13.10.2013gene13ab.ppt1 the pathogenesis of diseases from genetic and genomic point of view oliver...
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13.10.2013 gene13ab.ppt 1
The Pathogenesis of Diseases from The Pathogenesis of Diseases from Genetic and Genomic Point of ViewGenetic and Genomic Point of View
Oliver Oliver RRácz and František Ništiarácz and František Ništiar
Institute of Pathological PhysiologyInstitute of Pathological Physiology
Medical School, Šafárik UniversityMedical School, Šafárik University
2012/20132012/2013
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26th june 2000 26th june 2000 is neither the beginning nor the end of is neither the beginning nor the end of
the waythe 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 mappedThe genome is mapped** - now what ? - now what ?
Not a discovery!Not a discovery! A very important technological result and A very important technological result and
competition is always useful.competition is always useful. all is based on Mendel‘s and Watson‘s & Crick‘s all is based on Mendel‘s and Watson‘s & Crick‘s
discoveries in XIXth XXth centurydiscoveries in XIXth XXth century
*3*109 letters
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Mendel, Watson, Crick & the medical Mendel, Watson, Crick & the medical geneticsgenetics of XIX of XIXthth – XX – XXthth century century
Mendel‘s laws are valid also todayMendel‘s laws are valid also today
Watson & Crick provided the material basis of Watson & Crick provided the material basis of these laws (central dogma of molecular biology)these laws (central dogma of molecular biology)
Mendel‘s laws in medicine can be applied to Mendel‘s laws in medicine can be applied to monogenic diseases – long list, relatively raremonogenic diseases – long list, relatively rare
What is the genetics of diabetes, hypertension, What is the genetics of diabetes, hypertension, coronary heart disease, Alzheimer disease ?coronary heart disease, Alzheimer disease ?
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White flowerWhite flower fromfrom red red parents ???parents ???
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Quidditch ball for Harry Potter ?
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We discovered the secret of life, let’s have a beer!
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The classicsThe classics
Mendel G: Versuche Mendel G: Versuche über Pflanzen-über Pflanzen-Hybriden. Verh. Naturfortsch. Verein Hybriden. Verh. Naturfortsch. Verein Brünn, 4, 1866, 3-47Brünn, 4, 1866, 3-47
Watson.JD, Crick FHC: Watson.JD, Crick FHC: Molecular Molecular structure of nucleic acids. structure of nucleic acids. Nature 171, 1953, 737-738Nature 171, 1953, 737-738
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CentralCentral dogma dogma of of molemolecculularar biolbiolooggyy ( (cca. cca. 1965)1965)
ReplicationTranscriptionTranslationTransformation
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CentralCentral dogma dogma of of molemolecculularar biolbiolooggyy
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CentralCentral dogma dogma of of molemolecculularar biol biolooggyy? ? Epigenetics ? Role of RNA?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
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CentralCentral dogma dogma of of molemolecculularar biol biolooggyy
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CentralCentral dogma dogma of of molemolecculularar biolbiolooggyy ( (nownow)) – role of RNAs – role of RNAs
ReplicationTranscriptionTranslationTransformation
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It is a little more complicatedIt is a little more complicated
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We and our relativesWe and our relativesOrganismOrganism Genome sizeGenome size chromosomeschromosomes/ /
genesgenes
Homo sapiensHomo sapiens 3 000 000 0003 000 000 000 23/30 00023/30 000
Mus musculusMus musculus 2 600 000 0002 600 000 000 20/30 00020/30 000
D. MelanogasterD. Melanogaster 137 000 000137 000 000 4/13 0004/13 000
C. ElegansC. Elegans 97 000 00097 000 000 6/19 0006/19 000
S. CerevisiaeS. Cerevisiae 12 100 00012 100 000 18/6 00018/6 000
E. ColiE. Coli 4 600 0004 600 000 {1}3 200{1}3 200
HIV virusHIV virus 9 7009 700 99
A. ThalianaA. Thaliana 100 000 000100 000 000 ?/25 000?/25 000
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What is not What is not “genetic”“genetic” The number of human genes is as low as 30 000The number of human genes is as low as 30 000
the small worm C. elegans has 20 000 genesthe small worm C. elegans has 20 000 genes the mouse has as many genes as we, also with very similar the mouse has as many genes as we, also with very similar
functionfunction
The mystery is in complexity and networking: The mystery is in complexity and networking: 223000030000 >>>> 2 >>>> 220000 20000 (possible on/off states)(possible on/off states)
And a number of surprises around transcription And a number of surprises around transcription and translation (miRNA, tRNA modifications)and translation (miRNA, tRNA modifications)
It is mapped but do we understand it? It is mapped but do we understand it? GENETICS = HEREDITYGENETICS = HEREDITY GENOMICS = EVERYTHINGGENOMICS = EVERYTHING
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GGEENNESES A AND THE ENVIRONMENTND THE ENVIRONMENT
GENOME ENVIRONMENT
SEVERE MONOGENICDISEASES
LESS IMPORTANT MUTATIONS
GENETIC RISK
NEGATIVE ANDPOSITIVE
ENVIRONMENTAL FACTORS
physicalchemicalbiologicalnutritionlife style
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Genes and diseases in practical Genes and diseases in practical medicinemedicine
XIXth Century: symptom diagnosis
sugar in urine = diabetes
XXth Century: symptom etiopatogenesis diagnosis
autoimmune destruction of cells = dm Type 1
XXIst Century: symptom genes and environment
etiopatogenesis diagnosissusceptibility + overeating =
subtypes of dm Type 2
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New era of preventive medicineNew era of preventive medicine
Better understanding of disease Better understanding of disease pathogenesispathogenesis
Targeted, individualized preventionTargeted, individualized prevention Clear, unambiguous argumentsClear, unambiguous arguments Healthy genes Healthy genes healthy peoplehealthy people Genes sana in corpore sanoGenes sana in corpore sano
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MutationsMutations changes of genetic information changes of genetic information
THREE PRINCIPAL POSSIBILITIESTHREE PRINCIPAL POSSIBILITIES1.1. changes in genome not compatible with lifechanges in genome not compatible with life
2.2. development and diversitydevelopment and diversity
3.3. disease or disease or increased risk fo disease*increased risk fo disease*
THE BASIC DIFFERENCE FOR HEREDITY:THE BASIC DIFFERENCE FOR HEREDITY:– somatic and germ cell mutationssomatic and germ cell mutations
genome, chromosomal and genome, chromosomal and genegene mutations mutations
no genes for diseases! – sickle cell, Alzheimer, diabetes...
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Gene mutations and SNPs*Gene mutations and SNPs* Point mutations in exons Point mutations in exons
– silent, missense (AA change) and nonsense (stop)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 aberrationsBigger deletions – transition to chromosomal aberrations Mutations in regulatory parts, intronsMutations in regulatory parts, introns, genes for r-tRNA, genes for r-tRNA Variability of repeated sequences - markersVariability of repeated sequences - markers Dynamic mutations – triple repeat mutationsDynamic mutations – triple repeat mutations
*SINGLE NUCLEOTID POLYMORPHISM
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Monogenic diseases with mendelian Monogenic diseases with mendelian inheritanceinheritance
Autosomal recessiveAutosomal recessive– sickle cell disease thalassemia and other sickle cell disease thalassemia and other
hemoglobinopathieshemoglobinopathies– cystic fibrosiscystic fibrosis– enzymopathies (inborn errors of metabolism)enzymopathies (inborn errors of metabolism)
Autosomal dominantAutosomal dominant– familiar hypercholesterolemiafamiliar hypercholesterolemia
X chromosome linked diseasesX chromosome linked diseases– hemophilia A, B; daltonismhemophilia A, B; daltonism
and von Willebrand disease, factor V Leiden, and von Willebrand disease, factor V Leiden, hereditary hemochromatosis...hereditary hemochromatosis...
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Sickle cell diseaseSickle cell disease Clinical description Clinical description 1910, Hb abnormality, 19401910, Hb abnormality, 1940 Pauling / Ingram - 1 AA change in Pauling / Ingram - 1 AA change in chain chain Point mutation – Glu 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 – 20 % heterozygotes in some parts of Africa
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And the other haemoglobinopathies ?And the other haemoglobinopathies ?
