20.11.2007 gene2.ppt 1

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

Part 2Part 2©©Oliver Oliver RRácz ácz and František and František NištiarNištiar

Institute of Pathological PhysiologyInstitute of Pathological Physiology

Medical SchoolMedical School, Šafárik , Šafárik UniversityUniversity

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Expansion of trinucleotideExpansion of trinucleotiderepetitionsrepetitions((dynamicdynamicmutationsmutations))

�� Increased number of tripletsIncreased number of triplets (not (not insertionsinsertions) ) ⇒⇒⇒⇒⇒⇒⇒⇒diseasedisease ??????

�� Increase of repetition number from one Increase of repetition number from one generationgeneration toto the nextthe next ((anticipationanticipation))

�� DoesDoes not fit not fit into our concept of classical into our concept of classical geneticsgenetics, , molecular molecular biology biology and mutationsand mutations

�� More More repetitionsrepetitions, more severe , more severe diseasedisease

�� Only in humansOnly in humans ??????

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Expansion of trinucleotidExpansion of trinucleotidrepetitionsrepetitions((dynamic mutationsdynamic mutations))

�� sysy. Martin . Martin && Bell, Bell, mental retardationmental retardation, X , X -- linked linked

((fragilefragile X, FRAXA) X, FRAXA) men men 1/1000; 1/1000; women women 1/25001/2500–– FRAXE, FRAXE, mental retardationmental retardation(X) (X) less less severesevere

–– Myotonic dystrophyMyotonic dystrophy(m. (m. SteinertSteinert, AD), AD)

�� Huntington diseaseHuntington disease(AD) (AD) ataxia ataxia & & dementiadementia–– Spinal & Spinal & bulbar muscular atrophybulbar muscular atrophy(AR), (AR),

spinocerebellar atrophyspinocerebellar atrophytype 1, type 1, Haw River syHaw River sy, , MachadoMachado-- Joseph diseaseJoseph disease((allall AD)AD)

�� Friedreich ataxiaFriedreich ataxia(AR)(AR)

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sysy. Martin . Martin && BellBell�� Xq27, gene FMRXq27, gene FMR--1, 1,

�� chromosome chromosome breaksbreaks, , hypermethylationhypermethylation

�� CGC CGC repetitionsrepetitionsin in noncodingnoncoding55’’ regionregion

�� 6 6 -- 53 (53 (mostlymostly29)29) normnorm, , stablestable

�� 54 54 -- 200200 „„ premutationpremutation“ “ expansionexpansion

�� 200 200 -- 10001000 fullfull mutationmutation, ,

�� methylationmethylationaffetcs the promotor region affetcs the promotor region (OFF)(OFF)

�� AsymptomaticAsymptomaticmen pass themen pass thedefectdefectto to theirtheirdaughtersdaughters, , increaseincreaseofof triplettriplet numbernumber, , manifestationmanifestationin in thethenextnextgenerationgeneration

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ActiveActive and and inactiveinactive genegene, , block ofblock of gene gene expression expression and and protein bindingprotein binding

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29 ⇒ 29 ⇒ 29 ⇒ 29 ⇒ 29 ⇒ 29 ⇒ 29 ⇒ 29

55 ⇒ 75 ⇒ 95 ⇒ 120 ⇒ 200 ⇒ 325 ⇒ 500

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GroupGroupofof neurodegenerative neurodegenerative diseasesdiseases-- Huntington Huntington + 4+ 4

�� OnlyOnly nervous tissuenervous tissue, , deathdeathofof a a specificspecificgroupgroupofof cellscells; ; manifestationmanifestationin in earlyearlyadulthoodadulthood

�� repetitionsrepetitionsin in exonsexons-- > 35 CAG = > 35 > 35 CAG = > 35 GluGlu in in proteinprotein

�� Gain of functionGain of functionmutationsmutations

�� LotLot ofof unansweredunansweredquestionsquestions–– why in adulthoodwhy in adulthood??

–– why why a a specific group of cells specific group of cells ??

�� the expression of the expression of a (a (mutatedmutated) gene ) gene depends depends on a on a lot lot of other genesof other genes!!!!!!

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Friedreich ataxiaFriedreich ataxia

�� RareRareAR AR diseasedisease(1/50 000)(1/50 000)

�� MultisystemicMultisystemic-- besidebesidesymptomssymptomsofofnervousnervoussystemsystemcardiomyopathycardiomyopathyandanddisordersdisordersofof glucoseglucosemetabolismmetabolism

�� gene gene -- frataxinfrataxin

�� repetitionsrepetitionsGAA GAA inin thethe1st 1st intronintron–– normnorm7 7 -- 22, 22, diseasedisease200 200 -- 900 900 (splicing ???)(splicing ???)

