Paul L F Giangrande

Introduction: clinical features of hemophilia, 184 Inheritance of hemophilia, 185 Molecular basis of hemophilia A, 186 Molecular basis of hemophilia B, 188

Therapeutic applications of molecular biology to patient care, 190 Antenatal diagnosis of hemophilia, 191

Recombinant blood products, 193 Gene therapy for hemophilia, 195 Conclusions, 197

Hemophilia resources on the Internet, 197 Further reading, 197

Introduction: clinical features of hemophilia

Hemophilia is a congenital disorder of coagulation and affects approximately 1 in 10 000 males worldwide, with approximately 5000 patients with hemophilia in the United Kingdom. Hemophilia A is due to a deficiency of factor VIII in the circulating blood, and hemophilia B (also known as Christmas disease) is a clinically identical disorder caused by factor IX deficiency. Typical laboratory findings in hemophilia include a normal prothrombin time but prolonged activated partial thromboplastin time. The platelet count and bleeding time are normal. A specific factor assay is required to confirm the diagnosis.

The clinical severity (phenotype) is critically determined by the level of circulating factor VIII (or IX) in the blood, and severe hemophilia is defined by a clotting factor level of <1 IU/ dl (Table 16.1). The hallmark of severe hemophilia is recurrent and spontaneous hemarthrosis. Typically, hinge joints such as the knees, elbows and ankles are affected, but bleeds

Table 16.1 The relation of blood levels of factor VIII (or IX) to the

severity of hemorrhagic manifestations.

Level (IU/dl)

Hemorrhagic manifestations

50-100

Normal level, no bleeding problems

25-50

No problems in day-to-day life. Tendency to

bleed after major surgery

5-25

Mild hemophilia. Bleeding typically occurs only

after significant injury

2-5

Moderately severe hemophilia; occasionally

apparently spontaneous bleeds. Most bleeds as

sociated with injury, albeit often relatively minor

<1

Severe hemophilia with spontaneous and recur-

rent bleeding into muscles and joints

may also occur in the wrist or shoulder. Bleeding into the hip joint is unusual. The affected joint is swollen and warm, and held in a position of flexion (Figure 16.1), with no external discoloration or bruising around the joint. It is unusual for an infant to suffer spontaneous hemarthroses in the first few months of life, and the first joint to be affected tends to be the ankle as the child learns to crawl. The first sign of a hem-arthrosis in an infant will often be obvious discomfort and distress, accompanied by limping or reluctance to use a limb. Recurrent bleeds into a joint lead to synovitis and joint damage resulting in crippling arthritis (Figure 16.2). Bleeding into muscles is also a feature of hemophilia, but this is usually a consequence of direct injury, albeit often minor (Figure 16.3).

Fig. 16.1 Acute hemarthrosis in severe hemophilia

This usually arises in the absence of injury. The joints most frequently involved are the knees, elbows and ankles. The joint is swollen, warm and tender but there is no external bruising or discoloration.

Fig. 16.1 Acute hemarthrosis in severe hemophilia

This usually arises in the absence of injury. The joints most frequently involved are the knees, elbows and ankles. The joint is swollen, warm and tender but there is no external bruising or discoloration.

Hemophilia Bone Marrow Xray

Fig. 16.2 Radiograph of the knee of a patient with severe hemophilic arthropathy

Joint replacement surgery was subsequently carried out in this case.

Fig. 16.2 Radiograph of the knee of a patient with severe hemophilic arthropathy

Joint replacement surgery was subsequently carried out in this case.

ronif s

Fig. 16.3 Magnetic resonance imaging (MRI) scan showing bilateral iliopsoas hemorrhage

This bleed was associated with complete but transient paralysis in both legs, as the femoral nerve is located on the anterior surface of the muscle and may be compressed in such cases.

Fig. 16.3 Magnetic resonance imaging (MRI) scan showing bilateral iliopsoas hemorrhage

This bleed was associated with complete but transient paralysis in both legs, as the femoral nerve is located on the anterior surface of the muscle and may be compressed in such cases.

