Cariotipul

Aberatii cromozomiale
Citogenetica

• Studiul cromozomilor si a anomaliilor cromozomiale:

- in metafaza
- Normal 46 cromozomi: 22 perechi cromozomi
somatici +2 cromozomi sexuali XX sau XY

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

bandarea G a cromozomilor
 Modelul de benzi este cromozom-
specific

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Aranjarea cromozomilor in cariotip
Indicatii clinice pentru analiza
cromozomilor
• Deficitul cresterii pre-/postnatale
• Avort spontan/n.n. mort
• Sterilitate/infertilitate
• Istoric familial de rearanjament cromozomial
• Valori anormale ale markerilor serici
materni/anomalii fetale depistate ecografic
• Neoplazii
Frecventele unor anomalii
cromozomiale in avorturile
spontane
Prepararea cariotipului
FISH
Metoda FISH pe nuclei interfazici
Se folosesc probe oligonucleodidice marcate fluorecent pt.
diagnosticul aneuplidiilor cromozomilor 13, 18, 21, X, si Y

Nuclei de la
acelasi
produs de
conceptie

verde = cromozomul 13 bleu = cromozomul 18

rosu= cromozomul 21 verde = X
rosu = Y
• Translocatii

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 Syndrome

• Normal Karyotype
Semnificatia clinica a anomaliilor
cromozomiale

• Non-disjunctii in meioza

• Aberatii cromozomiale numerice

• Aberatii cromozomiale structurale

• Screening si diagnosticul prenatal

Anomalii cromozomiale
• Spermatozoizi 10%
• Ovocit matur 25%
• Avort spontan 50%
• N.n. viu 0.5-1%
• In general apar prin non-disjunctii in
meioza materna, in corelatie cu varsta
gravidei
Non-
disjunctii
cromozomiale
Aneuploidiile complete ale cromozomilor somatici sunt
in general letale.

Exceptii:
1. Sindromul Down - trisomia 21
2. Sindromul Patau - trisomia 13
3. SindromulEdwards - trisomia 18

4. Aneuploidiile cromozomilor sexuali

Originea parentala a
aneuplidiilor
Paterna % Materna %
Trisomia 13 15 85
Trisomia 18 10 90
Trisomia 21 5 95
45,X 80 20
47,XXX 5 95
47,XXY 45 55
47,XYY 100 0
Tipuri de anomalii cromozomiale în
avorturile spontane
Incidence %
Trisomia 13 2
Trisomia 16 15
Trisomia 18 3
Trisomia 21 5
Alte trisomii 25

Monosomia X 20
Triploidii 15
Tetraploidii 5
Alte aberaţii 10
Sindromul Down

• 95% liberă şi omogenă

• 1% mozaicuri
– Efectul vârstei materne

• 4% translocaţii
 Downs Syndrome
Boy

• 47,XY,+21
Trisomia 21 sau Sindromul Down

1. 47, XY, +21 or 47, XX, +21

– 1866 descris de John Langdon Down.
– Obstrucţia duodenului, atrezie de esofag,
duoden sau anus.
– 40% prezintă malformaţii cardiace: canal
atrioventricular , defect de sept ventricular .
– 95% : non-disjuncţii, restul sunt translocaţii.
– 90 - 95 % în meioza maternă
– 1 - 3 % mozaicuri.
– Regiunea 21 q22
 Sindromul Edward

• 47,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.
Trisomia 13

– 47, XY, +13

– Patau syndrome: 1/10000 n.n. vii .
– Malformations severe: cardiace, anencefalie,
holoprozencefalie, renale etc
– Despicătura de buză şi palat, microftalmie, polidactilie.
– Supravieţuire redusă
– 80% trisomie liberă, restul translocaţii .
– 95% din sarcinile cu trisomie13 sunt avortate.
Patau
Syndrome
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 gonosomale
 Deletion

Anomalii
cromozomiale
structurale Duplication

Homologous
chromosomes

Inversion

Reciprocal
translocatio
n

Nonhomologous
chromosomes Figure 8.23A, B
Translocatia Robertsoniana intre
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
Deletii
Interstitiale
Terminale
• Cri du chat, 5p15 • Williams, 7q11.2,
– microdeletion (FISH)
• Wolf-Hirschhorn,
4p36 • Retinoblastoma,
13q14
• Prader-Willi, 15q11.2
• Angelman, 15q11.2
• DiGeorge, 22q11.2
Deletii

