Copy number variations and

cancer

Presented by: Narjes Khatoon Shabani SadrProvided for : Cancer Genetics - Dr. Galedari

Agenda for this Lecture• Introduction of Copy number variations (CNV)• types of CNV :bi-allelic and multi-allelic CNVs. • Mechanism for the creation CNV• Role in disease• Define Cancer• CNV role in cancer development• Copy-number changes and cancer• CNVs and cancer predisposition:

first hits to the tumor genome• Common cancer CNVs• Rare cancer CNVs• Examples of cancer associated with CNVs• CVN and Pharmacogenetics in oncology• Conclusions

• The gene copy number (also "copy number variants" or CNVs) is the number of copies of a particular gene in the genotype of an individual.

• Recent evidence shows that the gene copy number can be elevated in cancer cells.

Introduction Copy number variations (CNV)

Introduction Copy number variations (CNV) :A brief history of CNVs

• The first CNV in humans – discovered in the early 1900s – was found to be widespread throughout all populations, with a range of striking but largely benign phenotypic consequences.

• However, the first large, systematic structural comparisons of the genomes of healthy humans were only carried out in 2004 and used hybridization to identify large numbers of CNVs that are present at significant frequency.

• Further studies have revealed well over a thousand CNVs, which at one time were believed to account for up to 15% of the human genome, a figure inflated somewhat by the limited spatial resolution of the methods used and consequent overestimation of the sizes of the genomic regions involved.

• Copy-number variations (CNVs)—a form of structural variation—are alterations of the DNA of a genome that results in the cell having an abnormal or, for certain genes, a normal variation in the number of copies of one or more sections of the DNA.

• CNVs correspond to relatively large regions of the genome that have been deleted (fewer than the normal number) or duplicated (more than the normal number) on certain chromosomes.

• For example, the chromosome that normally has sections in order as A-B-C-D might instead have sections A-B-C-C-D (a duplication of "C") or A-B-D (a deletion of "C").

• This variation accounts for roughly 13% of human genomic DNA and each variation may range from about one kilo base (1,000 nucleotide bases) to several megabases in size.

Introduction Copy number variations (CNV)

Introduction Copy number variations (CNV)

• DNA copy number variations (CNVs) are an important component of genetic variation, affecting a greater fraction of the genome than single nucleotide polymorphisms (SNPs).

• CNVs correspond to relatively large regions of the genome that have been deleted or duplicated on certain chromosomes.

• This variation accounts for roughly 13% of human genomic DNA and each variation may range from about 1 kb to several megabases in size.

• CNVs contrast with SNPs, which affect only one single nucleotide base.

What is a copy number variant?

• Human DNA has one copy of autosomal regions on each chromosome.

• However, as discovered by the Human Genome Project, many genetic regions display a variation in the number of copies (more or less than two copies).

• Alleles containing 0 –13 gene copies have been reported across the human population.

What is a copy number variant?

• These genetic variants are termed CNVs and are defined as DNA segments ranging in size from one kilo base to several mega bases among individuals owing to deletion, insertion, inversion, duplication, or complex recombination.

• Although the contribution of CNVs to the pathogenesis of common diseases is questionable, CNVs in some pharmacogenetic genes play a clear role in drug efficacy and toxicity.(especially in cancer)

Copy Number Variations in the Human Genome

Chromosome Position

Person 1

Person 2

Sig

na

lS

ign

al

Extra DNAMissing DNA

bi-allelic and multi-allelic CNVs.

Mechanism for the creation CNV

• Most CNVs are stable and heritable, so CNV between individuals is largely a product of genetic heritage, however, de novo CNVs arise through diverse mechanisms at various stages of development.

• Multiple homologous recombination reactions on each chromosome are required for the meiotic cell divisions that give rise to gametes, and although these events are of very high fidelity, occasional mistakes are inevitable.

• Therefore, most CNV in the human genome likely arises through non-allelic homologous recombination events in which unmatched regions of chromosomes are mistakenly recombined during meiosis.

• However, two lines of evidence suggest that this is not the whole story.

• Firstly, various studies have revealed extensive CNV between different cells in the same individuals; these CNVs must have arisen post-fertilization.

• Secondly, some complex genetic rearrangements cannot be readily reconciled with a non-allelic homologous recombination mechanism; these have been proposed to arise through rare replication defects resulting from broken DNA at one replication fork invading another fork, resulting in a template switch.

