Differentiation of leukemic blasts is not completely blocked in acute myeloid leukemia.

Hematopoiesis, the formation of blood cells, involves the hierarchical differentiation of immature blast cells into mature, functional cell types and lineages of the immune system. Hematopoietic stem cells precisely regulate self-renewal versus differentiation to balance the production of blood cells and maintenance of the stem cell pool. The canonical view of acute myeloid leukemia (AML) is that it results from a combination of molecular events in a hematopoietic stem cell that block differentiation and drive proliferation. These events result in the accumulation of primitive hematopoietic blast cells in the blood and bone marrow. We used mathematical modeling to determine the impact of varying differentiation rates on myeloblastic accumulation. Our model shows that, instead of the commonly held belief that AML results from a complete block of differentiation of the hematopoietic stem cell, even a slight skewing of the fraction of cells that differentiate would produce an accumulation of blasts. We confirmed this model by interphase fluorescent in situ hybridization (FISH) and sequencing of purified cell populations from patients with AML, which showed that different leukemia-causing molecular abnormalities typically thought to block differentiation were consistently present in mature myeloid cells such as neutrophils and monocytes at similar levels to those in immature myeloid cells. These findings suggest reduced or skewed, rather than blocked, differentiation is responsible for the development of AML. Approaches that restore normal regulation of hematopoiesis could be effective treatment strategies.

The ploidy conveyor of mature hepatocytes as a source of genetic variation.

Mononucleated and binucleated polyploid hepatocytes (4n, 8n, 16n and higher) are found in all mammalian species, but the functional significance of this conserved phenomenon remains unknown. Polyploidization occurs through failed cytokinesis, begins at weaning in rodents and increases with age. Previously, we demonstrated that the opposite event, ploidy reversal, also occurs in polyploid hepatocytes generated by artificial cell fusion. This raised the possibility that somatic 'reductive mitoses' can also happen in normal hepatocytes. Here we show that multipolar mitotic spindles form frequently in mouse polyploid hepatocytes and can result in one-step ploidy reversal to generate offspring with halved chromosome content. Proliferating hepatocytes produce a highly diverse population of daughter cells with multiple numerical chromosome imbalances as well as uniparental origins. Our findings support a dynamic model of hepatocyte polyploidization, ploidy reversal and aneuploidy, a phenomenon that we term the 'ploidy conveyor'. We propose that this mechanism evolved to generate genetic diversity and permits adaptation of hepatocytes to xenobiotic or nutritional injury.

Mosaic variegated aneuploidy in mouse BubR1 deficient embryos and pregnancy loss in human.

Chromosome aberrations (aneuploidies mostly) are the cause of the majority of spontaneous abortions in humans. However, little is known about defects in the underlying molecular mechanisms resulting in chromosome aberrations and following failure of preimplantation embryo development, initiation of implantation and postimplantation pregnancy loss. We suggest that defects of the spindle assembly checkpoint (SAC) are responsible for aneuploidy and the following abortions. To develop our hypothesis, we modeled this process in the mouse after inactivation of protein BubR1, one of the key players of SAC. We found that soon after implantation, more than 50 % of cells of BubR1 (-/-) embryos were aneuploid and had an increased level of premature sister chromatid separation (PSCS). Aneuploid cells do not have a predominant gain or loss of some specific chromosomes, but they have mosaic variegated aneuploidy (MVA), which is characterised by random mixture of different chromosomes. MVA leads to growth retardation, stochastic massive apoptosis, disruption of bilateral symmetry, and embryo death between embryonic days 7.5 to 13.5. Analysis published human data revealed that human recurrent pregnancy loss (RPL) embryos and rare infant patients carrying BubR1 mutations that have been described so far have the PSCS and MVA as in BubR1 deficient/insufficient mice. Based on this data, we predict that deficiency/insufficiency of BubR1 and other components of the SAC in human are responsible for a significant fraction of both early and late RPLs.

Comparison of chromosome breakage in non-mosaic and mosaic patients with Fanconi anemia, relatives, and patients with other inherited bone marrow failure syndromes.

