Background: Aberrations in DNA methylation patterns are well-described in human malignancies. However, the existence of the 'CpG island methylator phenotype' (CIMP) in human breast cancer is still controversial. Materials & methods: Illumina's HumanMethylation 450K BeadChip was used to analyze genome-wide DNA methylation patterns. Chromosomal abnormalities were determined by array-based CGH. Results: Invasive lobular breast carcinomas exhibit the highest number of differentially methylated CpG sites and a strong inverse correlation of aberrant DNA hypermethylation and copy number alterations. Nine differentially methylated regions within seven genes discriminating the investigated subgroups were identified and validated in an independent validation cohort and correlated to a better relapse-free survival. Conclusion: These results depict a clear difference between genetically and epigenetically unstable breast carcinomas indicating different ways of tumor progression and/or initiation, which strongly supports the association of CIMP with the lobular subtype and provide new options for detection and therapy.
Background: BRCA1 and BRCA2 mutation carriers are at substantially increased risk for developing breast and ovarian cancer. The incomplete penetrance coupled with the variable age at diagnosis in carriers of the same mutation suggests the existence of genetic and non-genetic modifying factors. In this study we evaluated the putative role of variants in many candidate modifier genes. Methods: Genotyping data from 15,252 BRCA1 and 8,211 BRCA2 mutation carriers, for known variants (n=3,248) located within or around 445 candidate genes, were available through the iCOGS custom-designed array. Breast and ovarian cancer association analysis was performed within a retrospective cohort approach. Results: The observed p-values of association ranged between 0.005-1.000. None of the variants was significantly associated with breast or ovarian cancer risk in either BRCA1 or BRCA2 mutation carriers, after multiple testing adjustments. Conclusion: There is little evidence that any of the evaluated candidate variants act as modifiers of breast and/or ovarian cancer risk in BRCA1 or BRCA2 mutation carriers. Impact: Genome-wide association studies have been more successful at identifying genetic modifiers of BRCA1/2 penetrance than candidate gene studies.
Two long and one truncated isoforms (termed LAP*, LAP, and LIP, respectively) of the transcription factor CCAAT enhancer binding protein beta (C/EBP?) are expressed from a single intronless Cebpb gene by alternative translation initiation. Isoform expression is sensitive to mammalian target of rapamycin (mTOR)-mediated activation of the translation initiation machinery and relayed through an upstream open reading frame (uORF) on the C/EBP? mRNA. The truncated C/EBP? LIP, initiated by high mTOR activity, has been implied in neoplasia, but it was never shown whether endogenous C/EBP? LIP may function as an oncogene. In this study, we examined spontaneous tumor formation in C/EBP? knockin mice that constitutively express only the C/EBP? LIP isoform from its own locus. Our data show that deregulated C/EBP? LIP predisposes to oncogenesis in many tissues. Gene expression profiling suggests that C/EBP? LIP supports a pro-tumorigenic microenvironment, resistance to apoptosis, and alteration of cytokine/chemokine expression. The results imply that enhanced translation reinitiation of C/EBP? LIP promotes tumorigenesis. Accordingly, pharmacological restriction of mTOR function might be a therapeutic option in tumorigenesis that involves enhanced expression of the truncated C/EBP? LIP isoform.
Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7 × 10(-3)) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3)). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.
Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a ?-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of ?-retroviral vectors is associated with a substantial risk of leukemogenesis.
Acute myeloid leukemia with complex karyotype (CK-AML) is a distinct biological entity associated with a very poor outcome. Since complex karyotypes frequently contain deletions of the chromosomal region 12p13 encompassing the tumor suppressor genes ETV6 and CDKN1B, we aimed to unravel their modes of inactivation in CK-AML.
Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a 20% risk of evolving into leukemia or myelodysplastic syndrome (MDS). Patterns of acquisition of leukemia-associated mutations were investigated using next-generation deep-sequencing in 31 CN patients who developed leukemia or MDS. Twenty (64.5%) of the 31 patients had mutations in RUNX1. A majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R mutations. In contrast to their high frequency in CN patients who developed leukemia or MDS, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric acute myeloid leukemia. A sequential analysis at stages prior to overt leukemia revealed RUNX1 mutations to be late events in leukemic transformation. Single-cell analyses in 2 patients showed that RUNX1 and CSF3R mutations were present in the same malignant clone. Functional studies demonstrated elevated granulocyte colony-stimulating factor (G-CSF)-induced proliferation with diminished myeloid differentiation of hematopoietic CD34(+) cells coexpressing mutated forms of RUNX1 and CSF3R. The high frequency of cooperating RUNX1 and CSF3R mutations in CN patients suggests a novel molecular pathway of leukemogenesis: mutations in the hematopoietic cytokine receptor (G-CSFR) in combination with the second mutations in the downstream hematopoietic transcription fator (RUNX1). The detection of both RUNX1 and CSF3R mutations could be used as a marker for identifying CN patients with a high risk of progressing to leukemia or MDS.
Methods for generating induced pluripotent stem cells (iPSCs) for disease modeling and cell therapies have progressed from integrating vectors to transient delivery of reprogramming factors, avoiding permanent genomic modification. A major limitation of unmodified iPSCs is the assessment of their distribution and contribution to adverse reactions in autologous cell therapy. Here, we report that polycistronic lentiviral vectors with single Flp recombinase (Flp) recognition target (FRT) sites can be used to generate murine iPSCs that are devoid of the reprogramming cassette but carry an intergenic 300-bp long terminal repeat sequence. Performing quantitative polymerase chain reaction on this marker, we could determine genetic identity and tissue contribution of iPSC-derived teratomas in mice. Moreover, we generated iPSCs carrying heterospecific FRT twin sites, enabling excision and recombinase-mediated cassette exchange (RMCE) of the reprogramming cassette for another expression unit of choice. Following screening of iPSCs for "safe harbor" integration sites, expression cassettes were introduced by RMCE into various previously silenced loci of selected single-copy iPSCs. Analysis of DNA methylation showed that RMCE reverted the local epigenetic signature, which allowed transgene expression in undifferentiated iPSCs and in differentiated progeny. These findings support the concept of creating clonotypically defined exchangeable and traceable pluripotent stem cells for disease research and cell therapy.
Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for studies on disease-related developmental processes and may serve as an autologous cell source for future treatment of many hereditary diseases. New genetic engineering tools such as zinc finger nucleases and transcription activator-like effector nuclease allow targeted correction of monogenetic disorders but are very cumbersome to establish. Aiming at studies on the knockdown of a disease-causing gene, lentiviral vector-mediated expression of short hairpin RNAs (shRNAs) is a valuable option, but it is limited by silencing of the knockdown construct upon epigenetic remodeling during differentiation. Here, we propose an approach for the expression of a therapeutic shRNA in disease-specific iPSCs using third-generation lentiviral vectors. Targeting severe ?-1-antitrypsin (A1AT) deficiency, we overexpressed a human microRNA 30 (miR30)-styled shRNA directed against the PiZ variant of A1AT, which is known to cause chronic liver damage in affected patients. This knockdown cassette is traceable from clonal iPSC lines to differentiated hepatic progeny via an enhanced green fluorescence protein reporter expressed from the same RNA-polymerase II promoter. Importantly, the cytomegalovirus i/e enhancer chicken ? actin (CAG) promoter-driven expression of this construct is sustained without transgene silencing during hepatic differentiation in vitro and in vivo. At low lentiviral copy numbers per genome we confirmed a functional relevant reduction (-66%) of intracellular PiZ protein in hepatic cells after differentiation of patient-specific iPSCs. In conclusion, we have demonstrated that lentiviral vector-mediated expression of shRNAs can be efficiently used to knock down and functionally evaluate disease-related genes in patient-specific iPSCs.
To study genomic imbalances potentially involved in disease development and/or progression of childhood MDS, array-based comparative genomic hybridization (aCGH) is a helpful tool. Copy number alterations (CNA) of subtle chromosomal regions containing potential candidate genes, e.g., TP53 or RUNX1 can be detected. However, characterizing small and/or heterogeneous tumor subpopulations by high-resolution aCGH within a majority of normal cells is a challenge in MDS and requires validation by independent methods like FISH or quantitative PCR. For the identification of tumor-relevant CNA, the analysis of DNA isolated from purified granulocytes or myeloid populations instead of DNA from whole bone marrow (BM) cells is helpful to overcome some of these limitations.
Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.
