Enhancement of Cytogenetic Diagnosis of Myeloid Disorders through Application of SNP-Array-Based Karyotyping.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 108-108 ◽  
Author(s):  
Lukasz P. Gondek ◽  
Ramon Tiu ◽  
Marcin Wlodarski ◽  
Christine O’Keefe ◽  
Michael McDevitt ◽  
...  
Keyword(s):  
Clinical Relevance ◽  
Germ Line ◽  
Copy Number Variants ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Disease Process ◽  
Normal Karyotype ◽  
Diagnostic Value ◽  
Differential Analysis ◽  
The Impact

Abstract Cytogenetic testing improves diagnosis in myeloid disorders; chromosomal (chr) aberrations have important clinical implications. SNP arrays (SNP-A) can be applied for karyotyping with a superb resolution of unbalanced defects and detection of uniparental disomy (UPD). We stipulated that SNP-A will enhance diagnostic value of metaphase cytogenetics (MC) and uncover new random/recurrent lesions. We applied 250K SNP-A to analysis of 76 controls and 318 patients, including 95 MDS, 64 AA, 20 PNH, 48 MDS/MPD, and AML both as primary (N=32) and secondary (N=59). Multiple samples were obtained in 13 patients. Minimal clonal size detectable by SNP-A was 25–50% by dilution studies. Repetitive testing resulted in congruent results; analysis of chr X in males showed >99% fidelity. To obtain reference, deletions and duplications seen in controls were analyzed. These abnormalities correspond to germ line encoded copy number variants (CNV). In patients such CNV were not deemed pathogenic. SNP-A confirmed 82% of unbalanced chr lesions detected by MC; discordant cases included defects involving smaller clones (<8/20 metaphases) and aberrations of Y. SNP-A allowed for detection of defects in 63% vs. 37% by MC, including 77% vs. 58% in MDS, 75% vs. 37% in MDS/MPD, 33% vs. 0% in AA, 30% vs. 0% in PNH, 59% vs. 31% in AML and 76% vs. 53% in sAML. New lesions were confirmed by paired SNP-A and microsatellite analysis. Concurrent analysis of blood and marrow showed concordant results suggesting utility of SNP-A performed on blood. Serially followed patients N=6, showed occurrence of new lesions (del(4)(q) and del(7)(q)) and earlier detection of the chr aberrations. In sAML, differential analysis of blasts and granulatocytes revealed occurrence of new lesions e.g., UPD6 or 7. In both MDS and AML, UPD of various chrs was present in 20% of patients and found in up to 35% of MDS/MPD (in addition to 9p involving also chrs 6,7,11 & 14). Other newly detected lesions included isolated/recurrent microdeletions and duplications involving genes such as AML1 or Ftl3 among others. Clinical utility of SNP-A depends on whether SNP-A karyotypig will have impact on disease parameters. In all groups tested the newly detected lesions showed impact on overall survival. While the detailed results will be a subject of our presentation, survival analysis in AML can illustrate our point; cases with a normal karyotype showed superior OS to those with newly detected defects (21 vs. 6 mo, p=.05). Similarly, new additional lesions worsen the survival as compared to those with confirmed MC (3 vs. 10 mo, p=.004). The impact on OS was also established for some of the new recurrent lesions such as UPD7q (3 vs. 39 mo, p=.002). Clinical relevance of SNP-A karyotyping is also demonstrated in AA; it may help to distinguish AA from hypocellular MDS (clonal chr. defects, including UPD, occur in 33% of AA patients), AA with normal SNP-A testing showed superior response to immunosuppression as compared to patients with a totally normal karyotype. Aside of the clinical relevance, new overlapping/recurrent lesions point towards genes involved in the disease process. We conclude that SNP-A karyotyping may enhance MC in diagnosis of chr. defects and allow for a better clinical correlations of the defects with the phnenotypic and clinical features.