Fanconi anemia (FA) is a rare inherited bone marrow failure syndrome (IBMFS). Affected individuals must be distinguished from relatives, patients with mosaicism must be identified, and patients with other IBMFS classified as non-FA. The diagnostic feature of FA is increased chromosomal breakage in blood lymphocytes cultured with diepoxybutane or mitomycin C. Here, we sought a method to uniquely identify patients with FA with mosaicism, using cells from participants in the National Cancer Institute IBMFS cohort. Lymphocytes were treated with diepoxybutane or mitomycin C, and metaphases scored for breaks and radials. Analyses included the percentage of cells with any aberration, breaks per cell, and breaks per aberrant cell. There were 26 patients with FA (4 mosaics), 46 FA relatives, and 62 patients with a non-FA IBMFS. By all analytic methods, patients with FA were abnormal compared with other groups. Those with FA mosaicism had more breakage than relatives or patients with non-FA IBMFS, but there was some individual overlap. The choices of clastogen are laboratory-dependent, but there was no method or analysis of lymphocytes that clearly distinguished all individuals mosaic for FA from relatives or patients with other IBMFS. Thus, genotyping remains the best method for providing absolute clarity.

Bloom syndrome radials are predominantly non-homologous and are suppressed by phosphorylated BLM.

Biallelic mutations in BLM cause Bloom syndrome (BS), a genome instability disorder characterized by growth retardation, sun sensitivity and a predisposition to cancer. As evidence of decreased genome stability, BS cells demonstrate not only elevated levels of spontaneous sister chromatid exchanges (SCEs), but also exhibit chromosomal radial formation. The molecular nature and mechanism of radial formation is not known, but radials have been thought to be DNA recombination intermediates between homologs that failed to resolve. However, we find that radials in BS cells occur over 95% between non-homologous chromosomes, and occur non-randomly throughout the genome. BLM must be phosphorylated at T99 and T122 for certain cell cycle checkpoints, but it is not known whether these modifications are necessary to suppress radial formation. We find that exogenous BLM constructs preventing phosphorylation at T99 and T122 are not able to suppress radial formation in BS cells, but are able to inhibit SCE formation. These findings indicate that BLM functions in 2 distinct pathways requiring different modifications. In one pathway, for which the phosphorylation marks appear dispensable, BLM functions to suppress SCE formation. In a second pathway, T99 and T122 phosphorylations are essential for suppression of chromosomal radial formation, both those formed spontaneously and those formed following interstrand crosslink damage.

FANCL ubiquitinates ß-catenin and enhances its nuclear function.

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of ß-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates ß-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, ß-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate ß-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer ß-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/ß-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

Frequent aneuploidy among normal human hepatocytes.

Murine hepatocytes become polyploid and then undergo ploidy reversal and become aneuploid in a dynamic process called the ploidy conveyor. Although polyploidization occurs in some types of human cells, the degree of aneuploidy in human hepatocytes is not known. We isolated hepatocytes derived from healthy human liver samples and determined chromosome number and identity using traditional karyotyping and fluorescence in situ hybridization. Similar to murine hepatocytes, human hepatocytes are highly aneuploid. Moreover, imaging studies revealed multipolar spindles and chromosome segregation defects in dividing human hepatocytes. Aneuploidy therefore does not necessarily predispose liver cells to transformation but might promote genetic diversity among hepatocytes.

In vivo selection of transplanted hepatocytes by pharmacological inhibition of fumarylacetoacetate hydrolase in wild-type mice.

Genetic fumarylacetoacetate hydrolase (Fah) deficiency is unique in that healthy gene-corrected hepatocytes have a strong growth advantage and can repopulate the diseased liver. Unfortunately, similar positive selection of gene-corrected cells is absent in most inborn errors of liver metabolism and it is difficult to reach the cell replacement index required for therapeutic benefit. Therefore, methods to transiently create a growth advantage for genetically modified hepatocytes in any genetic background would be advantageous. To mimic the selective pressure of Fah deficiency in normal animals, an efficient in vivo small molecule inhibitor of FAH, 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) was developed. Microarray analysis demonstrated that pharmacological inhibition of FAH produced highly similar gene expression changes to genetic deficiency. As proof of principle, hepatocytes lacking homogentisic acid dioxygenase (Hgd) and hence resistant to FAH inhibition were transplanted into sex-mismatched wild-type recipients. Time course analyses of 4-6 weeks of CEHPOBA administration after transplantation showed a linear relationship between treatment length and replacement index. Compared to controls, recipients treated with the FAH-inhibitor had 20-100-fold increases in liver repopulation. We conclude that pharmacological inhibition of FAH is a promising approach to in vivo selection of hepatocytes.

Limitations of gene editing assessments in human preimplantation embryos.

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.

Human FANCC is hypomorphic in murine Fancc-deficient cells.

