Molecular genetics of von Willebrand disease · Molecular Genetics of von Willebrand Disease By...

1992 79: 2507-2519

D Ginsburg and EJ Bowie Molecular genetics of von Willebrand disease

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REVIEW ARTICLE

Molecular Genetics of von Willebrand Disease

By David Ginsburg and E.J. Walter Bowie

ON WILLEBRAND disease (vWD) was first described V by Erik von Willebrand in 1926l in several members of a family from the h a n d archipelago in Finland. The proband, a 7-year-old girl, and 9 of her 11 siblings all had significant bleeding symptoms, four dying of hemorrhage between the ages of 2 and 4 and the proband herself dying of hemorrhage at the age of 13 at the time of her fourth menstrual period. von Willebrand coined the term “hereditary pseudohaemophilia” for the disease that subsequently bore his name. In retrospect, the first case of vWD may actually have been described by Minot and Lee in 1920,2 and a similar clinical disorder was reported independently by four American groups in 1928.3,4 In 1953, an association between decreased factor VI11 (FVIII) procoagulant activity and vWD was first described, leading to some confusion concerning the protein defects responsible for hemophilia A and v W D . ~ , ~ An explosion in the understanding of von Willebrand factor (vWF) and FVIlI began with the immu- nologic characterization of the proteins in the early 1970s, culminating in the cDNA cloning of FVIII in 19845,6 and vWF in 1985.7-10 Molecular defects responsible for hemophilia and vWD were first detected by Southern blot analysis in 198S1 and 1987,12,13 respectively. The subsequent identification of point mutations in DNAs from large numbers of hemophilic and vWD patients was made possible by the discovery of the polymerase chain reaction in 1985 and the application of Tuq polymerase in 1987.14J5 This review will focus on the molecular genetics of vWD. The biosynthesis, structure, and function of the vWF protein will only be briefly introduced. For a more detailed discussion of these latter topics, the interested reader is referred to several excellent recent reviews.16-20

vWF BIOSYNTHESIS

The vWF monomeric subunit of approximately 250 Kd in molecular weight is assembled into multimers containing up to 100 subunits with molecular weights in excess of 20 x loh daltons. vWF is synthesized exclusively in endothelial cells and megakaryocytes and has become a standard marker of endothelial cell origin for histochemical studies. vWF is first synthesized as a large prescursor form that initially dimerizes and subsequently multimerizes with coin- cident processing to the mature vWF subunit. Within the endothelial cell, vWF is secreted via both constitutive and regulated pathways. Dimerization of vWF and early carbohydrate processing begin in the endoplasmic reticulum with final carbohydrate processing and multimerization re- stricted to golgi and post-golgi compartments. A specific storage compartment for vWF has been identified within the endothelial cell termed the Weibel-Palade body. This structure contains densely packed vWF with a characteristic appearance under the electron microscope. The only other protein known to be contained within the Weibel- Palade body is the recently described selectin, GMP140

(P-selectin).21,22 Platelet vWF is stored within the a-gran- uole.’6

STRUCTURE AND FUNCTION OF vWF

In plasma, vWF serves two major function. Via specific binding to the platelet surface as well as to one or more discrete ligands within the subendothelium, vWF provides a major adhesive link between the platelet and the vessel wall at sites of vascular injury. In addition, vWF serves as the carrier for FVIII in plasma, conferring increased stability and localizing FVIII to sites of platelet plug and subsequent fibrin clot formation.

The various binding functions of vWF appear to be localized to discrete domains within the molecule as shown by studies of proteolytic vWF fragments (summarized in Fig 1 and reviewed in Ruggeri and Zimmerman,17 Girma et a1,’8 Ruggeri,19 and SadlerZo). The FVIII binding domain of vWF has been localized to a 272 amino acid tryptic fragment at the N-terminus of In addition, two monoclonal antibodies mapping to an epitope spanning amino acids 78 through 9625,26 were shown to block FVIII binding to vWF, suggesting a further localization for this function. Recent evidence suggests that the mature vWF N-terminus is required for FVIII binding,27 although the role of the vWF propeptide is c o n t r ~ v e r s i a l . ~ ~ ~ ~ ~ Regions involved in binding to platelet glycoprotein Ib (GPIb), heparin, and collagen have been localized to a 48/52-Kd tryptic fragment spanning Val449 through L y ~ 7 2 8 . ~ ~ - ~ ’ An additional potential collagen binding domain has been mapped to residues 911-1114.32,33 Finally, an RGDS sequence thought to function as a ligand for the GPIIb/IIIa platelet surface integrin is located at amino acids 1744- 1747.

THE vWF GENE AND cDNA

vWF cDNAs were independently isolated by four groups in 1985.7-1° The complete primary amino acid sequence of the mature vWF subunit was also independently deter- mined by Titani et aP4 using direct amino acid sequence analysis. In addition to the expected agreement with the

From the Howard Hughes Medical Institute, Departments of Human Genetics and Internal Medicine and Program of Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI; and the Mayo Clinic, Section of Metabolic and Hemuto- logic Biochemistry, Department of Laboratory Medicine & Patholoa, Rochester, MN.

Submitted November 12,1991; accepted February 28, 1992. Supported in part by National Institutes of Health Grant No.

I-ROlHL39693. D.G. is a Howard Hughes Medical Institute Investi- gator.

Address reprint requests to David Ginsbuig, MD, Howard Hughes Medical Institute, 1150 W Medical Center Dr, 4520 MSRBL Ann Arbor, MI 48109-0650. 0 1992 by The American Society of Hematology. 0006-4971 I921 7910-0039$3.0OlO

Blood, Vol79, No 10 (May 15). 1992: pp 2507-2519 2507




2508 GINSBURG AND BOWIE

_.._- I 1 - I

I I 1 7 U I mor I ’

272 U9 12.9 911 1114

Fig 1. Schematic of tho human vWF gene, mRNA, and protein. lha vWF gem end pseudogene are depicted nt the top with boxes repmaonting exons and the solid black line, introns. The vWF mRNA encoding the full prepro-vWF subunit is depicted in the middle as the stippled bar and lettered boxes. The location of signal peptide (sp) and propeptide (pro-) cleavage sites are indicated by anowheads, and the lettered boxes denote regions of internally repeated sequence. The approximate localizations for known vWF functional domains within the mature vWF sequence ere indicated at the bottom. Numbers undemeath the domains refer to amino acid residues within the mature vWF subunit. The clusters of mutations responsible for type IIA vWD, type IIB vWD, and the FVtll binding defects are indicated. (aa. amino acids; chr, chromosome.)

cDNA the direct protein analysis also identified the location of N- and 0-linked glycosylation sites within the mature vWF subunit.J4 Surprisingly, the vWF cDNA predicted a 309-Kd (2,813 amino acid) primary translation product with the sequence corresponding to the N-terminus of mature vWF beginning at codon 764. The N-terminal segment was shown to encode a typical hydrophobic signal peptide followed by sequence identical to von Willebrand antigen I1 (vWA~II)?.~~-’~.~” vWAglI is a plasma protein of previously unknown function, associated with, but distinct from VWF.~”,~’ The vWF propeptide (vWAgll) was subsequently shown to play an important role in the process of vWF mul t imer i za t i~n~~~ and targeting to the Weibel- Palade b0dy.4~ The vWF propeptide is removed by a single proteolytic cleavage between codon 763 and 764, following two basic amino acid residues (Lys-Arg). This cleavage has recently been shown to be mediated by a specific paired basic amino acid cleaving enzyme with sequence similarity to the subtilisin-like protease family, including the yeast protease Ke~2.4~

Analysis of the vWF cDNA sequence also shows a pattern of repeated homologous sequence domains, suggesting that the gene may have arisen via a complex process of gene segment dupl icat i~n.~~-’~*~~ The positions of these domains with respect to the various localized vWF functions are shown schematically in Fig 1. Comparison of vWF to other known sequences within the available protein and DNA databases shows limited, but potentially important, sequence similarities. This is most evident for the A domains, which share significant sequence similarity with the “L“ domains of the MAC-1 integrin receptor a subunit, chicken cartilage matrix protein, and type VI c0llagen.4~ A possible distant relationship between the C repeats and

segments of thrombospondin and procollagen has also been

The human vWF gene has been localized to chromosome 12~12 + pter.7*“’ Localization studies using a cDNA probe from the midportion of vWF identified not only the authentic gene on chromosome 12 but a second sequence on chromosome 22.12 The latter has recently been localized to 22q11.22-ql 1.23.49 The complete exon/intron structure of the vWF gene has been established by Mancuso et al.”)The 52 exons span 178 kb, approximately 0.1% of human chromosome 12. Exons range from 40 bp to 1.4 kb for the largest exon (exon 28). The latter encodes the entire AI and A2 repeats, a critical region of the molecule spanning several important functional domains (Fig 1) and also containing most of the mutations responsible for type IIA and type IIB vWD (see below). The vWF intron/exon structure shows little correlation with the homologous repeat structure described above. Although there is some limited correspondence of introns in the D domains, the A1 and A2 domains are contained in a single exon, whereas the A3 domain extends across 5 exons.” Nearly the entire vWF pseudogene on chromosome 22 has now been sequenceds1 and shows 97% homology to the authentic chromosome 12 gene, indicating a very recent evolutionary origin. The pseudogene represents a nonprocessed duplication spanning exons 23-34.5l The presence of multiple stop codons in the coding sequence provides conclusive evidence that this is not a functional gene in humans. Southern blot studies confirm the absence of this pseudogene in a number of other mammalian species.’U3 The location and high degree of sequence identity between the pesudogene and authentic gene present particularly difficult problems for the identification of mutations in vWD (see below).

Information on potential regulatory elements within the vWF gene includes the analysis of 2.2 kb of upstream sequence. Although there is a typical “TATA” box at -30, no “CCAAT” or “ G C box elements are Transfection studies using vWF promoter sequences have been difficult and little information is currently available to explain the extremely high level of expression of vWF in endothelial cells and megakaryocytes and its remarkable tissue specificity.

vWD

vWD is an extremely heterogeneous disorder, with over 20 distinct clinical subtypes described (Table 1).1755-s9 The numerous vWD variants generally all fit into one of two classes, characterized by either quantitative (types I and 111) or qualitative (type 11 and other variants) defects in vWF. For the former class, vWF of relatively normal appearing structure and function may be present, but in significantly decreased amounts. In type I, vWF levels are generally between 20% and 50% of normal, and in type 111, levels are extremely low or undetectable. The type I variant of vWD is by far the most common, accounting for at least 70% of clinical cases. Type 111 vWD is much less common with frequency estimated at approximately 1 per mil- lion.”“ Overall prevalence figures for all cases of vWD have ranged as high as 1% to 3% of the population$1*62




VON WILLEBRAND DISEASE 2509

Table 1. Summary of vWD Phenotypes

Clinical Molecular Subtype Frequency Features Diagnosis Basis

Mild to moderate bleeding; vWF:Ag, vWF:RCo, and Unknown.

Type IIA

Type llB

Types IIC-H. others

FVlll binding defects (vWF Normandy)

Platelet-type vWD ("pseudo-vWD)

1-3O:l.OOO; most common vWD variant (> 70% of vWD)

1-5: lo6

Approximately 10-15% of clinical vWD cases

Uncommon variant (<5% of clinical vWD)

Rare (case reports)

Rare (case reports)

Rare (case reports)

autosomal dominant; incomplete penetrance (approximately 60%).

Severe bleeding disorder;? autosomal recessive inheritance.

Mild to moderate bleeding disorder; autosomal dominant, more complete penetrance than type I ; generally poor response to DDAVP.

Mild to moderate bleeding disorder; autosomal dominant, more complete penetrance than type I;? DDAVP contraindicated.

generally autosomal dominant though some autosomal recessive (type IIC).

Variable bleeding disorder;

Variable bleeding disorder; homozygotes (or compound heterozygotes) may present as autosomal hemophilia A.

Similar to type llB vWD.

FVlllC all proportionately decreased (20-50%). Nor- mal multimer distribution.

Markedly decreased or undetectable vWF:Ag, vWF: RCo, and FVIIIC.

Variable decreased vWF:Ag, vWF:RCo, and FVIIIC; absent high and intermediate size vWF multimers with prominent satellite bands;

Variably decreased vWF antigen vWF:RCo and FVIIIC; loss of large multimers; enhanced RIPA; thrombocytopenia.

Variably decreased vWF:Ag, vWF:RCo, and FVIIIC. Diag. nosis generally based on unique abnormalities in multimer pattern.

Variable vWF:Ag and vWF: RCo. Disproportionately low FVIIIC. Generally normal multimers. Decreased or absent vWF binding to FVIII.

type llB by mixing studies with normal platelets and plasma.

Can be distinguished from

vWF gene deletions; nonsense mutation; other cis- defects in mRNA expression.

Missense mutations clustered within vWF A2 domain. Two subgroups: group 1-Defect in intracellular transport; group 2-? proteolysis in plasma after secretion.

Missense mutation clustered in vWF A1 domain result in increased or spontaneous binding to platelet GPlb.

Unknown, some may represent compound heterozygotes for other variants.

Missense mutations within the N-terminus of mature vWF which interfere with FVlll binding.

Missense mutation within GPlb a-chain probably resulting in increased or spontaneous binding to vWF.

although others suggest a considerably lower range of approximately 1:10,000.56,59 The sensitivity and specificity of the current standard diagnostic tests (vWF:Ag, vWF ristocetin cofactor activity [vWF:RCo], FVIII procoagulant activity [FVIIIC], and bleeding time) may be as low as 60%.63 Additional variability is contributed by a number of other factors, including blood group and estrogen leve1.56,M,65 Mean vWF:Ag levels can vary from 75% for blood type 0 individuals to 123% for type AB, when compared with a standard normal donor plasma pool. As a result, the diagnosis of vWD may be more readily established in patients who are blood type 0. Clinical symptoms are more common in women, most likely due to the hemostatic stresses of pregnancy and menstruation. The bewildering complexity of vWD classification and the low accuracy of the current diagnostic tests present major problems for the practicing clinical hematologist. These issues lend additional importance to the identification of vWF mutations, to eventually facilitate the precise diagnosis and classification of vWD at the molecular level.

The treatment of choice for mild type I vWD is DDAVP. Intravenous or intranasal administration of this vasopressin

analogue generally results in marked elevation of vWF and FVIIl levels, providing adequate hemostasis in the majority of patient^.^',^^ This treatment is much less effective in the type 111 and type I1 variants and may be contraindicated in type IIB In this latter group of patients, as well as type I vWD patients who have otherwise failed DDAVP, the treatment of choice is replacement of vWF either in the form of pooled plasma fractions (cryoprecipitate) or, more recently, partially purified FVIII concentrates that also contain intact vWF m~ltimers.~' These latter products are thought to be free of viral transfusion risks on the basis of heat or detergent treatment. Whether cryoprecipitate or one of the purified products should be the first line treatment for severe (type 111) vWD and type I1 variants remains controversial.

ANIMAL MODELS FOR vWD

In addition to its high prevalence in human populations, vWD has also been frequently encountered in other animal species, including dogs, rabbits, cats, mice, pigs, and horses.52,69-74 Heterogyzotes for porcine vWD are generally





asymptomatic, while homozygotes have less than 1% of normal vWF levels and a severe bleeding diathesis closely resembling type I11 human VWD.~* , ’~ -~~ vWD has been recognized in over 30 breeds of dogs, including examples of type 111, type I, and type I1 vWD. The prevalence of vWD is extremely high in several inbred dog strains (up to 65% of Doberman Pinchers and Airedale terrier^).^^ Murine vWD has been recently described in the RIIIS/J mouse, identified by a screen of 25 inbred mouse strains, again suggesting a high spontaneous frequency for this disorder among mammalian species. vWD in the RIIIS/J mouse appears to most closely resemble human type I vWD. It is autosomal dominant in inheritance associated with a partial defect in plasma vWF, prolonged bleeding time, and decreased platelet aggregation. Type I vWD in this animal model appears to be a “true dominant” disorder with homozygotes and heterozygotes having similar phenotype^.^^ These data also suggest that type I11 vWD is not simply the homozygous form of type I vWD (see below).

The reason for the high frequency of vWD in humans and other mammalian species remains unclear. vWD may have a protective effect against atherosclerosis in the porcine mode1,72,74,75 although no such effect has yet been shown in humans.7h However, given the generally late age of onset for clinically significant heart disease, a protective effect from vWD probably would not exert a strong evolutionary pressure and should be easily outweighed by even mild changes in hemorrhagic risk. Interestingly, vWD also appears to afford some resistance to bacterial endocarditis in the porcine In addition, a sequence similarity has recently been noted between the YopM outer membrane protein of the Yersinia pestis organism (the pathogenic bacteria of plague) with the a-chain of GPIb,78,79 the platelet surface receptor for vWF. YopM- mutants of Yersinia pestis show decreased virulence in mice.7y If an interaction between YopM and vWF is important for virulence, a potential relative protection from this or other pathogenic bacteria may have provided a strong selective pressure for vWD. The large size of the vWF gene may also afford an ample target for mutation, a proposed explanation for the high mutation rates observed in several other disorders associated with unusually large proteins and genes, such as hemophilia A and muscular dystrophy.8D

For any autosomal dominant disease, the proportion of cases representing new mutations should be directly related to the decrease in reproductive fitness associated with the disorder.s1 Because vWD is in general a mild disorder, the decrease in reproductive fitness would be expected to be small, and thus the relative proportion of new mutations in von Willebrand disease should be low. Consistent with this hypothesis only a few patients have been identified in whom vWD appears to have arisen as a new genetic event (see below).

GENETIC LINKAGE ANALYSIS IN vWD

Given the complexity of vWF biosynthesis, secretion, and function, defects at a variety of genetic loci could potentially result in a vWD phenotype. As an example, platelet- type vWD, clinically very similar to type IIB vWD, is now

known to be due to a molecular defect in the GPIba chain genes2 (see below). Indeed, genetic locus heterogeneity could be partially responsible for the extensive phenotypic heterogeneity observed in vWD. Such a mechanism could also explain the apparent discordance between the relative frequencies of type I and type 111 vWD; ie, if the true incidence of type I vWD is 1% to 3%,61362 this would imply a gene frequency of 0.015 to 0.005, which should translate into a homozygote frequency (expected to have severe vWD) of 1:4,000 to 1:40,000, much greater than the observed frequency of approximately 1:1,000,000.56~60 Simi- larly, the “true autosomal” pattern of murine vWD might also suggest a regulatory locus outside of the vWF gene.

In studies of porcine vWD, a restriction fragment length polymorphism (RFLP) identified within the porcine vWF gene was shown to be tightly linked to the inheritance of vWD with a LOD score of 5.3 (at 0 = 0). Southern blot analysis showed no evidence of gene deletion or rearrangement. Taken together, these observations suggest a point mutation or small insertion or deletion within the vWF gene as the molecular basis for porcine v W D . ~ ~ A large number of RFLPs have been identified within the human vWF gene, including a highly polymorphic tetranucleotide repeat in intron 40.83-85 For polymorphisms located within the midportion of the vWF gene, care must be taken to distinguish RFLPs within the authentic gene from pseudogene polymor- p h i s m ~ . ~ ~ ~ ~ I

Linkage analysis of type I vWD is complicated by problems inherent in the incomplete penetrance of this disorder and low sensitivity and specificity of conventional diagnostic tests. Tight linkage has been demonstrated for two human type IIA vWD pedigrees (LOD scores of 3.68h and 5.7*’). Although RFLP analyses in a number of other vWD pedigrees have been consistent with linkage,s3,ss-94 none have individually obtained statistical significance (LOD score > 3.0). Taken together with the recent identification of specific vWF gene mutations in several groups of patients, these data indicate that vWD is generally due to defects within the vWD gene itself. With the exception of platelet-type vWD, defects at other loci have not yet been identified.

GENE DELETIONS IN vWD

Gene delection or rearrangement is a common mechanism for human genetic disease. Once DNA probes for vWF became available, large numbers of patients were screened by Southern blot analysis for the presence of such a b n o r m a l i t i e ~ . ~ ~ ’ * J ~ ~ ~ ~ However, gene deletions have only been reported in six families, associated with type 111 vWD in five (Table 2).12J3,y5 The one exception is a patient with an apparent de novo deletion in the midportion of the vWF gene resulting in a type I1 vWD phenotype (Table 3).yh Although the numbers are small, there appears to be a correlation between the presence of vWF inhibitors (anti- vWF allo-antibody) and the presence of gene deletion. Similar correlations have been noted in the hemophilias, particularly hemophilia B.I2 Give the low incidence of type I11 vWD (see above) and the presence of deletions in only a small subset of these patients, this would appear to be a




VON WILLEBRAND DISEASE 251 1

Table 2. Type 111 vWD

No. of Amino Acid Molecular Independent Substitution Defect Families Reference

- lg gene deletion (? entire gene) 2 12 - lg gene deletion (? entire gene) 2 13 - 2.3 kb deletion (exon 42) 1 95 - cis-defect in vWF mRNA expression 1 84

Rpro365* C1093T 1 98 R1772’ C7603T 3 t 97 - cis-defect in vWF mRNA expression 3 t 97

‘Termination codon. t A l l 3 alleles have identical RFLP haplotypes.

rare mechanism for vWD. However, it is certainly possible that some deletions have been missed due to the large size of the vWF gene and cross-hybridization to the pseudogene. Southern blots spanning the entire vWF gene are necessar- ily very complex and deletions involving only one vWF allele could easily be missed, particularly in the region of the pseudogene duplication. In the latter instance, deletion would be manifested as only a 25% reduction in intensity of the relevant bands. Although 50% dose reduction has been detected by careful den~itometry,’~ such detailed quantitative analysis has not generally been performed on routine Southern blots.

