Successful bone marrow transplantation in a patient with DNA ligase IV deficiency and bone marrow failure

BACKGROUND: DNA Ligase IV deficiency syndrome is a rare autosomal recessive disorder caused by hypomorphic mutations in the DNA ligase IV gene (LIG4). The clinical phenotype shows overlap with a number of other rare syndromes, including Seckel syndrome, Nijmegen breakage syndrome, and Fanconi anemia. Thus the clinical diagnosis is often delayed and established by exclusion. METHODS: We describe a patient with pre- and postnatal growth retardation and dysmorphic facial features in whom the diagnoses of Seckel-, Dubowitz-, and Nijmegen breakage syndrome were variably considered. Cellular radiosensitivity in the absence of clinical manifestations of Ataxia telangiectasia lead to the diagnosis of DNA ligase IV (LIG4) deficiency syndrome, confirmed by compound heterozygous mutations in the LIG4 gene. At age 11, after a six year history of progressive bone marrow failure and increasing transfusion dependency the patient was treated with matched sibling donor hematopoetic stem cell transplantation (HSCT) using a fludarabine-based conditioning regimen without irradiation. RESULTS: The post-transplantation course was uneventful with rapid engraftment leading to complete and stable chimerism. Now at age 16, the patient has gained weight and is in good clinical condition. CONCLUSION: HSCT using mild conditioning without irradiation qualifies as treatment of choice in LIG4-deficient patients who have a matched sibling donor

Ex Vivo Expansion of Hematopoietic Progenitors from CD34+-Selected Cells on Mouse Embryonic Fibroblast Feeders in a Non-Contact Co-Culture Using Matrigel or Fibronectin-Coated Inserts.

Abstract Adult pluripotent stem cells can be characterized as cells with both the capacity to self-renew and the ability to differentiate into distinct hematopoietic cell lineages and also into other tissue types. Because of the important course to stem cell research and clinical application the development of ex vivo culture systems is crucial for an effective expansion of these hematopoetic progenitor cells. After positive selection, CD34+-cells from peripheral G-CSF stimulated blood of healthy donors were cultured for 2 weeks in a serum-free medium with 2% of cord blood serum and the addition of early-acting cytokines (SCF, IL-3, Flt3-ligand, LIF, and TPO). The cells were grown in 6-well inserts on a feeder layer of mouse embryonic fibroblastic cells (MEF). Both cell types were separated from each other by a microporous membrane allowing only diffusion of soluble factors like cytokines but not cell migration. Additionally, the membrane was coated with Matrigel, a basement membrane matrix equivalent (MG), or with fibronectin (FN). Cultures were sampled at days 4, 7, 10 and 14 for cell count, colony-forming units-assay and immunophenotyping by flow cytometry. After two weeks of culture, the highest proliferation of stem cells was found in the control (medium without MEF, Matrigel and FN; 364-fold) followed by MEF- and FN-cultures (341- and 306-fold, respectively). The lowest clonal production was estimated in the cultures with MG (MG alone: 124,4-fold and MEF+MG: 48,2-fold, respectively). Examining the cells for colony-forming units the highest number of colony-forming unit-granulocyte-erythrocyte-macrophage (CFU-GEM), burst-forming unit-erythrocyte (BFU-E), colony-forming unit-granulocyte-macrophage (CFU-GM) and colony-forming unit-erythrocyte (CFU-E) were found in cultures with MEF and MEF+FN. We found multipotential colonies (CFU-GEM and BFU-E) at the highest expansion rate with FN (13,3-fold) and MEF (8,2-fold) at the fourth day of culture and with both additives in a MEF+FN-culture at the seventh day (17,2-fold). In addition to these results, there were sporadic undifferentiated progenitor cell colonies in MEF+MG-culture up to the day 14, despite an initially lower growth rate than that on MEF alone. The percentages of CD34+-cells estimated by flow cytometry were downregulated continiously during the culture period. At day 7, the highest number of CD34+-cells was found in the cultures with MG (46,0%) and MEF+MG (42,0%). The maximum number of CD34+-cells expressing no CD38 but CD61 was found at day 4 in FN culture (20,4%) and MG culture (15,9 %), respectively. In summary, we recognized in cultures with MG as a factor similar to the natural microenvironment a correlation between the diminished proliferation rate of stem cells in favor of an increased generation of multipotential colonies combined with their delayed differentiation into granulocyte-macrophage and erythrocyte colonies. Appropiate feeder cells as well as matrix proteins like Matrigel or fibronectin may helpful in improving the ex vivo expansion of hematopoietic progenitor cells.</jats:p

In Acute Leukemia, the Polymorphism −211C&gt;T in the Promoter Region of the Multidrug Resistance-Associated Protein 3 (MRP3) Is Not Associated with the Expression Level of the Gene.

Abstract We could recently show that the expression of the multidrug resistance-associated protein 3 (MRP3; ABCC3) is an important prognostic factor in ALL and in AML (Blood2003; 102:4493–8 and Clin Cancer Res2003; 9:1083–6). Lang et al. showed that the single nucleotide polymorphism −211C&gt;T (NCBI SNP ID: rs4793665) in the promoter region of the MRP3 gene is associated with the expression of the gene in liver cells (Pharmacogenetics2004;14:155–64). They found that individuals who were homozygous for cytosine in position 211 showed higher levels of MRP3 mRNA and protein. Lang et al. also provided evidence that the polymorphism is located at a binding site for transcription factors and that the polymorphism affected the binding of such factors. This could explain the influence of the genotype on the transcription level of the gene. Because of these findings, we hypothesized that the SNP −211C&gt;T might also determine the expression of MRP3 in acute leukemia and therefore be an inborn determinant of drug resistance. In order to prove this hypothesis we analyzed this SNP in our previously described groups of ALL and AML patients. The genotype was determined using a ready to use TaqMan® SNP Genotyping Assay and the ABI PrismTM 7700 Sequence Detector (Assay ID: C_27829307_10; Applied Biosystems, Foster City, CA, USA). Samples from 139 patients were analyzed. The frequency of the genotypes was: TT = 32% (n=44), CC = 22% (n=31), and CT = 46% (n=64). We did not find any correlation between the genotype and the expression of MRP3 (p=0.44). The result was the same, when patients with AML, T-lineage ALL, and precursor B-lineage ALL were analyzed separately. We also did not find a correlation between the genotype and response to therapy or the chance of survival. In conclusion, the SNP −211C&gt;T in the promoter region of MRP3 does not have a major impact on the level of MRP3 gene expression in acute leukemia and it does not determine the response to therapy. Possibly, the putative transcription regulator which binds at this part of the MRP3 gene is not active in acute leukemia.</jats:p