Fancc suppresses cross-linker-induced genotoxicity, modulates growth-inhibitory cytokine responses, and modulates endotoxin responses. Although loss of the latter function is known to account for endotoxin-induced marrow failure in murine Fancc (mFancc)-deficient mice, some argue that cytokine and endotoxin hypersensitivities devolve simply from genomic instability. Seeking to resolve this question, we planned to ectopically express instructive human FANCC (hFANCC) mutants in murine Fancc-deficient hematopoietic stem cells. To first assure that hFANCC cDNA was competent in murine cells, we compared hFANCC and mFancc in complementation assays for cross-linking agent hypersensitivity and endotoxin hypersensitivity. We found that mFancc complemented murine Fancc-deficient cells in both assays, but that hFANCC fully suppressed only endotoxin hypersensitivity, not cross-linking agent hypersensitivity. These results support the notions that Fancc is multifunctional and that structural prerequisites for its genoprotective functions differ from those required to constrain endotoxin responses known to lead to marrow failure in Fancc-deficient mice.

Patients with Fanconi anemia and AML have different cytogenetic clones than de novo cases of AML.

Specific cytogenetic clones might distinguish patients with unrecognized Fanconi anemia (FA) who present with acute myeloid leukemia (AML) from those with sporadic AML. Cytogenetic reports in literature cases of FA and AML were compared with de novo cases enrolled on CCG-2961. Gain of 1q, gain of 3q, monosomy 7, deleted 7q, gain of 13q, and deleted 20q were more frequent in FA AML; t(8;21), trisomy 8, t(9;11), t(6;9), and inversion 16 were exclusive to de novo AML cases. Observation of the FA AML cytogenetic clonal patterns should raise suspicion of an underlying leukemia predisposition syndrome and influence management.

Complex cytogenetic analysis of early lethality mouse embryos.

An increasing interest in the molecular mechanisms governing cell division has resulted in the discovery of several groups of genes that participate in the regulation of mitosis and meiosis in eukaryotes. Inactivation of these genes in mice often leads to early embryonic lethality. To show direct causality between mutations of these genes, chromosomal instability and embryonic death, a technique enabling detailed cytogenetic analysis of embryonic cells is required. Here, we develop and test a comprehensive approach that allows complex analysis of individual early postimplantation embryos and combines polymerase chain reaction genotyping with the preparation and detailed karyotypic inspection of cells at the metaphase and anaphase stages. The method enables good chromosomal spreading and scattering of nuclei to perform routine cytogenetics (i.e., standard stain and G-banding). It also permits the application of specialized techniques such as fluorescence in situ hybridization to detect particular chromosomes and to verify the integrity of individual chromosomes. Utility of the new method is demonstrated by an analysis of embryonic day E7.5-E9.5 tissue from mice deficient in the spindle checkpoint gene Bub1b.

Ploidy reductions in murine fusion-derived hepatocytes.

We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH), and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ss-galactosidase and the Y-chromosome. Approximately 2-5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ss-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ss-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by >or=50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis.

Adult acute myeloid leukemia patients with NUP98 rearrangement have frequent cryptic translocations and unfavorable outcome.

Acute myeloid leukemia (AML) with NUP98 rearrangement (AML-NUP98) has been uncommonly reported in adults, and its incidence in our institution is ∼2.5%. There were four men and five women with a median age of 49 years, among which six cases were de novo AML and three were therapy-related. Five cases were AML with minimal differentiation or without maturation, followed by four with monocytic differentiation. NUP98 rearrangement was confirmed in all cases by FISH, and five cases showed cryptic translocations. The median overall survival (OS) was 13 months, shorter than that of AML-NPM1 (p < 0.05), and similar to that in AML-KMT2A patients in our institution. The unfavorable OS was further confirmed by comparing to AML patients in TCGA database. In conclusion, adult AML-NUP98 is associated with cryptic translocations and an unfavorable outcome. Our study suggests that incorporating the NUP98 probe into AML FISH panels are warranted to improve clinical management.

Section E9 of the American College of Medical Genetics technical standards and guidelines: fluorescence in situ hybridization.

This updated Section E9 has been incorporated into and supersedes the previous Section E9 in Section E: Clinical Cytogenetics of the 2008 Edition (Revised 02/2007) American College of Medical Genetics Standards and Guidelines for Clinical Genetics Laboratories. This section deals specifically with the standards and guidelines applicable to fluorescence in situ hybridization analysis.

Fanconi anemia-like presentation in an infant with constitutional deletion of 21q including the RUNX1 gene.

