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21.9.2015gene15ab1 The Pathogenesis of Diseases from Genetic and Genomic Point of View Oliver Rcz and Frantiek Nitiar Oliver Rcz and Frantiek Nitiar Institute of Pathological Physiology Medical School, afrik University 2015 Slide 2 21.9.2015gene15ab2 26th june 2000 is neither the beginning nor the end of the way 5 years before term (1990 - 2005) 5 years before term (1990 - 2005) The race is over, victory for Craig Venter. The race is over, victory for Craig Venter. The genome is mapped* - now what ? The genome is mapped* - now what ? Not a discovery! Not a discovery! A very important technological result and competition is always useful. A very important technological result and competition is always useful. all is based on Mendels and Watsons & Cricks discoveries in XIXth XXth century all is based on Mendels and Watsons & Cricks discoveries in XIXth XXth century *3*10 9 letters Slide 3 21.9.2015gene15ab3 Mendel, Watson, Crick & the medical genetics of XIX th XX th century Mendels laws are valid also today Mendels laws are valid also today Watson & Crick provided the material basis of these laws (central dogma of molecular biology) Watson & Crick provided the material basis of these laws (central dogma of molecular biology) Mendels laws in medicine can be applied to monogenic diseases long list, relatively rare Mendels laws in medicine can be applied to monogenic diseases long list, relatively rare What is the genetics of diabetes, hypertension, coronary heart disease, Alzheimer disease ? What is the genetics of diabetes, hypertension, coronary heart disease, Alzheimer disease ? Slide 4 21.9.2015gene15ab4 White flower from red parents ??? Slide 5 21.9.2015gene15ab5 Slide 6 21.9.2015gene15ab6 Quidditch ball for Harry Potter ? Slide 7 21.9.2015gene15ab7 We discovered the secret of life, lets have a beer! Slide 8 21.9.2015gene15ab8 Slide 9 21.9.2015gene15ab9 The classics Mendel G: Versuche ber Pflanzen- Hybriden. Verh. Naturfortsch. Verein Brnn, 4, 1866, 3-47 Mendel G: Versuche ber Pflanzen- Hybriden. Verh. Naturfortsch. Verein Brnn, 4, 1866, 3-47 Watson.JD, Crick FHC: Molecular structure of nucleic acids. Nature 171, 1953, 737-738 Watson.JD, Crick FHC: Molecular structure of nucleic acids. Nature 171, 1953, 737-738 Slide 10 21.9.2015gene15ab10 Central dogma of molecular biology (cca. 1965) Replication Transcription Translation Transformation Slide 11 21.9.2015gene15ab11 Slide 12 21.9.2015gene15ab12 Central dogma of molecular biology Slide 13 21.9.2015gene15ab13 Central dogma of molecular biology? Epigenetics ? Role of RNA? Regulation of transcription Transcription factors, etc. Methylation, acetylation Regulation of RNA editing Alternative splicing Regulation of RNA transport Regulation of translation siRNA, tRNA modifications Postsynthetic modifications Slide 14 21.9.2015gene15ab14 Central dogma of molecular biology Slide 15 21.9.2015gene15ab15 Central dogma of molecular biology (now) role of RNAs Replication Transcription Translation Transformation Slide 16 21.9.2015gene15ab16 It is a little more complicated Slide 17 21.9.2015gene15ab17 We and our relatives Organism Genome size chromosomes/ genes Homo sapiens 3 000 000 000 23/30 000 Mus musculus 2 600 000 000 20/30 000 D. Melanogaster 137 000 000 4/13 000 C. Elegans 97 000 000 6/19 000 S. Cerevisiae 12 100 000 18/6 000 E. Coli 4 600 000 {1}3 200 HIV virus 9 700 9 A. Thaliana 100 000 000 ?/25 000 Slide 18 21.9.2015gene15ab18 What is not genetic The number of human genes is as low as 30 000 The number of human genes is as low as 30 000 the small worm C. elegans has 20 000 genes the mouse has as many genes as we, also with very similar function The mystery is in complexity and networking: 2 30000 >>>> 2 20000 (possible on/off states) The mystery is in complexity and networking: 2 30000 >>>> 2 20000 (possible on/off states) And a number of surprises around transcription and translation (miRNA, tRNA modifications) And a number of surprises around transcription and translation (miRNA, tRNA modifications) It is