Bleeds into certain areas are particularly dangerous because of the risk of compression of neighboring structures. Patients with inhibitory antibodies are particularly at risk in this regard as bleeds may be more difficult to control. Bleeds in the tongue can obstruct the airway, and retroperitoneal bleeding within the iliopsoas muscle may result in femoral nerve compression, causing weakness and wasting of leg muscles (Figure 16.3). Bleeding from the gastrointestinal tract (melena) and bleeding into the urinary tract (hematuria) may also occur. There is also a significant risk of intracranial hemorrhage in severe hemophilia, which was a significant cause of mortality in the past when treatment was not so readily available. Higher levels of factor VIII (or IX) above 5 IU/dl are associated with a milder form of the disease, with no spontaneous joint bleeds but a definite risk of bleeding after even relatively minor injury.

Treatment of bleeding episodes involves the intravenous injection of coagulation factor concentrates; the total dose and frequency of treatment will be determined by the severity and site of bleeding. The great majority of joint bleeds will resolve with a single infusion of material if the bleed is recognized early and treated promptly. There is an increasing move to prophylactic therapy, in which the patient gives himself injections of coagulation factor concentrate two or three times a week to prevent bleeds rather than just treating on demand when bleeds occur. Patients on prophylactic therapy experience few or even no spontaneous bleeds, and thus progressive joint damage and arthritis can be avoided. The quality of life of patients on prophylaxis may be greatly enhanced, allowing them to lead much more independent lives.

Approximately 15% of patients with severe hemophilia A can be expected to develop inhibitory antibodies to factor VIII at some stage. Inhibitor development in hemophilia B is, by contrast, very rare and encountered in fewer than 1% of patients. The development of such antibodies poses considerable problems in treatment as these immunoglobulins (IgG) are capable of rapidly inactivating infused factor VIII; furthermore, the antibody titer may rise dramatically after a course of factor VIII. Very occasionally, acquired hemophilia may arise in a previously normal individual, due to the formation of autoantibodies directed against factor VIII, and both males and females may be affected. Hemarthrosis is unusual in acquired hemophilia, and the principal manifestations are usually extensive superficial purpura and muscle bleeds. Acquired hemophilia arises most often in the elderly, and there is an association with underlying malignant or autoimmune diseases.

Inheritance of hemophilia

The genes for factors VIII and IX are both located at the telo-meric end of the X chromosome and thus hemophilia is inherited as an X-linked recessive condition (Figure 16.4). The daughters of affected males are obligate carriers but the sons are normal. The phenotype remains constant within a family, so the daughter of a man with only mild hemophilia may

Fig. 16.4 The inheritance of hemophilia

The genes for factors VIII and IX are both on the X chromosome, and inheritance is thus sex-linked. The daughter of a man with hemophilia is an obligate carrier but the son of a hemophiliac will not be affected. Hemophilia may thus be transmitted to a grandson via a carrier daughter. (Color blindness and Duchenne muscular dystrophy are other examples of X-linked disorders.

Fig. 16.4 The inheritance of hemophilia

The genes for factors VIII and IX are both on the X chromosome, and inheritance is thus sex-linked. The daughter of a man with hemophilia is an obligate carrier but the son of a hemophiliac will not be affected. Hemophilia may thus be transmitted to a grandson via a carrier daughter. (Color blindness and Duchenne muscular dystrophy are other examples of X-linked disorders.

be reassured that she will not pass on a severe form of the condition. However, approximately one-third of all cases of hemophilia arise in the absence of a previous family history and are due to a new mutation. The most famous example is that of Queen Victoria, who had a hemophilic son (Leopold), and two daughters (Alice and Beatrice) who turned out to be carriers. There are instances of hemophilia affecting females due to inheritance of the defective gene from both parents, and there are also case reports of hemophilia in females with Turner's syndrome (XO karyotype) and androgen insensitiv-ity syndrome (testicular feminization, XY karyotype).