Induced pluripotent stem cells (iPSC) are important tools in regenerative medicine. Yet, it is becoming increasingly clear that the reprogramming process, including retroviral transduction with potent oncogenes like c-Myc and long-term cultivation, may induce genetic instability. Genetically altered iPS cells can grow out and dominate the cell culture. This review intends to comprehensively summarize the current knowledge on genetic instability of embryonic and iPSCs, with an emphasis on cytogenetic alterations, and compares these data with what is known from tumorigenesis.
The multidrug-resistance 1 (MDR-1) P-glycoprotein (Pgp) is a transmembrane transporter system, which actively pumps cytotoxic drugs out of the cell. MDR-1 acquired in vitro differs from MDR-1 acquired in vivo, but has important consequences on the cellular phenotype and metastatic behavior. Here we report that the human colonic cancer cell line HT29 (MDR-1 negative) is more malignant than its MDR-1 overexpressing variant (HT29 MDR-1 positive). HT29 MDR-1 negative cells produce undifferentiated signet ring carcinomas when implanted subcutaneously into SCID mice, while HT29 MDR-1 positive cells form tumors with tubular structures, but without signet ring cells. Immunohistochemical proliferation marker analysis revealed that the MDR-1 positive cells proliferate much more slowly than the MDR-1 negative cells. MDR-1 overexpression results in a less differentiated phenotype at the cellular level (absence of mucin producing cells) but in a more differentiated phenotype at the tissue level (tubule formation). In addition, lectin binding patterns including that of Helix pomatia agglutinin (HPA), an indicator of metastatic potential, differed between the two cell lines. HT29 MDR-1 positive cells had less HPA binding sites than HT29 MDR-1 negative counterparts and metastasized less frequently in SCID mice. As slow proliferation, low degree of differentiation and multidrug-resistance is a hallmark of cancer stem cells and all were present in MDR-1 positive tumors, it is attractive to speculate that they represent a stem cell rich tumor. As shown by global gene expression analyses, genes involved, e.g. in cell adhesion, glycosylation and signal transduction, were deregulated in MDR-1 positive tumors compared to MDR-negative tumors. Overexpression of E-cadherin and carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1) may provide clues to the mechanisms responsible for the reduced metastatic potential of MDR-1 overexpressing tumors. Since drug treatment shifted the cells towards a less metastatic phenotype in this in vivo model, it seems conceivable to achieve this using drug treatment also in a clinical situation.
Juvenile myelomonocytic leukaemia (JMML) is a unique myeloproliferative disorder of early childhood. Frequently, mutations in NRAS, KRAS, PTPN11, NF1 or CBL are found in these patients. Monosomy 7 is the most common cytogenetic aberration. To identify submicroscopic genomic copy number alterations, 20 JMML samples were analysed by comparative genomic hybridization. Ten out of 20 samples displayed additional submicroscopic alterations. In two patients, an almost identical gain of chromosome 8 was identified. In both patients, fluorescence in situ hybridization confirmed a constitutional partial trisomy 8 mosaic (cT8M). A survey on 27 cT8M patients with neoplasms showed that 21 had myeloid malignancies, and five of these had a JMML. Notably, the region gained in our cases is the smallest gain of chromosome 8 reported in cT8M cases with malignancies so far. Our results dramatically reduce the critical region to 8p11.21q11.21 harbouring 31 protein coding genes and two non-coding RNAs, e.g. MYST3, IKBKB, UBE2V2, GOLGA7, FNTA and MIR486--a finding with potential implications for the role of somatic trisomy 8 in myeloid malignancies. Further investigations are required to more comprehensively determine how constitutional partial trisomy 8 mosaicisms may contribute to leukaemogenesis in different mutational subtypes of JMML and other myeloid malignancies.
Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH?/? mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH?/?-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH?/? iPS cell lines, we aggregated FAH?/?-iPS cells with tetraploid embryos and obtained entirely FAH?/?-iPS cell-derived mice that were viable and exhibited the phenotype of the founding FAH?/? mice. Then, we transduced FAH cDNA into the FAH?/?-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell-derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.
