Inferring mechanisms of copy number change from haplotype structures at the human DEFA1A3 locus

Breast phyllodes tumour (PT) is a rare fibroepithelial tumour. The genetic alterations contributing to its tumorigenesis are largely unknown. To identify genomic regions involved in pathogenesis and progression of PTs we obtained genome-wide copy number profiles by array comparative genomic hybridization (CGH).DNA was isolated from fresh-frozen tissue samples. 11 PTs and 3 fibroadenomas, a frequently occurring fibroepithelial breast tumour, were analyzed. Arrays composed of 2464 genomic clones were used, providing a resolution of ~1.4 Mb across the genome. Each clone contains at least one STS for linkage to the human genome sequence.No copy number changes were detected in fibroadenomas. On the other hand, 10 of 11 PT (91%) showed DNA copy number alterations. The mean number of chromosomal events in PT was 5.5 (range 0–16) per case. A mean of 2.0 gains (range 0–10) and 3.0 losses (range 0–9) was seen per case of PT. Three cases showed amplifications. DNA copy number change was not related to PT grade. We observed recurrent loss on chromosome 1q, 4p, 10, 13q, 15q, 16, 17p, 19 and X. Recurrent copy number gain was seen on 1q, 2p, 3q, 7p, 8q, 16q, 20.In this study we used array CGH for genomic profiling of fibroepithelial breast tumours. Whereas most PT showed chromosomal instability, fibroadenomas lacked copy number changes. Some copy number aberrations had not previously been associated with PT. Several well-known cancer related genes, such as TP53 and members of the Cadherin, reside within the recurrent regions of copy number alteration. Since copy number change was found in all benign PT, genomic instability may be an early event in PT genesis.

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An Integrated Pharmacogenomic Strategy for the Definition of Thalidomide Response Signatures in Presenting Cases of Multiple Myeloma.

Blood ◽

10.1182/blood.v110.11.2493.2493 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2493-2493

Author(s):

Gareth J. Morgan ◽

Matthew W. Jenner ◽

Brian A. Walker ◽

David C. Johnson ◽

Paola A. Leone ◽

...

Keyword(s):

Gene Expression ◽

Gene Mapping ◽

Candidate Genes ◽

Copy Number ◽

Copy Number Change ◽

Plasma Cells ◽

Poor Response ◽

Expression Arrays ◽

Intensive Group ◽

Mapping Arrays

Abstract Whilst gene expression signatures have been defined that correspond to poor overall survival, the mechanism for deregulation of such genes is often elusive. We and others have described acquired copy number change as one potential mechanism of gene deregulation in myeloma. Other potential mechanisms exist that may influence the expression of myeloma-associated genes such as inherited SNPs and copy number variation (CNV). We have therefore embarked upon an integrated pharmacogenomic strategy to determine the importance of acquired and inherited genetic changes in determining response to therapy. We have carried out gene expression analysis on CD 138 selected bone marrow plasma cells from 231 newly diagnosed myeloma cases using Affymetrix U133 Plus 2.0 expression arrays and copy number analysis using 500K Gene Mapping arrays on a subset of 90 cases. Peripheral blood DNA has been genotyped using Affymetrix 500K Gene Mapping arrays and the BOAC chips. Cytogenetics was available in the majority of cases. Younger, fitter patients received either cyclophosphamide, thalidomide and dexamethasone (CTD) or cyclohosphamide-VAD (C-VAD), followed by high dose melphalan (HDM). Older, less fit patients received attenuated dose CTD or MP. Response was assessed before and after HDM in the intensive group and on completion of therapy in the non-intensive group using EBMT criteria plus the category of VGPR. We used a supervised approach to define a gene expression signature corresponding to high level response (CR, VGPR or PR) against poor response (NC, PD or MR) overall and for each of the three induction strategies, CTD/CTDA, CVAD and MP. We have combined the data from expression arrays together with mapping data from tumor DNA and 2 different SNP arrays performed on germline DNA. We defined a poor response expression signature initially and then identified the genomic loci of these genes and how they were affected by acquired copy number change. For each candidate gene we also examined the constitutional DNA to see if each fell within a region of inherited CNV and how this could be affected by acquired copy number change. In a similar fashion, we used the BOAC chip to define genes and SNPs associated with response. This is different as it utilized mostly functional cSNPs in candidate genes. We then looked at how CNV affected these genes. Although not all genes in which functional cSNPs are present would necessarily be expected to be expressed in plasma cells, this approach is a vital step in identifying the clinical relevance of such cSNPs in myeloma. We also took the alternate approach and designed an algorithm able to correlate acquired copy number change with paraprotein response. We then identified differentially expressed genes in these loci and their impact on response, narrowing the candidate genes down to define a signature which could be validated. Using this approach has allowed us to identify genes important in determining response and their relation to tumor-associated copy number change and inherited CNV. Overall, this methodology provides significant insight in to the factors that predict response to different chemotherapy regimens. Preliminary data will be presented.