mapped but do we understand it? It is mapped but do we understand it? GENETICS = HEREDITY GENETICS = HEREDITY GENOMICS = EVERYTHING GENOMICS = EVERYTHING Slide 19 21.9.2015gene15ab19 GENES AND THE ENVIRONMENT GENOMEENVIRONMENT SEVERE MONOGENIC DISEASES LESS IMPORTANT MUTATIONS GENETIC RISK NEGATIVE AND POSITIVE ENVIRONMENTAL FACTORS physical chemical biological nutrition life style Slide 20 21.9.2015gene15ab20 Genes and diseases in practical medicine XIX th Century: symptom diagnosis sugar in urine = diabetes XX th Century: symptom etiopatogenesis diagnosis autoimmune destruction of cells = dm Type 1 XXI st Century: symptom genes and environment etiopatogenesis diagnosis susceptibility + overeating = subtypes of dm Type 2 Slide 21 21.9.2015gene15ab21 New era of preventive medicine Better understanding of disease pathogenesis Better understanding of disease pathogenesis Targeted, individualized prevention Targeted, individualized prevention Clear, unambiguous arguments Clear, unambiguous arguments Healthy genes healthy people Healthy genes healthy people Genes sana in corpore sano Genes sana in corpore sano Slide 22 21.9.2015gene15ab22 Mutations changes of genetic information THREE PRINCIPAL POSSIBILITIES THREE PRINCIPAL POSSIBILITIES 1.changes in genome not compatible with life 2.development and diversity 3.disease or increased risk fo disease* THE BASIC DIFFERENCE FOR HEREDITY: THE BASIC DIFFERENCE FOR HEREDITY: somatic and germ cell mutations genome, chromosomal and gene mutations genome, chromosomal and gene mutations no genes for diseases! sickle cell, Alzheimer, diabetes... Slide 23 21.9.2015gene15ab23 Gene mutations and SNPs* Point mutations in exons Point mutations in exons silent, missense (AA change) and nonsense (stop) Frameshift mutation in exons (1,2,4,5...) Frameshift mutation in exons (1,2,4,5...) Small deletion of triplets (3,6...) Small deletion of triplets (3,6...) Bigger deletions transition to chromosomal aberrations Bigger deletions transition to chromosomal aberrations Mutations in regulatory parts, introns, genes for r- tRNA Mutations in regulatory parts, introns, genes for r- tRNA Variability of repeated sequences - markers Variability of repeated sequences - markers Dynamic mutations triple repeat mutations Dynamic mutations triple repeat mutations *SINGLE NUCLEOTID POLYMORPHISM Slide 24 21.9.2015gene15ab24 Monogenic diseases with mendelian inheritance Autosomal recessive Autosomal recessive sickle cell disease thalassemia and other hemoglobinopathies cystic fibrosis enzymopathies (inborn errors of metabolism) Autosomal dominant Autosomal dominant familiar hypercholesterolemia X chromosome linked diseases X chromosome linked diseases hemophilia A, B; daltonism and von Willebrand disease, factor V Leiden, hereditary hemochromatosis... and von Willebrand disease, factor V Leiden, hereditary hemochromatosis... Slide 25 21.9.2015gene15ab25 Sickle cell disease Clinical description 1910, Hb abnormality, 1940 Clinical description 1910, Hb abnormality, 1940 Pauling / Ingram - 1 AA change in chain Pauling / Ingram - 1 AA change in chain Point mutation Glu Point mutation Glu Val on 6th place (GAG/GTG) Decreased solubility of Hb in low pO 2 Rigid, deformed red cells in venous blood Thrombosis, decreased life span of Er, hemolysis, icterus, anemia - HYPOXIA Epidemiology: 8 % of black people in USA are heterozygotes; 1:400 homozygotes 5 20 % heterozygotes in some parts of Africa Slide 26 21.9.2015gene15ab26 The molecular structure of human Hb Slide 27 21.9.2015gene15ab27 Normal and sickled red cells Slide 28 21.9.2015gene15ab28 The pathogenesis of sickle cell disease Slide 29 21.9.2015gene15ab29 Occurrence of Hb S in the world Slide 30 21.9.2015gene15ab30 Slide 31 21.9.2015gene15ab31 And the other haemoglobinopathies ? Theoretical number astronomical Theoretical number astronomical Known very rare Known very rare Hb C = same point as Hb S but lysine, mild haemolysis. HbSC heterozygotes Hb C = same point as Hb S but lysine, mild haemolysis. HbSC heterozygotes Different types: labile, low and high