Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1 Grace X.Y. Lim, Yu.

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Presentation transcript:

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1 Grace X.Y. Lim, Yu Ling Loo, Farmaditya E.P. Mundhoﬁr, Ferdy K. Cayami, Sultana M.H. Faradz, Indhu-Shree Rajan-Babu, Samuel S. Chong, Yvonne Y. Koh, Ming Guan The Journal of Molecular Diagnostics Volume 17, Issue 3, Pages 302-314 (May 2015) DOI: 10.1016/j.jmoldx.2014.12.005 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Frequency of CGG repeat lengths in the clinical archive cohort of 528 samples. In samples with two alleles such as female heterozygous specimens or male mosaic samples, only the larger allele is considered in the analysis. FMR1, fragile X mental retardation 1 gene. The Journal of Molecular Diagnostics 2015 17, 302-314DOI: (10.1016/j.jmoldx.2014.12.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Melt curve profiles that show analytic sensitivity of the triplet repeat-primed PCR assay with melting curve analysis on the ABI 7500 Fast real-time PCR platform. Melt curve profiles of male (A) and female (B) Coriell DNA samples that cover four FMR1 allelic forms tested in varying amounts, from 10 ng to 100 ng per reaction. Each reaction was conducted in triplicates; representative profiles are shown. The resumed baseline −dF/dT temperature cutoffs refer to the GZ samples for each sex: GM20230 (A) and GM20236 (B). FM, full mutation; FMR1, fragile X mental retardation 1 gene; GZ, gray zone; NL, normal; NTC, no-template control; PM, premutation; resumed baseline −dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed. The Journal of Molecular Diagnostics 2015 17, 302-314DOI: (10.1016/j.jmoldx.2014.12.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Melt curve profiles that show the analytic specificity of the triplet repeat-primed PCR assay with melting curve analysis on the ABI 7500 Fast real-time PCR platform. Melt curve profiles of four female DNA samples from Coriell spiked with increasing amounts of non-relevant DNA (GM23378) that harbor expanded CTG repeats in the DMPK gene. Zero, 50, 100, or 150 ng of GM23378 was added to 50 ng of each female DNA sample in each reaction. Each reaction was conducted in triplicates; representative profiles are shown. The resumed baseline −dF/dT temperature cutoff refers to the GZ sample, GM20236. DMPK, dystrophia myotonica-protein kinase gene; FM, full mutation; GZ, gray zone; NL, normal; NTC, no-template control; PM, premutation; resumed baseline −dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed. The Journal of Molecular Diagnostics 2015 17, 302-314DOI: (10.1016/j.jmoldx.2014.12.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Melt curve profiles that show consistency of the resumed baseline −dF/dT temperature as a parameter across PCR platforms. A: Melt curve profiles of potential cutoff controls: GM06890 (30 CGG repeats), GM20244 (41 CGG repeats), and GM20230 (53 CGG repeats) on the Rotor-Gene Q HRM platform. B: Average resumed baseline −dF/dT temperatures of GM06890, GM20244, and GM20230 obtained on the Rotor-Gene Q HRM platform across nine valid runs operated independently at two sites. C: Intrarun average resumed baseline −dF/dT temperatures of GM06890, GM20244, and GM20230 on the ABI and the Rotor-Gene Q HRM platforms. Data are expressed as means ± SD. n = 9 (B); n = 96 (C, ABI); n = 12 (C, RGQ). ABI, ABI 7500 Fast Real-Time PCR; resumed baseline −dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed; RGQ, Rotor-Gene Q HRM platform. The Journal of Molecular Diagnostics 2015 17, 302-314DOI: (10.1016/j.jmoldx.2014.12.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Melt curve profiles of simulated mosaic samples relative to a 53 CGG control (GM20230). Melt curve profiles of a male normal sample (GM06890) mixed with a male PM sample (GM06906) (A) or a male FM sample (GM06852) (B) in varying proportions while maintaining total DNA input at 50 ng. Resumed baseline −dF/dT temperatures are compared with a 53 CGG control (GM20230) and a 30 CGG sample (GM06890). All reactions are performed in triplicates on the Rotor-Gene Q HRM; representative profiles are shown. A: 100% to 7.5% PM mosaic samples are in the top panel, whereas 5% to 1% PM mosaic samples are in the lower panel. B: 100% to 20% FM mosaic samples are in the top panel, whereas 10% to 1% FM mosaic samples are in the lower panel. FM, full mutation; PM, premutation; resumed baseline −dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed. The Journal of Molecular Diagnostics 2015 17, 302-314DOI: (10.1016/j.jmoldx.2014.12.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Melt curve profiles of male mosaic clinical samples. Male mosaic samples from the clinical archive were detected through melt curve analysis on the Rotor-Gene Q HRM platform when a 53 CGG control (GM20230) was used. Resumed baseline −dF/dT temperature cutoff refers to that of GM20230. NTC, no template control; resumed baseline −dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed. The Journal of Molecular Diagnostics 2015 17, 302-314DOI: (10.1016/j.jmoldx.2014.12.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 Results of female heterozygous clinical samples with expanded alleles. Representative melt curve profiles of female homozygous NL samples and heterozygous expanded samples relative to a male 53 CGG cutoff control (GM20230) on the Rotor-Gene Q HRM platform. FM, full mutation; NL, normal; PM, premutation; resumed baseline −dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed. The Journal of Molecular Diagnostics 2015 17, 302-314DOI: (10.1016/j.jmoldx.2014.12.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions