Genetic Instability

M.C.PrabhathUniversity of Sri JayawardenepuraSri Lanka 4th Year -1st Semester 17.2.20161Genetic Instability

Content2IntroductionGenetic InstabilityTypes of Genetic InstabilityCauses of Genetic Instability Methods for detection and analysis of genome instability Genome instability and tumor genesis Major mechanisms used to maintain genomic instability Discussion SummeryReferences

Introduction3The genome is an organisms complete set of DNA and is organized into chromosomes containing genes that encode for hereditary traits.As our cells grow reproduce and die, DNA is repeatedly replicated and repaired and bits and pieces of its sequences are changed in the process, thus producing mutations.These mutations create genetic variation and it is proven that genetic mutation is key to our evolution and survival.But ,mutations are not always beneficial, they can be harmful leading to genetic diseases.When these mutations occur in an increased levels genetic instability takes place.

Genetic Instability 4A range of genetic alterations from point mutations to chromosome rearrangements - Aguilera and Gonzales,2008An increased rate of genomic alteration although some use the term to describe the state of the altered cancer genome. - Kwei et al.,2010A variety of DNA alterations, encompassing single nucleotide to whole chromosome changes. - Pikor et al., 2013

5A transient or persistent state that increases the spontaneous mutation rate, leading to gross genetics alterations such as rearrangements and changes in chromosome number. - Pikor et al., 2013 Commonly , Genetic alterations Increased rates Nucleotides to Chromosomes

Types of Genetic Instability6Based on the level of disruption, 1) Nucleotide Instability 2) Microsatellite Instability 3) Chromosomal Instability - Pikor et al., 2013

01) Nucleotide Instability 7Due to replication errors and impairment of the base excision repair and nucleotide excision repair pathways.Subtle sequence changes involving only one or few nucleotides , such as Substitutions Deletions InsertionsMitochondrial genome also displays this.

Source- Crols classroom blog.genetics8

Dissorders associate with NIN9Xeroderma pigmentosum MYH associated polyposis

Detection of G>C varient encoding a Gly>Arg amino acid change by sanger sequencing in two lung cancers.Source Pikor et al.,201310

02)Microsatellite Instability 11Repetitive DNA sequences ,comprising 1-6 bp located throughout the genome.Size is highly variable.Results from defects in mismatch repair ,specifically alterations of the MLH1,MSH2,MSH6 and PMS2 genes, which causes deletions or random insertions and expansion of microsatellites and a hyper mutable phenotypes.

Defects in Mismatch repair lead to the expansion or contraction of microsatellites throughout the genome.Source-Pikor et al., 201312

Disorders associate with MIN13Gastric,Endometrial,Ovarian,Lung and Colorectal cancers.Lynch syndrome

03)Chromosomal Instability 14Most prominent form.90% of human cancers exhibiting chromosomal abnormalities and aneuploidy.An increase in the rate of gain or loss of segmental and whole chromosomes during cell divisions.CIN tumors are characterized by Aneuploidy Amplifications Deletions Translocations Inversions

15

16

17

- Inversion-Amplification- Translocations

18Also occur because of alterations in, Mitotic Timing Mitotic checkpoint control Microtubule or centrosome dynamics Double strand break repair These alterations lead to karyotypic instability and growth of tumor populations.

Disorders associate with CIN19Breast,prostate .non small cell lung cancer, leukaemia,Neuroblastoma,Hodgkins and non Hodgkins lymphoma,Head and Neck cancer.Angelmen syndrome,Williams Syndrome , Cri du chat syndrome.

20

Causes of genetic instability 21Replication dysfunction as a major source of instability . Low replication Initiation Density Untimely Initiation causing Re-replicationFaulty replication fork progression S phase checkpoint DysfunctionDefective Nucleosome Assembly and remodeling

22Failures in post replicative repair and homologous recombinationSite specific hotspots DNA repeats Fragile sitesNon B DNA structures ,G-Quadruplexes and TelomeresDNA-Protein barriers to replication fork progressionCell physiology and metabolismAging

Methods for the detection and analysis of genome instability 23Single cell approach Karyotyping Fluorescence insitu Hibridization Fish Single cell sequencing Multiple annealing Looping based amplification cycles

24

25Multi cell approach Flow cytometry measures cells in a suspension as they pass through a laser scatter light and emit fluorescence can be used to detect cellular aneuploidy. Array comparative genomic hybridization- Offers the ability to quantitatively detect and visualize whole and segmental chromosomal alterations . SNP arrays- enable more precise mapping of copy number alterations. PCR-Amplify microsatellite regions and lengths of tandem repeats.

