Expression of Phosphate Transporters during Dental Mineralization.

The importance of phosphate (Pi) as an essential component of hydroxyapatite crystals suggests a key role for membrane proteins controlling Pi uptake during mineralization in the tooth. To clarify the involvement of the currently known Pi transporters (Slc17a1, Slc34a1, Slc34a2, Slc34a3, Slc20a1, Slc20a2, and Xpr1) during tooth development and mineralization, we determined their spatiotemporal expression in murine tooth germs from embryonic day 14.5 to postnatal day 15 and in human dental samples from Nolla stages 6 to 9. Using real-time polymerase chain reaction, in situ hybridization, immunohistochemistry, and X-gal staining, we showed that the expression of Slc17a1, Slc34a1, and Slc34a3 in tooth germs from C57BL/6 mice were very low. In contrast, Slc34a2, Slc20a1, Slc20a2, and Xpr1 were highly expressed, mostly during the postnatal stages. The expression of Slc20a2 was 2- to 10-fold higher than the other transporters. Comparable results were obtained in human tooth germs. In mice, Slc34a2 and Slc20a1 were predominantly expressed in ameloblasts but not odontoblasts, while Slc20a2 was detected neither in ameloblasts nor in odontoblasts. Rather, Slc20a2 was highly expressed in the stratum intermedium and the subodontoblastic cell layer. Although Slc20a2 knockout mice did not show enamel defects, mutant mice showed a disrupted dentin mineralization, displaying unmerged calcospherites at the mineralization front. This latter phenotypical finding raises the possibility that Slc20a2 may play an indirect role in regulating the extracellular Pi availability for mineralizing cells rather than a direct role in mediating Pi transport through mineralizing plasma cell membranes. By documenting the spatiotemporal expression of Pi transporters in the tooth, our data support the possibility that the currently known Pi transporters may be dispensable for the initiation of dental mineralization and may rather be involved later during the tooth mineralization scheme.

Oxidative stress enhances and modulates protein S-nitrosation in smooth muscle cells exposed to S-nitrosoglutathione.

Among S-nitrosothiols showing reversible binding between NO and -SH group, S-nitrosoglutathione (GSNO) represents potential therapeutics to treat cardiovascular diseases (CVD) associated with reduced nitric oxide (NO) availability. It also induces S-nitrosation of proteins, responsible for the main endogenous storage form of NO. Although oxidative stress parallels CVD development, little is known on the ability of GSNO to restore NO supply and storage in vascular tissues under oxidative stress conditions. Aortic rat smooth muscle cells (SMC) were stressed in vitro with a free radical generator (2,2'-azobis(2-amidinopropane) dihydrochloride, AAPH). The cellular thiol redox status was reflected through levels of reduced glutathione and protein sulfhydryl (SH) groups. The ability of GSNO to deliver NO to SMC and to induce protein S-nitrosation (investigated via mass spectrometry, MS), as well as the implication of two redox enzymes involved in GSNO metabolism (activity of gamma-glutamyltransferase, GGT, and expression of protein disulfide isomerase, PDI) were evaluated. Oxidative stress decreased both intracellular glutathione and protein -SH groups (53% and 32% respectively) and caused a 3.5-fold decrease of GGT activity, while PDI expression at the plasma membrane was 1.7-fold increased without any effect on extracellular GSNO catabolism. Addition of GSNO (50 µM) increased protein -SH groups and protein S-nitrosation (50%). Mass spectrometry analysis revealed a higher number of S-nitrosated proteins under oxidative stress (83 proteins, vs 68 in basal conditions) including a higher number of cytoskeletal proteins (15, vs 9 in basal conditions) related with cell contraction, morphogenesis and movement. Furthermore, proteins belonging to additional protein classes (cell adhesion, transfer/carrier, and transporter proteins) were S-nitrosated under oxidative stress. In conclusion, higher levels of GSNO-dependent S-nitrosation of proteins from the cytoskeleton and the contractile machinery were identified under oxidative stress conditions. The findings may prompt the identification of suitable biomarkers for the appraisal of GSNO bioactivity in the CVD treatment.

Sclerostin Deficiency Promotes Reparative Dentinogenesis.

