Jayne Leggo

Genetic linkage and association analysis in type 1 von Willebrand disease: results from the Canadian type 1 VWD study.

Summary.  Background: von Willebrand disease (VWD) is the most common bleeding disorder known in humans, with type 1 VWD representing the majority of cases. Unlike the other variant forms of VWD, type 1 disease represents a complex genetic trait, influenced by both genetic and environmental factors. Aim: To evaluate the contribution of the von Willebrand factor (VWF) and ABO blood group loci to the type 1 VWD phenotype, and to assess the potential for locus heterogeneity in this condition, we have performed genetic linkage and association studies on a large, unselected type 1 VWD population. Method: We initially collected samples from 194 Canadian type 1 VWD families for analysis. After the exclusion of families found to have either type 2 or type 3 VWD, and pedigrees with samples from single generations, linkage and association analysis was performed on 155 type 1 VWD families. Results and conclusion: The linkage study has shown a low heterogeneity LOD score of 2.13 with the proportion of families linked to the VWF gene estimated to be 0.41. Linkage was not detected to the ABO locus in this type 1 VWD population. In the family‐based association test, significant association was found between the type 1 VWD phenotype, the quantitative traits, VWF:Ag, VWF:RCo, and FVIII:C and the ABO ‘O’ and ‘A’ alleles and the VWF codon 1584 variant. There was also weak association with the −1185 promoter polymorphism and VWF:Ag, VWF:RCo, and FVIII:C plasma levels. These studies provide further evidence to support the role for genetic loci other than VWF and ABO in the pathogenesis of type 1 VWD.

Frequency of Platelet type versus Type 2B von Willebrand Disease: An international registry-based study

Less than 50 patients are reported with platelet type von Willebrand disease (PT-VWD) worldwide. Several reports have discussed the diagnostic challenge of this disease versus the closely similar disorder type 2B VWD. However, no systematic study has evaluated this dilemma globally. Over three years, a total of 110 samples/data from eight countries were analysed. A molecular approach was utilised, analysing exon 28 of the von Willebrand factor (VWF) gene, and in mutation negative cases the platelet GP1BA gene. Our results show that 48 cases initially diagnosed as putative type 2B/PT-VWD carried exon 28 mutations consistent with type 2B VWD, 17 carried GP1BA mutations consistent with a PT-VWD diagnosis, three had other VWD types (2A and 2M) and five expressed three non-previously published exon 28 mutations. Excluding 10 unaffected family members and one acquired VWD, 26 cases did not have mutations in either genes. Based on our study, the percentage of type 2B VWD diagnosis is 44% while the percentage of misdiagnosis of PT-VWD is 15%. This is the first large international study to investigate the occurrence of PT-VWD and type 2B VWD worldwide and to evaluate DNA analysis as a diagnostic tool for a large cohort of patients. The study highlights the diagnostic limitations due to unavailability/poor application of RIPA and related tests in some centres and proposes genetic analysis as a suitable tool for the discrimination of the two disorders worldwide. Cases that are negative for both VWF and GP1BA gene mutations require further evaluation for alternative diagnoses.

The genetics of Canadian type 3 von Willebrand disease: further evidence for co-dominant inheritance of mutant alleles.

Type 3 von Willebrand disease (VWD) is the most severe form of the disease and is classically inherited in an autosomal recessive fashion.

DNA microarray analysis for the detection of mutations in hemophilia A

Summary.  Background: Congenital deficiency of factor (F) VIII results in the inherited X‐linked bleeding disorder hemophilia A. More than 900 different mutations are reported in the hemophilia A mutation database with the largest number of mutations being single nucleotide substitutions distributed throughout the gene. Complicating the molecular characterization of this disease is the complexity of the F8 gene, the mutational heterogeneity, and technical limitations of the current mutation detection techniques. Objective: Development of a DNA oligonucleotide microarray‐based technique for F8 gene analysis to detect hemophilia A mutations. Methods: To construct the oligonucleotide DNA microarray system: a total of 720, one base pair overlapping, 25‐mer perfect match probes were designed from six exons of the F8 gene. Twenty‐two different F8 gene mutations previously identified by CSGE and DNA sequence analysis were tested by using a loss‐of‐signal analysis approach. Differentially labeled wild type and hemophilic samples were co‐hybridized to the array. Sequence alterations were detected by quantifying relative losses of test sample hybridization signals to the perfectly matched probes. Results: A total of 22 different F8 mutations were tested. To test the sensitivity of the system, a blinded study was performed on 16 of the samples. F8 gene mutations can be detected with 96% efficiency with this microarray system. Conclusion: This proof‐of‐principle study has demonstrated that a F8 DNA microarray platform is an alternative gene mutation analysis approach that has a high sensitivity, and reproducibility. The methodology is, however, expensive and time consuming, and with the reduction in sequencing costs, direct sequencing is now the most cost and time efficient strategy for hemophilia A mutation analysis.

