PRENATAL AND POSTNATAL CYTOGENETIC

INVESTIGATION IN PATIENTS WITH

PRADER-WILLI SYNDROME

Conf. dr. Eusebiu Vlad Gorduza

Medical Genetics Department, “Gr. T. Popa” University of Medicine and Pharmacy Iaşi;
 Cytogenetic analysis are performed to
view the chromosomal material in nucleus
of cells;
Cytogenetic techniques was introduced
in practice in the middle of XX century
and after that was ameliorated by
application of chromosomal banding and
molecular cytogenetic techniques →
diagnosis of chromosomal diseases;
The major indications of chromosomal
analysis are: plurimalformative
syndromes, couples with reproductive
troubles, prenatal diagnosis and some
CHROMOSOMAL DISEASES
=
CHROMOSOMAL
ABNORMALITIES
ABNORMAL KARYOTYPE

GENOMIC CHROMOSOMAL
MUTATIONS MUTATIONS
 CHROMOSOMAL
ABNORMALITIES

NUMERICAL STRUCTURAL
 STRUCTURAL
ABNORMALITIES
BALANCED UNBALANCED
•deletions
•inversions
•duplications
•translocations
•Ring chrs.
•Dicentric chrs.
NORMAL PHENOTYPE
•isochromosomes

RISK FOR CHILDREN WITH UNBALANCED

STRUCTURAL ABNORMALITIES,
ABORTIONS, STERILITY DISEASES
 Cytogenetic techniques

CLASSICAL MOLECULAR
Analyse the Analyse the
chromosomes chromosomes in
in light UV microscopy
microscopy
FISH
 Without
banding

With banding

1950

1970

Evolution of cytogenetic techniques

FISH

1990
Evolution of modern cytogenetic techniques
growing-up of resolution

Control Signals

Region-Specific Signal

Metaphase Pro(meta)phase FISH

1 band – 10 Mb 1 band – 3-5 Resolution – some kb
Mb
Principles of classical
cytogenetics methodes
(1) Obtaining of cells in division:
 Cultures of cells:
Lymphocytes – short culture 72 hours
fetal cells – amniocytes – long cultures – 2-3 weeks

 direct analysis – tissues with high rate of divsion

(bone marrow)
(1) Blocking of division: colchicine
(2) Obtaining of chromosomal preparates:
hypotonic treatment, fixation, staining ±
banding.
(3) Analysis of chromosomes → karyotype
Principles of
FISH Molecular technique to
detection of chromosomal
abnormalities

F fluorescence
I in Hybridization between a
fluorescent probe and a
S situ target DNA sequence on
H patient’s chromosomes
hybridization

Molecular technique to localize

the gene’s sequences
 PRADER-WILLI SYNDROME
1/10.000-1/20.000 n.b.
microdeletion/deletion 15q11-q13
Clinical:
 Major criteria:
 Infantile central hypotonia;
 Infantile feeding problems/ failure to thrive;
 Rapid weight gain between 1-6 years;
 Characteristic facial features (narrowing of bifrontal diameter;
almond shape eyes);
 Hypogonadism (genital hypoplasia; pubertal deficiency);
 Developmental delay/ mental retardation;
 Minor criteria:
 Typical behavior problems;
 Sleep disturbances;
 Short stature;
 Hypopigmentation;
 Acromicria.
 PRADER-WILLI
SYNDROME

Crs 15
 Prader-Willi syndrome
GENETICS
Lack of expression of normally active
paternally inherited genes at
chromosome 15q11-q13 - an
imprinted region on chromosome 15;
75% of patients have a paternally
small deletion;
24% of patients have a maternally
uniparental disomy of chromosome 15
1% of patients have a defect of
imprinting center of chromosome 15
Prader-Willi syndrome
GENETICS

IC SPW SA mat

N IC SPW SA pat

ICIC SPW SA

Prader-Willi Syndrome
mat

Deletion
pat

IC SPW SA mat

DUP 15 IC SPW SA mat

IC SPW SA mat

IC mutation IC SPW SA patm

ACTIVE INACTIVE
 Postnatal investigation

Is correlate with presence of

clinical features of
Prader-Willi syndrome
 Prader-Willi syndrome
DIAGNOSIS
HIGH RESOLUTION KARYOTYPE

