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Acquired aplastic anemia (AA) is a bone marrow failure resulting from an autoimmune T-cell attack against hematopoietic stem and progenitor cells (HSPCs). Several escape mechanisms within the HSPC compartment have been reported: i) adaptive alterations causing loss of high-risk HLA allele of AA development [deletion or uniparental disomy (UPD) of chromosome 6p including HLA cluster region, and loss-of-function (LOF) mutations in HLA gene], ii) clonal expansion of paroxysmal nocturnal hemoglobinuria (PNH) clone resulting from PIGAmutation, and iii) presence of clonal hematopoiesis of indeterminate potential (CHIP) mutations (i.e., BCOR/ BCORL1, DNMT3A, ASXL1). Multiple types of alterations often co-occur, however due to the inherent limitations of conventional methods, it is not known if i) these alterations are in the same or independent clones, ii) what is the mutational hierarchy, and iii) what is the cell of origin. We addressed these questions using single-cell and bulk genomic sequencing.

Background:Infantile hemangioma (IH) is the most frequent vascular tumor of early childhood. Recently, propranolol, a nonselective ß1- and ß2-adrenoceptor inhibitor, was introduced into the therapy of severe proliferating IH with excellent results. However, the underlying mechanism of action of propranolol is still unclear.Methods:We performed immunohistochemistry for cluster of differentiation 31 (CD31), D2-40, glucose transporter-1 (GLUT-1), and Ki67 in order to characterize 21 vascular anomalies (nine IH, seven venous malformations (VMs), and five lymphatic malformations (LMs)). Furthermore, we analyzed the expression of ß1-, ß2-, and ß3-adrenoceptor mRNA in these specimens as well as in hemangioma-derived stem cells by quantitative real-time PCR (qPCR).Results:We show that the expression of ß1-adrenoceptor mRNA is 10.7-fold higher in IH independent of the proliferative or regressive phase as well as 2.5-fold higher in hemangioma-derived stem cells as compared with ß2-adrenoceptor mRNA. In LM, the expression of ß2-adrenoceptor mRNA was ninefold higher than that of ß1-adrenoceptor mRNA. VM showed low expression levels of all ß-adrenoceptor mRNAs, and ß3-adrenoceptor mRNA was hardly detectable in any specimens examined.Conclusion:These results provide the first evidence of distinctions between IH and vascular malformations with regard to ß-adrenoceptor subtype mRNA levels.

Juvenile myelomonocytic leukemia (JMML) is a malignant hematopoietic disorder of early childhood with myeloproliferative and myelodysplastic properties. The proliferative component is a result of RAS pathway deregulation caused by somatic mutation in the RASor PTPN11oncogenes (60% of cases) or by underlying neurofibromatosis type 1 (NF-1) with a germline NF1gene defect (clinically 11% of cases). To search for potential collaborating genetic abnormalities, we used Affymetrix GeneChip Mapping 50K arrays to analyze over 116,000 single nucleotide polymorphisms (SNPs) across the genome using DNA from bone marrow or peripheral blood granulocytes from 16 children with JMML and normal karyotype (mutant RAS, n=4; mutant PTPN11, n=7; NF-1, n=5). Quantitative evaluation of hybridization intensities at each SNP locus failed to identify recurrent allelic gains or losses in the 16 cases. We were specifically interested in chromosome 7 because monosomy 7 or interstitial/terminal 7q deletions are found in about 30% of JMML cases and it is conceivable that submicroscopic 7q lesions occur in the other cases but remain undetected by standard techniques. However, at the resolution provided by the arrays used here, we saw no evidence for genomic copy number alterations on chromosome 7. Interestingly, evaluation of the SNP allelotypes identified large regions of copy-neutral loss of heterozygosity (LOH) on chromosome 17q, including the NF1locus, in 4 of the 5 samples from patients with JMML and NF-1. The LOH region spanned a genomic range of approximately 55 Mbp in each case and included more than 1,400 contiguous SNPs. Allelic copy numbers were normal within the homozygous regions, indicating uniparental isodisomy (UPD). Compatible with isodisomy, 17q was normal in the corresponding conventional karyotypes. By contrast, the array data provided no evidence for 17q UPD in any of the 12 JMML cases without NF-1. In all four cases with 17q UPD, the recombination breakpoints appeared to be confined to a 400-kbp region on 17q11.1–17q11.2; however, lack of parental or nonleukemic DNA precluded definitive mapping of the breakpoints. Of note, the 17p chromosomal arm retained heterozygosity in all cases, indicating that the p53 tumor suppressor was not affected by the UPD event. We confirmed 17q disomy in the four NF-1 samples using matrix-based comparative genomic hybridization and are currently verifying homozygosity of multiple microsatellites spaced across chromosome 17. Furthermore, NF1mutational analysis is under way to show that the individual lesion within this tumor suppressor gene is biallelic in leukemic cells from patients with JMML and NF-1. In summary, we assume that a mitotic recombination event in an early hematopoietic progenitor cell led to UPD involving the NF1locus. Our observations provide strong confirmatory evidence that it is indeed the NF1gene that is responsible for the increased incidence of JMML in NF-1 patients. In addition, our data support the emerging role of mitotic recombination as a second hit in leukemogenesis and corroborate the concept that RAS pathway deregulation is central to JMML pathogenesis.

