Ian R. Peake

Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor

Summary.  von Willebrand disease (VWD) is a bleeding disorder caused by inherited defects in the concentration, structure, or function of von Willebrand factor (VWF). VWD is classified into three primary categories. Type 1 includes partial quantitative deficiency, type 2 includes qualitative defects, and type 3 includes virtually complete deficiency of VWF. VWD type 2 is divided into four secondary categories. Type 2A includes variants with decreased platelet adhesion caused by selective deficiency of high‐molecular‐weight VWF multimers. Type 2B includes variants with increased affinity for platelet glycoprotein Ib. Type 2M includes variants with markedly defective platelet adhesion despite a relatively normal size distribution of VWF multimers. Type 2N includes variants with markedly decreased affinity for factor VIII. These six categories of VWD correlate with important clinical features and therapeutic requirements. Some VWF gene mutations, alone or in combination, have complex effects and give rise to mixed VWD phenotypes. Certain VWD types, especially type 1 and type 2A, encompass several pathophysiologic mechanisms that sometimes can be distinguished by appropriate laboratory studies. The clinical significance of this heterogeneity is under investigation, which may support further subdivision of VWD type 1 or type 2A in the future.

Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia.

Genomic DNA from 97 cases of adult de novo acute myeloid leukaemia (AML) was screened using polymerase chain reaction (PCR) and conformation‐sensitive gel electrophoresis (CSGE) for FLT3 exon 20 mutations. Initial sequencing of four cases, representing the spectrum of CSGE abnormalities, revealed changes affecting codon Asp835 in three cases and also an intron 20 A to G change. In order to identify all possible Asp835 alterations, as well as the frequency of the intronic change nucleotide 2541 + 57 A→G, the patient PCR products were digested with EcoRV and NlaIII respectively. Seven cases (7·2%) possessed a mutation affecting Asp835; these were identified, following DNA sequencing, as Asp835Tyr (n = 5), Asp835His (n = 1) and Asp835del (n = 1). Alterations affecting Asp835 were not found in 80 normal control DNA samples. In contrast, the nucleotide 2541 + 57 A→G change was shown to be a polymorphism, with an allelic frequency of 0·24 for the G and 0·76 for the A allele. This study reports, for the first time, point mutations in the human FLT3 gene that, because of their homology with other class III receptor tyrosine kinase mutations, probably result in constitutive activation of the receptor.

Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias

Summary. DNA from 110 adult de novo acute myeloid leukaemia (AML) patients exhibiting either inv(16) (n = 63) or t(8;21) (n = 47) was screened for mutations in the c‐KIT (exon 8 and Asp816) and FLT3 (ITD and Asp835) genes. c‐KIT exon 8 mutations were found in 15/63 (23·8%) inv(16) patients and 1/47 (2·1%) t(8;21) patients. c‐KIT Asp816 mutations were present in 5/63 (7·9%) inv(16) AML and 5/47 (10·6%) t(8;21) AML. FLT3 mutations were identified in five patients (7·9%) with inv(16) and three patients (5·6%) with t(8;21) AML. All mutations were mutually exclusive; 40% of inv(16) AML patients possessed either a c‐KIT or FLT3 mutation. c‐KIT exon 8 mutations were shown to be a significant factor adversely affecting relapse rate.

FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group.

Genomic DNA from 106 cases of adult de novo acute myeloid leukaemia (AML) was screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3 internal tandem duplication (ITD) mutations within the juxtamembrane (JM) domain. FLT3 mutations were detected in 14 cases (13·2%) and occurred in FAB types M1 (4 out of 14 cases), M3 (1 out of 10 cases), M4 (5 out of 37 cases) and M5 (4 out of 11 cases). Sequence analysis of four cases with abnormal PCR electrophoretic patterns revealed in frame duplications in the region of exon 11 of between 27 and 111 base pairs. Three are predicted to result in the tandem duplication of adjacent amino acid residues and one to result in a tandem duplication plus insertion of a novel amino acid motif. Statistical analysis showed the FLT3 mutations to be a strong prognostic factor, with patients lacking the mutation surviving significantly longer from diagnosis (mean 29·1 months) than those with an ITD (mean 12·8 months; P = 0·0002). Thirteen of the 14 patients with FLT3 mutations died within 18 months of diagnosis. FLT3 mutations were of prognostic significance in good risk disease (P = 0·04), as well as in patients with standard risk disease (P = 0·0096). This study demonstrates that the FLT3 ITD mutation occurs in a significant percentage of adult AML cases and is an important adverse prognostic factor that appears independent of conventional karyotypic findings.

c-kit proto-oncogene exon 8 in-frame deletion plus insertion mutations in acute myeloid leukaemia.

