CariotipulCariotipulAberatii cromozomialeAberatii cromozomiale

CitogeneticaCitogenetica

• Studiul cromozomilor si a anomaliilor Studiul cromozomilor si a anomaliilor cromozomiale:cromozomiale:

- in metafaza - in metafaza - Normal 46 cromozomi: 22 perechi cromozomi - Normal 46 cromozomi: 22 perechi cromozomi

somatici +2 cromozomi sexuali XX sau XYsomatici +2 cromozomi sexuali XX sau XY

• anomaliile cromozomiale: prevalenta de 1/150 anomaliile cromozomiale: prevalenta de 1/150 n.n. viin.n. vii

bandarea G a cromozomilor

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

Modelul de benzi este Modelul de benzi este cromozom-specificcromozom-specific

Aranjarea cromozomilor in Aranjarea cromozomilor in cariotipcariotip

Indicatii clinice pentru Indicatii clinice pentru analiza cromozomiloranaliza cromozomilor• Deficitul cresterii pre-/postnatalDeficitul cresterii pre-/postnatalee• Avort spontan/n.n. mortAvort spontan/n.n. mort• Sterilitate/infertilitateSterilitate/infertilitate• Istoric familial de rearanjament Istoric familial de rearanjament

cromozomialcromozomial• Valori anormale ale markerilor serici Valori anormale ale markerilor serici

materni/anomalii fetale depistate ecograficmaterni/anomalii fetale depistate ecografic• NeoplaziiNeoplazii

Frecventele unor anomalii Frecventele unor anomalii cromozomiale in avorturile cromozomiale in avorturile spontanespontane

Prepararea cariotipuluiPrepararea cariotipului

FISHFISH

Metoda FISH pe nuclei interfazici

verde = cromozomul 13rosu= cromozomul 21

bleu = cromozomul 18verde = Xrosu = Y

Nuclei de la acelasi produs de conceptie

Se folosesc probe oligonucleodidice marcate fluorecent pt. diagnosticul aneuplidiilor cromozomilor 13, 18, 21, X, si Y

• TranslocatiiTranslocatii

Figure 8.23Bx

FISH: Fluorescent IN SITU hybridization green = probe for end of

chromosome 4

red = control probe for centromere of the X chromosome & another probe for end of chromosome X

William's SyndromeWilliam's Syndrome

• Normal KaryotypeNormal Karyotype

Semnificatia clinica a Semnificatia clinica a anomaliilor cromozomialeanomaliilor cromozomiale

• Non-disjunctii in meiozaNon-disjunctii in meioza

• Aberatii cromozomiale numericeAberatii cromozomiale numerice

• Aberatii cromozomiale structuraleAberatii cromozomiale structurale

• Screening si diagnosticul prenatalScreening si diagnosticul prenatal

Anomalii cromozomiale• SpermatozoiziSpermatozoizi 10%10%• Ovocit maturOvocit matur 25%25%• Avort spontanAvort spontan 50% 50%• N.n. viuN.n. viu 0.5-1%0.5-1%• In general apar prin non-disjunctii in In general apar prin non-disjunctii in

meioza materna, in corelatie cu meioza materna, in corelatie cu varsta gravideivarsta gravidei

Non-disjunctiicromozomiale

Aneuploidiile complete ale cromozomilor somatici sunt in general letale.

Exceptii:1. Sindromul Down - trisomia 212. Sindromul Patau - trisomia 133. SindromulEdwards - trisomia 18

4. Aneuploidiile cromozomilor sexuali

Originea parentala a Originea parentala a aneuplidiiloraneuplidiilor

Paterna %Paterna % Materna %Materna %Trisomia 13Trisomia 13 1515 8585Trisomia 18Trisomia 18 1010 9090Trisomia 21Trisomia 21 55 959545,X45,X 8080 202047,XXX47,XXX 55 959547,XXY47,XXY 4545 555547,XYY47,XYY 100100 00

Tipuri de anomalii cromozomiale Tipuri de anomalii cromozomiale în avorturile spontane în avorturile spontane

Incidence %Incidence %TrisomTrisomiaia 13 13 22TrisomTrisomiaia 16 16 1515TrisomTrisomiaia 18 18 33TrisomTrisomiaia 21 21 55Alte Alte ttrisomrisomiiii

2525

MonosomMonosomiaia X X 2020TriploidTriploidiiii 1515TetraploidTetraploidiiii 55Alte aberaţiiAlte aberaţii 1010

