RNDr Z.POLÍVKOVÁ
LECTURE NO 421– COURSE : HEREDITY

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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

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
; 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 …)

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

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
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(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 G A → 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
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-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)
Biologica l –viruses - viral nucleic acid integrates into the genome of host cell

– 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 !

Forms: sarcomas – mesenchymal tissue carcinomas – epithelial tissue hematopoetic and lymphoid malignancies
(leukemias, lymphomas)
Uncontrolled growth – invasivity, metastases
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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

=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

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…

→ 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

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

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
1.st step also heritable change – mutation on 5q –in polyposis coli,
Gardner sy
Normal cell
Increased proliferation
DNA hypomethylation
Adenoma I
Mutation K-ras oncogene 12p
Adenoma II Adenoma III
Carcinoma metastasis

:
• 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 genom ics
+ informations from presentation http://dl1.cuni.cz/course/view.php?id=324