Adipose-derived stem cells (ASCs) are reported to display multilineage differentiation potential, including neuroectodermal pathways. The aim of the present study was to critically re-evaluate the potential neurogenic (trans-)differentiation capacity of ASCs using a neurogenic induction protocol based on the combination of isobutylmethylxanthine (IBMX), indomethacin and insulin. ASCs isolated from lipo-aspirate samples of five healthy female donors were characterized and potential neurogenic (trans-)differentiation was assessed by means of immunohistochemistry and gene expression analyses. Cell proliferation and cell cycle alterations were studied, and the expression of CREB/ATF transcription factors was analyzed. ASCs expressed CD59, CD90 and CD105, and were tested negative for CD34 and CD45. Under neurogenic induction, ASCs adopted a characteristic morphology comparable to neur(on)al progenitors and expressed musashi1, ?-III-tubulin and nestin. Gene expression analyses revealed an increased expression of ?-III-tubulin, GFAP, vimentin and BDNF, as well as SOX4 in induced ASCs. Cell proliferation was significantly reduced under neurogenic induction; cell cycle analyses showed a G2-cell cycle arrest accompanied by differential expression of key regulators of cell cycle progression. Differential expression of CREB/ATF transcription factors could be observed on neurogenic induction, pointing to a decisive role of the cAMP-CREB/ATF system. Our findings may point to a potential neurogenic (trans-)differentiation of ASCs into early neur(on)al progenitors, but do not present definite evidence for it. Especially, the adoption of a neural progenitor cell-like morphology must not automatically be misinterpreted as a specific characteristic of a respective (trans-)differentiation process, as this may as well be caused by alterations of cell cycle progression.
We describe the clinical course and have characterised anatomically and genetically a unique case of a newborn with bilateral hypoplasia of pulmonary arteries, consecutive extremely hypoplastic lung tissue and associated unilateral renal agenesis. Intrauterine oxygenation by the placenta seemed to have allowed normotrophic body maturity but immediately after delivery, in the third trimester, progressive hypoxemia developed and the newborn succumbed to acute respiratory failure. Genetic analysis by array-based comparative genomic hybridisation and quantitative PCR revealed duplication of 1p21, which, however, might not be the disease causing aberration. This case might represent an extreme form of previously reported, rare cases with simultaneous dysorganogenesis of lungs and kidneys.
Cyclin E is often overexpressed in cancer tissue, leading to genetic instability and aneuploidy. Cullin 3 (Cul3) is a component of the BTB-Cul3-Rbx1 (BCR) ubiquitin ligase that is involved in the turnover of cyclin E. Here we show that liver-specific ablation of Cul3 in mice results in the persistence and massive expansion of hepatic progenitor cells. Upon induction of differentiation, Cul3-deficient progenitor cells underwent substantial DNA damage in vivo and in vitro, thereby triggering the activation of a cellular senescence response that selectively blocked the expansion of the differentiated offspring. Positive selection of undifferentiated progenitor cells required the expression of the tumor suppressor protein p53. Simultaneous loss of Cul3 and p53 in hepatic progenitors turned these cells into highly malignant tumor-initiating cells that formed largely undifferentiated tumors in nude mice. In addition, loss of Cul3 and p53 led to the formation of primary hepatocellular carcinomas. Importantly, loss of Cul3 expression was also detected in a large series of human liver cancers and correlated directly with tumor de-differentiation. The expression of Cul3 during hepatic differentiation therefore safeguards against the formation of progenitor cells that carry a great potential for transformation into tumor-initiating cells.
To evaluate whether copy number alterations (CNAs) are present that may contribute to disease development and/or progression of childhood myelodysplastic syndromes (MDS), 36 pediatric MDS patients were analyzed using array-based comparative genome hybridization (aCGH). In addition to monosomy 7, the most frequent chromosome aberration in childhood MDS, novel recurrent CNAs were detected. They included a loss of 3p14.3-p12.3, which contains the putative tumor suppressor gene FHIT, a loss of 7p21.3-p15.3, a loss of 9q33.3-q34.3 (D184) and microdeletions in 17p11.2, 6q23 containing MYB, and 17p13 containing TP53. In this small patient cohort, patients without CNA, patients with monosomy 7 only and patients with one CNA in addition to monosomy 7 did not differ in their survival. As expected, all patients with complex karyotypes, including two patients with deletions of TP53, died. A challenge inherent to aCGH analysis of MDS is the low percentage of tumor cells. We evaluated several approaches to overcome this limitation. Genomic profiles from isolated granulocytes were of higher quality than those from bone marrow mononuclear cells. Decreased breakpoint calling stringency increased recognition of CNAs present in small clonal populations. However, further analysis using a custom-designed array showed that these CNAs often did not confirm the findings from 244k arrays. In contrast, constitutional CNVs were reliably detected on both arrays. Moreover, aCGH on amplified DNA from distinct myeloid clusters is a new approach to determine CNAs in small subpopulations. Our results clearly emphasize the need to verify array-CGH results by independent methods like FISH or quantitative PCR.
