The Molecular Pathology oC Hemophilia A David LiIIicrap

UR UNDERSTANDING of the molecular abnormalities that affect the coagulation cofactor, factor VIII, and give rise to the inherited clotting disorder, hemophilia A, has increased impressively over the past decade. This previously frustrating area of medical research took on a new lease of life in 1984 with the simultaneous publication by two biotechnology companies of the factor VIII gene sequence. 1 ,2 This information has made possible many studies that have brought us closer to understanding the role that this protein plays in normal hemostasis. This review will focus on several selected areas of progress relating to the molecular pathology of hemophilia A, and reference will be made to additional literature to supplement this article as deemed appropriate,

O

THE FACTOR VIII GENE

The gene that encodes factor VIII is situated at the telomeric end of the long arm of the X chromosome at band Xq28. A number of other important genetic loci, including the deutan and protan color vision genes, the gene encoding glucose6-phosphate dehydrogenase, and the abnormal genes implicated in adrenoleukodystrophy, Emery-Dreifuss muscular dystrophy and Hunter's syndrorne, are also found in this area of the genome. The gene encompasses 186 kilobases (kbp) of germ line DNA on the X chromosome (0.1 % of the DNA sequence on this chromosome) and comprises 26 exons ranging in size from 69 basepairs (bp) to 3.1 kbpl (Fig 1). AlI the invariant splice donor and acceptor sequences conform to the 5' GT/AG 3' rule, and the flanking sequences are in general agreement with other reported nucleotide frequencies. A GATAAA sequence is present 30 bp 5' of the presumed transcriptional start site and probably represents an alternative TATA sequence for transcriptional initiation. From the Departments oj Pathology and Medicine, Queen's University, Kingston. Ontario, Canada. Supported in part by the Ontario Ministry oj Health and the Canadian Medical Research Council. Address reprint requests to David Lillicrap. MD, Departments oj Pathology and Medicine, Queen' s University. Richardson Laboratory, Stuart St, Kingston, Ontario. Canada K7L 3N6. Copyright © 1991 by W.B. Saunders Company 0887-796319110503 -0008$3 .0010

196

There is no CAAT sequence in the -70 bp region. At the 3' end of the gene is a large 1.8 kbp Untranslated sequence that has a classic AATAAA polyadenylation signal 19 bp 5' of the messenger RNA (mRNA) cleavage site. The gene encodes a mature factor VIII mRNA of 9 kbp and the synthesis of the unprocessed transcript may be expected to take up to 3 hours. THE FACTOR VIII PROTEIN

The primary translation product of the factor VIII gene is a single chain polypeptide of 2,351 amino acids (Fig 1).3 The amino terminal 19 residues of this protein constitute a typical signal sequence with a central hydrophobic region. As with other secreted proteins, this domain is essential for the translocation of the nascent primary translation product across the membrane of the endoplasmic reticulum (ER) before its cleavage by a signal peptidase. The factor VIII protein sequence comprises several repeat domains that are homologous to sequences found in the other coagulation cofactor, factor V, and in some seemingly unrelated proteins. 3,4 The three A domains of approximately 350 amino acids share 30% internal homology and a similar homology with both factor V and the copper-binding protein, ceruloplasmin. The amino-terminal Al and A2 domains are separated from the A3 domain by the 980 amino acid B domain that is encoded exclusively by the large 3.1 kbp exon 14. The function of this connecting peptide, which contains 19 of the potential N-linked glycosylation sites in factor VIII, remains unknown. 5 At the carboxyl terminus of the protein are two approximately 150 residue C domains. These sequences share a 20% homology with the protein discoidin I, a ga1actose-binding lectin from the slime mold Dictyostelium. This similarity suggests that these sequences may play a role in the binding of factor VIII to phospholipid surfaces. THE BIOSYNTHETIC PROCESSING OF FACTORVIII

The cellular site of synthesis of factor VIII has long been a subject of debate. The combination of mRNA detection studies, immunohistochemistry, and the results of liver transplantation all indicate Transfusion Medicine Reviews, Vol

v,

No 3 (July), 1991: pp 196-206

MOLECULAR PATHOLOGY OF HEMOPHILlA A

FACrOR ~ GENE

197

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s.P. Fig 1. The factor VIII gene and protein. The 186 kbp factor VIII gene encodes a single-chain glycoprotein of molecular weight 265 kd. The primary translation product comprises a 19 amino acid signal peptide (SP) and a secreted protein that has three types of domain structures. designated A. B. and C. During the processing of factor VIII. the central B domain is released by proteolytic cleavage. resulting in a metal cation bridged complex of an N-terminal heavy chain and carboxyl-terminal light chain. which subsequently undergo further limited proteolysis by thrombin to achieve fuli cofactor expression.

that celIs within this organ are an important site of factor VIII production. 6-9 Further to this statement, it would appear that the hepatocyte is the best candidate celI within the liver parenchyma for factor VIII synthesis. 8 Nevertheless, in addition, there is good evidence for several sites of extrahepatic factor VIII production inc1uding kidney, spleen, lymph nodes, and placenta. 6 As detailed previously, the primary translation product is cotranslationally transported to the lumen of the ER and the signal peptide that directs this translocation is c1eaved from the secreted protein. High mannose oligosaccharrides are added in the ER, and it appears that a large percentage of the synthesized protein complexes with the glucose regulated protein GRP78 (also referred to as immunoglobulin binding protein [BiPJ) and remains sequestered in this organelle. 10 The purpose of this binding activity is unresolved and may either retard or retain improperly conformed factor VIII or may in some way facilitate transport through the ER. The protein that reaches the Golgi complex undergoes modification of the N-linked glycosylation sites and addition of carbohydrate at O-linked sites. In addition, tyrosine sulphation takes place at essential sites in the acidic regions between domains Al and A2 and in the amino terminal region of the A3 domain. 11 ,12 These same acidic regions, which are not found in factor V, mayaiso play a role in the interaction of factor VIII with von Willebrand factor (vWF). The N-terminal acidic region extends from residue 336, the inactivation c1eavage site for protein C, to residue 372, one of the two activating thrombin

c1eavage sites. 13.14 In the light chain, the second acidic region extends from the N-terminal residue 1,649 to the second thrombin c1eavage site at residue 1,689. 13 • 14 Site-directed mutagenesis studies have shown that deletion of the light chain acidic region abolishes binding of vWF to factor VIII, whereas deletion of the heavy chain region abolishes VIIIa cofactor activity but retains vWF binding capability. J 1 Therefore at present, it appears that the heavy and light chains of factor VIII are secreted from the celI of synthesis and that their stable association, in a metal ion bridged complex, is promoted by the binding of vWF to probab1y both the light and heavy chain acidic domains. HEMOPHILlA A MUTATIONS

The c10ning of the factor VIII gene in 1984 opened the way for a systematic study of the molecular genetic pathology that underlies this disorder. This review will not provide a detailed list of the mutations that have been identified to date, as this is available in several recent reviews on factor VIII molecular pathology. 15·20 Instead, this article will focus on some of the themes that are emerging from the studies that have been performed in laboratories around the world on c10se to 1000 hemophilic genes. The initial studies performed after the characterization of the factor VIII gene involved Southem blotting with complementary DNA (cDNA) probes. These investigations demonstrated that approximately 5% of patients had gross genetic deletions involving their factor VIII genes, and a fur-

DAVID L1LLlCRAP

198

ther 5% had point mutations that coincided with one of the commonly used restriction endonuclease target sequences (Table 1). Gene Deletions

The gene deletions resulting in hemophilia A vary in size, from one report of a complete deletion of the factor VIII sequence to multiple reports of defects that remove from 1 to 22 exons from the gene. AlI but one of these patients has severe hemophilia A. The exception to this finding was a patient who has a 5.5 kbp deletion removing exon 22 but preserving the normal reading frame for the protein. 21 The truncated protein retains some factor VIII activity, and the phenotype in this patient is of only moderate severity. At the time that these deletion mutants were first identified, a similar colIection of studies had been reported in hemophilia B patients. In the hemophilia B patients, these initial reports had alluded to an association between genetic deletions as a form of mutation and the occurrence of acquired antifactor IX alloantibodies folIowing treatment with clotting factor concentrates. 22 This association has subsequently been disproved, and similarly, in hemophilia A, there is no direct correlation between gross gene deletions and the development of factor VIII alloantibodies. The incidence of deletion mutants in hemophiIia Ais likely, at least in part, to be a function of the size of the gene. For example, in Duchenne type muscular dystrophy, partial, gross deletions of the large dystrophin gene (>2.3 megabases) comprise 60% to 70% of the total mutations at this 10cus. 23 Other aspects of the genetic sequence, including the presence of repetitive elements and the chromosomal 10cation of the gene, also appear to influence the frequency of deletion mutations in disorders such as a-thalassemia24 and steroid sulphatase defidency. 25 Gene lnsertions

Kazazian et a1 26 at Johns Hopkins University have reported two de novo mutations in patients with severe hemophilia A, involving the insertion of LI repetitive elements into exon 14 of the factor VIII gene. Both these insertions (3.8 and 2.3 kbp) contained regions of the 3' sequence from an Li element, including a poly A tract, and in each case, at least 12 or 13 nucleotides of the factor VIII sequence had been duplicated at the site of inser-

Table 1. Types o, Mutation Resulting in Hemophilia A Detectable by Southern analysis Gross deletions or insertions (= 5% oftotał) Com mon restriction site mutations (= 5% o, totał) Subtle mutations requiring PCR-based characterization* 90% to 95% ot all hemophilia A mutations * See Table 2.

tion. As the sequence of Li elements includes two open reading frames, the second of which encodes a potential protein with homology to reverse transcriptase, LI elements may be implicated in insertional mutagenesis elsewhere by retrotransposition via an RNA intermediate. Point Mutations

The initial point mutations reported in the factor VIII gene coincided with the target sequence, TCGA, for the restriction endonuclease Taql. 27-29 This sequence contains a CpG dinucleotide in which the cytosine residue is frequently modified as 5-methylcytosine. Deamination of 5-methylcytosine to thymine has been documented as a frequent cause of spontaneous mutation in the human genorne, and CpG dinucleotides have now been recognized as hypermutable sequences in several genes. 30·32 In the factor VIII gene, there are seven such sequences in the coding region. Five of these sequences have CGA as a codon for arginine, and missense or nonsense mutations have been detected at each of these sites. In addition, independent occurrence of several of these mutations has been documented, providing further evidence in support of the hypermutability of CpG sequences. Based on accumulated data for these mutations, the Johns Hopkins group has suggested that the extent of this hypermutation in factor VIII is 10 to 20 fold higher than the average mutation rate in this gene. 28 More recent studies for factor VIII mutations have used amplification of specific areas of the gene by the polymerase chain reaction (PCR). Because of the large size of the factor VIII gene and the constraints on target size for PCR, most investigations have focused on only smalI, functionally important areas of the gene, such as those that encode the acidic regions of the protein containing the thrombin cleavage sites. 33·37 Analysis of the PCR-amplified sequences has involved one of several techniques including alIele-specific oligonucleotide (ASO) probing, direct nucleotide sequenc-

