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A Multicentre Technology Assessment of the Abbott Fragile X Assay

A Multicentre Technology Assessment of the Abbott Fragile X Assay. CMGS Spring Meeting 3 rd 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)

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A Multicentre Technology Assessment of the Abbott Fragile X Assay

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  1. A Multicentre Technology Assessment of the Abbott Fragile X Assay CMGS Spring Meeting 3rd 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 Genemapper 5-70 repeats (1hr) 10-25ng DNA 17uL PCR (4hrs) 2% agarose gel (2hrs) 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 - F35 = 20.31; P = 8.05 x 10-9)

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

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

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

  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.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

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