In humans, the SOST gene encodes sclerostin, an inhibitor of bone growth and remodeling, which also negatively regulates the bone repair process. Sclerostin has also been implicated in tooth formation, but its potential role in pulp healing remains unknown. The aim of this study was to explore the role of sclerostin in reparative dentinogenesis using Sost knockout mice ( Sost-/-). The pulps of the first maxillary molars were mechanically exposed in 3-mo-old Sost-/- and wild-type (WT) mice ( n = 14 mice per group), capped with mineral trioxide aggregate cement, and the cavities were filled with a bonded composite resin. Reparative dentinogenesis was dynamically followed up by micro-computed tomography and characterized by histological analyses. Presurgical analysis revealed a significantly lower pulp volume in Sost-/- mice compared with WT. At 30 and 49 d postsurgery, a large-forming reparative mineralized bridge, associated with osteopontin-positive mineralization foci, was observed in the Sost-/- pulps, whereas a much smaller bridge was detected in WT. At the longer time points, the bridge, which was associated with dentin sialoprotein-positive cells, had expanded in both groups but remained significantly larger in Sost-/- pulps. Sclerostin expression in the healing WT pulps was detected in the cells neighboring the forming dentin bridge. In vitro, mineralization induced by Sost-/- dental pulp cells (DPCs) was also dramatically enhanced when compared with WT DPCs. These observations were associated with an increased Sost expression in WT cells. Taken together, our data show that sclerostin deficiency hastened reparative dentinogenesis after pulp injury, suggesting that the inhibition of sclerostin may constitute a promising therapeutic strategy for improving the healing of damaged pulps.

Minimal intervention dentistry II: part 3. Management of non-cavitated (initial) occlusal caries lesions--non-invasive approaches through remineralisation and therapeutic sealants.

Epidemiological data on dental caries show that prevention and treatment needs have evolved significantly over the past two decades. In younger patients the distribution of caries lesions is mainly found on the occlusal surfaces. The treatment approaches utilised by dentists must evolve to integrate preventive and treatment solutions tailored to the care needs, which are straightforward to implement in the dental office and whose effectiveness is underpinned by scientific evidence. This article aims to describe the principles of non-invasive management of non-cavitated (initial) occlusal caries lesions, based on evidence from recent studies published in the international literature.

Minimal intervention dentistry: part 4. Detection and diagnosis of initial caries lesions.

The detection of carious lesions is focused on the identification of early mineral changes to allow the demineralisation process to be managed by non-invasive interventions. The methods recommended for clinical diagnosis of initial carious lesions are discussed and illustrated. These include the early detection of lesions, evaluation of the extent of the lesion and its state of activity and the establishment of appropriate monitoring. The place of modern tools, including those based on fluorescence, is discussed. These can help inform patients. They are also potentially useful in regular control visits to monitor the progression or regression of early lesions. A rigorous and systematic approach to caries diagnosis is essential to establish a care plan for the disease and to identify preventive measures based on more precise diagnosis and to reduce reliance on restorative measures.

Tooth dentin defects reflect genetic disorders affecting bone mineralization.

Several genetic disorders affecting bone mineralization may manifest during dentin mineralization. Dentin and bone are similar in several aspects, especially pertaining to the composition of the extracellular matrix (ECM) which is secreted by well-differentiated odontoblasts and osteoblasts, respectively. However, unlike bone, dentin is not remodelled and is not involved in the regulation of calcium and phosphate metabolism. In contrast to bone, teeth are accessible tissues with the shedding of deciduous teeth and the extractions of premolars and third molars for orthodontic treatment. The feasibility of obtaining dentin makes this a good model to study biomineralization in physiological and pathological conditions. In this review, we focus on two genetic diseases that disrupt both bone and dentin mineralization. Hypophosphatemic rickets is related to abnormal secretory proteins involved in the ECM organization of both bone and dentin, as well as in the calcium and phosphate metabolism. Osteogenesis imperfecta affects proteins involved in the local organization of the ECM. In addition, dentin examination permits evaluation of the effects of the systemic treatment prescribed to hypophosphatemic patients during growth. In conclusion, dentin constitutes a valuable tool for better understanding of the pathological processes affecting biomineralization.

