Inborn chromosomal Inborn chromosomal abnormalitiesabnormalities

5th year 5th year

RNDr Z.PolívkováRNDr Z.Polívková

Clinical cytogeneticsClinical cytogeneticsChromosome abnormalitiesChromosome abnormalities in: in:

1/150 live birth1/150 live birth

50% of first-trimester spontaneous abortions 50% of first-trimester spontaneous abortions

20% of second-trimester spont. abortions20% of second-trimester spont. abortions

4.5% of all recognized pregnancies 4.5% of all recognized pregnancies

50% of all human zygotes50% of all human zygotes

CHA = leading cause of CHA = leading cause of mental retardation and mental retardation and

pregnancy losspregnancy loss

important cause of important cause of morbidity and mortalitymorbidity and mortality

Down syndrome - free trisomy 21, 47,XX,Down syndrome - free trisomy 21, 47,XX,+21 +21

DS with translocation form of trisomy (Robersonian translocation

14/21)

46,XX,der(14;21)(q10;q10),+21

Carrier of balanced Robertsonian translocation 14/21

45,XX,der(14;21)(q10;q10)

DS with translocation form of trisomy (homologous translocation 21/21)

46,XX,der(21;21)(q10;q10)+21

47,XX,+18 – Edwards syndrome

47,XX,+13 – Patau syndrome

45,X - Turner syndrome

Isochromosome X - 46,X,i(Xq) in TS (fertility is possible)

47,XXY – Klinefelter syndrome

Terminal deletion Xp -small stature,cubiti valgi - in 3 generations of women

Terminal deletion Xq - amenorrhea, gonadal dysgenesis

Ring chromosome X- in mosaic with 45,X – Turner sy

Terminal deletion 4p - Wolf- Hirschhorn sy

Terminal deletion 4p

dysmorphic features, microcephaly, micrognathia,

cleft palate, heart defect, PMR

Terminal deletion 6p

Terminal deletion 6p

dysmorphic features, cleft, dystrophy of cornea, PMR

Interstitial deletion 3p

Interstitial deletion 3p

hypotony, atypic cry, simian creases, pedes calcaneovalgus, dysplasia of kidney, heart defect, PMR

Interstitial duplication 2q

Interstitial duplication 2q

mild facial dysmorphy, wide neck, irregular dentition, oligophrenia

Abnormal chromosome 6 derived from reciprocal translocation(4;6)-dysmorphic features, hydrocephalus, microphthalmos, dextrocardia, clubbed fingers,absence of reflexes

balanced reciprocal translocation t(4;6) in mother

Reciprocal translocation 8q/22q-balanced

Reciprocal translocation 8q/22q

2 x SA, heart defect in a child

Reciprocal translocation 6p/18p-balanced

4xSA, congenital malformations in fetus with der(18)-hydrops fetalis

Abnormal-recombinant-chromosome 2 (from maternal inversion)

pericentric inversion of chromosome 2 in mother-balanced

Pericentric inversion 2

Recombinant chromosome 2

in AMC for anencephalus

Recombinant chromosome 6

Pericentric inversion of chromosome 6

Inversion of chromosome 6 in mother

Recombinant chromosome 6

hyperdolichocephalus, epicanthus, hypertelorism, simian creases, PMR

Paracentric inversion on chromosome 15 - balanced

Paracentric inversion 15

2 x SA

Insertion of a part of 14q to 10q - balanced

Insertion of 14q to 10q

balanced aberrationfacial dysmorphy, PMR

Derivative chromosome 18 from maternal CCR –

facial dysmorphy, hypertelorism, epicanthus, retrognathia, wide nasal bridge, PMR

Complex balanced rearrangement of chromosomes 1,8,18

Complex chromosome rearrangement – chrom.1,6,14,18

Partial karyotype – CCR(1,6,14,18)

balancedhypospadia, dysmorphic features, dolichocephalus, small nose and mandible, low set ears, short neck, conical fingers

Complex structural rearrangement chromosomes Nos 3, 8, 10 – FISH

balanced karyotype of mother

Phenotype of a child with unbalanced form of anomaly

der(8),der(10)t(3;8;10)mat

Indications for chromosome analysis Indications for chromosome analysis

(postnatal):(postnatal):• Known or suspected chromosome abnormalityKnown or suspected chromosome abnormality

