J.Gras Aneuploidy 2011
1
COMPARISON OF 3 MOLECULAR METHODS TO DETECT ANEUPLOIDIES IN PRENATAL DIAGNOSIS Jeremie Gras*, Nathalie Lannoy, Xavier Pepermans, Marie Ravoet, Marianne Philippe Center for Human Genetics, UCL Saint-Luc University, Brussels (*Now at Clinique Saint-Luc Bouge) 1.Background • In prenatal diagnostics, chromosomal abnormalities are diagnosed using FISH, array CGH and caryotyping • These techniques are long, costly, and require a considerable amount of expertise • The purpose of this work is to evaluate the performance of three molecular techniques (MLPA from MRC-Holland, MAQ from Multiplicon and BoBs from PerkinElmer) for the detection of chromosomal abnormalities as well as their sensitivities in the detection of mosaics 2. Design • From April to September 2010, a total of 30 AF and 17 CVS samples were prospectively collected • These samples were firstly analyzed by FISH • Then, DNA was purified using a homebrew phenol chloroform method, and samples were analyzed using the three molecular technologies • Artificial mosaics were also obtained by dilutions of pathologic DNA into a negative DNA pool • Mosaics with 50%, 40%, 30%, 20%, 10% of abnormal cells were obtained for the following chromosomal abnormalities: +21, +18, XXY, XYY, XO, XXX Luminex Genescan Genescan Separation of amplified products Yes NO NO CE Marking Yes NO NO Detection of micro deletions Yes Yes NO Triploid analysis +13, +18, +21 Aneusomies X,Y +13, +18, +21 Aneusomies X,Y +13, +18, +21 Aneusomies X,Y Aneuploidies detected PerkinElmer Multiplicon MRC-Holland Manufacturer Bacs-on-Beads MAQ assay MLPA Examples of results 47,XY,+18 using MLPA 47,XX,+21 using MAQ 47,XX,+21 using BoBs 3. Results: comparison with FISH Legend: Not. Int: result that could not be interpreted Sample µ : insufficient sample [DNA] µ: insufficient DNA concentration Normal 46,XY 46,XY 46,XY AF 10 Normal 46,XY 46,XY 46,XY AF 9 Normal 46,XY 46,XY 46,XY AF 8 Normal 46,XX 46,XX 46,XX AF 7 Normal 46,XY sample µ 46,XY AF 5 Normal 46,XX 46,XX 46,XX AF 4 Normal 46,XX 46,XX 46,XX AF 3 Normal 46,XY 46,XY 46,XY AF 2 FISH BoBs MAQ MLPA Normal 46,XY [ADN] µ [ADN] µ AF 20 Normal 46,XX Not int. 46,XX AF 19 +21 47,XY,+21 47,XY,+21 Not int. AF 18 Normal 46,XX 46,XX 46,XX AF 17 XXY 47,XXY 47,XXY 47,XXY AF 16 Normal 46,XY 46,XY 46,XY AF 15 Normal 46,XY 46,XY 46,XY AF 14 Normal 46,XY 46,XY 46,XY AF 13 Normal 46,XX 46,XX 46,XX AF 12 Normal 46,XX sample µ 46,XX AF 11 FISH BoBs MAQ MLPA Normal 46,XX 46,XX 46,XX AF 29 Normal 46,XX Not int. 46,XX AF 28 Normal 46,XY 46,XY 46,XY AF 27 Normal 46,XY 46,XY 46,XY AF 26 Normal sample µ sample µ 46,XX AF 25 Normal 46,XY Not int. 46,XY AF 24 Normal 46,XY 46,XY 46,XY AF 23 Normal 46,XY 46,XY 46,XY AF 22 Normal 46,XX 46,XX 46,XX AF 21 FISH BoBs MAQ MLPA Normal 46,XX 46,XX 46,XX AF 36 Normal 46,XY 46,XY 46,XY AF 35 Normal 46,XY 46,XY 46,XY AF 34 FISH BoBs MAQ MLPA Normal 46,XX 46,XX 46,XX CVS 10 47,XX,+21 47,XX,+21 47,XX,+21 Not. Int. CVS 9 Normal 46,XY 46,XY 46,XY CVS 8 Normal 46,XY 46,XY 46,XY CVS 7 Normal 46,XY 46,XY 46,XY CVS 6 Normal 46,XY 46,XY 46,XY CVS 5 47,XX,+21 47,XX,+21 47,XX,+21 47,XX,+21 CVS 4 45,XO 45,XO 45,XO 45,XO CVS 3 Normal 46,XX Not. Int. 46,XX CVS 2 47,XY, +18 47,XY, +18 Not. Int. 47,XX,+18 CVS 1 FISH BoBs MAQ MLPA Normal 46,XY 46,XY 46,XY CVS 17 Normal 46,XY 46,XY Not. Int. CVS 16 47,XX,+21 47,XX,+21 47,XX,+21 47,XX,+21 CVS 15 Normal 46,XX 46,XX 46,XX CVS 14 47,XY,+18 47,XX,+18 47,XX,+18 47,XX,+18 CVS 13 47,XY,+18 47,XY,+18 47,XY,+18 47,XY,+18 CVS 12 47,XX,+21 47,XX,+21 47,XX,+21 47,XX,+21 CVS 11 FISH BoBs MAQ MLPA 4. Discussion • MLPA produced 6,4 % of uninterpretable results, MAQ gave 10,6 % • BoBs produced no uninterpretable results, its concordance with FISH was 