for
the mother.
Cloned DNA probes derived from parts of the gene for factors VIIIC or IX offer
an alternative and more reliable method of diagnosing carriers. Such clones can also
be used for prenatal diagnosis at an early stage in pregnancy, using chorionic
trophoblast or amniotic cell samples. Such procedures have been pioneered for the
antenatal diagnosis of sickle cell anaemia and some forms of thalassaemia. In these
cases, gene probes can identify the mutation directly causing the gene defect
(reviewed by Weatherall, 1982). In contrast, haemophilia A and B gene defects are
not fixed and maintained in the population by virtue of heterozygote advantage (as is
believed to be the case for sickle cell anaemia). Hence mutations are believed (see
below) to be caused by a variety of defects of recent and independent origin. For this
reason, it is not practical to develop mutation-specific probes for every affected
pedigree, so that an indirect diagnosis relying on genetic linkage to a polymor­
phism identified within or very close to a gene becomes the method of choice.
Polymorphisms in DNA can be detected by different methods, but those that result
in the appearance of one or other of two bands of differing size in a restriction enzyme
digest of DNA on an agarose gel are the easiest to work with experimentally. These
polymorphisms are referred to as restriction fragment length polymorphisms
(RFLP). Three distinct such RFLPs have been identified and used for carrier
diagnosis in haemophilia B pedigrees (Giannelli et al. 1984; Winship, Anson, Rizza
& Brownlee, 1984; Giannelli, 1986). Two such polymorphisms are known at present
for haemophilia A (Gitschier et al. 1985a; Antonarakis et al. 1985).
One current limitation of the linkage analysis with RFLPs is that the frequencies of
these polymorphisms in the general population, which appear to mimic that in the
affected pedigrees, is such that carrier diagnosis is possible as yet on an average of
only 68 % of known Caucasian haemophilia B pedigrees and about 50 % of Caucasian
haemophilia A pedigrees. Moreover, as the assays are indirect, they rely on the
availability of blood samples not only from the potential carrier under investigation
but also from a known patient within the pedigree, preferably a brother, as well as
from the mother and father. Fig. 2 illustrates the example of such an RFLP linkage
study taken from Winship et al. (1984). The person under investigation III3 was
shown to have a genotype aiai. Given the genotypes of her mother ( a ^ ) , father (ai)
and affected brother (ai), we concluded she must be a carrier. If she had been aia2,
we would have excluded her as a carrier. The chances of error in this method,
omitting issues like paternity and technical errors in the assay due to mixing up
samples, are the chance that recombination occurred in the female oocyte at meiosis between the actual disease mutation (somewhere in the factor IX gene) and
the RFLP (also located and at a known position within the factor IX gene).
Recombination probably occurs non-randomly in chromosomes and we do not know
whether this is more frequent or less frequent than average near the factor IX locus;
but for the purposes of argument, if we assume there is a possibility of three cross­
overs per X chromosome and that the X chromosome is 200X 106 bases long, and
449
Haemophilia A and B
2
1
4
o-
1
I
al
o
-©
©
□
5
□
□
E
I
I
e 2 m2 m3 I,
_______11________ ii________ i i________ ;i----------
(11.5 )
mm
sr
• •
tBtB
Fig. 2. Carrier diagnosis (below) of a haemophilia B pedigree (above) using an X m n I
R FLP in the factor IX gene and a subgenomic probe V III that gives ai ( 1 1 - 5 x 1 0 3 base)
and a2 (6 -5 X 10 3 base) alleles (from Winship et al. 1984, with permission).
knowing the maximum distance between the polymorphism and gene defect cannot
be more than 3 0X103 bases, there will be an even chance of the linkage being broken
in the ratio of 3 0 x 3 : 2 0 0 x l 0 3 or —1 in 2 x l 0 3 times. In percentage terms this is
0-05% chance. For such reasons, we concluded that the indirect linkage analysis
w ith intragenic R F LP s is 99-9% accurate for factor IX (W inship et al. 1984). For
factor V IIIC with a gene of approxim ately five times the length of the factor IX gene,
the corresponding value is 99-7% . In the clinical situation, where fam ilies and
450
G. G. Brownlee
carriers may be counselled, one must remember that this is an average probability
and only an approximate calculation, and that there must be a chance, as more
families are counselled as a result of these new methods of analysis, that cases will
arise in which the linkage is broken and therefore a wrong diagnosis is made.