Review: behaviour of endothelial cells faced with hypoxia.

Hypoxia is a diminution of oxygen quantity delivered to tissue for cellular need to product energy. Hypoxia derives from two major conditions in health diseases: anemia and ischemia. Anemic hypoxia comes from damage to O(2) transport like red blood cells diminution or disease. Ischemic hypoxia is a diminution of blood flow following a diminution of blood volume after a hemorrhagic shock. After hypoxia, vessels dilate to increase blood flow allowing a better oxygenation of peripheral tissues. This vasodilation appears immediately after the beginning of hypoxia and can be maintained during several hours. Today, the molecular mechanisms of this vasodilation stay unclear. But it seems that potassic channels, ATP concentration and medium acidification in addition to vasodilator/vasoconstrictor balance play a great role to facilitate the oxygenation of the ischemic areas.As endothelial cells (EC) are lining the vasculature, they are always in contact with blood, which carries, amongst other compounds, oxygen. In this way, they are the first target for an oxygen partial pressure (PO(2)) diminution. EC, through different mechanosensors, can sense a variation in PO(2) and adapt their metabolism to maintain ATP production. Under hypoxia, EC switch into hypoxic metabolism, leading to the production of reactive oxygen species (ROS). Indeed, when PO(2) is low, the respiratory chain in the mitochondria runs slower. Furthermore, cytochrome C capacity to trap O(2) is reduced; this phenomenon alters the cellular redox potential and leads to the accumulation of electrons that induce the formation of ROS.This review presents an overview of the behaviour of endothelial cells face to hypoxia. We propose to focus on nitric oxide, hypoxia inducible factor (HIF), lactate and ROS productions. Then we present the different mode of culture of EC under hypoxia. Finally, we conclude on the difficulty to study hypoxia because of the various types of system developed to reproduce this phenomenon and the different signalling ways that can be activated.

In vitro impact of physiological shear stress on endothelial cells gene expression profile.

In the vascular system, the shear applied to the vascular wall activates mechano-sensors located on endothelial cells (ECs) leading to a modification in the gene expression profile. We applied laminar shear stress at 1 Pa on ECs for 6 h and measured by quantitative real time PCR the expression modulation of genes implied in inflammation (ICAM-1 and E-selectin), oxidative stress sensing (HO-1) and vascular tone modulation (eNOS). We showed that all these genes are shear stress inducible. ICAM-1 is more up-regulated than E-selectin suggesting different levels of implication in inflammatory responses and different modes of induction (SSRE, cytokine). Laminar shear stress induces an oxidative stress translated into HO-1 up-regulation, and a possible vasodilatation through the induction of eNOS. Our laminar shear stress system opens a novel and interesting frame in the evaluation of the impact on ECs and blood cells of new pharmacological substances injected in the bloodstream.

Type 2M vWD resulting from a lysine deletion within a four lysine residue repeat in the A1 loop of von Willebrand factor.

Type 1 von Willebrand disease is characterized by a decreased plasma concentration of functionally normal von Willebrand factor (vWF) whereas type 2M is characterised by an abnormal vWF displaying decreased affinity for platelets. In these two types of patients, the multimeric structure of vWF is normal. We report here the identification, in two unrelated families from the UK and Algeria, of an in-frame 3 bp deletion, at the heterozygous state, resulting in the deletion of a lysine residue within a four lysine repeat at position 642-645 of the mature vWF subunit (del K 1405-1408 in pre-pro vWF). The patients who have a discrepancy between vWF antigen level and vWF ristocetin cofactor activity exhibited decreased ristocetin-induced binding but only a slight decrease in the percentage of high molecular weight (HMW) multimers in plasma. Recombinant vWF harbouring this deletion did not bind to platelet GPIb in the presence of ristocetin or botrocetin although the protein is multimerized. Consequently, this lysine deletion was considered as a type 2M vWD mutation.

Conformational changes in the D' domain of von Willebrand factor induced by CYS 25 and CYS 95 mutations lead to factor VIII binding defect and multimeric impairment.

