Download Genetic variability

Genetic determination of
diseases
Heritability
Genetic variability (mutations 
polymorphism)
Monogenic  complex diseases
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Genetics, genomics
 genetics
– specialised field of biology focusing on variability
and heritability in living organism
 human genetics
 clinical genetics
 genetics of pathological states, diagnostics, genetic counselling
and prevention (family members)
– cytogenetics
 chromosome alterations
– molecular genetics
 study of the structure and function of isolated genes
– population genetics
 study of variability in populations
– comparative and evolutionary genetics
 inter-species comparisons and evolution of species
 genomics
– study of the structure and function of genomes by means of
genetic mapping, sequencing and functional analysis of genes
– aims to understand entire information contained in DNA
 structural genomics = structure of genomes
 construction of detail genetic, physical and transcriptional maps of genomes
with ultimate aim to complete entire DNA sequence (e.g. HUGO project)
 functional genomics = function of genes and other parts of genome
 understanding of the function of genes; very often using model organisms
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(mouse, yeast, nematodes, Drosophila etc.) as an alternative to higher
organisms (many generations in relatively short time)
Nucleoside  nucleotide  base  DNA
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DNA replication
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Gene
 DNA contains defined

regions called genes – basic
unit of heritability
gene = segment of DNA
molecule containing the code
for (m r t) RNA sequence and
necessary regulatory
sequences for the regulation
of gene expression
– promoter (5’-flanking region)
 binding sites for transcription
factors
– exons
– introns
– 3’ untranslated region (UTR)
 transcription creates RNA
– 1) hnRNA is complementary to
the entire gene (1. exon 
poly-A tail)
– 2) mRNA formed by slicing of
introns from hnRNA
 translation forms proteins
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RNA splicing
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Translation
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Translation – tRNA / amino acid
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Genetic code
 determines the
sequence of AA
in protein
– universal
 Similar/the same
principle in most
living organisms
– triplet
 combination of 3
out 4 available
nucleotides (A, C,
G, T)
– degenerated
 43 = 64, but only
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21 AA
Chromatin  chromatide  chromosome
 DNA is organised in
chromosomes
– chromatin + chromosomal
proteins (histones)
 chromosome = linear sequence


of genes interspaced by noncoding regions
chromatin is in a relaxed form in
the nucleus in non-dividing cells
it becomes highly
organised/condensed into visible
chromosomes in dividing cells
– prometaphase/metaphase
 structure of chromosome
– centromere/telomeres
– arms
 long - q
 short – p
 2 copies of a given chromosome
after replication (before
cytokinesis) = sister
chromatides
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Human karyotype
 set of chromosomes characteristic for a
given eukaryote species (number and
morphology)
– human
 somatic cells are diploid (46 chromosomes)
 22 pairs of homologous autosomes
 1 pair of gonosomes (44XX or 44XY)
 gametes (oocyte, spermatide) 23 – haploid
– mouse 40 chromosomes
– crayfish 200 chromosomes
– fruit flies 8 chromosomes
 examination of karyotype (karyogram)
– synchronising of cell division in metaphase by
colchicin
– staining by dyes (e.g. Giemsa) leads to the
characteristic band pattern
– standard classification by numbering according
to the size
 assessment and interpretation of karyogram
– manual – most often lymphocytes or fetal cells
from amniotic fluid obtained by amniocentesis
 photography and manual pairing
– automatic (microscopy + software)
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Cell division
 mitosis
– 1 cycle of DNA replication followed by
chromosome separation and cell division
 prophasis  prometaphasis  metaphasis
 anaphasis  telophasis  cytokinesis
– 2 daughter cells with diploid number of
chromosomes
 meiosis (“to make small”)
– 1 cycle of replication followed by 2 cycles
of segregation of chromosomes and cell
division
 1. meiotic (reduction) division –
separation of homologous chromosomes
 significant! – meiotic crossing-over

