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The Pathogenesis of Diseases from Genetic and Genomic Point of View Oliver Rácz and František Ništiar Institute of Pathological Physiology Medical School, Šafárik University 2015 21.9.2015 gene15ab 1 26th june 2000 is neither the beginning nor the end of the way 5 years before term (1990 - 2005) The race is over, victory for Craig Venter. The genome is mapped* - now what ? Not a discovery! A very important technological result and competition is always useful. all is based on Mendel‘s and Watson‘s & Crick‘s discoveries in XIXth XXth century *3*109 letters 21.9.2015 gene15ab 2 Mendel, Watson, Crick & the medical genetics of XIXth – XXth century Mendel‘s laws are valid also today Watson & Crick provided the material basis of these laws (central dogma of molecular biology) Mendel‘s 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 ? 21.9.2015 gene15ab 3 White flower from red parents ??? 21.9.2015 gene15ab 4 21.9.2015 gene15ab 5 Quidditch ball for Harry Potter ? 21.9.2015 gene15ab 6 We discovered the secret of life, let’s have a beer! 21.9.2015 gene15ab 7 21.9.2015 gene15ab 8 The classics Mendel G: Versuche über PflanzenHybriden. Verh. Naturfortsch. Verein Brünn, 4, 1866, 3-47 Watson.JD, Crick FHC: Molecular structure of nucleic acids. Nature 171, 1953, 737-738 21.9.2015 gene15ab 9 Central dogma of molecular biology (cca. 1965) Replication Transcription Translation Transformation 21.9.2015 gene15ab 10 21.9.2015 gene15ab 11 Central dogma of molecular biology 21.9.2015 gene15ab 12 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 21.9.2015 gene15ab 13 Central dogma of molecular biology 21.9.2015 gene15ab 14 Central dogma of molecular biology (now) – role of RNAs Replication Transcription Translation Transformation 21.9.2015 gene15ab 15 It is a little more complicated 21.9.2015 gene15ab 16 We and our relatives Organism Homo sapiens Mus musculus D. Melanogaster C. Elegans S. Cerevisiae E. Coli HIV virus A. Thaliana 21.9.2015 Genome size 3 000 000 000 2 600 000 000 137 000 000 97 000 000 12 100 000 4 600 000 9 700 100 000 000 gene15ab chromosomes/ genes 23/30 000 20/30 000 4/13 000 6/19 000 18/6 000 {1}3 200 9 ?/25 000 17 What is not “genetic” 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: 230000 >>>> 220000 (possible on/off states) And a number of surprises around transcription and translation (miRNA, tRNA modifications) It is mapped but do we understand it? GENETICS = HEREDITY GENOMICS = EVERYTHING 21.9.2015 gene15ab 18 GENES AND THE ENVIRONMENT GENOME ENVIRONMENT SEVERE MONOGENIC DISEASES NEGATIVE AND POSITIVE ENVIRONMENTAL FACTORS physical chemical biological nutrition life style LESS IMPORTANT MUTATIONS GENETIC RISK 21.9.2015 gene15ab 19 Genes and diseases in practical medicine XIXth Century: symptom diagnosis sugar in urine = diabetes XXth Century: symptom etiopatogenesis diagnosis autoimmune destruction of b cells = dm Type 1 XXIst Century: symptom genes and environment etiopatogenesis diagnosis susceptibility + overeating = subtypes of dm Type 2 21.9.2015 gene15ab 20 New era of preventive medicine Better understanding of disease pathogenesis Targeted, individualized prevention Clear, unambiguous arguments Healthy genes healthy people Genes sana in corpore sano 21.9.2015 gene15ab 21 Mutations changes of genetic information 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: – somatic and germ cell mutations genome, chromosomal and gene mutations no genes for diseases! – sickle cell, Alzheimer, diabetes... 