Sdr. “Cri du Chat “

-laringe hipoplazic
-microcefalie
-dismorfii faciale
Cri du Chat

• Deleţie terminală
– 5p15

• Plânset asemănător
mieunatului pisici

• Retard mental
Wolf-Hirschhorn
Syndrome

Internet
Link
Diagnosticul prenatal

• Teste de SCREENING  Teste de

DIAGNOSTIC
Diagnosticul prenatal

• Teste de Diagnostic • Teste de screening

– ecografie – ecografie
– amniocenteza • translucencta nucala
(NT)
– CVS
– Markerii serici materni
– cordocenteza
• Trim II
• Trim I
– Depistarea celulelor
fetale in sangele
matern (in viitor)
Screening

• În trim. I şi II

• Markerilor serici materni

• Ecografic

• Rezultate anormale: teste invazive de diagnostic

Screening al markerilor biochimici

• Screening în I trimestru:
– Vârsta gravidei
– free -hCG
– Pregnancy associated plasma protein-A
(PAPP-A)
• nu în practica curentă
Screening prenatal : I trimestru

• Test de screening : translucenţa nucală(NT)

– Se efectueaza intre săptămânile 10-14
– Depistează 54-72% din feţii cu Sindrom Down
(DS)
– Alte anomalii cromozomiale: trisomia 13,18,
triploidii, sdr. Turner (45X)
– ≥3,5mm NT frecvent la feţii cu malformaţii
cardiace, hernie diafragmatică, malf. renale,
defecte de perete abdominal
 Screening ecografic
• Translucenta nucala (NT) and osul nazal (NB)
– Se utilizeaza impreuna cu screening seric din trim I pt. sdr. Down.
• NT (>3.5mm) se asociaza cu un risc crescut pt::
– Aberatii cromozomiale
– Defecte cardiace majore, DTN, alte anomalii structurale
– Anumote sdr. genetice
– Intarzierea cresterii intrauterine
• Cariotip normal dar >NT:
– Triplu test
– Morfologie fetala 18-22 weeks

• Morfologie fetala – sapt. 18-22

• Nu este perfecta: un rezultat normal nu inseamna intotdeauna un

copil sanatos
Screening prenatal : II trimestru

– Varsta gravidei
– Alpha-fetoproteina (AFP)
– hCG (total, , free-, free-)
– estriolul (uE3)*
– +/-Inhibina
• Triplu test: rezultate fals pozitive 5%
– Down syndrome: 60-70%
– Trisomia 18: 60%
– DTN: 75-80%
Malformatii congenitale
• Majore: 2-3%
• Minore: 14%
– Sunt implicate in 20% din decesele din prima sapt de
viata extrauterina.
– Etiologie %
Necunoscuta 65-75%
Genetica 15-25%
Factori de mediu 10%
– Afectiuni materne 4%
– Afenti infectiosi 3%
– Ag. Chimici, radiatii ~1%
– Agenti mecanici 1-2%
Malformatii congenitale

• Aberatii cromozomiale numerice

– Sdr. Down syndrome (trisomia 21)
• 1/ 600-700 n.n.vii
– Sdr.Edward (trisomia 18)
– Sdr.Patau (trisomia 13)
– Aneuploidii gonosomale
• Sdr.Turner (45,X)
• Sdr. Klinefelter (47,XXY)
• 47,XXX; 47,XYY
Malformatii congenitale

• Afectiuni monogenice-example:
Transmitere A.D, A.R, X-linkat

– Sdr. Marfan
– Fragile-X
– Acondroplazia
• etc etc
Anomalii congenitale

• Afectiuni materne:
– Diabet
– fenilcetonuria
– alcoolism

• Agenti infectiosi: rubeola, toxoplasma,

v.herpetic etc
Metode de diagnosic prenatal

• Biopsia de vilozităţi coriale

– Se efectueaza intre săptămânile 10-12
– Abord transabdominal sau transcervical
– Tesutul placentar este utilizat:
• cariotip
• Depistarea afecţiunilor monogenice (defect genic
cunoscut)
– Risc de avort: 1%
Prenatal diagnosis: chorionic villi sampling (CVS)

• sampling cells from placenta

• usually done 10-12 weeks
Teste invazive de diagnostic: biopsia de
vilozitati coriale

Test Risc de avort Moment optim Rezultat

• CVS 1/100 11 sapt. 11-12sapt
Rezultate neconcludente
(CVS)

• 1% CVS se obtin rezultate ambigui

• Contaminare cu celulele materne
• Mozaicism placentar (CPM)
• Mozicism adevarat al fatului
• Repetarea testului prin amniocenteza
sau cordocenteza
Metode de diagnosic prenatal