Molecular basis of hemophilia A

Factor VIII is an essential cofactor for the activation of factor X by activated factor IXa (see Chapter 15). Factor VIII must itself undergo proteolytic cleavage at two distinct sites through the action of thrombin before it becomes physiologically active. It circulates in plasma as a large glycoprotein bound non-

covalently to the larger protein, von Willebrand factor. The factor VIII gene was first cloned in 1984. It is 186 kb in length and is situated on the long arm of the X chromosome at Xq28 (Figure 16.5). The factor VIII gene consists of 26 exons, of which exon 14 is the largest, and 25 introns. The mature factor VIII protein is made up of 2332 amino acids.

By far the commonest single genetic defect causing severe hemophilia is an inversion in intron 22, which is encountered in as many as 45% of people with severe hemophilia in all ethnic groups (Figure 16.6). The inversion mechanism involves an intronless gene of unknown function, designated F8A. Two copies of this gene are located near the tip of the X chromosome and there is another copy within intron 22 of the factor VIII gene itself. During meiosis, either of the two telomeric copies may cross over with the intronic copy, resulting in a division of the gene into two halves facing in opposite directions and separated by approximately 500 kb. Cross-over with the distal copy is much more common than cross-over with the proximal copy, and accounts for approximately 80% of all inversions. It is now recognized that inversion almost

Fig. 16.5 The factor VIII gene

The factor VIII gene was cloned in 1984 and is located towards the telomeric end of the long arm of the X chromosome (Xq28). It maps distal to the gene encoding glucose-6-phosphate dehydrogenase (G6PD), about 1 Mb from the Xq telomere. The gene is composed of 26 exons, of which exon 14 is the largest. The large intron 22 contains a nested intronless gene termed F8A (the function of the gene and its transcript are unknown). There are two further copies of F8A located ~400 kb telomeric to the factor VIII gene. The spliced factor VIII mRNA is ~9 kb in length and the mature factor VIII molecule is composed of 2332 amino acids.

X chromosome q28

qter 100 kb

10 kb

Factor VIII

G6PD

1 2 3456 78910111213 141516171819 20 21 22 23 24 25 26

Exon 22

F8A F8B

always occurs during a male meiosis. It is believed that the presence of a large region of non-homology between the X and Y chromosomes during meiotic pairing may favor a misalignment, and the presence of a second X chromosome with a complementary region may act as a stabilizing factor. An important clinical consequence of this observation is that, when an apparently new and spontaneous case of hemophilia is diagnosed in which the gene inversion is identified, it is likely that the defect arose in the maternal grandfather's allele; thus, the mother can generally be assumed to be a carrier and at risk of having another affected male child. The resulting truncated protein product is presumably unstable, resulting in severe hemophilia. The inversion is not found in individuals with mild forms of hemophilia.

The inversion is easily detected and the identification of this defect as the commonest cause of severe hemophilia has simplified both screening for carriers and the antenatal diagnosis of hemophilia (Figure 16.7). Using the restriction enzyme BclI, three bands are identified in normal individuals. The 21.5-kb band is contributed by intron 22 and the 14- and 16-kb bands are derived from the proximal and distal repeats, respectively. The inversion may be identified and classified according to the appearance of abnormal bands representing the products of recombination (Figure 16.7). More recently, inversions in intron 1 of the factor VIII gene have been identified as a cause of severe hemophilia. On the basis of the limited data currently available, this abnormality appears to be responsible for approximately 5% of all cases of severe hemophilia.

Developments in molecular biology have permitted the more rapid identification of defects in hemophilia. Southern blotting has been superseded by methods involving polymerase chain reaction (PCR) amplification of either patient DNA or material derived from the reverse transcription of mRNA (RT-PCR). Although automated sequence analyzers

Fig. 16.6 'Flip tip inversion'

This unique inversion of the tip of the X chromosome is now recognized to be responsible for about half of all cases of severe hemophilia in all ethnic groups. Cross-over occurs between a copy of F8A within the factor VIII gene and one of the two telomeric copies. Cross-over with the distal copy is more common, occurring in approximately 80% of cases where an inversion is identified.

cen S-i q28

FVIII-

F8B F8A

F8A F8A

tel

tel tel

Normal

Proximal inversion

Distal inversion

21.5 kb

Inversions within intron 22 of the factor VIII gene are by far the commonest cause of severe hemophilia, and are detected in almost half of all cases. This figure shows the results of Southern blot analysis using the BclI restriction enzyme. The normal pattern is shown on the left. A female carrier with an inversion will show a combination of the normal and either the proximal or the distal inversion pattern.