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The Impact of Constitutional Copy Number Variants in Myeloma

Blood ◽

10.1182/blood.v112.11.496.496 ◽

2008 ◽

Vol 112 (11) ◽

pp. 496-496

Author(s):

Matthew W Jenner ◽

David C Johnson ◽

Paola E Leone ◽

Brian A Walker ◽

David Gonzalez ◽

...

Keyword(s):

Copy Number ◽

Copy Number Change ◽

Copy Number Variations ◽

Genomic Variation ◽

Chromosome 21 ◽

Nucleotide Polymorphisms ◽

Copy Number Changes ◽

The Impact ◽

First Time ◽

Tumor Dna

Abstract Single nucleotide polymorphisms (SNPs) have been long regarded as being important in determining variation and disease predisposition. Recently, chromosomal structural variation in the form of deletions, insertions and duplifications have been identified frequently in the genome of the general population. Such copy number variations (CNVs) have been shown to contribute to a range of human diseases. In recent studies we have utilized Affymetrix 50K and 500K arrays to identify acquired copy number change in myeloma tumor samples. In those studies we had access to paired constitutional DNA and in the present study have been able to report for the first time a CNV map of the constitutional genome of myeloma patients. Affymetrix 500K mapping arrays were used to identify copy number changes in 63 paired samples using DNA from peripheral blood and CD138 selected plasma cells. Tumor samples were analyzed in CNAG using both a paired and unpaired analysis to distinguish between inherited and acquired copy number change. Constitutional DNA was analyzed by both CNAG and GEMCA using 90 Caucasian samples from the Hapmap database as a reference set. For maximum calling accuracy, only those regions identified by both algorithms were called as CNVs. As with similar studies, overlapping CNVs identified using this approach were merged to generate a list of CNV regions (CNVRs) characteristic of the constitutional DNA of these myeloma cases. Using this approach, we identified 292 CNVs across 63 cases, with a median of 4 regions per sample. There were 155 discrete CNVRs, of which 46 were recurrent. The recurrent CNVRs were found most frequently in the pericentric regions of chromosome 14 and 15 in keeping with other studies. We then compared these recurrent CNVRs with a comparable dataset of normal individuals generated using Affymetrix 500K arrays. In this analysis, 25/46 recurrent CNVRs in the myeloma cases were novel. The two most frequent novel CNVRs in the myeloma cases were gains on chromosome 21 and 15. We also compared the characteristics of the constitutional CNVs with the acquired copy number changes in the corresponding tumor samples and identified that the constitutional CNVs were generally considerably smaller. However, using unpaired analysis it was possible to determine the presence of the constitutional CNV in the tumor sample, providing validation of the CNVs. We were also able to demonstrate that acquired copy number change in the tumor cells can either exaggerate or ameliorate the effect of the inherited CNV in the tumor genome, such as cases with acquired trisomy 15 and deletion or gain of regions of 15q in the constitutional DNA. These findings also reinforce the need for paired non-tumor DNA when undertaking copy number analysis of tumor DNA using SNP arrays. In this study we have been able to identify for the first time the presence of CNVs in the constitutional genome of individuals with myeloma. We have been able to systematically catalogue these CNVRs. These results provide the basis for future studies aimed at identifying how this type of genomic variation may influence the development of and outcome of myeloma and a broad range of other hematological conditions.

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