More distantly related RFLP markers, e.g. anonymous DNA probes, have been
advocated by some experimentalists for carrier and prenatal diagnosis, particularly in
the case of factor VIII (Gitschier et al. 1985a) in which few intragenic RFLPs are
known to date. However, a problem with these markers, e.g. D X13 and S tl4 , at a
close, but still uncertain (Janco et al. 1986), distance from the disease locus, is that
the recombination fraction is not known with sufficient accuracy to make them as
reliable diagnostically as specific gene probes. Only if and when 200 to 300 total
meioses are studied and the recombination fraction is shown to be less than 1 %
would I advocate their use clinically.
If obligatory carriers fail to show heterozygosity for any of the known gene-specific
polymorphisms, of course, no accurate information is forthcoming for potential
carriers - a situation hardly reassuring for the family under investigation, as they
would have to fall back on classical diagnostic methods or somewhat less-reliable
diagnostic markers such as D X13 or S tl4 in the case of haemophilia A. One may
calculate that if 95 % of all families with haemophilia A or B are to be counselled, a
knowledge of at least five different gene-specific RFLPs will be required even if we
assume that (1) each RFLP is distributed in the haemophiliac population at the
theoretical maximum frequency of 50% , and (2) each RFLP segregates indepen­
dently. In practice, not all RFLPs are favourably distributed and because of the
tendency of adjacent regions of DNA to be inherited in a linked way some of the
RFLPs will not be distributed independently of each other in the population at large.
This linkage disequilibrium can severely reduce the value of additional RFLPs
(as shown for the XmnI polymorphism in factor IX; Winship et al. 1984) when
information from a Taql RFLP is already known. Because of such difficulties, which
have also been described for the /3-globin locus (Weatherall, 1982), a realistic
estimate of the number of RFLPs required in order to ‘catch’ 95 % of the affected
pedigrees is nearer seven to ten. This value will be difficult to achieve in the short
term, but in the medium term, say the next 3 -5 years, it should be possible. Of
course, a highly polymorphic ‘minisatellite’ type of sequence such as that occurring
near the ar-globin locus (Weatherall, Higgs, Wood & Clegg, 1984) would be ex­
tremely valuable, but to date there is no evidence of this near the factor VIIIC or IX
loci. The current position on factor IX is that a fourth RFLP involving an M nll
RFLP is under investigation in Oxford (P. R. Winship, personal communication).
Similarly, I am sure that further RFLPs will be found in the much longer factor
VIIIC gene. So far, only a few prenatal diagnoses have been performed on foetal
chorionic villi or amniotic samples using RFLPs in haemophilia A and B, but as the
modern techniques for carrier diagnosis become more available and potential carriers
in affected families find out their status, I predict an increasing demand for such
antenatal information.
Haemophilia A and B
451
M O L E C U L A R D E F E C T S IN H A E M O P H I L I A A A N D B
Evidence of heterogeneity exists in both haemophilia A and B as there is variation
in the clinical severity of the disease in different pedigrees, as well as variation in
laboratory tests of clotting activity and the antigen concentration measured in
samples of blood taken from different patients. We therefore expect a wide range of
molecular defects and indeed a precedent for this exists in the variety of mutations
already known in the haemoglobin disorders (Weatherall et al. 1984). Haemophilia
patients may be conveniently subdivided into those patients in whom protein is
present as detected by immunological methods, referred to as antigen positive, and
those in whom it is absent, referred to as antigen negative. The former might be
expected to have point mutations in that region of the gene encoding amino acid
residues of the protein and studies of such patients can clearly pinpoint critical
functional regions. The latter are more likely to be mutants involving deletions of
reasonably large regions of the molecule or critical point mutations involved in the
biosynthesis of mRNA (e.g. splice junction sequences), or of protein. Critical point
mutations involved in protein synthesis might be changes in the translation initiation
or termination codons, or indeed the introduction of such nonsense codons in
incorrect coding positions, or mutations affecting protein secretion and maturation.