Uniparental Disomy May Be Associated with NPM Mutations in AML with a Normal Karyotype.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2310-2310
Author(s):  
Elena Serrano ◽  
Vanesa Orantes ◽  
Camino Estivill ◽  
Adriana Lasa ◽  
Salut Brunet ◽  
...  
Keyword(s):  
Complete Remission ◽  
Copy Number ◽  
Germ Line ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Normal Karyotype ◽  
Copy Number Analysis ◽  
Complex Germ ◽  
Flt3 Mutations ◽  
Tandem Duplications

Abstract Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by an abnormal proliferation of the myeloid precursors and a maturation block. A large proportion of AML cases have either a normal karyotype or non-recurrent chromosomal alterations. Underlying genetic lesions of some of these cases have been characterized with the discovery of MLL-internal tandem duplications, activating FLT3 mutations and NPM mutations. Loss of heterozygosity (LOH) derives from the loss of one of the two alleles at a given locus and can be a sign of inactivation of tumor-suppressor genes. We performed a high-resolution genotype analysis on DNA obtained from 19 AML patients with a normal karyotype, both at diagnosis and in samples obtained in complete remission(assessed by multiparametric flow cytometry) using the 10K SNP Array (Affymetrix). Both LOH and copy number analysis, as well as visualization of these analysis were performed by means of the dChip software (M. Lin et al., Bioinformatics (2004), 20:1233–40). A mean call rate of 96.8%. SNP array-based LOH analysis revealed that 4 patients presented large regions of homozygosity at diagnosis which were absent from samples in complete remission. In all four patients copy number analysis indicated no gross chromosomal losses or gains, as was confirmed by conventional cytogenetic analysis. Therefore, it can concluded that the LOH observed in these four patients was due to the presence of uniparental disomy. Simultaneous analysis of FLT-3 internal tandem duplications (FLT-3/ITD), FLT3- D835 mutations, NPM mutations and MLL rearrangements was performed using conventional molecular methods. Two of these patients (UPN2 and UPN12) had FLT-3/ITD in association with NPM mutations. UPN4 had a mutated form of NPM whereas in patient UPN16 FLT-3 and NPM genes were in the germ line configuration. All four cases were negative for MLL rearrangements and FLT-3-D835 mutations. These results suggest that NPM and FLT3 mutations may be associated with acquired somatic recombinations. It remains to be investigated whether there are loci preferentially involved by these events. Uniparental disomy and genetic lesions in normal karyotype AML Patient LOH FLT3 NPM D835 MLL UPN2 13q Mutated Mutated Germ line Germ line UPN4 6pter-p12.212q13.12-qter Germ line Mutated Germ line Germ line UPN12 2p Mutated Mutated Germ line Germ line UPN16 complex Germ line Germ line Germ line Germ line

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.

Constitutional Segmental Uniparental Disomy in a Twin Pair with t(12;21) Positive Acute Lymphoblastic Leukemia Characterized by the Same Prenatal Clone and Divergent Clonal Evolution.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4549-4549
Author(s):  
Giovanni Cazzaniga ◽  
Julie Irving ◽  
Marco Citterio ◽  
Silvia Bungaro ◽  
Roxane Tussiwand ◽  
...  
Keyword(s):  
Germ Line ◽  
Twin Pair ◽  
Rapid Determination ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Monozygotic Twin ◽  
Nucleotide Polymorphisms ◽  
Overt Leukemia

