Medical Genetics Kateřina Staňo Kozubík, Michael Doubek zakladni_EN Miroslav Barták Why medical genetics ØGenome role in diagnostics, therapy and prevention = application in medical practice It is possible to implement into practice only what I know and what I have in mind What you should already know Ø What is a gene genes: structural for functional RNAs - housekeeping genes - gene expression - exones, intrones, non-transcribed regions, promotors Ø ØInformational macromolecules Ø ØTranscription, alternative splicing, translation Ø ØChromosomes What is a DNA? cold ethanol + salt + detergent > 1 m DNA Věda je zábava, Svojtka & Co, 2015 Kočárek, 2013 Cavendish Laboratory and The Eagle Pub Watson + Crick + Wilkins + Franklin Image result for dna discovery cambridge Cavendish Laboratory and The Eagle Pub Watson + Crick + Wilkins + Franklin Image result for dna discovery cambridge ? Using results without permit Eugnenics Black men’s IQ ØGenetics: study of genes, genetic variation, and heredity in living organism Ø ØGenome: complete set of DNA within a single cell of an organism Ø ØGenomics: focuses on the structure, function, evolution, and mapping of genomes Terminology ØGenetics Ø ØGenome Ø ØGenomics Genome is more than just a sum of genes Terminology ØGenetics Ø ØGenome Ø ØGenomics - Structural (DNA, chromosomes) - Functional (RNA, gene expression) - Comparative Terminology ØGenetics Ø ØGenome Ø ØGenomics Ø ØMicrobiome Ø ØTranscriptome Ø ØEpigenetics Terminology ØGenetics Ø ØGenome Ø ØGenomics Ø ØMicrobiome Ø ØTranscriptome Ø ØEpigenetics Community of microorganisms inhabiting a particular environment Terminology ØGenetics Ø ØGenome Ø ØGenomics Ø ØMicrobiome Ø ØTranscriptome Ø ØEpigenetics Set of all RNA molecules in one cell or a population of cells in certain time Terminology ØGenetics Ø ØGenome Ø ØGenomics Ø ØMicrobiome Ø ØTranscriptome Ø ØEpigenetics Terminology Study of heritable changes in gene function that do not involve changes in the DNA sequence What is a genome? Human genome: 3.2 x 109 bp, ~ 20,000 genes karyotypes.jpg What is a genome? Human genome: 3.2 x 109 bp, ~ 20,000 genes karyotypes.jpg Genome Variability § Nucleotide polymorphism - Single Nucleotide Polymorphisms - SNP § Structural variations - Copy Number Variations – CNV - Short Tandem Repeats – STR (2-5) Human genome was published in 2001 Individual sequences of human genomes were published in 2007 and 2008 220px-James_D_Watson_Genome_Image 220px-Craigventer2 J. D. Watson C. Venter 220px-James_D_Watson_Genome_Image 220px-Craigventer2 J. D. Watson Difference in 7648 amino acid substitutions C. Venter Individual sequences of human genomes were published in 2007 and 2008 220px-James_D_Watson_Genome_Image 220px-Craigventer2 J. D. Watson The 1000 genome project published in 2010 C. Venter Individual sequences of human genomes were published in 2007 and 2008 Moore’s law (1965): „The number of transistors (hence the processing power) that can be squeezed onto a silicon chip of a given size will double every 18 months”. Postgenomic era ØGenomes were described Ø ØOngoing genomes annotations Genetics today from phenotype to genotype from genotype to phenotype DNA RNA protein fenotype Modern techniques of genome analysis intron exon protein DNA strand gene whole genome all exons = exome < 1.5% of whole genome Whole-Genome Sequencing vs. Whole-Exome Sequencing •human Genome •= 3.2 * 109 bp •~ 20 000 genes • •Exome = < 1.5% of human genome •contains ~ 85% of known disease causing mutations NGS – flexibility whole genome exome targeted genes or hotspots 3 200 000 000 bp 30 x coverage 20 000 genes 100 x coverage < 100 genes ≥ 1000 x coverage Generating a Person’s Genome Sequence Break genome into small pieces Generate millions of sequence reads Align sequence reads to establish reference sequence Reference Sequence Deduce starting sequence and identity differences from reference sequence Genomic DNA Genomic DNA Reference Sequence Whole-Genome Sequencing Whole-Exome Sequencing Capture library Mutation vs. human genome variability •Every 1000th base could be mutated Þ 3.2 x 106 variants •One men has approx. 0.5 x 106 variants •Exome analysis (1.5% of genome) Þ tens thousands of variants • Which of the found variants is the disease causing one? Mutation vs. human genome variability •Every 1000th base could be mutated Þ 3.2 x 106 variants •One men has approx. 