A small subgroup of patients of the antigen negative subgroup are referred to as
‘inhibitor’ patients because they have specific anti-factor-VIIIC or -IX antibodies in
their plasma, which arise in response to therapy with injected normal clotting factors.
Only a few defects of patients have been characterized fully at the molecular level
using recombinant DNA methods and the results are summarized in Table 1. (For
completion, I include one factor IX antigen positive patient, factor IX ChapeiHiii
(Noyes et al. 1983), whose defect has been characterized by a study of the abnormal
protein using the methods of amino acid sequencing.) An antigen positive patient,
haemophilia Boxford3 i has been investigated by recombinant DNA methods (Bentley
et al. unpublished data) and this patient possesses a point mutation at.an amino acid
in the propeptide precursor domain of the protein, specifically at amino acid —4,
(i.e. 4 amino acids preceding the N-terminal tyrosine of the mature factor IX ), which
results in an abnormally long factor IX protein with an N-terminal extension of 18
amino acids accumulating in plasma. This material is inactive in clotting for reasons
that are unknown. Nevertheless, it clearly shows the need for accurate protein
processing, which is obviously critically dependent on the arginine residue at amino
acid —4 and defines a processing intermediate that has not previously been charac­
terized. Another antigen positive patient, factor IXAiabama> has been characterized as
a change at amino acid 47, which affects calcium binding (Davis et al. 1984, and
personal communication). Among the factor IX antigen negative patients, two
patients, haemophilia 0 0
from independent pedigrees have been shown to
have a defect in different splice—donor junction sequences. One is at the G-T donor at
the 3' end of exon f (exon f is the sixth of the 8 coding regions of the gene), which is
changed to T-T (Fig. 3; and Rees, Rizza & Brownlee, 1985). The other involves the
G-T at the 3' end of exon c (the third of the 8 coding regions of the gene), which
B
xf
r d ia n d 2 ,
452
Table 1. Characterized molecular defects in haemophilia
Haemophilia B
Subgroup
Haemophilia A
Defect
Factor I X A l a b a m a
Factor I X C h a p e l H i l l
Haemophilia B0xfc,rd3
Asp—>Gly (47)
Arg—» His (145)
Arg—» Gin (- 4 )
Antigen negative
Haemophilia B
0 x f 0r d 1
Haemophilia B
0 x f 0r d 2
Inhibitors
4 patients from independent
pedigrees in UK
Patient
Defect
G-T —» T-T, donor splice
junction of exon f
G-T —> G-G, donor splice
junction of exon c
H22
Arg (2307) —» TGA, chain termination
giving truncated protein
Deletion of 22X103bases of last exon
(no. 26) and extending 3' to the gene,
giving truncated (2282 amino acid long)
protein
Partial and complete gene
deletions; exact length
of deletion not known
Family A
80 X103 base deletion within gene
Family B
Arg (1941)—» TGA, chain termination
giving truncated protein
Arg (2209) — » TGA, chain termination
giving truncated protein
Deletion of 39X103 bases within gene,
excising exons 23—25
H51
H2
H96
The numbers in parentheses refer to the amino acid residues of the protein.
G. G. Brownlee
Patient
Antigen positive
Haemophilia A and B
453
is changed to G-G (P. R. Winship, unpublished). These mutations are very
reminiscent of the kind of defect that occurs in the /3-globin gene in /3° thalassaemias
(Weatherall et al. 1984) and it is instructive to note how critical a single point
mutation in a vital processing pathway can be, causing as it does clinically severe
haemophilia in both affected patients.