Abstract We previously reported (Tussiwand R. et al., ASH 2002) the case of a monozygotic twin pair with concordant ALL aged 3.0 years at diagnosis, with only 13 days difference in latency. Twin 1 was classified as pre-B ALL and twin 2 as common-ALL, based on standard immunophenotyping criteria. A large screening for TcR and Ig gene rearrangements was performed, resulting in only one common VKII-Kde rearrangement, the others being not related to each others. Highly sensitive RQ-PCR was performed for all markers in both twins. The result of the crossed analysis was consistent with the hypothesis that after a prenatal event resulting in a preleukemic clone, at least a second independent event must have occurred before overt leukemia. To further identify markers of the clonal evolution, high-resolution single nucleotide polymorphisms (SNP) genotype analysis was performed on the DNA using the 10K SNP array (Affymetrix). Remission bone marrow was taken as a germ line samples. Array-based analysis of SNPs allows the rapid determination of genome-wide allelic information at high density, including the identification of submicroscopical copy number changes and/or loss of heterozygosity (LOH). SNP array analysis have been so far successfully applied to demonstrate allelic imbalance in ALL and AML blasts. A 12p12-13 deletion was observed on twin 2. The deletion of the TEL allele not involved in the t(12;21) was confirmed in twin 2 by FISH and microsatellite analyses. The twin 1 did not show any 12p deletion. Similar observations have been made in late-relapses occurring in ALL carrying the t(12;21) translocation: the predominant clone did not correspond to the same clone observed at diagnosis, but represented a second, independent transformation event within the fetal pre-leukemic clone, even when in the presence of the same genetic background. More interestingly, we found by SNP array a 13Mb area of LOH in remission and presentation samples of both twins involving the 2q13-14.3 region. As further confirmed by FISH with 2q probes, LOH was not associated with chromosomal loss, implying a recombination event resulting in Uniparental Isodisomy (UPD). The UPD area includes 57 known genes, several of them implicated in oncogenesis; they include translin, a gene involved in the control of chromosomal translocation and implicated in lymphoid malignancy. UPD of this region has not been reported in other tumors or in remission samples of leukemia; in genetic diseases few cases have been reported with maternal or paternal UPD 2, never associated with haematological disorders. By contrast, it has been shown that a transmitted deletion of 2q13 to 2q14.1 causes no phenotypic abnormalities. This is the first report on constitutional UPD in leukemia patients. Further analyses are necessary to understand the clinical meaning of this chromosomal abnormality; one hypothesis could be that the twins were born with a genetic predisposition to develop leukaemia. In this context, t(12;21) and additional events (i.e. TEL deletion) may be responsible for the overt leukemia.

scholarly journals Prenatal Diagnosis of Complete Paternal Uniparental Isodisomy for Chromosome 3: A Case Report

2021 ◽  
Author(s):  
xiufen bu ◽  
Xu Li ◽  
Shihao Zhou ◽  
Liangcheng Shi ◽  
Xuanyu Jiang ◽  
...  
Keyword(s):  
Genetic Counseling ◽  
Prenatal Diagnosis ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Rare Condition ◽  
Normal Karyotype ◽  
Chromosome 3 ◽  
Uniparental Isodisomy ◽  
Normal Ultrasound ◽  
Physical Findings

Abstract Background Paternal uniparental disomy (UPD) of chromosome 3 is a very rare condition. At present, only 5 cases of paternal UPD(3) has been reported. This was the second ascertained paternal UPD(3) with no apparent disease phenotype.Case presentation We hereby reported a case of a fetus with normal karyotype and normal ultrasound features at the whole gestation. A copy neutral regions of homozygosity on chromosome 3 was indentified by Single Nucleotide Polymophism array (SNP array). Subsequent SNP array data of parent–child trios showed the fetus has carried complete paternal uniparental isodisomy (isoUPD) of chromosome 3. The parents decided to continue the pregnancy after genetic counseling. The neonate had normal physical findings at birth and develops normally after 1.5 years. Conclusions The findings could provide further evidence to confirm that there was no important imprinted genes causing serious diseases on paternal chromosome 3 and provided a reference for the prenatal diagnosis and genetic counseling of UPD(3) in the future.

