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

17uL PCR (4hrs)

10-25ng DNA

1 9 10 18

X- 203bp

Y- 170bp

100bp

ladder

2% agarose gel (2hrs)

Genemapper 5-70 repeats (1hr)

Genemapper 70-250 repeats (2hrs)

X-amplicon N-allele (32rpts)

I-allele (56rpts)

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 Samples analysed

No of Failures

Failure Rate

03 Plate 9700/Aluminium 78 0 0% 04 Strips 9700/Aluminium 78 2 2.6% 05 Strips 9700/Silver 78 8 10.3% 07 Strips 9700/Aluminium 78 0 0% 08 Strips 9700/Silver 69 1 1.4% 10 Plate 9700/Aluminium 178 13 7.3% 11 Strips/Plate (0.4/0.6) MJ Tetrad 78 5 6.4% 12 Plate 9700/Aluminium 78 24 30.8% Overall - - 715 53 7.4%

• Variability between centres

• ~1/12 failure rate

Results - sizing

Reference Control Centre 1 2 3 4 6 03 30 22,31 39 30 73/75 04 30 23,31 39 30 73 05 30 22,31 39 30 73* 07 30 22,31 39 30 73 08 31 23,31 39 30 74 10 30 22,31 39 30 73 11 Fail 22,31 39 30 73 12 31 Fail Fail Fail 74 Actual 30 22,31 39 30 73

Reference Control Sample Measure 1 2 (allele 1) 2 (allele 2) 3 4 6 Mean 283.68 259.93 286.42 310.21 283.29 412.77 Actual 283 259 286 310 283 412 Deviation +0.68 +0.93 +0.42 +0.21 +0.29 +0.77

Analysis of 6 sequenced alleles from reference control samples by 8 centres

Range 23 to 73 repeats

Slight tendency to overestimate (+0.21 to +0.93bp)

Significant differences between centres (ANOVA - F35 = 20.31; P = 8.05 x 10-9)

Allele Sizing Accuracy

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6 7

Allele

Dev

iatio

n (b

p)

03

04

05

07

08

10

11

12

Mean 259bp 283bp 283bp 286bp 310bp 412bp

Precision within +/-1 repeat up to 73 repeats

Results – sizing precision

Precision of allele sizing

-3

-2

-1

0

1

2

3

15 25 35 45 55 65 75 85

No. of repeats

Dev

iati

on

(b

p)

Precision of allele sizing +/-1.96 standard deviations (SD)

Results – determination of hetero/homozygosity

TR/X ratio TR - zygosity <0.85 Heterozygous ≥0.85 and ≤1.0 Undetermined/Inconclusive >1.0 Homozygous

Abbott Molecular suggested TR/X ratio ranges

X XFMR-1FMR-1

30,30 30,FM

Variability in TR/X ratios – reference control samples

Centre 05TR/X = 0.12

Centre 08TR/X = 1.25

TR/X Ratios

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 1 2 3 4 5 6 7 8 9

Centre

TR/X

ratio

Hom - 30rpts

Het - 22,31rpts

Het - 30rpts,PM-m

Het - 39rpts,FM

03 04 05 07 08 10 11 12

Ho

m(0

4)

He

t(0

4)

Ho

m(0

5)

He

t(0

5)

Ho

m(0

7)

He

t(0

7)

Ho

m(0

8)

He

t(0

8)

Ho

m(1

0)

He

t(1

0)

Ho

m(1

1)

He

t(1

1)

Ho

m(1

2)

He

t(1

2)

Z yg o s ity ( C e n tr e )

0

1

2

3

4

5

6

7

TR

/X R

ati

o

Variability in TR/X ratios – prospective samples

Significant overlap between TR/X ratio of homozygotes and heterozygotes at all centres

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

analysisAgarose 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

• 6/11 male mosaics identified

• 2/3 female mosaics detected

• 5 further female mosaics identified on blind testing

Agarose Long run (raw) data

Agarose Long run (raw) data

Results – mosaicism

Agarose Short run (GM) data Short run – close up

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

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