1. 13.10.2013gene13ab.ppt1 The Pathogenesis of Diseases from Genetic and Genomic Point of View Oliver Rácz and František Ništiar  Oliver Rácz and František Ništiar Institute of Pathological Physiology Medical School, Šafárik University 2012/2013

2. 13.10.2013gene13ab.ppt2 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 Mendel‘s and Watson‘s & Crick‘s discoveries in XIXth XXth century all is based on Mendel‘s and Watson‘s & Crick‘s discoveries in XIXth XXth century *3*10 9 letters

3. 13.10.2013gene13ab.ppt3 Mendel, Watson, Crick & the medical genetics of XIX th – XX th century Mendel‘s laws are valid also today Mendel‘s 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) Mendel‘s laws in medicine can be applied to monogenic diseases – long list, relatively rare 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 ? What is the genetics of diabetes, hypertension, coronary heart disease, Alzheimer disease ?

4. 13.10.2013gene13ab.ppt4 White flower from red parents ???

5. 13.10.2013gene13ab.ppt5

6. 13.10.2013gene13ab.ppt6 Quidditch ball for Harry Potter ?

7. 13.10.2013gene13ab.ppt7 We discovered the secret of life, let’s have a beer!

8. 13.10.2013gene13ab.ppt8

9. 13.10.2013gene13ab.ppt9 The classics Mendel G: Versuche über Pflanzen- Hybriden. Verh. Naturfortsch. Verein Brünn, 4, 1866, 3-47 Mendel G: Versuche über Pflanzen- Hybriden. Verh. Naturfortsch. Verein Brünn, 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

10. 13.10.2013gene13ab.ppt10 Central dogma of molecular biology (cca. 1965) Replication Transcription Translation Transformation

11. 13.10.2013gene13ab.ppt11

12. 13.10.2013gene13ab.ppt12 Central dogma of molecular biology

13. 13.10.2013gene13ab.ppt13 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

14. 13.10.2013gene13ab.ppt14 Central dogma of molecular biology

15. 13.10.2013gene13ab.ppt15 Central dogma of molecular biology (now) – role of RNAs Replication Transcription Translation Transformation

16. 13.10.2013gene13ab.ppt16 It is a little more complicated

17. 13.10.2013gene13ab.ppt17 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

18. 13.10.2013gene13ab.ppt18 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

19. 13.10.2013gene13ab.ppt19 GENES AND THE ENVIRONMENT GENOMEENVIRONMENT SEVERE MONOGENIC DISEASES LESS IMPORTANT MUTATIONS GENETIC RISK NEGATIVE AND POSITIVE ENVIRONMENTAL FACTORS physical chemical biological nutrition life style

20. 13.10.2013gene13ab.ppt20 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

21. 13.10.2013gene13ab.ppt21 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

22. 13.10.2013gene13ab.ppt22 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...

23. 13.10.2013gene13ab.ppt23 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

24. 13.10.2013gene13ab.ppt24 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...

25. 13.10.2013gene13ab.ppt25 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

26. 13.10.2013gene13ab.ppt26 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

27. 13.10.2013gene13ab.ppt27 Occurrence of Hb S in the world

28. 13.10.2013gene13ab.ppt28

29. 13.10.2013gene13ab.ppt29 The molecular structure of human Hb

30. 13.10.2013gene13ab.ppt30 Normal and sickled red cells

31. 13.10.2013gene13ab.ppt31 The pathogenesis of sickle cell disease

32. 13.10.2013gene13ab.ppt32 Globin genes 16p - alfa family 11p - beta family  G  A  

33. 13.10.2013gene13ab.ppt33 Globin genes - ontogenesis  G  A   Gower 1     Portland     Gower 2     Fetal    

34. 13.10.2013gene13ab.ppt34 Globin genes - adult  G  A   A =     (95%) A  =     (1%) 3 exons and 2 introns

35. 13.10.2013gene13ab.ppt35 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

36. 13.10.2013gene13ab.ppt36 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

37. 13.10.2013gene13ab.ppt37 Alpha thalassemias  G  A   2-krát

38. 13.10.2013gene13ab.ppt38 Beta thalassemia  G  A  

39. 13.10.2013gene13ab.ppt39 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

40. 13.10.2013gene13ab.ppt40 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

41. 13.10.2013gene13ab.ppt41 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

42. 13.10.2013gene13ab.ppt42 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)

43. 13.10.2013gene13ab.ppt43 The structure of chloride transporter coded by CFTR gene

44. 13.10.2013gene13ab.ppt44 The structure of chloride transporter coded by CFTR gene

45. 13.10.2013gene13ab.ppt45 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 !!!

46. 13.10.2013gene13ab.ppt46 CFTR is a chloride channel BUT Its functions are tissue specific

47. 13.10.2013gene13ab.ppt47 Sweat glands – salty sweat Cooperation with ENaC, NaCl in sweat low No CFTR, salty sweat

48. 13.10.2013gene13ab.ppt48 Other exocrine glands thick secrets Cooperation with ENaC, influences of water transport (and ofj bicarbonate) No CFTR, thick acid secrets

49. 13.10.2013gene13ab.ppt49 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 !!!

50. 13.10.2013gene13ab.ppt50 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

51. 13.10.2013gene13ab.ppt51 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

52. 13.10.2013gene13ab.ppt52 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

53. 13.10.2013gene13ab.ppt53 Clinical manifestation (Spišák, Feketeová) 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 Spišák B, Feketeová A: Cystická fibróza Pediatria 1, 2006,, 194-198

54. 13.10.2013gene13ab.ppt54 Hemophilia A Talmud Talmud Queen Victoria and her descendants Queen Victoria and her descendants 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

55. 13.10.2013gene13ab.ppt55 Hemophilia A

56. 13.10.2013gene13ab.ppt56 Structure of factor VIII and IX genes and proteins (with vWf) F VIIIF IX

57. 13.10.2013gene13ab.ppt57 Other coagulopathies Haemophilia B - similar to A Haemophilia B - similar to A 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)

58. 13.10.2013gene13ab.ppt58 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

59. 13.10.2013gene13ab.ppt59 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 Dalton’s retina – 1992 Gene analysis from Dalton’s retina – 1992

60. 13.10.2013gene13ab.ppt60 Disroder of color vision