NONDELETION vWF DEFECTS IN TYPE 111 vWD

Once deletion has been excluded as a molecular mechanism, the search for subtle mutations resulting in quantitative decreases in vWF presents a formidable problem. Such defects could lie anywhere within the 178-kb vWF gene (on either of two alleles) or potentially in distant regulatory sequences located hundreds of kilobases upstream or down- stream of the gene. Direct sequence analysis of such a large region of DNA is currently impractical. A method for indentifying the presence of a subset of such defects has recently been described.84 In this approach, DNA sequence polymorphisms, identified within vWF exons, are assessed

Table 3. Miscellaneous Defects

No. of Amino Acid Nucleotide Independent Functional Substitution Substitution Families Studies References

FVlll binding mutants R19W C2344T 1 + 133 T28M C2372T 1 +132 127,132 R53W C2446T 2 + l X 130,134 R91Q G2561A 4 + 128.129 128- 130

Other variants Gene deletion

- (exons 26-34) 1 96 G561S G3970A 1 + 140

139 F6061 T4105A 1 -

Platelet type (“pseudo-vWD) G233V (GPlb a-chain) 1 82

141 M239V (GPlb a-chain) 1

- -

by polymerase chain reaction (PCR) from both genomic DNA and platelet vWF messenger RNA (mRNA). Any molecular defect resulting in loss of vWF mRNA expression from one allele would result in detection of both alleles at the level of genomic DNA but only the normal allele in platelet mRNA. Defects that could be detected in this way include any cis-acting mutation affecting mRNA transcription, nuclear processing, or mRNA stability. Given the complexity of the vWF gene (51 introns), aberrant splicing might present a particularly likely mechanism for vWD. By this analysis, Nichols et als4 demonstrated defective vWF mRNA expression from both maternal and paternal alleles in one type I11 vWD pedigree. All offspring inheriting both aberrant alleles were affected with type I11 vWD, whereas both parents (obligate carriers) and five heterozygous siblings were asymptomatic and had normal vWF plasma levels. In additional preliminary studies, a similar “null” allele has been identified in another type I11 vWD family. Analysis in three type I vWD pedigrees demonstrated equal expression of both vWF alleles (W.C. Nichols and D. Ginsburg, unpublished data). In a preliminary report, defective mRNA expression was detected from 2 different alleles in the Dutch population by a similar approach, with one allele also associated with a nonsense mutation.97 A nonsense mutation has also been identified in another type I11 vWD pedigree by screening 18 families at 2 potential sites.98 The currently identified molecular defects in type I11 vWD are summarized in Table 2.

TYPE 111 VERSUS TYPE I vWD

Type I11 and type I vWD are both quantitative defects, the former characterized by total or near complete absence of vWF and the latter by partial vWF reduction of 50% or more. In the simplest model, these two disorders could be viewed as the homozygous and heterozygous states for the same defect. Mutations within the vWF gene resulting in loss of expression would manifest as type I vWD in the heterozygote and type I11 in the homozygote. In this model, vWD would be more properly classified as an autosomal codominant disorder. However, several lines of evidence argue against this model. Although some investigators use the term type I11 vWD synonymously with severe vWD, others require clear autosomal recessive inheritance as a condition for this classification. Of note, for the majority of severely affected vWD patients, both parents, who would be assumed to be obligate heterozygotes, are completely asymptomatic. In the three reported type 111 vWD gene deletion fa mi lie^^*.*^^^^ and the nondeletion RNA expression defect family,84 parents, as well as sibling carrier, were all asymptomatic with essentially normal plasma vWF levels. Al- though there are occasional patients with type I11 vWD for whom one or both parents appear to have a mild form of vWD, this presentation is less common. Similarly, in the animal models described above, two patterns seem to emerge. The porcine model shows a recessive pattern of inheritance quite similar to human type 111 vWD and by linkage is due to a defect within the vWF gene.52 By contrast, the murine model shows a pure dominant inheritance, potentially analogous to type I vWD, with both




251 2 GINSBURG AND BOWlE

heterozygotes and presumed homozygotes having identical vWD phenotypes.70 Taken together, these observations suggest that type 111 and type I vWD may arise via different molecular mechanisms.

Despite considerable progress in characterizing vWD mutations in a variety of vWD subtypes, the molecular defects responsible for type I vWD remain unknown. Gene deletions, and presumably other defects resulting in complete loss of vWF expression from the vWF gene, appear to result in recessive type I11 vWD. If loss of vWF expression from one allele results in a silent carrier for type 111 vWD, an alternative mechanism must be proposed for the autosomal pattern of type I vWD inheritance. Type I vWD could be due to defects within the vWF gene giving rise to an abnormal protein whose function interferes with the normal allele in a "dominant-negative'' manner. The multimeric nature of vWF provides a plausible mechanism for such an interaction because an abnormal protein product from one allele could affect not only its own secretion, assembly, or function, but also the product of the normal allele with which it forms heteromultimers. Similar interac- tions between mutant and normal a1 subunits in the trimeric type I collagen molecule account for the autosomal dominance of osteogenesis imperfecta type II.99 Alterna- tively, type I vWD could be due to a defect(s) at another genetic locus, perhaps in a gene involved in vWF biosynthesis, processing, or secretion.

