Heterogeneous lengths of copy number mutations in human coagulopathy revealed by genome-wide high-density SNP array

Abstract Sézary Syndrome (SS) is a rare and aggressive form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving blood and skin and whose etiology and molecular pathogenesis are still unclear. Conventional cytogenetics studies have shown that most SS patients have chromosome aberrations; however allelotyping studies and genome-wide surveys for chromosome imbalances in this tumour are still very limited (Mao X. et al. 2003 Genes Chromosome Cancer 36:250–260). High-density single nucleotide polymorphism (SNP) arrays allow high-resolution and genome-wide detection of both loss of heterozygosity (LOH) and copy number (CN) abnormality. Therefore we used Affymetrix 10K SNP mapping array containing 11,560 tiled SNPs to investigate genomic aberrations of 13 individuals affected by SS. Genotype calls and signal information were obtained using GeneChip Operating Software (GCOS 1.4) and GeneChip DNA Analysis Software (GTYPE4.0). SNP calls were exported to be analysed with DNA-chip Analyser (dChip v1.3+) genotyping software which allows the simultaneous measurement of DNA copy number changes and LOH events (Zhao X. et al. 2004 Cancer Res. 64:3060–71; Lin M et al. 2004 Bioinformatics 20:1233–40). Our findings indicate that chromosomes 17p, 10/10q and 9 are most frequently affected by LOH events, while gains of CN were observed more often for chromosome 17q and 8/8q. Among our patients almost all individuals showing loss of the 17p arm have also gain of the 17q, suggesting the presence of the isochromosome 17q, a frequently reported abnormality in SS. In addition to this, we characterised the chromosome LOH pattern identifying seven regions of significant loss shared by multiple tumours. Sample clustering based on significant LOH regions identified two groups of patients: one of them consists of 4 patients with a lower rate of chromosomal losses while the other contains 9 patients mainly characterised by the co-occurrence of LOH at chromosome 17 and chromosome 10. The frequency and pattern of chromosomal changes in our group of 13 SS patients are in substantial agreement with previously described results using more conventional techniques, demonstrating the feasibility of the 10K SNP mapping array system to assess allelic imbalance in this tumour. The genome-wide approach and SNP high density allowed the identification of a larger number of LOH regions, including, however, those already described (chromosome 9p, 10q and 17p). Even though no significant statistical association can be observed due to the low number of cases available, we observed a lower overall survival in the group of 9 patients showing simultaneous LOH events at chromosome 17 and 10.

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Genome-wide copy number profiling using high-density SNP array in chickens

Animal Genetics ◽

10.1111/age.12267 ◽

2015 ◽

Vol 46 (2) ◽

pp. 148-157 ◽

Cited By ~ 12

Author(s):

G. Yi ◽

L. Qu ◽

S. Chen ◽

G. Xu ◽

N. Yang

Keyword(s):

Copy Number ◽

Snp Array ◽

High Density ◽

Genome Wide

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Genome-wide detection of copy number variation in Chinese indigenous sheep using an ovine high-density 600 K SNP array

Scientific Reports ◽

10.1038/s41598-017-00847-9 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 16

Author(s):

Qing Ma ◽

Xuexue Liu ◽

Jianfei Pan ◽

Lina Ma ◽

Yuehui Ma ◽

...

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Snp Array ◽

High Density ◽

Indigenous Sheep ◽

Genome Wide ◽

Number Variation

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A high-density SNP genome-wide linkage search of 206 families identifies susceptibility loci for chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2007-05-091561 ◽

2007 ◽

Vol 110 (9) ◽

pp. 3326-3333 ◽

Cited By ~ 53

Author(s):

Gabrielle S. Sellick ◽

Lynn R. Goldin ◽

Ruth W. Wild ◽

Susan L. Slager ◽

Laura Ressenti ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Familial Aggregation ◽

