Genome-Wide Analysis of MDS/MPD Disclosed Frequent Homozygous C-Cbl mutations Tightly Associated with 11q-UPD

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 855-855 ◽  
Author(s):  
Sanada Masashi ◽  
Shih Lee Yung ◽  
Takahiro Suzuki ◽  
Motohiro Kato ◽  
Mamiko Yanagimoto Sakata ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Point Mutations ◽  
Loss Of Function ◽  
Hematopoietic Progenitors ◽  
Gain Of Function ◽  
Gene Target ◽  
Genome Wide ◽  
Homozygous Mutations

Abstract Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic progenitors characterized by ineffective hematopoiesis and high propensity to leukemias. Although a number of gene targets have been identified, in many MDS cases, particular genetic targets are unknown. In this study, we performed genome-wide profiling of copy number (CN) abnormalities and allelic imbalances in MDS genomes in order to clarify the distribution of LOH (loss of heterozygosity) and to identify their gene targets. We analyzed a total of 171MDS and MDS/MPD specimens, including 7 RA/RARS, 23 RCMD/RCMD-RS, 6 5q-syndrome, 30 RAEB-1, 40 RAEB-2, 4 therapy related-MDS/AML, 5 MDSu, 17 CMML-1, 16 CMML-2, 24 overt AML, using high-density SNP arrays. The data were analyzed by CNAG/AsCNAR software, which enabled allele-specific CN analysis and sensitive LOH detection. MDS showed characteristic CN profiles in SNP array analysis. Of particular interest is the finding of high frequency of CN-neutral LOH (Uniparental disomy,UPD) observed in 51 of 171 (30%) MDS cases. They preferentially involved 1p, 1q, 4q, 7q, 11q, 17p and other chromosomal segments, which were associated with homozygous mutations of both loss-of-function mutations and gain-of function mutations of tumor suppressor genes and cellular oncogenes, including TP53 (17p UPD), AML1/RUNX1 (21q UPD), Nras and cMPL (1p UPD), JAK-2 (9p UPD), and FLT3 (13q UPD). Next we tried to identify a new gene target in 11q UPD, which was most common UPD region in this study and many of these cases were CMML with a normal karyotype. The minimum 11q UPD segment is about 2Mb which existed in 11q23. We sequenced coding exons of c-cbl and detected homozygous mutations in 8 of 9 MDS cases with 11q UPD (CMML=5, RAEB=3, overt leukemia=1), but very rare in cases without 11q UPD (1/162), demonstrating that the mutation is tightly linked to 11q UPD. These mutations were 8 point mutations and 1 micro-deletion, they were accumulated in the linker or RING domain. These c-cbl mutants transformed NIH3T3 in a dominant fashion, in which they were phosphorylated and activate PI3K-Akt pathway. To investigate the functions of these mutants in hematopoietic cells, we introduced these mutants into c-kit(+)Sca1(+)Lin(−) murine bone marrow cells, it prolonged replating capacity of these hematopoietic progenitors, suggesting involvement of aberrant c-cbl functions in the myeloproliferative phenotypes frequently found in 11q-UPD positive cases. In conclusion, UPD is an important mechanism of development of MDS, in which both gain-of-function and loss-of-function mutations are duplicated with exclusion of wild-type allele. Analysis of 11q UPD disclosed novel gain-of-function mutations. Identification of the targets of UPDs in 1q, 4q and 7q should also be important to gain a novel insight into the pathogenesis of MDS.

