vlad gorduza - zalau testare prenatal si postnatal a prader willi
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8/8/2019 Vlad Gorduza - Zalau Testare Prenatal Si Postnatal A Prader Willi
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Conf. dr. Eusebiu Vlad Gorduza
Medical Genetics Department, “Gr. T. Popa” University of Medicine and Pharmacy Iaşi;
PRENATAL AND POSTNATAL CYTOGENETICPRENATAL AND POSTNATAL CYTOGENETIC
INVESTIGATION IN PATIENTS WITHINVESTIGATION IN PATIENTS WITHPRADER-WILLI SYNDROMEPRADER-WILLI SYNDROME
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Cytogenetic analysis are performed toview the chromosomal material in nucleusof cells;Cytogenetic techniques was introduced
in practice in the middle of XX centuryand after that was ameliorated byapplication of chromosomal banding andmolecular cytogenetic techniques →
diagnosis of chromosomal diseases;The major indications of chromosomal
analysis are: plurimalformativesyndromes, couples with reproductive
troubles, prenatal diagnosis and some
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CHROMOSOMAL DISEASES
CHROMOSOMAL
ABNORMALITIES
=
GENOMIC
MUTATIONSCHROMOSOMAL
MUTATIONS
ABNORMAL KARYOTYPE
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CHROMOSOMAL
ABNORMALITIES
NUMERICAL STRUCTURAL
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STRUCTURAL
ABNORMALITIESBALANCED UNBALANCED
•inversions
•translocations
•deletions
•duplications
•Ring chrs.
•Dicentric chrs.
•isochromosomes
RISK FOR CHILDREN WITH UNBALANCED
STRUCTURAL ABNORMALITIES,
ABORTIONS, STERILITY DISEASES
NORMAL PHENOTYPE
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Cytogenetic techniques
CLASSICALAnalyse the
chromosomes
in lightmicroscopy
MOLECULARAnalyse the
chromosomes in
UV microscopy
FISH
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1950
1970
1990
Without
banding
With banding
FISH
Evolution of cytogenetic techniques
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Evolution of modern cytogenetic techniques
growing-up of resolution
Control Signals
Region-Specific Signal
Metaphase
1 band – 10 Mb
Pro(meta)phase
1 band – 3-5
Mb
FISH
Resolution – some kb
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(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 classical
cytogenetics methodes
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Principles of
FISH
F f luorescence
I inS situ
Hhybridization
Molecular technique to
detection of chromosomal
abnormalities
Molecular technique to localize
the gene’s sequences
Hybridization between afluorescent probe and a
target DNA sequence on
patient’s chromosomes
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PRADER-WILLI SYNDROME1/10.000-1/20.000 n.b.
microdeletion/deletion 15q11-q13Clinical:
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.
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PRADER-WILLISYNDROME
Crs 15
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Prader-Willi syndromeGENETICS
Lack of expression of normally activepaternally inherited genes atchromosome 15q11-q13 - animprinted region on chromosome 15;75% of patients have a paternallysmall deletion;24% of patients have a maternally
uniparental disomy of chromosome 151% of patients have a defect of imprinting center of chromosome 15
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Prader-Willi syndromeGENETICS
mat
patN
mat
pat
mat
mat
mat
patm
IC SPW SA
IC SPW SA
ICIC SPW SA
DUP 15
IC SPW SA
IC SA
Deletion
IC SPW SA
IC SAIC mutation
SPW
SPW
P r a d e r - Wi l l i S yn d r om e
ACTIVE INACTIVE
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Prader-Willi syndromeDIAGNOSIS
HIGH RESOLUTION KARYOTYPE
NORMAL
FISH
ABNORMAL De novo 15q11-13
deletion
Unbalanced translocation
Karyotype in both parents
Microdeletion NORMAL
Methylation test, gene mutation analysisMaternal 15 UPD
Defect in imprinting center
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PRADER-WILLI SYNDROME
15q11-13 chromosomal deletion
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PRADER-WILLI SYNDROME
Abnormalchromosome
15
Normal
chromosome
15
15q11-13 chromosomal microdeletion
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Prenatal investigation
Is correlate with presence of
a case of Prader-WilliPrader-Willi
syndromesyndrome in family
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PRADER WILLI SYNDROMEGENETIC COUNSELING
The risk of parents that have a child with PWsyndrome 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 – 5-5-
20%20% depending of chromosomes and parental origin →major indication for prenatal cytogenetic investigationmajor indication for prenatal cytogenetic investigation;
In the case of a mutation in imprinting center on 15paternally inherited chromosome (mutation providingfrom paternal grandmother) – 50% - the mutation istransmitted in an autosomal dominant pattern → needneedprenatal molecular investigationprenatal molecular investigation
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Prenatal diagnosticprenatal diagnostic is a complex medical act that
allows identification of some congenital defects andgenetic diseases at embryone or foetus.
need a multidisciplinare colaborration betweengeneticist, obstetrician, cytogeneticist,ultrasonographist, biochemistrician → essential roleof geneticist for evaluation and genetic counselling.
identification of couples with risk can be make:
between conception – one of the members of couplehave balanced chromosomal abnormality;early in pregnancy
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Technics in Prenatal diagnosis
ultrasonography .
amniocentesis
chorionic villussampling.
cordonocentesis.
CChhromosomromosomalal
analysisanalysis
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FISH – hybridization between DNA targetsequence from chromosome and a specific probefluorescent labeled;
Advantages in prenatal diagnosis:
Short times - results in 2-3 days → ↓ anxiety of couple;
Can be apply also on interphasic cells (no need thecell harvest)
High precision
Disavantages of FISH technique: High level of technicity
Expensive price of technique
PRENATAL FISH
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METHODS TO OBTAINMETHODS TO OBTAIN
EMBRYONIC CELLSEMBRYONIC CELLS
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Prenatal diagnosis
chorionic villus samplingWeek 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
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Chorionic villus sampling
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Prenatal diagnosis -
amniocentesisWeek 12-18Aspiration of 10 – 20 ml amniotic fluidUnder sonografical control
Chromosomal analysis after cell harvesting → 14-21days: 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.
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Amniocentesis
P t l di i
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Prenatal diagnosis -chordonocentesis
Week 20-24Aspiration of 3 -5 ml of fetal bloodUnder sonografical control
Limitated aplication to chromosomal diagnosis – toconfirm/infirm an mosaicism confined to placentaAvantage:
Results in 3-4 days;Cells with long chromosome → facile to examen;
Problems:Late diagnosis – 20-24 week of gestation →
problems with finished pregnancy.
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Chordonocentesis
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Conclusion
Thw cytogenetic investigation in Prader-Willi syndrome is different before and
after the birth;Before the birth the chromosomalanalysis by classical techniques or byFISH technique is correlate with thepresence of clinical features of syndrome;
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Conclusion
In PWS prenatal chromosomaldiagnosis is difficult and impose
the use of FISH technique;
After obtain the results, is
essential to give a correctgenetic counselling → family cantake an informed decision
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