Susceptibility Gene ◽

Snp Array ◽

High Density ◽

Lymphocytic Leukemia ◽

Nonparametric Model ◽

Model Free ◽

Genome Wide ◽

A Genome

Abstract Chronic lymphocytic leukemia (CLL) and other B-cell lymphoproliferative disorders display familial aggregation. To identify a susceptibility gene for CLL, we assembled families from the major European (ICLLC) and American (GEC) consortia to conduct a genome-wide linkage analysis of 101 new CLL pedigrees using a high-density single nucleotide polymorphism (SNP) array and combined the results with data from our previously reported analysis of 105 families. Here, we report on the combined analysis of the 206 families. Multipoint linkage analyses were undertaken using both nonparametric (model-free) and parametric (model-based) methods. After the removal of high linkage disequilibrium SNPs, we obtained a maximum nonparametric linkage (NPL) score of 3.02 (P = .001) on chromosome 2q21.2. The same genomic position also yielded the highest multipoint heterogeneity LOD (HLOD) score under a common recessive model of disease susceptibility (HLOD = 3.11; P = 7.7 × 10−5), which was significant at the genome-wide level. In addition, 2 other chromosomal positions, 6p22.1 (corresponding to the major histocompatibility locus) and 18q21.1, displayed HLOD scores higher than 2.1 (P < .002). None of the regions coincided with areas of common chromosomal abnormalities frequently observed in CLL. These findings provide direct evidence for Mendelian predisposition to CLL and evidence for the location of disease loci.

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Can Genomic Copy Number Variants Be a Part of Complex Genetic Traits Predisposing to Marrow Failure?.

Blood ◽

10.1182/blood.v110.11.106.106 ◽

2007 ◽

Vol 110 (11) ◽

pp. 106-106

Author(s):

Christine O’Keefe ◽

Lukasz Gondek ◽

Marcin Wlodarski ◽

Judith Karp ◽

Michael McDevitt ◽

...

Keyword(s):

Copy Number ◽

Complex Disease ◽

Disease Susceptibility ◽

Disease Risk ◽

Global Analysis ◽

Copy Number Variants ◽

Snp Array ◽

High Density ◽

Genetic Traits ◽

Disease Predisposition

Abstract Individual variability, including disease susceptibility, is determined by the interaction of inherited single base differences (single nucleotide polymorphisms, SNPs) and copy number variants (CNVs) of large genomic regions. A complex combination of these factors may result in a genetic background predisposing to disease. Regions of CNV account for approximately 12% of the human genome, including coding sequences and can range in size from kilobases to megabases. Recent studies have investigated the correlation between CNVs and complex conditions, including mental retardation, lupus and cardiovascular disease. While SNPs have been intensely investigated in many diseases, the influence of CNVs on disease susceptibility is only poorly understood. With the advent of high-throughput, high density array technology, global analysis of complex disease predisposition traits, including CNVs, can be performed. We have applied high-density SNP arrays (SNP-A) for the analysis of somatic chromosomal defects in various hematologic disorders. During our studies we noted a high frequency of germ-line CNVs, complicating our analysis of somatic defects. This observation lead us to the hypothesis that CNVs can themselves constitute predisposition factors to disease and chose to systematically investigate their type and frequency in myeloid disorders including aplastic anemia (AA; N=65), myelodysplastic syndrome (MDS; N=145) and primary and secondary (non-core binding factor) acute myeloid leukemia (AML; N=75). We performed whole genome scanning in patients and a cohort of healthy controls (N=79) using the Affymetrix 250K SNP array. We first identified and catalogued CNVs in controls; their frequency was compared to those reported in the Database of Genomic Variants (http://projects.tcag.ca/variation/) and found to be similar. The CNVs ranged in size from 245.6 Kb to 2.32 Mb (average 805.9 Kb) and were identified on all chromosomes except 5, 13, 16, 18 and 21. We next analyzed copy number changes in patients with myeloid disorders. Using controls (both our cohort and those in the literature) as a reference we determined the frequencies of recurrent CNVs in patients. For most of the CNVs the frequency was <10% within the individual patient groups, similar to what was seen in controls. Nonetheless, four regions (2 distinct loci in the pericentromeric region of 14q, pericentromeric 15q and a locus on 17q21.31) were identified in over 15% of samples studied. We then determined whether a distinct CNV is associated with specific disease risk. While for most CNVs the frequencies found in patients were similar to those in controls, two regions, 3q29 and 14q11.2, were more frequently encountered in patients with AML (3q29, 27/75 vs. 13/79 in controls, p=0.01; 14q11.2, 20/75 vs. 8/79 in controls, p=0.014). The region at 3q29 contains several genes and is a common breakpoint region for hematologic malignancies including MDS and AML, suggesting that this chromosomal area sensitive to physical rearrangement. The locus at 14q11.2 is a known hypervariable region, containing T cell receptor genes. In sum, in addition to SNPs, CNVs may be a part of complex genetic traits in patients with AA, MDS and AML and constitute disease predisposition factors. Beyond their potential role in disease, CNVs have to be excluded in SNP array-based analysis of somatic chromosomal lesions.