Sindromul DownSindromul Down• 95% 95% liberă şi liberă şi

omogenăomogenă• 1% mo1% mozaicurizaicuri

– Efectul vârstei Efectul vârstei maternematerne

• 4% transloca4% translocaţiiţii

Downs Downs SyndromeSyndromeBoyBoy

• 47,XY,+2147,XY,+21

TrisomTrisomiaia 21 21 sausau S Siindromndromul ul DownDown1.1. 47, XY, +21 or 47, XX, +21 47, XY, +21 or 47, XX, +21

– 1866 1866 descris dedescris de John Langdon Down. John Langdon Down. – Obstrucţia duodenului, Obstrucţia duodenului, atreatreziezie dede eso esoffag, ag,

duoden duoden sausau anus. anus. – 40% 40% prezintă malformaţii cardiace: canal prezintă malformaţii cardiace: canal

aatrioventricular trioventricular ,, defect de sept ventricular defect de sept ventricular ..– 95% 95% :: non-disjunc non-disjuncţiiţii, re, restul sunt translocaţiistul sunt translocaţii..– 90 - 95 % 90 - 95 % în meioza maternăîn meioza maternă– 1 - 3 % 1 - 3 % mozaicurimozaicuri..– RegiRegiuneaunea 21 q22 21 q22

Sindromul Edward Sindromul Edward

• 47,XX,+1847,XX,+18

Trisomia 18 ( Edward syndrome)

• 47, XY, +18 .

• a II-a trisomie ca frecvenţă

• Deficitul creşterii prenatale, trăsături faciale caracteristice. Defecte cardiace congenitale similare cu cele din sdr. Down.

• Mortalitate crescută:doar 10% supravieţuiesc mai mult de 1 an.

TrisomTrisomiaia 13 13

– 47, XY, +13 47, XY, +13 – Patau syndromePatau syndrome:: 1/10000 1/10000 n.n. vii n.n. vii . . – Malformations Malformations severe: cardiace, anencefalie, severe: cardiace, anencefalie,

holoprozencefalie, renale etcholoprozencefalie, renale etc– Despicătura de buză şi palat,Despicătura de buză şi palat, micro microftalmieftalmie, ,

polidactiliepolidactilie..– SuSupravieţuire redusăpravieţuire redusă– 80% 80% trisomie liberătrisomie liberă, rest, restul translocaţiiul translocaţii . .– 95% 95% din sarcinile cudin sarcinile cu ttrisomrisomieie13 13 sunt avortatesunt avortate..

Patau Patau SyndromSyndromee

Turner syndrome (45, X)

-1 /2000 n.n. sex feminin

- întârzierea dezvoltării sexuale

- Fenotip: hipostatură, pterygyum colli, dezvoltare sexuală incompletă, ovare transformate într-o bandeletă fibroasă

- - 1% din produşii de comcepţie sunt 45,X- 99% din feţii 45X sunt avortaţi- 75% din cazuri: cromozomul X lipsă este patern

Aneuploidii gonosomaleAneuploidii gonosomale

Figure 8.23A, B

Deletion

Duplication

Inversion

Homologouschromosomes

Reciprocaltranslocatio

n

Nonhomologouschromosomes

Anomalii cromozomiale structurale

Translocatia Robertsoniana Translocatia Robertsoniana intre chromosomii 13 si 14intre chromosomii 13 si 14

Segregarea cromozomilor la un purtator de translocatie 14/21

Purtatorii de translocatie:

- Risc de a produce gameti neechilibrati genetic

- Moartea produsului de conceptie purtator de aberatie cromozomiala

- Diagnostic frecvent in familiile in care exista cazuri de malformatii congenitale si avorturi spontane repetate

DeletiiDeletii

TerminaleTerminale• Cri du chat, 5p15Cri du chat, 5p15• Wolf-Hirschhorn, Wolf-Hirschhorn,

4p364p36

InterstitialeInterstitiale• Williams, 7q11.2,Williams, 7q11.2,

– microdeletion (FISH)microdeletion (FISH)• Retinoblastoma, Retinoblastoma,

13q1413q14• Prader-Willi, Prader-Willi,

15q11.215q11.2• Angelman, 15q11.2 Angelman, 15q11.2 • DiGeorge, 22q11.2DiGeorge, 22q11.2