The third group of patients, which have been studied in detail in haemophilia B
and less extensively in haemophilia A, are those that produce inhibitors. We argued
that the reason these patients made antibodies was that their immune system had not
been made tolerant to normal factor IX and therefore such patients were likely to
have gene deletions. Of course, critical point mutations involving RNA processing or
nonsense mutations affecting translation may also have the same effect as gene
deletion by preventing normal protein synthesis. Of the six known U K patients, five
had evidence of partial or complete gene deletion (Giannelli et al. 1983; Peake,
Furlong & Bloom, 1984). Presumably, the one non-deletion inhibitor patient
described by Giannelli et al. (1983) has a critical point mutation elsewhere. Other
examples of factor IX inhibitor patients are under study and three Italian patients
seem to have at least partial gene deletions whereas an Australian patient may be
of the non-deletion type. In haemophilia A, four inhibitor patients have been
characterized in detail. Only one (family A, Table 1) has an extensive gene deletion
estimated as greater than 80X 103 bases. The others either have much shorter
deletions near the 3' end of the gene (patient H96) or point mutations generating an
aberrant stop codon, which would effectively produce a truncated factor VIIIC
protein in family B and patient H2 (Gitschier et al. 19856; Antonarakis et al. 1985).
The postulate still remains valid, however, that the basic reason for the development
of the inhibitor status, a clinical complication second only in importance to AID S
contamination of concentrates, is the same in both haemophilia A and B, i.e. that the
patient’s tolerance has not been induced to critical epitopes of the protein (Giannelli
et al. 1983). However, the position of these critical epitopes in the protein, and the
probable differences in detail in the development of tolerance to factor VIIIC and IX
remain unknown. Two other clinically severe haemophilia A patients H22 and H51
(see Table 1) have been characterized at the molecular level. One is caused by
another point mutation, generating an aberrant translation stop codon, and a second
is caused by a short deletion at the 3' terminus of the gene (Gitschier et al. 19856).
Neither is an inhibitor patient, but Giannelli & Brownlee (1986) note that the
probable effect of these latter two mutations on the protein would be expected to be
----------------------------E x o n
f
------------------------------------------------------- ►
190
V
V
G
G
E
D
A
K
P
G
Q
F
P
W
Q
TTGTT GGTGG AG AAG ATG CCAAACCAG GT CA ATT C C CTTG G CA G GTACTTTATACTG AT GGTGTGTCA AA
21130
21140
21150
21160
^21170
21180
T
Fig. 3. Sequence surrounding the 3' end of exon f of the normal factor IX gene showing
the G—»T mutation in haemophilia B0xf0rdi. One-letter symbols are used for the amino
acids (from Rees et al. 1985, with permission).
21190
454
G. G. Brownlee
Xq
25
-
_
_
_
HPRT
26
27 [_J
28 * ------- ^
FIX
FS
HEMA
G6PD
CB
ALD
Fig. 4. Diagram of the terminal section of the long arm of the human X chromosome
showing some disease loci. For abbreviations, see the text. H PRT is the locus for
hypoxanthine guanine phosphoribosyl transferase.
less than the mutations discussed above in the two patients with inhibitors. To date
no patients have been described with defects in the promoter or in the A-A-U-A-A-A
polyadenylation region of the gene.
Further studies of antigen positive patients should give valuable information about
the critical regions for function in both the factor V IIIC and the factor IX molecules.
Factor IX and its activated form IXa have to interact with at least five other
molecules in the m iddle stage in the intrinsic clotting pathway, so we m ust expect
m any parts of the molecule to be critical for function and, or, correct folding of the
protein. Sim ilar argum ents apply to factor V IIIC , although in this case we sus­
pect that the central carbohydrate-rich portion of this very large molecule is less
im portant functionally than the rest of the molecule, as it has diverged extensively in
amino acid sequence among mammals (Orr et al. 1985).