oxygen affinity, methemoglobin formation, etc. Different types: labile, low and high oxygen affinity, methemoglobin formation, etc. Why is sickle cell disease relative common? Why is sickle cell disease relative common? Plasmodia malariae do not like Hb S! AA dies on malaria, SS on sickle cell disease AS have relative advantage for survival Slide 32 21.9.2015gene15ab32 Globin genes 16p - alfa family 11p - beta family G A Slide 33 21.9.2015gene15ab33 Globin genes - ontogenesis G A Gower 1 Portland Gower 2 Fetal Slide 34 21.9.2015gene15ab34 Globin genes - adult G A A = (95%) A = (1%) 3 exons and 2 introns Slide 35 21.9.2015gene15ab35 Globin genes regulation! G A A = (95%) A = (1%) 3 exons and 2 introns LCR = locus control region with promoter and 2 enhancers before the cluster LCR deletion no gama/delta/beta chain synthesis Slide 36 21.9.2015gene15ab36 Thalassemias Deletion of smaller or bigger parts of or gene region Deletion of smaller or bigger parts of or gene region (or mutation in regulatory parts, nonsense mutation and intron splicing mutations) (or mutation in regulatory parts, nonsense mutation and intron splicing mutations) deletions back to embryonal Hb F if possible deletions back to embryonal Hb F if possible deletions 2*2 = 4 genes! deletions 2*2 = 4 genes! norm, healthy norm, healthy 1 deletion, clinically not manifest 1 deletion, clinically not manifest 2 deletions, clinically mild/not manifest 2 deletions, clinically mild/not manifest thalassemia Hb Bart = 4 thalassemia Hb Bart = 4 no hydrops fetalis no hydrops fetalis Slide 37 21.9.2015gene15ab37 Alpha thalassemias G A 2-krt Slide 38 21.9.2015gene15ab38 Beta thalassemia G A Slide 39 21.9.2015gene15ab39 Cystic fibrosis Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon must die Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon must die Anders, 1938 cystic fibrosis of pancreas Anders, 1938 cystic fibrosis of pancreas Farber, 1945 - mukoviscidosis Farber, 1945 - mukoviscidosis SR 3 centres BA, BB, KE (cca 60) SR 3 centres BA, BB, KE (cca 60) 1/26 heterozygotes, 1/2736, (1/676 marriages) 1/26 heterozygotes, 1/2736, (1/676 marriages) Increased NaCl is sweat, thick secrets of glands in bronchi, pancreas, GIT - organ failure, death Increased NaCl is sweat, thick secrets of glands in bronchi, pancreas, GIT - organ failure, death Disorder of reverse chloride and water transport Disorder of reverse chloride and water transport Slide 40 21.9.2015gene15ab40 Clinical manifestation (Spik, Feketeov) TYPICAL SPTs TYPICAL SPTs Progressive bronchopulmonal disease Nasal polyps Pancreas insufficiency Meconium ileus Male infertility Malnutrition Growth retardation Rectal prolaps ATYPICAL SPTs Icterus Distal gut obstruction Liver and bile tract malfunction Pankreatitis Chronic rhinosinusitis Diabetes mellitus Spik B, Feketeov A: Cystick fibrza Pediatria 1, 2006,, 194-198 Slide 41 21.9.2015gene15ab41 Cystic fibrosis competition Willamson et al., London 1987 - miss Lap-Chee Tsui a spol, Toronto, 1989 - hit Very different situation compared to Hb S Very different situation compared to Hb S The faulty protein was not known The localization of the gene was unkown Genetic linkage with an enzyme polymorphism - chromosome 7 (classical genetics) Genetic linkage with an enzyme polymorphism - chromosome 7 (classical genetics) Further markers, narrowing down of the region Further markers, narrowing down of the region 4 clones, 1 complementary with cDNA* of a protein from sweat gland 4 clones, 1 complementary with cDNA* of a protein from sweat gland Localization and sequenation of the gene Localization and sequenation of the gene *cDNA = mirror of mRNA for a synthesized protein Slide 42 21.9.2015gene15ab42 Cystic fibrosis Different from haemoglobinopathies: Different from haemoglobinopathies: No known protein involved Unknown site for mutation Genetic linkage with an enzyme polymorphism located on ch. 7 Genetic linkage with an enzyme polymorphism located on ch. 7 Further markers in the region Further markers in the region 4 clones, 1 is complementary to a sequence