Theoretical number – astronomicalTheoretical number – astronomical Known Known very rarevery rare Hb C = same point as Hb S but lysine, mild Hb C = same point as Hb S but lysine, mild
haemolysis. HbSC heterozygoteshaemolysis. HbSC heterozygotes Different types: labile, low and high oxygen affinity, Different types: labile, low and high oxygen affinity,
methemoglobin formation, etc.methemoglobin formation, etc. Why is sickle cell disease relative common?Why is sickle cell disease relative common? Plasmodia malariae do not like Hb S!Plasmodia malariae do not like Hb S! AA dies on malaria, SS on sickle cell diseaseAA dies on malaria, SS on sickle cell disease AS have relative advantage for survivalAS have relative advantage for survival
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Occurrence of Hb S in the worldOccurrence of Hb S in the world
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The molecular structure of human HbThe molecular structure of human Hb
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Normal and sickled red cellsNormal and sickled red cells
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The pathogenesis of sickle cell diseaseThe pathogenesis of sickle cell disease
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Globin genesGlobin genes
16p - alfa family
11p - beta family
GA
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Globin genes - ontogenesisGlobin genes - ontogenesis
GA
Gower 1
Portland
Gower 2
Fetal
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Globin genes - adultGlobin genes - adult
GA
A = (95%)
A = (1%)
3 exons and 2 introns
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Globin genes – Globin genes – regularegulattion!ion!
GA
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
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ThalassemiasThalassemias Deletion of smaller or bigger parts of Deletion of smaller or bigger parts of or or gene gene
region region (or mutation in regulatory parts, nonsense mutation (or mutation in regulatory parts, nonsense mutation
and intron splicing mutations)and intron splicing mutations) deletions – back to embryonal Hb F if possibledeletions – back to embryonal Hb F if possible deletions – 2*2 = 4 genes!deletions – 2*2 = 4 genes! norm, healthynorm, healthy 1 deletion, clinically not manifest1 deletion, clinically not manifest 2 deletions, clinically mild/not manifest2 deletions, clinically mild/not manifest thalassemia Hb Bart = thalassemia Hb Bart = 44
no no hydrops fetalishydrops fetalis
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Alpha thalassemiasAlpha thalassemias
GA
2-krát
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Beta thalassemiaBeta thalassemia
GA
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Cystic fibrosisCystic fibrosis
Woe to that child which when kissed on the Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon forehead tastes salty. He is bewitched and soon must diemust die
Anders, 1938 – cystic fibrosis of pancreasAnders, 1938 – cystic fibrosis of pancreas Farber, 1945 - mukoviscidosisFarber, 1945 - mukoviscidosis SR 3 centres – BA, BB, KE (cca 60)SR 3 centres – BA, BB, KE (cca 60) 1/21/266 heterozygot heterozygoteses, 1/2, 1/2736, (1/676 marriages)736, (1/676 marriages) Increased NaCl is sweat, thick secrets of glands in Increased NaCl is sweat, thick secrets of glands in
bronchi, pancreas, GIT - organ failure, deathbronchi, pancreas, GIT - organ failure, death Disorder of reverse chloride Disorder of reverse chloride and water and water transporttransport
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Cystic fibrosis competitionCystic fibrosis competitionWillamson et al., London Willamson et al., London 1987 1987 - miss- miss
Lap-Chee Tsui a spol, Toronto, 1989 - hitLap-Chee Tsui a spol, Toronto, 1989 - hit