Deletii

Sdr. “Cri du Chat “

-laringe hipoplazic-microcefalie-dismorfii faciale

Cri du ChatCri du Chat• Deleţie tDeleţie terminalerminalăă

– 5p155p15

• Plânset asemănător Plânset asemănător mieunatului pisicimieunatului pisici

• Retard mRetard mental ental

Wolf-Hirschhorn Wolf-Hirschhorn SyndromeSyndrome

Internet Link

Diagnosticul prenatalDiagnosticul prenatal

• Teste de SCREENING Teste de SCREENING Teste de Teste de DIAGNOSTICDIAGNOSTIC

Diagnosticul prenatalDiagnosticul prenatal• Teste de Teste de

Diagnostic Diagnostic – ecografieecografie– amniocentezaamniocenteza– CVSCVS– cordocentezacordocenteza

• Teste de screening Teste de screening – ecografieecografie

• translucencta nucala translucencta nucala (NT)(NT)

– Markerii serici Markerii serici maternimaterni• Trim IITrim II• Trim ITrim I

– Depistarea celulelor Depistarea celulelor fetale in sangele fetale in sangele matern (in viitor)matern (in viitor)

ScreeningScreening• În trim. I şi IIÎn trim. I şi II

• Markerilor sMarkerilor sererici materniici materni

• EcograficEcografic

• Rezultate anormale: teste invazive de Rezultate anormale: teste invazive de diagnosticdiagnostic

SScreeningcreening al markerilor al markerilor biochimicibiochimici• Screening în I trimestru:Screening în I trimestru:

– Vârsta gravideiVârsta gravidei– free free -hCG-hCG– Pregnancy associated plasma protein-A Pregnancy associated plasma protein-A

(PAPP-A)(PAPP-A)• nnu în practica curentău în practica curentă

Screening prenatal : I Screening prenatal : I trimestrutrimestru• Test de sTest de screening : translucencreening : translucenţaţa nucalănucală(NT)(NT)

– Se efectueaza intreSe efectueaza intre săptămânile săptămânile 10-1410-14– Depistează Depistează 54-72% 54-72% din feţii cudin feţii cu S Siindromndrom Down Down

(DS)(DS)– Alte anomalii cromozomiale: trisomia 13,18, Alte anomalii cromozomiale: trisomia 13,18,

triploidii, sdr. Turner (45X)triploidii, sdr. Turner (45X) – ≥≥33,5,5mmmm NT NT frecvent la feţii cu malformaţii frecvent la feţii cu malformaţii

cardiace, hernie diafragmatică, malf. cardiace, hernie diafragmatică, malf. rrenale, enale, defecte de perete abdominaldefecte de perete abdominal

Screening ecograficScreening ecografic• Translucenta nucala (NT) and osul nazal (NB) Translucenta nucala (NT) and osul nazal (NB)

– Se utilizeaza impreuna cu screening seric din trim I pt. sdr. Se utilizeaza impreuna cu screening seric din trim I pt. sdr. Down.Down.

• NT (>3.5mm) se asociaza cu un risc crescut pt::NT (>3.5mm) se asociaza cu un risc crescut pt::– Aberatii cromozomialeAberatii cromozomiale– Defecte cardiace majore, DTN, alte anomalii structuraleDefecte cardiace majore, DTN, alte anomalii structurale– Anumote sdr. geneticeAnumote sdr. genetice– Intarzierea cresterii intrauterineIntarzierea cresterii intrauterine

• Cariotip normal dar >NT: Cariotip normal dar >NT: – Triplu testTriplu test– Morfologie fetala 18-22 weeksMorfologie fetala 18-22 weeks

• Morfologie fetala – sapt. 18-22 Morfologie fetala – sapt. 18-22

• Nu este perfecta: un rezultat normal nu inseamna Nu este perfecta: un rezultat normal nu inseamna intotdeauna un copil sanatosintotdeauna un copil sanatos

Screening prenatal : II Screening prenatal : II trimestrutrimestru

– Varsta gravideiVarsta gravidei– Alpha-fetoproteinAlpha-fetoproteinaa (AFP) (AFP)– hCG (total, hCG (total, , free-, free-, free-, free-))– estriolestriolulul (uE3)* (uE3)*– +/-+/-InhibinInhibina a

• Triplu test: rezultate fals pozitive 5%Triplu test: rezultate fals pozitive 5%– Down syndrome: 60-70%Down syndrome: 60-70%– Trisomia 18: 60%Trisomia 18: 60%– DTN: 75-80%DTN: 75-80%

Malformatii congenitaleMalformatii congenitale• Majore: 2-3%Majore: 2-3%• Minore: 14%Minore: 14%

– Sunt implicate in 20% din decesele din prima Sunt implicate in 20% din decesele din prima sapt de viata extrauterina.sapt de viata extrauterina.