26

27

Genetic Instability and Tumor genesis28Cancer is a genetic disease.Tumor cells contain multiple mutations ,ranging from single nucleotide sequences changes and numerical alterations of chromosomes.Collectively , these mutations are referred to as genome instability ,which may be predisposed through inherited ,germline mutations as in the case of p53,BRCA1,BRCA2 or acquired as somatic mutations throughout an individuals lifetime. - Chen ,2015

29

Acquisition of some form of inherent genomic instability is a hallmark of tumor genesis. - Sieber etal ., 2013

Arguments for genomic instability as the engine of tumor genesis 30Tumors harbor too many mutations to be explained by anything other than underlying genomic instability.The probability of a tumor acquiring enough mutations for the full, malignant phenotype is too low unless the cells have an unstable genome.Humans and model organisms with inherent genomic instability are prone to tumors.In some tumors , there is direct evidence that some pathways that are involved in maintaining genomic integrity are defective.

Genetic instability accelerate carcinogenesis. - Beckmann and Loeb, 201531

32DNA polymerases and genetic instability-In mammals it is limited.Genetic instability in proofreading of DNApolymerases increases the incidences of lymphoma and epithelial tumors in mice, so can be in humans.

DNA repair enzymes and genetic instability Xeroderma pigmentosum patients- defect in nucleotide excision repair pathway

33Chromosomal instability Aneuploidy ,gross chromosomal translocations and molecular loss of heterozygocity without grossly visible karyotypic changes are common in all type of tumors.

Cell cycle checkpoints and genetic instability The most extensively checkpoint genes p53 and pRb are among the most frequently mutated in human cancers.Other checkpoint genes include the ATM gene, which is mutated in Ataxia telangiectasia .There is also evidence that BRCA1,BRCA2 inherited breast and ovarian cancer .

34Could the clinical appearance of cancer be prevented by decreasing the rate of genetic change?

35Cancer typically occurs late in life and evolves over decades.Amodest reduction in the rate of carcinogenesis could delay the onset of cancer by decades.Enhanced genetic stability could slow down carcinogenesis in a meaningful way ,leading to PREVENTION by DELAY.Prevention by delay may be particularly applicable for cancers associated with prolonged chronic inflammation due to either bacterial infection, viral infection, immunological insufficiencies or unknown etiology.

36A reduction in the rate of mutation accumulation by only two fold ,could delay the clinical appearance of the tumor from age 50 to age 90.

Compensation of the altered DNA mechanisms or altered DNA polymerases in promoting genetic instability would be more challenging with current technologies , as it might require comprehensive gene therapy.

Role of genetic instability in relation to therapy 37Genetic instability may play a role in the mechanism of action of chemotherapy.

Genetic instability not only increases the rate of acquiring mutations which may be essential for carcinogenesis,but also accelerates the acquasition of deleterious mutations which reduces the fitness of clones potentially leading to their extinction.

38Beyond a critical value, further increases in genetic instability are likely to exceed the optimum and lead to extinction of clones.The majority of chemotherapeutic agents interfere with aspects of DNA metabolism and are mutagens.If the cancer consists of genetically unstable cells, further increased mutagenesis due to chemotherapy could lead to accumulate deleterious mutations and extinction of malignant clones.Some scientists have suggested that in addition to directly causing mutations ,chemotherapy may select for genetic instability in normal tissues, this is because of secondary malignancies as a side effect of chemotherapy.

Major mechanisms used to maintain genetic stability39Normal mammalian cells mainly resort to four mechanisms to maintain their genomic stability during cell division.(Shen,2011)1)High fidelity of DNA replication in S phase2)Accurate distribution of chromosomes among daughter cells during mitosis3)Error-free repair of sporadic DNA damage throughout the cell cycle.4)Cell cycle progression and checkpoint control.

40

1)High fidelity of DNA replication in S phase

41High fidelity of base pairing and proofreading activities by DNA polymerases.Mismatch repair macinery to correct not only mismatched bases ,but also secondery DNA structures resulted from replication slippage.Timely resolution of stalled replication forks.Maturation of okazaki fragments.Replication licensing mechanisms to ensure that the entire genome is duplicated completely .Coordinated reassembly of chromosomes from newly synthesized DNA.Telomere maintenance

2)Accurate distribution of chromosomes among daughter cells during mitosis

42During mitosis ,the sister chromatids are equally distributed in daughter cells .This is mainly coordinated by many processes.Chromosome condensationSister chromatid cohesionCentrosome duplication and separationKinetochore assembly and attachment Spindle formation and checkpointChromosome segregation Cytokinesis Deregulation of above processes cause chromosomal instability.

3)Error-free repair of sporadic DNA damage throughout the cell cycle.

43Throughout the cell cycle ,the genome encounters various forms of spontaneous and induced DNA damages.These damages are repaired by severel well defined repair pathways.Error prone repair- Completion of some of the repair processes to fix chemical damages to the DNA double helix may cause alterations or rearrangements.Error free repair- Fix the damage to the DNA,but also preserve the original genome structure.