• This was subsequently superseded by a more general micro homology-mediated break-induced replication (MMBIR) model.

Mechanism for the creation CNV

Non-allelic homologous recombination (NAHR)

Non-homologous end joining (NHEJ) can result in a genomic rearrangement that immediately or eventually results in the gain or loss of genetic material after multiple double-strand DNA breaks occur and are repaired.

Non-homologous end joining (NHEJ) can result in a genomic rearrangement that immediately or eventually results in the gain or loss of genetic material after multiple double-strand DNA breaks occur and are repaired.

Role in disease

• Like other types of genetic variation, some CNVs have been associated with susceptibility or resistance to disease.

• Gene copy number can be elevated in cancer cells. For instance, the EGFR copy number can be higher than normal in non-small cell lung cancer.

• In addition, a higher copy number of CCL3L1 has been associated with lower susceptibility to HIV infection, and a low copy number of FCGR3B (the CD16 cell surface immunoglobulin receptor) can increase susceptibility to systemic lupus erythematosus and similar inflammatory autoimmune disorders. Copy number variation has also been associated with autism, schizophrenia, and idiopathic learning disability.

Define Cancer• Cancer is a term used to describe a large group

of diseases that are characterized by a cellular malfunction.

• Healthy cells are programmed to “know what to do and when to do it”.

• Cancerous cells do not have this programming and therefore grow and replicate out of control.

• They also serve no physiological function. These cells are now termed a neoplasm.

• This neoplasmic mass often forms a clumping of cells known as a tumor.

• Although the exact pathogenic mechanisms leading to many cancers are unclear, the consensus view is that cancer results from dysregulation of the activity or expression of genes that control cell growth and differentiation, leading to abnormal cell proliferation.

• CNVs have been reported in many kinds of cancers, and presumably contribute to this dysregulation.

Define Cancer

Copy-number changes and cancer

• CNVs have clearly been shown to have the potential to indirectly influence a healthy individual’s susceptibility to cancer, for example by varying the gene dosage of tumour suppressors or oncogenes .

• A more direct and immediate role of copy-number change is seen in cancerous cells themselves, which frequently display CNVs that are absent in the patient’s normal cells in characteristic (although cancer-type specific) parts of the genome.

• Arguably, such copy-number changes in cancer cells should not be called ‘variants’ because they do not fall within the spectrum of normal human variation.

• Aneuploidy, double-minutes and nonreciprocal translocations have long been recognized in many cancers and are one form of CNV.

• But cancers have also been shown to gain additional copies of certain smaller genomic regions.

• Such gains are of particular interest when they are found in many patients with a given cancer type or in the early stages of cancer because they can then be inferred to harbor so-called ‘driver genes’ that favor the growth of abnormal cells.

Copy-number changes and cancer

• By contrast, in the later stages of many cancers, ‘genomic chaos’ often ensues, which makes it difficult to determine which copy-number changes are significant or causative and which are purely incidental or of minor importance.

Copy-number changes and cancer

CNVs and cancer predisposition: first hits to the tumor genome

• The goal of cancer genetics is to discover all variant alleles that predispose to neoplasms.

• To this end, SNPs have been the most widely studied form of genetic variation and, by using massive GWA studies, many common SNPs have been shown to be associated with cancer and other complex traits.

• However, the results of these efforts have not explained much of the heritability of disease.

• This is perhaps because GWA studies have mostly ignored the

inter-individual genetic variation provided by CNVs, which affect more than 10% of the human genome.

• CNVs, especially smaller variants, have been essentially hidden from view until recently; thus, only a handful of studies have found an association of CNVs with cancer.

• Once these CNVs have been identified, one can only assume that CNVs will explain a larger portion of the genetic basis of cancer.

• Once identified, common and rare CNVs should be considered separately, as they may have very different roles in cancer.

CNVs and cancer predisposition: first hits to the tumor genome

Common cancer CNVs: Distribution of common cancer CNVs in the human genome

As with SNPs, CNVs that are found frequently in the healthy population (common CNVs) are very likely to have a role in cancer etiology.

Common cancer CNVs

• Rad51L1:this is essential for DNA repair by homologous recombination and has been shown by a GWA study to contain a SNP that is strongly associated with breast cancer.

• MLLT4:that seems to be associated with the Li-Fraumeni cancer predisposition disorder (LFS)

• Mtus1 :a small deletion in Mtus1 is associated with a decreased risk of familial and high-risk breast cancer

Rare cancer CNVs

• There are over 200 cancer syndromes and although most arise infrequently, they account for 5-10% of all cancer cases.

