Mutation = permanent heritable change of genetic material

= change in nucleotide sequence or arrangement of DNA in the genome

Genetic changes are frequent Most of genetic changes are functionally

insignificant, some have minor effect on phenotype, some are deleterious (cause genetic disorder or miscarriages)

Some genetic variants are called polymorphisms

Mutations x polymorphisms

Many genes have only one normal version = wild type allele

Other genes exhibit polymorphism (many forms) in population

Normal variants (alleles) are relatively common in population

Variant allele found in more than 1% in population = polymorphism;

this definition is independent of functional or pathogenetic relevance of alteration – most of common variants (polymorphisms) are without effect on human health, but some can modify the risk of common diseases (as tumors…)

Alleles with frequencies of less than 1% are rare variants

Mutant alleles are rare variants – identified through clinically significant disorder (disease-causing variants)

More mutant alleles at same locus (each capable of producing an abnormal phenotype)= allelic heterogeneity

But some of rare variants appear to have no deleterious effect,

i.e. there is „grey zone“ between definitions used for mutation

and polymorphism

Types of polymorphism:

Single nucleotide polymorphisms (SNPs)90 % of all DNA variants are exchanges of single nucleotide bases

- in coding or noncoding regions of human genome

Frequent SNPs are mostly without effect or have subtle effect (alter disease susceptibility)

Occurence 1/1000 base pairs

Other variants: deletions, duplications, multiplications of several nucleotides or larger genome segments

Different alleles are due to variable numbers of repetitions at particular location

Effect: some are without effect to human health, some are pathogenetic, some can modify risk of disease

Mutations:

Mutations: spontaneous (errors in replication)

induced (by mutagens)

Mutations: somatic – consequences: tumors, ageing

(accumulation of mutations)

gametic – consequences in next generation: genetic disorder or carrier of mutation

Mutations:

• genome mutations – changes in chromosome number:

a) euploid change = multiplication of haploid chromosome set

(triploidy, tetraploidy)

b) aneuploidy = additional chromosome (trisomy) or missing

chromosome (monosomy)

• chromosome mutations= structural aberrations of chromosomes

=consequences of breaks and abnormal rearrangement of

chromosomal segments – detected in light microscope

submicroscopic deletions or duplications of large genomic

segments = copy number variants CNVs (consequences:benign

or pathogenic, or risk modifiers for common diseases)

• gene mutations= qualitative or quantitative changes in DNA coding

sequences

point mutation affects one single base pair

GENE mutations

mutation without any change of amino acid (degeneration of genetic code)

• MISSENSE mutation.........replacement of one amino acid by different amino acid in protein

• NONSENSE mutation.........mutation generates one of three „stop“ codons → premature termination of translation

• ELONGATION mutations.....change of stop codon to amino acid coding triplet

• FRAMESHIFT mutations......insertions, deletions of coding nucleotides in a number not divisible by three

Mutations in rRNA and tRNA genes - error in translation

Mechanisms of mutations

Single nucleotide SUBSTITUTION = base exchange

– (transition = purine (A,G) for purine, pyrimidine (C,T) for pyrimidine or

transversion = purine for pyrimidine or vice versa)

e.g. base alkylation, oxidation, deamination leads to change of pairing

properties and change of nucleotide during replication

i.e. it alters triplet code

→ replacement of one amino acid by another in the gene product

(missense mutation)

consequences: enzyme inactivity or changed specificity

altered properties or structure of protein

→ stop codon (nonsense mutation) – loss of protein function

→ elongation mutations - loss of protein function

INSERTION

- frame shift mutations (number of bases involved is not divisible by three)

DELETION - alters translational reading frame→ premature stop codon

Mutation in promotor region affect gene expression

- impaired binding of transcription factors leads to reduced transcription

Mutation on the boundary of exons and introns interfere with proces of splicing

Mutations leading to loss of enzyme function

mostly express itself as recessive

Mutations leading to gain of abnormal function

or to origin of abnormal structural protein –

mostly express itself as dominant

Examples of mutations:A) SUBSTITUTION (alkylation, methylation, hydroxylation→error in base pairing)

nucleotide substitution = replacement of one amino acid by another → MISSENSE MUTATION a) Change inside coding sequences

• in sickle cell disease G A G → G T G in β-globin gene–replacement of amino acid glu → val HbA → HbS

b) Mutation outside coding sequences • in hemophilia B:

change A → G in promotor of gene for antihemophylic factor IX = prevention of transcription factor binding → decrease in the amount of product