We describe a newborn female with a de novo interstitial deletion of chromosome 21q21.1-22.12 including the RUNX1 gene who had developmental delay, multiple congenital anomalies, tetralogy of Fallot, anemia, and chronic thromobocytopenia requiring frequent platelet transfusions from birth. Because of her physical and hematologic abnormalities, she was tested for Fanconi anemia (FA). Lymphocytes and fibroblasts from this patient demonstrated increased chromosome breakage with exposure to the clastogen mitomycin C, but not, in contrast to most FA patients, to diepoxybutane. Further testing by Western analysis and complementation testing did not show a defect in the function of known Fanconi proteins. Her constitutional deletion was later found to span 13.2 Mb by chromosome microarray analysis, encompassing the RUNX1 gene that has been implicated in thrombocytopenia and predisposition to acute myelogenous leukemia (AML) when in the haploinsufficient state. We compare her phenotype to other individuals with similar 21q deletions and thrombocytopenia, as well as those with FA. We suggest that deletion of RUNX1 or another critical gene within the deleted region may result in chromosomal instability similar to that seen in FA.

A Review of Recent Developments in Turner Syndrome Research.

Turner syndrome is a rare disorder resulting from complete or partial loss of the second sex chromosome. Common manifestations include delayed growth, premature ovarian failure, congenital heart defects, endocrine disorders, lymphedema, and webbed neck. People with Turner syndrome have significantly increased mortality risk primarily due to cardiovascular abnormalities. The mechanisms that lead to these defects are not completely understood and are obscured by the significant variability of both karyotype and phenotype without consistent correlation between the two. This paper presents a review of the recent literature surrounding the symptoms, mechanisms, diagnosis, and treatment of Turner syndrome with a focus on cardiovascular manifestations. With technological advancements in genetics, the molecular processes of Turner syndrome have begun to be dissected. Certain genes on the X chromosome that typically escape inactivation have been implicated in both specific manifestations and broader risk categories. Recently identified genome-wide epigenetic changes may help explain the variability in presentation. It remains unclear as to how the combination of these factors results in the overall clinical picture, but advances in genomic, genetic, epigenetic, and -omics technology hold promise for providing insights that will improve the medical management of individuals with Turner syndrome.

Characterizing the HER2/neu status and metastatic potential of breast cancer stem/progenitor cells.

INTRODUCTION: Treatment resistance, long latency, and high recurrence rates suggest that breast cancers arise from defective breast stem cells. HYPOTHESIS: Within cancers, subpopulations of cells will demonstrate differences in stem/progenitor potential, HER2/neu amplification, and gene expression. Related cells will be found in normal breast tissue. METHODS: ER-/PR-/HER2/neu + breast cancer cells were flow-sorted into subpopulations: (A) CD49f(+) CD24(-), (B) CD49f(+)CD24(+), (C) CD49f CD24(-), and (D) CD49f(-)CD24(+). Gel matrix cell invasion, fluorescence in situ hybridization (FISH) HER2/neu amplification, and qRT-PCR gene expression were measured in all groups. Cells from sorted groups were implanted into rat brains. Resultant tumors were analyzed by immunohistochemistry (IHC) and FISH. Normal breast tissue was examined by IHC. RESULTS: Tumor development varied among sorted groups (25-75%), but was highest in group A. Tumor cells were mostly CD49f(-)CD24(-), with variable fractions of other stem/progenitor cells. Tumors showed HER2/neu amplification, but fewer chromosome 17 per cell than inoculates. Group A tumors exhibited cells with normal chromosome 17 copy number and near normal HER2/neu amplification. Cell invasion was 61% higher in unsorted cells and 34-42% in sorted groups compared with controls. Sorted groups showed significantly different expression of development, proliferation, and invasion associated genes. In normal breast tissue, CD49f(+) cells were identified in CD14(+) CK19(-) basal epithelial layers of mammary glands; these were 95% CD24(+) and 60% CD44(+). CONCLUSIONS: Breast cancer stem/progenitor cell populations differ in tumor-initiating potential but are not solely responsible for metastasis. Cancer stem/progenitor cells are less polyploid than cancer cells in general and may not be HER2/neu amplified. In normal breast tissue, breast stem/progenitor cell-like populations are present.

DNA interstrand crosslink repair in mammalian cells.

DNA damage by agents crosslinking the strands presents a formidable challenge to the cell to repair for survival and to repair accurately for maintenance of genetic information. It appears that repair of DNA crosslinks occurs in a path involving double strand breaks (DSBs) in the DNA. Mammalian cells have multiple systems involved in the repair response to such damage, including the Fanconi anemia pathway that appears to be directly involved, although the mechanisms and site of action remain elusive. A particular finding relating to deficiency of the Fanconi anemia pathway is the observation of chromosomal radial formations after ICL damage. The basis of formation of such chromosomal aberrations is unknown although they appear secondarily to DSBs. Here we review the processes involved in response to DNA interstrand crosslinks which might lead to radial formation and the role of the nucleotide excision repair gene, ERCC1, which is required for a normal response, not just to DNA crosslinks, but also for DSBs at collapsed replication forks caused by substrate depletion.