• Multiple congenital anomalies and/or growth and mental Multiple congenital anomalies and/or growth and mental

retardation and/or dysmorphic featuresretardation and/or dysmorphic features

• Disorders of sexual development (amenorrhea, pubertal Disorders of sexual development (amenorrhea, pubertal

failure)failure)

• Small stature in females, oedema in newbornsSmall stature in females, oedema in newborns

• Undiagnosed mental retardation (esp. X-linked mental Undiagnosed mental retardation (esp. X-linked mental

retardation)retardation)

• Fertility problems (repeated spontaneous abortions, Fertility problems (repeated spontaneous abortions,

sterility)sterility)

• Selected hematologic malignanciesSelected hematologic malignancies

• Selected Mendelian disorders (microdeletion syndromes, Selected Mendelian disorders (microdeletion syndromes,

syndromes associated with chromosome instability)syndromes associated with chromosome instability)

• Family history of structural chromosomal aberrationFamily history of structural chromosomal aberration

1. What is the risk of repeated abnormality in the family in which the child with free trisomy 21 was born? What factor does the risk depend on?

2. What is the mechanism of origin of trisomy, monosomy?

3. The child with translocation form of trisomy 21 was born in a family. Both parents have normal karyotypes. What is the risk of same abnormality in the next pregnancy?

4. The child with translocation form of trisomy 21 was born. in a family The father is carrier of balanced translocation 14/21. What is the risk of same abnormality in the next pregnancy? Is this risk same if mother is carrier of this balanced translocation?

5. In one family the child with translocation form of trisomy 21 was born (in this case it is homologous translocation 21/21). The mother is carrier of balanced translocation. What is the risk of DS or abortus for the next pregnancy?

6. One parent is a carrier of balanced reciprocal translocation. Is the risk of unbalanced abnormality same in child if father is carrier as in case of mother-carrier. In case of negative answer, why?

7. Explain the mechanism of origin of unbalanced aberration in fetus, if mother is carrier of balanced pericentric inversion?

8. What are consequences of balanced chromosomal aberration?

9. What are causes of structural chromosomal aberrations?

Prenatal cytogenetic diagnosisPrenatal cytogenetic diagnosis

Indications:Indications:

1.1. Increased maternal ageIncreased maternal age

2.2. Abnormal values of biochemical markers Abnormal values of biochemical markers - - risk risk

1: 3501: 350

„„triple test“:triple test“: - AFP = - AFP = αα-fetoprotein -fetoprotein

hCG = choriogonadotropine hCG = choriogonadotropine

uE3 = estriol uE3 = estriol

AFP AFP = neural tube defects = neural tube defects

AFP AFP uE3 uE3 hCG hCG = risk of +21 = risk of +21

AFP AFP uE3 uE3 hCG hCG = risk of +18 = risk of +18

= screening in the 2= screening in the 2ndnd trimester trimester - from peripheral blood – - from peripheral blood –

can can detect about 60-70 % of DSdetect about 60-70 % of DS

!!! high % of false positive results !!! high % of false positive results (9-14%)(9-14%)

Screening in the 1st trimesterScreening in the 1st trimester: : markers – PAPP-Amarkers – PAPP-A

(pregnancy associated plasma protein A), free , free -hCG-hCG

Combined screening Combined screening in the 1in the 1st st trimester - trimester - PAPP-A, free PAPP-A, free

-hCG -hCG + US (nuchal translucency, nasal bone)+ US (nuchal translucency, nasal bone)

1010thth-13-13thth week week ((1010thth-11-11thth -blood collection, 12 -blood collection, 12thth-13-13thth – US) – US)

85% effectivity (false positive results in 3.8-6.8%)85% effectivity (false positive results in 3.8-6.8%)

Integrated test: Integrated test: biochemical markers 1biochemical markers 1st st trimester + trimester +

US + US + biochem.markers of 2biochem.markers of 2nd nd trimester trimester (15(15thth-17 -17 th th week)week)

94%94% effectivity (false positivity 0.8-1.2)effectivity (false positivity 0.8-1.2)

Serum integrated testSerum integrated test: biochemical markers 1: biochemical markers 1stst + 2 + 2ndnd

trimestertrimester

85% effectivity (false positivity 2.7-5.2%)85% effectivity (false positivity 2.7-5.2%)

33. . Pathology on US screeningPathology on US screening: :

IUGRIUGR (intrauterinne growth retardation) (intrauterinne growth retardation)

fetal malformationsfetal malformations

abnormal amount of amniotic fluidabnormal amount of amniotic fluid

Specific featuresSpecific features: : cystic hygroma/hydrops fetalis - 45,X; cystic hygroma/hydrops fetalis - 45,X;

+21+21

duodenal atresia - +21duodenal atresia - +21

US markers (small):US markers (small): nuchal translucency nuchal translucency

nasal bone, nasal bone, length of bones ….length of bones ….

Integrated detection of US markers + biochem. Integrated detection of US markers + biochem. screening (1screening (1stst + 2 + 2nd nd trimesters) trimesters) high effectivity, high effectivity, low degree of false positivitylow degree of false positivity

4. One parent is carrier of balanced CHA 4. One parent is carrier of balanced CHA – – risk risk of of unbalanced CHA in progenyunbalanced CHA in progeny

5.5. Psychologic indicationsPsychologic indications – previous pregnancy with – previous pregnancy with

trisomy, TStrisomy, TS

Methods of prenatal cytogenetic diagnosis:Methods of prenatal cytogenetic diagnosis:

AMC – amniocentesisAMC – amniocentesis - cultivation of amniotic cells –- cultivation of amniotic cells –

14th-18th week of pregnancy14th-18th week of pregnancy = = standard AMCstandard AMC

cultivation and cytogenetic analysis = result till 14 days cultivation and cytogenetic analysis = result till 14 days

after after collectioncollection

high degree of safety (risk of fetal loss only 0.5%)high degree of safety (risk of fetal loss only 0.5%)

very reliable resultsvery reliable results

10th – 13th week10th – 13th week = early AMC = early AMC -- (risk of fetal loss higher (risk of fetal loss higher

2-3%)2-3%)

longer cultivation (small amount of amniotic fluid) longer cultivation (small amount of amniotic fluid)

amniotic cells in culture

CVS – chorionic villus samplingCVS – chorionic villus sampling – 10– 10thth –13 –13thth week week

of of pregnancypregnancy

Direct methodDirect method – – cells on the surfice of chorionic villicells on the surfice of chorionic villi

Cultivation methodCultivation method – long term cultivation – cells of – long term cultivation – cells of

mesodermal mesodermal core of villicore of villi

fetal loss in 1% fetal loss in 1%

less reliable – less reliable – risk of karyotype discrepancyrisk of karyotype discrepancy!!!!!!

CVS=extraembryonal tissue - CVS=extraembryonal tissue - false positive or false negative false positive or false negative

resultsresults

It is necessary to It is necessary to combine direct and cultivation methodscombine direct and cultivation methods or or

to verify pathological findingsto verify pathological findings by other method by other method (if only one (if only one

CVS method is used, esp. direct method)CVS method is used, esp. direct method)

Chorionic villi and amniotic cell collection

Cell lineages arising in early embryogenesis

Placental biopsyPlacental biopsy : in the late 2: in the late 2ndnd or 3 or 3rdrd trimester trimester

direct method or short cultivationdirect method or short cultivation

same disadvantages as in CVSsame disadvantages as in CVS

Fetal bloodFetal blood : from umbilical cord: from umbilical cord

risk of fetal loss 2-5%risk of fetal loss 2-5%

qquick and reliableuick and reliable method – cytogenetic result in 3 method – cytogenetic result in 3

daysdays

suitable: for late detection of abnormalities on USsuitable: for late detection of abnormalities on US

for verification of discrepancies in AMC, for verification of discrepancies in AMC,

pathology pathology detected in CVS samples detected in CVS samples

Rapid karyotypingRapid karyotyping : - without cultivation: - without cultivation

FISHFISH on interphase cells (amniotic) on interphase cells (amniotic)

QFPCRQFPCR – quantitative fluorescent – quantitative fluorescent

PCRPCR

can detect only specific aneuploidiescan detect only specific aneuploidies !!!

Chromosomal abnormalities detected in prenatal cytogenetic examination: aneuploidies, structural aberrations

Rule: pathology should be verified in the 2nd parallel culture (in AMC),

by combination of two different CVS methods (direct + cultivation)

or by another collection (fetal blood after CVS), detection of

abnormality on ultrasound…

Problematic findings:Mosaics: must be verified in both parallel cultures = true mosaicism = very probably real pathology

Probably normal phenotype : mosaicism only in one culture = pseudomosaicism

single cell mosaicism (cultivation artefact)

Mosaicism in CVS –possibly confined placental mosaicism – must be verified (by another tissue examination)!!