Abstract 403: CRISPR/Cas9 genome-wide sgRNA libraries for loss-of-function and gain-of-function genetic screens

2017 ◽  
Author(s):  
Donato Tedesco ◽  
Paul Diehl ◽  
Mikhail Makhanov ◽  
Sylvain Baron ◽  
Alex Chenchik
Keyword(s):  
Loss Of Function ◽  
Genetic Screens ◽  
Gain Of Function ◽  
Genome Wide

An Increased Frequency of Abnormalities Including Uniparental Disomy Is Detected by SNP Array Compared with Conventional Karyotype Analysis in Acute Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 97-97 ◽  
Author(s):  
Manoj Raghavan ◽  
Rosemary E. Gale ◽  
Spyros Skoulakis ◽  
Tracy Chaplin ◽  
Gael Y. Molloy ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosomal Abnormalities ◽  
Germ Line ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Data Set ◽  
Array Data ◽  
The Mean ◽  
Homozygous Mutations ◽  
Acute Myeloid

Abstract SNP array technology permits the simultaneous analysis of copy number and allelotype data. This approach has revealed the somatic acquisition of uniparental disomy (UPD) in approximately 20% of acute myeloid leukemias (AMLs) (Raghavan et al. Cancer Res2005;65:375–378). UPD, which mostly appears to be the consequence of mitotic recombination, cannot be detected by conventional analysis. We have conducted a pilot investigation of samples from the UK MRC AML 10 trial in order to confirm these findings in a larger data set from a large clinical trial. The Affymetrix 10K GeneChip Mapping Array was used to type DNA from 100 AML blast samples of which 87 produced arrays with call rates in excess of 90%. Analysis was performed using the genome orientated laboratory file (GOLF) system, an in-house software package designed to interpret SNP array data. Control germline DNA was not available for each AML and GOLF was used to create a control copy number experiment from the mean of 52 array data sets from normal tissue (blood and remission marrow). The copy number ratio was calculated for each SNP for each sample. In 44 samples the karyotype concurred with the SNP array results (excluding balanced translocations which cannot be detected by SNP array). In the other samples, 54 abnormalities were detected that were not seen in the karyotype (6 samples had no karyotype information). Nine were amplifications, 12 were deletions and 31 were UPDs (35%). Of the UPDs, 12 were either whole chromosome or extended to the most telomeric SNP, with the others therefore being interstitial changes. The mean size of an interstitial LOH was 13.3 Mb, with the smallest detected being 3.2 Mb. Recurrent chromosomal abnormalities not detected by giemsa banding are listed in table 1. Twenty samples that were regarded as normal karyotype by gene banding had abnormalities by SNP array. In three examples numerical karyotypic abnormalities were not seen on SNP array analysis (add4q, −8 and a hypodiploid AML). This may be because a minority clone of the AML cells had this karyotype. UPD is known to be associated with homozygous mutations in AML (Fitzgibbon et al. Cancer Res 2005; in press). In this series, two patients had UPD13, both with biallelic FLT3 internal tandem duplication mutations. However, this study has identified several new areas to look for potential homozygous mutations. Given that in this study germ-line comparison has not been made one should not rule out the possibility of some of the smaller abnormalities being copy number polymorphisms (Bailey et al. Science2005;297;1003–1007). However, consanguinity can probably be ruled out given the lack of widespread homozygosity. This study confirms the frequency of UPD and illustrates the potential of SNP arrays for highlighting novel genetic events in AML. Table 1: Recurrent chromosomal abnormalities not detected by gene banding Previously Undetected Abnormality Number UPD1p 2 Amplified 1p 2 UPD2p 2 Del7q 3 UPD8p 2 UPD11p 2 UPD11q 3 Amplified 12 3 Amplified 13 2 UPD16p 4 UPD16q 2 Del20q 2

Infant ALL Patients Carrying t(4;11) Have a Different Genotypic Profile Than Older ALL Children.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1436-1436
Author(s):  
Michela Bardini ◽  
Lilia Corral ◽  
Eleonora Mangano ◽  
Roberta Spinelli ◽  
Grazia Fazio ◽  
...  
Keyword(s):  
Copy Number ◽  
Critical Role ◽  
Snp Array ◽  
Point Mutations ◽  
Latency Period ◽  
Older Children ◽  
Available N ◽  
Childhood All ◽  
Genome Wide ◽  
The Individual