Use of SNP-array-based karyotyping for cytogenetic prognostication in unclassified cases of myelodysplasia and associated overlap disorders

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7016-7016 ◽  
Author(s):  
B. Bhatnagar ◽  
R. V. Tiu ◽  
L. P. Gondek ◽  
C. O'Keefe ◽  
J. Huh ◽  
...  
Keyword(s):  
Chromosomal Abnormalities ◽  
Neutral Loss ◽  
Copy Number Variants ◽  
Snp Array ◽  
Normal Karyotype ◽  
Myeloproliferative Disorders ◽  
Categorical Variables ◽  
International Prognostic Scoring System ◽  
Exact Test ◽  
Chromosomal Defects

7016 Background: Myeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS) often have overlapping features resulting in unclassifiable cases (MDS-U and MDS/MPD-U). Chromosomal abnormalities impact prognosis, but 50% of cases show normal karyotype by metaphase cytogenetics (MC). Single nucleotide polymorphism arrays (SNP-A) are novel karyotyping tools with superior resolution and ability to detect copy neutral loss of heterozygosity, a defect not detected by MC. Methods: MDS-U (N = 17) and MDS/MPD-U (N = 61) patients were selected from an MDS database (N = 720, median age = 76, median follow-up = 42 mos). SNP-A was performed on 67 patients and 751 controls. An algorithm for identification of somatic lesions was designed: 1) Lesions detected by MC and SNP-A required no further analysis; 2) Micro-duplications/ deletions overlapping with copy number variants (CNV) were excluded. Lesions not in CNV databases were confirmed by CD3 lymphocytes; 3) UPD <25 Mb were unlikely somatic and excluded. Telomeric and interstitial (≥ 25 Mb) UPD were considered somatic. International Prognostic Scoring System (IPSS) was used to assess routine risk. Fisher's exact test was used for categorical variables. Overall (OS) and event-free (EFS) survival defined by the MDS working group criteria were analyzed by Kaplan Meier analysis (log-rank or Wilcoxon and 2-sided significance). Results: SNP-A yielded superior detection rate for chromosomal defects compared to MC (71% vs 47%, p = 0.008). UPD was seen in 17 patients and frequently involved chromosomes 1, 3, 6, 8, 11, 17. MDS/MPD-U and MDS-U patients had similar OS and EFS (OS = 42 vs. 45 mos, p = 0.13; EFS = 42 vs. 45 mos p = 0.63). SNP-A revealed a more complex karyotype in patients with advanced MDS. Furthermore, SNP-A karyotyping resulted in prognostic refinement of previously assigned IPSS: Unclassified cases = 6% versus 0%, int-1 = 45% versus 53%, int-2 = 6% versus 19%, high = 5% versus 8%. Overall, patients with new SNP-A lesions had worse OS and EFS (OS = 41 mos vs NR, p = 0.07; EFS = 32 vs 112 mos, p = 0.07). Conclusions: SNP-A karyotyping complements MC in detecting chromosomal defects in MDS-U and MDS/MPD-U. This technology will be helpful in refining diagnosis based on characteristic recurrent chromosomal lesions including UPD. No significant financial relationships to disclose.

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

Analysis of Loss of Heterozygosity in Adult Patients with De Novo Acute Myeloid Leukemia and Normal Karyotype.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 806-806 ◽  
Author(s):  
Christian Schon ◽  
Lars Bullinger ◽  
Frank G. Rucker ◽  
Konstanze Dohner ◽  
Hartmut Dohner
Keyword(s):  
Acute Myeloid Leukemia ◽  
Signal Intensity ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Normal Karyotype ◽  
Intensity Data ◽  
Chromosome 13 ◽  
Acute Myeloid

