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Application of Genomic and Expression Arrays for Identification of new Cancer Genes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Genomics. (Díaz de Ståhl)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Copy number variation (CNV) comprises a recently discovered kind of variation involving deletion and duplication of DNA segments of variable size, ranging from a few hundred basepairs to several million. By altering gene dosage levels or disrupting proximal or distant regulatory elements CNVs create human diversity. They represent also an important factor in human evolution and play a role in many disorders including cancer. Array-based comparative genomic hybridization as well as expression arrays are powerful and suitable methods for determination of copy number variations or gene expression changes in the human genome. In paper I we established a 32K clone-based genomic array, covering 99% of the current assembly of the human genome with high resolution and applied it in the profiling of 71 healthy individuals from three ethnic groups. Novel and previously reported CNVs, involving ~3.5% of the genome, were identified. Interestingly, 87% of the detected CNV regions overlapped with known genes indicating that they probably have phenotypic consequences. In papers II through IV we applied this platform to different tumor types, namely two collections of brain tumors, glioblastoma (paper II) and medulloblastoma (paper III), and a set of bladder carcinoma (paper IV) to identify chromosomal alterations at the level of DNA copy number that could be related to tumor initiation/progression. Tumors of the central nervous system represent a heterogeneous group of both benign and malignant neoplasms that affect both children and adults. Glioblastoma and medulloblastoma are two malignant forms. Glioblastoma often affects adults while the embryonal tumor medulloblastoma is the most common malignant brain tumor among children. The detailed profiling of 78 glioblastomas, allowed us to identify a complex pattern of aberrations including frequent and high copy number amplicons (detected in 79% of samples) as well as a number of homozygously deleted loci. These regions encompassed not only previously reported oncogenes and tumor suppressor genes but also numerous novel genes. In paper III, a subset of 26 medulloblastomas was analyzed using the same genomic array. We observed that alterations involving chromosome 17, especially isochromosome 17q, were the most common genomic aberrations in this tumor type, but copy number alterations involving other chromosomes: 1, 7 and 8 were also frequent. Focal amplifications, on chromosome 1 and 3, not previously described, were also detected. These loci may encompass novel genes involved in medulloblastoma development. In paper IV we examined for the presence of DNA copy number alterations and their effect on gene expression in a subset of 21 well-characterized Ta bladder carcinomas, selected for the presence or absence of recurrences. We identified a number of novel genes as well as a significant association between amplifications and high-grade and recurrent tumors which might be clinically useful.

The results derived from these studies increase our understanding of the genetic alterations leading to the development of these tumor forms and point out candidate genes that may be used in future as targets for new diagnostic and therapeutic strategies.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 550
Keywords [en]
Array-CGH, Expression array, Copy number variation, Glioblastoma, Medulloblastoma, Bladder carcinoma, Oncogenes, Tumor suppressor genes
National Category
Medical Genetics Medical Genetics Cell and Molecular Biology Genetics
Research subject
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-121957ISBN: 978-91-554-7775-2 (print)OAI: oai:DiVA.org:uu-121957DiVA, id: diva2:306953
Public defence
2010-05-18, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-04-21 Created: 2010-03-31 Last updated: 2018-01-12Bibliographically approved
List of papers
1. Profiling of copy number variations (CNVs) in healthy individuals from three ethnic groups using a human genome 32 K BAC-clone-based array
Open this publication in new window or tab >>Profiling of copy number variations (CNVs) in healthy individuals from three ethnic groups using a human genome 32 K BAC-clone-based array
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2008 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 29, no 3, p. 398-408Article in journal (Refereed) Published
Abstract [en]

To further explore the extent of structural large-scale variation in the human genome, we assessed copy number variations (CNVs) in a series of 71 healthy subjects from three ethnic groups. CNVs were analyzed using comparative genomic hybridization (CGH) to a BAC array covering the human genome, using DNA extracted from peripheral blood, thus avoiding any culture-induced rearrangements. By applying a newly developed computational algorithm based on Hidden Markov modeling, we identified 1,078 autosomal CNVs, including at least two neighboring/overlapping BACs, which represent 315 distinct regions. The average size of the sequence polymorphisms was approximately 350 kb and involved in total approximately 117 Mb or approximately 3.5% of the genome. Gains were about four times more common than deletions, and segmental duplications (SDs) were overrepresented, especially in larger deletion variants. This strengthens the notion that SDs often define hotspots of chromosomal rearrangements. Over 60% of the identified autosomal rearrangements match previously reported CNVs, recognized with various platforms. However, results from chromosome X do not agree well with the previously annotated CNVs. Furthermore, data from single BACs deviating in copy number suggest that our above estimate of total variation is conservative. This report contributes to the establishment of the common baseline for CNV, which is an important resource in human genetics.

Keywords
genetic variation, array-CGH, genetics, population, polymorphism, human genome, gene dosage
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-102483 (URN)10.1002/humu.20659 (DOI)000253837300009 ()18058796 (PubMedID)
Note

De två (2) sista författarna delar sistaförfattarskapet.

Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2017-12-13Bibliographically approved
2. Characterization of novel and complex genomic aberrations in glioblastoma using a 32K BAC array
Open this publication in new window or tab >>Characterization of novel and complex genomic aberrations in glioblastoma using a 32K BAC array
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2009 (English)In: Neuro-Oncology, ISSN 1522-8517, E-ISSN 1523-5866, Vol. 11, no 6, p. 803-818Article in journal (Refereed) Published
Abstract [en]

Glioblastomas (GBs) are malignant CNS tumors often associated with devastating symptoms. Patients with GB have a very poor prognosis, and despite treatment, most of them die within 12 months from diagnosis. Several pathways, such as the RAS, tumor protein 53 (TP53), and phosphoinositide kinase 3 (PIK3) pathways, as well as the cell cycle control pathway, have been identified to be disrupted in this tumor. However, emerging data suggest that these aberrations represent only a fraction of the genetic changes involved in gliomagenesis. In this study, we have applied a 32K clone-based genomic array, covering 99% of the current assembly of the human genome, to the detailed genetic profiling of a set of 78 GBs. Complex patterns of aberrations, including high and narrow copy number amplicons, as well as a number of homozygously deleted loci, were identified. Amplicons that varied both in number (three on average) and in size (1.4 Mb on average) were frequently detected (81% of the samples). The loci encompassed not only previously reported oncogenes (EGFR, PDGFRA, MDM2, and CDK4) but also numerous novel oncogenes as GRB10, MKLN1, PPARGC1A, HGF, NAV3, CNTN1, SYT1, and ADAMTSL3. BNC2, PTPLAD2, and PTPRE, on the other hand, represent novel candidate tumor suppressor genes encompassed within homozygously deleted loci. Many of these genes are already linked to several forms of cancer; others represent new candidate genes that may serve as prognostic markers or even as therapeutic targets in the future. The large individual variation observed between the samples demonstrates the underlying complexity of the disease and strengthens the demand for an individualized therapy based on the genetic profile of the patient.

Keywords
amplification, array-CGH, cancer, deletion, glioblastoma
National Category
Clinical Medicine
Identifiers
urn:nbn:se:uu:diva-113074 (URN)10.1215/15228517-2009-013 (DOI)000272974100010 ()19304958 (PubMedID)
Available from: 2010-01-25 Created: 2010-01-25 Last updated: 2017-12-12Bibliographically approved
3. Novel amplicons in pediatric medulloblastoma identified by high-resolution genomic analysis: Genetic aberrations in medulloblastoma
Open this publication in new window or tab >>Novel amplicons in pediatric medulloblastoma identified by high-resolution genomic analysis: Genetic aberrations in medulloblastoma
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Medulloblastoma (MB) is an aggressive and invasive embryonal CNS tumor that mainly affects children. Despite treatment, ~30% of the patients die within 2 years from diagnosis. MB patients are currently categorized into high- or standard-risk based on the clinical criteria, with high-risk group including patients <3 years, with incomplete tumor resection or with concomitant metastatic disease at presentation. However, these clinical parameters do not always predict patient outcome and additional biomarkers are desirable. In this study we have profiled a series of 25 MB samples with a high-resolution 32K BAC-array covering 99% of the current assembly of the human genome for the identification of genetic copy number alterations. The most frequent observed alteration was the combination of 17p loss and 17q gain, indicative of an isochromosome 17q, which was identified in 40% of the patients. This aberration was detected in both high- and standard-risk groups and was not associated with worse outcome. We also defined minimal overlapping regions of aberrations, including 16 regions of gains and 18 regions of loss in different chromosomes. Noteworthy, are a few very narrow amplified loci identified on autosomes 1, 3 and 8, aberrations that were verified with an alternative platform (Illumina 610Q chips). Several genes as CYR61, LMO4, EOMES, and MLH1 encompassed within these loci were also found to present with transcript up-regulation. These genes represent novel candidate genes most probably involved in MB development.

 

Keywords
Medulloblastoma
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-121954 (URN)
Available from: 2010-03-31 Created: 2010-03-31 Last updated: 2010-03-31
4. Focal amplifications are associated with high grade and recurrences in stage Ta bladder carcinoma
Open this publication in new window or tab >>Focal amplifications are associated with high grade and recurrences in stage Ta bladder carcinoma
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2010 (English)In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 126, no 6, p. 1390-1402Article in journal (Refereed) Published
Abstract [en]

Urinary bladder cancer is a heterogeneous disease with tumors ranging from papillary noninvasive (stage Ta) to solid muscle infiltrating tumors (stage T2+). The risk of progression and death for the most frequent diagnosed type, Ta, is low, but the high incidence of recurrences has a significant effect on the patients' quality of life and poses substantial costs for health care systems. Consequently, the purpose of this study was to search for predictive factors of recurrence on the basis of genetic profiling. A clinically well characterized cohort of Ta bladder carcinomas, selected by the presence or absence of recurrences, was evaluated by an integrated analysis of DNA copy number changes and gene expression (clone-based 32K, respectively, U133Plus2.0 arrays). Only a few chromosomal aberrations have previously been defined in superficial bladder cancer. Surprisingly, the profiling of Ta tumors with a high-resolution array showed that DNA copy alterations are relatively common in this tumor type. Furthermore, we observed an overrepresentation of focal amplifications within high-grade and recurrent cases. Known (FGFR3, CCND1, MYC, MDM2) and novel candidate genes were identified within the loci. For example, MYBL2, a nuclear transcription factor involved in cell-cycle progression; YWHAB, an antiapoptotic protein; and SDC4, an important component of focal adhesions represent interesting candidates detected within two amplicons on chromosome 20, for which DNA amplification correlated with transcript up-regulation. The observed overrepresentation of amplicons within high-grade and recurrent cases may be clinically useful for the identification of patients who will benefit from a more aggressive therapy.

Keywords
Amplification, Array-CGH, Cancer, Deletion, Expression arrays, Metabolism of xenobiotics, Oncogenes, Ta bladder cancer, Tumor suppressor genes
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-121019 (URN)10.1002/ijc.24954 (DOI)000275434100010 ()19821490 (PubMedID)
Available from: 2010-03-18 Created: 2010-03-18 Last updated: 2017-12-12Bibliographically approved

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