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The Pathogenesis of Diseases from The Pathogenesis of Diseases from Genetic and GenomicGenetic and GenomicPoint Point of Viewof View

OliverOliver RRácz ácz and František and František NištiarNištiar

Institute of Pathological PhysiologyInstitute of Pathological Physiology

Medical SchoolMedical School, Šafárik , Šafárik UniversityUniversity

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26th 26th junejune 2000 2000 isis neither the beginning neither the beginning nor nor the the end end of of

the waythe way�� 5 5 yearsyearsbeforebefore term term (1990 (1990 -- 2005) 2005)

�� The race isThe race isover, over, victory for Craig Ventervictory for Craig Venter ..

�� The genome is mappedThe genome is mapped** -- now whatnow what ??

�� Not Not a a discoverydiscovery!!

�� A A veryvery importantimportant technologicaltechnologicalresult result and and competitioncompetitionisis alwaysalwaysusefuluseful..

�� allall isis basedbasedon on MendelMendel‘s and ‘s and WatsonWatson‘s & ‘s & CrickCrick‘s ‘s discoveries discoveries in in XIXth XIXth XXth centuryXXth century

*3*109 letters

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Mendel, Watson, Crick & the medical Mendel, Watson, Crick & the medical geneticsgeneticsofof XIXXIX thth –– XXXX thth centurycentury

�� MendelMendel‘s ‘s lawslaws areare validvalid alsoalso todaytoday

�� WatsonWatson & & CrickCrick providedprovided thethe materialmaterial basisbasisofofthesetheselawslaws ((centralcentral dogma dogma ofof molecularmolecular biologybiology))

�� MendelMendel‘s ‘s lawslaws in in medicinemedicinecancan bebe appliedapplied to to monogenicmonogenicdiseasesdiseases–– longlong list, list, relativelyrelatively rarerare

�� WhatWhat isis thethe geneticsgeneticsofof diabetes, diabetes, hypertensionhypertension, , coronarycoronary heartheart diseasedisease, , Alzheimer diseaseAlzheimer disease??

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Biely kvet od červených rodičov?Biely kvet od červených rodičov?

Biely kvet od červených rodičov?Biely kvet od červených rodičov?

White flowerWhite flower fromfromredred parentsparents ??????

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Biely kvet od červených rodičov?Biely kvet od červených rodičov?

Biely kvet od červených rodičov?Biely kvet od červených rodičov?

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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 Mendel G:G: VersucheVersucheüber Pflanzenüber Pflanzen--Hybriden. Verh. Naturfortsch. Verein Hybriden. Verh. Naturfortsch. Verein Brünn, 4, 1866, 3Brünn, 4, 1866, 3--4747

�� Watson.JD, Crick FHC: Watson.JD, Crick FHC: Molecular Molecular structure of nucleic acidsstructure of nucleic acids. . Nature Nature 171, 171, 1953, 7371953, 737--738738

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CentralCentraldogma dogma of of molemoleccululararbiolbiolooggyy ((ccacca. . 1965)1965)

ReplicationTranscriptionTranslationTransformation

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CentralCentraldogma dogma of of molemoleccululararbiolbiolooggyy

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CentralCentraldogma dogma of of molemoleccululararbiolbiolooggyy

•Regulation of transcription

•Transcription factors, etc.

•Methylation

•Regulation of RNA editing

•Alternative splicing

•Regulation of RNA transport

•Regulation of translation

•siRNA

•Postsynthetic modifications

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CentralCentraldogma dogma of of molemolecculularar biolbiolooggyyand and epiepi??geneticsgenetics

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It is a little more complicatedIt is a little more complicated

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We We and and our relativesour relatives

?/25 000?/25 000100 000 000100 000 000A. A. ThalianaThaliana

999 7009 700HIV virusHIV virus

{1}3 200{1}3 2004 600 0004 600 000E. E. ColiColi

18/6 00018/6 00012 100 00012 100 000S. S. CerevisiaeCerevisiae

6/19 0006/19 00097 000 00097 000 000C. C. ElegansElegans

4/13 0004/13 000137 000 000137 000 000D. D. MelanogasterMelanogaster

20/30 00020/30 0002 600 000 0002 600 000 000Mus musculusMus musculus

23/30 00023/30 0003 000 000 0003 000 000 000Homo sapiensHomo sapiens

chromosomeschromosomes/ / genesgenes

Genome sizeGenome sizeOrganismOrganism

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WhatWhat isis notnot “genetic”“genetic”

�� TheThenumbernumberofof humanhumangenesgenesisis asaslowlow as as 30 00030 000�� thethesmallsmallwormworm C. C. eleganseleganshas 20 000 has 20 000 genesgenes

�� thethemousemousehas has asasmanymanygenesgenesasaswewe, , also with very similar also with very similar functionfunction

�� TheThemysterymysteryisis in in complexitycomplexityand and networkingnetworking: : 223000030000>>>> 2>>>> 220000 20000 (possible on/off states)(possible on/off states)

�� It is mapped but do we understand it? It is mapped but do we understand it?