Abstract A large proportion of acute myeloid leukemia (AML) exhibits a normal karyotype in which the underlying pathomechanisms still have to be determined. Novel techniques like arrayCGH or single nucleotide polymorphism (SNP) chip analysis allow the identification and characterization of molecular rearrangements at the sub-megabase level. Recently, the application of genome-wide SNP array technology revealed frequent uniparental disomy (UPD) in approximately 20% of AML suggesting that UPD represents a nonrandom event in leukemogenesis. Uniparental disomy is acquired by somatic recombination and therefore not accessible by conventional cytogenetic methods or arrayCGH. In this study we analyzed DNA from AML patients with normal karyotype for the presence of LOH. SNP analysis was performed on the Mapping 100k GeneChip (Affymetrix, Santa Clara, CA). DNA was extracted from paired samples of 56 de novo AML patients with normal karyotype at diagnosis and in complete remission, respectively. Signal intensity data were analyzed by the GCOS GeneChip analysis software and statistical analysis of SNP call data was performed by the dChipSNP software. In addition, standard mutation screening of the genes encoding NPM1, FLT3, CEBPA, MLL and NRAS was performed in all cases. Using the 100k SNP array, a mean SNP call rate of 98.2% was reached, resulting in &gt; 110,000 SNP genotype calls per sample. Signal intensity data analysis revealed submicroscopic chromosomal deletions resulting in hemizygosity in three patients. Patient 1 had a single 2 Mb deletion in chromosomal band 3p14.1, patient 2 had two small deletions affecting chromosome 12q23 and 12p13, the latter encompassing the ETV6 locus, and patient 3 had two small deletions within the long arm of chromosome 8. Besides these small chromosomal regions of copy number alterations, we found 4 large stretches of somatically acquired homozygosity without numeric alterations, affecting chromosome 6 (6p21 to 6 pter and 6q26 to 6 qter), chromosome 11 (11p12 to 11pter) and chromosome 13 (13q11 to 13qter). Noteworthy, in the case with uniparental disomy of chromosome 13, we could detect a homozygous FLT3-ITD mutation, supporting the findings that acquired isodisomy for chromosome 13 is common in AML, and associated with FLT3-ITD mutations (Griffiths et al., Leukemia, 2005). In summary, high resolution SNP assay technology in AML patients with normal karyotype allowed the identification of distinct chromosomal regions affected by UPD, supporting the postulated nonrandom mechanism of acquired mitotic recombination events in AML. Besides known chromosomal regions known to be affected by genomic aberrations in AML, we found additional submicroscopic chromosomal aberrations in cases with normal karyotype. Analysis of larger patient series will allow the identification of novel regions of interest harboring genes that might be involved in the pathogenesis of AML.

Molecular Diversity Detected by Whole Exome Sequencing in Chronic Myelomonocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 310-310
Author(s):  
Basel Rouphail ◽  
Kenichi Yoshida ◽  
Holleh D Husseinzadeh ◽  
Edward P Evans ◽  
Satoru Miyano ◽  
...  
Keyword(s):  
Research Funding ◽  
Germ Line ◽  
Target Therapy ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Gene Mutations ◽  
Molecular Pathogenesis ◽  
Common Mutation ◽  
Mutational Status ◽  
Recurrent Mutations