TYPE HA vWD

Identifying single point mutations within the vWF gene is a difficult problem. As noted above, because of its large size, direct sequence analysis of the entire vWF gene is not currently a practical approach. Concentrating on qualitative vWF variants, in which a mutation within the coding sequence might be expected, narrows the search to the coding exons (or mRNA). DNA sequence analysis is also complicated by the autosomal dominant nature of most vWD variants, requiring distinction between the single mutant allele and the other normal allele. In addition, mutations located within the mid portion of the vWF gene must be distinguished from the two pseudogene alleles. The pseudogene problem can be circumvented by directly analyzing vWF mRNA1" or, alternatively, using allele- specific PCR strategies to amplify only the authentic gene.51,84,86JM)-102 The latter approach is facilitated by knowl- edge of the authentic and pseudogene sequences deter- mined by Mancuso et al.50951

The first point mutations responsible for vWD were reported for the type IIA variant.lo0 A 176-Kd proteolytic fragment present in normal plasma and localized to the C-terminus of the mature vWF subunit has been observed to be markedly increased in the plasma of type IJA vWD patients.Io3 A similar proteolytic fragment and the associated vWF multimer satellite bands were also shown to result from proteolytic degradation in studies of cultured human umbilical vein endothelial cells derived from a patient with type IIA vWD.IM The hypothesis that increased sensitivity to proteolysis in the vicinity of a type IIA vWD mutation might account for the 176-Kd fragment

focused attention on the corresponding segment of vWF in exon 28. PCR sequence analysis of vWF mRNA obtained from platelets or directly from exon 28 genomic DNA sequences has led to the identification of a number of mutations in type IIA vWD patients, generally all clustered within the A2 homologous repeat (Table 4 and Fig 1).

Expression of mutant vWF sequences by transfection in mammalian cells has provided important insights into the molecular basis of type IIA vWD. Transfection results allow subclassification of type IIA vWD patients into two distinct s u b g r o ~ p s . ~ ~ J ~ ~ In group 1, the associated point mutation leads to a defect in intracellular transport with vWF observed to be retained within the endoplasmic reticu- l ~ m . ' ~ ~ In the heterozygous state, increased retention of the larger multimers (more likely to contain one or more variant subunits) could result in relatively more efficient secretion of the smaller multimers, accounting for the characteristic pattern observed in type IIAvWD plasma. By contrast, expression of recombinant vWF from group 2 results in normal appearing vWF multimers in tissue culture cells.105 One of these mutations (Arg834 -+ Trp) has been identified on at least two distinct genetic back- grounds in six unrelated type IIA vWD patients DNAs (Table 4).

The characteristic loss of large multimers in group 2 appears to occur via a second mechanism. Several studies have reported that large multimer loss in some type IIA vWD patients results from proteolysis occurring after synthesis and secretion1MJ06-1w mediated by a plasma or platelet specific protease, possibly a calpain.lOsJ1O The site of proteolysis within vWF generating the characteristic 176-Kd fragment has recently been localized to Tyr842- Met843, in close proximity to many of the identified type IIA mutations (Table 4).lI0 A model for loss of large vWF multimers as a result of this single proteolytic cleavage has been proposed.I1l Collection of blood from some type IIA patients into a cocktail of protease inhibitors results in relative preservation of multimer s t r u c t ~ r e . ~ ~ ~ J ~ ~ This group of patients may well correspond to the group 2 mutations. In support of this hypothesis, a close correlation was observed between preservation of vWF multimer structure

Table 4. Type IIA vWD Mutations


V551F G3940T 1 - 143 G742R G4513C 1 + 105 G742E G4514A 1 + 105 S743L C4517T 2 + l o 5 105,144 L777P T4619C 1 - 105 V802L G4693T 1 - 143 R834W C4789T 6* +".'O5 100,105,144,145 V844D T4820A 1 + 100,105 S850P T4837C 1 + 146 1865T T4883C 1 - 86 E875K G4912A 1 - 143

There are a total of 11 mutations in 17 families *Greater than or equal to 2 independent alleles, based on haplotype

analysis.lo5





in platelet vWF (protected from proteases within the platelet a-granule) and expression of intact multimers in in vitro transfection studies.105 Interestingly, in 1983 Weiss et a1 first suggested a subdivision of type IIA vWD based on platelet multimer pattern,Il2 a subclassification that probably will correspond to the subgroups now suggested on the basis of molecular pathogenesis.105 The list of currently reported potential type IIA mutations is shown in Table 4. In all, 11 mutations have been identified in 17 families. In one study, mutations were identified in 9 of 11 patients studies.lo5 Thus, it is likely that a panel of mutations accounting for the vast majority of type IIA vWD may eventually be established, permitting accurate diagnosis and classification at the DNA level for this common vWD subtype.

