Variability and changes of genetic Variability and changes of genetic information, mutationsinformation, mutations

RNDr Z.PolívkováRNDr Z.Polívková

Lecture No 69 - GIELecture No 69 - GIE

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)

(variant allele found in more than 1% = polymorphism;

alleles with frequencies of less than 1% are rare variants)

Mutant alleles are rare – identified through clinically

significant disorder

more mutant alleles at same locus (each capable of

producing an abnormal phenotype)= allelic heterogeneity

Mutation = permanent heritable change of genetic material = change in nucleotide sequence or arrangement of DNA in the genome

Mutations: spontaneous

induced

Mutations: somatic – consequences: tumors, ageing

gametic – consequences in next generation:

genetic disorder or mutation carrier

Mutations:• genome mutations – changes in chromosomal number:

a) euploid change = multiplication of haploid

chromosomal set (triploidy, tetraploidy)

b) aneuploidy = additional chromosome (trisomy) or

missing chromosome (monosomy)• chromosome mutations= structural chromosomal

aberrations =consequences of breaks and abnormal

rearrangement of chromosomal segments

• gene mutations= qualitative or quantitative changes in DNA

sequences

GENE mutations • mutation without any change of amino acid (degeneration of genetic code)• MISSENSE mutation...........replacement of one amino

acid by another• NONSENSE mutation .........mutation generates one of

three „stop“ codons• ELONGATION mutations......change of stop codon to

amino acid coding triplet• FRAME SHIFT mutations........insertions, deletions

Mutations in rRNA and tRNA genes - error in translation

Mechanisms of mutationsSingle nucleotide SUBSTITUTION (point mutation)

alters triplet code → replacement of one amino acid by

another in the gene product→ enzyme inactivity or changed specificity of enzyme

(alkylating agents)

INSERTION - frame shift mutations (number of bases

involved is not a multiple of three)DELETION - alters translational reading frame

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 –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 exone and

introne

in Tay-Sachs disease

mutation in hexosamidase A gene - intron between 12. and

13. exone is not removed

Defect 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 a multiple of 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 hexosaminidase A

enzyme 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 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)

MutagensPhysical: radiation• UV (ultraviolet radiation) → T-T, C-C, T-C dimers =

error in replication and transcription• ionizing (rtg, γ)

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

Chemical – alkylating agents - adducts - base analogs – error in base pairing - acridine dyes – insertions

- nitric acid – base deamination – error in base pairing

direct mutagens indirect mutagens – reactive product arises after metabolic

activation (cytochrom dependent oxygenases)

Biological –viruses - viral nucleid 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

Clonal nature of tumors – from single cell

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

Clonal evolution of cancer

Genetic change in one cell and division of 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 increased 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 → 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

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 – 2 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 (individual is heterozygote)

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

b) Sporadic form : unilateral

both somatic mutation (of both alleles) 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

• if DNA damage is unrepaired it starts apoptosis

Mutation of TP53 in many tumorsLi Fraumeni syndrome = heritable mutation of TP53 = tumor families = tumors in young people in family

Mutator genes

Genes of DNA repair -

Mutation - 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 transcribtion with host genome

mRNA protooncogene transcript after introns splicing is

„picked up“ by virus altogether with viral genome

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

Rous sarcoma virus- cancers in chickens

4 genes: gag = capside protein

pol = reverse transcriptase

env= viral protein envelope

src = oncogene – membrane protein kinase

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

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

Mutation + immunosuppression

Family with inherited mutation of TP53

Normal cellIncreased

proliferation Adenoma I

Adenoma II Adenoma III Carcinoma

Muta

tion/d

ele

tion

tu s

u g

ene M

CC

5q

DNA hypomethylation Mutation K-ras

oncogene 12pM

uta

tion/d

ele

tion,

chro

m.loss

tu s

u g

ene D

CC

18

q

Muta

tion/d

ele

tion

chro

m.loss

tu

su g

ene p

53

on 1

7p

metastasis

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

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