Dept. of Medical Genetic and Fetal Medicine, University Hospital, Olomouc, Czech Republic

Consideration of the effectivity and application of methods of molecular

diagnosis in prenatal diagnosis

Santavy J., Dhaifalah I., Capkova P., Vrbicka D., Vrtel R., Vodicka R.

jiri.santavy@fnol.cz

1967 AC with detection of chromosomal aberrations (Jacobson a Barter)

1970 Banding techniques (Caspersson)1982 Cordocenthesis (Rodeck)1983 CVS (Brambati a Simoni)1980s FISH techniques (especially interphasic

FISH) in prenatal diagnosis with high resolution

1990s QF PCR2000s micro array CGH

Prenatal cytogenetics

Methods of molecular diagnosticReplace karyotyping?

QF PCR in prenatal diagnostic (21, 13, 18, X and Y)MLPA (21, 13, 18, X a Y)Molecular karyotyping – array CGHNon-invasive methods of prenatal diagnostic Fetal cells in maternal circulation detected by

FISH (Bianchi a Klinger, 1992) Extracellular fetal DNA in maternal circulation

detected by RT-PCR (Lo, 1997) DNA microarray (oligo-array with high resolution)

using different metylation between mother’s blood cells and fetus’ placental cells in studying sequences (Chim, 2004).

Karyotyping and FISH

by Michael Kress Russick

Patient PatientControl

Samples mixture

Clone DNA coupling

Laser scanner

Duplication DuplicationDeletion Deletion

Initial signal Normalized signal Integrated signal

DNA microarray

Chromosomal abnormalities detected by karyotyping and array CGH

Duplication

Karyotyping Array CGHBoth methods

by Michael Kress-Russick

QF-PCR in prenatal diagnosis in our department

Objective: to assess the implications of a change in prenatal diagnosis from full karyotype to rapid testing by PCR for women referred primarily for increased risk of Down Syndrome

Design and populationretrospective collection and review of data for pregnant women (4221) having invasive prenatal diagnosis for

1998 – 2006 at the clinical genetic and fetal medicine dept. Olomouc.

Methodology

Abnormal karyotype detected and total number ofsamples referred for:

- raised maternal age- positive triple test (including increased hCG

and positive TT + US markers)- positive combined first trimester screening

were collected and assessed for their clinical significance.

Interpretation of results

TRIALELIC FORM OF TRISOMY DIALELIC FORM OF TRISOMY

D21S1411

D21S1414

physiological finding

trisomy D21S1414

Methodology

Indications as US and FH were removed from the study as full karyotype is recommended in these cases

Abnormal karyotype detected by QF PCR were also removed

Measure outcomes: proportion of prenatal samples with abnormal karyotype that would not have been detected by rapid testing and those which may have clinical effect

Detection of chromosomal abnormalities 1998-2006

0

100

200

300

400

500

600

1 2 3 4 5 6 7 8 9

year 1998-2006

invasive tests

chrom. abn.

Year Total invasive tests Detected aneupl. Structure aneupl. Mosaics Total abnormalities

1998 353 5 1 0 6 (1,70 %)

1999 389 13 0 0 13 (3,34 %)

2000 459 9 8 1 18 (3,92 %)

2001 529 15 9 3 27 (5,10 %)

2002 460 13 5 6 24 (5,22 %)

2003 430 10 5 6 21 (4,88 %)

2004 560 17 9 2 28 (5,00 %)

2005 558 20 3 5 28 (5,02 %)

2006 483 18 9 2 29 (5,99 %)

4221 120 49 25 194 (4,5%)

0

50

100

150

200

1 2 3 4 5 6 7 8

Detection of CA and TT 3,25 %

0

50

100

150

200

250

300

350

1 2 3 4 5 6 7 8 9

Detection of CA and age 2,64 %

total number detection

CA = chromosomal abnormalities TT = triple test

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8 9

Detected CA and US 14,10 % Detected CA and FH 10,90 %

total num detection

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7 8 9

total numberdetection

CA = chromosomal abnormalities US = ultrasound FH = family history

Year TT Not detected Age Not detected 1st Trim.Not

detected%of non

det.

1998 112 0 157 0

1999 173 0 154 0

2000 211 1 137 2

2001 219 2 174 3

2002 201 0 156 2

2003 198 2 133 2

2004 282 3 152 4 39 1

2005 222 2 185 1 48 0

2006 212 2 143 2 57 0

3367

1830 12(0,66 %) 1391 16(1,15 %) 144 1(0,69%) 29(0,86%)

Number of abnormalities undetected by QF- PCR for each indication

TT age 1st. Trim.

1998 0 0 01 case +21

translocation

1999 0 0 01 case +21

translocation

2000 der(13;14),t(14;12);

mosaic t(3;4) 3

2001mosaic t(2;7),

inv(8)der(12); inv(10)

mosaic +20 5

2002 0 inv.(7), inv(16) 2

2003 t(5;15), inv(Y)inv.(10), mosaic.

der.20 4

2004t(6;19), der(15;21),

+16inv(12); inv(10);

inv(7); inv(2)mosaic

+20 8spont ab. in +16

and +20

2005t(17;19),

mosaic.marker mos.t(4;5) 3

2006 t(Y;15),i(Yq)t(7;16)inv(3),

marker 4spont ab. in

marker

Year Not detected by QF PCR Total Clin. results

29 (4) 0,29%

Results and conclusion

Number of chromosomal abnormalities that will not be detected by using QF- PCR only in the above defined indications equals 29 (0,86%) and those which showed clinical effect are 4 (0,29%)

Non detected translocation in fetus = non detected translocation in family

For distinguishing the type of trisomy under question it is necessary to karyotype the couple (most of the aneuploidies show US markers that weight more for full Karyotype)

Reliability of QF-PCR for mosaic detection is still questionable?

In case of late detection of US markers or non-interpreted QF-PCR it is necessary to repeat the test.

Uses of capillary electrophoresis in prenatal diagnosis

Uniparental disomy

Binding analysis

Detection of gene mutation

Detection of aneuploidy and gender

Cytogenetic vs. QF PCR

Time Labor intensityCostInformation gained

Prenatal detection of aneuploidy

Pathologies

11,8%11,8%

7,8%

5,9%

3,9%

3,9%

3,9%

2,0%3,9%3,9%

3,9%37,3%

trisomy 21 - Down syndrom

trisomy 18 - Edwards syndrom

autosomal mosaicism

triploidy

Turner syndrom

gonosomal mosaicism

autosomal deletion

balanced translocation

translocation

inversion

47,XYY

trisomy 13 - Patau syndrom

Prenatal diagnosis of chromosomal abnormalities in 2006 in Olomouc

15 % - 21 %

Thank you