0.5 x 106 variants •Exome analysis (1.5% of genome) Þ tens thousands of variants • • Which of the found variants is the disease causing one? mutation frequency 1.1 – 1.3 x 10-8 Mutation vs. human genome variability Mutation vs. human genome variability •Every 1000th base could be mutated Þ 3.2 x 106 variants •One men has approx. 0.5 x 106 variants •Exome analysis (1.5% of genome) Þ tens thousands of variants • • Which of the found variants is the disease causing one? mutation x polymorfisms Mutations: spontaneous vs. induced gene vs. chromosomal Mutations: missense nonsense (terminating triplet) same sense frameshift Mutation vs. human genome variability Mutation vs. human genome variability Mutation vs. human genome variability Microsatelites (STR) Mutation vs. human genome variability Sickle-cell anemia Mutation vs. human genome variability Sickle-cell anemia Thalassemia Leiden mutation of f. V Hemochromatosis Cystic fibrosis Positive mutations File:Portulaca grandiflora mutant1.jpg mutation Germinal vs. somatic mutations Germinal vs. somatic mutation Germinal mutation Somatic mutation Are we Homo sapiens? Reich a kol. Nature 2010 Denisova hominins, 41 000 years ago mtDNA Are we Homo sapiens? https://3c1703fe8d.site.internapcdn.net/newman/gfx/news/hires/2016/aworldmapofn.jpg Europe 1 – 3% of the genome Some diseases of Neanderthal origin – depression, skin disorders? Are we Homo sapiens? Ancient genomes analysis Broushaki a kol., Science 2017 Genome from the Younger Stone Age and Iron Age - Zagros, Iran Broushaki a kol., Science 2017 Two ancient genomes in modern humans Famous ancient genomes Őtzi Cheddar man https://upload.wikimedia.org/wikipedia/commons/e/ee/Otzi-Quinson.jpg Origin based on mitochondrial DNA Mitochondrial and Y-inheritance father mother The seven daughters of Eve Katrine 16 000 years Tara 17 000 years Helena 20 000 years Velda 17 000 years Ursula 30 – 50 000 years Xenia 25 000 years Jasmine 43 000 years The seven daughters of Eve Bryan Sykes, 2004 Ø mitochondrial Eve (140 000 years ago in Ethiopia) Ø Ø 7 main mitochondrial haplotypes in Europe Ø Ø 29 haplotypes worldwide Ø Ø ØResults are not as accurate as from other methods – mtDNA is very similar offspring of the mitochondrial Eve may still live The seven daughters of Eve in the Czech population ØHelena 43.51% (dominant lineage from Polland and european part of Russia) ØUrsula 17.6% (mainly UK and Scandinavia) Ø ØTara 11.17% Ø ØJasmine 8.78% Ø ØKatrine 5.89% (Ashkenazi Jews) Ø ØVelda 4% Ø ØXenia 3% The seven daughters of Eve after the Ice Age Katrina 16 000 years Tara 17 000 years Helena 20 000 years Velda 17 000 years Ursula 30 – 50 000 years Xenia 25 000 years Jasmine 43 000 years The seven daughters of Eve after the Ice Age Katrina 16 000 years Tara 17 000 years Helena 20 000 years Velda 17 000 years Ursula 30 – 50 000 years Xenia 25 000 years Jasmine 43 000 years ØMendelian hereditary diseases 8% Ø ØMultifactorial 90% Ø ØOthers 2% The role of genome in the disease onset ØMendelian hereditary diseases 8% Ø ØMultifactorial 90% Ø ØOthers 2% Ø Ø Þ genetic background plays almost always a role in the disease onset The role of genome in the disease onset Inheritance types Inheritance types Mendelian monogenic: one gene Þ one feature X-linked and Y-linked (sex-linked disorders) Polygenic several genes Þ one feature Mitochondrial Environmental factors What is the procedure of hereditary diseases tracing? Ø family studies: pedigree monozygous twins odds ratio relative risk Ø Ø disease frequency in population Ø Ø molecular biology methods Ø Ø genetic linkeage and functional tests Common ancestor •two common ancestors in previous generation: parents •4 grandparents, 8 great-grandparents •the number of ancestors in generation n is 2n • • • • • • • • • •40th generation (1000 years back): 240 = 1.09 x 1012 •so many people didn’t live on this planet (7.0 x 109) Pedigree Mitochondrial inheritance Y-chromosome inheritance What Y-chromosome carries on? Y-chromosome inheritance Autosomal dominant inheritance Autosomal dominant inheritance Autosomal recessive inheritance Environmental factors Beer vepřo2 Bachovy esence a stres Monogenic disorders How many monogenic disorders exist? ≈ 1 000 ≈ 10 000 ≈ 100 000 ≈ 1 000 ≈ 10 000 ≈ 100 000 How many monogenic disorders exist? Recessive disorders Ø hemochromatosis (1:10) Ø mutation of factor V Leiden (1:20) Ø Ø cystic fibrosis (1:25) Ø Ø spinal muscular atrophy (1:40) Ø deafness Ø Ø polydactyly Ø Ø Huntington's Chorea Ø Ø Li-Fraumeni syndrome Ø breast and ovarian cancer Ø Dominant disorders Origin by mutation type ØFounder effect Ø ØSmall closed populations: Ashkenazi Jews franco-Canadiens Iceland surroundings od Maracaibo lake… Ø ØMarriages of relatives http://www.consang.net/images/c/c4/Globalcolourlarge.jpg Consanguinity map http://www.consang.net/images/c/c4/Globalcolourlarge.jpg Consanguinity map Mandatory genetic testing of partners: Bahrajn Saudi Arabia (Iceland) Consanguinity example Consanguinity example Homozygous mutation BLM gene c.1642C>T, p.