ABNORMAL De novo 15q11-13 NORMAL

deletion

Unbalanced translocation

FISH
Karyotype in both parents

Microdeletion NORMAL

Maternal 15 UPD Methylation test, gene mutation analysis

Defect in imprinting center

 PRADER-WILLI SYNDROME

15q11-13 chromosomal deletion

PRADER-WILLI SYNDROME
Abnormal
chromosome
15

Normal
chromosome
15

15q11-13 chromosomal microdeletion

 Prenatal investigation

Is correlate with presence of

a case of Prader-Willi
syndrome in family
 PRADER WILLI SYNDROME
GENETIC COUNSELING

The risk of parents that have a child with PW

syndrome to have another affected child is:
 In the case of a de novo chromosomal deletion < 1%;
 In the case of a de novo chromosomal microdeletion <
1%;
 In the case of a de novo unbalanced translocation < 1%;
 In the case of an inherited unbalanced translocation – 5-
20% depending of chromosomes and parental origin →
major indication for prenatal cytogenetic investigation;
investigation
 In the case of a mutation in imprinting center on 15
paternally inherited chromosome (mutation providing
from paternal grandmother) – 50% - the mutation is
transmitted in an autosomal dominant pattern → need
prenatal molecular investigation
 Prenatal diagnostic
prenatal diagnostic is a complex medical act that
allows identification of some congenital defects and
genetic diseases at embryone or foetus.

need a multidisciplinare colaborration between

geneticist, obstetrician, cytogeneticist,
ultrasonographist, biochemistrician → essential role
of geneticist for evaluation and genetic counselling.

identification of couples with risk can be make:

between conception – one of the members of couple
have balanced chromosomal abnormality;
early in pregnancy
Technics in Prenatal diagnosis
 ultrasonography.

amniocentesis

chorionic villus Chromosomal

sampling. analysis

cordonocentesis.
PRENATAL FISH
FISH – hybridization between DNA target
sequence from chromosome and a specific probe
fluorescent labeled;
Advantages in prenatal diagnosis:
Short times - results in 2-3 days → ↓ anxiety of
couple;
Can be apply also on interphasic cells (no need the
cell harvest)
High precision
Disavantages of FISH technique:
 High level of technicity
 Expensive price of technique
METHODS TO OBTAIN
EMBRYONIC CELLS
Prenatal diagnosis
chorionic villus sampling
Week 8-12
Under sonografical control
Chromosomal analysis direct or after cell harvesting:
 Direct analysis → results after 24-36 hours.
 Final results after harvesting
Avantage – prenatal diagnosis in first trimester of pregnancy
Problems:
 Cells with small chromosomes → difficults to evaluate the kyryotype
 Chromosomal mosaicism - 1-3% cases – real or confined to placenta →
amniocentesis → another karyotype.
 3-5% incidents: malformations of limbs, spontaneous abortion,
gestational bleeding, infection
Chorionic villus sampling
Prenatal diagnosis -
amniocentesis
Week 12-18
Aspiration of 10 – 20 ml amniotic fluid
Under sonografical control
Chromosomal analysis after cell harvesting → 14-21
days:
 To reduce the anxiety → screening by FISH.
Avantage:
 diagnosis of all chromosomal abnormalities;
 Cells with long chromosome → facile to examen;
 Small risks (0,5-1%) for spontaneous abortion, gestational
bleeding, infection
Problems:
 Late diagnosis – 16-20 week of gestation → problems with
finished pregnancy.
Amniocentesis
Prenatal diagnosis -
chordonocentesis
Week 20-24
Aspiration of 3 -5 ml of fetal blood
Under sonografical control
Limitated aplication to chromosomal diagnosis – to
confirm/infirm an mosaicism confined to placenta
Avantage:
Results in 3-4 days;
Cells with long chromosome → facile to examen;
Problems:
Late diagnosis – 20-24 week of gestation →
problems with finished pregnancy.
Chordonocentesis
Conclusion
Thw cytogenetic investigation in Prader-
Willi syndrome is different before and
after the birth;
Before the birth the chromosomal
analysis by classical techniques or by
FISH technique is correlate with the
presence of clinical features of syndrome;
Conclusion
In PWS prenatal chromosomal
diagnosis is difficult and impose
the use of FISH technique;
After obtain the results, is
essential to give a correct
genetic counselling → family can
take an informed decision
Thank you for atention
I will wait you in the capital of Moldavia region