Children with neurofibromatosis type 1 (NF-1), being constitutionally deficient for one allele of the NF1 gene, are at greatly increased risk of juvenile myelomonocytic leukemia (JMML). NF1 is a negative regulator of RAS pathway activity, which has a central role in JMML. To further clarify the role of biallelic NF1 gene inactivation in the pathogenesis of JMML, we investigated the somatic NF1 lesion in 10 samples from children with JMML/NF-1. We report that two-thirds of somatic events involved loss of heterozygosity (LOH) at the NF1 locus, predominantly caused by segmental uniparental disomy of large parts of chromosome arm 17q. One-third of leukemias showed compound-heterozygous NF1-inactivating mutations. A minority of cases exhibited somatic interstitial deletions. The findings reinforce the emerging role of somatic mitotic recombination as a leukemogenic mechanism. In addition, they support the concept that biallelic NF1 inactivation in hematopoietic progenitor cells is required for transformation to JMML in children with NF-1.
Array-based comparative genomic hybridization (array-CGH) is an emerging high-resolution and high-throughput molecular genetic technique that allows genome-wide screening for chromosome alterations. DNA copy number alterations (CNAs) are a hallmark of somatic mutations in tumor genomes and congenital abnormalities that lead to diseases such as mental retardation. However, accurate identification of amplified or deleted regions requires a sequence of different computational analysis steps of the microarray data.
The early diagnosis of chronic organ rejection after lung transplantation (LTx) is currently hampered by the lack of reliable diagnostic markers. The present study aims to establish the procedure of gene expression profiling in bronchial epithelial cells for the identification of candidate genes that might prove useful in the early diagnosis.
To ensure targeted treatment, it would be useful to know at the time of diagnosis whether a BRCA mutation is causally related to an individual breast cancer. The aim of this study was to investigate in an unselected series of breast cancer patients the value of incorporating morphological and immunohistochemical features for the selection of patients who may benefit from BRCA1 genetic testing. In a retrospective approach, histopathological results of tumors from 897 women were reevaluated regarding age at diagnosis, subtype of cancer, tumor grade, and estrogen (ER), progesterone (PR), and Her2/neu receptor status, as well as p53 and Ki67 status. In all, 142 tumors fulfilled morphological criteria indicative of a BRCA1 mutation. Of the 59 women willing to participate, 26 women concomitantly showed a positive family history. Pathogenic BRCA1 germline mutations were detected in 7 of 18 women (39%) (95% confidence interval = 0.17-0.64). All BRCA1-associated tumors were of high grade, invasive-ductal subtype, and PR and Her2/neu negative, and 91% of the tumors were negative for ER; 60% of the tumors showed a high expression of p53 and 60% a high expression of Ki67. There was a significant difference with respect to grading (P = 0.001 for G3), ER negativity (P = 0.0075), Ki67 > or = 65% (P = 0.0039), and triple negativity (i.e., ER(-), PR(-), Her2/neu(-)) (P = 0.0019) between tumors of mutation carriers and noncarriers.
Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E-cadherin. ILBCs also lack beta-catenin expression and show aberrant cytoplasmic localization of the E-cadherin binding protein p120-catenin. The lack of a well-characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH-926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH-926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH-926 expressed various epithelial cell markers but lacked expression of E-cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH-926 from this particular tumour. IPH-926 also lacked beta-catenin expression and showed aberrant cytoplasmic localization of p120-catenin. Array-CGH analysis of IPH-926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH-926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH-926 cells were anti-cancer drug-resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH-926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH-926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo.
The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into safe harbor sites in the genomes of autologous, patient-derived iPS cells.