– Etiologie %Etiologie %Necunoscuta 65-75%Necunoscuta 65-75%Genetica 15-25%Genetica 15-25%Factori de mediu 10%Factori de mediu 10%– Afectiuni materne 4%Afectiuni materne 4%– Afenti infectiosi 3%Afenti infectiosi 3%– Ag. Chimici, radiatii ~1%Ag. Chimici, radiatii ~1%– Agenti mecanici 1-2%Agenti mecanici 1-2%

Malformatii congenitaleMalformatii congenitale• Aberatii cromozomiale numericeAberatii cromozomiale numerice

– Sdr. Down syndrome (trisomia 21) Sdr. Down syndrome (trisomia 21) •1/ 600-700 n.n.vii1/ 600-700 n.n.vii

– Sdr.Edward (trisomia 18)Sdr.Edward (trisomia 18)– Sdr.Patau (trisomia 13)Sdr.Patau (trisomia 13)– Aneuploidii gonosomaleAneuploidii gonosomale

•Sdr.Turner (45,X)Sdr.Turner (45,X)•Sdr. Klinefelter (47,XXY)Sdr. Klinefelter (47,XXY)•47,XXX; 47,XYY47,XXX; 47,XYY

Malformatii congenitaleMalformatii congenitale• Afectiuni monogenice-example:Afectiuni monogenice-example: Transmitere A.D, A.R, X-linkatTransmitere A.D, A.R, X-linkat

– Sdr. MarfanSdr. Marfan– Fragile-XFragile-X– AcondroplaziaAcondroplazia

•etc etcetc etc

Anomalii congenitale Anomalii congenitale • Afectiuni materne:Afectiuni materne:

– DiabetDiabet– fenilcetonuriafenilcetonuria– alcoolismalcoolism

• Agenti infectiosi: rubeola, toxoplasma, Agenti infectiosi: rubeola, toxoplasma, v.herpetic etcv.herpetic etc

Metode de diagnosic Metode de diagnosic prenatalprenatal• Biopsia de vilozităţi corialeBiopsia de vilozităţi coriale

– Se efectueaza intreSe efectueaza intre săptămânile 10-12 săptămânile 10-12– Abord transabdominal sau transcervicalAbord transabdominal sau transcervical– Tesutul placentar este utilizat:Tesutul placentar este utilizat:

•cariotipcariotip•Depistarea afecţiunilor monogeniceDepistarea afecţiunilor monogenice ( (defect defect

genic cunoscutgenic cunoscut))– RisRisc de avortc de avort: 1%: 1%

Prenatal diagnosis: chorionic villi sampling (CVS)

• sampling cells from placenta• usually done 10-12 weeks

Teste invazive de diagnostic: Teste invazive de diagnostic: biopsia de vilozitati corialebiopsia de vilozitati coriale

Test Risc de avort Moment optim Rezultat• CVS 1/100 11 sapt. 11-12sapt

Rezultate neconcludenteRezultate neconcludente(CVS)(CVS)

• 1% CVS se obtin rezultate ambigui1% CVS se obtin rezultate ambigui• Contaminare cu celulele materneContaminare cu celulele materne• Mozaicism placentar (CPM)Mozaicism placentar (CPM)• Mozicism adevarat al fatuluiMozicism adevarat al fatului• Repetarea testului prin Repetarea testului prin

amniocenteza sau cordocentezaamniocenteza sau cordocenteza

Metode de diagnosic Metode de diagnosic prenatalprenatal• AmniocentezaAmniocenteza

– Se efectueaza intreSe efectueaza intre săptămânile săptămânile 15-20 15-20 – 10-20 ml10-20 ml L.A L.A

•CCarariotipiotip•Dozarea Dozarea Alpha-fetoproteinAlpha-fetoproteineiei (AFP) (AFP) •Alte teste biochimice: bilirubina, Alte teste biochimice: bilirubina,

acetilcolonesterazaacetilcolonesteraza•QF-QF-PCR PCR sausau FISH FISH pt. diagnosticul citogenetic rapidpt. diagnosticul citogenetic rapid

– RRisc de avortisc de avort: ~: ~0,50,5%%

Amniocenteza: sapt 15-18

Teste invazive de diagnostic: Teste invazive de diagnostic: amniocentezaamniocenteza

Test Risc de avort Moment optim Rezultat• Amniocenteza 1/200 16 sapt. 18-22sapt.

