Molecular Basis of Disease

The VWF gene is located near the tip of the short arm of chromosome 12,spans 180 kb,and consists of52 exons.The intron-exon boundaries roughly correlate with the VWF domains. The presence of an unprocessed partial pseudogene located on chromosome 22q11.2 corresponds to exons 23 through 34 of the VWF gene. VWF is synthesized in endothelial cells and megakaryocytes and is secreted from storage sites (platelet alpha granules and endothelial cell Weibel-Palade bodies) into the plasma. VWF is a 2,813 amino acid peptide consisting of a 22 amino acid signal peptide, a 741 amino acid propeptide, and a mature 2,050 amino acid peptide (Figure 12-5). After removal of the signal peptide, the pro-VWF dimerizes by disulfide bonds at the C-terminal ends and further polymerizes at the N-terminal ends, resulting in multimers ranging in size from 0.5 to more than 10 million daltons. Binding sites for various ligands have been localized to different domains of the VWF subunit.

VWD can be divided into two broad categories based on quantitative (types 1 and 3) or qualitative (type 2) abnormalities of plasma VWF. Quantitative abnormalities

Signal peptide Propeptide

1 23

A1 A2 A3

FVlll

GPlb Botrocetin Heparin Sulfatide Multimer S-S

Collagen

2813

A1 A2 A3

FVlll

Collagen

GPllb-llla

RGDS I

GPllb-llla

Dimer S-S

Figure 12-5. Von Willebrand peptide domains. FVIII, factor VIII; SS, disulphide bridge; GPIb, Glycoprotein Ib; VWF, von Willebrand factor; GPIIb—IIIa, glycoprotein IIb-IIIa; RGDS, arginine (R), glycine (G), aspartic acid (D), serine (S) binding sequence.(Reprinted with permission from Sadler AJE. Von Willebrand disease. In: Scriver CR, Beaudet AL, Valle D, et al.,eds. The Metabolic and Molecular Bases of Inherited Disease.Copy-right 2001 McGraw-Hill.]

High M.W. multimers

Low M.W. multimers which may be due to defects in multimer synthesis or secretion.

High M.W. multimers

Low M.W. multimers

Figure 12-6. Von Willebrand factor multimer distribution. Types 1, 2, and 3 refer to types of von Willebrand disease. M.W., molecular weight. (Reprinted with permission from Bleeding disorders: an overview and clinical practice. In: Tefferi A, ed. Primary Hematology. Totowa, NJ: Humana Press; 2001:303. By permission of Mayo Foundation for Medical Education and Research.)

Normal Type 1 Type 2 A,B Type 3 Normal

Figure 12-6. Von Willebrand factor multimer distribution. Types 1, 2, and 3 refer to types of von Willebrand disease. M.W., molecular weight. (Reprinted with permission from Bleeding disorders: an overview and clinical practice. In: Tefferi A, ed. Primary Hematology. Totowa, NJ: Humana Press; 2001:303. By permission of Mayo Foundation for Medical Education and Research.)

include a mild reduction of qualitatively normal VWF (type 1) or absent VWF (type 3). In type 2 VWD,the plasma VWF exhibits defective structure and function, and typically results in absence or reduction of the larger VWF multimers (Figure 12-6).

Mutations and polymorphisms in the VWF gene are currently being cataloged in an international database, which can be accessed via the Internet. References for the amino acid number system used below can be accessed at this website (http://www.vwf.group.shef.ac.uk/index.html).

Type 3 von Willebrand Disease

Type 3 VWD is autosomal recessive and is characterized by a severe reduction in VWF antigen, ristocetin cofactor activity, and a concordant reduction in F8 activity, resulting in a more severe phenotype. Type 3 VWD mutations include frameshifts, deletions, and nonsense mutations. Although most patients are typically compound heterozygous for such VWF mutations, homozygosity has been demonstrated in a few consanguineous families. Although most patients with type 3 VWD appear to have two defective VWF alleles, many have clinically unaffected parents. This circumstance poses a challenge in providing genetic counseling.

The prevalence of type 3 VWD is 0.5 to 3 per million and, based on the Hardy Weinberg equilibrium, heterozygotes should occur at an expected frequency of at least 1,400 to 3,500 per million population. However, the currently estimated prevalence of type 1 VWD is at least 10-fold lower. Although VWF levels may be lower in parents of patients with type 3 VWD, there is clearly variable expression, with most parents being asymptomatic.

Type 2 Von Willebrand Disease

Type 2 variants of VWD are characterized by qualitatively abnormal VWF with defective stability, function, or multimer distribution and include types 2A, B, M, and N.