Abstract Abstract 310 CMML is characterized by monocytic proliferation, cytomorphologic dysplasia, and frequent progression to AML. Heterogeneity in or subtlety of presentation can make diagnosis challenging. Recent advances in molecular technology set the stage for systematic study of genetic and genomic lesions associated with CMML. Initially, RAS and RUNX1 mutations were identified in CMML; subsequently, mutations in TET2, CBL, ASXL1 and EZH2 have been discovered. Most recently, recurrent mutations in various genes of the spliceosomal machinery have been added, with SRSF2 likely the most common mutation in this condition. We hypothesized that more precise analysis of molecular lesions in CMML may allow for better categorization of this condition according to molecular pathogenesis and may provide clues to target therapy of this rather refractory condition. We identified 136 patients with CMML or secondary AML (sAML) with antecedent CMML: 87 CMML-1, 20 CMML-2 and 29 post-CMML sAML. The original cohort has been expanded by an additional 53 patients since first reported. The mean follow up period was 16 months (range, 0–114). Abnormal cytogenetics were found in 50% of the cohort by both metaphase and SNP-array-based karyotyping. In a representative subset of 27 patients, we have applied whole genome sequencing (WES) for which paired tumor/germ line DNA was used. To minimize false positives and focus on the most prevalent/relevant somatic events, we implemented a rational bioanalytic filtering approach and results were aligned using Burrows-Wheeler Aligner and variants detected using the GATK pipeline (Best Practice Variant Detection from Broad Institute). We focused on somatic defects with a frequency of >5% of the cohort. For the most commonly affected genes, results were validated using an expanded panel of 18 genes in 72 additional patients and, thus, for the most relevant genes a cohort of 95 patients was studied. The most frequently mutated genes were TET2 (48%), SRSF2 (35%), ASXL1 (17%) and RUNX1 (17%), whereas CBL (13%), EZH2 (13%), UTX (8%) and U2AF1 (8%), SETPB1 (10%), and RIT1 (9%) were less frequent. We also found TP53 and RUNX1 mutations in 5% and 16% of patients, respectively. A JAK2 V617F mutation was present in one case of seemingly typical CMML. BCOR and STAG2 mutations were found in 13% and 9% of patients, respectively; KRAS/NRAS mutations were in 10%. Spliceosomal gene mutations seem to be mutually exclusive, but were frequently associated with other non-spliceosomal gene mutations examined. Within the cohort of 28 SRSF2 mutant cases, 15 had coexisting TET2 mutations, 22 had ASXL1 mutations, 7 had RUNX1 and 5 had CBL mutations. Among 10 U2AF1 mutant cases, 3, 5 and 2 had TET2, ASXL1, and RUNX1 mutations, respectively. SETBP1 mutations were present in 34% of CMML-1/2 and frequently associated with RUNX1, SRSF2, CBL (approximately 2% each) and ASXL1 (4%) mutations. Cohesin mutations were less frequent (10%) because RAD21 and SMC mutations were absent. Mutations of PTPN11 and NF1 were less frequent in adult CMML than those reported in JMML. We also identified several less-recurrent gene mutations that likely modify pathogenesis or clinical outcomes of specific cases. Serial studies performed on 6 cases showed insight into the clonal architecture, producing a series of putative ancestral and secondary events, including uniparental disomy and acquisition of KRAS/NRAS or SETPB1 mutations. Association between mutational status and overall survival (OS) was assessed using Kaplan-Meier statistics. While all permutations were tested, we highlight here only significant positive and relevant negative results. In the whole cohort, presence of CBL mutations conferred worse OS (p=.018; HR 2.44, 95%CI 1.18–4.69). Median OS was 16 months for CMML-1, 6 months for CMML-2, and 14-months for sAML. In subgroup analyses, CBL mutations were also significant worse prognostic factor in CMML-1 cohort (p=.037; HR 3.23, 95%CI 1.07–8.04). In sum, WES provides intricate information on the molecular pathogenesis of CMML and the wide mutational spectrum correlates with the clinical diversity. Expert-based analysis of the genomic data may be supplanted by unsupervised and unbiased approaches which would cluster patients based on molecular similarities. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Makishima:Scott Hamilton CARES Initiative: Research Funding.

scholarly journals Inherited unbalanced translocation (4p16.3p15.32 duplication/8p23.3p23.2deletion) in the four generation pedigree with intellectual disability/developmental delay

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Dongmei Hao ◽  
Yajuan Li ◽  
Lisha Chen ◽  
Xiliang Wang ◽  
Mengxing Wang ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Intellectual Disability ◽  
Developmental Delay ◽  
Copy Number Variants ◽  
Snp Array ◽  
Normal Karyotype ◽  
Clinical Syndrome ◽  
Chromosome 8 ◽  
Fish Analysis ◽  
Balanced Translocation

AbstractChromosomal copy number variants (CNVs) are an important cause of congenital malformations and mental retardation. This study reported a large Chinese pedigree (4-generation, 76 members) with mental retardation caused by chromosome microduplication/microdeletion. There were 10 affected individuals with intellectual disability (ID), developmental delay (DD), and language delay phenotypes. SNP array analysis was performed in the proband and eight patients and found all of them had a microduplication of chromosome 4p16.3p15.2 and a microdeletion of chromosome 8p23.3p23.2. The high-resolution karyotyping analysis of the proband had unbalanced karyotype [46, XY, der(8)t(4;8)(p15.2;p23.1)mat], his mother had balanced karyotype [46, XX, t(4;8) (p15.2;p23.1)], whereas his father had normal karyotype [46,XY]. Fluorescence in situ hybridization (FISH) analysis further confirmed that the proband’s mother had a balanced translocation between the short arm terminal segment of chromosome 4 and the short arm end segment of chromosome 8, ish t(4;8)(8p + ,4q + ;4p + ,8q +). In conclusion, all the patients inherited chromosomes 8 with 4p16.3p15.2 duplication and 8p23.3p23.2 deletion from their parental balanced translocation, which might be the cause of the prevalence of intellectual disability. Meanwhile, 8p23.3p23.2 deletion, rather than 4p16.3p15.2 duplication might cause a more severe clinical syndrome.