MOLECULAR PATHOLOGY OF HEMOPHILlA A

ing, and denaturing gradient gel electrophoresis (DGGE). Mutations have been found in these "targeted" searches, but the diagnostic yield has been low. Four recent reports illustrate the use of this type of mutation detection strategy. In a survey of 215 patients for mutations affecting the arginine codons at residues 336, 372, 1,648, and l ,689, Gitschier et ae 3 noted oniy two (I %) changes, a nonsense mutation in the activated protein C cleavage site at residue 336 and a missense mutation resulting in the substitution of cysteine for arginine in the thrombin activation site at amino acid 1,689. The same investigator used a different technique, DGGE, to screen codons 318 through 385 (which encode the N-terminal acidic do main) in 228 unselected hemophilia A patients. 34 Three mutations (1.5%) were identified in this study: missense mutations at residues 329 and 326 and a four-nucleotide deletion, the latter resulting in a frameshift change in a third severe hemophiliac patient. In another study using DGGE of a larger number of selected regions of factor VIII (exons 8, 17, 18, 24, and the 3' end of exon 14), Traystman et ae 7 identified three missense mutations in 52 patients (6%). Finally, in a recent investigation, Pattinson et ae 5 screened 793 hemophilia A patients for mutations at eight CpG dinucleotide sequences in the factor VIII sequence by PCR followed by ASa probing. 35 Using this strategy, mutations were found in 16 (2%) of the patients. Recurrent mutations were documented at codons 336 and 1,689 and single mutations were identified at codons - 5, 372,427, 583, 795, and 1,696. These studies not only provide important insights into structure/function correlates for factor VIII but also illustrate the practical difficulty in identifying the large number of subtle mutations that result in hemophilia A. This topic will be addressed in more detail later in this review. The final point that will be made in this section on mutation characterization relates to the optimal maintenance and dissemination of hemophilia A mutational information. The number of defined factor VIII mutations has increased steadily over the past 5 years and has now reached a level that is probably beyond the useful incorporation of these data into reviews on this subject. This subject has been managed by the collection of hemophilia B mutations into an international data base that will be published and updated on an annual basis for several years. 38 The establishment of a similar re-

199

porting system for hemophilia A mutations would prove a valuable asset for everyone involved in this area of biologic research. THE ORIGIN OF HEMOPHILlA A MUTATIONS

As J. B. S. Haldane predicted some 55 years ago, approximately one third of the cases of hemophilia A represent de novo mutations. The origin of these new mutations has long been an area of interest, and with the introduction of recombinant DNA techniques over the past several years, there are now data that provide at least a partial answer to this question. Studies using the linkage of restriction fragment length polymorphisms and coagulation test data to derive probabilities of carrier status for hemophilia A indicate that approximately 90% of mothers of sporadic hemophiliac children are carriers, and that in most of these women, the mutation has been transmitted through the germ line of their unaffected fathers. 39 This bias towards a male germline origin for new mutations may well be explained by differences that are known to occur in the development of the male and female gametes. 40 lf spontaneous mutations occur as a result of mistakes made at the time of DNA replication or because of defects in the postreplication repair mechanisms, one would expect that the number of divisions experienced by a cell would be an important factor in determining its risk of acquiring new mutations. Female gametes undergo approximately 30 mitotic divisions up to the time of birth, at which point they remain "suspended" in meiotic prophase until proceeding to the two meiotic divisions in postpubertal development. In contrast, the male germ line continues to undergo mitotic divisions in adult life, and by age 35 the spermatogonia may have undergone more than 500 mitoses. Therefore, unless the control of the replication mechanisms are different between mitosis and meiosis, one would expect the majority of new mutations to occur in association with mitosis and thus to be more often associated with the male germ line. The other fact that is becoming apparent from recent studies is that these new mutations may not always be events that are restricted to single gametes. Several reports have now documented germ line and somatic ceB mosaicism for new mutations that have arisen in the mothers of sporadic hemophiliac infants. 41 - 44 These mutations have presum-

200

DAVID L1LLlCRAP

ably oeeurred during a mitotie eell division involving a progenitor eell that gives rise to eells of both germ line and somatie lineage. These observations provide further support for the origin of new mutations in mitosis and also present an important eounseling dilemma. When new sporadic cases of hemophilia are identified, they can no longer be eonfident1y regarded as being unique mutational events. The possibility of germinal mosaicism must be kept in mind, although, until more practica! methods are available for the direct detection of factor VIII mutations, there is no way to definitively confirm or refute this possibility. DIRECT MUTATION DETECTION STRATEGIES FOR HEMOPHILlA A

The size and complexity of the factor VIII gene poses a significant challenge to the direct identification of the greater than 90% mutations that involve only minor changes to the genome strueture. The studies of Gitschier et al,33 Kogan and Gitschier,34 Pattinson et al, 35 Higuchi et al, 36 and Traystman et al,37 all represent a considerable output of time and effort for what is a relatively smali diagnostic yield. In addition, it must be remembered that in each of these investigations, onły a small part of the factor VIII sequence has been screened. Two types of strategy are now being pursued in an attempt to overcome these limitations. The first type of investigation involves the application of one of severa1 mutation screening methods that uses PCR-amplified DNA as their test material (Table 2).45.47 Each of these teehniques has different advantages and drawbacks, but to date, most experience has been obtained with DGGE. In a recent1y reported study using this teehnique to analyze 99% of the coding sequence of factor VIII and 41 of 50 splice junctions, the Johns Hopkins group made two interesting Table 2. Methods tor the Oetection ot Heterogeneous, Subtle Mutations in the Genome RNase c1eavage Denaturing gradient gel electrophoresis (DGGE) Single strand conformation analysis Chemical modification and cleavage Direct nucleotide sequencing NOTE. Ali use PCR-amplified DNA as the test materia!.