Inversions within intron 22 of the factor VIII gene are by far the commonest cause of severe hemophilia, and are detected in almost half of all cases. This figure shows the results of Southern blot analysis using the BclI restriction enzyme. The normal pattern is shown on the left. A female carrier with an inversion will show a combination of the normal and either the proximal or the distal inversion pattern.

have been developed, gene sequencing of the entire factor VIII gene is both expensive and labour-intensive. Methods have been developed to identify restricted areas of abnormal DNA in patients with hemophilia, which may then be targeted for specific attention. These methods include amplification and mismatch detection (AMD), conformation sensitive gel electrophoresis (CSGE) and denaturing gradient gel electrophoresis (DGGE). Approximately 4% of cases of hemophilia are the consequence of gene deletions, which have been reported throughout the gene and which are very variable in size. As with the intron 22 inversion, most deletions are associated with a severe clinical phenotype. Frameshift mutations resulting from insertions or small deletions have also been identified as a cause of severe hemophilia. Most other cases of both severe and mild hemophilia are associated with single point mutations, and approximately 200 missense mutations have been identified as causing hemophilia A. A full list is outside the scope of this chapter, but a list of Further reading is provid ed at the end. Such mutations affect RNA processing, mRNA translation or the fine structure of factor VIII itself. Nonsense mutations result in the formation of stop conditions and the production of truncated factor VIII molecules devoid of any functional activity; for example, TGG(Trp)^TGA(Stop) at nucleotide 255 in exon 7, and CGA(Arg)^TGA at nucleotide 1941 in exon 18. Missense mutations that involve critical sites will also result in hemophilia A; for example, the Arg372^His or Cys and Ser373^Leu mutations, which involve a thrombin cleavage site. Approximately 40% of all missense mutations arise at CG dinucleotide sites, resulting in a change to TG or CA sequences. It is generally believed that CG nucleotides represent genomic hotspots. Cytosine is predominantly methylated in human DNA, but this is relatively unstable and 5-methylcytosine is prone to spontaneous deamination to yield a GT mismatch which is inefficiently repaired. It is also of interest that a missense mutation may be associated with varying degrees of clinical severity. Thus a C^T mutation at nucleotide 1689 has been reported in association with both severe and moderately severe phenotypes. Similarly, a Val326^ Leu substitution has been reported in individuals with either a severe or a moderately severe phenotype.

Molecular basis of hemophilia B

The factor IX gene is also located on the long arm of the X chromosome at band Xq27, and is encoded by a stretch of DNA approximately 34 kb long which contains eight exons (Figure 16.8). The basic structure of the gene is similar in organization to those of protein C and coagulation factors VII and X, and it is likely that they all originated in the distant past from a common ancestral gene by duplication. Factor IX is a polypeptide of 415 amino acids, and is made up of a glutamic acid-rich sequence (Gla domain) and two epidermal growth

30 kb

Exons

3947

128145 180

F.IX gene

H EGF-type B EGF-type A

Catalytic

F.IX protein

Fig. 16.8 The factor IX gene

The factor IX gene was first cloned in 1982. It is located on the long arm of the X chromosome at band Xq27. The gene spans 34 kb and contains eight exons (exons are shown as shaded boxes, and dotted lines between the gene and protein indicate protein domains encoded by each exon). The signal peptide and propeptide sequences are cleaved during the processing and activation of factor IX.

factor (EGF)-like domains separated from the serine protease domain by an activation region. The 12 glutamic acid residues in the Gla domain undergo post-translational y-carbox-ylation, which is necessary for binding of calcium, and exon 2 encodes a recognition site for the carboxylase. Exon 1 encodes the signal peptide necessary for transport into the endoplas-mic reticulum. Exon 6 encodes the activation peptide that is cleaved off during the activation of factor IX by either factor XI or a complex of tissue factor and factor VII. Exons 7 and 8 encode the catalytic regions of factor IX, which are responsible for the subsequent activation of factor X in the coagulation cascade. The gene is controlled by a promoter.