21.9.2015 gene15ab 22 Gene mutations and SNPs* Point mutations in exons – silent, missense (AA change) and nonsense (stop) Frameshift mutation in exons (1,2,4,5...) Small deletion of triplets (3,6...) Bigger deletions – transition to chromosomal aberrations Mutations in regulatory parts, introns, genes for rtRNA Variability of repeated sequences - markers Dynamic mutations – triple repeat mutations *SINGLE NUCLEOTID POLYMORPHISM 21.9.2015 gene15ab 23 Monogenic diseases with mendelian inheritance Autosomal recessive – sickle cell disease thalassemia and other hemoglobinopathies – cystic fibrosis – enzymopathies (inborn errors of metabolism) Autosomal dominant – familiar hypercholesterolemia X chromosome linked diseases – hemophilia A, B; daltonism and von Willebrand disease, factor V Leiden, hereditary hemochromatosis... 21.9.2015 gene15ab 24 Sickle cell disease Clinical description 1910, Hb abnormality, 1940 Pauling / Ingram - 1 AA change in b chain Point mutation – Glu Val on 6th place (GAG/GTG) Decreased solubility of Hb in low pO2 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 – 21.9.2015 20 % heterozygotes in some gene15ab parts of Africa 25 The molecular structure of human Hb 21.9.2015 gene15ab 26 Normal and sickled red cells 21.9.2015 gene15ab 27 The pathogenesis of sickle cell disease 21.9.2015 gene15ab 28 Occurrence of Hb S in the world 21.9.2015 gene15ab 29 21.9.2015 gene15ab 30 And the other haemoglobinopathies ? Theoretical number – astronomical Known 500,very rare Hb C = same point as Hb S but lysine, mild haemolysis. HbSC heterozygotes Different types: labile, low and high oxygen affinity, methemoglobin formation, etc. 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 21.9.2015 gene15ab 31 Globin genes zYzYaa2a1 16p - alfa family eGgAgYbdb 11p - beta family 21.9.2015 gene15ab 32 Globin genes - ontogenesis zYzYaa2a1 eGgAgYbdb Gower 1 z2e2 Portland z2g2 21.9.2015 Gower 2 a2e2 Fetal a2g2 gene15ab 33 Globin genes - adult zYzYaa2a1 eGgAgYbdb A = a2b2 (95%) 3 exons and 2 introns A2 = a2d2 (1%) 21.9.2015 gene15ab 34 Globin genes – regulation! LCR = locus control region with promoter and 2 enhancers before the cluster LCR deletion – no gama/delta/beta chain synthesis eGgAgYbdb A = a2b2 (95%) 3 exons and 2 introns A2 = a2d2 (1%) 21.9.2015 gene15ab 35 Thalassemias Deletion of smaller or bigger parts of a or b gene region (or mutation in regulatory parts, nonsense mutation and intron splicing mutations) b deletions – back to embryonal Hb F if possible a deletions – 2*2 = 4 genes! aaaa aaa aa a no a 21.9.2015 norm, healthy 1 deletion, clinically not manifest 2 deletions, clinically mild/not manifest thalassemia Hb Bart = g4 hydrops fetalis gene15ab 36 Alpha thalassemias zYzYaa2a1 2-krát eGgAgYbdb 21.9.2015 gene15ab 37 Beta thalassemia zYzYaa2a1 eGgAgYbdb 21.9.2015 gene15ab 38 Cystic fibrosis 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 Farber, 1945 - mukoviscidosis SR 3 centres – BA, BB, KE (cca 60) 1/26 heterozygotes, 1/2736, (1/676 marriages) Increased NaCl is sweat, thick secrets of glands in bronchi, pancreas, GIT - organ failure, death Disorder of reverse chloride and water transport 21.9.2015 gene15ab 39 Clinical manifestation (Spišák, Feketeová) 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 Spišák B, Feketeová A: Cystická fibróza Pediatria 1, 2006,, 194-198 21.9.2015 gene15ab 40 Cystic fibrosis competition Willamson et al., London 1987 - miss Lap-Chee Tsui a spol, Toronto, 1989 - hit 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) Further markers, narrowing down of the region 4 clones, 1 complementary with cDNA* of a protein from sweat gland Localization and sequenation of the gene *cDNA = mirror of mRNA for a synthesized protein 21.9.2015 gene15ab 41 Cystic fibrosis Different from haemoglobinopathies: – No known protein involved – Unknown site for mutation Genetic linkage with an enzyme polymorphism located on ch. 7 Further markers in the region 4 clones, 1 is complementary to a sequence from sweat gland the gene is found and sequenced 21.9.2015 gene15ab 42 Cystic fibrosis CFTR (cystic fibrosis conductance regulator gene) 1989, chromosome 7 - a big gene with 24 exons Codes a big transmembrane