100 % • BoBs allowed the analysis of one sample that could not be performed using MLPA and MAQ due to insufficient DNA concentration (sample AF20) 5. Sensitivity for mosaic detection +21 mosaic study using BoBs % of abnormal cells detected 30 % 30 % 40 % XXY mosaic 30 % 50% at the limit 50 % not detected XXX mosaic 30 % 40 % 50 % not detected XO mosaic 30 % 30 % 30 % XYY mosaic 30 % 50 % 50 % not detected +18 mosaic 20 % 50 % 50 % not detected +21 mosaic BoBs MAQ MLPA T21 – 50 % T21 – 40 % T21 – 30 % T21 – 20 % 6. Detection of micro deletion syndromes using BoBs BoBs is the only of the three methods to allow the detection of 9 micro deletion syndromes 5 frozen peripheral blood DNA samples with MDS were selected from our bio bank were selected: Di George, Williams-Beurens, Prader-Willi, Angelman, Smith-Magenis All were successfully identified using BoBs technology BoBs, Di George BoBs, Williams Beurens 7. Conclusions In this work, BoBs gave no uninterpretable results, with a total concordance with FISH; BoBs gave also the better results regarding the sensitivity to detect mosaics BoBs is the only of the three techniques to allow the detection of nine micro deletion syndromes; in this study, all tested micro deletions syndromes were correctly identified In our study, MLPA produced to many uninterpretable results; its capability to detect mosaics was insufficient MAQ is a fast and technique that could be particularly interesting on an operational point of view; however, too many samples are not interpretable despite numerous attempts to improve the quality of results Further operational studies are needed to assess the potential impact of BoBs implementation in terms of cost reduction and turn around time improvement, especially in comparison with FISH Notably, it could be interesting to analyze BoBs results generated with samples purified using fast DNA extraction techniques to reduce answer time Contact: [email protected]
Transcript of J.Gras Aneuploidy 2011
COMPARISON OF 3 MOLECULAR METHODS TO DETECT ANEUPLOIDIES IN PRENATAL DIAGNOSIS
Jeremie Gras*, Nathalie Lannoy, Xavier Pepermans, Marie Ravoet, Marianne Philippe
Center for Human Genetics, UCL SaintLuc University, Brussels (*Now at Clinique SaintLuc Bouge)
1.Background• In prenatal diagnostics, chromosomal abnormalities are
diagnosed using FISH, array CGH and caryotyping
• These techniques are long, costly, and require a considerable amount of expertise
• The purpose of this work is to evaluate the performance of three molecular techniques (MLPA from MRCHolland, MAQ from Multiplicon and BoBs from PerkinElmer) for the detection of chromosomal abnormalities as well as their sensitivities in the detection of mosaics
2. Design• From April to September 2010, a total of 30 AF and 17 CVS
samples were prospectively collected
• These samples were firstly analyzed by FISH
• Then, DNA was purified using a homebrew phenol chloroform method, and samples were analyzed using the three molecular technologies
• Artificial mosaics were also obtained by dilutions of pathologic DNA into a negative DNA pool
• Mosaics with 50%, 40%, 30%, 20%, 10% of abnormal cells were obtained for the following chromosomal abnormalities: +21, +18, XXY, XYY, XO, XXX
LuminexGenescanGenescanSeparation of amplified products
YesNONOCE Marking
YesNONODetection of micro deletions
YesYesNOTriploid analysis
+13, +18, +21
Aneusomies X,Y
+13, +18, +21
Aneusomies X,Y
+13, +18, +21
Aneusomies X,Y
Aneuploidies detected
PerkinElmerMultipliconMRCHollandManufacturer
Bacs-on-BeadsMAQ assay MLPA
Examples of results
47,XY,+18 using MLPA 47,XX,+21 using MAQ
47,XX,+21 using BoBs
3. Results: comparison with FISH
Legend:
Not. Int: result that could not be interpreted