Abstract Mice models and prenatal studies indicate that in childhood ALL the individual genetic lesions alone are insufficient to generate a full leukemic phenotype, and cooperating oncogenic lesions are required. Recently, multiple genome-wide studies on childhood ALL (1–18 years) identified deletions at several loci, mainly affecting genes that play a critical role in regulating B cell development and differentiation. By contrast, the prenatal and postnatal steps in the pathogenesis of Infant ALL (less than 1 year at diagnosis) are not defined. Infant ALL is a very aggressive disease, with t(4;11)/MLL-AF4 fusion representing the major subgroup. Although the very short latency period suggests that leukemogenic events occur prenatally, mice models indicates that MLL-AF4 alone is not sufficient to induce leukemia, and additional mutations may occur. Also unclear is whether the molecular pathways needed for lymphoid cell differentiation are altered in cases with an MLL rearrangement and, if so, whether these alterations differ between the leukemia of infants and older children. Aim of this study was to detect MLL-cooperating aberrations, undetectable by conventional techniques, by using genome-wide single nucleotide polymorphism (SNP) genome wide analysis (100K SNP human mapping, Affymetrix). More specifically, we searched for Loss of Heterozygosity (LOH) associated or not to copy number alteration. The identification of these lesions could help identifying leukemia pathogenesis, as well as providing the basis for targeted therapy. We have analyzed 28 cases of Infant ALL with t(4;11) at diagnosis and their corresponding samples at remission, when available (n=18). SNP data were analyzed by using dChip software, and confirmed by CNAG 2.0. A more dense SNP array analysis (250K) has been applied in selected cases to confirm LOH and precisely dissect the affected chromosomal regions. Compared to older childhood ALL patients, a far limited number of deletions/amplifications has been found; only 2/28 patients showed deletions, namely 1p36.33-p36.31 in 1 patient and 3p11.1-p12.2 plus 7q22.1-q22.2 in another patient, while 26/28 Infant ALL did not present any visible structural variation. Different from older children, several segmental copy-number neutral (CNN) LOH have been detected by dChip. The extension and prevalence of the affected regions was variable; among them 6p21.32 (4/28 cases), 7q31.33-q32.1 (3/28), 8q21.12-q21.3 (2/28), 8q24.11 (2/28) and 14q21.2 (2/28). Overall, these results confirm that Infant ALL with t(4;11)/MLL-AF4 fusion represents a biologically unique disease, different from other type of leukemia occurring in older children. While in older children a multistep mechanism (with the involvement of several genes) is required for the full leukemic phenotype, MLL rearrangements per se might play a major role on the leukemogenesis. By this approach it could not be excluded that different mechanisms could cooperate with MLL in transforming cells, including point mutations. The functional role of CNN-LOH still needs to be understood: they could either reflect the duplication of oncogenic mutations, or be related to epigenetic mechanisms.

SNP-Array-Based Karyotyping Has Impact on Cytogenetic Diagnosis and Prognosis of Non-Core Binding Factor Primary and Secondary AML.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 597-597
Author(s):  
Ramon V. Tiu ◽  
Lukasz P. Gondek ◽  
Andrew J. Dunbar ◽  
Mikkael A. Sekeres ◽  
Matt E. Kalaycio ◽  
...  
Keyword(s):  
Copy Number Variants ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Point Mutations ◽  
Clinical Value ◽  
Core Binding Factor ◽  
Secondary Aml ◽  
Mutational Status ◽  
Binding Factor ◽  
The Impact

