Molecular Diversity Of

von Willebrand disease is an extremely heterogeneous disorder, and results from phenotypic and genotypic tests form the basis of the diagnosis and classification of different types of VWD. The numerous variants of VWD generally fit in the original classification of either type I (partial) and III (total) reduction or type II with qualitative defect of VWF. Multimeric analysis of von Willebrand antigen (VWF:Ag) by SDS-gel agarose gel electrophore-sis[7] results in identification of many different type II variants that have minor structural differences. The latest classification of VWD, however, reduced the total number of type 2 to four variants of 2A, 2B, 2M, and 2N on the basis of their structure/function (Table 1).[8] As type 1 VWD is now considered to be purely quantitative disorders, some previously designated variants have now been reclassified as type 2, e.g., type 2M (Table 1). The molecular basis of these six new subtypes with clear pathophysiological mechanisms has now been further defined with identification mutations in some of these

Fig. 1 Schematic presentation of the human VWF gene, pseudogene, mRNA, and protein. Numbers below the boxes representing signal peptide (sp), propeptide, and mature VWF in the upper row (in red) are that of mature VWF amino acid numbers and below them are the amino acid numbers from the ATG initiation codon (residue 1) to the carboxy-terminal lysine (residue 2813) of the pre-pro-VWF (in blue). The lettered boxes denote the series of the repeated homologous segments. The approximate localization for known VWF functional domains within the mature VWF sequence is indicated below. The general location of mutations associated with the most common type 2 variants of VWD is depicted below the domain structure of VWD together with the associated exons. (View this art in color at www.dekker.com.)

Fig. 1 Schematic presentation of the human VWF gene, pseudogene, mRNA, and protein. Numbers below the boxes representing signal peptide (sp), propeptide, and mature VWF in the upper row (in red) are that of mature VWF amino acid numbers and below them are the amino acid numbers from the ATG initiation codon (residue 1) to the carboxy-terminal lysine (residue 2813) of the pre-pro-VWF (in blue). The lettered boxes denote the series of the repeated homologous segments. The approximate localization for known VWF functional domains within the mature VWF sequence is indicated below. The general location of mutations associated with the most common type 2 variants of VWD is depicted below the domain structure of VWD together with the associated exons. (View this art in color at www.dekker.com.)

defects. A database of VWD lists all the point mutations, insertions, and deletions identified in this type. This site (http://www.shef.ac.uk/vwf), which has been created on behalf of the ISTH VWF Scientific and Standardization Committee, is maintained by Sheffield University and contains up-to-date information on the latest mutations and polymorphisms in VWF gene. In the recent 2003 XIX Congress of the International Society on Thrombosis and Hemostasis in Birmingham, United Kingdom, several presentations emphasized the need for a new classification of VWD based on molecular diagnosis and improved multimeric analysis of VWF. With so many unclassifiable patients with type 1 VWD group, it has now been suggested that such an improved multimeric method would help in future classification of these patients.

Type 1 VWD patients have reduced VWF:Ag level with normal multimer composition (Fig. 2). This is the commonest VWD type, accounting for about 70% of the clinical cases, with patients having mild to moderate bleeding. It has dominant mode of transmission with incomplete penetrance of approximately 60%. While lowering of VWF:Ag could be the result of a mutation in VWF gene, additional variations such as ABO blood group, race, and age complicate the diagnosis of this type of VWD. A broad range of VWF levels suggests several distinct molecular mechanisms could be responsible for this type of VWD, and in many affected families possession of a single mutant allele does not consistently cause symptoms, with some patients being asymptomatic and only transmitter of the disease. In some other families, exceptionally lowered VWF levels and with dominant inheritance trait and very high penetrance may be caused by dominant-negative missense mutations that impair the intracellular transport and secretion of normal VWF