Quantitative Fluorescent Quantitative Fluorescent PCRPCR Diagnosticul prenatal rapid al trisomiilor

Ratio: 1 : 1 : 1

Marker polimorfic ADN de pe cz. 21

Ratio: 1 : 2

Utilizeaza polimorfisme ADN pt. a Utilizeaza polimorfisme ADN pt. a defini nr. de copiidefini nr. de copii

CVS vs amniocentezaCVS vs amniocenteza• CVSCVS

– Săpt.Săpt.10-12 10-12 – RiscRisc de avortde avort: 1 : 1 //100100– Nu diagnosticul Nu diagnosticul NTDNTD– Analize ADNAnalize ADN – rerezultatzultat: 2: 2săptsăpt; ;

FISH:FISH: 72h72h– momozzaicism:1-2%aicism:1-2%– +/-+/- D&C D&C

• AmniocenteAmniocentezaza– Săpt.Săpt.15-20 15-20 – 1 1 // 200 200– (Open NTD): (Open NTD): dada– mai dificilmai dificil– 22săptsăpt; ; FISH:FISH: 72h72h – rarrar– Avort terapeuticAvort terapeutic

Cromozomul"Philadelphia " Cromozomul"Philadelphia " Translocatie 9:22 Translocatie 9:22

Tipuri de anomalii cromozomiale Tipuri de anomalii cromozomiale în avorturile spontane în avorturile spontane

Incidence %Incidence %TrisomTrisomiaia 13 13 22TrisomTrisomiaia 16 16 1515TrisomTrisomiaia 18 18 33TrisomTrisomiaia 21 21 55Alte Alte ttrisomrisomiiii

2525

MonosomMonosomiaia X X 2020TriploidTriploidiiii 1515TetraploidTetraploidiiii 55Alte aberaţiiAlte aberaţii 1010

Anomalii cromozomialeAnomalii cromozomiale

• Exista 2 tipuri importante:Exista 2 tipuri importante:– Modificari ale numarului de cromozomiModificari ale numarului de cromozomi1.1. Multiplicarea numarului de seturi haploide Multiplicarea numarului de seturi haploide

(poliploidii)(poliploidii)2.2. Prezenta de cromozomi suplimentari Prezenta de cromozomi suplimentari

(2n+1=47) sau in minus (2n-1=45), (2n+1=47) sau in minus (2n-1=45), denumite trisomii si respectiv monosomii; denumite trisomii si respectiv monosomii; apar prin non-disjunctii in special in timpul apar prin non-disjunctii in special in timpul meiozeimeiozei

– Modificari ale structurii cromozomilorModificari ale structurii cromozomilor

Mecanisme de aparitie a Mecanisme de aparitie a poliploidiilorpoliploidiilorTriploidia (3n=69 cromozomi):Triploidia (3n=69 cromozomi):• fecundarea unui ovul care n-a expulzat al II-lea fecundarea unui ovul care n-a expulzat al II-lea

globul polar de catre un spermatozoid (diginie)globul polar de catre un spermatozoid (diginie)• fecundarea unui ovul de catre 2 spermatozoizi fecundarea unui ovul de catre 2 spermatozoizi

(diandrie) sau de catre un spermatozoid purtator a 2 (diandrie) sau de catre un spermatozoid purtator a 2 seturi haploide (formula cromozomiala 46 yy sau 46 seturi haploide (formula cromozomiala 46 yy sau 46 xx) in urma unor erori aparute in cursul xx) in urma unor erori aparute in cursul spermatogenezeispermatogenezei

Tetraploidia (4n=92 cromozomi):Tetraploidia (4n=92 cromozomi):• eroari in prima diviziune mitotica a zigotului: eroari in prima diviziune mitotica a zigotului:

duplicarea materialului genetic nu este insotita de duplicarea materialului genetic nu este insotita de diviziunea nucleului.diviziunea nucleului.

Mecanismul aneuploidiilorMecanismul aneuploidiilor

• Erori in desfasurarea meiozei I sau meiozei Erori in desfasurarea meiozei I sau meiozei II prin non-disjunctii ale cromozomilor II prin non-disjunctii ale cromozomilor omologi sau cromatidelor surori.omologi sau cromatidelor surori.

• Se formeaza gameti cu nulisomie (22 cz) Se formeaza gameti cu nulisomie (22 cz) sau disomie (24 cz) care, prin unire cu sau disomie (24 cz) care, prin unire cu gameti normali formeaza zigoti cu gameti normali formeaza zigoti cu monosomie (45 cz) sau trisomie (47 cz) monosomie (45 cz) sau trisomie (47 cz)

• Non-disjunctiile aparute in cursul Non-disjunctiile aparute in cursul diviziunilor mitotice ale embrionului au ca diviziunilor mitotice ale embrionului au ca rezultat aparitia mozaicurilor cromozomialerezultat aparitia mozaicurilor cromozomiale

Non-disjunctii in meioza INon-disjunctii in meioza I

Non-disjunctii in meioza IINon-disjunctii in meioza II

Non-disjunctiicromozomiale

Aneuploidiile complete ale cromozomilor somatici sunt in general letale.