observations. 48 First, in patients with mild or moderate factor VIII deficiency, they were able to doeument deleterious nucleotide substitutions in 41 of 47 (87%) cases studied. In contrast, they found mutations in onIy 18 of 31 (58%) severe hemophi1iac patients. They had tested the sensitivity of their DGGE method on a population of 26 previously charaeterized factor VIII mutations and had been able to identify all 26 single nucleotide changes. Therefore, a1though it appears that most mutations resulting in mild and moderate hemophilia A occur within the coding sequence and splice sites of the factor VIII gene, a significant proportion of the mutations leading to severe disease may be located in sequences elsewhere within the gene or at an adjacent locus. Once again, it is worthwhile noting that this study represents the conclusions of a large amount of work and that the 45 sets of primer pairs used in these experiments make this type of approach prohibitive for all but the best-endowed laboratories. The other development that bears mention in terms of direct mutation detection is that relating to the establishment of studies that use a source of factor VIII mRNA as the test substrate. Recent reports indicate that a minimaI "background" level of transcription of all genes is identifiable in all tissue sources using PCR analysis. 49 ,50 Therefore, access to tissue-specific mRNAs, such as that for factor VIII, may not require procedures such as liver biopsy. A recent study from David Cooper's group in London indicates that "ectopically" transcribed factor VIII mRNA from blood leukocytes can indeed be used as the template for PCR amplification of factor VIII sequences. 51 This finding opens up the possibility of amplifying and screening larger regions of the factor VIII coding sequence in a few initia! PCR experiments. However, it will of course be a less advantageous strategy if 30% of the severe mutations occur outside of this sequenee. CARRIER AND PRENATAL DIAGNOSIS IN HEMOPHILlA A

It will be apparent from the preceding text that the time has not yet arrived when direct mutation detection is a feasible prospect in most cases of hemophilia A. Therefore, in laboratories requested to provide routine carrier and prenatal diagnostic studies, the use of restriction fragment length poly-

MOLECULAR PATHOLOGY OF HEMOPHILlA A

201

improve the yield with intragenic polymorphisms to between 60% and 70%. This polymorphism can also be studied with PCR, but, as will be noted later, this area of the factor VIII gene is duplicated on the X ehromosome in two other sites outside the gene and these sequences are coamplified in the PCR reaction along with the intragenic polymorphic site. This eomplicates the interpretation of data in females and usually requires analysis to be earried out by Southem blotting to provide definitive information. Almost all the other factor VIII intragenie polymorphisms that have been reported (je, for HindIII and MspI) are in strong linkage disequilibrium with the Bell and XbaI RFLPs and therefore not of further benefit in diagnostie studies. The only other sites that are worth analyzing are the BglI site adjacent to exon 26, which is sometimes informative in non-white families,57 and a nonrestriction polymorphic site in intron 7. 34 This latter polymorphism can be studied by ASa probing of PCR-amplified DNA and will be informative in approximately 5% to 10% of those females who

morphism (RFLP) analysis remains the strategy of choice. 52 Carrier Diagnosis Using Intragenic Polymorphisms The high frequency factor VIII intragenic and adjacent intergenic polymorphisms are detailed in Table 3.34.53-56 Unfortunately, to further complicate the analysis of this gene, there appears to be very little polymorphic sequence variability at this locus. The BclI polymorphism in intron 18 can now be studied using PCR and is the first site that should be studied. Approximately 40% of families will be informative with this polymorphism, which, because of its intragenic situation, would be expected to recombine with the factor VIII mutation less than once in 10,000 meioses. There is no doubt that when using an intragenic polymorphism for diagnostic purposes the largest source of error is not the risk of genetic recombination but administrative and laboratory errors and the possibility of false pedigree information. The addition of the XbaI RFLP to the diagnostic analysis will

Table 3. Oiagnostically Usetul Polymorphic Sites in and Adjacent to the Factor VIII Gene

Probe

Enzyme Intragenic Bcll Xbal

BgII

Intergenic BstXI

Polymorphic Site

Size ot Polymorphic Fragments

p.114.12

Intran 18

p.482.6

Intran 22

ASO

Intron 7

F VIII/cONA.C

Adjacent to exon 26

1.2 kbp 0.8 kbp 6.2 kbp 4.8 kbp Guanine Adenine 20.0 kbp 5.0 kbp

767

OXSl15 2 cM tram OXSl15 2 cM trom OXS115 2 cM trom OXSl15 2 cM tram OXS33 3 cM tram OXS52 4 cM trom OXS15 6 cM trom

6.4 kbp 4.3 kbp 1.8 kbp 1.75 kbp 11.8 kbp 6.0 + 5.8 kbp 9.0 kbp 4.0 kbp 10.0 kbp 8.0 kbp 12 alleles 6.6 - 1.4 kbp 2.8 kbp 5.8 kbp

Pstl

767

Mspl

767

Accl

767

BgII

MN12

Taql

St14

BgIII

OX13

F VIII F VII' F VIII F VIII F VIII F VIII F VII'

Abbreviations: F, tactor; cM, centimorgans; PIC, polymorphism intormation content.

