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The Pathogenesis of Diseases from Genetic and Genomic Point of View II. Oliver Rácz Institute of Pathological Physiology Medical School, Šafárik University 2009 – 2016 26.9.2016 gene16ab 1 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 – 26.9.2016 20 % heterozygotes in some gene16ab parts of Africa 2 The molecular structure of human Hb 26.9.2016 gene16ab 3 Normal and sickled red cells 26.9.2016 gene16ab 4 The pathogenesis of sickle cell disease 26.9.2016 gene16ab 5 Occurrence of Hb S in the world 26.9.2016 gene16ab 6 26.9.2016 gene16ab 7 Treatment 2016 Symptomatic??? Antisickling agents (toxic) Almost healthy sickle child??? – TWO MUTATIONS: Hb S AND PERSITENCE OF FETAL Hb! – Cca 20% is enough to prevent sickling – Artificial induction of F Hb? CRISPR repair of zygote? 26.9.2016 gens16b 8 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 26.9.2016 gene16ab 9 Globin genes z Yz Ya a2 a1 16p - alfa family e Gg Yb Ag d b 11p - beta family 26.9.2016 gene16ab 10 Globin genes - ontogenesis z e Gower 1 z2e2 Portland z2g2 26.9.2016 Gower 2 a2e2 Yz Gg Ya Yb Ag a2 d a1 b Fetal a2g2 gene16ab 11 Globin genes - adult z e Yz Gg A = a2b2 (95%) Ya Yb Ag a2 d a1 b 3 exons and 2 introns A2 = a2d2 (1%) 26.9.2016 gene16ab 12 Globin genes – regulation! LCR = locus control region with promoter and 2 enhancers before the cluster LCR deletion – no gama/delta/beta chain synthesis e Gg Ag Yb d A = a2b2 (95%) b 3 exons and 2 introns A2 = a2d2 (1%) 26.9.2016 gene16ab 13 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 26.9.2016 norm, healthy 1 deletion, clinically not manifest 2 deletions, clinically mild/not manifest thalassemia Hb Bart = g4 hydrops fetalis gene16ab 14 Alpha thalassemias z Yz Ya a2 a1 2-krát e 26.9.2016 Gg Yb Ag gene16ab d b 15 Beta thalassemia z e 26.9.2016 Yz Gg Ya Yb Ag gene16ab a2 d a1 b 16 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 26.9.2016 gene16ab 17 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 26.9.2016 gene16ab 18 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 26.9.2016 gene16ab 19 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 26.9.2016 gene16ab 20 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) 26.9.2016 gene16ab 21 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 26.9.2016 gene16ab 22 The structure of chloride transporter coded by CFTR gene 26.9.2016 gene16ab 23 The structure of chloride transporter coded by CFTR gene 26.9.2016 gene16ab 24 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 !!! 26.9.2016 gene16ab 25 CFTR is a chloride channel BUT Its functions are tissue specific 26.9.2016 gene16ab 26 Sweat glands – salty sweat Cooperation with ENaC, NaCl in sweat low No CFTR, salty sweat 26.9.2016 gene16ab 27 Other exocrine glands thick secrets Cooperation with ENaC, influences of water transport (and ofj bicarbonate) No CFTR, thick acid secrets 26.9.2016 gene16ab 28 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 !!! 26.9.2016 gene16ab 29 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 26.9.2016 gene16ab 30 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 26.9.2016 1,8 y gene16ab 31 Cystic fibrosis and personalized medicine? Ivacaftor, N-(2,4-di-tert-butyl-5-hydroxyphenyl)1,4-dihydro-4-oxoquinoline-3-carboxamide The G551D-CFTR mutation results in defective channel opening, present in 4% of patients with CF, affecting approximately 1,200 people in the US. In vitro studies utilizing CF human bronchial epithelial cells positive for G551D have shown that ivacaftor increases stimulated chloride secretion and also reduces excessive sodium and fluid absorption PRICE 26.9.2016 ??? OTHER CHILDREN ??? gens16b 32 Hemophilia A Talmud Queen Victoria and her descendants ? 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 26.9.2016 gene16ab 33 Hemophilia A 26.9.2016 gene16ab 34 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) 26.9.2016 gene16ab 35 26.9.2016 gene16ab 36 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 26.9.2016 gene16ab 37 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 26.9.2016 gene16ab 38 Structure of factor VIII and IX genes and proteins (with vWf) F VIII 26.9.2016 F IX gene16ab 39 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) 26.9.2016 gene16ab 40 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 26.9.2016 gene16ab 41 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 26.9.2016 gene16ab 42 Disorder of color vision 26.9.2016 gene16ab 43 Dominant heredity ? Simple? Example: polydactylia Gene mathematician (5) Stupid in math (6) 26.9.2016 gens16b 44 Polydactylia in cats of sailors and of Ernest Hemingway Sonic hedgehog – an important gene regulating development of different body parts – not only extremities and fingers. (discovered in drosophila and working also in fish) Coding a small signal molecule Point mutation in the regulatory region of the gene makes a small mistake in us, in cats, chicken, mice, horses... Homozygotes – serious disorders of body development Gene knockaut: no development of limbs at all 26.9.2016 gens16b 45