Abstract Non-core binding factor AML has heterogeneous clinical phenotypes, likely due to various modifying genetic lesions (i.e. point mutations such as Flt3, c-kit, or and nucleophosmin). Using metaphase cytogenetics (MC), chromosomal (chr.) abnormalities are found in only 50% of newly diagnosed patients with primary AML (pAML) and secondary AML (sAML) arising from MDS or MPD. Previously, we have demonstrated that SNP arrays (SNP-A) can detect previously cryptic lesions (including uniparental disomy, UPD) and enhance the clinical value of MC in patients with MDS. Here, we hypothesize that SNP-A will improve cytogenetic analysis in AML as well. Our study included 79 healthy control marrows and 103 AML cases; 36 pAML (FAB M0=3, M1=10, M2=10, M4=6, M5=6, M7=1; mean age 53y) and 67 sAML (from MDS, N=40 and MPD/MDS, N=27; mean age 63y). Normal MC was present in 69% and 45% of pAML and sAML, respectively. First, we investigated technical aspects of SNP-A karyotyping. Dilution studies showed that SNP-A can detect clones spanning 25–50% as well as LOH calls >99% of the time as shown X chr. analysis in males. Repetitive/serial testing demonstrated congruent results and somatic derivation of randomly selected lesions was confirmed by microsatellite and SNP-A of non-clonal cells. Copy number variants (CNV) encountered in controls or described in public databases were excluded. Using SNP-A, new cytogenetic abnormalities were found in 52% (28% UPD) and 59% (33% UPD) of pAML and sAML with normal MC, respectively. Moreover, 80% and 88% of pAML and sAML with previously abnormal MC harbored lesions detected by SNP-A. Examples of microdeletions/duplications include regions harboring known leukemia susceptibility genes, such as AML1. Segmental UPD involved regions often affected by deletion, including 5q, 7q, and 11q among others. Results of SNP-A can help characterize recurrent or minimally shared lesions, map their location, or identify causative genes. However, clinical utility of this technology is best demonstrated by the impact of the new defects on survival and other clinical parameters. In both pAML and sAML patients, we found that those with both normal MC and normal SNP-A had a better overall survival (OS) and event-free survival (EFS) as compared to those showing normal MC but abnormal SNP-A. (pAML: OS: p=.04, 21 vs. 5mo; EFS: p=.03, 19 vs. 6mo; sAML: OS: p=.04, 15 vs. 4mo; EFS: p=.04, 10 vs. 4mo). A subset analysis of those sAML patients derived from MDS showed similar results (OS: p=.02, 20 vs. 4mo; EFS: p=.03, 16 vs. 6mo). Most significantly, new lesions detected by SNP-A in AML patients with previously abnormal MC corresponded to a worse prognosis (OS: p=.0004, 10 vs. 3mo). For frequently encountered lesions, we performed survival analysis. For example, the presence of UPD7q negatively affected clinical outcomes (5 patients with UPD7 had equally poor survival to patients with del7/7q, N=10). Subset analyses (e.g., AML with normal MC) also indicated that chr. lesions detected by SNP-A impact stratification schemes independent of known risk factors such as Flt3 mutational status. In summary, SNP-A karyotyping allows for detection of previously cryptic cytogenetic lesions that together with routine MC may aid not only in diagnosis but prognosis in patients with both pAML and sAML.

Rapid Diagnosis of Imprinting Disorders Involving Copy Number Variation and Uniparental Disomy Using Genome-Wide SNP Microarrays

2015 ◽  
Vol 146 (1) ◽  
pp. 9-18 ◽  
Author(s):  
Weiqiang Liu ◽  
Rui Zhang ◽  
Jun Wei ◽  
Huimin Zhang ◽  
Guojiu Yu ◽  
...  
Keyword(s):  
Copy Number ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Copy Number Variations ◽  
Chromosomal Microarray ◽  
Chromosomal Microarray Analysis ◽  
Imprinting Disorders ◽  
Genome Wide ◽  
Number Variation ◽  
Efficient Alternative

Imprinting disorders, such as Beckwith-Wiedemann syndrome (BWS), Prader-Willi syndrome (PWS) and Angelman syndrome (AS), can be detected via methylation analysis, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), or other methods. In this study, we applied single nucleotide polymorphism (SNP)-based chromosomal microarray analysis to detect copy number variations (CNVs) and uniparental disomy (UPD) events in patients with suspected imprinting disorders. Of 4 patients, 2 had a 5.25-Mb microdeletion in the 15q11.2q13.2 region, 1 had a 38.4-Mb mosaic UPD in the 11p15.4 region, and 1 had a 60-Mb detectable UPD between regions 14q13.2 and 14q32.13. Although the 14q32.2 region was classified as normal by SNP array for the 14q13 UPD patient, it turned out to be a heterodisomic UPD by short tandem repeat marker analysis. MS-MLPA analysis was performed to validate the variations. In conclusion, SNP-based microarray is an efficient alternative method for quickly and precisely diagnosing PWS, AS, BWS, and other imprinted gene-associated disorders when considering aberrations due to CNVs and most types of UPD.