VVery different situation compared to Hb Sery different situation compared to Hb S– The faulty protein was not knownThe faulty protein was not known– The localization of the gene was unkownThe localization of the gene was unkown
Genetic linkage with an enzyme polymorphism - Genetic linkage with an enzyme polymorphism - chromosome 7 (classical genetics)chromosome 7 (classical genetics)
Further markers, narrowing down of the regionFurther markers, narrowing down of the region 4 clones, 1 complementary with cDNA4 clones, 1 complementary with cDNA** of a of a
protein from sweat glandprotein from sweat gland Localization and sequenation of the geneLocalization and sequenation of the gene
*cDNA = mirror of mRNA for a synthesized protein
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Cystic fibrosisCystic fibrosis
Different from haemoglobinopathies:Different from haemoglobinopathies:– No known protein involvedNo known protein involved– Unknown site for mutationUnknown site for mutation
Genetic linkage with an enzyme polymorphism Genetic linkage with an enzyme polymorphism located on ch. 7located on ch. 7
Further markers in the regionFurther markers in the region 4 clones, 1 is complementary to a sequence from 4 clones, 1 is complementary to a sequence from
sweat glandsweat gland the gene is found and sequencedthe gene is found and sequenced
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Cystic fibrosisCystic fibrosis
CFTR (cystic fibrosis conductance regulator gene)CFTR (cystic fibrosis conductance regulator gene) 1989, chromosome 7 - a big gene with 24 exons1989, chromosome 7 - a big gene with 24 exons Codes a big transmembrane protein - Cl channelCodes a big transmembrane protein - Cl channel More than More than 101000 mutations found in the gene00 mutations found in the gene
BUTBUT 60 % patients have a triplet deletion - omission a 60 % patients have a triplet deletion - omission a
Phe on 508th place of proteinPhe on 508th place of protein additional 15 % 8 other mutations (also in introns)additional 15 % 8 other mutations (also in introns)
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The structure of chloride transporter coded by The structure of chloride transporter coded by CFTR geneCFTR gene
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The structure of chloride transporter coded by The structure of chloride transporter coded by CFTR geneCFTR gene
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CCFTR proteinFTR 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 RRegulation 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 !!!
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CFTR is a chloride channelCFTR is a chloride channelBUTBUT
Its functions are tissue specificIts functions are tissue specific
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Sweat glands – salty sweatSweat glands – salty sweat
Cooperation with ENaC, NaCl in sweat low
No CFTR, salty sweat
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Other exocrine glandsOther exocrine glandsthick secretsthick secrets
Cooperation with ENaC, influences of water transport
(and ofj bicarbonate)
No CFTR, thick acid secrets
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How many How many cystic fibrcystic fibroses we haveoses we have? ? Typical monogenous disease (?)Typical monogenous disease (?) The number of known mutations in CFTR gene The number of known mutations in CFTR gene >>
13130000 6666 % % of patients have a deletion of aof patients have a deletion of a triplet triplet inin
1010thth ex exoonn = = deletion ofdeletion of Phe 508 Phe 508, the protein is , the protein is degraded in the endoplasmic reticulumdegraded in the endoplasmic reticulum
2020 otherother mut mutationsations (a (also inlso in intron intronss) – ) – otherother 15 % 15 % patientspatients
6 6 different classes of mutationsdifferent classes of mutations – – different cldifferent clinicinicalal symptoms of also withoutsymptoms of also without
MiMixedxed heterozygot heterozygotes es !!!!!!