• These are caused by base-pair-sized germline mutations in many central

• tumor suppressor genes - such as: TP53, APC, BRCA1, BRCA2, PTEN, and RB1

• and (fewer) oncogenes, including HRAS and RET.

Proposed model for CNVs in tumorigenesis.

CNVs and tumor genomes: Copy number alterations(CNA)

• Genome-scale analyses have found many formerly invisible CNAs.

• In an analysis of 371 lung adenocarcinoma samples, Weir et al. identified seven recurrent homozygous deletions and 24 recurrent amplifications.

• The most significant amplification, at 14q13.3 and containing the novel oncogene NKX2-1.

• Mullighan et al found copy number changes in PAX5, a gene within the B-cell development pathway, in 57 of 192 ALL cases.

• In glioblastoma, CNA information, mRNA expression levels and methylation changes have been measured and nucleotide mutational analyses have been carried out.

• Integrative analysis has shown that over 70% of tumors carry alterations in the retinoblastoma, p53 and receptor tyrosine kinase pathways.

• Although cancer is driven primarily by alterations of the genome, CNA profiles can be combined with other high-throughput data to create insights that are 'greater than the sum of their parts'.

CNVs and tumor genomes: Using CNAs to define the key pathways of a tumor

• CGH analysis has also shown that CNVs are associated with prostate cancer, breast cancer, and colorectal cancer.

• Tse et al identified eight regions with CNVs including six deletions (on chromosomes 3, 6, 7, 8 and 19), and two duplications (on chromosomes 7 and 12) that were significantly overrepresented in nasopharyngeal carcinoma (NPC) patients compared with healthy controls.

Examples of cancer associated with CNVs

• Among these CNVs, MICA and HCP5 gene deletions on chromosome 6p21.3 showed the highest association signal.

• Examining 51 BRCA1-associated ovarian cancer patients, and 47 healthy women via Affymetrix Genome-Wide Human SNP Array 6.0, Yoshihara et al identified germline CNVs in BRCA1 associated ovarian cancer patients.

Examples of cancer associated with CNVs

Potential mechanism of the UGT2B17 CNV in prostate cancer pathogenesis.

Single-nucleotide polymorphisms(SNPs) effect on Pharmacogenomics

Single Nucleotide Polymorphism (SNP):

GAATTTAAG

GAATTCAAG

SNPs are defined as Single base-pair positions in genomic DNA that vary among individuals in one or several populations.

SNPs are believed to underlie susceptibility to such common diseases as cancer, diabetes, and heart disease and to contribute to the traits that make individuals unique.

SNPs are used as genomic biomarkers.

Hence SNP analysis can be used to enhance drug discovery and development.DNA molecule 1 differs from

DNA molecule 2 at a single base-pair location (a C/T polymorphism)

Examples of drugs where pharmacogenomics testing is useful are listed in this Table

CVN and Pharmacogenetics in oncology

Pharmacogenetics focused on the effects of genetics in cancer treatment.

Pharmacogenetics And selection of anticancer drugs:• Thiopurine• Irinotecan،• Tamoxifen

Thiopurine such as : 6-mercaptopurine (6-MP) thioguanine and azathioprine (TPMT)

Metabolized by: thiopurine-S-methyl transferase (TPMT)

CVN and Pharmacogenetics in oncology

Metabolism of 6-mercaptopurin to 6-methylmercaptopurin by the methylator TPMT.

Various alleles of the gene TPMT and their SNPs

The metabolic pathway of azathioprine 

Irinotecan

Irinotecan

7-ethyl-10- hydroxycamptothecin (SN-38)

(UGT1A1) UDP- glucuronosyl transferase

Active metabolite

Detoxification

Irinotecan

Tamoxifen (estrogen receptor-positive breast cancer)

Tamoxifen is a prodrug

endoxifen (4-hydroxy-N-desmethyl-tomoxifer)

Tamoxifen

By CYP2D6

Conclusions

• The study of cancer and CNVs is in its infancy but is maturing quickly.

• In considering the effect of this form of genetic variation on cancer predisposition, cancer gene expression and tumor genome profiling, there is much to learn from past studies on genomic disorders.

• Denser micro-arrays, next-generation sequencing and integrative informatics analyses are around the corner and promise to uncover new CNVs and CNAs.

