Transcript A Multicentre Technology Assessment of the Abbott Fragile

A Multicentre Technology
Assessment of the Abbott
Fragile X Assay
CMGS Spring Meeting
3rd April 2008 - Liverpool
Outline of test – key features
• Abbott Fragile X kit (part no:
6L4301)
– Analyte specific reagent (ASR)
– Accurate allele sizing (<71+/-1;
71-230+/-3)
– Amplification and detection of
large expansions (up to 645
repeats)
– X specific/FMR allele ratio –
potential to differentiate between
hetero/homozygosity
– Gender determination
– Reduction in Southern Blotting
Testing workflow
10-25ng DNA
Genemapper 5-70 repeats (1hr)
17uL PCR (4hrs)
X-amplicon
N-allele (32rpts)
I-allele (56rpts)
100bp
ladder
2% agarose gel (2hrs)
1
9 10
18
Genemapper 70-250 repeats (2hrs)
X- 203bp
Y- 170bp
Aims of study
• Test kit performance
– Accuracy of allele sizing
– Differentiation between hetero and
homozygosity in females
– Detection of large expansions/full mutations
– Detection of mosaicism
– Ease of use in diagnostic setting
– Reproducibility
Design of study
•
13 laboratories (10 UKGTN – 3
Eurogentest)
– 8 ‘Testing labs’ used kit & provided
samples
– 5 ‘Sample labs’ provided samples
•
577 samples analysed
– 6 reference control samples
• All 8 testing centres
• Test for consistency and robustness
– 196 retrospective samples
• Analysed blind and unblind
• Full range of genotypes
– 375 prospective samples
• Analysed alongside routine samples
• Typical spread of genotypes in normal
use
Results - reliability
Centre
Plasticware
PCR/Block
03
04
05
07
08
10
11
12
Overall
Plate
Strips
Strips
Strips
Strips
Plate
Strips/Plate (0.4/0.6)
Plate
-
9700/Aluminium
9700/Aluminium
9700/Silver
9700/Aluminium
9700/Silver
9700/Aluminium
MJ Tetrad
9700/Aluminium
-
•
Variability between centres
•
~1/12 failure rate
Samples
analysed
78
78
78
78
69
178
78
78
715
No of
Failures
0
2
8
0
1
13
5
24
53
Failure Rate
0%
2.6%
10.3%
0%
1.4%
7.3%
6.4%
30.8%
7.4%
Results - sizing
Analysis of 6 sequenced alleles from reference control samples
by 8 centres
Range 23 to 73 repeats
Measure
Mean
Actual
Deviation
1
30
30
30
30
31
30
Fail
31
30
1
283.68
283
+0.68
2
22,31
23,31
22,31
22,31
23,31
22,31
22,31
Fail
22,31
2 (allele 1)
259.93
259
+0.93
Reference Control
3
39
39
39
39
39
39
39
Fail
39
4
30
30
30
30
30
30
30
Fail
30
Reference Control Sample
2 (allele 2)
3
286.42
310.21
286
310
+0.42
+0.21
Allele Sizing Accuracy
6
73/75
73
73*
73
74
73
73
74
73
4
283.29
283
+0.29
2.5
2
Deviation (bp)
Centre
03
04
05
07
08
10
11
12
Actual
6
412.77
412
+0.77
03
04
05
07
1.5
1
0.5
0
-0.5
08
10
11
-1
12
-1.5
Mean
0
259bp
1
283bp
286bp
310bp
412bp
2 283bp
3
4
5
6
Allele
Slight tendency to overestimate (+0.21 to +0.93bp)
Significant differences between centres (ANOVA - F35 = 20.31;
P = 8.05 x 10-9)
7
Results – sizing precision
Precision of allele sizing
3
Deviation (bp)
2
1
0
15
25
35
45
55
65
75
85
-1
-2
-3
No. of repeats
Precision of allele sizing +/-1.96 standard deviations (SD)
Precision within +/-1 repeat up to 73 repeats
Results – determination of hetero/homozygosity
30,FM
30,30
X
X
FMR-1
FMR-1
Abbott Molecular suggested TR/X ratio ranges
TR/X ratio
<0.85
≥0.85 and ≤1.0
>1.0
TR - zygosity
Heterozygous
Undetermined/Inconclusive
Homozygous
Variability in TR/X ratios –
reference control samples
TR/X Ratios
1.4
Centre 05
TR/X = 0.12
1.2
TR/X ratio
1
Hom - 30rpts
0.8
Het - 22,31rpts
Het - 30rpts,PM-m
0.6
Het - 39rpts,FM
0.4
0.2
0
0
1
03
2
04
053
074
5
08
Centre
6
10
7
11
128
9
Centre 08
TR/X = 1.25
Variability in TR/X ratios –
prospective samples
7
Significant overlap between
TR/X ratio of homozygotes
and heterozygotes at all
centres
6
4
3
2
Z y g o s it y ( C e n t r e )
H e t( 1 2 )
H om ( 12)
H e t( 1 1 )
H om (11)
H e t( 1 0 )
H om (10)
H e t( 0 8 )
H om (08)
H e t( 0 7 )
H om (07)
H e t( 0 5 )
H om (05)
H e t( 0 4 )
H om (04)
1
0
T R /X R a t io
5
TR/X too unreliable to be
used diagnostically
Results – large expansions
• 57/58 (98.3%) of full mutation males detected on blind
analysis
• 48/54 (88.9%) of full mutation females detected on blind
analysis
Agarose
Long Run (GeneMapper) Data
Visible most consistently on raw data (beyond largest size standard!)
Results - mosaicism
Mosaicism consistently represented between centres
However kit only detects size mosaicism NOT
methylation mosaicism
Results – mosaicism
• Concordance between in
house genotype and kit
low
Agarose
Long run (raw) data
• 6/11 male mosaics
identified
• 2/3 female mosaics
detected
• 5 further female mosaics
identified on blind testing
Agarose
Long run (raw) data
Results – mosaicism
Male sample genotyped in house as Normal/Intermediate (N/I) mosaic
Abbott genotype Intermediate (I)
Close inspection of data showed a low level Normal (N) allele of
correct size
Agarose
Short run (GM) data
Short run – close up
Is the ‘in house’ PCR assay selectively amplifying the normal allele
more strongly?
May account for some of the non-concordance between mosaicism
reported on in house and Abbott testing
Conclusions
•
Accurately sizes alleles through critical Normal –
Small premutation range
•
Routinely amplifies majority of full mutations (but
not all)
•
TR/X ratio too variable to be used diagnostically to
determine hetero/homozygosity
•
Size mosaicism only detected – may not
correspond with ‘in house’ PCR/Southern data
•
Superior to ‘in house’ PCR alone -useful for urgent
cases/PNDs
•
Use would not significantly reduce the Southern
blotting workload
•
Full report available online www.ngrl.org.uk
Acknowledgments
• Yogen Patel
• Co-authors
– D Barton, PA van Bunderen, J Duncan, J Dunlop, S Man, J
MacPherson, G Monaghan, J McLuskey, G Norbury, H
Powell, V Race, M Sweeney, E Thompson, R Treacy, MM
Weiss, N Williams, HE White, B Wymer
• Participating Laboratories
– Birmingham, Cambridge, Dublin, Edinburgh, Glasgow, GOS,
Leiden, Leuven, Newcastle, NGRL(Wessex), Oxford,
Sheffield
• Abbott Molecular
– Jonathan Bradshaw & John Norton