Variability and changes of
genetic information, mutations
RNDr Z.Polívková
Lecture 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 mutations
Single 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 GA → 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 gene
d) 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)
Mutagens
Physical: 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 cancers
Forms: 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 cancers
5% 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 tumors
Li 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
Mutation +
immunosuppression
Viruses
Complex origin of tumors
Family with inherited mutation of TP53
Multistep origin of colon cancer - multiple genetic changes
Mutation/deletion
tu su gene MCC 5q
1.st step also heritable change– mutation on 5q –in polyposis
coli, Gardner sy
Increased
proliferation
Adenoma III
Mutation K-ras
oncogene 12p
Adenoma I
Mutation/deletion
chrom.loss
tu su gene p53 on 17p
Mutation/deletion,
chrom.loss
tu su gene DCC 18q
Normal cell
Adenoma II
DNA
hypomethylation
Carcinoma
metastasis
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