Exceptii:1. Sindromul Down - trisomia 212. Sindromul Patau - trisomia 133. SindromulEdwards - trisomia 18

4. Aneuploidiile cromozomilor sexuali

Segregarea cromozomilor la un purtator de translocatie 14/21

Purtatorii de translocatie:

- Risc de a produce gameti neechilibrati genetic

- Moartea produsului de conceptie purtator de aberatie cromozomiala

- Diagnostic frecvent in familiile in care exista cazuri de malformatii congenitale si avorturi spontane repetate

Malformatii congenitaleMalformatii congenitale• Aberatii cromozomiale numericeAberatii cromozomiale numerice

– Sdr. Down syndrome (trisomia 21) Sdr. Down syndrome (trisomia 21) •1/ 600-700 n.n.vii1/ 600-700 n.n.vii

– Sdr.Edward (trisomia 18)Sdr.Edward (trisomia 18)– Sdr.Patau (trisomia 13)Sdr.Patau (trisomia 13)– Aneuploidii gonosomaleAneuploidii gonosomale

•Sdr.Turner (45,X)Sdr.Turner (45,X)•Sdr. Klinefelter (47,XXY)Sdr. Klinefelter (47,XXY)•47,XXX; 47,XYY47,XXX; 47,XYY

Distribution of non-Distribution of non-disjunctiondisjunction

  Meiosis I Meiosis II

Mitosis

Maternal 21, 15, 16 18 15, 18, 21, 8

Paternal - 18, 21 18, 21

AneuploidyAneuploidy• As women age As women age

– some chromosomes exhibit non-disjunction in oocytessome chromosomes exhibit non-disjunction in oocytes– Many theories whyMany theories why

• 13, 18, 2113, 18, 21 associated with age associated with age• 1616 and and XX only first meiotic division associated only first meiotic division associated

with age with age • Most chromosome abnormalities incompatible Most chromosome abnormalities incompatible

with lifewith life• Will miscarryWill miscarry

Maternal age specific estimates of Maternal age specific estimates of trisomy trisomy among all clinically recognisable among all clinically recognisable pregnanciespregnancies

Hassold et al., 1985

Production line hypothesis Production line hypothesis (PLH)(PLH)

• Henderson and Edward (1968) Henderson and Edward (1968)

• Germ cells committed to meiosis sequentially in Germ cells committed to meiosis sequentially in fetal life fetal life

• Released as mature ova in sequence enter meiosisReleased as mature ova in sequence enter meiosis• Chiasmata fewer in ova laid down late in fetal lifeChiasmata fewer in ova laid down late in fetal life• Leads to increased number of univalents Leads to increased number of univalents • Thus aneuploid offspring in older femalesThus aneuploid offspring in older females

• Evidence both supporting (Polani and Crolla, 1991) Evidence both supporting (Polani and Crolla, 1991) and refuting (Speed and Chandley, 1983)and refuting (Speed and Chandley, 1983)

Depleted oocyte hypothesis Depleted oocyte hypothesis (DOH)(DOH)

• Warburton (1989)Warburton (1989)

• as women ageas women age

– decreasing number of antral stage follicles per cycledecreasing number of antral stage follicles per cycle

– thus increased likelihood of ovulating sub-optimal thus increased likelihood of ovulating sub-optimal oocytesoocytes

– may include those with aberrant recombinationmay include those with aberrant recombination

Parental origin of Parental origin of aneuploidyaneuploidy

Paternal %Paternal % Maternal %Maternal %Trisomy 13Trisomy 13 1515 8585Trisomy 18Trisomy 18 1010 9090Trisomy 21Trisomy 21 55 959545,X45,X 8080 202047,XXX47,XXX 55 959547,XXY47,XXY 4545 555547,XYY47,XYY100100 00

Down syndrome typeDown syndrome type• 95% standard 95% standard

trisomytrisomy• 1% mosaics1% mosaics

– Due to increase in Due to increase in maternal agematernal age

• 4% translocations 4% translocations – no age effectno age effect

Chromosome abnormalities in humans

• SpermatozoaSpermatozoa10%10%• Mature oocytesMature oocytes 25%25%• Spontaneous miscarriageSpontaneous miscarriage 50%50%• Live birthsLive births 0.5-1%0.5-1%• Most due to maternal meiotic non Most due to maternal meiotic non

disjunctiondisjunction• Strongly related to maternal ageStrongly related to maternal age• Natural selection at workNatural selection at work