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Genome-Wide DNA Copy Number Analysis by SNP Arrays of B-Cell Chronic Lymphocytic Leukemia: Correlation with Known Biological and Molecular Prognostic Markers.

Blood ◽

10.1182/blood.v112.11.1061.1061 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1061-1061

Author(s):

Laura Mosca ◽

Sonia Fabris ◽

Giovanna Cutrona ◽

Luca Agnelli ◽

Serena Matis ◽

...

Keyword(s):

High Resolution ◽

Copy Number ◽

Prognostic Markers ◽

Snp Array ◽

Lymphocytic Leukemia ◽

Array Analysis ◽

Genome Wide ◽

Trisomy 12 ◽

Snp Array Analysis ◽

Cell Chronic Lymphocytic Leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is a genetically heterogeneous disease with a variable clinical course. Chromosomal changes have been identified by FISH in approximately 80% of patients, and the presence of specific lesions, such as trisomy 12 and 13q14, 11q23, 17p13.1 and 6q23 deletions represent prognostic markers for disease progression and survival. In order to characterize further the complexity of B-CLL genomic lesions, we performed high density, single nucleotide polymorphism (SNP) array analysis in highly purified neoplastic cells (>92%) from a panel of 100 untreated, newly diagnosed patients (57 males and 43 females; age, median 63 years, range 30–87) in Binet stage A. All patients were investigated by FISH for the presence of trisomy 12 (21 cases); 13q14 deletion (44 cases, 34 as the sole abnormality); 11q22.3, 17p13.1 and 6q23 (15, 7 and 2 patients, respectively). In addition, ZAP-70 and CD38 expression resulted positive in 42 and 46 patients, whereas IgVH genes were mutated in 45 patients. Genome-wide DNA profiling data were generated on GeneChip® Human Mapping 250K NspI arrays (Affymetrix); copy number alterations (CNA) were calculated using the DNA copy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS) (Olshen et al, 2004). A total of 782 CNAs (ranging from 1 to 31 per sample, mean and median values 7.82 and 7, respectively) were detected; DNA losses (365/782=46.67% loss; 194/782=24.81% biallelic deletion) were found to be more frequent than gains (148/782=18.93% gain; 75/782=9.59% amplification). The most recurrent alterations detected by FISH were all confirmed by SNP array analysis, strengthening further the good reliability of such high-resolution technology. We identified 12 minimally altered regions (MARs) larger than 100 kb with a frequency higher than 5%. Among well known alterations, the largest was represented by chromosome 12 trisomy, followed by 6q, 17p and 11q23 deletions (32.87, 19.09 and 10.43 Mb, respectively) and 13q14 deletion (635 kb). Gain of 2p25.3 involves a common region of 4.39 Mb region in 7 patients, although it was extended to the whole short arm of chromosome 2 in 3 cases. Among those alterations previously described in B-CLL, we found losses at 14q32.33 (12 pts) and 22q11.2 (5 pts) involving the IGH and IGLλ loci, respectively. With regard to novel regions, we identified losses at 4q35.2 (5 pts) and 11q25 (6 pts). In addition we found a high frequency of losses/gains at 14q11.2 (42 pts) and 15q11.2 (33 pts), two genomic regions reported to be affected by DNA copy number variations in normal individuals. As regards correlations between CNAs and biological markers, we found that the number of CNAs is significantly higher in cases with unmutated IgVH (9.4; range 2–31) as compared with mutated IgVH (6; range 1–13) (p=0.002), while neither CD38 nor ZAP-70 expression showed significant correlation. In addition, a significant higher number of either CNAs (p=0.001), total MARs (p<0.0001) or even only novel MARs (p=0.009) was significantly associated with cases with 17p deletion or multiple cytogenetic aberrations as evaluated by FISH analysis. Our data indicate that genetic abnormalities involving chromosomal gains and losses are very common in early-stage B-CLL and further support the application of high resolution SNP array platforms in the characterization of genetic changes in the disease. In addition, we detected novel altered chromosomal regions that warrant further investigations to better define their pathogenetic and prognostic role in B-CLL.