Genomic DNA from 60 cases of acute myeloid leukaemia (AML) was screened for mutations in the c‐kit gene. DNA from all 21 exons was subjected to polymerase chain reaction (PCR) amplification and analysis by conformation sensitive gel electrophoresis (CSGE); exons showing altered CSGE patterns were then sequenced. Mutations were identified only in those patients with inv(16) (3/7 cases) or t(8;21) (1/2 cases) and comprised three in‐frame deletion plus insertion mutations (exon 8) and one point mutation (exon 10, GTA → ATA, Val530Ile). Exons 8 and 10 were then analysed in 31 further cases of inv(16) (n = 14) and t(8;21) (n = 17), revealing four additional exon 8 in‐frame deletion plus insertion mutations, all of which were in cases of inv(16). All exon 8 in‐frame deletion plus insertion mutations (n = 7) involved the loss or repacement of the codon for Asp419 which is highly conserved cross species and is located in the receptors extracellular domain. The high frequency of the c‐kit proto‐oncogene exon 8 deletion plus insertion mutations in AML suggests an essential role for this region of the receptors extracellular domain. The association with inv(16) invites speculation as to the link between these two changes in the pathogenesis of AML.

High prevalence of a mutation in the factor V gene within the U.K. population: relationship to activated protein C resistance and familial thrombosis

Summary. Recent findings have indicated the importance of factor V (FV) in causing resistance to activated protein C (APC) in a high proportion of patients with venous thrombosis. This prompted us to investigate whether resistance could be due to defective inactivation of FVa by APC. Consequently, we amplified a 3.2 kb fragment of the FV gene sequence encoding the heavy chain APC cleavage site. DNA analysis showed a guanine to adenine transition at nucleotide 1691 in all affected members of two families with inherited APC resistance associated with thrombosis and confirmed suspected homozygosity in two individuals. The mutation, in heterozygous form, was also found in ˜3.5% of our normal population (n = 144) and correlated with low APC resistance. The high prevalence of this mutation suggests that it may be a major contributory factor in early thrombosis.

Identification of type 1 von Willebrand disease patients with reduced von Willebrand factor survival by assay of the VWF propeptide in the European study: Molecular and Clinical Markers for the Diagnosis and Management of Type 1 VWD (MCMDM-1VWD)

The decreased survival of von Willebrand factor (VWF) in plasma has been implicated as a mechanism in a subset of type 1 von Willebrand disease (VWD) patients. We have previously reported that the ratio of plasma levels of VWF and its propeptide (VWFpp) can be used to identify patients with reduced VWF survival. In this study, we report the assay of VWFpp and VWF:Ag in 19 individuals recruited from 6 European centers within the MCMDM-1VWD study. Eight individuals had a VWF:Ag level less than 30 IU/dL. Seven of these patients had a robust desmopressin response and significantly reduced VWF half-life that was predicted by a markedly increased steady-state plasma VWFpp/VWF:Ag ratio. VWF mutations previously associated with reduced VWF survival were identified in each of the 7 individuals. Thus, a substantially increased ratio of steady-state VWFpp/VWF:Ag predicted a reduced VWF half-life in patients with markedly decreased VWF:Ag levels. These data indicate that a reduced VWF survival is found in a subpopulation of patients with type 1 VWD. The systematic assay of both plasma VWF and the VWF propeptide in moderately severe type 1 VWD patients may identify patients with a reduced VWF survival phenotype.

Somatic Mosaicism in Hemophilia A: A Fairly Common Event

Mutations in the large gene of clotting factor VIII (FVIII) are the most common events leading to severe human bleeding disorder. The high proportion of de novo mutations observed in this gene raises the possibility that a significant proportion of such mutations does not derive from a single germ cell but instead should be attributed to a germline or somatic mosaic originating from a mutation during early embryogenesis. The present study explores this hypothesis by using allele-specific PCR to analyze 61 families that included members who had sporadic severe hemophilia A and known FVIII gene defects. The presence of somatic mosaicisms of varying degrees (0.2%-25%) could be shown in 8 (13%) of the 61 families and has been confirmed by a mutation-enrichment procedure. All mosaics were found in families with point mutations (8 [25%] of 32 families). In the subgroup of 8 families with CpG transitions, the percentage with mosaicism increased to 50% (4 of 8 families). In contrast, no mosaics were observed in 13 families with small deletions/insertions or in 16 families with intron 22 inversions. Our data suggest that mosaicism may represent a fairly common event in hemophilia A. As a consequence, risk assessment in genetic counseling should include consideration of the possibility of somatic mosaicism in families with apparently de novo mutations, especially families with the subtype of point mutations.