from sweat gland 4 clones, 1 is complementary to a sequence from sweat gland the gene is found and sequenced the gene is found and sequenced Slide 43 21.9.2015gene15ab43 Cystic fibrosis CFTR (cystic fibrosis conductance regulator gene) CFTR (cystic fibrosis conductance regulator gene) 1989, chromosome 7 - a big gene with 24 exons 1989, chromosome 7 - a big gene with 24 exons Codes a big transmembrane protein - Cl channel Codes a big transmembrane protein - Cl channel More than 1000 mutations found in the gene More than 1000 mutations found in the geneBUT 60 % patients have a triplet deletion - omission a Phe on 508th place of protein 60 % patients have a triplet deletion - omission a Phe on 508th place of protein additional 15 % 8 other mutations (also in introns) additional 15 % 8 other mutations (also in introns) Slide 44 21.9.2015gene15ab44 Cystic fibrosis Norm: ATC TTT = Ileu Phe Norm: ATC TTT = Ileu Phe Deletion: ATC TTT Deletion: ATC TTT ATT = Ileu, deleted CTT but lack of TTT ATT = Ileu, deleted CTT but lack of TTT Prenatal diagnostics direct? Prenatal diagnostics direct? Indirect something is wrong with the gene Indirect something is wrong with the gene NaCl in sweat > 60 mmol/l NaCl in sweat > 60 mmol/l Slide 45 21.9.2015gene15ab45 The structure of chloride transporter coded by CFTR gene Slide 46 21.9.2015gene15ab46 The structure of chloride transporter coded by CFTR gene Slide 47 21.9.2015gene15ab47 CFTR protein 2 transmembrane domains (Cl transport) 2 transmembrane domains (Cl transport) 2 nucleotide binding domains (for ATP) 2 nucleotide binding domains (for ATP) Regulation domain R Regulation domain R REGULATION ALSO OF OTHER CHANNELS !!! REGULATION ALSO OF OTHER CHANNELS !!! Different functions in sweat and other glands !!! Different functions in sweat and other glands !!! Slide 48 21.9.2015gene15ab48 CFTR is a chloride channel BUT Its functions are tissue specific Slide 49 21.9.2015gene15ab49 Sweat glands salty sweat Cooperation with ENaC, NaCl in sweat low No CFTR, salty sweat Slide 50 21.9.2015gene15ab50 Other exocrine glands thick secrets Cooperation with ENaC, influences of water transport (and ofj bicarbonate) No CFTR, thick acid secrets Slide 51 21.9.2015gene15ab51 How many cystic fibroses we have? Typical monogenous disease (?) Typical monogenous disease (?) The number of known mutations in CFTR gene > 1300 The number of known mutations in CFTR gene > 1300 66 % of patients have a deletion of a triplet in 10th exon = deletion of Phe 508, the protein is degraded in the endoplasmic reticulum 66 % of patients have a deletion of a triplet in 10th exon = deletion of Phe 508, the protein is degraded in the endoplasmic reticulum 20 other mutations (also in introns) other 15 % patients 20 other mutations (also in introns) other 15 % patients 6 different classes of mutations different clinical symptoms of also without 6 different classes of mutations different clinical symptoms of also without Mixed heterozygotes !!! Mixed heterozygotes !!! Slide 52 21.9.2015gene15ab52 Cystic fibrosis Gene therapy insertion of the gene with viral vectors into airway epithelial cells not a real success Gene therapy insertion of the gene with viral vectors into airway epithelial cells not a real success Treatment of infections, dilution of mucus, improvement of the digestion Treatment of infections, dilution of mucus, improvement of the digestion Physiotherapy Physiotherapy Psychosocial care Psychosocial care Lung transplantation Lung transplantation SURVIVAL 1975 10 15 years, today > 40 SURVIVAL 1975 10 15 years, today > 40 Slide 53 21.9.2015gene15ab53 Mixed heterozygotes 508/508508/otherOther/other No15111725 %52408 Panreas insuficiency 99%72%36% Pancreas ok 1%28%64% Age at dg 1,8 y 4,4 y 8,4 y Slide 54 21.9.2015gene15ab54 Hemophilia A Talmud Talmud Queen Victoria and her descendants A? Queen Victoria and her descendants A? Family of the last Russian Czar Nicolaus (Alexandra - 4 daughters and one affected son) Family of the last Russian Czar Nicolaus (Alexandra - 4 daughters and one affected son) Absolute