R E G I O N A L L O C A L I Z A T I O N ON THE X C H R O M O S O M E
Fig. 4 shows the localization of the factor IX locus (labelled FIX ) to band q27 near
to the tip of the long arm of the X chromosome. The localization was discovered
using cloned factor IX probes (e.g. see Boyd et al. 1984) and has been more recently
refined to band q27.1 by in situ hybridization to extended early metaphase
chromosomes (V. J. Buckle, personal communication). T he short cytogenetic
distance from the locus at q27.3 for mental retardation with macro-orchidism
associated with fragility (F S) is clear. U nfortunately, however, there is still too much
recombination (20 %) between the factor IX locus and the FS for the factor IX
probes to have any real value in carrier diagnosis of this rather common and
Haemophilia A and B
455
depressing disease (e.g. see Choo et al. 1984). The haemophilia A locus (HEMA) is
known to be closely linked to a group of three other markers; glucose-6-phosphate
dehydrogenase (G6PD), colour blindness (CB) and adrenoleukodystrophy (ALD),
but again the recombinational fraction between haemophilia A and FS is too great for
the factor VIII probes to be useful in diagnosis of the mental retardation syndrome.
Many other anonymous gene markers are now known for this region of the X
chromosome, but those that have been tested so far are no closer to the FS locus than
factor IX. A closely linked probe or a specific gene probe is urgently needed.
G E N E T I C A L L Y E N G I N E E R E D F A C T O R S VI I I C A N D IX
Haemophilia A and B patients need regular injections of factors VIIIC and IX if
haemorrhage is to be avoided and controlled. Unfortunately, there is risk of viral
contamination introduced in the donor blood used for these preparations, which has
not been completely removed in the purification procedure. Recently, AID S has
superseded hepatitis B and the non-A, non-B hepatitis as the most hazardous
complication of therapy. Although the AID S virus is fortunately rather heat-labile,
we do not yet know how successful the heat treatment (instituted in 1985) will be and
whether other viruses might appear from time to time that cannot be so easily
inactivated. It is therefore highly desirable to produce the required proteins from a
genetically engineered source non-contaminated with viruses. This should in
addition give a well-standardized product and manufacturers would no longer need
to rely on blood donors for their starting material.
The ability to produce factor VIII in vitro from cloned DNA was first described
late in 1984 by two genetic engineering companies (Wood et al. 1984; Toole et al.
1984). They introduced modified cDNA clones into mammalian cells, i.e. kidney
cells or T lymphoma cells, and showed that biologically active factor VIIIC was
secreted into the medium, as assayed by highly specific in vitro tests. In 1985, three
papers, including one from my own laboratory, reported similar successes with factor
IX, also in mammalian cells (Anson, Austen & Brownlee, 1985; de la Salle et al.
1985; Busby et al. 1985). Both liver and kidney cells successfully produced active
material, although the specific activity of the factor IX protein was higher in the liver
cell, presumably reflecting the fact that this cell tissue had correctly modified and
processed the factor IX. As liver is the tissue in which factor IX is normally
synthesized, it might be expected to be appropriate for correct expression and pro­
duction of factor IX that is indistinguishable from the raw material. Nevertheless,
in all these studies, of both factor VIIIC and IX, minute yields of product are
reported. One of the best yields of approaching 1 /¿g of factor IX per ml of medium
was reported in one of the papers on factor IX (Busby et al. 1985). Even with this,
I estimate it would be necessary to culture hundreds of thousands of litres of cells to
obtain enough material for the U K requirement of 50 g of factor IX for one year.