Chromosome abnormalities in Chromosome abnormalities in miscarriagesmiscarriages

Incidence %Incidence %Trisomy 13Trisomy 13 22Trisomy 16Trisomy 16 1515Trisomy 18Trisomy 18 33Trisomy 21Trisomy 21 55Other TrisomyOther Trisomy 2525

Monosomy XMonosomy X 2020TriploidyTriploidy 1515TetraploidyTetraploidy 55Other Other 1010

Chromosome abnormalities in Chromosome abnormalities in newbornsnewborns

Incidence / 10,000 birthsIncidence / 10,000 birthsTrisomy 13Trisomy 13 22Trisomy 18Trisomy 18 33Trisomy 21Trisomy 21 1515

45,X45,X 1147,XXX47,XXX 101047,XXY47,XXY 101047,XYY47,XYY 1010UnbalancedUnbalanced 1010BalancedBalanced 3030TotalTotal 9090

• TriploidyTriploidy

• Trisomy 16Trisomy 16

• Trisomy 13 Trisomy 13 &18&18

• Trisomy 21 Trisomy 21

• KlinefeltersKlinefelters

• 45X 45X

→rare at birth – lethalrare at birth – lethal

→Most common in spontaneous Most common in spontaneous miscarriagesmiscarriages

→Completely lethal. Cause unknown Completely lethal. Cause unknown

→95% miscarry 95% miscarry

→80% miscarry 80% miscarry

→50% miscarry 50% miscarry

→1% at conception1% at conception→98% miscarry, probably mosaic survive98% miscarry, probably mosaic survive

Chromosome abnormalitiesChromosome abnormalities

Each probe is specific to one region of a chromosome (pair), and is labeled with fluorescent molecules throughout it's

length.

Each microscope slide contains many metaphases. Each metaphase consists of the complete set of chromosomes, one small segment of which each probe will seek out and bind

itself to

Step 1 - break apart (denature) the double strands of DNA in both the probe DNA and the chromosome DNA so they can bind to each other.

This is done by heating the DNA in a solution of formamide at a high temperature.                                                                          

Step 2 - the probe is placed on the slide and a glass coverslip is placed on top.

The coverslip is sealed with rubber cement. The slide is then placed in a 37 C incubator overnight to allow the probe to hybridize with the target chromosome.

Example of chromosome enumeration:

Here, an interphase cell shows three pink signals and two green signals. This is the case in the

detection of trisomy 21, where the chromosome 21 probe would be labeled pink and a control probe

(13) is labeled green.

Example of detection of deletion using control probe:

In this case, the probe for the region of interest is labeled green, and another probe (control) which binds to the same chromosome is labeled pink. The left-hand

chromosome is intact, but the other is deleted in the region where the probe was supposed to bind. If the

control probe was not used, the deleted chromosome would not have been easily singled out from all of the

others in the metaphase because it would not have contained a fluorescent signal.

Centromere for X chromosome 48,XXXX

FISH with Y probe showing YY male - metaphase and interphase

FISH on interphase nuclei withX and Y probes - show mosaicism

cells with XY and XXY

green = X / red = Y

Prenatal Aneuploidy FISHPrenatal Aneuploidy FISH

Rapid identification of a fetus or newborn • aids in the decision making phase regarding management (3-6 hrs)

PrenatallyPrenatally FISH can serve

as a stand-alone testWhen:Abnormal U/SLate gestation

AMAanxiety

FISH DNA Probes The AneuVysion assay from Vysis, Inc.  Centromeric

CEP 18 - 18Z1 / CEP X - DXZ1 / CEP Y - DYZ3

  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Unique DNA sequences

LSI 13 21 - 13q14 region (RB1 locus)LSI 21 21 - 21q22.13 to 21q22.2 region

Assay performed according to manufactures instructions  

Cytogenetic analyses - standard G-banding methods  

Molecular Cytogenetic (FISH) TestsConstitutional studies

Aneuploidy 13, 18, 21, X and Y

Wolf Hurshorn 4p-Cri du Chat 5p-Williams 7q22-Retinoblastoma 13q14- Angelman 15q12-Prader-Willi 15q12-Miller-Dieker 17p13-Smith-Magenis 17p11-DiGeorge/VCF 22q11-STS Xp22.3-SRY Xp22.3- Kallmans Xp22.3-Sex X and Y