Conclusions

• There are, therefore, many exciting questions to be addressed: • what role do CNVs have in cancer predisposition and how can we

use this newly discovered form of genetic variation to identify those most at risk?

• Which cancer-related genes are affected by CNVs and, of these changes, which are both necessary and sufficient to cause neoplastic growth?

• Can incipient cancer cells use these constitutional deletions and duplications to induce or accelerate tumorigenesis and tumor proliferation?

• As these questions are resolved, the potential value of cancer CNVs as novel biomarkers of cancer susceptibility and initiation, and of cancer progression and metastases, will become apparent. Whether cancer CNVs offer insight into genes that might be targets for novel drug development remains to be determined.

Conclusions

References

• Adam Shlien and David Malkin. (16 June 2009). Copy number vvariations and cancer. Genome Medicine, 11:: 62.

• Ana CV Krepischi,Maria Isab el W Achatz,Erika MM San tos,, Silvia S Costa,Bianca CG Lisboa,Helena Brentani,. (2012). Germline DNA copy number variation in familial and early-onset breast cancer. Krepischi et al . Breast Cancer Research, 14-24.

• Andrew N. Shelling,Lynnette R. Ferguson. (2007). Genetic variation in human disease and a new role for copy number variants. Mutation Research 622, 33–41.

• Antonis C Antoniou and Georgi a Chenev ix-Trench. (2010). Common genetic variants and cancer risk in Mendelian cancer syndromes. Current Opinion in Gen etics & Development, 299 –307.

• Bin Liu,ei Yang,Binf ang Huang,Mei C heng,Hui Wang,Yinyan Li,Do ngshen g Huang. (August 10, 2012). A Functional Copy-Number Variation in MAPKAPK2. The American Journal of Human Genetics 91, 384–390.

• Charles Lee , Courtney Hyland , Arthur S. Lee , Shona Hislop and Chunhwa Ihm. (2010). Copy Number Variation and Human Health. In C. H. Charles Lee, Essentials of Genomic and Personalized Medicine by Ginsburg & Willard (p. CHAPTER 5).

• Colin C . Pritchard, Stephen J. Salipante ,. (January 2014). Validation and Implem entation of Targeted Capture and Sequencing for the Detection of Actionable

• Mutation, Copy Number Variation, and Gene Rearrangemen t in Clinical Cancer Specimens. The Journal of Molecular Diagnostics.

• Dear, P. H. (July 2009). Copy-number variation: the end of the human genome? Trends in Biotechnology, 448-456.

• Elsa Vanh ecke,Alexan der Valen t,Ximing Tang,Philippe Vielh,Luc Frib oulet,Tao Tang,Aïcha Goubar,Yua nyuan Li,. (2013). 19q13- ERCC1 Gene Copy Number Incre ase in Non e Small-Cell Lung Cancer. Clinical Lung Cancer, 549-57.

• Iuliana Ionita-Laza,Angela J . Rogers,, Christoph Lange,, Benjamin A . Raby,Charles Lee. (2009). Genetic association analysis of copy-number variation (CNV) in human disease pathogenesis. Genomics 93 , 22-26.

• Roland P Kuiper,Marjolijn JL Ligten berg,Nicoline Hoogerb rugge,and Ad Geurt s van Kessel. (2010). Germline copy number variation and cancer risk. Current Opinion in Gen etics & Development, 282 – 289.

References

• Tie-Lin Yang, Yan Guo,Christopher J. Papasian and Hong-Wen Deng. (2013). Copy Number Variation. In Y. G.-W. Tie-Lin Yang, Genetics of Bone Biology and Skeletal Disease (p. chapter 9).

• Xiaosu Zhao,Qi Wu,Xinrong Fu,Bo Yu,Yong Shao,Hong Yang,Ming Guan,Xiaojun Huang,. (2010). Examination of copy number variations of CHST9 in multiple types of hematologic malignancies. Cancer Genetics and Cytogenetics 203 , 176 e179.

• Yijing He, Janelle M. Hoskins and Howard L. McLeod. (May 2011). Copy number variants in pharmaco genetic genes. Trends in Molecular Medicine.

References

“Human beings are ultimately nothing but carriers-passageways- for genes. They ride us into the ground like racehorses from generation to generation. Genes don't think about what constitutes good or evil. They don't care whether we are happy or unhappy. We're just means to an end for them. The only thing they think about is what is most efficient for them.”

Haruki Murakami, 1Q84

Thanks for your attention