We report 2 new mutations identified in 3 patients and characterized by the markedly decreased affinity of von Willebrand factor (vWF) for factor VIII (FVIII). Patients 2 and 3, who have a typical type 2N phenotype, were found to be compound heterozygous for Arg91Gln and Cys25Tyr or Cys95Phe, respectively. Patient 1, who is the first cousin of patient 2, had an FVIII binding defect of vWF, low levels of vWF, and multimeric impairment. She was found to be compound heterozygous for the mutations Cys25Tyr and a stop codon (D93ter) in exon 4. Transient expression of recombinant vWF (rvWF) containing either Cys25Tyr or Cys95Phe mutations resulted in mutated rvWF with markedly reduced FVIII binding ability, multimeric structure impairment, and a significant decrease in the vWF expression level. Moreover, the use of anti-vWF monoclonal antibodies that inhibit the FVIII binding showed that these 2 mutations likely induce a conformational change in the D' domain. These results show that the native conformation of the D' domain of vWF is not only required for FVIII binding but also for normal multimerization and optimal secretion.

Association between high von willebrand factor levels and the Thr789Ala vWF gene polymorphism but not with nephropathy in type I diabetes. The GENEDIAB Study Group and the DESIR Study Group.

BACKGROUND: A genetic susceptibility for diabetic kidney disease is suspected since diabetic nephropathy occurs in only 30 to 40% of type I diabetic patients. As elevated von Willebrand factor (vWF) plasma concentrations have been reported to precede the development of microalbuminuria in type I diabetes, we addressed a possible implication of vWF as a genetic determinant for diabetic nephropathy. METHODS: Three known vWF gene polymorphisms were genotyped in a group of 493 type I diabetic subjects, all showing proliferative retinopathy, but with various stages of renal involvement, which ranged from no microalbuminuria, despite a mean duration of diabetes of 31 years, to advanced nephropathy (GENEDIAB Study): Thr789Ala (Rsa I), M-/M+ (Msp I) (intron 19), and Ala1381Thr (Hph I). Plasma vWF and factor VIII (F VIII) levels were also measured in this population. RESULTS: Plasma vWF and F VIII levels were increased in diabetic subjects with nephropathy (P < 0.001) or with coronary heart disease (CHD; P < 0.001), but there was no interaction of both conditions on plasma levels. The Msp I polymorphism (M-/M+) was weakly associated with nephropathy (P = 0. 04), but this association was not more significant when other risk factors were used in a logistic regression analysis. The vWF Thr789Ala polymorphism was associated with CHD (P = 0.002) and with plasma vWF levels. Logistic regression analysis indicated an independent and codominant effect of the Thr789Ala polymorphism on CHD, but not on nephropathy, with a maximal risk for Ala/Ala homozygotes (OR = 4.2, 95% CI, 1.8 to 9.9, P = 0.0008). CONCLUSION: It is unlikely that polymorphisms in the vWF gene contribute to the risk for nephropathy in type I diabetic patients. However, the Thr789Ala polymorphism might affect the risk for CHD in this population through modulation of plasma vWF levels.

Identification of new type 2B von Willebrand disease mutations: Arg543Gln, Arg545Pro and Arg578Leu.

We report the identification in five patients (three families) affected with type 2B von Willebrand disease (VWD) of three heterozygous nucleotide substitutions at the codon for arginine 543, 545 and 578 of the mature von Willebrand factor (VWF) subunit resulting in a glutamine, proline and leucine substitution, respectively. These mutations are located in the A1 loop where prevalent type 2B mutations (Arg543Trp, Arg545Cys and Arg578Gln) have been already identified at the same positions. By in vitro mutagenesis of full-length cDNA of VWF and transient expression in Cos-7 cells, we have shown that the six corresponding mutated recombinant VWFs (Gln543, Trp543, Cys545, Pro545, Leu578 and Gln578 rVWF) exhibited quantitatively normal expression and normal multimeric pattern but increased ristocetin- and botrocetin-induced binding to platelets as compared with that for wild-type rVWF. The two mutations at position 545 induced the greatest reactivity for GPIb of corresponding rVWFs as compared to the two mutations at positions 543 and 578.

