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VARIABILITY AND CHANGES
OF GENETIC INFORMATION,
MUTATIONS
RNDr Z.POLÍVKOVÁ
LECTURE NO 421– COURSE: HEREDITY
 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
DELETION
- frame shift mutations (number of bases involved
is not divisible by three)
- 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 GA → T G A
arg
→ stop
NF1 = tumor supressor gene
premature termination of translation
RNA SPLICING mutation – on boundary between exon and intron
in Tay-Sachs disease
mutation in hexosamidase A gene - intron between 12. and 3. exon 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 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 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 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
Mutagens
Physical: 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 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
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 nature of tumors – origin from single cell
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 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
Mutagenic, carcinogenic,
immunosuppressive
efects
Complex origin of tumors
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
+ informations from presentation
http://dl1.cuni.cz/course/view.php?id=324