TYPE llB vWD

Type IIB vWD is a relatively uncommon variant characterized by a unique “gain of function.” In this disorder, vWF demonstrates an increased reactivity with its platelet receptor GPIb, resulting in spontaneous vWF/platelet interaction and subsequent clearance from the circulation. The larger vWF multimers are more reactive with platelets and are thus selectively cleared, resulting in the characteristic low molecular weight multimer pattern observed in type IIB vWD plasma. Since the vWF GPIb binding domain had been localized to a peptide fragment within vWF exon 28, several groups have analyzed DNA sequence from type IIB vWD patients, concentrating on this region. By this approach, seven specific single amino acid substitutions (and one insertion) have been identified in type IIB vWD patients (see Table 5) all clustered within a small segment of the vWF A1 repeat (Fig 1). Expression of the Trp550 -+

Cys mutation in a recombinant vWF fragment resulted in increased binding to platelets, consistent with the type IIB vWD phenotype.Il3 Full-length recombinant vWF containing the Arg543 - Trp mutation also demonstrated increased platelet binding, most marked for the large multimers, accounting for the characteristic vWF multimer

Table 5. Type llB vWD Mutations


540insM 39lOinsATG 1 - 147 R543W C3916T 8* +114 101,102,114,117,119,144 R545C C3922T 8t - 101,102 W550C G3939C 1 + 113 V551L G3940C I* - 119 V553M G3946A 79 - 101,102,117,120 P574L C4010T I + 116 R578Q G4022A 2 + I 1 5 101.1 15.1 17

There are a total of 7 mutations in 28families. *Greater than or equal to two independent alleles based on haplo-

type analysis (in two different s t ~ d i e s ~ ~ ~ , ~ ~ ~ ) . tGreater than or equal to three independent alleles based on

haplotype analysis.102 *New mutation. §Greater than or equal to three independent alleles based o n

haplotype analysis, including two new m ~ t a t i o n s . ~ ~ ~ ~ ’ 1 ~

pattern observed in type IIB vWD plasma.Il4 Preliminaq expression studies have also demonstrated increased platelet binding as a result of the Arg.578 + Gln and Pro574 + Leu substitutions, confirming their identify as authentic type IIB vWD muta t ion~ .~~~J l6

Four mutations account for nearly 90% of the type IIB vWD patients studied to date (Table 5). Several of these mutations have been demonstrated to be recurrent independent genetic events on the basis of RFLP haplotype analysis101,102 and in at least three cases, the amino acid substitution appears to be a new m~tation.’~*J l7-Il9 In one of these cases, the mutation was shown to have originated on one allele during the development of the germ-line in the founder and was subsequently passed on to a subset of his offspring inheriting that allele (germ-line mosaicism).lZ0 Five of the eight substitutions represent C + T transitions at CpG dinucleotides, proposed hot spots for mutation within the human genome.101J02 Thus, in the case of type IIB vWD, screening for a small panel of mutations should have a high sensitivity for the detection of type IIB vWD, and should facilitate precise diagnosis and classification of this disorder at the DNA level.

vWF/FVlll INTERACTION AND AUTOSOMAL HEMOPHILIA

As discussed above, vWF and FVIII are closely associated in plasma as a noncovalent molecular complex. vWF is critical for FVIII transport and stability in plasma and the decreased vWF levels resulting from most vWD variants are also generally associated with a proportional decrease in FVIII antigen and procoagulant activity. In patients with severe or type 111 vWD, FVIII levels are markedly reduced and contribute significantly to the resulting bleeding diathesis.56,58 The close association of vWF and FVIII led to initial difficulties in distinguishing classic hemophilia A from “pseudohemophilia” (VWD). ’ .~~~

Hemophilia A is an X-linked disorder due to mutations within the FVIII gene on the X chromosome long arm. In the past few years, over 100 distinct mutations responsible for the hemophilia phenotype have been identified, most through the use of PCR techniques.Iz1 Female patients affected with hemophilia are rarely encountered, often ascribed to unequal lyonization in a carrier. However, in several pedigrees this unusual form of hemophilia appears to be inherited in an autosomal f a s h i ~ n . ~ ~ ~ - ~ ~ ~ In several cases, plasma vWF was demonstrated to have a markedly decreased binding capacity for FVITI.124-126 One of these original reports suggested the name vWD “Normandy” (the patient’s province of origin) for vWF variants defined by deficient FVIII binding without other qualitative or quantitative a b n ~ r m a l i t i e s . ~ ~ ~ With the identification of additional patients with similar defects from a number of locations in the United States as well as Europe, a more general terminology should probably be a d ~ p t e d . I ~ ~ - * ~ ’

Recently, specific mutations within the vWF genes have been identified as the apparent explanation for the decreased vWF FVIII binding (Table 3). A single amino acid substitution, Thr28 + Met, has been identified in one of the original vWD “Normandy” p e d i g r e e ~ . l ~ ~ J ~ ~ Recombinant vWF containing this substitution demonstrated markedly





decreased FVIII binding.132 A study of three additional patients with low FVIII levels (6% to 22%) identified a homozygote for an Arg91 + Gln substitution, a homozygote for Arg53 + Trp, and a third patient who was a compound heterozygote for both of these latter muta- t i o n ~ . ~ ~ ~ The Arg91- Gln mutation was also identified on 1 vWF allele in each of two unrelated mild type I vWD patients with disproportionately low FVIII levels.128J29 Plasma vWF from one of these patients had previously been shown to exhibit an abnormal interaction with FVIII.131