Allelic Frequencies

0.29 0.71 0.41 0.59 0.79 0.21 0.10 0.90

0.86 0.14 0.77 0.23 0.86 0.14 0.10 0.90 0.08 0.92 PIC> 0.9 0.5 0.5

202

DAVID L1LLlCRAP

2

are homozygous for the absence of the Bell polymorphic site. Carrier Diagnosis Using Adjacent Intergenic Polymorphisms Polymorphisms at four adjacent intergenic sites are of benefit if intragenic RFLPs are uninformative. These sites are from 2 to approximately 6 centimorgans from factor VIII, and therefore, diagnoses made with these markers are always associated with a recombination probability .58 One of the polymorphisms, at DXS52, is a variablenumber tandem-repeat sequence with 12 alleles, and the use of this latter, highly informative marker in combination with the other intragenic and intergenic polymorphisms results in conclusive studies in more than 90% of families where there is a prior history of hemophilia A. One of the problems associated with the use of RFLP analysis in hemophilia A carrier diagnosis is the 20% to 30% incidence of sporadic disease (Fig 2). In these cases, polymorphism analysis can only be used to exclude carrier status, because the origin of the mutation within the pedigree cannot usually be defined with confidence. This is one of the instances in which coagulation studies on the potential carrier females are still extremely important. 59-62 The universal discriminant, reported several years ago by an international group of investigators, appears to be the phenotypic strategy of choice. 59 This method uses the measurement of factor VIII and vWF:Ag and includes age and ABO blood group data, which improve the efficacy of the discriminant function. Previous reports have indicated that approximately 90% of females who have sporadic hemophiliac sons are likely to be carriers of the factor VIII mutation and that in the majority of these women the mutant allele has been transmitted from a nonhemophilic father. 39 The other issue that needs to be considered in these cases and that has been addressed previously is whether the mutation has occurred as a unique meiotic event or (as now ~eems increasingiy likely) has occurred in mitosis and has resulted in germ line mosaicism. Of course, this influences the recurrence risk for these mutations; unfortunately, until more practical, methods are available for direct mutation detection in hemophilia A, there is no way to either coMirm or quanI tify this possibility.

1

Bell.F8

2.2

II

2.1

2.1

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2.2

Bell. F8

Fig 2. Hemophilia A carrier diagnosis in a family with a single affected male. The male (111.1 l is the only hemophiliac in this family. Carrier diagnosis is sought for three potential carrier females (11.2, 11.3 and 111.21. The analysis has been performed with the intragenic Bc11 polymorphism. The X chromosome bearing the mutant factor VIII gene (linked to the Bc11 'T' allelel has originated from the nonhemophilic grandfather. Thus, the hemophilic mutation has occurred at one of four possible sites, in either mitosis or meiosis in the grandfather or in mitosis or meiosis in the mother of the hemophiliac. No matter where the mutation has occurred, the sister of the hemophiliac can be excluded from being a carrier of the mutation, as she has inherited the normaI factor VIII gene marked by the "2" allele from her mother. The carrier status of the two second generation females can be assessed in two ways. Ił the factor Vlll/vWF:Ag levels in either female are indicative of her being a carrier, we know that the mutation has arisen in the grandfather (1.1 l. Similarly, if the mutation could be detected directly in 11.2 and 11.3 (not usually practical), we would have definitive evidence of the grandparental origin of the mutation. In both these instances, the possibility of a mitotic origin for the mutation in the grandfather with subsequent germ line mosaicism must be considered.

Prenatal Diagnosis oj Hemophilia A Prenatal analysis for hemophilia A is usually now performed by one of two techniques, chorionie villus sampling (CVS) or amniocentesis. The former method has several advantages in terms of subsequent DNA analysis. CVS can be performed as early as the eighth week of pregnancy before the presence of the pregnancy is known to anyone other than the parents. Amniocentesis has traditionally been performed at about the 16th week of pregnancy but is now, in some centers, being car-

203

MOLECULAR PATHOLOGY OF HEMOPHILlA A

ried out as early as week 12. The other major advantage of CVS is that these sampIes can be anaIyzed directly by DNA testing, in contrast to those obtained during amniocentesis in which a period of 2 to 3 weeks of amniocyte culture is required to obtain sufficient material for study. The risk of procedure-related miscarriage seems to be similar for both methods. The other advance that is now possible through PCR analysis of CVS material is rapid, early fetal sexing. The use of Y chromosome-specific sequences from a region of the chromosome involved in sex determination (ZFY, zinc finger Y or SRY, sex-determining region Y) can assign fetal

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F CV M

sex as early as 9 weeks (Fig 3). This technique has also proved feasible using a maternal blood sample as the test material,63 and in the future, we are very likely to see further pursuit of this type of noninvasive prenatal analysis. A GENE WITHIN THE FACTOR VIII GENE

The final section of this review concerns a fascinating and as yet unexplained observation that has been reported from Dr Gitschier's laboratory at the University of California. This group has been searching for new genes in the Xq28 region by screening for unmethylated CpG-rich sequences (CpG islands) that are associated with the regula-

D X 488bp Y 340bp

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B F CV M

00 142bp 99 bp

43 bp

Fig 3. Prenatal testing in hemophilia A. (A) PCR analysis of a CVS obtained at the 10th week of pregnaney. DNA from the fetus (CV) and the mother (M) and father (F) have been amplified with primers eorresponding to the regions ZFX and ZFY on the X and Y ehromosome, respeetively. The amplified material has been analyzed on an 8% polyaeryamide gel and visualized by staining with ethidium bromide. Female and male DNA sam pies are ineluded as eontrols in lanes 1 and 5, respeetively. The fetal sample has no evidenee of a Y ehromosomal sequenee and is thus shown to be a female. (B) PCR analysis of the factor VIII intragenie Be11 RFLP. The two polymorphie alleles shown on this ethidium bromide stained gel are at 142 bp (A1) and 99 + 43 bp (A2). Sampies from the fetus (CV), mother (M), father (F) and hemophilie brother (B) have been tested along with a normai male and a female. The female fetus is shown to be heterozygous for the Be11 marker and has inherited the polymorphie A2 allele, linked to the normai factor VIII gene, from her mother.