�� We know the keys of the piano but are we able to We know the keys of the piano but are we able to play even simple composition on it?play even simple composition on it?

�� GENETICS = HEREDITYGENETICS = HEREDITY

�� GENOMICS = EVERYTHINGGENOMICS = EVERYTHING

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GGEENNESES AAND THE ENVIRONMENTND THE ENVIRONMENT

GENOME ENVIRONMENT

SEVERE MONOGENICDISEASES

LESS IMPORTANT MUTATIONS

GENETIC RISK

NEGATIVE ANDPOSITIVE

ENVIRONMENTALFACTORS

physicalchemicalbiologicalnutritionlife style

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MutationsMutationschanges of genetic informationchanges 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 increased risk fofo disease*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 genegenemutationsmutations

no genes for diseases! – sickle cell, Alzheimer, diabetes...

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Gene mutations and Gene mutations and SNPsSNPs**

�� Point mutations in Point mutations in exons exons –– silent, silent, missensemissense(AA change) and nonsense (stop)(AA change) and nonsense (stop)

�� FrameshiftFrameshiftmutation in mutation in exonsexons(1,2,4,5...)(1,2,4,5...)�� Small deletion of triplets (3,6...)Small deletion of triplets (3,6...)�� Bigger deletions Bigger deletions –– transition to chromosomal transition to chromosomal

aberrationsaberrations�� Mutations in regulatory parts, Mutations in regulatory parts, intronsintrons, , genes for genes for rr--

tRNAtRNA�� Variability of repeated sequences Variability of repeated sequences -- markersmarkers�� Dynamic mutations Dynamic mutations –– triple repeat mutationstriple repeat mutations

*SINGLE NUCLEOTID POLYMORPHISM

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Monogenic Monogenic diseases with diseases with mendelianmendelianinheritanceinheritance

�� AutosomalAutosomalrecessiverecessive–– sickle cell disease sickle cell disease thalassemiathalassemiaand other and other

hemoglobinopathieshemoglobinopathies–– cystic fibrosiscystic fibrosis–– enzymopathiesenzymopathies(inborn errors of metabolism)(inborn errors of metabolism)

�� AutosomalAutosomaldominantdominant–– familiar familiar hypercholesterolemiahypercholesterolemia

�� X chromosome linked diseasesX chromosome linked diseases–– hemophilia A, B; hemophilia A, B; daltonismdaltonism

�� and von and von WillebrandWillebranddisease, factor V disease, factor V LeidenLeiden, , hereditary hereditary hemochromatosishemochromatosis......

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Sickle cell diseaseSickle cell disease�� Clinical description Clinical description ≈ 1910, Hb abnormality, 19401910, Hb abnormality, 1940�� PaulingPauling/ Ingram / Ingram -- 1 AA change in 1 AA change in ββ chainchain�� Point mutation Point mutation –– Glu 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 And the other haemoglobinopathieshaemoglobinopathies??

�� Theoretical number Theoretical number –– astronomicalastronomical�� Known Known ≈≈ 500, 500, very rarevery rare�� Hb C = same point as Hb S but lysine, mild Hb C = same point as Hb S but lysine, mild

haemolysishaemolysis. . HbSC heterozygotesHbSC heterozygotes�� Different types: labile, low and high oxygen affinity, Different types: labile, low and high oxygen affinity,

methemoglobinmethemoglobinformation, etc.formation, etc.�� Why is sickle cell disease relative common?Why is sickle cell disease relative common?�� Plasmodia Plasmodia malariaemalariaedo not like Hb S!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 ofOccurrence ofHbHb S in the worldS in the world

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The molecular structure of humanThe molecular structure of humanHbHb

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Normal and Normal and sickledsickledred cellsred 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

ε Gγ Aγ Ψβ δ β

ζ Ψζ Ψα α2 α1

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Globin genes Globin genes -- ontogenesisontogenesis

ε Gγ Aγ Ψβ δ β

ζ Ψζ Ψα α2 α1

Gower 1 ζ2ε2Portland ζ2γ2Gower 2 α2ε2

Fetal α2γ2

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Globin genes Globin genes -- adultadult