Noonan syndrome is characterized by short stature, facial dysmorphism, and cardiac defects. We and our colleagues discovered novel de novo germline KRASmutations that introduce V14I, T58I, or D153V amino acid substitutions in individuals with NS and a P34R alteration in an individual with cardio-facio-cutaneous (CFC) syndrome, which has overlapping phenotypic features with NS. Recombinant V14I and T58I K-Ras proteins display defective intrinsic GTP hydrolysis and impaired responsiveness to the GTPase activating proteins (GAPs) p120 GAP and neurofibromin. We also found that V14I and T58I K-Ras render primary hematopoietic progenitors hypersensitive to growth factors and deregulate signal transduction in a cell lineage specific manner (Nature Genetics 38, 331, 2006). We recently began interrogating the P34R and D153V K-Ras mutant proteins and a novel CFC-associated F156L K-Ras mutant protein. Both P34R and D153V K-Ras display normal levels of intrinsic GTP hydrolysis. In contrast, F156L K-Ras displays defective intrinsic GTP hydrolysis that resembles oncogenic G12D K-Ras. P34R K-Ras is completely resistant to both GAPs, which is intriguing as these data suggest that the ability of GAPs to down-regulate Ras-GTP levels is dispensible for development. In contrast, D153V K-Ras is responsive to both GAPs. We expressed mutant Ras proteins in COS-7 monkey kidney cells to investigate activation of Ras and downstream effectors. Cells expressing P34R, D153V, F156L, and G12D K-Ras demonstrate elevated levels of Ras-GTP and phospho-MEK in basal and serum-starved conditions. We conclude that germline KRASmutations that cause human disease encode proteins with distinct mechanisms of biochemical activation. Further investigation of this allele series will provide insights about the relative importance of the intrinsic Ras GTPase, GAPs, and other biochemical mechanisms in controlling the growth of different cell lineages.

Neonatal thrombocytopenia occurs in about 1% of all newborns. Inherited forms like 11q- or Jacobsen syndrome are rare. However, they may remain undetected with karyotyping because the deleted regions in 11q often involve small subtelomeric regions. Here we report on the detection of deletions in 11q in two newborns with normal routine karyotypes who were shown to carry subtelomeric deletions in 11q by means of fluorescence in situhybridization (FISH) using a subtelomeric 11q probe (Abbott, Diagnostics, Wiesbaden, Germany). Both children showed thrombocytopenia (18.000/μl and 26.000/μl, respectively) and dysmegakaryopoiesis (absence of normal megakaryocytes and presence of micromegakaryocytes) associated with facial dysmorphism, cardiac defects and psychomotoric retardation. In the second case, the mother and the grandmother also showed mild thrombocytopenia. In both patients, FISH analyses on peripheral blood and bone marrow showed the loss of the telomere-associated region of 11q distal of the MLL gene. In the first patient, the deletion of 11q resulted from an unbalanced complex rearrangement with duplication of 11p. As the source of this chromosomal aberration, a paternal pericentric inversion of chromosome 11 was identified. The partial monosomy 11q and the partial trisomy 11p in the first patient were confirmed by comparative genomic hybridization (CGH) analysis. Array/matrix CGH assisted in determining the breakpoints at 11p15.1 and 11q24.1. No structural aberrations of 11q were found in the mother of the second patient, but further investigations are under way. These findings give further evidence that small subtelomeric deletions of 11q and probably mutations of genes located therein cause thrombocytopenia. Since it can be very difficult to detect these deletions by karyotyping, FISH using a subtelomeric 11q probe seems to be an extremely useful new diagnostic tool. This new method should be applied in children with congenital thrombocytopenia, in particular if they have additional complex dysmorphic features.