Given that purification of proteins on a large scale necessarily entails losses, it is clear
that more efficient small-scale synthesis has to be developed before an investment
in a large-scale industrial process is made. Fortunately, there is an indication that
456
G. G. Brownlee
a protein related to factor IX (the anti-coagulant protein C) can be successfully
produced in high yields in Chinese hamster ovary cells using recombinant DNA
methods. In these cells the protein C gene copy number can be amplified by linkage
of the clone to the gene for dihydrofolate reductase followed by growth of cells in
methotrexate. Thus it may be possible to produce cell lines giving at least 10 times
more factor IX. For factor VIIIC, there is a similar requirement for maximization of
the yields of genetically engineered material on a small scale.
I estimate that it will be 3 -5 years before the necessary industrial processes for
factors VIIIC and IX will be developed and the material adequately characterized
and tested for clinical application to human beings. Nevertheless, the product should
be safer, free of viruses, and, I suggest, will supersede traditional products made
from blood with all their inherent problems.
I thank the Medical Research Council for support.
REFERENCES
D. S ., A ust en , D. E. G. & B row nlee , G. G. (1985). Expression of active human clotting
factor IX from recombinant D N A clones in mammalian cells. N ature, Lond. 315, 683-686.
A n so n , D. S ., C hoo , K . H ., R e e s , D. J. G ., G ian n e lli , F ., G o u l d , K ., H u d d le sto n , J. A . &
B r o w n l e e , G. G. (1984). Gene structure of human anti-haemophilic factor IX. EMBO J . 3,
1053-1064.
A n t o n a r a kis , S . E., W a b e r , P. G., K ittur , S . D ., P a te l , A . S ., K a z a z ia n , H. H., J r , M e l l is ,
M . A ., C o un ts , R. B ., S tamatoyannopoulos , G., B o w ie , E. J . W ., F a s s , D . N ., P ittman ,
D . D ., W osn e y , J . M . & T oole , J . J . (1 9 8 5 ). Hemophilia A : detection of molecular defects and
of carriers by D N A analysis. N. E n g.J. M ed. Oct. 3, 8 4 2 - 8 4 8 .
A u st e n , D. E. G. & R h ym es , I. L. (1975). A L aboratory M anual o f B lood C oagulation. Oxford:
Blackwell Scientific.
B a r r o w , E. S ., M iller , C . H., R eisner , H. M . & G ra h a m , J. B. (1982). Genetic counselling in
haemophilia by discriminant analysis 1975-1980. .7- m ed. Genet. 19, 26.
B o y d , Y ., B u c k l e , V . J., M unro , E. A., C hoo , K . H., M igeon , B. R . & C r a ig , I. W . (1984).
Assignment of the haemophilia B (factor IX) locus to the q26—qter region of the X chromosome.
Ann. hum. G enet. 48, 145-152.
B u s b y , S., K u m a r , A., J oseph , M., H a l f p a p , L ., I n sl e y , M., B erkner , K ., K u r ach i , K . &
W o o d bu r y , R. (1985). Expression of active human factor IX in transfected cells. N ature, Lond.
316, 271-273.
A n so n ,
C hoo , K . H ., G eorge , G ., F ilby , G ., H a l l id a y , J . L ., L e ve r sh a , M ., W ebb , G . & D a n k s ,
D . M . (1984). Linkage analysis of X-linked mental retardation with and without fragile X using
factor IX gene probe. L ancet ii Aug., 349.
K. H., G o u l d , K. G ., R e e s , D. J. G . & B ro w n lee , G . G . (1982). Molecular cloning of
the gene for human antihaemophilic factor IX. N ature, Lond. 299, 178-180.
C hoo ,
D a v is , L . M ., M c G r a w , R . A ., G r a h a m , J . B ., R oberts , H. R . & S tafford , D . W . (1 9 8 4 ) .
Id entification of th e genetic defect in facto r IXAiabama' D N A sequence reveals a G ly su b stitution
fo r A s p 4 6 . B lood 64 (S u p p l. 1), 262a.
de l a S a l l e , H ., A ltenburger , W ., E l k a im , R ., D ott, K ., D ieterle , A ., D rillien , R .,
C a se n a v e , J.-P., T olstoshev , P. & L ecocq , J.-P. (1985). Active y-carboxylated human factor
IX expressed using recombinant D N A techniques. N ature, Lond. 316, 268-270.