deficiency of factor VIII Absolute deficiency of factor VIII 1/10000 boys, one third are new mutations in their ancestors (during meiosis) 1/10000 boys, one third are new mutations in their ancestors (during meiosis) High number of mutations, the most common form is an inversion with 0 activity of factor High number of mutations, the most common form is an inversion with 0 activity of factor Slide 55 21.9.2015gene15ab55 Hemophilia A Slide 56 Epidemiology and the Queen 172 000 worldwide 172 000 worldwide 60% severe, classic (gene inversion) 15% moderate (1-5 % activity) 25% mild (6-30%) Moderate and mild, more B and other Queen Victoria 1819 1901, 9 children Carriers Alice and Beatrice (2/5) Affected Leopold, 1853 1884 (1/4) Edward VII healthy, etc George Alice, Alexandra and Alexei (1904 1918) 5.12.2015koage.ppt56 Slide 57 5.12.2015koage.ppt57 Slide 58 The Queen and her Russian grand-grandson Queen Victoria 1819 1901, 9 children Carriers Alice and Beatrice (2/5) Alices daughter, Alexandra/Nicholas 4 daughters Olga, Tatiana, Maria, Anastasia and affected Alexei (1904 1918) 1918 Tobolsk, execution THEY ALL HAD MUTATION OF FACTOR IX = HEMOPHILIA B 5.12.2015koage.ppt58 Slide 59 History of treatment 1840 first attempt with transfusion 1840 first attempt with transfusion 1923 plasma replacement therapy 1923 plasma replacement therapy 1960 70 cryoprecipitate, 40 000 HIV INFECTIONS 1960 70 cryoprecipitate, 40 000 HIV INFECTIONS 1989 Genetic engineering, pure VIII and IX 1989 Genetic engineering, pure VIII and IX 2014 Extended life of factors 2014 Extended life of factors In progress Gene therapy In progress Gene therapy Not only queens but also dogs have haemophilia Not only queens but also dogs have haemophilia Treatment of immune reaction and joint damage Treatment of immune reaction and joint damage 5.12.2015koage.ppt59 Slide 60 21.9.2015gene15ab60 Structure of factor VIII and IX genes and proteins (with vWf) F VIIIF IX Slide 61 21.9.2015gene15ab61 Other coagulopathies Haemophilia B - similar to A, not so serious Haemophilia B - similar to A, not so serious Haemophilia C - AR heredity Haemophilia C - AR heredity All other factors - very rare All other factors - very rare Von Willebrand disease - AD; mild or asymptomatic, heterogeneous Von Willebrand disease - AD; mild or asymptomatic, heterogeneous vW factor is a big protein with multiple function - stabilizes factor VIII vW factor is a big protein with multiple function - stabilizes factor VIII Bleeding when associated with other circumstances (acetylsalicylic acid) Bleeding when associated with other circumstances (acetylsalicylic acid) Slide 62 21.9.2015gene15ab62 An upside down coagulopathy Hereditary thrombophilia Point mutation in factor V (Leiden) Point mutation in factor V (Leiden) The protein is resistant to thrombolytic inactivation. The protein is resistant to thrombolytic inactivation. Part of common european heritage (2-7 %) Part of common european heritage (2-7 %) Elevated risk of venous thrombosis: Elevated risk of venous thrombosis: VV = 1; vV = 7; vv = 80; VV = 1; vV = 7; vv = 80; Manifestation in association with other circumstances Manifestation in association with other circumstances Slide 63 21.9.2015gene15ab63 Disorder of color vision daltonism Francis Dalton, Manchester, fyzik (1776-1844) Francis Dalton, Manchester, fyzik (1776-1844) Did not understand why he perceived the colors differently as other people and let his eyes conserved in formaline Did not understand why he perceived the colors differently as other people and let his eyes conserved in formaline 4 photoreceptors (G-proteins, Guiness recored in sensitivity), vitamin A 4 photoreceptors (G-proteins, Guiness recored in sensitivity), vitamin A Genes for red and green opsins are on the X, 98 % homolog, polymorph Genes for red and green opsins are on the X, 98 % homolog, polymorph 8 % white men (no selection pressure) 8 % white men (no selection pressure) Gene analysis from Daltons retina 1992 Gene analysis from Daltons retina 1992 Slide 64 21.9.2015gene15ab64 Disorder of color vision