�� AlsoAlso classicclassicmutationsmutationsleadleadto to diseasedisease

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Imprinting Imprinting -- absolutely against Mendelabsolutely against Mendel

�� MendelMendel: : Homologous autosomesHomologous autosomes((mothermother= = fatherfather) are ) are equivalentequivalent((globinglobin αα chainschains))

�� Imprinting Imprinting -- different expression of genesdifferent expression of geneson on homologous chromosomeshomologous chromosomes((mothermother≠ fatherfather))

�� OddityOddity? ? PraderPrader-- WilliWilli & & Angelman syAngelman sy..�� PWS: PWS: Muscle hypotonyMuscle hypotony, , mental retardationmental retardation, , bulimia bulimia --

eat everything what they findeat everything what they find, , also from garbagealso from garbage

�� AS: AS: MentalMental& motor & motor retardationretardation, , agressivityagressivity, do not , do not sleep sleep („(„ happy puppet happy puppet syndrome“)syndrome“)

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PWS & AS are PWS & AS are different diseasesdifferent diseases

�� Caused Caused by by deletions of the deletions of the 15th 15th chch..

�� PWS PWS -- deletiondeletionon CH15on CH15fromfrom fatherfather

�� AS AS -- deletion deletion on CH15on CH15fromfrom mothermother

P PM M

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And And this is only the beginningthis is only the beginning!!Uniparental disomyUniparental disomy

P P M M

AS ! PWS !

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Different expression of genes Different expression of genes on on paternal paternal and and maternal chromosomesmaternal chromosomes

P M

ABCDE

ABCDE

The same genes are present on both chromosomes

For normal function we needexpression of one set ABCDE

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Different expression of genes Different expression of genes on on paternal paternal and and maternal chromosomesmaternal chromosomes

P M

ABCDE

ABCDE

For normal function we needexpression of one set ABCDE

This can be achieved by imprinting(block) of some genes on paternal and maternal chromosome

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ExplanationExplanation

�� for for normal development normal development gene gene cooperation is cooperation is necessarynecessary

�� some genes expressed only some genes expressed only on on maternalmaternal, , others others on on paternal paternal chromosomechromosome

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Deletions of maternal on paternal chromosomeDeletions of maternal on paternal chromosome

P M

ABCDE

ABCDE

P M

ABCDE

ABCDE

P M

ABCDE

ABCDE

ABCDE AC BDE

UniparentalUniparental disomydisomy

P P

ABCDE

ABCDE

ABCDE

ABCDE

M M

BBDDEEAACC

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ExplanationExplanation

�� for for normal development normal development gene gene cooperation is cooperation is necessarynecessary

�� some genes expressed only some genes expressed only on on maternalmaternal, , others others on on paternal paternal chromosomechromosome

�� AC m +AC m + BDE p = ABCDE BDE p = ABCDE ((O.K.)O.K.)�� DeletionDeletion„m“„m“ expressionexpressionBDE notBDE notenoughenough�� DeletionDeletion„p“„p“ expressionexpressionAC notAC not enoughenough�� twotwo „p“„p“ expressionexpressionBBDDEE notBBDDEE notenoughenough�� twotwo „m“„m“ expressionexpressionAACC notAACC not enoughenough

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MechanismMechanism

�� methylationmethylationof regulatory regions of genesof regulatory regions of genes

�� catchcatch22 22 -- what is occuring in the next what is occuring in the next generationgeneration??

AC BDEBDE AC

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We should something We should something to do to do with itwith it

SPERMIUM AC & BDE OVUM AC & BDE

& &

PROBLEM WITH THEIR COMBINATION50 % children with PWS or AS

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We should something We should something to do to do with itwith it

SPERMIUM AC & BDE OVUM AC & BDE

& &

Removal of the old sign introduction of new imprinting according to the sex

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We should something We should something to do to do with itwith it

SPERMIUM OVUM

& &

Removal of the old sign introduction of new imprinting according to the sex

FATHER MOTHER

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Parthenogenesis is Parthenogenesis is not not workingworking

�� The development of zygotesThe development of zygotes46 XX 46 XX oror46 XY 46 XY is normal only when the is normal only when the chromosomes chromosomes are are from from both parentsboth parents

�� ExperimentsExperimentson on micemice

�� 46 XX 46 XX fromfrom mothermother-- teratomateratoma

�� 46 XY 46 XY fromfrom fatherfather-- mola mola hydatydosahydatydosa

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IsIs itit reallyreally soso rarerare ??

�� About mice and menAbout mice and men

�� GrowthGrowth, obesity, , obesity, energetic metabolismenergetic metabolism

�� gene for IGF2gene for IGF2is active paternalis active paternalCH7 CH7 inin mousemouse

�� gene for IFG2gene for IFG2receptor receptor isis activeactiveon on maternalmaternalCH17CH17

�� Pathogenesis ofPathogenesis ofType 2 diabetes ???Type 2 diabetes ???

�� The cooperation of paternal and maternal The cooperation of paternal and maternal part part of of genome is genome is not not so peacso peaceeful ful ??????

