Faisel M. Abu-Duhier

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.

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

Mutational analysis of class III receptor tyrosine kinases (C-KIT, C-FMS, FLT3) in idiopathic myelofibrosis.

Summary. Genomic DNA from patients with idiopathic myelofibrosis (IMF) was screened by polymerase chain reaction (PCR) and conformation sensitive gel electrophoresis (CSGE) for mutations in the C‐KIT gene (60 patients), as well as the C‐FMS and FLT3 genes (40 patients). Intronic primers were used to amplify the entire coding region of both the C‐KIT and C‐FMS genes, and selected regions of the FLT3 gene. CSGE and direct DNA sequencing detected all previously reported as well as several novel polymorphisms in each of the genes. A novel c‐fms exon 9 mutation (Gly413Ser) was detected in two patients. Its functional significance remains to be determined. The c‐kit mutation Asp52Asn, previously described in two of six IMF patients in Japan, was not detected in this study. In addition, the reported c‐fms mutations involving codons 301 and 969 were not identified. Therefore, in contrast to acute myeloid leukaemia, mutations in RTKs class III do not appear to play a significant pathogenetic role in idiopathic myelofibrosis.

Sequencing, Analysis, and Annotation of Expressed Sequence Tags for Camelus dromedarius

Despite its economical, cultural, and biological importance, there has not been a large scale sequencing project to date for Camelus dromedarius. With the goal of sequencing complete DNA of the organism, we first established and sequenced camel EST libraries, generating 70,272 reads. Following trimming, chimera check, repeat masking, cluster and assembly, we obtained 23,602 putative gene sequences, out of which over 4,500 potentially novel or fast evolving gene sequences do not carry any homology to other available genomes. Functional annotation of sequences with similarities in nucleotide and protein databases has been obtained using Gene Ontology classification. Comparison to available full length cDNA sequences and Open Reading Frame (ORF) analysis of camel sequences that exhibit homology to known genes show more than 80% of the contigs with an ORF>300 bp and ∼40% hits extending to the start codons of full length cDNAs suggesting successful characterization of camel genes. Similarity analyses are done separately for different organisms including human, mouse, bovine, and rat. Accompanying web portal, CAGBASE (http://camel.kacst.edu.sa/), hosts a relational database containing annotated EST sequences and analysis tools with possibility to add sequences from public domain. We anticipate our results to provide a home base for genomic studies of camel and other comparative studies enabling a starting point for whole genome sequencing of the organism.