The gene for factor IX, which was cloned in 1982, is considerably smaller than that for factor VIII, and patients with hemophilia B have been studied more extensively than those with hemophilia A. The first defects identified in hemophilia B were gross deletions, detected by Southern blotting. However, it is now recognized that gene deletions account for only approximately 3% of all cases of hemophilia B. No equivalent of the factor VIII gene inversion has been encountered in hemophilia B and it is now clear that point mutations account for the vast majority of cases of hemophilia B; over 500 have been described from families around the world. The great majority involve single base changes, which have been identified in all domains of the protein. The unusually high frequency of mutations at CG dinucleotide sites in hemophilia B probably reflects the high number of CG dinucleotides at critical sites in the factor IX gene.

Mutations involving the glutamic acid residues within the Gla domain (residues 1-38) result in severe hemophilia, emphasizing their functional importance; for example, Glu7^ Val and Glu17^Lys. Mutations within exon 6 result in hemophilia through disruption of the activation of the factor IX molecule; for example, CGT^TGT at position 20 413 results in Arg145^Cys. Most cases of hemophilia B are attributable to mutations within exons 7 or 8, resulting in impaired catalytic activity; for example, a change from AGT to AGA at nucleo-tide 31 216 results in a change from Ser365 to Arg within the active site. The original case of Christmas disease has been identified as a TGT^TCT mutation at nucleotide 31 170 resulting in a change from Cys206 to Ser within exon 8. The creation of nonsense mutations will lead to severe hemophilia B due to the production of ineffective, truncated proteins; for example, CGA(Arg)^TGA(Stop) at nucleotide 30 863 and TGG(Trp)^TGA(Stop) at nucleotide 31 051 in exon 8.

A few patients with hemophilia B have been described in whom the level of factor IX rises significantly after puberty, and this is associated with loss of the bleeding tendency. Several point mutations have been reported in association with this interesting variant, referred to as the hemophilia B Leiden phenotype. All are located in the promoter region of the factor IX gene; for example, TTG^TAG at -20 and G^

A at nucleotide -6. Most of these mutations have been shown to be located in regions which contain binding sequences for liver-enriched transcription factors, which are presumably influenced by androgenic steroids.

Inhibitor formation: etiology and clinical implications

A minority of patients with hemophilia will develop immu-noglobulins directed against infused factor VIII (or IX) after exposure to these blood products for treatment of bleeding episodes. This is potentially very serious, as patients will be refractory to conventional doses of coagulation factor concentrates and bleeding will be difficult to control. Porcine factor VIII (Hyate:C), prothrombin complex concentrates (e.g. FEIBA) and recombinant activated factor VIIa (Novo Seven; Novo Nordisk) are valuable therapeutic materials in controlling bleeding in patients with inhibitory antibodies. Another important strategy in the management of patients who develop inhibitory antibodies is immune tolerance, which involves the daily administration of coagulation factor concentrate over a period of some months. This usually results in the eventual disappearance of the antibody, as the body becomes tolerant of the protein and inhibitor formation is suppressed.

Inhibitory antibodies interfere with the normal function of factor VIII in a number of different ways. The most frequent site of inhibitor binding occurs within the A2 and C2 domains. Inhibitors may thereby block the ability of activated factor VIIIa to bind and activate factors IXa and X, or inhibit the binding of factor VIII to von Willebrand factor or negatively charged phospholipid surfaces. Inhibitors may also hinder the activation of factor VIII by thrombin, or the subsequent release of factor VIII from von Willebrand factor. Proteolysis of factor VIII has recently been identified as a novel additional mechanism of inactivation in some cases.