4)Cell cycle progression and checkpoint control44Cell cycle checkpoints are built to ensure that progression from one phase to the next under a condition of minimum risk of genomic alteration.This is accomplished by delaying the entry into the next phase until the risk factors are removed.Another important function of cell cycle checkpoint is to effectively trigger some processes to eliminate the severely damaged or high risk cells.

45The G1/S checkpoint is to restrict damage cells entering S phase .The G2/M checkpoint prevents cells from premature entry into mitosis.The intra S checkpoint helps to delay the fringe of replication of origins or slows down DNA duplication during S phase.The mitotic spindle checkpoint ensures normal spindle function in order to minimize chromosome segregation errors.The post mitotic checkpoint can prevent daughter cells of abnormal mitosis from entering the next interphase.

Discussion46Genetic Instability is a hall mark of cancer.Nucleotide instability is the rarest among three types of genetic instabilities.Ionizing radiation, dietary factors, lack of nutrition will also can be considered as causes of genetic instability.Detection methods of genetic instabilities exhibit both advantages and disadvantages. Those techniques must be developed.It is an argument .the role of genetic instability in cancer.

Summary 47Genetic Instability Various definitions, but have common features.Three types of genetic Instability.Causes of Genome Instability .Single cell and multi cell approaches in methods for the detection and analysis of genomic instability.Genetic Instability as a major reason for cancer.Four major mechanisms used to maintain genomic stability.

References 48Abdel-Rahman, W. M., Katsura, K., Rens, W., Gorman, P. A.,Sheer, D., Bicknell, D.(2001). Spectral karyotyping suggests additional subsets of colorectal cancers characterized by pattern of chromosome rearrangement. Proceedings of the National Academy of Sciences of the United States of America,98(5), 25382543.Anderson,G.R.(2001)Genomic Instability in Cancer.Current Science,18(5):501-507Beeckmann,A.R. and Loeb,A.L.(2005) Genetic onstability in Cancer:Theory and experiment.Seminars in Cancer Biology,15:423-435.Aguilera,A.and Gonzales,B.G.(2008) Genomic Instability: a mechanistic view of its causes and consequences.Nature,9:204-217.Aguilera,A. and Garcia-Muse,T.(2013) Causes of Genome Instability.Annual Review of Genetics,47:19-50.

49Charames,G.S. and Bopat,B.(2003) Genomic Instability and Cancer.Current Molecular Medicine,3 : 589-596.Chen,H.Maxwell,C. Connel,M. (2015) The Generation,Detection and Prevention of Genomic Instability During Cancer Prognosis and Metastasis.Cancer Metastasis,20:15-26.Dworaczek, H., and Xiao, W. (2007). Xeroderma pigmentosum: a glimpse into nucleotide excision repair, genetic instability, and cancer. Critical Reviews in Oncogenesis, 13(2), 159177.Gagos, S., and Irminger-Finger, I. (2005). Chromosome instability in neoplasia: chaotic roots to continuous growth. The International Journal of Biochemistry & Cell Biology, 37(5), 10141033.Kwei,K.A. Kung,Y. Salari,K. Holcomb,N.I. Pollock,J.R. (2010) Genomic Instability in breast cancer: Pathogenesis and clinical impications.Molecular Biology, 4 : 255-266.Leach, F. S., Nicolaides, N. C., Papadopoulos, N., Liu, B., Jen,J., Parsons, R., et al. (1993). Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer.Cell, 75(6), 12151225.

50Lengauer,C. Kizler,K.W. Vogelestein, B.(1998) Genetic Instabilities in Human cancers.Nature,396:643-649.Muresu, R., Sini, M. C., Cossu, A., Tore, S., Baldinu, P., Manca,A.(2002). Chromosomal abnormalities and microsatellite instability in sporadic endometrial cancer. European Journal of Cancer, 38(13), 18021809.Pikor,L. Thu,K. Vucic,E. Lam,W.(2013) The detection and implication of genome instability in cancer.Cancer Metastasis,32:341-352.Rao,C. and Yamada,H.Y.(2013) Genomic Instability and colon carcinogenesis;from the perspective view of genes.Frontiers in Oncology,3:1-13.Schvartsman,J.M. Sotillo,R. Benzera,R.(2010) Mitotic Chromosomal Instability and cancer :Mouse modelling of the human disease.Nature.10:102-115. Shen,S.(2011) Genomic Instability and Cancer:an Introduction.Journal of Molecular Biology,3:1-3.

51Sieber,M.O. Heinmann,K.Tomlinson,P.M.(2003) Genomic Instability ,The engine of Tumorigenesis?.Cancer,3:701-707.Tassan, N., Chmiel, N. H., Maynard, J., Fleming, N.,Livingston, A. L., Williams, G. T.,(2002). Inherited variants of MYH associated with somatic G:CT:A mutations in colorectal tumors. Nature Genetics, 30(2), 227232.

52

53