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Genetic determination of diseasesHeritabilityGenetic variability (mutations ×polymorphism)Monogenic × complex diseases

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Genetics, genomicsgenetics– specialised field of biology focusing on variability

and heritability in living organismhuman geneticsclinical genetics

genetics of pathological states, diagnostics, genetic counselling and prevention (family members)

– cytogeneticschromosome alterations

– molecular geneticsstudy of the structure and function of isolated genes

– population geneticsstudy of variability in populations

– comparative and evolutionary geneticsinter-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 genomesconstruction 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 genomeunderstanding of the function of genes; very often using model organisms (mouse, yeast, nematodes, Drosophila etc.) as an alternative to higher organisms (many generations in relatively short time)

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Nucleoside × nucleotide × base × DNA

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DNA replication

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GeneDNA contains defined regions called genes – basic unit of heritabilitygene = segment of DNA molecule containing the code for AA 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 codedetermines the sequence of AA in protein– universal

similar principle in most living organisms

– tripletcombination of 3 out 4 available nucleotides (A, C, G, T)

– degenerated 43 = 64, but only 21 AA

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Chromatin × chromatide × chromosomeDNA is organised in chromosomes– chromatin + chromosomal

proteins (histones)chromosome = linear sequence of genes interspaced by non-coding regions chromatin is in a relaxed form in the nucleus in non-dividing cellsit becomes highly organised/condensed into visible chromosomes in dividing cells– prometaphase/metaphase

structure of chromosome– centromere/telomeres– arms

long - qshort – p

2 copies of a given chromosome after replication (before cytokinesis) = sister chromatides

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Human karyotypeset of chromosomes characteristic for a given eukaryote species (number and morphology) – human

somatic cells are diploid (46 chromosomes) 22 pairs of homologous autosomes1 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 sizeassessment and interpretation of karyogram– manual – most often lymphocytes or fetal cells

from amniotic fluid obtained by amniocentesisphotography and manual pairing

– automatic (microscopy + software)

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Cell divisionmitosis– 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 – non-disjunction - e.g. polyploidy, trisomy, …

2. meiotic division – separation of sister chromatides

– humansoogonia → oocyte + 3 polar bodies

very long period of completion, thus vulnerable

spermatogonia → 4 spermscontinually

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Mitosis - detail

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Crossing-over and recombinationeach 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 CHpdue 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 × phenotypegene – basic unit of heritability– gene families

sequence similarity among genes formed e.g. by duplication during evolution

hemoglobin chains, immunoglobulins, some isoenzymes, …

– pseudogenessimilar 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 locus in paternal and maternal chromosomes in diploid genomehaplotype – linear combination of alleles in a single ch. of homologous pairphenotype – 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

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Human genomeHuman 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 proteinsthe rest are RNAs and others regulators

– ~75% formed by unique (non-repetitive) sequence, the rest are repetitions

function is not clear, could be structure effects or evolutionary reserve types of repetitions

tandem » microsatellites» minisatellites

Alu-repetitionsL1-repetitions

density of genes in and between each chromosome is quite heterogeneousmitochondrial DNA– several tens of genes coding proteins

involved in mitochondrial processesrespiratory chain

– inherited from mother!

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Microsatellites

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Genetic variabilityDNA sequence of coding as well as non-coding regions of genome is variable in each individualgenetic variability = v existence of several variants (alleles) with various frequency for a given gene in populationsources:

1) sexual reproduction2) recombination (meiotic crossing-over)3) mutations de novo

“error” 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 (reproductive success)genetic (allelic) drift = random selection of alleles (entirely from chance)

» “founder” effect20

Evolution – selection for continually changing environment??

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Types of DNA substitutions1) 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 variantsrepetitions (microsatellites! (e.g. CA12)deletions (1bp – MB)insertions + duplicationsinversions 22

Mutation vs. polymorphismbased 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 regions5’ 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 variabilityphysiological 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– “monofactorial” incl. monogenic – “multifactorial” incl. polygenic (complex)

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Genetic determination of diseasepractically 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 virulentinfections

– monogenic diseasessingle 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» prevalence in families of

affected probands >>> prevalence in general population 26

Complex diseasesdiseases developing due to the ethiopathogenic “complex“ of genetic, epigenetic and environmental factors

– 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

– effect of non-genetic factors is a necessary modifierdiet, physical activity, smoking, ….

– genes interact between themselvestypical features of complex diseases

– incomplete penetrance of pathological phenotypesome subjects eho inherited predisposing alelles never become ill

– existence of phenocopiespathological 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 syndromcan develop as a consequence of various loci (= locus heterogeneity) in which there could be several variants (= allelic heterogeneity)

– polygenic inheritancepredisposition to disease is significantly increased only in thepresence of the set of several risk alleles (polymorphisms), hence their high population frequency

in isolated occurrence the effect is mild

– other modes of transmissionmitochondrial, imprinting (<1% of all alleles in genome)

examples of complex diseases: essential hypertension, diabetes (type 1 and 2), dyslipidemie, obesity, atopy, Alzheimer disease, …

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Genetic epidemiologythere 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 studiescompare frequencies of genetic marker(s) (most often SNPs) between phenotypically disparate groups of unrelated subjects

case x controlselection 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 SNPssubtypes of studies

cross-sectional retrospectiveprospective 28