Table 1 Previous and revised classification of VWD

Revised classification of VWD

Type 1 VWD

Partial quantitative deficiency of VWF Type 3 VWD

Virtually complete deficiency of VWF Type 2 VWD

Qualitative abnormalities of VWF Type 2A

Qualitative variants with decreased platelet-dependent function that is associated with absence of high molecular weight multimers Type 2B

Qualitative variants with increased affinity for platelet GP1b Type 2M

Qualitative variants with decreased platelet-dependent function not caused by the absence of high molecular weight multimers Type 2N

Qualitative variants with markedly decreased affinity for factor VIII

Previous Classification

Type I, I ''platelet normal," I ''platelet low'' IA, I-1, I-2, I-3

Type III

Type IIA, B, C, D, E, F, G, H, I and I ''platelet discordant''

Type IIB, I New York, I Malmo, I Sydney Type B, IC, ID, Vincenza

Normandy subunits. Presently, a European study into molecular and clinical markers for diagnosis and management of type 1 VWD has been reporting large number of mutations associated with this type of VWD.[9]

Severe type 3 VWD has autosomal recessive inheritance with a prevalence of about one per million. Molecular analysis of this type has been more difficult, with no possibility of targeting for mutation detection. Reported

Fig. 2 VWF:Ag multimer patterns in normal and different types of VWD plasma. The plasma samples were electrophoresed in 1.2% (panel A) and 1.8% (panel B) agarose gel with sodium dodecyl sulphate, and the bands were visualized by 125I-labeled anti-VWF monoclonal antibody followed by autoradiography. Lane 1 is a type IIA phenotype; lane 2, IID phenotype; lane 3, type 2B; lane 4, normal; and lane 5, type 1 VWD plasma. The triplet structure of multimer band is demonstrated in lane 3 (panel B). The minor satellite bands ''a'' and "b" can be best seen in the band 1 of plasma from phenotype IIA VWD in lane 1. Arrow at the top of the gel points to the line between the stacking and separating gels. Direction of electrophoresis is from top to the bottom.

Fig. 2 VWF:Ag multimer patterns in normal and different types of VWD plasma. The plasma samples were electrophoresed in 1.2% (panel A) and 1.8% (panel B) agarose gel with sodium dodecyl sulphate, and the bands were visualized by 125I-labeled anti-VWF monoclonal antibody followed by autoradiography. Lane 1 is a type IIA phenotype; lane 2, IID phenotype; lane 3, type 2B; lane 4, normal; and lane 5, type 1 VWD plasma. The triplet structure of multimer band is demonstrated in lane 3 (panel B). The minor satellite bands ''a'' and "b" can be best seen in the band 1 of plasma from phenotype IIA VWD in lane 1. Arrow at the top of the gel points to the line between the stacking and separating gels. Direction of electrophoresis is from top to the bottom.

mutations for type 3 VWD are either homozygous or compound heterozygous, and although total or partial VWF deletions have been reported, this is uncommon. A large number of nonsense, frame shift, or missense mutations that predict the loss of VWF protein expression, or a markedly truncated or disrupted protein, have been identified.[10,11] Multiple substitutions in the VWF gene that mimic the pseudogene sequences have also been reported to cause type 1 and 3 VWD.[12] Haplotyping with a large battery of RFLPs including a highly polymorphic tetranucleotide repeat in intron 40 in families with type 1 and 3 VWD can have an important role in determining the inheritance of disease in these families.[13] Gene tracking of the affected allele can be used to identify the transmitters or the asymptomatic carriers of VWD, and this methodology has been found to be an essential initial step in studying such families prior to more comprehensive investigations and mutation detection.[14]