ε Gγ Aγ Ψβ δ β

ζ Ψζ Ψα α2 α1

A = α2β2 (95%)

A2 = α2δ2 (1%)

3 exons and 2 introns

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ThalassemiasThalassemias

�� Deletion of smaller or bigger parts of Deletion of smaller or bigger parts of αα or or ββ gene gene region region

�� (or mutation in regulatory parts, nonsense (or mutation in regulatory parts, nonsense mutation and mutation and intronintron splicing mutations)splicing mutations)

�� ββ deletions deletions –– back to back to embryonalembryonalHb F if possibleHb F if possible�� αα deletions deletions –– 2*2 = 4 genes!2*2 = 4 genes!�� αααα αααα norm, healthynorm, healthy�� αααααα 1 deletion, clinically not manifest1 deletion, clinically not manifest�� αααα 2 deletions, clinically mild/not manifest2 deletions, clinically mild/not manifest�� αα thalassemiathalassemiaHb Bart = Hb Bart = γγ44

�� no no αα hydrops fetalishydrops fetalis

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Alpha thalassemiasAlpha thalassemias

ε Gγ Aγ Ψβ δ β

ζ Ψζ Ψα α2 α1

2-krát

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Beta thalassemiaBeta thalassemia

ε Gγ Aγ Ψβ δ β

ζ Ψζ Ψα α2 α1

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Cystic fibrosisCystic fibrosis

�� Woe Woe to to that child which when kissed that child which when kissed on on the the forehead tastes forehead tastes salty. salty. He is bewitched and soon He is bewitched and soon must diemust die

�� AndersAnders, 1938 , 1938 –– cystic fibrosiscystic fibrosisofof pancreaspancreas�� FarberFarber, 1945 , 1945 -- mukoviscidosismukoviscidosis�� SR 3 SR 3 centrescentres–– BA, BB, KE (cca 60)BA, BB, KE (cca 60)�� 1/21/266 heterozygotheterozygoteses, 1/2, 1/2736, (1/676 736, (1/676 marriagesmarriages))�� Increased NaCl is sweatIncreased NaCl is sweat, , thick secrets of glands in thick secrets of glands in

bronchibronchi, , pancreaspancreas, GIT , GIT -- organ organ failurefailure, , deathdeath�� Disorder of Disorder of reverse chloride reverse chloride and and water water transporttransport

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Cystic fibrosisCystic fibrosiscompetitioncompetitionWillamson etWillamson etalal., London ., London 1987 1987 -- missmiss

LapLap--Chee Tsui Chee Tsui a spol, Toronto, 1989 a spol, Toronto, 1989 -- hithit

�� VVery different situation compared ery different situation compared to Hb Sto Hb S–– TheThefaultyfaulty protein protein waswasnot not knownknown–– The localization of the The localization of the gene gene was unkownwas unkown

�� Genetic linkage with an Genetic linkage with an enzyme enzyme polymorphism polymorphism --chromosome 7 (chromosome 7 (classicalclassicalgeneticsgenetics))

�� Further markersFurther markers, , narrowing down of the narrowing down of the regionregion�� 4 4 clonesclones, 1 , 1 complementarycomplementarywith cDNAwith cDNA** of of a a

protein protein from sweat glandfrom sweat gland�� LocalizationLocalizationand sequenation of the and sequenation of the genegene

*cDNA = mirror of mRNA for a synthesized protein

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Cystic fibrosisCystic fibrosis

�� Different from haemoglobinopathiesDifferent from haemoglobinopathies::

–– No No known known protein protein involvedinvolved

–– Unknown site Unknown site for for mutationmutation

�� Genetic linkage with an Genetic linkage with an enzyme enzyme polymorphism polymorphism located located on on chch. 7. 7

�� Further markers in theFurther markers in theregionregion

�� 4 4 clonesclones, 1 , 1 is complementary is complementary to a to a sequence from sequence from sweat glandsweat gland

�� the the gene gene is foundis foundandandsequencedsequenced

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Cystic fibrosisCystic fibrosis

�� CFTR (CFTR (cystic fibrosis conductance regulator cystic fibrosis conductance regulator gene)gene)

�� 1989, chromosome 7 1989, chromosome 7 -- a a big big gene gene with with 24 24 exonsexons

�� Codes Codes a a big transmembrane big transmembrane protein protein -- Cl channelCl channel

�� More More than than 101000 00 mutations found in the mutations found in the genegene

