FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.

MicroRNAs (miRNAs) regulate the expression of multiple proteins in a dose dependent manner. We hypothesized that increased expression of miRNAs encoded on chromosome 21 (chr 21) contribute to the leukemogenic role of trisomy 21. The levels of chr 21 miRNAs were quantified by qRT-PCR in four types of childhood ALL characterized by either numerical (trisomy or tetrasomy) or structural abnormalities of chr 21. Suprisingly high expression of the hsa-mir-125b-2 cluster, consisting of three miRNAs, was identified in leukemias with the structural ETV6/RUNX1 abnormality and not in ALLs with trisomy 21. Manipulation of ETV6/RUNX1 expression and chromatin immunoprecipitation studies demonstrated that the high expression of the miRNA cluster is an event independent of the ETV6/RUNX1 fusion protein. Overexpression of hsa-mir-125b-2 conferred a survival advantage to Ba/F3 cells following IL-3 withdrawal or a broad spectrum of apoptotic stimuli through inhibition of caspase 3 activation. Conversely, knockdown of the endogenous miR-125b in the ETV6/RUNX1 leukemia cell line REH increased apoptosis after Doxorubicin and Staurosporine treatments. P53 protein levels were not altered by miR-125b. Together these results suggest that the expression of hsa-mir-125b-2 in ETV6/RUNX1 ALL provides survival advantage to growth inhibitory signals in a p53 independent manner.

The purpose of this study was the appraisal of the clinical and functional consequences of germline mutations within the gene for the IL-2 inducible T-cell kinase, ITK. Among patients with Epstein-Barr virus-driven lymphoproliferative disorders (EBV-LPD), negative for mutations in SH2D1A and XIAP (n ¼ 46), we identified two patients with R29H or D500T,F501L,M503X mutations, respectively. Human wild-type (wt) ITK, but none of the mutants, was able to rescue defective calcium flux in murine Itk À/À T cells. Pulse-chase experiments showed that ITK mutations lead to varying reductions of protein half-life from 25 to 69% as compared with wt ITK (107 min). The pleckstrin homology domain of wt ITK binds most prominently to phosphatidylinositol monophosphates (PI(3)P, PI(4)P, PI(5)P) and to lesser extend to its double or triple phosphorylated derivates (PIP2, PIP3), interactions which were dramatically reduced in the patient with the ITK R29H mutant. ITK mutations are distributed over the entire protein and include missense, nonsense and indel mutations, reminiscent of the situation in its sister kinase in B cells, Bruton's tyrosine kinase.

IntroductionFanconi anemia (FA) is a complex recessive inherited disorder that is clinically characterized by variable congenital abnormalities, progressive bone marrow (BM) failure, and a high propensity to develop myeloid and epithelial malignancies. 1-5 On a cellular level, FA is characterized by a profound hypersensitivity upon exposure to DNA cross-linking agents such as mitomycin-C (MMC) or diepoxybutane (DEB). [6][7][8][9] Genetically, germ-line mutations in 13 genes (FANCA/B/C/D1/ D2/E/F/G/I/J/L/M/N) result in the clinical phenotype of FA. 2,8,[10][11][12][13][14] Spontaneous genetic correction of a germ-line mutation leading to repopulation of the entire hematopoietic system with normal progeny has been identified in a few FA patients. [15][16][17][18][19] These observations, in combination with the fact that the hematopoietic system can be functionally corrected in mice with targeted disruptions of FA genes by retroviral vectors expressing human analogues of the targeted mouse genes in stem cells, [20][21][22] have led to 3 clinical stem cell gene therapy phase 1 studies in FA-A and FA-C patients. So far, neither long-term marking/correction of cells nor clinical benefits for the patients were observed. 23,24 Due to the biologic characteristics of the gammaretroviral vectors used for transduction of stem cells, 25,26 optimal gene transfer protocols for delivery of genes to mammalian stem cells require a prestimulation period of 1 to 2 days with cytokines that promote the proliferation and survival of stem/progenitor cells. This is followed by a 2-to 3-day exposure of the target cells to vector containing supernatant on the recombinant fibronectin fragment 28 This gene transfer protocol was successful in transducing hematopoietic stem cells in humans, primates/monkeys, and mice. [29][30][31] However, in murine FA models, prolonged in vitro culture of Fancc Ϫ/Ϫ BM results in a length-of-culture-dependent reduction in myeloid progenitors and repopulating ability, 32,33 and the surviving untransduced Fancc Ϫ/Ϫ repopulating cells have an increased risk of developing cytogenetic abnormalities and myeloid malignancies. 22 Therefore, limiting the in vitro culture would be predicted to enhance both the efficacy and safety for genetic therapies of FA stem cells.Wild-type foamy viruses are the only retroviruses that are not associated with any disease in their natural hosts or in accidentally infected human beings. [34][35][36] It has been shown that vectors based on the prototype (formerly human) foamy virus (FV) can efficiently transduce hematopoietic stem cells from mice, 37 dogs, 38 and nonobese diabetic/ severe combined immunodeficiency (NOD/SCID) repopulating human cells. [39][40][41] Further, FV vectors are at least equally efficient at transduction of CD34 ϩ umbilical cord blood cells engrafting in NOD/SCID mice as lentiviral vectors based on HIV-1. 40 In the present study, we demonstrated for the first time the ability of FV vectors encoding the human FANCC transgene to completely correct Fancc Ϫ/Ϫ myelo...

The reciprocal translocation t(12;21)(p13;q22), the most common structural genomic alteration in B-cell precursor acute lymphoblastic leukaemia in children, results in a chimeric transcription factor TEL-AML1 (ETV6-RUNX1). We identified directly and indirectly regulated target genes utilizing an inducible TEL-AML1 system derived from the murine pro B-cell line BA/F3 and a monoclonal antibody directed against TEL-AML1. By integration of promoter binding identified with chromatin immunoprecipitation (ChIP)-on-chip, gene expression and protein output through microarray technology and stable labelling of amino acids in cell culture, we identified 217 directly and 118 indirectly regulated targets of the TEL-AML1 fusion protein. Directly, but not indirectly, regulated promoters were enriched in AML1-binding sites. The majority of promoter regions were specific for the fusion protein and not bound by native AML1 or TEL. Comparison with gene expression profiles from TEL-AML1-positive patients identified 56 concordantly misregulated genes with negative effects on proliferation and cellular transport mechanisms and positive effects on cellular migration, and stress responses including immunological responses. In summary, this work for the first time gives a comprehensive insight into how TEL-AML1 expression may directly and indirectly contribute to alter cells to become prone for leukemic transformation.