Sample µ : insufficient sample
[DNA] µ: insufficient DNA concentration
Normal46,XY46,XY46,XYAF 10
Normal46,XY46,XY46,XYAF 9
Normal46,XY46,XY46,XYAF 8
Normal46,XX46,XX46,XXAF 7
Normal46,XYsample µ46,XYAF 5
Normal46,XX46,XX46,XXAF 4
Normal46,XX46,XX46,XXAF 3
Normal46,XY46,XY46,XYAF 2
FISHBoBsMAQMLPA
Normal46,XY[ADN] µ[ADN] µAF 20
Normal46,XXNot int.46,XXAF 19
+2147,XY,+2147,XY,+21Not int.AF 18
Normal46,XX46,XX46,XXAF 17
XXY47,XXY47,XXY47,XXYAF 16
Normal46,XY46,XY46,XYAF 15
Normal46,XY46,XY46,XYAF 14
Normal46,XY46,XY46,XYAF 13
Normal46,XX46,XX46,XXAF 12
Normal46,XXsample µ46,XXAF 11
FISHBoBsMAQMLPA
Normal46,XX46,XX46,XXAF 29
Normal46,XXNot int.46,XXAF 28
Normal46,XY46,XY46,XYAF 27
Normal46,XY46,XY46,XYAF 26
Normalsample µsample µ46,XXAF 25
Normal46,XYNot int.46,XYAF 24
Normal46,XY46,XY46,XYAF 23
Normal46,XY46,XY46,XYAF 22
Normal46,XX46,XX46,XXAF 21
FISHBoBsMAQMLPA
Normal46,XX46,XX46,XXAF 36
Normal46,XY46,XY46,XYAF 35
Normal46,XY46,XY46,XYAF 34
FISHBoBsMAQMLPA
Normal46,XX46,XX46,XXCVS 10
47,XX,+2147,XX,+2147,XX,+21Not. Int.CVS 9
Normal46,XY46,XY46,XYCVS 8
Normal46,XY46,XY46,XYCVS 7
Normal46,XY46,XY46,XYCVS 6
Normal46,XY46,XY46,XYCVS 5
47,XX,+2147,XX,+2147,XX,+2147,XX,+21CVS 4
45,XO45,XO45,XO45,XOCVS 3
Normal46,XXNot. Int.46,XXCVS 2
47,XY, +1847,XY, +18Not. Int.47,XX,+18CVS 1
FISHBoBsMAQMLPA
Normal46,XY46,XY46,XYCVS 17
Normal46,XY46,XYNot. Int.CVS 16
47,XX,+2147,XX,+2147,XX,+2147,XX,+21CVS 15
Normal46,XX46,XX46,XXCVS 14
47,XY,+1847,XX,+1847,XX,+1847,XX,+18CVS 13
47,XY,+1847,XY,+1847,XY,+1847,XY,+18CVS 12
47,XX,+2147,XX,+2147,XX,+2147,XX,+21CVS 11
FISHBoBsMAQMLPA
4. Discussion• MLPA produced 6,4 % of uninterpretable results, MAQ gave 10,6 %
• BoBs produced no uninterpretable results, its concordance with FISH was 100 %
• BoBs allowed the analysis of one sample that could not be performed using MLPA and MAQ due to
insufficient DNA concentration (sample AF20)
5. Sensitivity for mosaic detection +21 mosaic study using BoBs
% of abnormal cells detected
30 %30 %40 %XXY mosaic
30 %50% at the limit
50 % not detected
XXX mosaic
30 %40 % 50 % not detected
XO mosaic
30 %30 %30 %XYY mosaic
30 %50 %50 % not detected
+18 mosaic
20 %50 %50 % not detected
+21 mosaic
BoBsMAQMLPA
T21 – 50 % T21 – 40 %
T21 – 30 % T21 – 20 %
6. Detection of micro deletion syndromes using BoBs BoBs is the only of the three methods to allow the detection of 9 micro deletion syndromes
5 frozen peripheral blood DNA samples with MDS were selected from our bio bank were selected:
Di George, WilliamsBeurens, PraderWilli, Angelman, SmithMagenis
All were successfully identified using BoBs technology BoBs, Di George BoBs, Williams Beurens
7. Conclusions In this work, BoBs gave no uninterpretable results, with a total concordance with FISH; BoBs gave also the better results regarding the sensitivity to detect mosaics
BoBs is the only of the three techniques to allow the detection of nine micro deletion syndromes; in this study, all tested micro deletions syndromes were correctly identified
In our study, MLPA produced to many uninterpretable results; its capability to detect mosaics was insufficient
MAQ is a fast and technique that could be particularly interesting on an operational point of view; however, too many samples are not interpretable despite numerous attempts to improve the quality of results
Further operational studies are needed to assess the potential impact of BoBs implementation in terms of cost reduction and turn around time improvement, especially in comparison with FISH
Notably, it could be interesting to analyze BoBs results generated with samples purified using fast DNA extraction techniques to reduce answer time
Contact: [email protected]
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