A novel mutation in the D3 domain of von Willebrand factor markedly decreases its ability to bind factor VIII and affects its multimerization.

In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient's vWF gene showed, at heterozygous state, a G-->A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C-->T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient's vWF. The monoclonal antibody 31H3 against D' domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D' domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.

Type 2N von Willebrand disease due to Arg91Gln substitution and a cytosine deletion in exon 18 of the von Willebrand factor gene.

Two members of a family previously classified as type 1 von Willebrand disease (VWD), showed a quantitative defect in von Willebrand factor (VWF) antigen and ristocetin cofactor activity and an abnormal capacity of VWF to bind FVIII. Sequencing of the VWF gene region coding for the FVIII binding domain revealed the most frequent type 2N mutation: a single nucleotide change (G2811A) in exon 20, resulting in substitution of glutamine (Gln) for arginine (Arg) 91 in the mature VWF protein in one allele. The other allele contained a cytosine deletion (2680delC) in exon 18, introducing a premature stop codon at position 79 (Val79X) which produced a quantitative defect in VWF levels. The Arg91Gln defect is usually not evident in heterozygotes; however, in these patients it was expressed due to the lack of VWF production from the other allele. This is the first report of type 2N VWD in Italy.

A new candidate mutation (N528S) within the von Willebrand factor propeptide identified in a Japanese patient with phenotype IIC of von Willebrand disease.

Phenotype IIC of von Willebrand disease (vWD) is a subtype of type 2A vWD characterized by recessive inheritance and an impaired multimerization of von Willebrand factor (vWF) molecules beyond dimers. The 5 patients with phenotype IIC whose vWF gene defect has been characterized so far are either homozygous or double heterozygotes for mutations localized in exons 11, 12, 14 or 15. We report here the identification of a new candidate mutation in a previously described Japanese patient affected with phenotype IIC vWD. The propositus is homozygous for the A1833G nucleotide substitution, in exon 14 of vWF gene, responsible for the N528S mutation within the vWF propeptide. This finding is in agreement with the consanguineous origin of the propositus, whose parents are first cousins. Six patients' relatives who are asymptomatic were studied and found heterozygous for the N528S mutation. The screening of the whole vWF gene, either by SSCP or sequencing, did not reveal any other deleterious sequence alteration in the patient. Furthermore, the N528S nonconservative substitution identified is located in the vWF propeptide region, where the other phenotype IIC mutations described so far are clustered. The N528S candidate mutation characterized is, therefore, most probably responsible for the multimerization defect of vWF observed in this patient.

Type 2N von Willebrand disease: rapid genetic diagnosis of G2811A (R854Q), C2696T (R816W), T2701A (H817Q) and G2823T (C858F)--detection of a novel candidate type 2N mutation: C2810T (R854W).

The majority of patients with type 2N von Willebrand disease (VWD type 2N) have mutations in the region of the von Willebrand factor (VWF) gene encoding the factor VIII binding domain of VWF. Two mutations predominate among VWD type 2N patients: G2811A and C2696T, which respectively bring about the amino acid substitutions R854Q and R816W in VWF. Several other mutations have been found in VWD type 2N, including T2701A (H817Q) and G2823T (C858F). We have developed a genetic test which permits rapid screening for these four mutations in a single polymerase chain reaction (PCR). The test employs induced heteroduplex formation using two universal heteroduplex generators, one of which detects G2811A (R854Q) and G2823T (C858F), the other detects C2696T (R816W) and T2701A (H817Q). The allele frequency of the common G2811A (R854Q) mutation was investigated in the local (S. Wales) population by examination of 216 VWF genes (108 individuals) and was found to be 0.01. The heteroduplex-based test additionally detected a novel candidate type 2N mutation, C2810T (R854W) and a previously described polymorphism, G2805A (R852Q). The polymorphism showed allele frequencies of 0.92 (G nucleotide) and 0.08 (A nucleotide) in the population study.

Molecular genetics of von Willebrand disease.

