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 CYTOGENETIC PRENATAL AND POSTNATAL CYTOGENETIC INVESTIGATION IN PATIENTS WITH INVESTIGATION IN PATIENTS WITH PRADER-WILLI SYNDROME PRADER-WILLI SYNDROME

Transcript of 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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Thank you for atention

I will wait you in the capital of Moldavia region