→ NONSENSE MUTATION - generates stop codon → abnormal product

• in neurofibromatosis - NF1 gene C G A → T G A arg → stop NF1 = tumor supressor gene premature termination of translation

RNA SPLICING mutation – on boundary between exon and intronin Tay-Sachs disease mutation in hexosamidase A gene - intron between 12. and 3. exon is not removedDefect of hexosamidase A enzyme

B) DELETION, INSERTION

(deletion of 1 or more base-pairs, deletion of a part of gene, deletion of whole gene, or deletion of several genes = microdeletion syndromes)

a) small number of base-pairs (not divisible by three)

frameshift mutation• in ABO blood groups

deletion G T G → single base-pair deletion at the ABO locus alters reading frame (allele A → allele O)

• in Tay – Sachs disease

4 base-pairs insertion → frameshift leading to the origin of premature stop codon = deficiency of enzyme hexosaminidase A

b) 3 or a multiple of 3 bases

• in cystic fibrosis

the most frequent mutation = 3 base-pair deletion → 1 amino acid is missing (delta F 508 = fenylalanin is missing)

c) Total gene deletion• in X- linked ichtyosis

deletion of steroid sulphatase gened) Large deletion within gene

• in: Duchenne muscular dystrophy large deletion within dystrophin gene (in 60 % of cases)

Origin of large deletion and insertions:•Unequal crossing over or exchange between misaligned sister chromatids or homologous chromosomes (aberrant recombination)

• deletion of -globin gene in -thalasemia• deletions of pigment genes in X-linked defect in green and red color perception• deletion of retinoblastoma gene (Rb1)• Error in replication

MutagensPhysical: radiation

• UV (ultraviolet radiation) → T-T, C-C, T-C dimers (covalent bonds) → error in replication and transcription• ionizing (rtg, gamma)

direct effect → DNA breaks indirect effect = ionization of molecules → DNA breaks

Chemical – alkylating agents – adducts – base substitution - base analogs – error in base pairing - acridine dyes – insertions – frame shift mutations - nitric acid – base deamination – error in base pairing

direct mutagens

indirect mutagens – reactive product arises after metabolic activation (cytochrom dependent oxygenases)

Biological –viruses - viral nucleic acid integrates into the genome of host cell

Dynamic mutations

– gradual origin

= amplification of triplet repeats - in fragile X syndrome,

Huntington disease…

Origin through premutation in previous generation

This type of mutation is not caused by the environmental mutagens !

Genetics of cancersForms: sarcomas – mesenchymal tissue

carcinomas – epithelial tissue

hematopoetic and lymphoid malignancies (leukemias, lymphomas)

Uncontrolled growth – invasivity, metastases

Tumor cells in tissue culture:• loss of contact inhibition

• changes in surface antigens

• chromosomal changes

• unlimited number of cell generations

Genetic nature of cancers5% familiar (AD with reduced penetrance)

multifactorial

All cancers – mutations of specific genes in somatic

cells (growth controlling genes):

1. protooncogenes

2. tumor suppressor genes

3. mutator genes = genes involved in reparation→ increased

frequency of mutations

CARCINOGENESIS =multistep process – genetic + environmental factors

Multiple mutations (growth controlling genes)

Multiple causes and mechanisms

Environmental factors:

• chemical carcinogens• UV, ionizing radiation• tumor viruses – RNA, DNA viruses

Mutations – role in iniciation of carcinogenesis

Genetic change in one cell and division of cell

Clonal nature of tumors – origin from single cell

Protooncogenes: control of cell proliferation,

differentiation

Protooncogenes products:

role in cell communications

in transport of signal from cell surfice to the genes which

regulate cell cycle

Protooncogenes: signal molecules, their receptors,

tyrosin kinases, transcription factors, cell cycle

regulation proteins…

Change of protooncogenes to oncogenes →

abnormal cell division

Mechanisms:

1.gene mutation

2. translocation

3. retroviral insertion

4. amplification – double minutes, homogenously staining

regions = amplified copies of protooncogenes

5. error in gene methylation (gene expression) =

epigenetic changes

Consequences of change of protooncogene to oncogene

• synthesis of abnormal product

• increased synthesis of normal product

Dominant character of mutation of protooncogene (change in one allele)

Examples of chromosomal translocations involving protooncogenes:

CML = chronic myelogenous leukemia

Ph1 chromosome = t(9q;22q) = translocation of protooncogene c-abl from 9q to 22q near to protooncogene bcr → fused gene bcr/abl →abnormal protein with stable tyrosinkinase activity = abnormal stimulation of cell division