Genome-wide profiling of copy number alterations by array-based high resolution comparative genomic hybridization (array-CGH) is an important method to ensure the genomic integrity of cells in diverse conditions. We observed that the analysis of genomic profiles, in particular of fast-dividing murine leukemia cell lines, is challenging due to characteristic patterns oscillating around the array-CGH baseline. Here we show array-CGH data can be drastically improved by reducing proliferation rates of cultured cells using deprivation protocols or cell cycle inhibitors. Arresting cell cycle in the G1 phase leads to smoother genomic profiles, and hence to a more reliable detection of copy number alterations.
BRD7 (bromodomain 7), a subunit of poly-bromo-associated BRG1-associated factor (PBAF)-specific Swi/Snf chromatin remodeling complexes, has been proposed as a tumour suppressor protein following its identification as an important component of both functional p53 and BRCA1 (breast cancer 1, early onset) pathways. As low BRD7 expression levels have been linked to p53-wild-type breast tumour cells, we hypothesized an implication of BRD7 germline alterations in the pathogenesis of hereditary breast cancer similar to that of TP53 in Li-Fraumeni syndrome. We performed sequence analysis of the BRD7 gene on 61 high-risk individuals with hereditary or very-early-onset breast cancer and 100 healthy controls. Four potentially disease-causing single-nucleotide alterations were detected within the cohort of breast cancer patients (one listed as a rare single-nucleotide polymorphism (SNP) in the NCBI (National Center for Biotechnology Information) SNP database). Two of the detected variants were also each found once within the control collective. Segregation analysis on both families of those carrying the remaining two variants revealed segregation of these BRD7 alterations independent of breast cancer. In conclusion, it seems that the BRD7 variants we detected represent rare polymorphisms and mainly rule out BRD7 as a frequent high-penetrance breast cancer susceptibility gene. However, further analyses in larger cohorts of women with hereditary breast cancer should clarify the role of BRD7 in breast cancer predisposition.
In recent years, several new white matter diseases have been identified based on magnetic resonance imaging and clinical findings. For most newly defined disorders the genetic basis has been identified. However, there is still a large group of patients without a specific diagnosis. Hypomyelinating leukodystrophies are the largest group among them. In some disorders characterized by hypomyelination only central nervous system involvement is observed, but in some disorders involvement of other organs is observed as well, such as eyes or teeth. Pelizaeus-Merzbacher-like disease (PMLD) is an autosomal recessive hypomyelinating disorder of the central nervous system characterized by nystagmus, ataxia, and progressive spasticity. The disease is caused by mutations in GJC2, the gene that encodes the gap junction protein connexin 47. Here we describe hypomyelination and Müllerian agenesis syndrome in a girl who is homozygous for a novel mutation in the GJC2 gene. It is an open question whether this is an association by chance or a feature of PMLD not previously noted.
The small cell ovarian carcinoma of the hypercalcemic type (SCCOHT) represents an aggressive tumor with poor prognosis predominantly affecting young women and so far, no cell line or animal model is available to investigate this devastating disease. Biopsy material from a recurrent SCCOHT was subjected to an explant culture to obtain an adherent and continuously proliferating cell population. Morphological and functional characterization revealed a heterogeneous population (SCCOHT-1) of about 13 µm in diameter and approximately 36 h of doubling time. Flow cytometric analysis of surface markers demonstrated the expression of CD15, CD29, CD44 and CD90 paralleled by the presence of cytokeratins and vimentin. Cytogenetic analysis and high-resolution oligo-array comparative genomic hybridization (aCGH) demonstrated a stable karyotype including deletions of the PARK2, CSMD1, GRIN2B and ATF7IP genes. Following lentiviral transduction with a GFP vector, the labeled SCCOHT-derived cells were subjected to CCE to separate distinct subpopulations as evidenced by cell cycle analysis. Subcutaneous injection of these subpopulations into NOD/SCID mice exhibited hypercalcemia and a tumor development in 100% of the mice. Re-cultivation of the mouse tumors revealed an outgrowth of SCCOHT-derived phenotypes and all cell populations expressed high telomerase activity. Moreover, histopathological evaluation demonstrated close similarities between the mouse tumors and the original patient tumor. In conclusion, SCCOHT-1 cells provide a study platform to investigate this rare disease and to examine effective and sufficient therapeutic strategies for this rather unknown type of cancer.