Mosaic maternal uniparental disomy of chromosome 15 in Prader-Willi syndrome: Utility of genome-wide SNP array

2012 ◽  
Vol 161 (1) ◽  
pp. 166-171 ◽  
Author(s):  
Kosuke Izumi ◽  
Avni B. Santani ◽  
Matthew A. Deardorff ◽  
Holly A. Feret ◽  
Tanya Tischler ◽  
...  
Keyword(s):  
Snp Array ◽  
Uniparental Disomy ◽  
Chromosome 15 ◽  
Prader Willi Syndrome ◽  
Maternal Uniparental Disomy ◽  
Genome Wide

Genome Wide SNP Analysis Reveals Frequent Cryptic Clonal Chromosomal Aberrations Including Uniparental Disomy (UPD) in Waldenstrom's Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3932-3932 ◽  
Author(s):  
Stephanie Poulain ◽  
Christophe Roumier ◽  
Meyling Cheok ◽  
Sylvie Zoutina ◽  
Agnes Daudignon ◽  
...  
Keyword(s):  
Research Funding ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Tumour Suppressor ◽  
Cgh Array ◽  
Resolution Method ◽  
Genome Wide

Abstract Abstract 3932 Poster Board III-868 Background Waldenstrom's macroglobulinemia (WM) is a rare lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells that secrete monoclonal IgM antibody. Approximately 50% of patients (pts) with WM exhibit a normal karyotype using either conventional chromosome banding analysis (CBA) or FISH approach. However, CBA is a low resolution method and FISH only target previously described abnormalities. Comparative genomic hybridization (CGH) array delineated the minimal deleted region on 6q deletion, the most frequent aberration in WM, and pointed out the role of NFKB pathway key regulators genes. However, tumour suppressor gene or oncogene involved in WM physiopathology is not known, to date. Partial uniparental disomy (UPD) induced by copy neutral loss of heterozygosity (LOH) are important mechanisms for tumour suppressor gene inactivation or oncogene activation in cancer. Recently, Single Nucleotide Polymorphism (SNP) based arrays had been described as a powerful high resolution method allowing both the detection of LOH and copy number alteration (CNA) analysis in the same experiment, a major advantage over CGH array. Our aim was to identify new CNA and LOH involved in WM pathogenesis using SNP arrays analysis. Material and Methods. BM samples of 12 pts with WM (7 males, mean age: 67 years, 7 symptomatic pts) were analysed. DNA was extracted following CD19 B cells selection. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both LOH and CNA. In 6 pts, paired samples (tumor / normal T lymphocytes) were used as an intra-individual reference to identify germline polymorphisms. In the 6 left pts, CN polymorphisms were excluded using copy-number variants database (http://projects.tcag.ca/variation) and the reference genotyping data from the HapMap project. Size, position and location of genes were identified with UCSC Genome Browser HG18 assembly, LOH and CNA using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. FISH analysis was performed to detect deletion 6q; 13q14, 11q22, TP53, trisomy 4 and 12 chromosomal aberrations using Vysis probes. Results SNP array detected 35 CNA (23 gains, 12 losses) in 9/12 pts, with a mean of 2.9 abnormalities per patient (range 0 to 10). 91% of CNA were observed in symptomatic pts. 50% of CNA were < 5 mb in size, the lower limit of detection by CBA. 15 cryptic aberrations were identified among the somatic CNA spread over 7 pts. These 11 gains and 4 losses are identified by *. Deletions were observed on chromosomal segments 1p36*, 3p21.*, 6q16-q27 (2pts), 8p21*, 8p23-q24, 9p21*, 11q22 (2pts), 16p13*, 19p13*. One cryptic homozygous deletion in 13q14 was identified. Gains were observed on chromosomal segments 3p26, 4p16-q35 (3pts), 4q24*, 5p15 (2pts)*, 5p14, 7p22, 12p13-q24, 16p13*, 17q11*, 18p11-q23, 19p13-q13, Xq21* (5pts), Xq22*, Xq25*, Xp22-q28, Xq27-q28*(2pts). We also identified 42 LOH in 9/12 pts (mean of 3.5 per genome, range 0 to 7) dispersed on 17/23 chromosomes. The LOH observed in the absence of CNA loss are consistent with UPD in 35 cases (83%). 59% of LOH were observed in symptomatic pts. The interstitial or telomeric UPD regions varied in size, from 0.4 to 154mb. Two recurrent regions were identified on chromosome 4 (1.2mb involving genes DCLK2 and LRBA) and 13 (0.8mb involving genes KPNA3, ARL11 and SETDB2), probably subsequent to mitotic recombination. No UPD was observed on chromosome 6q. SNP arrays detected all FISH and CBA findings, except in one pt with subclonal deletion of TP53. Of the 5 pts with normal CBA or FISH, SNP array detected a CNA and 4 UPD in one patient and 7 UPD in an other patient. The remaining 3 cases (25%) had no detected CNA or LOH. Overall, SNP array detected a total of 77 genetic aberrations (CNA + LOH) (6.4 per genome, range 0 -16) in this cohort allowing the selection of 976 relevant genes (159 implicated in cellular growth and proliferation, 165 in cancer, 69 in cell cycle). Conclusion New cryptic clonal chromosomal lesions were detected using high resolution SNP array in this study. Several abnormalities were recurrent. We described a high frequency of UPD in WM, that might contribute to the inactivation of tumour suppressor genes by mutations or epigenetic alterations and subsequently to the regulation of tumor progression in WM. Further confirmation of the role of these candidate genes is underway. Disclosures: Leleu: Celgene: Research Funding; Janssen Cilag: Research Funding; Chugai: Research Funding.