G ian n e lli , F . (1 9 8 6 ). The contribution of gene specific probes to the genetic counselling of
families segregating for haemophilia B (factor IX deficiency). In B ari Int. Conf. on F a ctor
VIII/von W illebrand F a cto r -B io lo g ica l a n d C linical A dvances (ed. N. Ciavarella, Z. Ruggieri
& T. S. Zimmerman). Milan: Wichtig (in press).
G ian n e lli , F . & B ro w n le e , G . G . (1986). N ature, Lond. (in press).
Haemophilia A and B
457
G., B o y d , Y., R i z z a , C . R . & B r o w n l e e , G. G.
(1983). Gene deletions in patients with haemophilia B and anti-factor IX antibodies. N ature,
Lond. 303 , 181-182.
G ia n n e l l i , F . , C h o o , K . H ., W in s h ip , P . R ., A n s o n , D . S., R e e s , D . J . G . , F e r r a r i , N.,
R i z z a , C. R . & B r o w n l e e , G . G . (1984). Characterisation and use of an intragenic polymorphic
marker for detection of carriers of haemophilia B (factor IX deficiency). L ancet i, 239-241.
G i t s c h i e r , J ., D r a y n a , D., T u d d e n h a m , E. G . D., W h i t e , R. I. & L a w n , R. M. (1985a).
Genetic mapping and diagnosis of haemophilia A achieved through a Bell polymorphism in the
factor VIII gene. N ature, Lond. 314 , 738-740.
G i t s c h i e r , J., W o o d , W . I., G o r a l k a , T . M., W i o n , K. L ., C h e n , E . Y., E a t o n , D. H.,
V e h a r , G . A . , C a p o n , D. J. & L a w n , R. M. (1984). C h a ra c te riz a tio n o f th e h u m a n fa c to r V I I I
g e n e . N ature, Lond. 312 , 326-330.
G it sc h ie r , J., W o o d , W . I., T u d d e n h a m , E. G . D., S h u m a n , M. A . , G o r a l k a , T . M., C h e n ,
E. Y. & L a w n , R. M. (19856). Detection and sequence of mutations in the factor VIII gene of
haemophiliacs. N ature, Lond. 315 , 427-430.
G r a h a m , J. B., F l y e r , P., E l s t o n , R. C. & K a s p e r , C. K . (1979). Statistical study of genotype
assignment (carrier detection) in haemophilia B. Thromb. Res. 15 , 69.
G i a n n e l l i , F ., C h o o , K . H ., R e e s , D . J .
J a n c o , R . L . , P h il l ip s , J . A . , O r l a n d o , P ., D a v i e s , K . E ., O l d , J . & A n t o n a r a k is , S . E.
(1986). Carrier testing in haemophilia A . L ancet i, 148.
J a y e , M., d e l a S a l l e , H., S c h a m b e r , F ., B a l l a n d , A., K o h l i , V., F i n d e l i , A., T o l s t o s h e v ,
P. & L e c o c q , J .- P . (1983). Isolation of a human anti-haemophilic factor IX cDNA clone using a
unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine
factor IX. N ucl. Acids R es. 11 , 2325—2335.
K a t a y a m a , K . , E r i c s s o n , L. H., E n f i e l d , D . L ., W a l s h , K . A., N e u r a t h , H., D a v ie , E . W . &
TlTANI, K. (1979). Comparison of amino acid sequence of bovine coagulation factor IX
(Christmas factor) with that of other vitamin K-dependent plasma proteins. P roc. natn. Acad.
S ci. U.SA. 76 , 4990-4994.
K u r a c h i , K . & D a v ie , E . W . (1982). Isolation and characterization of a cDNA coding for human
factor IX. P roc. natn. Acad. Sci. U.SA. 79 , 6461-6464.
