Genetic determination of diseases

1. Heritability Genetic variability (mutations  polymorphism) Monogenic  complex diseases Genetic determination of diseases

2. 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 genomesby 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 (mouse, yeast, nematodes, Drosophila etc.) as an alternative to higher organisms (many generations in relatively short time)

3. Nucleoside  nucleotide  base  DNA

4. DNA replication

5. 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

6. RNA splicing

7. Translation

8. Translation – tRNA / amino acid

9. 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 21 AA

10. Chromatin  chromatide  chromosome • DNA 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 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

12. 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)

13. 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 – non-disjunction - 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

14. Mitosis - detail

15. 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)

16. 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 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

17. 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 (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-repetitions • L1-repetitions • density of genes in and between each chromosome is quite heterogeneous • mitochondrial DNA • several tens of genes coding proteins involved in mitochondrial processes • respiratory chain • inherited from mother!

18. Microsatellites

19. 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 (reproductive success) • genetic (allelic) drift = random selection of alleles (entirely from chance) • “founder” effect

20. Evolution – selection for continually changing environment??

21. 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 repetitions (microsatellites! (e.g. CA12) deletions (1bp – MB) insertions + duplications inversions Types of DNA substitutions

22. 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

23. Missense and frameshift substitutions

24. 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 • “monofactorial” incl. monogenic • “multifactorial” incl. polygenic (complex)

25. 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 prevalence in families of affected probands >>> prevalence in general population Genetic determination of disease

26. Complex diseases • diseases 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 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 • 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, …

27. 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 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