In the latest classification of the VWD, type 2 variants with qualitative abnormalities of VWF are divided into four groups of 2A, 2B, 2M, and 2N (Table 1). They are classified on the bases of phenotypic data and functional assays. These categories have distinct pathophysiological mechanisms, and their reported mutations are located in specific functional regions of the VWF gene. Each group has distinct clinical features and with specific therapeutic need. FVIII:C and VWF:Ag concentration, together with multimer distribution, ristocetin-induced platelet aggregation, and FVIII binding assay are the minimum phenotypic assays, which are needed for these classifications. Type 2A VWD patients have decreased platelet-dependent function with selective absence of high and intermediate molecular weight multimers (Fig. 2). Both recessive and dominant mode of inheritance have been reported; however, dominant inheritance accounts for the majority of cases. The reported mutations are all singlebase substitutions with five in the A1 domain, and the rest in the A2 domain. However, mutations for some of the type 2A phenotypes that are now classified in this group (formally designated as type IIC, IID, and IIE VWD; Table 1) are located in other regions of VWF gene. Most of the point mutations for type 2A VWD were identified in exon 28 where VWF pseudogene is also located. Using allele-specific PCR strategies for amplification of this exon circumvents this problem.[15] Arg1597Try mutation accounts for about 1/3 of type 2A VWD patients. The mechanisms for these mutations can be divided into two categories.[16] Group 1 mutation is the result of improper folding of the VWF multimers, resulting in impairment of the assembly, storage, and retention in the endoplasmic reticulum, both in the plasma and in the platelet compartments. In group 2, the mutations do not interfere with VWF assembly or secretion, but they do render normal VWF multimers more susceptible to a specific

VWF cleaving metalloproteinase or ADAMST13 present in the plasma.[17] ADAMST13 cleaves normal VWF multimer between the proteolytic site Tyr1605 and Met1606, and generates a 176-kDa fragment localized to the C-terminus, and a 140-kDa to the N-terminus of the mature VWF.[18]

In the very rare recessive IIC, IID, and IIE phenotypes, several missense, small insertions, or deletion mutations have been reported in the D2 domain. The common pathophysiological mechanism involved in all of these VWD type 2 A mutations is the absence of high molecular weight multimers and, consequently, the reduction in in vivo VWF-dependent platelet adhesion.

Type 2B VWD is characterized by increased affinity of the mutant VWF for platelet and reduction in high molecular weight multimers (Fig. 2). The remaining VWF multimers are not hemostatically effective and cause bleeding and thrombocytopenia in the patients that can be exacerbated during physical exercise, stress, and pregnancy. This defect is identified in the laboratory by enhanced ristocetin-induced platelet aggregation. Type 2B is inherited as an autosomal dominant and accounts for less than 20% of all type 2 VWD. Aside from a single amino acid insertion, the genetic defect is generally found to be a point mutation in the A1 domain of VWF gene that contains the glycoprotein Ib (GpIb) binding domain and results in a gain of function. The most frequent mutations are Arg1306Trp, Arg1308Trp, Val1316Met, and Arg1314Gln accounting for ~ 90% of the subtype.

Type 2M variants are characterized by decreased platelet-dependent function not caused and associated with loss of high molecular weight multimers, and their phenotype can be caused by mutations that inactivate specific binding sites for the ligands in connective tissue or on the platelet surface, without affecting multimer assembly or stability. Most of these variants were previously grouped with type I VWD, including type B with absent ristocetin—but normal botrocetin-cofactor activity and normal multimers and ''Vincenza'' variant where unusually ultrahigh molecular weight multimers are present.[19] In the past few years, mutations have been identified and are now listed in the database. Some of these mutations, including Ala1374Cys and Ala1374His, have been confirmed by mutation recombinant VWF studies. A type 2M ''Vincenza'' mutation, heterozygous Arg1205His, is the latest to be reported for type 2M VWD identified in all of the affected members of seven Italian families and in one German patient.[20]

Type 2N VWD or ''Normandy VWD'' results from defective binding ability of VWF with FVIII, and these patients show disproportionately reduced levels of FVIII and normal VWF multimers.[21] As a result of description of this type of VWD, FVIII binding assay should be the differential diagnosis test for patients with mild to

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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