A case of non‐resolving hemophilia B Leyden in a 42‐year‐old male (F9 promoter + 13 A>G)

Hemophilia B (inherited factor IX deficiency) is a bleeding disorder resulting from mutations in the F9 gene on the long arm of the X chromosome, at Xq27 [1]. Many different single nucleotide substitutions, deletions and insertions within the gene can result in the hemophilia B phenotype [2]. A subset of hemophilia B patients with mutations in the promoter region of the F9 gene spanning nucleotides ) 40 to+ 20 (also known as the Leyden specific region) exhibit a spontaneous improvement of the phenotype following puberty, characterized by increasing FIX levels in the postpubertal period and normal or near-normal FIX levels throughout adulthood [3]. Hemophilia B Leyden was first described by Veltkamp and colleagues in 1970[4] and then again, in more detail, in 1982 [5]. A number of different mutations clustered around the F9 transcription start site and resulting in the hemophilia B Leyden phenotype have been described (http://www.kcl.ac.uk/ ip/petergreen/haemBdatabase.html). The recovery mechanism after the onset of puberty in hemophilia B Leyden is complex and has been the subject of much investigation. Two specific mechanisms have been postulated for the phenotypic recovery, both based on the concept that before puberty, F9 gene expression is regulated by constitutive binding of transcription factors to cis-acting FIX promoter elements [CCAAT/ enhancer binding protein (C/EBP), HNF-4 and others]. Mutations within those elements disrupt binding of the relevant transcription factors, and hemophilia B results. At puberty, and thereafter, it is assumed that testosterone-dependent transcription, mediated by an FIX promoter androgen response element (ARE) and possibly binding of the D-site binding protein (DBP) to an upstream sequence, becomesmore important [6]. With specific reference to the subject described here, nucleotide substitutions at + 13 have been shown to disrupt C/EBP binding, and in all previous reports have been associated with the amelioration of the phenotype following puberty [7–9]. In general, these reports describe cases with clotting factor activities rising from 0.03 IU mL to 0.40 IU mL after puberty, at a rate of 0.05 IU mL per year. A 42-year-old male (who gave written informed consent for the publication of his information) was diagnosed with hemophilia B at the age of 1 year after a fall and cut to his lip resulted in prolonged bleeding. His baseline FIX level was 0.08 IU mL. He was treated with plasma for intermittent bleeding episodes throughout his childhood. At the age of 16 years, he was involved in a motor vehicle accident and was hospitalized and treated with FIX concentrate replacement. His FIX level was recorded as 0.17 IU mL at the time of that admission. Laboratory reports showed his peak FIX level to be 0.40 IU mL at the age of 21 years; however, subsequent FIX levels are all lower than this, with a number of levels < 0.20 IU mL after the age of 30 years (Fig. 1). It is possible that the FIX peak in his late teens represents a response to high levels of testosterone around the time of

Different clinical phenotype in triplets with haemophilia A

Summary.   Although many patients with haemophilia may have exactly the same residual clotting factor level, the clinical disease phenotype may vary greatly. This variation may be related to different genetic mutations responsible for haemophilia, environmental influences and co‐inheritance of polymorphisms affecting the coagulation system. The study of siblings with haemophilia offers the opportunity to examine additional factors, other than genetic mutation and environment that may impact on the clinical phenotype of haemophilia. We present the unusual case of haemophilia occurring in fraternal triplets. Each of the triplets had a slightly different pattern of bleeding and response to treatment.