1. A Multicentre Technology Assessment of the Abbott Fragile X Assay CMGS Spring Meeting 3 rd April 2008 - Liverpool

2. 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

3. Testing workflow 17uL PCR (4hrs) 10-25ng DNA 2% agarose gel (2hrs) Genemapper 5-70 repeats (1hr) Genemapper 70-250 repeats (2hrs)

4. 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

5. 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

6. Results - reliability Variability between centres ~1/12 failure rate

7. Results - sizing 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 - F 35 = 20.31; P = 8.05 x 10 -9 )

8. Precision within +/-1 repeat up to 73 repeats Results – sizing precision Precision of allele sizing +/-1.96 standard deviations (SD)

9. Results – determination of hetero/homozygosity Abbott Molecular suggested TR/X ratio ranges X X FMR-1 30,30 30,FM

10. Variability in TR/X ratios – reference control samples Centre 05 TR/X = 0.12 Centre 08 TR/X = 1.25

11. 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

12. 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 Visible most consistently on raw data (beyond largest size standard!)

13. Results - mosaicism Mosaicism consistently represented between centres However kit only detects size mosaicism NOT methylation mosaicism

14. 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

15. 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 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

16. 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.ukwww.ngrl.org.uk

17. 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