Data from the UK registry show that 14% of all patients with severe hemophilia A have developed antibodies at some time, but it is quite likely that this figure underestimates the true prevalence as transient and low-titer inhibitors may not be detected. As a general rule, if an individual is susceptible to inhibitor development, this will become apparent at a fairly young age. Data from prospective studies involving recombinant factor VIII products suggest a median of approximately 10 exposure days for inhibitor development if this is to occur. It is now clear that the major factor that determines the predisposition to inhibitor development is the underlying molecular defect. However, there is also additional evidence from family and twin studies that other subtle genetic factors play a role, although no associations with specific human leukocyte antigens (HLA) or other linkages have been conclusively identified. Race may influence the risk of inhibitor development,

HIGH RISK

multi domain

Large deletions

Light chain Nonsense mutations Single domain ^n 22

Heavy chain .

inversions

LOW RISK

Non A-run

Small C1-C2-junction deletions Missense

A-rUn Non C1-C2-junction

Splice site mutations and several studies have shown that people of Afro-Caribbean origin are more susceptible to inhibitor formation.

Certain types of gene defects in hemophilia are undoubtedly associated with a significantly increased risk of inhibitor development (Figure 16.9). The risk of inhibitor development in patients with severe molecular defects, such as large deletions, nonsense mutations and the intron-22 inversion, is 7-10 times higher than in patients with other defects, such as missense mutations, small deletions and splice site mutations. The overall risk of inhibitor development in patients with the common intron-22 inversion is approximately 30%. Further studies will be required to determine the risk of inhibitor development in association with the newly reported intron-1 inversion, although 1 of 10 (10%) subjects in the original publication was known to have developed an inhibitor.

Inhibitor development in hemophilia B is a relatively rare event, occurring in probably fewer than 1% of patients, and these patients often have an underlying large gene deletion. By contrast with the immunoglobulin inhibitors in patients with hemophilia A, inhibitory antibodies in patients with hemophilia B are often capable of fixing complement proteins. The administration of coagulation factor concentrate in such cases may therefore trigger severe, often anaphylactic, allergic reactions. The development of nephrotic syndrome has also been reported in patients with hemophilia B and inhibitors who are undergoing immune tolerance induction treatment with factor IX concentrates.

Therapeutic applications of molecular biology to patient care

Carrier testing

Ideally, carriers of hemophilia should be identified before a

Fig. 16.9 Mutation types and the risk of inhibitor development pregnancy, and offered counseling. The inheritance of hemophilia is sex-linked, as it is for other disorders, such as color blindness and Duchenne muscular dystrophy. The daughters of men with hemophilia are thus obligate carriers of the condition, with a 50:50 chance of passing on the condition to a son, and there is a similar chance that a daughter of a carrier will also herself be a carrier of the condition. No special genetic tests are therefore required to determine the carrier status of daughters of men with hemophilia, although the results of DNA-based studies are likely to be useful for subsequent antenatal diagnostic procedures. The phenotype of hemophilia remains constant within a family, so that the daughter of a man with only mild hemophilia may be reassured that she can only transmit a similarly mild form of the condition. However, a more common problem is to be confronted with a woman with only a vague history of a bleeding disorder in a distant relative. National patient registers are particularly useful in such circumstances and may help to establish the type and severity of bleeding disorder of an affected relative. It may seem logical to initiate carrier testing to determine carrier status as soon as possible in girls with a family history of the condition, as this would facilitate the management of pregnancy in the case of an early and possibly unexpected pregnancy. It is clear that there are significant differences of opinion between geneticists and hematologists with regard to the timing of testing of children for genetic disorders. Clinical geneticists in the UK usually take the view that it is unethical to test healthy young children to determine carrier status for inherited disorders for conditions which have no immediate implications for their own health. There are also legal implications in the UK, as testing of young children ignores the rights of children with respect to the Children Act (1989), and testing cannot be considered to have been obtained with the informed consent of the individual child concerned. By contrast, guidelines from the Genetics Working Party of the UK Haemophilia Centre

Directors' Organisation (UKHCDO) conclude that carrier testing in young children may be appropriate, but that the issues must be discussed openly with the family.