BUTBUT

�� 60 % 60 % patients have patients have a triplet a triplet deletion deletion -- omission omission a a Phe Phe on 508th on 508th place of place of proteinprotein

�� additionaladditional15 % 8 15 % 8 other mutations other mutations ((also in intronsalso 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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ClinicalClinical manifestation manifestation ((SpišákSpišák, , FeketeováFeketeová))

�� TYPICAL TYPICAL SPTsSPTs–– Progressive bronchopulmonalProgressive bronchopulmonal

diseasedisease

–– Nasal Nasal polypspolyps

–– Pancreas insufficiencyPancreas insufficiency

–– Meconium ileusMeconium ileus

–– Male infertilityMale infertility

–– MalnutritionMalnutrition

–– GrowthGrowthretardationretardation

–– Rectal prolapsRectal prolaps

�� ATYPICAL ATYPICAL SPTsSPTs–– IcterusIcterus

–– DistalDistal gut obstructiongut obstruction

–– LiverLiver and and bilebile tracttractmalfunctionmalfunction

–– PankreatitisPankreatitis

–– Chronic rhinosinusitisChronic rhinosinusitis

–– Diabetes mellitusDiabetes mellitus

Spišák B, Feketeová A: Cystická fibrózaPediatria 1, 2006,, 194-198

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HemophiliaHemophiliaAA

�� Talmud Talmud

�� Queen VictoriaQueen Victoriaandandher her descendantsdescendants

�� Family of the last Russian Czar NicolausFamily of the last Russian Czar Nicolaus((Alexandra Alexandra -- 4 4 daughters and one affected sondaughters and one affected son))

�� Absolute deficiency of factor Absolute deficiency of factor VIIIVIII

�� 1/10000 1/10000 boysboys, , one third one third are are new mutations in their new mutations in their ancestors ancestors ((during meiosisduring meiosis))

�� High number of mutationsHigh number of mutations, , the the most most common common form is an form is an inversion inversion with with 0 0 activity of factoractivity of factor

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HHemophiliaemophiliaAA

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Structure of factor VIII and IX genes and proteins Structure of factor VIII and IX genes and proteins (with (with vWfvWf))

F VIII F IX

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Other coagulopathiesOther coagulopathies

�� Haemophilia Haemophilia B B -- similar similar to Ato A

�� Haemophilia Haemophilia C C -- AR heredityAR heredity

�� All other factors All other factors -- very rarevery rare

�� Von Willebrand disease Von Willebrand disease -- AD; AD; mild or mild or asymptomaticasymptomatic, , heterogeneousheterogeneous

�� vW factor is vW factor is a a big big protein protein with multiple function with multiple function --stabilizes factor stabilizes factor VIIIVIII

�� Bleeding when associated with other Bleeding when associated with other circumstances circumstances ((acetylsalicylic acidacetylsalicylic acid))

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An An „„ upside downupside down“ “ coagulopathycoagulopathy

Hereditary thrombophiliaHereditary thrombophilia�� Point Point mutation in factormutation in factorV (V (LeidenLeiden))�� TheTheproteinproteinis is resistantresistanttoto thrombolytic thrombolytic

inactivationinactivation. . �� Part Part of common european heritage of common european heritage (2(2--7 %)7 %)�� Elevated Elevated risk risk of venous thrombosisof venous thrombosis::�� VV = 1; VV = 1; vV vV = 7; v= 7; vvv = 80; = 80; �� Manifestation in association with other Manifestation in association with other

circumstancescircumstances

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DisorderDisorderofof colorcolor visionvision –– daltonismdaltonism

�� Francis DaltonFrancis Dalton, Manchester, fyzik (1776, Manchester, fyzik (1776--1844)1844)�� DidDid notnot understandunderstandwhywhy heheperceivedperceivedthe colorsthe colors

differentlydifferently asasotherotherpeople people and let and let hishis eyeseyesconservedconservedin in formalineformaline

�� 4 4 phphotoreceptorsotoreceptors(G(G--proteinsproteins, , GuinessGuinessrecoredrecoredin in sensisensittivityivity ), ), vitaminvitamin AA

�� GenesGenesfor redfor redand and green opsinsgreen opsinsareareon on thetheX, 98 % X, 98 % homologhomolog, , polymorphpolymorph

�� 8 % 8 % whitewhite menmen(no (no selection pressureselection pressure))�� Gene analysisGene analysisfrom Daltonfrom Dalton’s’s retinretinaa –– 19921992

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Disroder Disroder of color visionof color vision