204

DAVID L1LLlCRAP

• 14

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INTRON 22 TRANSCRIPTS

18 Kbp

2.5 Kbp

F8-A

FS- B

tory regions of some transcribed genes. In their analysis of the genorne around factor VIII, they discovered a CpG region in intron 22 of the gene, marked by the presence of a Sadl restriction site. 64 Genomie fragments from this region of the intron were hybridized to Northem blots prepared from several cell types, and a 1.8 kbp mRNA was identified in a number of these preparations. Therefore, there is evidence of a gene in intron 22 of factor VIII that is transcribed in several diverse tissues and cell lines (including liver, erythroleukemia cells, and cervical carcinoma cells). The finding of transcribed sequences within the introns of a gene has also been noted recently in the large, human neurofibromatosis type 1 gene. 65 A comparison of the cDNA and genomic sequence of the original factor VIII intron 22 gene has shown that the transcribed gene, factar VIII-associated-gene A (F8A), has no introns. The intron 22 gene sequence is also present in two other copies elsewhere on the X chromosome and at least one of these two other gene copies is expressed. 64 The two homologous sequences outside factor VIII appear to be situated at the locus DXSI 15, approximately l Mbp 5' offactor VIII. 66 In addition, these sequences also represent the nonfactor VIII regions that complicate the interpretation of the Xbal polymorphism data from within intron 22. 67 ,68 The analysis of the intron 22 sequence has become increasingly complex (Fig 4). The initially identified 1.8 kbp mRNA is transcribed in the opposite direction to factor VIII and appears to encode a protein that is expressed at high levels in many tissues. The amino acid sequence is not homologous to any other protein and its putative



Fig 4. The genes within intron 22 of the faetor VIII gene. Levinson et al identified two transeripts (---> direetion of tran. seriptionl, F8·A and F8- B, arising from intron 22 of the faetor VIII gene. 69 F8-A is an intronless gene that is duplieated in two additional eopies approximately 1 Mbp 5' of faetor VIII. The F8-B produet is a ehimerie transeript eomprising a short 5' exon embedded in intron 22 splieed to faetor VIII exons 23 through 26.

function remains unknown. In addition, Levinson et al have now described a second transcript from intron 22. 69 This 2.5 kbp mRNA (factor VIIIassociated-gene B, F8-B) is transcribed in the opposite direction to F8-A (ie, in the same direction as factor VIII) and comprises a chimeric sequence derived from a new exon embedded in intron 22 (encoding the first 8 amino acids of the protein) and factor VIII exons 23 through 26. This transcript has been found in tissues not involved in the expression of factor VIII. Once again, the functional significance of this product remains unclear, although the majority of the sequence encodes the CI and C2 domains of factor VIII, which may play a role in binding to phospholipid surfaces. CONCLUSION

Although we have leamed a great deal about the molecular pathology of hemophilia A in the 6 years since the cloning of the factor VIII gene, there are still many questions that remain unanswered. Although we knowalot about the structure of this protein, we still do not understand its precise cofactor function. We are still some way from developing tests that can reliably, and in a practical manner, detect the mutations that cause hemophilia A. Even more of a challenge appears to be that approximately 30% of severe hemophilia mutations are not found after extensive screening of the factor VIII coding sequence and splice junctions. The site of these changes will prove highly informative. Furthermore, the relevance, if any, of the intron 22 genes to hemophilia A remains to be clarified. With these issues in mind, the molecular study of hemophilia A will continue to attract the attention of scientists for some years to come.

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ACKNOWLEDGEMENT The author acknowledges Drs Higuchi, Kazazian, and Brocker-Vriends for sharing their unpublished data. Thanks to Dr Jane Gitschier for sharing her enthusiasm and expertise

relating to the factor VIII gene. My thanks to Hermina Wensing for her assistance with the preparation of this manuscript and to Suzanne Hoyle for the art work. Finally, my thanks to all of my immediate colleagues for their continued support and curiosity.

REFERENCES I. Gitschier J, Wood WI, Goralka TM, et al: Characterisation of the human factor VIII gene. Nature 312:326-330, 1984 2. Toole H, Knopf JL, Wozney JM, et al: Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 312:342-347, 1984 3. Vehar GA, Keyt B, Eaton D, et al: Structure of human factor VIII. Nature 312:337-342, 1984 4. VeharGA, Eaton DL: Factor VIII structure and function, in Zimmerman TS, Ruggeri ZM (eds): Coagulation and Bleeding Disorders: The Role of Factor VIII and von Willebrand Factor. New York, NY, Dekker, 1989, pp 1-21 5. Toole H, Pittman DD, Orr EC, et al: A large region (approx. 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. Proc Nad Acad Sci USA 83:59395942, 1986 6. Wion KL, Kelly D, Summerfield JA, et al: Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature 317:726-728, 1985 (1etter) 7. Lewis JH, Bontempo PA, Spero JA, et al: Liver transplantation in a hemophiliac. N Engl J Med 312:1189-1190, 1985 8. Ingerslev J, Christiansen S, Heickendorff L, et al: Synthesis of factor VIII in human hepatocytes in culture. Thromb Haemost 60:398-391, 1988 9. Kelly DA, Summerfield JA, Tuddenham EGD: Localization of factor VIII:C antigen in guinea-pig tissues and isolated liver celi fractions. Br J Haematol 56:535-543, 1984 10. Kaufman RJ, Wasley LC, Dorner AJ: Synthesis, processing and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 263:6352-6360, 1988 II. Pittman DD, Kaufman RJ: Structure-function relationships of factor VIII elucidated through recombinant DNA technology. Thromb Haemost 61:161-165, 1989 12. Leyte A, Van Schijndel HB, Niehrs C, et al: Sulfation of Tyr 1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor. J Biol Chem 266:740-746, 1991 13. Pittman DD, Kaufman RJ: Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII). Proc Nad Acad Sci USA 85:2429-2433, 1988 14. Eaton D, Rodriguez H, Vehar GA: Proteolytic processing of human factor VIII. Correlation of specific cleavages of thrombin, factor Xa and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 25:1986-1990, 1986 15. Antonarakis SE: The molecular genetics of hemophilia A and B in man: Factor VIII and factor IX deficiency. Adv Hum Genet 17:27-60, 1988 16. White GC II, Shoemaker CB: Factor VIII gene and hemophilia A. Blood 73:1-12, 1989 17. Gitschier J: The molecular genetics of hemophilia A, in