Genomic structure of human FLT3: implications for mutational analysis.

The FLT3 gene encodes a class III receptor tyrosine kinase (RTKIII) that has the same overall structure as c-kit, c-fms and the platelet-derived growth factor receptors a and b. The genomic loci encoding RTKIII receptors share overall conservation of exon size, number, sequence and exon/ intron boundary positions, suggesting that they have arisen from a common ancestral gene (AgneÁs et al, 1994). Interest in FLT3 has been heightened by reports linking exonic mutations to the pathogenesis of acute myeloid leukaemia (AML). Nakao et al (1996) documented unexpectedly long FLT3 transcripts in patients with AML that resulted from internal tandem duplications (ITDs) involving exons 11 and 12. Subsequently, ITDs have been reported to occur in approximately 20% of adult AML cases and are an important adverse prognostic factor, independent of conventional karyotype (Abu-Duhier et al, 2000). Interestingly, Fenski et al (2000) produced data suggesting that other mechanisms of FLT3 activation probably exist, possibly from mutations elsewhere in the gene. Clearly, a systematic screening of the entire FLT3 gene is required, although this approach has been hindered by the lack of knowledge of its complete genomic structure. AgneÁs et al (1994) reported the organization of the downstream part and arbitrarily numbered the 11 exons according to the analogous exons of c-kit (exons 10±21). However, the structure of the region coding for the extracellular and transmembrane domains has remained unclear. We have used the basic logical alignment search tool provided by the National Center for Biotechnology Information (NCBI, 2000) to search for genomic sequences complimentary to human FLT3 mRNA (406322). Exon and intron sizes were determined and potential transcription factor sites located using gene jockey ii and patsearch v1.1 (Heinemeyer et al, 1998). We determined that human FLT3 is encoded by 24 exons, rather than the assumed 21, which span approximately 100 kb and range in size from 83 bp to 562 bp. Seven exons encode the first three immunoglobulin-like repeats, compared with four for c-kit, including a unique 126 bp exonic sequence straddling the 3 0 and 5 0 end of exons 2 and 3, respectively, together with an intervening intron of 8434 bp (Fig 1). The first exon contains the signal sequence, the second to fourth exons encode the first Iglike repeat, the fifth and sixth exons encode the second Iglike repeat, while the seventh and eighth exons encode the third Ig-like repeat (Fig 1). The remainder of the FLT3 gene is split into the same number of exons (16) as c-kit, which are highly conserved in size, sequence and exon/intron boundary positions. Our findings indicate that FLT3 ITD mutations are located in exons 14 and 15, and not 11 and 12 as previously reported. The availability of the complete genomic structure of FLT3 will facilitate mutational screening of the entire coding and promoter regions. Such an undertaking has assumed greater importance following the work of Fenski et al (2000) who suggested that other mechanisms of activation exist; a concept supported by the finding of novel FLT3 Asp835 mutations (Abu-Duhier et al, 2001). Sequence analysis for consensus transcription factor binding sites, using patsearch v1 ́1, failed to detect typical TATA and CAAT boxes in the 5 0 flanking sequence. British Journal of Haematology, 2001, 113, 1076±1089

A STUDY OF WILSON DISEASE MUTATIONS IN BRITAIN

Wilson disease (WD) is an autosomal recessive disease of copper transport. The disease is caused by a large number of mutations in the ATP7B gene, some of which appear to be population specific, whereas others are found in probands from a variety of different ethnic backgrounds. This study presents the results of screening the ATP7B gene by SSCP and sequencing in order to define the spectrum of mutations seen in British referrals for WD. The 52 patients screened included 10 with a non‐British mixed ethnicity origin. This study identified 19 novel mutations and 18 mutations that had been previously described. The novel mutations included seven nonconservative missense mutations, eight small insertions, or deletions causing frameshift, two nonsense mutations, and two splice‐site mutations. Seven of the 10 mixed ethnicity patients harboured homozygous mutations, whereas only four of the larger British group were homozygotes. The detection rate by SSCP analysis in the British group of 42 consecutive unrelated WD probands was 70%. However, SSCP screening of just three exons (exons 8, 14, and 18) is predicted to identify 60% of mutations present in WD referrals. Hum Mutat 14:304–311, 1999.