(Gln548*) Genetic diseases in Czech population Nijmegen breakage syndrome = Seeman syndrome (NBS) NBN gene for nibrin in 8q21 Heterozygotes 1:130-150 Common ancestor Seemanová, 1985 Czech dysplasia COL2A1 gene absence of ocular and orofacial anomalies shortening of third and/or fourth toes Neumann, 2003 How do we search for new genetic disorders? Molecular biology methods Ø Analysis of known disease-associated genes Ø Ø Comparing genetic information of healthy and affected family members Ø Ø Looking for new variants and “new” genes Ø Ø Verification by functional tests DNA variants and disorders Ø genes together with non-coding regions Li-Fraumeni Syndrome DNA repair impairment Immunodeficiency NBS, BS, AT, SDS, CMMRD Epigenetic dysregulation Sotos, RSTS, Weaver Chromosomal aberrations +21, +8 (mosaic), rob. t(15;21) RASopathies Noonan syndrome Transcriptional factors impairment IKZF, PAX5, ETV6, CEBPA, RUNX1, GATA2 Twins Ø Genetic vs. nongenetic influences - monozygotic: 100% of identical alelleles - dizygotic: twins/siblings 50% of identical alelleles ØGenetic influence: (concordance in MZ and DZ twins): Diabetes mellitus Schizophrenia Lupus Cleft Sclerosis multiplex Are monozygotic twins genetically identical? Clinical cases Clinical case: thrombocytopenia analyzed samples ? ? cancer ? Breast cancer MPN – JAK2 p.V617F hyperdiploid c-ALL, allo PBSC •Exome sequencing – exome comparison of healthy and affected family members • variant in gene ETV6: p.W380R Obrázek1.png Adjusted from Moriyama et al., 2015 ETV6 - Mapping on a reference sequence (BWA-mem) - build-up of two variant files (Samtools mpileup): 1. file – affected family members 2. file – healthy family members Exclusion of population and familial variants (VarScan): Selection of variants present only in affected family members Identification of potentionally causal variants: variant anotation (Annovar) filters: coverage > 20 left only exonic, ncRNA exonic, downstream and upstream variants left out synonymous variants excluded variants annotated in dbSNP dtb. with rsXXXXX ID Bioinformatics and biostatistics Bioinformatics and biostatistics 10522.png sick-people-clipart-png-39.png 14696-200.png blood-analysis_318-61819.jpg 532018579.jpg 19375-200.png flaticon_crowd.png 169217-200.png ray2.png Variant effect on the protein structure Annotated mutations Variant frequency in population 14696-200 – adfadfsdfds.png 14696-200 – kopie.png Obrázek2.png Functional analysis of ETV6 : fluorescence microscopy Functional analysis of ETV6 : fluorescence microscopy Obrázek2.png CYCS: exon 2, p.T20I analyzed samples plt: 47.7*109/l plt: 23.3*109/l unavailable sample plt: 54.6*109/l plt:74.7*109/l ? 50 years ? 92 years ? Thr20 Lys26 Asn32 His19 Fe CYCS: p.T20I What are the skills of clinical geneticist? Ø complex examination Ø gene/s analysis indication - exome sequencing - genome sequencing - functional tests Ø results interpretation (from practitioners to clinical geneticists) Ø therapeutic and preventive intervenation proposal - respecting wishes of affected individuals together with ethical aspects What are the skills of clinical geneticist? Ø complex examination Ø gene/s analysis indication - exome sequencing - genome sequencing - functional tests Ø results interpretation (from practitioners to clinical geneticists) Ø therapeutic and preventive intervenation proposal - respecting the desire of affected individuals together with ethical aspects Complex information analysis – complex is more than the sum of individual parts Why genetics? Ø Disease diagnostics: prenatal preimplantational genetic counselling Ø Therapy: pharmacogenetics pharmacogenomics immunogenetics Ø Prevention Ø Gene therapy, genome editing CRISPR-Cas9 Made-to-order children?