Zimmerman TS, Ruggeri ZM (eds): Coagulation and Bleeding Disorders: The Role of Factor VIII and von Willebrand Factor. New York, NY, Dekker, 1989, pp 23-46 18. Furie B, Furie BC: Molecular basis of hemophilia. Semin Hematol 27:270-285, 1990 19. Antonarakis SE, Kazazian HH Jr: Recent advances in hemophilia care. The molecular basis of hemophila A (factor VIII deficiency) in man: Progress report from the Johns Hopkins University Hemophilia Projecl. Prog Clin Biol Res 324:112, 1990 20. Lozier JN, High KA: Molecular basis of hemophilia. Hematol Pathol 4:1-26, 1990 21. Youssoufian H, Antonarakis SE, Aronis S, et al: Characterisation of five partial deletions of the factor VIII gene. Proc Nad Acad Sci USA 84:3772-3777, 1987 22. Giannelli F, Choo KH, Rees DJG, et al: Gene deletions in patients with haemophilia B and anti-factor IX antibodies. Nature 303:181-182, 1983 23. Chamberlain JS, Gibbs RA, Ranier JE, et al: Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 16: 11141-11156, 1988 24. Thein SL, Weatherall DJ: The thalassaemias, in Hoffbrand AV (ed): Recent Advances in Haematology Number 5 (ed 1). New York, NY, Churchill Livingston, 1988, pp 43-74 25. Ballabio A, Ranier JE, Chamberlain JS, et al: Screening for steroid sulfatase (STS) gene deletions by multiplex DNA amplification. Hum Genet 84:571-573, 1990 26. Kazazian HH Jr, Wong C, Youssoufian H, et al: Haemophilia A resuiting from de novo insertion of LI sequences represents anovel mechanism for mutation in man. Nature 332:164-166, 1988 27. Gitschier J, Wood WI, Tuddenham EGD, et al: Detection and sequence of mutations in the factor VIII gene of haemophiliacs. Nature 315:427-430, 1985 28. Youssoufian H, Antonarakis SE, Bell W, et al: Nonsense and missense mutations in hemophilia A: Estimate of the relative mutation rate at CpG dinucleotides. Am J Hum Genet 42:718-725, 1988 29. Levinson B, Lehesjoki A-E, De la Chapelle A, et al: Molecular analysis of hemophilia A mutations in the Finnish population. Am J Hum Genet 46:53-62, 1990 30. Cooper DN, Youssoufian H: The CpG dinucleotide and human genetic disease. Hum Genet 78:151-155, 1988 31. Cooper DN, Krawczak M: The mutational spectrum of single base-pair substitutions causing human genetic disease: Pattems and predictions. Hum Genet 85:55-74, 1990 32. Koeberl DD, Bottema CDK, Ketterling RP, et al: Estimate of the rates of spontaneous transitions, transversions and deletions in the human germ1ine. Am J Hum Genet 47:202-217, 1990