True mosaicism +20 – mostly cells from kidney, urine

bladder = somatic mosaicism very probably is not

connected with fetal pathology !

+i(12p) – isochromosome 12p – great risk of phenotypic

abnormalities, even in a case od pseudomosaicism (Pallister-

Klillian sy)

+i(20p) – probably without fetal pathology

Additional marker chromosome: examination of parental karyotypes

familial marker : risk is not increased if parent-carrier is normal

„de novo“ marker – increased risk of abnormalities, MR

detection of marker origin by FISH is necessary (centromeric probes)

Risk figures for unknown marker:

15% for nonsatellited marker

11% for markers from acrocentrics (with satellites on both ends)

The risk is dependent on the size of marker, presence of euchromatin,

pesence of satellites

Precise risk known for : inv dup (15), i(18p), inv dup(22) – risk=100%

small marker from chromosomes No 15, X,Y – risk cca 5%

Structural balanced CHA (unexpected)

Familial abnormality and parent-carrier is without phenotypic

consequences

→ child will be probably normal

CHA“de novo“→ increased risk of abnormalities (isolated defect or

MR)

Average risk figures: for“de novo“ reciprocal translocations - 6.1%

for“de novo“ Robertsonian translocations - less than 1%

for“de novo“ inversions - 9.4%

Common risk of abnormalities = 3%

For X/A translocations – risk of gonadal dysgenesis (break in critical region

of Xq)

risk of XR diseases (woman heterozygote, break in gene locus of

standard allele)

Noninvasive prenatal diagnosis

Examination of cell-free fetal DNA from maternal circulation

Source of fetal DNA: apoptosis of cells of trophoblast and fetal erythrocytes

10-15% of free DNA in maternal circulation during the late first

and early second trimesters is fetal in origin

Used for: prenatal detection of sex, Rh group when mother is Rh-, prenatal detection of paternity

Prenatal diagnosis of aneuploidies (in the 1st trimester): • on the basis of relative frequency of chromosome specific fragments Method = massively parallel sequencing (MPS)

compute a distribution of fetal and maternal DNA fragments• on the basis of changes in proportions of alleles in known heterozygous tandem single nucleotide polymorphisms (SNPs) specific to chromosomes of interest

Method based on digital PCR and capillary electrophoresis

= only screening test – confirmation of positive test with CVS or AMC!!

Preimplantation diagnosis:Material examined: • polar bodies

• 1-2 blastomeres from 6-10 cells embryo

(from 3rd days embryo - collection of 1 blastomere, from 5th

days embryo - several blastomeres)

FISH method (centromeric, subtelomeric probes) can detect:• aneuploidies, • sex chromosomes (X-linked disorders)• structural abnormalities: Robertsonian, reciprocal translocations, pericentric inversion

1. Compare advantages and disadvantages of cytogenetic examination of amniotic cells and chorionic villi cells.

2. How long approximately does the cultivation of amniotic cells take?

3. Compare methods of standard and early amniocenthesis.

4. Compare method of direct examination of chorionic villi cells and method of their cultivation .

5. What will be your next step in the case of finding of balanced chromosomal aberration in amniotic cells?

6. What will be your next step in the case of finding of of additional marker chromosome in amniotic cells?

7. What is the cause of possible discrepancy between karyotype of chorionic villi and karyotype of fetus?

8. Why is the examination of uncultivated amniocytes supplemented with the examination of karyotype of cultivated cells?

9. Why can you find more pathologies during examination of chorionic willi than during examination of amniotic cells?

10. Which chromosomal abnormalities are influenced by mother´s age?

11. Is the examination of free fetal DNA diagnostic or sreening method?

CHA and pregnancy loss and infertilityCHA and pregnancy loss and infertilitySpontaneous abortions (SA): Spontaneous abortions (SA): 10-15% of all recognized 10-15% of all recognized

pregnancies pregnancies spontaneously abortedspontaneously aborted

CHACHA in 30% of all abortionsin 30% of all abortions

in 60% of early abortions (8in 60% of early abortions (8thth-15-15thth week) week)