Subtelomeres (46 probes)M-FISH 23 chromosomes paints

Microdeletion syndrome probes

Prader-Willi del(15q12)

normal

Williams del(7q11)

Subtelomeric probes

a powerful new tool in detecting

cryptic telomericchromosomal rearrangements

Chromosomesappear normal

For mental retardation /

developmentaldelay

M-FISH (SKY)

FISH in Leukemia

•Translocations•Aneuploidy

•Rearrangements•Amplifications

•Etc.,

AML1/ETO t(8;21) PROBE PanelsPML/RARA t(15;17) CEP X/Y Sex mismatch transplantRARA t(11;17), t(15;17), 17q21 CLL 11q-, +12, 13q-, 17p-CBFB t(16;16), inv(16), del(16) ALL 4/10/17 Hyperdiploidy MLL t(11q23) Her 2 Neu Breast Cancer BCR/ABL t(9;22) UroVysion 3, 7, 17, 9p-CF1R -5/5q- Multiple myeloma (4;16), 11q, 13q, 17p EGR-1 -5/5qD7S486 -7/7q-LSI D7S522 -7/7q-CEP 8 +8LSID20S108 20q-TEL/AML1 t(12;21)LSI p16 del 9p21IGH/MYC t(8;14)MYC t(2;8), t(8;22), t(8;14) D13S25 del 13q14D13S319 del 13q14P53 del 17pALK t(2;5)BCL6 t(3q27)IGH/CCND1 t(11;14)IGH/BCL2 t(14;18)MALT t(18q21) Synovial t(X;18)

Major Cancer chromosome anomalies

Trisomy 4/10 of pre B-ALLTrisomy 4 and 10 - ALL

CML

Philadelphiatranslocation

Relatively good prognosis in CML Relatively poor prognosis in ALLAdditional chromosome changes: +8, iso(17q), +19,+Ph1, - blast crisis = poor prognosis

9;22 - CML

BCR - 22q in green

ABL - 9q in red

when fused - yellow

Chromosome 16 inversion

Examples of double minutes (dm) and homogeneous staining regions (hsr) - forms of gene amplification.- Prognosis is poor

Gene amplification of myc oncogene

Green is chromosome 17 centromere / Red is Her-2 gene

Gene amplification

76 year old male with 76 year old male with history of bladder cancerhistory of bladder cancer

6R 8G 3A 2Y 4R 3G 4A 2Y

Extra copies of chromosome 3(R), 7(A), 17(G)- 2 copies of 9p (normal)

Diploid numbers of some commonly studied organisms(as well as a few extreme examples)

Homo sapiens (human) 46

Mus musculus (house mouse) 40

Zea mays (corn or maize) 20

Drosophila melanogaster (fruit fly) 8

Xenopus laevis (South African clawed frog) 36

Caenorhabditis elegans (roundworm) 12

Saccharomyces cerevisiae (budding yeast) 32

Canis familiaris (domestic dog) 78

Arabidopsis thaliana (mustard plant) 10

Muntiacus reevesi (Chinese muntjac, a deer) 23

Muntiacus muntjac (its Indian cousin) 6

Myrmecia pilosula (an ant) 2

Parascaris equorum var. univalens (parasitic roundworm) 2

Cambarus clarkii (a crayfish) 200

Equisetum arvense (field horsetail, a plant) 216

Comparison of human (46),

Chimpanzee (48), Gorilla (48), andOrangoutang's (48) chromosomes

Evolution in action

Telomeres - Centromeres

In VitroIn Vitro Fertilization Fertilization

• Ovarian stimulation Ovarian stimulation • Transvaginal ultrasound guided aspirationTransvaginal ultrasound guided aspiration

Jeffrey Deaton, M.D Jeffrey Deaton, M.D Center for Reproductive MedicineCenter for Reproductive Medicine

Follicular Follicular DevelopmentDevelopment

Mature Mature OocyteOocyte

Insemination for PGDInsemination for PGD

• Intracytoplasmic Sperm Injection (ICSI)Intracytoplasmic Sperm Injection (ICSI)

J. David Wininger, Ph.D; Lab Director J. David Wininger, Ph.D; Lab Director Center for Reproductive MedicineCenter for Reproductive Medicine

Sperm Sperm ImmobilizationImmobilization Sperm InjectionSperm Injection

Embryo DevelopmentEmbryo DevelopmentPronuclear EmbryoPronuclear Embryo

4 Cell Embryo4 Cell Embryo

8 Cell Embryo8 Cell Embryo