• Amniocenteza
– Se efectueaza intre săptămânile 15-20
– 10-20 ml L.A
• Cariotip
• Dozarea Alpha-fetoproteinei (AFP)
• Alte teste biochimice: bilirubina, acetilcolonesteraza
• QF-PCR sau FISH pt. diagnosticul citogenetic rapid
– Risc de avort: ~0,5%
Amniocenteza:
sapt 15-18
 Teste invazive de diagnostic:
amniocenteza

Test Risc de avort Moment optim Rezultat

• Amniocenteza 1/200 16 sapt. 18-22sapt.
Quantitative Fluorescent PCR
Diagnosticul prenatal rapid al trisomiilor
Utilizeaza polimorfisme ADN pt. a defini nr. de
copii

Marker polimorfic
ADN de pe cz. 21

Ratio: 1 : 1 :
1

Ratio: 1 : 2
CVS vs amniocenteza

• CVS • Amniocenteza
– Săpt.10-12 – Săpt.15-20
– Risc de avort: 1 /100 – 1 / 200
– Nu diagnosticul NTD – (Open NTD): da
– Analize ADN – mai dificil
– rezultat: 2săpt; FISH: – 2săpt; FISH: 72h
72h – rar
– mozaicism:1-2% – Avort terapeutic
– +/- D&C
Cromozomul"Philadelphia "
Translocatie 9:22
Tipuri de anomalii cromozomiale în
avorturile spontane
Incidence %
Trisomia 13 2
Trisomia 16 15
Trisomia 18 3
Trisomia 21 5
Alte trisomii 25

Monosomia X 20
Triploidii 15
Tetraploidii 5
Alte aberaţii 10
 Anomalii cromozomiale

• Exista 2 tipuri importante:

– Modificari ale numarului de cromozomi
1. Multiplicarea numarului de seturi haploide
(poliploidii)
2. Prezenta de cromozomi suplimentari
(2n+1=47) sau in minus (2n-1=45), denumite
trisomii si respectiv monosomii; apar prin non-
disjunctii in special in timpul meiozei
– Modificari ale structurii cromozomilor
Mecanisme de aparitie a
poliploidiilor
Triploidia (3n=69 cromozomi):
• fecundarea unui ovul care n-a expulzat al II-lea globul
polar de catre un spermatozoid (diginie)
• fecundarea unui ovul de catre 2 spermatozoizi (diandrie)
sau de catre un spermatozoid purtator a 2 seturi
haploide (formula cromozomiala 46 yy sau 46 xx) in
urma unor erori aparute in cursul spermatogenezei
Tetraploidia (4n=92 cromozomi):
• eroari in prima diviziune mitotica a zigotului: duplicarea
materialului genetic nu este insotita de diviziunea
nucleului.
Mecanismul aneuploidiilor

• Erori in desfasurarea meiozei I sau meiozei II

prin non-disjunctii ale cromozomilor omologi sau
cromatidelor surori.
• Se formeaza gameti cu nulisomie (22 cz) sau
disomie (24 cz) care, prin unire cu gameti
normali formeaza zigoti cu monosomie (45 cz)
sau trisomie (47 cz)
• Non-disjunctiile aparute in cursul diviziunilor
mitotice ale embrionului au ca rezultat aparitia
mozaicurilor cromozomiale
Non-disjunctii in meioza I
Non-disjunctii in meioza II
Non-
disjunctii
cromozomiale
Aneuploidiile complete ale cromozomilor somatici sunt
in general letale.

Exceptii:
1. Sindromul Down - trisomia 21
2. Sindromul Patau - trisomia 13
3. 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 congenitale

• Aberatii cromozomiale numerice

– Sdr. Down syndrome (trisomia 21)
• 1/ 600-700 n.n.vii
– Sdr.Edward (trisomia 18)
– Sdr.Patau (trisomia 13)
– Aneuploidii gonosomale
• Sdr.Turner (45,X)
• Sdr. Klinefelter (47,XXY)
• 47,XXX; 47,XYY
Distribution of non-disjunction

  Meiosis I Meiosis Mitosis

II
Maternal 21, 15, 16 18 15, 18,
21, 8
Paternal - 18, 21 18, 21
Aneuploidy

• As women age
– some chromosomes exhibit non-disjunction in oocytes
– Many theories why
• 13, 18, 21 associated with age
• 16 and X only first meiotic division associated with
age
• Most chromosome abnormalities incompatible with
life
• Will miscarry
Maternal age specific estimates of trisomy
among all clinically recognisable pregnancies

Hassold et al., 1985

Production line hypothesis (PLH)
• Henderson and Edward (1968)

• Germ cells committed to meiosis sequentially in fetal

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

• Evidence both supporting (Polani and Crolla, 1991)

and refuting (Speed and Chandley, 1983)
 Depleted oocyte hypothesis
(DOH)
• Warburton (1989)

