Download Genetic determination of diseases

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