BL = Burkitt lymphoma – t (8q;14q)

Protooncogene c-myc transfered from 8q to 14q near to immunoglobuline genes (IgH) → abnormal transcriptional activity of protooncogene in a new position → increased synthesis of normal product

Cme.medscape.com

Fused gene bcr/abl in CML detected by locus specific probe (FISH)

Wysis 1996/97

Fused gene bcr/abl in CML

Translocation 8q/14q in Burkitt lymphoma

ncbi.nlm.nih.gov

Cytogenetic manifestation of amplification:• „double minutes“ – free copies of oncogene• HSR=homogenously staining regions= copies

tandemly integrated to chromosome• or copies inserted to different sites of chromosome

Tumor suppressor genes

Products - suppress cell division and abnormal proliferation

loss of function of both alleles→ malignant transformation

= recessive character of mutation

Example: RB – retinoblastoma – two-step origin of cancer

a) Hereditary tumor: bilateral

1st step = germline mutation (or deletion) of one allele of Rb1 gene = heritable or „de novo“ origin in one germ cell of parent (mutation in all cells of body = individual is heterozygote)

2nd step: somatic mutation of the 2nd allele in one cell of retina = loss of heterozygosity (LOH)

b) Sporadic form : unilateral

mutation of both alleles are somatic - in one cell of retina

tumor suppressor gene Rb1 gene on chromosome No 13

Wilms tumor: embryonal tumor of kidney

– tumor suppressor gene on 11p

Tumor suppressor gene TP53 – protein p53

Manager of genes involved in DNA reparation and apoptosis

• blocks cell cycle and starts reparation in G1 or G2 (cell cycle checkpoints)

• if DNA damage is unrepaired it starts apoptosis

Mutation of TP53 in many tumors

Li Fraumeni syndrome = heritable mutation of TP53 = tumor families = tumors in young people in family

Mutator genes

Genes responsible for DNA repair -

Mutations have recessive character

Example: heritable nonpolyposis colon cancer

Role of viruses in tumorigenesis

Neoplastic proliferation:

1. Integration of viral promoters („enhancers“) to the host

genome near the cell protooncogenes → increased expression

of the cell protooncogenes = latent tumor viruses

2. Insertion of viral oncogenes = acute tumor viruses

DNA viruses - oncogene = viral oncogene

RNA viruses – transmit cell protooncogene

Retroviruses = RNA tumor viruses

Their oncogenes – homologous to cell protooncogenes

viral oncogenes – without introns

Probable origin = from cell protooncogenes =

Integration of virus (DNA after reverse transcription) to host

genome, replication and transcription with host genome

mRNA protooncogene transcript after introns splicing is

„picked up“ by virus altogether with viral genome and

transfered to other cell (e.g. Rous sarcoma virus)

Viral infection:

Viral RNA → DNA (by reverse transcriptase)

integration to the host genome

replication, transcription with the host genome

translation – complete viral particules

oncogene product → cell transformation

Other factors of carcinogenesis

Different ability of metabolisation of mutagenic and carcinogenic compounds

Example:

enzyme aryl hydrocarbone hydroxylase (family of cytochrome P450 genes)

genetic polymorphisms in drug metabolisation

polycyclic hydrocarbons (from cigarette smoke) are converted to epoxydes (carcinogenic metabolites)

Individuals with high-inducible allele and smokers = great risk of lung cancer

Recessive homozygotes – resistant

Individuals with variant alleles – different activity of enzyme

DNA reparation – gene polymorphisms – differenet ability to repair DNA damage

Immunity

T lymphocytes – cell immunity – cytotoxic effect

defect in immunity, inborn or acquired(AIDS)→risk of tumors

Chemical carcinogens

Radiation

Viruses

Complex origin of tumors

Mutagenic, carcinogenic, immunosuppressive efects

Normal cellIncreased

proliferation Adenoma I

Adenoma II Adenoma III Carcinoma

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Multistep origin of colon cancer - multiple genetic changes

1.st step also heritable change– mutation on 5q –in polyposis coli, Gardner sy

Genotoxic effects:

• mutagenic

• carcinogenic

• teratogenic – affects embryonal development

• immunosuppressive

• allergenic

Each mutagen = possible carcinogen

But not all carcinogens are mutagenic (nongenotoxic carcinogens)

Thompson &Thompson: Genetics in medicine,7th ed. Chapter 9: Genetic variation in individuals and population: Mutation and polymorphism (till page 184)Chapter 16: Cancer genetics and genomics

+ informations from presentation

http://dl1.cuni.cz/course/view.php?id=324