(recombination) – none of the gametes is
identical!
abnormalities of segregation – nondisjunction - e.g. polyploidy, trisomy, …
 2. meiotic division – separation of sister
chromatides
– humans
 oogonia  oocyte + 3 polar bodies
 very long period of completion, thus
vulnerable
 spermatogonia  4 sperms
 continually
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Mitosis - detail
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Crossing-over and recombination
 each gamete formed receives randomly 1 ch. of the homologous pair
of chromosomes - paternal (CHp) or maternal (CHm)
– given 23 ch. pairs there is theoretically 223 possible combinations (=
8,388,608 different gametes)
 in fact, each gamete contains a mixture of homologous CHm and CHp
due to the process during 1st meiotic division = crossing-over and
recombination
 thus alleles originally coming from different grandparents can appear in one
chromosome
– creates much greater number of combinations than 8 millions
 however, probability of recombination is not the same in all parts
of DNA, it depends on the distance (linkage disequilibrium / haplotype
block)
– the closer the genes are, the lesser is the probability of recombination
 such length is expressed in centiMorganes (1cM = 1% probability of
recombination)
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Gene  allele  genotype  phenotype
 gene – basic unit of heritability
– gene families
 sequence similarity among genes formed e.g. by
duplication during evolution
 hemoglobin chains, immunoglobulins, some isoenzymes, …
– pseudogenes
 similar to functional genes by non-functional
 each gene occupies particular site in the
chromosome = locus (e.g. 12q21.5)
– localisation of genes in the same in species but
sequence is not!
 allele – sequence variant of gene
– vast majority of genes in population has several
variants (= alleles) with variable frequency =
genetic polymorphism
 genotype – combination of alleles in a given


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locus in paternal and maternal chromosomes in
diploid genome
haplotype – linear combination of alleles in a
single ch. of homologous pair
phenotype – expression of genotype
– trait –measurable, very often continuous variable
 QTL – quantitative trait locus (e.g. weight, height, …)
– phenotype – set of traits
– intermediate phenotype – similar to trait but not
always continuous
Human genome
 Human Genome Project (HUGO)
– ~3.3109 bp in haploid genome
– only ~3% coding sequences
– ~30 000 genes expressed in variable
periods of life
 ~25 000 proteins
 the rest are RNAs and others regulators
– ~75% formed by unique (nonrepetitive) sequence, the rest are
repetitions
 function is not clear, could be structure

effects or evolutionary reserve
types of repetitions
 tandem


» microsatellites
» minisatellites
Alu-repetitions
L1-repetitions
 density of genes in and between each

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chromosome is quite heterogeneous
mitochondrial DNA
– several tens of genes coding proteins
involved in mitochondrial processes
 respiratory chain
– inherited from mother!
Microsatellites
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Genetic variability
 DNA sequence of coding as well as
non-coding regions of genome is
variable in each individual
 genetic variability = v existence
of several variants (alleles) with
various frequency for a given
gene in population
 sources:
 1) sexual reproduction
 2) recombination (meiotic

crossing-over)
3) mutations de novo
 “errors” during DNA replication
» proof-reading of DNA
polymerase is not 100%
 effect of external mutagens
 4) effects on the population level
(evolution) – Hardy/Weinberg law
 natural selection = adaptive
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(reproductive success)
 genetic (allelic) drift = random
selection of alleles (entirely from
chance)
» “founder” effect
Evolution – selection for continually
changing environment??
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Types of DNA substitutions
 1) genome
– number of chromosomes (trisomy,
monosomy)
– sets of chromosomes (aneuploidy,
polyploidy)
 2) chromosomal (aberrations)
– significant structural change of
particular chromosome
 duplication, deletion, insertion,
inversion, translocation, …
 3) gene
– shorter (1 – thousands of bp) = the
true source of population genetic
variability
 point variants (transitions and
transversions)
 often bi-allelic single nucleotide
polymorphisms (SNPs) ~ 6 000 000 in
human genome (HapMap project)
 length variants
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 repetitions (microsatellites! (e.g. CA12)
 deletions (1bp – MB)
 insertions + duplications
 inversions
Mutation vs. polymorphism
 based on population frequency !!!
– mutation = minor allele population frequency (MAF) <1%
– polymorphism = existence of several (at least 2) alleles for given gene with MAF  1%
 sometimes are mutations vs. polymorphisms classified according to the functional impact
(mutations = significantly pathogenis, polymorphisms = mild or neutral)
 functional effects of substitutions – depends on the localisation in the gene!
– coding regions (exons)
 none (“silent”)
 new stop-codon and lack of protein (“nonsense”) – e.g. thalasemia, …
 AA exchange (“missense”) – e.g. pathological haemoglobins, …
 shift of the reading frame (“frameshift”) – e.g. Duchenne muscular dystrophy, Tay-Sachs, …
 expansion of trinucleotide repetition – e.g. Huntington disease, …
 deletion of protein – e.g. cystic fibrosis
 alternative splicing – qualitative (structure) as well as quantitative effect (affinity, activity,
stability)
– non-coding regions
 5’ UTR (promoters) = quantitative effect (e.g. variable transcription)
 introns - qualitative effect (splicing sites) or quantitative effect (binding of repressors or
enhancers)
– 3’ UTR - effect on mRNA stability (“gene-dosage effect”)
 pathologic consequences
– gametes  genetically determined (inherited) diseases
– somatic cells  tumors
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Missense and frameshift substitutions
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Interindividual variability
 physiological interindividual
variability of phenotypes/traits is a
consequence of genetic variability
– the more independent factors affect the
given trait the more “normal” the
population distribution is
– if the effect of one factor dominates over
the others or there are significant
interactions the distribution becomes
asymmetrical, discontinuous etc.
 interindividual variability of a given
trait is present in whole population
incl. healthy as well as diseases
subjects
– disease as a “continuous function of the
trait”
 aetiology of diseases
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– “monofactorial” incl. monogenic
– “multifactorial” incl. polygenic (complex)
Genetic determination of disease
 practically every diseases (i.e. onset, progression and outcome)
is, to some extent, modified by genetic make-up subject;
however, under the different mode
 with except of trauma, serious intoxications and highly virulent
infections
– monogenic diseases
 single critical “error” (allele) of a single gene is almost entirely