CHA in SACHA in SA: trisomy 60% (+16 is the most frequent): trisomy 60% (+16 is the most frequent)

monosomy 20 % (mostly 45,X, autosomal monosomy 20 % (mostly 45,X, autosomal

monosomies)monosomies)

polyploidy 15 %polyploidy 15 %

structural CHA 5 %structural CHA 5 %

effect of mother ageeffect of mother age: in all trisomies (except +16): in all trisomies (except +16)

no effect on 45,Xno effect on 45,X

no effect on polyploidyno effect on polyploidy

InfertilityInfertility = = inability to achieve conception or inability inability to achieve conception or inability

to sustain a pregnancy through to livebirthto sustain a pregnancy through to livebirth

Repeated SA-examination of parental Repeated SA-examination of parental

chromosomeschromosomes:: in 3-30 % of couples - one member in 3-30 % of couples - one member

is carrier of balanced CHA, or other CHA (gonosomal is carrier of balanced CHA, or other CHA (gonosomal

mosaic)mosaic)

2 and more spont.abortion, sterility= 2 and more spont.abortion, sterility=

indication for cytogenetic examination of indication for cytogenetic examination of

both partners !!!both partners !!!

Chromosomally abnormal abortion in Chromosomally abnormal abortion in

chromosomally normal parents= minimal chromosomally normal parents= minimal

or no increased risk for future or no increased risk for future

pregnancy !!!pregnancy !!!

CHA and infertility:CHA and infertility: XXY or XY/XXY in malesXXY or XY/XXY in males

45,X in females45,X in females

XX males, XY females - rareXX males, XY females - rare

structural CHA balanced – structural CHA balanced – in in

males males often connected often connected

with sterilitywith sterility

Male sterility: Male sterility: azoospermia azoospermia – 8-15% CHA – 8-15% CHA

(mostly gonosomal aneuploidy)(mostly gonosomal aneuploidy)

oligospermiaoligospermia - 4% CHA - 4% CHA (mostly autosomal (mostly autosomal

abnormality) abnormality)

male sterilitymale sterility: : gonosomal aneuploidygonosomal aneuploidy (47,XXY) (47,XXY)

structural CHA balancedstructural CHA balanced

gene mutation or deletiongene mutation or deletion - -DAZ1 DAZ1

gene=gene=ddeleted ineleted in

azazoospermia - on Yq1123 = oospermia - on Yq1123 = candidate for AZF–candidate for AZF–

(azoospermia factor)(azoospermia factor)

DAZ = multigene family DAZ = multigene family

about 10% of men with azoospermia have about 10% of men with azoospermia have

deletion deletion

CFTR gene CFTR gene mutation, or polymorphism in introne mutation, or polymorphism in introne

of CFTR geneof CFTR gene

in women trombophile mutation connected with repeated

SA

Hydatiform moleHydatiform moleabnormal pregnancy – hyperplasia of trophoblast- abnormal pregnancy – hyperplasia of trophoblast-

degenerative hydropic changes of chorionic villidegenerative hydropic changes of chorionic villi

• complete molecomplete mole == completely completely paternal karyotypic paternal karyotypic

originorigin (UPD)(UPD) - - diploid diploid constitution with two haploid paternal two haploid paternal

sets of chrom.sets of chrom.

Origin: fertilization of „empty egg“ – no fetal elementsOrigin: fertilization of „empty egg“ – no fetal elements

presents as vaginal bleedingpresents as vaginal bleeding

at at the begining and end of reproductive life in femalethe begining and end of reproductive life in female

small risk of recurrencesmall risk of recurrence

risk of malignityrisk of malignity – – choriocarcinoma!!!choriocarcinoma!!!

• partial mole partial mole = triploidy with additional male set= triploidy with additional male set of of

chromosomes chromosomes (1maternal + 2 paternal sets)(1maternal + 2 paternal sets)

origin: dispermy or fusion of normal ovum with diploid origin: dispermy or fusion of normal ovum with diploid

spermsperm

focal hyperplasia and hydatiform.changes of some villi, placenta focal hyperplasia and hydatiform.changes of some villi, placenta

abnormally largeabnormally large

presents as threatened, missed abortionpresents as threatened, missed abortion

CPM = confined placental mosaicismCPM = confined placental mosaicism - - triploidy only in triploidy only in

placentaplacenta – chromosomally normal fetus – chromosomally normal fetus

TriploidyTriploidy with 2 maternal sets with 2 maternal sets = nonmolar product= nonmolar product with severe with severe

growth retardation- only rarely survive to the birth (stillbirth or death growth retardation- only rarely survive to the birth (stillbirth or death

early after birth)early after birth)

Different phenotypes of triploidies – parental Different phenotypes of triploidies – parental

imprinting imprinting effecteffect

1. What are chromosomal causes of sterility in women?

2. What are chromosomal causes of sterility in men?

3. What is the karyotype of ovarial teratoma, what is origin of this pathology?

4. Explain origin of complete hydatiforme mole and partial hydatiforme mole.

5. What is the difference between phenotype of triploidy with additional set of paternal chromosomes and triploidy with additional set of maternal chromosomes? Explain.