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Epigenetic processesEpigenetic processes

�� Dynamic mutationsDynamic mutations–– control of control of triplet triplet number number -- reparatory reparatory

mechanisms coded mechanisms coded in in GENESGENES

�� Imprinted genesImprinted genes–– sign for sign for imprintingimprinting CODECODE

–– regulationregulation GENESGENES

–– methylation methylation ⇒ enzymes coded in enzymes coded in GENESGENES

–– removal of methyls removal of methyls ⇒ enzymes coded inenzymes coded inGENESGENES

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Mitochondrial geneticsMitochondrial genetics

�� In In thethe cellscells there isthere is oneone nucleusnucleus withwith twotwosets ofsets of chromosomes chromosomes (= (= MendelMendel’s laws)’s laws)

�� In the cells there is a In the cells there is a great number great number ofofmitochondrimitochondriaa in each of them in each of them more more copiescopies of circular twoof circular two--chainchain DNA (16569 DNA (16569 bpbp) ) -- HETEROPLAHETEROPLASSMIAMIA

�� MaternMaternaal l geneticsgenetics ((EvEve from Bible?e from Bible?))

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Functions and pathology Functions and pathology of of mitochondrimitochondriaa

�� CitrCitraattee cycyclecle

�� TerminTerminaal oxidl oxidationation

�� Damaged in hypoxia, Damaged in hypoxia, repefusion repefusion damage (release of free radicals)damage (release of free radicals)

�� Mitochondria and agingMitochondria and aging

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The sThe strutruccttuurre ofe of mtDNAmtDNA

�� 16959 16959 bpbp

�� HeavyHeavy & Light chain & Light chain and Dand D--loop (triple)loop (triple)

�� Both chains code genesBoth chains code genes((withoutwithout intrintroonneses))

�� 13 13 proteinsproteins

�� 2 2 rRNArRNAss ((!)!)

�� 22 22 tRNAtRNAss

�� Small Small noncoding noncoding regionsregions

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CharacteristicsCharacteristics of mtDNAof mtDNA

�� WithoutWithout histhistoonneses

�� 800 800 –– 1000 1000 copiescopies ((upupto to 10 000) in a 10 000) in a cellcell

�� Maternal heredityMaternal heredity

�� Higher mutations rate Higher mutations rate as in as in nDNAnDNA (ROS, (ROS, weakweakreparareparation tion mechanisms mechanisms and also tissue and also tissue specificityspecificity

�� RepliRepliccatatiivvee segregsegregationation

�� UGA = stop (Trp)UGA = stop (Trp)

�� AUA = AUA = Ile Ile (Met)(Met)

�� AGA/AGG = AGA/AGG = ArgArg (stop)(stop)

�� RiboRibososommes es similar as insimilar as inE.E.coli coli ((chloramfenichloramfeniccololsensitivitysensitivity))

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GGeenneses of of terminterminaal oxidl oxidationation

�� CCOMPLEXOMPLEX

�� I. NADH I. NADH dehydrogenasedehydrogenase

�� II. II. Succinate Succinate dehydrogenasedehydrogenase

�� III. III. UbichinoneUbichinone: : cytochromecytochrome CC--oxidoreductaseoxidoreductase

�� IV. IV. CytochromoxidaseCytochromoxidase�� V. ATP V. ATP synthasesynthase

�� SUBUNBITS IN SUBUNBITS IN nDNAnDNAand and mtDNAmtDNA

�� I.I. 35 35 –– 77

�� II.II. 4 4 –– 00

�� III.III. 9 9 –– 11

�� IV.IV. 13 13 –– 33

�� V.V. 12 12 –– 2 2

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MitochondrialMitochondrial diseasesdiseases�� Mutations of mtDNA Mutations of mtDNA -- nonmendeliannonmendelian

–– Sporadic Sporadic („(„somaticsomatic“), “), deletionsdeletions, , duplicationsduplications, , affecting protein coding genes affecting protein coding genes or or tRNAstRNAs

–– Maternal Maternal („(„gameticgametic“), point “), point mutationsmutations or or microdeletionsmicrodeletions

–– The combination of both The combination of both (!)(!)

�� Mutations Mutations in in genes of nDNA coding genes of nDNA coding mitochondrial proteins mitochondrial proteins –– mendelianmendelian

�� Defects ofDefects of intergenomic signalingintergenomic signaling

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Mitochondrial diseases Mitochondrial diseases ((mtDNAmtDNA))

�� Not very common with long namesNot very common with long names–– KSS KSS –– KearnsKearns--Sayre sySayre sy–– LHON LHON –– Leber hereditary optic neuropathyLeber hereditary optic neuropathy–– MERFF MERFF –– Myoclonic epilepsyMyoclonic epilepsy, , ragged red fibersragged red fibers–– MELAS MELAS –– MyopathyMyopathy, , encephalopathyencephalopathy, , lactic acidosislactic acidosis, apoplexia, apoplexia

�� MutationsMutations in in thethe genesgenes for tRNAs for tRNAs (?!) (?!) �� ForgetForget itit –– rememberremember only thatonly that most most ofof thethe

symptomessymptomes are consequences ofare consequences of alteredaltered energyenergyproductionproduction in in tissuestissues withwith highhigh energy demandenergy demand ––musclesmuscles, , heartheart, , brainbrain, , sensessenses

�� Do Do notnot forgetforget itit –– 22 y. 22 y. oldold manman withwith symptomssymptoms ofofstrokestroke, CT , CT negativenegative ??? MELAS !??? MELAS !

�� Accumulation of mutationsAccumulation of mutations –– explanation of agingexplanation of aging??