responsible for the development of disease (phenotype)
characteristic pedigree (segregation of phenotype ) due to the mode of
inheritance (recessive x dominant)
– chromosomal aberrations - inborn but nor inherited!
– complex (polygenic) diseases
 genetic dispositions + effect of non-genetic factors
 combination of several
alleles in several loci
 what indicates that disease is,
at least partly, genetically
conditioned ??
 familiar aggregation
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» prevalence in families of
affected probands >>>
prevalence in general
population
Complex diseases

diseases developing due to the ethiopathogenic
“complex“ of genetic, epigenetic and environmental
factors
–

“predisposing genes/alleles” increase probability
to become affected, however, do not determine
unequivocally its development
–

phenotype does not follows Mendel rules (dominant or
recessive mode of inheritance)
–
effect of non-genetic factors is a necessary modifier
 diet, physical activity, smoking, ….
genes interact between themselves
typical features of complex diseases
–
–
–
–
incomplete penetrance of pathological phenotype
 some subjects eho inherited predisposing alelles never
become ill
existence of phenocopies
 pathological phenotype can develop in subjects not
predisposed, entirely due to the non-genetic factors
genetic heterogeneity (locus and allelic)
 manifestation (clinical) is not specific but the same syndrom
can develop as a consequence of various loci (= locus
heterogeneity) in which there could be several variants (=
allelic heterogeneity)
polygenic inheritance
 predisposition to disease is significantly increased only in the
presence of the set of several risk alleles (polymorphisms),
hence their high population frequency
–

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 in isolated occurrence the effect is mild
other modes of transmission
 mitochondrial, imprinting (<1% of all alleles in genome)
examples of complex diseases: essential
hypertension, diabetes (type 1 and 2), dyslipidemie,
obesity, atopy, Alzheimer disease, …
Genetic epidemiology
 there are a lot of methods available suitable
for different problems
– positional mapping - linkage studies
 follows the transmission of genetic marker (most
often microsatellite) and phenotype (affected vs.
unaffected subjects)
 group of related subjects (family)
 trios of both parents and affected child (transmission

disequilibrium test, TDT)
sibling pairs
» concordant (both affected)
» discordant (1. yes, 2. no)
 parametric = known/estimated model of
inheritance (suitable for monogenic diseases)
 non-parametric = unknown mode of inheritance
(suitable for some complex diseases)
 association studies
 compare frequencies of genetic marker(s) (most
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often SNPs) between phenotypically disparate
groups of unrelated subjects
 case x control
 selection of genes is either pathogenetically based
(hypothesis-driven) or random (hypothesis-free)
 number of genes/alleles studied – 1 to n
 whole genome association (WGA) ~ 500 000 SNPs
 subtypes of studies
 cross-sectional
 retrospective
 prospective
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