6. What is the role of imprinted genes at the begining of embryonal life?

7. Which chromosomal abnormalities can be cause of repeated spontaneous abortions?

1. What does the term „dynamic mutation“mean?

2. What is the cause of fragile X syndrome?

3. What is the cytogenetic manifestation of fragile X syndrome, what cultivation conditions are needed for detection of it?

4. What is the cause of „ anticipation“ (or Sherman paradox) in fragile X syndrome ?

5. In which part of FMR1 gene does amplification occure in syndrome of fragile X and what is the consequence of it?

6. In which part of gene does amplification occure in Huntington disease and what is the consequence of it?

7. What is the efect of parental transmission on manifestation of mutation in fragile X syndrome, in Huntington disease?

8. Which phase of onthogenesis does amplification of triplets probably occur in?

9. What is the difference between premutation and full mutation in fragile X syndrome?

10. What triplet is amplified in fragile X; in Huntington disease?

Fragile X syndromeFragile X syndrome= = X-linked mental retardationX-linked mental retardation - 1:1500 of males - 1:1500 of males

cytogenetic manifestation – cytogenetic manifestation – fragile site Xq27.3fragile site Xq27.3 = FRAXA = FRAXA

Clinical signsClinical signs: mental retardation, macroorchidism (large : mental retardation, macroorchidism (large

testicles),testicles),

long face, large mandible, large everted earslong face, large mandible, large everted ears

mothers od affected males = carriersmothers od affected males = carriers

but: 30% of woman =carrier - mentally retardedbut: 30% of woman =carrier - mentally retarded

20% fraX men mentally normal20% fraX men mentally normal

deterioration of manifestation through deterioration of manifestation through

generation generation (Sherman paradox)(Sherman paradox)

Fragile X patients

Fra X

Unstable triplet repeats (CCG)n in FMR1 Unstable triplet repeats (CCG)n in FMR1

genegene

in normal population 6-50 copiesin normal population 6-50 copies

premutation (without MR) 50-200 copiespremutation (without MR) 50-200 copies

full mutation (with MR) 200-2000 copiesfull mutation (with MR) 200-2000 copies

DNA methylation (promoter region) DNA methylation (promoter region) FMR1 is FMR1 is

not not transcribedtranscribedabsence of proteinabsence of proteinMRMR

Premutation=unstable Premutation=unstable

premutationpremutationfull mutation = full mutation = only through mother only through mother

carriercarrier ((originorigin in oogenesis or early in embryonal in oogenesis or early in embryonal

life)life)

man with premutation man with premutation length of element is not length of element is not

increased in the increased in the next generationnext generation

length of amplification correlates with cytogenetic length of amplification correlates with cytogenetic

expressionexpression

gene function ?? – protein expressed in tissues, higer gene function ?? – protein expressed in tissues, higer

levels in levels in brain and testisbrain and testis

gradual origin of mutation = dynamic gradual origin of mutation = dynamic

mutationmutation

Dynamic mutationsDynamic mutationsInitial change of DNA produce another changeInitial change of DNA produce another change

- gradual expansion of triplet repeats- gradual expansion of triplet repeats

Main featuresMain features::

• homogenityhomogenity – no more alleles – no more alleles

• somatic variability-somatic variability- different number of copies in different different number of copies in different

tissuestissues

• effect of parental origin on manifestationeffect of parental origin on manifestation

• difference from mendelian principlesdifference from mendelian principles (low penetrance) (low penetrance)

• no new mutationsno new mutations – gradual arise through premutation, – gradual arise through premutation,

familialfamilial

• expresivity depends on number of copiesexpresivity depends on number of copies

• anticipationanticipation=deterioration of clinical signs through =deterioration of clinical signs through

generationsgenerations

Postzygotic originPostzygotic origin of amplification on chromosome of specific of amplification on chromosome of specific

parental originparental origin

determined in gametogenesisdetermined in gametogenesis

2 groups of mutations:2 groups of mutations:

