A novel deletion mutation is recurrent in von Willebrand disease types 1 and 3.

Direct sequencing of VWF genomic DNA in 21 patients with type 3 von Willebrand disease (VWD) failed to reveal a causative homozygous or compound heterozygous VWF genotype in 5 cases. Subsequent analysis of VWF mRNA led to the discovery of a deletion (c.221-977_532 + 7059del [p.Asp75_Gly178del]) of VWF in 7 of 12 white type 3 VWD patients from 6 unrelated families. This deletion of VWF exons 4 and 5 was absent in 9 patients of Asian origin. We developed a genomic DNA-based assay for the deletion, which also revealed its presence in 2 of 34 type 1 VWD families, segregating with VWD in an autosomal dominant fashion. The deletion was associated with a specific VWF haplotype, indicating a possible founder origin. Expression studies indicated markedly decreased secretion and defective multimerization of the mutant VWF protein. Further studies have found the mutation in additional type 1 VWD patients and in a family expressing both type 3 and type 1 VWD. The c.221-977_532 + 7059del mutation represents a previously unreported cause of both types 1 and 3 VWD. Screening for this mutation in other type 1 and type 3 VWD patient populations is required to elucidate further its overall contribution to VWD arising from quantitative deficiencies of VWF.

An investigation of the von Willebrand factor genotype in UK patients diagnosed to have type 1 von Willebrand disease.

Forty families diagnosed by UK centres to have type 1 VWD were recruited. Following review, six families were re-diagnosed to have type 2 VWD, one to have a platelet storage pool disorder, and one family was determined to be unaffected. Direct DNA sequencing of the promoter region and all exons and intronic boundaries of the VWF gene identified six mutations likely to be causative of VWD in index cases of nine of the 32 (28%) confirmed type 1 VWD families. These included R1205H (3614G > A) VWD Vicenza, P1648fsX45 (4944delT), D141G (422A > G) and three splice site mutations: 3108 + 5G > A, 7437 + 1G > A and 3379 + 1G > A. The Y1584C (4751A > G) polymorphism was present in eight additional families. No significant VWF gene mutation or polymorphism was identified in 15 of the 32 type 1VWD index cases (47%). Haplotype studies were performed using a panel of VWF polymorphisms to investigate the segregation in families of VWD phenotype with the VWF gene. In 13 of the 32 families it was likely that VWD segregated with the VWF gene. In eight families (25%) VWD clearly did not segregate with the VWF gene. We suggest that mutation screening of the VWF gene has limited general utility in genetic diagnostic and family studies in type 1 VWD. If genetic studies are performed, the incomplete penetrance and variable expressivity of type 1 VWD must be taken into account. Unless linkage of VWD phenotype with the VWF gene can be clearly demonstrated, the results of any genetic family studies should be interpreted with caution.

Long-PCR amplification of human genomic DNA.

Standard polymerase chain reaction (PCR) protocols amplify relatively small fragments precluding the use of this approach when examining gross rearrangements of DNA. By using combinations of DNA polymerases, which feature either good polymerase activity or error-correction abilities, it is now possible to extend the length of DNA fragment that can be amplified. These "long-PCR" protocols have allowed the development of more rapid and convenient ways to analyse large-scale rearrangements of DNA and in many cases has superseded alternative approaches such as Southern blotting. The protocol described in this chapter illustrates some of the key points to be considered when developing a long PCR protocol.

Use of robotics in high-throughput DNA sequencing.

Until relatively recently, full sequencing of genes consisting of more than several exons was not considered practicable within a routine diagnostic context. As a result, many approaches to unknown mutation detection in a specific gene involved a mutation pre-screening step to limit the amount of DNA sequencing required. Protocols to pre-screen for mutations and limit the amount of DNA sequencing may not localise every base change present and/or require considerable levels of manual intervention. Advances in technology, allied with careful protocol design, now permit direct DNA sequencing to be applied to larger areas of gene sequence, allowing unequivocal mutation identification in the area of a gene being analysed. The protocol described below utilises robotic systems, allied to custom-designed PCR primers, to facilitate rapid DNA sequencing of multiple gene targets. The general approach is amenable to adaptation for use with multi-channel pipettes.