Type 1 Von Willebrand Disease

Type 1 VWD accounts for approximately 70% of cases of VWD and is typically autosomal dominant. Diagnosis is established by assays of VWF demonstrating proportionate reduction in plasma VWF antigen, ristocetin cofactor activity, and F8 activity, with a normal distribution of VWF multimers. Although type 1 is the most commonly diagnosed variant of VWD, little is known of the molecular pathogenesis. Currently, apart from a few sporadic reports, few mutations in type 1 VWD have been characterized. Although type 1 VWD appears to be linked to the VWF locus, animal data is suggestive of locus heterogeneity as an explanation for the mild quantitative deficiency of VWF associated with type 1 VWD (e.g., defects in glycosylation of the VWF protein).15 However, selected patients with type 1 VWD are a result of heterozygous inheritance of a type 3 VWD defect, which includes nonsense, frameshift, or deletion mutations.

Type 1 VWD has heterogeneous clinical manifestations with variable penetrance that may be due to concordant or discordant reductions in platelet VWF. Some pedigrees are characterized by significant reductions in VWF levels,

Type 2A Von Willebrand Disease

Type 2A VWD is autosomal dominant and accounts for approximately 75% of all type 2 VWD. Type 2A VWD patients have a variable reduction in VWF antigen, with a discordant reduction in ristocetin cofactor activity, indicative of a qualitative VWF abnormality. The higher and intermediate plasma VWF multimers are reduced or absent, and the lower-molecular-weight multimers are relatively increased or have an abnormal infrastructure. Plasma and platelet multimer abnormalities may be concordant or discordant depending on the underlying molecular defect.

Missense mutations resulting in type 2A VWD occur predominantly in the A2 domain of the VWF gene and result in abnormal VWF patterns by two distinct mechanisms. The first group includes mutations impairing the assembly and secretion of normal VWF multimers, resulting in decreased higher-molecular-weight VWF multimers in both plasma and platelets. The second group includes mutations that result in normal assembly and secretion of VWF; however, the mutant VWF has an increased sensitivity to proteolytic degradation in plasma, resulting in decreased plasma high-molecular-weight multimers but a normal platelet VWF multimer pattern. The cleavage site in a subset of patients with type 2A VWD was shown to be the Tyr842-Met843 bond, where mutations may result in a conformational change, resulting in increased sensitivity to proteolysis.

Type 2B Von Willebrand Disease

Type 2B VWD is autosomal dominant and accounts for approximately 20% of all type 2 VWD. Type 2B VWD patients have a variable reduction in the VWF antigen, a discordant reduction in ristocetin cofactor activity, with a loss of the higher and intermediate plasma VWF multimers, but normal distribution of platelet VWF multimers. The type 2B variant is distinguished from type 2A VWD by the presence of mild-to-moderate thrombocytopenia. This occurs as a result of an increased affinity of VWF for platelet GPIb, resulting in spontaneous binding of VWF to platelets and rapid clearance of the platelet-bound larger multimers from plasma. In addition,platelet aggregation in response to ristocetin demonstrates an exaggerated response. The few cases described of normal multimer distribution with hyperresponsiveness to ristocetin appear to represent a mild form of type 2B VWD. Causative missense mutations occur in the A domain of the VWF gene and result in a dominant gain-of-function phenotype. Mutations in the A domain likely disrupt a regulatory site that normally inhibits the binding of the A1 domain to platelet GPIb.

Type 2B VWD must be distinguished from a pseudo-VWD or platelet-type VWD, which is similar in presentation. Patients with platelet-type VWD have a primary platelet defect resulting from mutations in the platelet GPIb/IX receptor.

Type 2M Von Willebrand Disease

In type 2M VWD patients, although the VWF antigen and the distribution of VWF multimers are normal, the risto-cetin cofactor activity is reduced, reflecting a functional defect of the VWF multimers. There may be uncleaved proVWF or ultrahigh-molecular-weight multimers. Type 2M mutations occur in the A1 domain of the VWF gene and result in decreased binding affinity of VWF for platelet GPIb.

Type 2N (Normandy) Von Willebrand Disease

Mutations in the F8 binding domain of VWF result in suboptimal binding of F8 to VWF. This binding defect results in a shorter half life of plasma F8, and thus plasma F8 activity is reduced. Levels of VWF antigen and ristocetin cofactor activity are normal, as is the VWF multimer distribution. The type 2N subtype mimics mild HA but has an autosomal recessive pattern of inheritance rather than the

X-linked recessive pattern of HA. In a recent international survey, VWD Normandy was detected in 4.8% (58 of 1198) of patients previously diagnosed as having mild HA. Three VWF gene mutations (Thr791Met, Arg816Trp, and Arg854Gln) account for 96% of type 2N patients.16 Type 2N VWD should be considered in patients with a diagnosis of "mild HA" with a non-X-linked inheritance pattern. Typically, heterozygotes have normal F8 levels and homozygotes have reduced F8 activity. However, apparent heterozygotes with low F8 levels typically have inherited a second allele, resulting in VWD type 1 (compound heterozygotes).

0 0

Post a comment