Integrating vectors developed on the basis of various retroviruses have demonstrated therapeutic potential following genetic modification of long-lived hematopoietic stem and progenitor cells. Lentiviral vectors (LV) are assumed to circumvent genotoxic events previously observed with ?-retroviral vectors, due to their integration bias to transcription units in comparison to the ?-retroviral preference for promoter regions and CpG islands. However, recently several studies have revealed the potential for gene activation by LV insertions. Here, we report a murine acute B-lymphoblastic leukemia (B-ALL) triggered by insertional gene inactivation. LV integration occurred into the 8th intron of Ebf1, a major regulator of B-lymphopoiesis. Various aberrant splice variants could be detected that involved splice donor and acceptor sites of the lentiviral construct, inducing downregulation of Ebf1 full-length message. The transcriptome signature was compatible with loss of this major determinant of B-cell differentiation, with partial acquisition of myeloid markers, including Csf1r (macrophage colony-stimulating factor (M-CSF) receptor). This was accompanied by receptor phosphorylation and STAT5 activation, both most likely contributing to leukemic progression. Our results highlight the risk of intragenic vector integration to initiate leukemia by inducing haploinsufficiency of a tumor suppressor gene. We propose to address this risk in future vector design.
Noonan syndrome (NS) is a common autosomal dominant condition characterized by short stature, congenital heart defects, and dysmorphic facial features caused in approximately 50% of cases by missense mutations in the PTPN11 gene. NS patients are predisposed to malignancies including myeloproliferative disorders or leukemias. We report a female NS patient carrying a PTPN11 germline mutation c.417 G?>?C (p.E139D), who developed in her second year of life an acute lymphoblastic leukemia (ALL) and after remission, she developed at 4 years of age a juvenile myelomonocytic leukemia (JMML). Molecular genetic analysis of lymphoblastic blasts at the time of the ALL diagnosis revealed the germline mutation in a heterozygous state, while in the myelomonocytic blasts occurring with JMML diagnosis, the mutation p.E139D was found in a homozygous state due to a uniparental disomy (UPD). These findings lead to the suggestion that the pathogenesis of ALL and JMML in our patient is due to different mechanisms including somatically acquired secondary chromosomal abnormalities.
The molecular pathophysiology of myeloproliferative neoplasms (MPNs) remains poorly understood. Based on the observation that the transcription factor NF-E2 is often overexpressed in MPN patients, independent of the presence of other molecular aberrations, we generated mice expressing an NF-E2 transgene in hematopoietic cells. These mice exhibit many features of MPNs, including thrombocytosis, leukocytosis, Epo-independent colony formation, characteristic bone marrow histology, expansion of stem and progenitor compartments, and spontaneous transformation to acute myeloid leukemia. The MPN phenotype is transplantable to secondary recipient mice. NF-E2 can alter histone modifications, and NF-E2 transgenic mice show hypoacetylation of histone H3. Treatment of mice with the histone deacetylase inhibitor (HDAC-I) vorinostat restored physiological levels of histone H3 acetylation, decreased NF-E2 expression, and normalized platelet numbers. Similarly, MPN patients treated with an HDAC-I exhibited a decrease in NF-E2 expression. These data establish a role for NF-E2 in the pathophysiology of MPNs and provide a molecular rationale for investigating epigenetic alterations as novel targets for rationally designed MPN therapies.
Rationale: Hereditary pulmonary alveolar proteinosis (hPAP) due to granulocyte-macrophage colony-stimulating-factor receptor ?chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signalling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein-degradation in hPAP alveolar macrophages remains poorly understood. Objective: To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cells (PAP-iPSCs) derived monocyte/macrophages. Methods: Patient-specific PAP-iPSCs were generated from CD34+ bone marrow cells of a CSF2RA-deficient PAP patient. We assessed pluripotency, chromosomal integrity and genetic correction to established iPSC lines. Upon hematopoietic differentiation, genetically corrected or uncorrected monocytes/macrophages were investigated in GM-CSF dependent assays. Measurements and Main Results: While monocytes/macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF dependent functions such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation following GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired upon lentiviral gene transfer of a codon-optimized CSF2RA-cDNA. Conclusions: These data establish PAP-iPSC-derived monocytes/macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes/macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to hPAP patients could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.
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