Von Willebrand disease (vWD), the most common congenital bleeding disorder in man, is related to quantitative and/or qualitative abnormalities of von Willebrand factor (vWF). This multimeric glycoprotein serves as carrier protein of factor VIII, an essential cofactor of coagulation in plasma, and promotes platelet adhesion to the damaged vessel and platelet aggregation. Distinct abnormalities of vWF are responsible for the three types of vWD. Types 1 and 3 are characterized by a quantitative defect of vWF whereas type 2, comprising subtypes 2A, 2B, 2M and 2N, refers to molecular variants with a qualitative defect of vWF. The knowledge of the structure of the vWF gene and the use of Polymerase Chain Reaction (PCR) have led to the identification of the molecular basis of vWD in a significant number of patients. Type 2A is characterized by a decreased platelet-dependent function of vWF associated with the absence of high molecular weight (HMW) multimers of vWF. Most of the type 2A mutations have been identified in the A2 domain of vWF which contains a proteolytic site, while a few others have been found within the propeptide and the C-terminal part of vWF which are involved in its multimerization and dimerization, respectively. In type 2B, defined by an increased affinity of vWF to platelet glycoprotein Ib (GPIb), various amino-acid (aa) substitutions or insertion have been localized within the A1 domain containing the GPIb binding site. In the latter domain have been also identified the few molecular abnormalities described in type 2M which is defined by a decreased platelet-dependent function not caused by the absence of HMW multimers. In type 2N, characterized by a defective binding of vWF to factor VIII, several aa substitutions have been identified within the factor VIII-binding domain in the N-terminal part of vWF. The identification of gene defects remains difficult in types 1 and 3. Whereas various abnormalities (total, partial or point deletions, point insertions, nonsense mutations) have already been identified in type 3, the molecular basis of type 1 is still unresolved in most cases. The characterization of the molecular basis of vWD is of fundamental interest in providing further insight into the structure-function relationship and the biosynthesis of vWF.

Gene defects in 150 unrelated French cases with type 2 von Willebrand disease: from the patient to the gene. INSERM Network on Molecular Abnormalities in von Willebrand Disease.

Type 2 vWD is defined by qualitative defects of vWF and is subdivided into four subtypes: 2N, 2B, 2A and 2M. The characterization of 150 unrelated French cases with type 2 vWD emphasizes the heterogeneity of this group. In 51 cases of type 2N vWD, new mutations were found not only in the D' domain (Cys25Tyr and Cys95Phe) but also in the D3 domain (Asp116Asn and Cys297Arg). In 42 cases of type 2B vWD, no new mutation was detected. In 45 cases with type 2A phenotype, three new candidate mutations were found in the A2 domain: Gln793Arg, Val841Phe and Leu876Pro. In addition, four new candidate mutations were detected in the A1 domain: Cys509Gly, Arg545His, Arg552Cys and Cys695Tyr. Finally, five new candidate mutations were identified in 12 patients with 2M (or unclassified) phenotype: Leu513Pro, Gly561A1a, Glu596Lys, Arg611Leu and IIe662Phe. For all candidate mutations, expression studies are in progress. This study of a large number of French variants of vWD brings further insight into the relationship between phenotype and genotype.

Rapid mutation screening in type 2A von Willebrand's disease using universal heteroduplex generators.

Patients with type 2A von Willebrand's disease (VWD) commonly have missense mutations in the A2 domain of the von Willebrand factor (VWF) protein. This domain is encoded by the 3' region of VWF gene exon 2 8 and the large majority of patients have heterozygous mutations clustered in the sequence between codons 742 and 909. We describe a DNA-based diagnostic technique which enables at least 10 previously described mutations to be rapidly identified. The method involves polymerase chain reaction (PCR) amplification of two exon 28 gene segments between codons 717-788 and 803-893, respectively. Each fragment is then hybridized with a synthetic complementary DNA molecule of similar size, termed a Universal Heteroduplex Generator (UHG). The UHG contains base deletions contiguous to the sites of known mutations and, following hybridization, allele-specific heteroduplexes are generated which can be detected by simple polyacrylamide gel electrophoresis and ethidium bromide staining. A small panel of UHG molecules covering the 3' region of exon 28 should enable the large majority of type 2A VWD patients to be rapidly diagnosed by genotype.