protein - Cl channel More than 1000 mutations found in the gene BUT 60 % patients have a triplet deletion - omission a Phe on 508th place of protein additional 15 % 8 other mutations (also in introns) 21.9.2015 gene15ab 43 Cystic fibrosis Norm: ATC TTT = Ileu Phe Deletion: ATC TTT ATT = Ileu, deleted CTT but lack of TTT Prenatal diagnostics – direct? Indirect – something is wrong with the gene NaCl in sweat > 60 mmol/l 21.9.2015 gene15ab 44 The structure of chloride transporter coded by CFTR gene 21.9.2015 gene15ab 45 The structure of chloride transporter coded by CFTR gene 21.9.2015 gene15ab 46 CFTR protein 2 transmembrane domains (Cl transport) 2 nucleotide binding domains (for ATP) Regulation domain R REGULATION ALSO OF OTHER CHANNELS !!! Different functions in sweat and other glands !!! 21.9.2015 gene15ab 47 CFTR is a chloride channel BUT Its functions are tissue specific 21.9.2015 gene15ab 48 Sweat glands – salty sweat Cooperation with ENaC, NaCl in sweat low No CFTR, salty sweat 21.9.2015 gene15ab 49 Other exocrine glands thick secrets Cooperation with ENaC, influences of water transport (and ofj bicarbonate) No CFTR, thick acid secrets 21.9.2015 gene15ab 50 How many cystic fibroses we have? Typical monogenous disease (?) 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 20 other mutations (also in introns) – other 15 % patients 6 different classes of mutations – different clinical symptoms of also without Mixed heterozygotes !!! 21.9.2015 gene15ab 51 Cystic fibrosis 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 Physiotherapy Psychosocial care Lung transplantation SURVIVAL 1975 10 – 15 years, today > 40 21.9.2015 gene15ab 52 Mixed heterozygotes 508/508 508/other Other/other No 151 117 25 % 52 40 8 Panreas 99% insuficiency Pancreas ok 1% 72% 36% 28% 64% Age at dg 4,4 y 8,4 y 21.9.2015 1,8 y gene15ab 53 Hemophilia A Talmud Queen Victoria and her descendants A? Family of the last Russian Czar Nicolaus (Alexandra - 4 daughters and one affected son) Absolute deficiency of factor VIII 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 21.9.2015 gene15ab 54 Hemophilia A 21.9.2015 gene15ab 55 Epidemiology and the Queen 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) 23. 5. 2017 koage.ppt 56 23. 5. 2017 koage.ppt 57 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 23. 5. 2017 koage.ppt 58 History of treatment 1840 – first attempt with transfusion 1923 – plasma replacement therapy 1960 – 70 cryoprecipitate, 40 000 HIV INFECTIONS 1989 – Genetic engineering, pure VIII and IX 2014 – Extended life of factors In progress – Gene therapy Not only queens but also dogs have haemophilia Treatment of immune reaction and joint damage 23. 5. 2017 koage.ppt 59 Structure of factor VIII and IX genes and proteins (with vWf) F VIII 21.9.2015 F IX gene15ab 60 Other coagulopathies Haemophilia B - similar to A, not so serious Haemophilia C - AR heredity All other factors - very rare Von Willebrand disease - AD; mild or asymptomatic, heterogeneous vW factor is a big protein with multiple function stabilizes factor VIII Bleeding when associated with other circumstances (acetylsalicylic acid) 21.9.2015 gene15ab 61 An „upside down“ coagulopathy Hereditary thrombophilia Point mutation in factor V (Leiden) The protein is resistant to thrombolytic inactivation. Part of common european heritage (2-7 %) Elevated risk of venous thrombosis: VV = 1; vV = 7; vv = 80; Manifestation in association with other circumstances 21.9.2015 gene15ab 62 Disorder of color vision – daltonism 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 4 photoreceptors (G-proteins, Guiness recored in sensitivity), vitamin A Genes for red and green opsins are on the X, 98 % homolog, polymorph 8 % white men (no selection pressure) Gene analysis from Dalton’s retina – 1992 21.9.2015 gene15ab 63 Disorder of color vision 21.9.2015 gene15ab 64