- - amplification in noncoding(nontranslated) region of amplification in noncoding(nontranslated) region of

genegene (promoters, introns) (promoters, introns) loss of functionloss of function

fra X (CCG/GGC), myotonic dystrophy (CTG), Friedreich ataxia fra X (CCG/GGC), myotonic dystrophy (CTG), Friedreich ataxia

(GAA)(GAA)

- - amplification in exons (usually CAG repeatsamplification in exons (usually CAG repeats) ) genes genes

are are transcribed transcribed abnormal proteinabnormal protein

Huntington disease-HD - (abnormal protein huntingtin Huntington disease-HD - (abnormal protein huntingtin

inactivates inactivates associated proteins), spinocerebellar ataxia type 1associated proteins), spinocerebellar ataxia type 1

Expansions depends on the sex of transmitting Expansions depends on the sex of transmitting

parentparent

Fra X, myotonic dystrophy – expansion if Fra X, myotonic dystrophy – expansion if

disease is disease is inherited from motherinherited from mother

HD - expansion - if inherited from father - HD - expansion - if inherited from father -

earlier earlier onset of diseaseonset of disease

1. What does gene imprinting mean?

2. What is the basis of inactivation of imprinted allele?

3. What is the evidence for existence of imprinting?

4. Which of syndromes are connected with an error of gene imprinting?

5. What mechanisms of origin of Prader-Willi syndrome do you know?

6. What mechanisms of origin of Angelman syndrome do you know?

7. The same or very similar deletion on chromosome No 15 is manifested in one case as Prader Willi syndrome, in other case as Angelman syndrome? What is the reason for it?

8. What mechanisms of dysregulation of imprinted region IGF2/H19 do you know in Beckwith-Wiedemann syndrome?

9. What does uniparental disomy mean and what is the most frequent mechanism of origin of UPD?

10. What is the consequence of polymorphisms of imprinting of tumor suppressor genes?

11.How can be the error of imprinting connected with origin of tumors?

Genomic imprinting = mechanism of Genomic imprinting = mechanism of

regulation of gene expressionregulation of gene expression

functional differences between paternal and functional differences between paternal and

maternal maternal allelesalleles

expression of only one allele of specific parental expression of only one allele of specific parental

originorigin

active allele active allele x x silent (imprinted) allelesilent (imprinted) allele

imprinting - connected with methylation and imprinting - connected with methylation and

rearrangement of rearrangement of chromatin to inactive statechromatin to inactive state

Error in imprinting = human pathologies and tumorsError in imprinting = human pathologies and tumors

Evidence of imprinting: Evidence of imprinting:

1. 1. triploidytriploidy

2 paternal sets 2 paternal sets partial mole = hyperplasia of partial mole = hyperplasia of trophoblasttrophoblast

2 maternal sets2 maternal sets small placenta small placenta

2. 2. parthenogenesis parthenogenesis

ovarial teratoma – division of ovum without ovarial teratoma – division of ovum without fertilizationfertilization

complete mole – division of only male complete mole – division of only male pronucleuspronucleus

3.3. different expressivity different expressivity in some genetic diseases - in some genetic diseases - dependent on the sex of trasmitting dependent on the sex of trasmitting

parentparent

4. 4. chromosomal deletions chromosomal deletions – PWS x AS– PWS x AS

5. 5. UPD = uniparental disomy UPD = uniparental disomy – PWS x AS– PWS x AS

Prader- Willi sy (PWS)

MR, short stature ,obesity, hypotonia, characteristic facies, small feet and hand, hypogonadism

Angelman sy (AS)

MR, absence of speech, seizures, jerky gait, inappropriate laughter, dysmorphic features

PWS ASPWS AS

deletion 15q11-13

on paternal chromosome on maternal chromosome

UPD (uniparental disomy)

maternal paternal

mutation maternal active allele

imprinting error maternal imprint paternal imprinton both chromosomes on both chromosomes in PWS region in AS region

UPD = both chromosomes 15 from one parent

PWS AS

proximal region of chromosome 15 – two groups of reciprocally imprinted genes

PWS region – active paternal elleles

AS region - active maternal allele

loss of function of active alleles in PWS region (pat)

loss of function of active allele in AS region (mat)