Genome-Wide Micro-Deletions and Amplifications Acquired at Relapse in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 166-166 ◽  
Author(s):  
Manoj Raghavan ◽  
Manu Gupta ◽  
Tracy Chaplin ◽  
Sabah Khalid ◽  
T. Andrew Lister ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Normal Karyotype ◽  
P Value ◽  
Copy Number Changes ◽  
Acute Myeloid

Abstract Abstract 166 Recurrence of acute myeloid leukemia AML has a poor prognosis with only 20% of adults surviving to 5 years. Therefore it is of importance to identify molecular changes that explain the pathogenesis of relapsed AML. Previous studies had not identified consistently acquired cytogenetic changes at relapse. Recently, acquired uniparental disomy due to mitotic recombination was described in 40% of relapsed AML (Raghavan et al 2008). Most of the events lead to homozygosity for FLT3 mutations. This study aimed to discover if there are further genetic abnormalities acquired at disease recurrence that cannot be identified by conventional cytogenetics, i.e. microdeletions or gains. Twenty-one presentation and relapse paired AML patient blood and marrow samples were stored with consent at St Bartholomew's Hospital, London. Eleven patient samples had a normal karyotype at diagnosis, two had favourable prognosis cytogenetics (inv(16) and t(8;21)) and others had varying numerical cytogenetic abnormalities and rearrangements associated with an intermediate prognosis. DNA from the samples was analysed by array based high-resolution single nucleotide polymorphism (SNP) genotyping (Affymetrix Human SNP array 6.0). Data was analysed using Partek Genome Browser (Partek, MO). In all cases, the leukemia infiltrate of the marrow or blood was greater than 60% and most cases were greater than 90% allowing accurate identification of DNA copy number changes. Abnormalities of a size that would be identified by cytogenetics were disregarded. Using segmentation analysis using a p-value less than 0.001, over 400 microdeletions and gains were detected that were acquired at relapse in the 21 pairs. Each of the copy number changes was less than 2 megabases in size. One AML sample with a normal karyotype at diagnosis and trisomy 8 and add(9)(q34) at relapse had not acquired any microdeletions or gains. In contrast, in other samples as many as 69 microdeletions/gains were detected. There was no correlation between increased complexity of the karyotype of the leukemia and the number of microdeletions/gains. Several of the acquired microdeletions/gains were in regions containing genes known to be involved in AML, including a deletion of 234Kb at 13q12.2 involving FLT3 and CDX2, and an acquired deletion at 21p11.2 of 150Kb involving exons encoding the runt domain of RUNX1. Another copy number gain was detected at the MLL locus, suggestive of partial tandem duplication. Other detected locations are in Table 1.Table 1Location by cytobandCopy number changeSize / KbP valueGene13q12.2Deletion23410−33FLT3, CDX221q22.12Deletion15010−13RUNX111q23.3Gain5.10.0099MLL11p15.4Gain830.00001NUP9817q21.31Deletion8.00.0007BRCA1The results indicate that recurrent AML may be associated with the deletion or gain of several genes involved in leukaemogenesis. Many other locations are involved throughout the genome, suggesting at least some of these are also involved in the clonal evolution of the leukaemia at recurrence. Further studies should identify novel genes from these regions involved in the pathogenesis of AML. Disclosures: No relevant conflicts of interest to declare.

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