From a theoretical point of view, the ideal approach to identifying carriers is to characterize the precise genetic defect responsible for hemophilia within a family. Once the molecular defect has been identified in an individual with hemophilia A or B, direct screening for that defect could be applied in subsequent generations for both carrier testing and antenatal diagnosis. This direct method is clearly the method of choice but if the gene defect is unknown and it is not possible to establish this with the facilities available, indirect methods can still be used to determine carrier status. This involves the tracking of intragenic or extragenic polymorphisms in families which act as convenient markers for the molecular defect causing hemophilia within a family. The method is based on the fact that there are some genetic polymorphisms which represent natural variations of the genome sequence but have no adverse impact on the function of the molecule. Ideally, such polymorphic sequences should be intragenic, but closely linked extragenic polymorphisms may also be exploited. If extragenic markers are relied on, the small possibility of recombination may also result in erroneous diagnoses. The polymorphisms are detected by cleavage of patient DNA with restriction enzymes, which generate fragments of varying size according to the presence or absence of the polymorphism (Table 16.2). The fragments are detected by either PCR-based methods or Southern blotting. Figure 16.10 shows the results of restriction fragment length polymorphism (RFLP) analysis in one family, and illustrates how the method may permit identification of carriers. Limitations of this approach include the fact that samples have to be taken from several members to permit the tracking of the mutant gene responsible for hemophilia in the family. Blood from an affected family member will be required, and this may not be possible in some cases where early death from complications such as HIV infection has occurred. Furthermore, non-paternity may confound attempts to track the gene in families. It should also be ap preciated that there is ethnic variation in the allelic frequencies of the various polymorphisms (more so with factor IX than factor VIII), and this may influence the choice of probes used in some family studies. For example, the allelic frequencies with BclI and HindIII are reversed in African-Americans compared with Caucasians. BglI is more likely to be informative in people of Afro-Caribbean origin but it is not likely to be informative in families of Chinese origin. RFLP analysis is likely to be informative in the majority of families, but occasionally carrier status cannot be determined even with multiple probes. The commonest problem is homozygosity for all markers in the proband's mother. However, the recent identification of hypervariable dinucleotide tandem repeats (VNTR) in introns 13 and 22 has also helped to identify a greater proportion of carriers. Once the carrier status has been determined and DNA markers have been identified, it is then possible to offer antenatal diagnosis of hemophilia to pregnant women. In the past, measurement of the ratio of the factor VIII level to that of von Willebrand factor antigen has been used to estimate the probability of a woman being a carrier of hemophilia. Such tests no longer have a role in the modern era of genetic testing, although it may certainly be useful to establish the level of factor VIII in a potential carrier; if it is low, this would have implications for management in the setting of surgery and other invasive procedures.

Antenatal diagnosis of hemophilia

As a general rule, it is the practice in the UK to perform antenatal procedures to determine whether a fetus has hemophilia only when a termination is being contemplated. The general experience in the UK has been that only a minority of women subsequently take up the offer of antenatal diagnosis with a view to termination if an affected fetus is identified. This may well reflect the fact that many women with affected relatives appreciate that major advances in treatment in recent years, such as the introduction of recombinant products for children

Table 16.2 Factor VIII intragenic DNA polymorphisms.

Site

Restriction enzyme

Southern blot allele

Allelic frequency

5' flanking region

TaqI

9.5 kb

72%

4.0 kb

28%

Intron 18

BclI

1.1 kb

29%

0.88 kb

71%

Intron 22

Xbal

6.2 kb

41%

4.8 + 1.4 kb

59%

3' of exon 26

BglI

20 kb

10%

5 kb

90%

Bcl I RFLP Xba I RFLP Bgl II RFLP

II:1 Robin

Bcl I RFLP Xba I RFLP Bgl II RFLP

0.88

II:2 Anne

I:2 Mary

II:3 Brian

II:2 Anne

II:3 Brian

11:4 Helen

Bcl I RFLP Xba I RFLP Bgl II RFLP

1.1

0.88

0.88

1.1

0.88

1.1

4.8

4.8

6.2

6.2

4.8

1 4.8

5.8

5.8

_

_

2.8

_

2.8

5.8

_

5.8

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