Patients with Down syndrome (DS) frequently develop 2 kinds of clonal megakaryocytosis: a common, congenital, spontaneously resolving, transient myeloproliferative disorder (TMD) and, less commonly, childhood acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in exon 2 of GATA1, an X-linked gene encoding a transcription factor that promotes megakaryocytic differentiation, were described in 6 DS patients with AMKL. The mutations prevent the synthesis of the full-length GATA1, but allow the synthesis of a shorter GATA1 protein (GATA1s) that lacks the transactivation domain. To test whether mutated GATA1 is involved in the initiation of clonal megakaryoblastic proliferation or in the progression to AMKL, we screened 35 DS patients with either AMKL or TMD and 7 non-DS children with AMKL for mutations in exon 2 of GATA1. Mutations were identified in 16 of 18 DS patients with AMKL, in 16 of 17 DS patients with TMD, and in 2 identical twins with AMKL and acquired trisomy 21. Analysis revealed various types of mutations in GATA1, including deletion/insertions, splice mutations, and nonsense and missense point mutations, all of which prevent the generation of full-length GATA1, but preserve the translation of GATA1s.We also show that the likely mechanism of generation of GATA1 isoforms is alternative splicing of exon 2 rather than, or in addition to, alternative translation initiation, as was proposed before. These findings suggest that acquired intrauterine inactivating mutations in GATA1 and generation of GATA1s cooperate frequently with trisomy 21 in initiating megakaryoblastic proliferation, but are insufficient for progression to AMKL. (Blood. 2003; 102:981-986)

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Background: Autosomal dominant familial hypercholesterolemia (FH) attributable to mutations in the LDL receptor (LDLR) gene is one of the most common genetic disorders associated with significant morbidity and mortality. Definitive diagnosis would help to initiate appropriate treatment to prevent premature cardiovascular disease. Currently, clinical diagnosis of FH is imprecise, and molecular diagnosis is labor-intensive and expensive because of the size of the LDLR gene and number of coding exons. Methods: We used PCR to amplify all exons, including exon/intron boundaries, and the promoter of the LDLR gene. Nine individuals from five families with typical findings for a clinical diagnosis of heterozygous FH, 2 heterozygous FH cell lines, and 50 control individuals were screened for mutations by denaturing HPLC (DHPLC) followed by direct sequencing of aberrantly migrating fragments. Results: Mutations that were previously reported to be disease causing were identified in eight of nine individuals with FH and both cell lines (V502M, C146X, E207X, C660X, C646Y, and delG197), but none were found in controls. The one individual with FH in whom no mutation was found had a previously unreported change in the 5′-untranslated region of unknown significance. In addition, we identified several previously reported polymorphism both in controls and individuals with FH. Conclusions: DHPLC can be used to detect mutations causing FH. On the basis of our current experience with DHPLC, this method combined with confirmatory DNA sequencing is likely to be sensitive and efficient.

Additional copies of chromosome (chr) 21 are the most common chromosomal aneuploidy in childhood leukemia. Patients with DS have a markedly increased risk for both AML and ALL. Thus trisomy 21 is leukemogenic. DS-AML is uniquely characterized by an acquired mutation in the chr X gene GATA1. The existence of a similar unique collaborating mutation in DS-ALL has been postulated but remained elusive. To identify such mutations, we performed a large mutational screen in 81 diagnostic samples of B cell precursor DS-ALL, stored in central labs of 9 European childhood ALL protocols (representing more than 8000 samples of childhood ALL). Mutations in JAK2 were identified in 16 (19.7%) DS-ALL patients. The mutations are different from those observed in myeloproliferative disorders. In fifteen patients a point mutation resulted in substitution of the conserved Arginine at position 683. In one patient an in-frame insertion caused displacement of the same amino-acid. The mutations cause constitutive activation of JAK2 and are predicted to disrupt a critical interaction between the pseudokinase and the SH2 domains. The mutations are acquired as they do not exist in remission samples. The mutated RNA is expressed. They are unique to DS-ALL as screening of over 300 non DS-ALL, as well as DS-AMKL, has revealed only one instance with a similar mutation. Strikingly, that patient had an ALL with an iso21q abnormality! The clinical presentation and survival data of DS-ALL patients with and without JAK2 mutation is currently analyzed and will be presented in the meeting. Our data demonstrate that novel mutations of JAK2 cooperate with trisomy 21 in at least 20% of ALLs in Down Syndrome. Thus it seems that, like DS-AML, at least 20% of DS-ALLs are different from sporadic childhood leukemias and characterized by unique acquired mutations in genes located outside chr 21 (GATA1 on the chr X and JAK2 on chr 9). Beyond the obvious therapeutic implications, these observations raise the hypothesis that JAK2 is important in B cell development and that constitutive activation of JAK2 in B cell precursors provides a survival advantage in the presence of a germline trisomy 21.