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High Density SNP Array Analysis of Tyrosine Kinase Inhibitor (TKI) Resistant Chronic Myeloid Leukemia (CML) Shows Secondary Genomic Alterations

Blood ◽

10.1182/blood.v112.11.3182.3182 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3182-3182

Author(s):

Daniel Nowak ◽

Norihiko Kawamata ◽

Tadayuki Akagi ◽

Ryoko Okamoto ◽

Nils Thoennissen ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Copy Number ◽

Myeloid Leukemia ◽

Snp Array ◽

High Density ◽

Array Analysis ◽

Snp Arrays ◽

Tki Resistance ◽

Snp Array Analysis

Abstract Despite the success story of tyrosine kinase inhibitors (TKIs) for the treatment of Chronic Myeloid Leukemia (CML), patients can develop resistances against the drugs. The main known causes for resistance are mutations or over-expression of the BCR/ABL fusion protein, reduced bioavailability of the drugs and activation of compensatory molecular pathways. It is hypothesized that during disease progression, genomic instability of CML cells increases, which may lead to new genomic lesions harboring additional mechanisms of resistance. In this context, we studied genomic DNA profiles of 32 Imatinib resistant CML patients with high density 250K SNP arrays (Affymetrix). Molecular allelokaryotyping for allele specific copy number and loss of heterozygosity analysis was performed with the CNAG software. Single DNA samples from 27 patients were extracted after they had acquired resistance to Imatinib or alternative TKIs such as Nilotinib or Dasatinib. DNA from 12 patients could be analyzed in sequential samples from the initial diagnosis timepoint and a second timepoint upon the emergence of TKI resistance. All patients were positive for BCR/ABL by PCR and FISH. 10 relapse patient samples had known BCR/ABL mutations of which two were T315I mutations. High density allelokaryotyping confirmed pre-existent data on unbalanced translocations, amplifications and deletions from routine cytogenetics: 5 samples displayed a genomic duplication of the BCR/ABL fusion gene, 4 samples had trisomy 8, 1 sample showed deletion of chromosome 17p, 1 sample had heterozygous deletion of chromosome 9. Apart from this, SNP array analysis revealed numerous new submicroscopic genomic lesions. After exclusion of genomic copy number polymorphisms (CNPs) by comparison to recorded CNPs in the UCSC Genome Browser (http://genome.ucsc.edu/) the following results were obtained: Two patients displayed common heterozygous microdeletions of the reciprocal ABL/BCR fusion product. Furthermore, single samples displayed heterozygous micro-deletions on chromosomes 1, 2, 10, 12, 15, 17, and 22 or microduplications on chromosomes 2,3,6, 8, 9, 11, 12, 14, 15, 22. The affected regions contained potentially interesting genes in respect to resistance to therapy such as tumor suppressor candidate MBP-1, apoptosis related protein RERE, metastasis associated gene MTA3, nuclear body associated gene SP100, alpha-T-catenin (CTNNA3), Cbl-interacting protein Sts-1 and the DNA repair associated gene RAD51. As a new genomic alteration in CML, we detected acquired uniparental disomy (UPD) in 5 samples with a common site of UPD on chromosome 19q in 2 patients. In conclusion, in 14 out of 39 TKI resistant cases, high density SNP arrays enabled us to identify submicroscopic copy number lesions and regions of UPD containing promising candidate genes, which merit further research as sites conferring TKI resistance.