M c K e e , P . A . (983). Haemostasis and disorders o f blood coagulation. In The M etabolic B asis o f
In h erited D isease, 5th edn (ed. J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, J. L.
Goldstein & M . S. Brown), pp. 1531-1560. New York: McGraw-Hill.
M i b a s h a n , R . S., G ia n n e l l i , F., P e m b r e y , M . E. & R o d e c k , C. H. (1986). The antenatal
diagnosis of clotting disorders. In A d van ced M edicine, vol. 20 (ed. M . J. Brown). London:
Pitman (in press).
N o y e s , C. M., G r i f f i t h , M. J., R o b e r t s , H. R . & L u n d b l a d , R . L . (1983). Identification of the
molecular defect in factor IX (Chapel H ill): Substitution of histidine for arginine at position
145. P roc. natn. Acad. Sci. U.SA. 80 , 4200-4202.
O r r , E., W o z n e y , J., B u e c k e r , J., P it t m a n , D., K a u f m a n , R. & T o o l e , J. (1985). “ S p a c e r”
fu n c tio n im p lie d fo r th e h e a v ily g ly c o s y la te d re g io n o f fa c to r VIII. Thromb. H aem ostas. 54 , 54.
P e a k e , I. R ., F u r lo n g , B. L . & B loom , A . L . (1 9 8 4 ). C arrier detection b y gene analysis in a
fa m ily w ith haem ophilia B (factor I X deficiency). L ancet i, 24 2.
D. J. G., R i z z a , C. R . & B r o w n l e e , G. G. (1985). Haemophilia B caused by a point
mutation in a donor splice junction of the human factor IX gene. N ature, Lond. 316 , 643-645.
T o o l e , J. J., K n o p f , J. L ., W o s n e y , J. M ., S u l t z m a n , L. A . , B u e c k e r , J. L., P it t m a n , D . D .,
K a u f m a n , R. J., B r o w n , E., S h o e m a k e r , C ., O r r , E. C ., A m p h le t t , G. W ., F o s t e r , B . W .,
C o u , M . L ., K n u t s o n , G. J., F a s s , D . N . & H e w ic k , R. M . (1984). M o le c u la r c lo n in g o f a
c D N A e n c o d in g h u m a n a n ti-h a e m o p h ilic fa c to r. N ature, Lond. 312 , 342-347.
W e a t h e r a l l , D. J. (1982). The N ew G enetics a n d C linical P ra ctice. London: Nuffield Provincial
Hospitals Trust.
W e a t h e r a l l , D. J., H ig g s , D. H ., W o o d , W . G. & C l e g g , J. B. (1984). Genetic disorders of
human haemoglobin as models for analysing gene regulation. Phil. Trans. R. S oc. Lond. B 307,
247-259.
W in s h ip , P. R ., A n s o n , D. S . , R iz z a , C. R . & B r o w n l e e , G. G. (1984). Carrier detection in
haemophilia B using two further intragenic restriction fragment length polymorphisms. Nucl.
A cids R es. 12 , 8861-8872.
R ees,
458
G. G. Brownlee
W . I . , C a p o n , D . J., S im o n se n , C . C ., E a t o n , D . L . , G it sc h ie r , J., K e y t , B ., S e e b u r g ,
H ., S m it h , D. H ., H o l l in g s h e a d , P., W io n , K . L . , D e l w a r t , E ., T u d d e n h a m , E . G . D.,
V e h a r , G . A . & L a w n , R. M. (1984). Expression of active human factor V I I I from recombinant
D N A clones. N ature, Lond. 312, 330-337.
Y o sh t t a k e , S . , S c h a c h , B. G., F o st e r , D . C., D a v i e , E. W. & K u r a c h i , K . (1985). Nucleotide
W
ood,
P.
sequence of the gene for human factor IX (antihaemophilic factor B). B iochem istry 24, 3736.