206

33. Gitschier J, Kogan S, Levinson B, et al: Mutations of factor VIII cleavage sites in hemophilia A. Blood 72: 10221028, 1988 34. Kogan S, Gitschier J: Mutations and a polymorphism in the factor VIII gene discovered by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 87:2092-2096, 1990 35. Pattinson JK, Millar DS, McVey JH, et al: The molecular genetic analysis of hemophilia A: A direeted search strategy for the detection of point mutations in the human factor VIII gene. Blood 76:2242-2248, 1990 36. Higuchi M, Wong C, Kochhan L, et al: Characterisation of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA. Genomics 6:65-71, 1990 37. Traystman MD, Higuchi M, Kasper CK, et al: Use of denaturing gradient gel electrophoresis to detect point mutations in the factor VIII gene. Genomics 6:293-301, 1990 38. Giannelli F, Green PM, High KA, et al: Haemophilia B: Database of point mutations and short additions and deletions. Nucleic Acids Res 18:4053-4059, 1990 39. Brocker-Vriends AHJT, Rosendaal FR, van Houwelingen JC, et al: Sex ratio of the mutation frequencies in haemophilia A: Coagulation assays and RFLP analysis. 1991 (unpublished observation) 40. Vogel F, Motulsky AG: Mutation, in Vogel F, Motulsky AG (eds): Human Genetics: Problems and Approaches. New York, NY, Springer-Verlag, 1986, pp 334-429 41. Gitschier J, Levinson B, Lehesjoki A-E, et al: Mosaicism and sporadic haemophilia: Implications for carrier determination. Lancet 1:273-274, 1989 42. Higuchi M, Kochhan L, Olek K: A somatic mosaic for hemophilia A detected at the DNA level. Mol Biol Med 5:2327, 1988 43. Gitschier J: Maternal duplication associated with gene deletion in sporadic hemophilia. Am J Hum Genet 43:274-279, 1988 44. Brocker-Vriends AHJT, Briet E, Dreesen JCFM, et al: Somatic origin of inherited hemophilia A. Hum Genet 85:288292, 1990 45. Rossiter BJF, Caskey CT: Molecular scanning methods of mutation deteetion. J Biol Chem 265:12753-12756, 1990 46. Forrest S, Cotton RGH: Methods of deteetion of single base substitutions in clinical genetic practice. Mol Biol Med 7:451-459, 1990 47. Grompe M, Gibbs RA, Chamberlain JS, et al: Detection of new mutation in man and mouse. Mol Biol Med 6:511-521, 1989 48. Higuchi M, Antonarakis SE, Kasch LM, et al: Molecular characterisation of hemophilia A: Detection rate of deleterious mutations is 87% in mild to moderate disease but only 58% in severe disease. 1991 (unpublished observation) 49. Sarkar G, Sommer SS: Access to an mRNA sequence or its protein product is not limited by tissue or species specificity. Science 244:331-334, 1989 50. Chelly J, Concordet J-P, Kaplan J-C, et al: lllegitimate transcription: Transcription of any gene in any celi type. Proc Natl Acad Sci USA 86:2617-2621, 1989 51. Berg L-P, Wieland K, Millar DS, et al: Deteetion of a novel point mutation causing haemophilia A by PCRJdirect sequencing of ectopically-transcribed factor VIII mRNA. Hum Genet 85:655-658, 1990

DAVID L1LLlCRAP

52. Lillicrap DP, Bridge PJ, Giles AR, et al: Organisation of a hemophilia genetic screening program. Prog Clin Biol Res 324:19-28, 1990 53. Gitschier J, Drayna D, Tuddenham EGD, et al: Genetic mapping and diagnosis of haemophilia A achieved through a BclI polymorphism. Nature 314:738-740, 1985 54. Wion KL, Tuddenham EGD, Lawn RM: A new polymorphism in the faetor VIII gene for prenatal diagnosis of haemophilia A. Nucleic Acids Res 14:4535-4542, 1986 55. Mandel J-L, Willard HF, Nussbaum RL, et al: Report of the committee on the genetic constitution of the X chromosome. Cytogenet Celi Genet 51:384-437, 1989 56. Jedlicka P, Greer S, Millar OS, et al: Improved carrier detection of haemophilia A using novel RFLPs at the DXS 115 (767) locus. Hum Genet 85:315-318,1990 57. Antonarakis SE, Youssoufian H, Kazazian HH Jr: Molecular genetics of hemophilia A in man (factor VIII deficiency). Mol Biol Med 4:81-94, 1987 58. Peake IR, Lillicrap OP, Liddell MB, et al: Linked and intragenic probes for haemophilia A. Lancet 2:1003-1004, 1985 (abstr) 59. Green PP, Mannucci PM, Briet E, et al: Carrier detection in haemophilia A: A cooperative international study. II. The efficacy of a universal discriminant. Blood 67:1560-1567, 1986 60. Graham JB, Green PP, McGraw RA, et al: Carrier detection in the haemophilias. Some limitations of the DNA methods. Blood 66:759-763, 1985 61. Lillicrap DP, Holden JJA, Giles AR, et al: Carrier detection strategy in haemophilia A: The benefits of combined DNA marker analysis and coagulation testing in sporadic haemophilic families. Br J Haematol 70:321-326, 1988 62. Brocker-Vriends AHJT, Briet E, Quadt R, et al: Carrier detection of haemophilia B by using an intragenic restriction fragment length polymorphism. Thromb Haemost 54:506-509, 1985 63. Lo Y-MD, Patel P, Wainscoat JS, et al: Prenatal sex deterrnination by DNA amplification from maternal peripheral blood. Lancet 2:1363-1365, 1989 64. Levinson B, Kenwrick S, Lakich D, et al: A transcribed gene in an intron of the factor VIII gene. Genomics 7:1-11, 1990 65. Cawthon RM, Weiss R, Xu G, et al: A major segment of the neurofibromatosis type I gene: cDNA sequence, genomic structure, and point mutations. Celi 62:193-201, 1990 66. Patterson M, Gitschier J, Bloomfield J, et al: An intronic region within the human factor VIII gene is duplicated within Xq28 and is homologous to the polymorphic locus DXS1l5 (767). Am J Hum Genet 44:679-685, 1989 67. Chan V, Tong TMF, Chan TPT, et al: Multiple XbaI polymorphisms for carrier detection and prenatal diagnosis of haemophilia A. Br J Haematol 73:497-500, 1989 68. Lillicrap DP, Taylor SAM, Schuringa PCR, et al: Variation of the non-factor VIII sequences detected by a probe from intron 22 of the factor VIII gene. Blood 75:139-143, 1990 69. Levinson B, Kenwrick S, Garnel P, et al: Two divergent transcripts, one containing factor VIII exons, originating in a CpG island in an intron of the factor VIII gene. Am J Hum Genet 47:A1l5, 1990 (abstr)

The molecular pathology of hemophilia A.

The Molecular Pathology oC Hemophilia A David LiIIicrap UR UNDERSTANDING of the molecular abnormalities that affect the coagulation cofactor, factor...
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