Recombinant vWF containing the Arg91-+ Gln substitution demonstrated markedly decreased capacity for FVIII binding.128,L29 Interestingly, an amino acid polymorphism was identified on the other vWF allele from one of the type I patients,lZ9 located just 2 amino acids upstream and resulting in the similar substitution, Arg89 + Gln. Expres- sion of vWF containing this latter substitution resulted in FVIII binding indistinguishable from ~ i1d- type . l~~ The functional significance of the Arg53 + Trp mutation, along with a fourth substitution (Argl9 + Trp) have also recently been confirmed by recombinant expression s t ~ d i e s . ~ ~ ~ , ~ ~ ~

All of these vWF mutations are located within the N-terminal portion of vWF, the region implicated in binding to FVIII. As noted above, two independent monoclonal antibodies that block FVIII binding to vWF have been localized to a 19 amino acid peptide (Thr78-Thr96) with the N-terminal ~ e g m e n t . * ~ % ~ ~ Interestingly, the Arg91 + Gln substitution is localized in the middle of this small epitope. Together, these data provide strong evidence that this region plays a critical role in the FVIII/vWF interaction. The corresponding segment within FVIII that binds vWF has been localized to an acidic peptide, spanning amino acids 1677 to 1684.135 The Thr78-Thr96 epitope is markedly basic, and the Arg91 substitution results in a loss of one of these basic residues. However, the finding that Arg89 +

Gln has no effect on FVIII binding indicates that this interaction is not simply based on charge.129

Thus, these mutations at the vWF N-terminus (Table 3) all interfere with the ability of vWF to bind FVIII and define a new variant of vWD. In the homozygote (or compound heterozygote) this defect results in an autosomal form of hemophilia characterized by decreased FVIII levels and a clinical pattern indistinguishable from mild to moderate classic hemophilia A.127J30,132J34 In the heterozygote, this defect is generally silent and only detected by inciden- tal screening, or when it occurs in conjunction with a coexistent type I or other vWD variant.128J29s131J33 Coinher- itance of a FVIII binding defective vWF could account for some of the phenotypic heterogeneity occasionally observed among hemophilia A patients with identical FVIII gene m u t a t i o n ~ . ~ ~ ~ - ’ 3 ~

OTHER vWD VARIANTS

A large number of other vWD variants have been described, most as single case reports, generally identified by subtle abnormalities in vWF multimeric structure (Table l).I9 Diagnosis of these variants requires high quality mulimer analysis, generally available at only a few reference laboratories. Thus, their frequency may be underestimated.

Some of these variants may represent compound heterozy- gosity for other known subtypes. Preliminary reports of potential mutations responsible for several rare variants have recently appeared. 139~140

Several families have been reported with a unique disorder termed platelet-type or “pseudo”-vWD. This condition is remarkably similar to type IIB vWD, with decreased large vWF multimers, thrombocytopenia, and increased vWF/platelet interaction. However, unlike type IIV vWD, in which the defect has been shown to lie within the GPIb binding domain of vWF (Table 5), the defect in platelet type vWD is in the platelet itself, specifically within the GPIb/IX complex. The two disorders can be distinguished by platelet and plasma mixing studies. Point mutations within the GPIba chain gene potentially responsible for platelet-type vWD in two families have recently been reported (Table 3).82,141

PRENATAL DIAGNOSIS

With the dramatic advances in our understanding of vWD molecular genetics, powerful tools are now available for potential prenatal diagnosis. In those cases in which the precise molecular defect is known (Tables 2 through 5 ) , reliable and accurate diagnosis can easily be achieved from amniotic fluid or chorionic villus biopsy by PCR techniques, as now regularly applied for a variety of genetic disor- d e r ~ . ~ ~ , ~ ~ ~ As noted above, a large number of polymorphisms within the vWF gene have now been identified including a highly informative variable number tandem repeat (VNTR) in intron 40.83-85,xs,89 Using these markers, prenatal diagnosis has been successfully performed by genetic linkage a n a l y s i ~ . ~ ~ . ~ ~ , ~ ~ However, given the potential for locus heterogeneity in vWD (ie, vWD resulting from defects in genes other than vWF; see above), caution should be exercised in interpreting these results.

CONCLUSIONS

Since the cloning of vWF cDNA in 19857,xJ0,13 and characterization of the complete genomic structure of the vWF gene in 1989:O considerable progress has been made in characterizing the specific molecular defects responsible for the heterogeneous disorder known as vWD. A large number of specific molecular defects have now been identified and precise characterization may now be possible in the majority of type IIA, type IIB, and potentially also type 111 vWD cases. However, the most common variant, type I vWD, still remains a major challenge. Continued progress in this area will not only improve our understanding of the pathogenesis of vWD, but should also lead to more rapid and precise diagnosis and classification for this common disorder. The problems of incomplete vWD penetrance and poor diagnostic sensitivity and accuracy for the currently available clinical laboratory tests provide strong incentives for the development of DNA-based diagnostics. In addition, prenatal diagnosis is now possible either at the level of single point mutations or by RFLP analysis (assuming linkage to the vWF gene) and will probably be applied with increasing frequency. Understanding the molecular basis of vWD also has important implications for vWF structure and function and is helping to define critical binding domains





within the vWF molecule. Insights gained from these studies may lead to improved therapeutic approaches not only for VWD, but for a variety of other genetic and acquired hemorrhagic and thrombotic disorders.

ACKNOWLEDGMENT

We thank J.E. Sadler and members of our labs for thoughtful comments and discussion and S.E. Labun for careful preparation of the manuscript.

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SUPPI 1)





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