• as women age
– decreasing number of antral stage follicles per cycle

– thus increased likelihood of ovulating sub-optimal

oocytes

– may include those with aberrant recombination

Parental origin of aneuploidy
Paternal % Maternal %
Trisomy 13 15 85
Trisomy 18 10 90
Trisomy 21 5 95
45,X 80 20
47,XXX 5 95
47,XXY 45 55
47,XYY 100 0
Down syndrome type

• 95% standard trisomy

• 1% mosaics
– Due to increase in
maternal age

• 4% translocations
– no age effect
Chromosome abnormalities in
humans
• Spermatozoa 10%
• Mature oocytes 25%
• Spontaneous miscarriage 50%
• Live births 0.5-1%
• Most due to maternal meiotic non
disjunction
• Strongly related to maternal age
• Natural selection at work
Chromosome abnormalities in
miscarriages
Incidence %
Trisomy 13 2
Trisomy 16 15
Trisomy 18 3
Trisomy 21 5
Other Trisomy 25

Monosomy X 20
Triploidy 15
Tetraploidy 5
Other 10
Chromosome abnormalities in
newborns
Incidence / 10,000 births
Trisomy 13 2
Trisomy 18 3
Trisomy 21 15

45,X 1
47,XXX 10
47,XXY 10
47,XYY 10
Unbalanced 10
Balanced 30
Total 90
Chromosome abnormalities
• Triploidy →rare at birth – lethal

• Trisomy 16 →Most common in spontaneous miscarriages

→Completely lethal. Cause unknown

→95% miscarry
• Trisomy 13 &18
→80% miscarry
• Trisomy 21
→50% miscarry
• Klinefelters
→1% at conception
• 45X →98% miscarry, probably mosaic survive
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 with

X and Y probes - show mosaicism

cells with XY and XXY

green = X / red = Y
 Prenatal Aneuploidy FISH
Rapid identification of a fetus or newborn
• aids in the decision making phase regarding
management (3-6 hrs)
Prenatally

FISH can serve

as a stand-alone test

When:
Abnormal U/S
Late gestation
AMA
anxiety
 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) Tests
Constitutional studies
Aneuploidy 13, 18, 21, X and Y
Wolf Hurshorn 4p-
Cri du Chat 5p-
Williams 7q22-
Retinoblastoma 13q14-
Angelman 15q12-
Prader-Willi 15q12- Microdeletion syndrome probes
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
 normal

Prader-Willi del(15q12)
Williams del(7q11)
 Subtelomeric
probes

a powerful new tool

in detecting
cryptic telomeric
chromosomal rearrangemen
 For mental
retardation /
developmental
delay

Chromosomes
appear
normal
M-FISH
(SKY)
FISH in Leukemia

•Translocations
•Aneuploidy
•Rearrangements
•Amplifications
•Etc.,
 Major Cancer chromosome anomalies
AML1/ETO t(8;21) PROBE Panels
PML/RARA t(15;17) CEP X/Y Sex mismatch transplant
RARA 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 -5/5q
D7S486 -7/7q-
LSI D7S522 -7/7q-
CEP 8 +8
LSID20S108 20q-
TEL/AML1 t(12;21)
LSI p16 del 9p21
IGH/MYC t(8;14)
MYC t(2;8), t(8;22), t(8;14)
D13S25 del 13q14
D13S319 del 13q14 Trisomy44/10
Trisomy andof
10pre B-ALL
- ALL
P53 del 17p
ALK t(2;5)
BCL6 t(3q27)
IGH/CCND1 t(11;14)
IGH/BCL2 t(14;18)
MALT t(18q21)
Synovial t(X;18)
 CML

Philadelphia
translocation

Relatively good prognosis in CML Relatively poor prognosis in ALL

Additional 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
 Gene amplification

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

76 year old male with
history 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

2
(parasitic roundworm)

Cambarus clarkii (a crayfish) 200

Equisetum arvense (field horsetail, a plant) 216

 Comparison of human (46),

Chimpanzee (48), Gorilla (48), and

Orangoutang's (48) chromosomes
Evolution in action

Telomeres - Centromeres
In Vitro Fertilization
Jeffrey Deaton, M.D
Center for Reproductive Medicine
• Ovarian stimulation
• Transvaginal ultrasound guided aspiration
Follicular Mature Oocyte
Development
Insemination for PGD
J. David Wininger, Ph.D; Lab Director
Center for Reproductive Medicine

• Intracytoplasmic Sperm Injection (ICSI)

Sperm Immobilization Sperm Injection
 Embryo Development
Pronuclear Embryo

4 Cell Embryo

8 Cell Embryo