scholarly journals Identification of rare de novo epigenetic variations in congenital disorders

2018 ◽  
Author(s):  
Mafalda Barbosa ◽  
Ricky S. Joshi ◽  
Paras Garg ◽  
Alejandro Martin-Trujillo ◽  
Nihir Patel ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Point Mutations ◽  
Ctcf Binding ◽  
Loss Of Function ◽  
Structural Variations ◽  
Genomic Technologies ◽  
Genome Wide ◽  
Rnaseq Data ◽  
Population Controls

AbstractCertain human traits such as neurodevelopmental disorders (NDs) and congenital anomalies (CAs) are believed to be primarily genetic in origin. With recent dramatic advances in genomic technologies, genome-wide surveys of cohorts of patients with ND/CAs for point mutations and structural variations have greatly advanced our understanding of their genetic etiologies1,2. However, even after whole genome sequencing (WGS), a substantial fraction of such disorders remain unexplained3. In contrast, the possibility that constitutive epigenetic variations (epivariations) might underlie such traits has not been well explored. We hypothesized that some cases of ND/CA are caused by aberrations of DNA methylation that lead to a dysregulation of normal genome function. By comparing DNA methylation profiles from 489 individuals with ND/CAs against 1,534 population controls, we identified epivariations as a frequent occurrence in the human genome. De novo epivariations were significantly enriched in cases when compared to controls. RNAseq data from population studies showed that epivariations often have an impact on gene expression comparable to loss-of-function mutations. Additionally, we detected and replicated an enrichment of rare sequence mutations overlapping CTCF binding sites close to epivariations. Thus, some epivariations occur secondary to cis-linked mutations in regulatory regions, providing a rationale for interpreting non-coding genetic variation. We propose that epivariations likely represent the causative genomic defect in 5-10% of patients with unexplained ND/CAs. This constitutes a yield comparable to CNV microarrays, and as such has significant diagnostic relevance.

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