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High Density SNP Array Analysis of Acute Promyelocytic Leukemia (APL) Detects New Common Genomic Copy Number Alterations as Possible Cooperating Lesions

Blood ◽

10.1182/blood.v116.21.2721.2721 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2721-2721

Author(s):

Daniel Nowak ◽

Marion Klaumuenzer ◽

Benjamin Hanfstein ◽

Maximilian Mossner ◽

Florian Nolte ◽

...

Keyword(s):

Bone Marrow ◽

Acute Promyelocytic Leukemia ◽

Copy Number ◽

Genomic Dna ◽

Snp Array ◽

Promyelocytic Leukemia ◽

High Density ◽

Copy Number Alterations ◽

Array Analysis ◽

Snp Array Analysis

Abstract Abstract 2721 Introduction: Acute Promyelocytic Leukemia (APL) is characterized by the typical chromosomal translocation t(15;17)(q22;q21) leading to the fusion product PML-RARA, which blocks granulocytic differentiation in the promyelocyte stage. Several experimental in vitro and in vivo studies have demonstrated that PML-RARA is necessary but not sufficient for the generation of APL. This circumstance has motivated the search for additional leukemogenic and cooperating molecular lesions. Patients and Methods: We have analyzed 101 APL patient bone marrow samples with high density Genome-Wide Human SNP 6.0 arrays, which interrogate >900.000 SNPs and >900.000 non-polymorphic copy number markers throughout the genome (Affymetrix, Santa Clara, CA, USA) in search for copy number alterations (CNAs) potentially relevant in the pathogenesis of APL. Genomic DNA from samples at initial diagnosis of 94 patients was analyzed. Furthermore, DNA from 11 samples at relapse was available, whereby 4 of these relapse samples also had paired DNA from initial diagnosis. Data analysis was carried out with the CNAG 3.3 software using anonymous references. For exclusion of copy number polymorphisms, all detected CNAs were compared with the databases of known copy number polymorphisms in the UCSC genome browser. For data validation, putatively acquired CNAs and regions of copy number neutral loss of heterozygosity (CNLOH) were confirmed by hybridization of DNA from paired normal samples when the patients were in remission, by quantitative real time PCR of genomic DNA and by direct sequencing of informative SNPs. Results: The high density SNP array analysis detected a total of 120 heterozygous deletions, 97 duplications or amplifications and 7 regions of telomeric CNLOH leading to an average of 2.3 CNAs per sample (range 0–30). The most common numerical and large structural aberrations were found on chromosome (chr.) 8 with either trisomy 8 (n=11) or duplication of regions on chr. 8q (n=10) followed by heterozygous deletions of chr. 7q (n=5) and chr. 16q (n=5). Furthermore, unbalanced translocations of chr. 15 and 17 involving PML and RARalpha were detected in five cases leading to duplication of the PML-RARA fusion or deletion of genomic regions flanking either PML or RARalpha. Recurrent microlesions (<1Mbp) were found in several regions as heterozygous deletions on chr. 1q31.3 containing the micro RNAs MIR181B1 and MIR181A1 (n=5), on chr. 2q32.3 containing serine/threonine kinase 17b (STK17B) (n=5) or chr. 3p24.3 containing ankyrin repeat domain 28 (ANKRD28) (n=5). One recurrent region of telomeric CNLOH was found on chr. 19q in two samples. Of note, besides the few regions of telomeric CNLOH a large number of intrachromosomal CNLOH regions (n=265) was identified, with recurrent regions on chr. 6p21.1 (n=10) or chr. 5q23.3-5q31.1 (n=6) containing genes relevant in hematopoiesis such as IL3, CSF2 or DNA damage repair such as RAD50. Although these CNLOH regions were not somatically acquired they may possibly harbor genetic predispositions for disease. Conclusions: We describe a detailed high density SNP array genomic profiling of bone marrow DNA from patients with APL, which has led to the identification of several new cryptic recurrent genomic lesions. These genomic alterations point to candidate genes, which could be cooperating factors in addition to PML-RARA. Therefore, our data helps to provide a better understanding of the molecular mechanisms underlying the development of APL. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Lengfelder:Cephalon: Research Funding.

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