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Cystic fibrosisCystic fibrosis
Norm: Norm: ATC TTT = Ileu PheATC TTT = Ileu Phe Deletion: Deletion: ATATC TTC TTT T ATT = Ileu, ATT = Ileu, deleteddeleted CTT CTT but lack ofbut lack of TTT TTT PrenatPrenataal diagnostil diagnosticscs – – direct?direct? IndirectIndirect – – something is wrong with the something is wrong with the
genegene NaCl NaCl in sweatin sweat > 60 mmol/l> 60 mmol/l
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Cystic fibrosisCystic fibrosis
GGeennee t thheraperapyy – in – insserertion of the gene with viraltion of the gene with viral vevecctortorss into airway into airway epitepithhelelial cells – not a real ial cells – not a real successsuccess
Treatment ofTreatment of infec infectionstions, , dilution of mucusdilution of mucus, , improvement of the digestionimprovement of the digestion
PhPhyyssiotiothheraperapyy PsychosociPsychosocialal carecare Lung tLung transplantransplantationation SURVISURVIVAVALL 1975 10 – 15 years, today > 401975 10 – 15 years, today > 40
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Mixed heterozygotesMixed heterozygotes
508/508508/508 508/other508/other Other/otherOther/other
NoNo 151151 117117 2525
%% 5252 4040 88
Panreas Panreas insuficiencyinsuficiency
99%99% 72%72% 36%36%
Pancreas okPancreas ok 1%1% 28%28% 64%64%
Age at dgAge at dg 1,8 y1,8 y 4,4 y4,4 y 8,4 y8,4 y
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Clinical manifestation Clinical manifestation (Spišák, Feketeová)(Spišák, Feketeová)
TYPICAL SPTsTYPICAL SPTs– Progressive bronchopulmonal Progressive bronchopulmonal
diseasedisease
– Nasal polypsNasal polyps
– Pancreas insufficiencyPancreas insufficiency
– Meconium ileusMeconium ileus
– Male infertilityMale infertility
– MalnutritionMalnutrition
– Growth retardationGrowth retardation
– Rectal prolapsRectal prolaps
ATYPICAL SPTsATYPICAL SPTs– IcterusIcterus
– Distal gut obstructionDistal gut obstruction
– Liver and bile tract Liver and bile tract malfunctionmalfunction
– PankreatitisPankreatitis
– Chronic rhinosinusitisChronic rhinosinusitis
– Diabetes mellitusDiabetes mellitus
Spišák B, Feketeová A: Cystická fibróza Pediatria 1, 2006,, 194-198
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Hemophilia AHemophilia A
Talmud Talmud Queen Victoria and her descendantsQueen Victoria and her descendants Family of the last Russian Czar Nicolaus Family of the last Russian Czar Nicolaus
((Alexandra - 4 daughters and one affected son)Alexandra - 4 daughters and one affected son) Absolute deficiency of factor VIIIAbsolute deficiency of factor VIII 1/10000 boys, one third are new mutations in their 1/10000 boys, one third are new mutations in their
ancestors (during meiosis)ancestors (during meiosis) High number of mutations, the most common High number of mutations, the most common
form is an form is an inversion inversion with 0 activity of factorwith 0 activity of factor
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HHemophilia Aemophilia A
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Structure of factor VIII and IX genes and proteins Structure of factor VIII and IX genes and proteins (with vWf)(with vWf)
F VIII F IX
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Other coagulopathiesOther coagulopathies
Haemophilia B - similar to AHaemophilia B - similar to A Haemophilia C - AR heredityHaemophilia C - AR heredity All other factors - very rareAll other factors - very rare Von Willebrand disease - AD; mild or Von Willebrand disease - AD; mild or
asymptomatic, heterogeneousasymptomatic, heterogeneous vW factor is a big protein with multiple function - vW factor is a big protein with multiple function -
stabilizes factor VIIIstabilizes factor VIII Bleeding when associated with other Bleeding when associated with other
circumstances (acetylsalicylic acid)circumstances (acetylsalicylic acid)
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An „upside down“ coagulopathyAn „upside down“ coagulopathy
Hereditary thrombophiliaHereditary thrombophilia Point mutation in factor V (Leiden)Point mutation in factor V (Leiden) The protein is The protein is resistant resistant to thrombolytic to thrombolytic
inactivation. 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; vVV = 1; vV = 7; vvv = 80; = 80; Manifestation in association with other Manifestation in association with other
circumstancescircumstances
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Disorder of color vision – daltonismDisorder of color vision – daltonism
Francis Dalton, Manchester, fyzik (1776-1844)Francis Dalton, Manchester, fyzik (1776-1844) Did not understand why he perceived the colors Did not understand why he perceived the colors
differently as other people and let his eyes conserved differently as other people and let his eyes conserved in formalinein formaline
4 4 phphotoreceptors (G-proteins, Guiness recored in otoreceptors (G-proteins, Guiness recored in sensisensittivity), vitamin Aivity), vitamin A
Genes for red and green opsins are on the X, 98 % Genes for red and green opsins are on the X, 98 % homolog, polymorphhomolog, polymorph
8 % white men (no selection pressure)8 % white men (no selection pressure) Gene analysis from DaltonGene analysis from Dalton’s’s retin retinaa – 1992 – 1992
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Disroder of color visionDisroder of color vision