→ functional nullisomy

Imprinted genes on chromosome No 15 normal situation

pat mat

SNRPN

ZNF127 }}

PWS genes

AS gene

active paternal ellele active maternal allele

„silent“= imprinted paternal allele

„silent“=imprinted maternal allele

UBE3A

Deletion in PWS and AS

pat mat pat mat

PWS AS

Deletion of paternal active alleles in PWS

Deletion of maternal active alleles in AS

Deletion 15q11-13Deletion 15q11-13

UPD - Uniparental disomy in PWS and AS

mat mat pat pat

UPD

Uniparental disomy

maternal in PWS

paternal in AS

PWS AS

Mutation in AS

AS

mutated active maternal allele in AS

Imprinting error

pat mat pat mat

PWS AS

maternal imprint of PWS genes on both chromosomes in PWS

paternal imprint of AS gene on both chromosomes in AS

UPD = uniparental disomyUPD = uniparental disomy= = both chromosomes (homologs) are from one both chromosomes (homologs) are from one

parentparent

Main mechanism of Main mechanism of originorigin::

loss of one chromosome from trisomic zygoteloss of one chromosome from trisomic zygote

Evidence:Evidence: trisomy 15 in CVS, normal karyotype from fetal trisomy 15 in CVS, normal karyotype from fetal

bloodbloodchild with PWSchild with PWS

transmission of hemophilia from father to suntransmission of hemophilia from father to sun

increased parental age in UPD casesincreased parental age in UPD cases

UPDUPDabnormal development if imprinted genes are abnormal development if imprinted genes are

presentpresent

Origin of uniparental disomy from trisomic zygote

trisomic zygote

loss of chromosomeloss of chromosome

uniparental disomy

normal

Imprinting in Beckwith-Wiedeman syndromeEMG sy - exomphalos-macroglossia-gigantismClinical signs: macroglossia, omfalocoele, visceromegaly, abnorml growth, hypoglycaemy in neonatal period

Risk of tumors(Wilms tu ...)

imprinted genes on 11p15 :near tel:

IGF2 – growth factor – expressed from paternal allele dysregulated in many tumors

H19 – nontranslated mRNA – expressed from maternal allele

Second imprinted region connected with BWS

near centromere:

IPL – maternal expression

CDKN1C = Cdk inhibitor (p57KIP2)

overexpression → cell cycle arrest in G1

reduced expression (mutation, LOI=loss of imprinting → abnormal growth (BWS)

gene transcribed from maternal allele, interindividual and tissue specific polymorfism of imprinting

KCNQ1 – K channel – maternal expression

normal

BWS region 11p15

pat mat

pat mat

1. paternal duplication

active and imprinted paternal alleles

active and imprinted maternal alleles

IGF2

IGF2

IGF2H19

H19

H19

IGF2

H19

IGF2/H19 region

pat pat pat mat

2. paternal UPD

3. del,transl.,mutation of maternal allele H19 expression of maternal IGF2

pat mat

4. Imprinting error

biallelic expression of

IGF2

IGF2

H19

IGF2

IGF2

H19

BWS – changes in region of IGF2/H19 on 11p15

1. paternal duplication on 11p (2 x IGF2)

2. paternal UPD (2 x IGF2)

3. deletion or translocation of maternal active allele H19

→ activation of maternal allele IGF2 (IGF2 and H19 genes use common “enhancer“)

4. imprinting error = biallelic expression IGF2

patogenesis of disease – double contribution of IGF2 product (growth factor), or deficiency of H19? role of other other genes (CDKN1C, KCNQ1) ?

Imprinting and tumorsTumors: - inhereted or induced mutations of protooncogenes, tumor suppressor genes

- epigenetic changes = changes in methylation (imprinting) of these genes

Imprinted protooncogenes – error in imprinting →

activation of imprinted allele (biallelic expression) =

oncogenes

Imprinted tumor suppressor genes– loss of

function of one allele only (active allele) = loss of gene

function

only 1 step= increased sensitivity to tumors

Polymorfism of imprinting of some genes in

population

tumor suppressor genes WT1(11p13), IGF2R (=receptor for

intracellular degradation of IGF2 on 6q26)

in most people - biallelic expression, in some people -

monoallelic expression (i.e. imprinted)

imprinting of these genes = predisposition tu tumors

Methylation = reversible process – possibility of therapy of tumors caused by aberrant methylation??