A novel methylation PCR that offers standardized determination of FMR1 methylation and CGG repeat length without southern blot analysis
| Autor: | Marina Grasso, Elles M. J. Boon, Andrew G. Hadd, Stela Filipovic-Sadic, Ru Cao, Gary J. Latham, Patrick A. van Bunderen, Elena Gennaro, Domenico A. Coviello |
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| Přispěvatelé: | Human genetics |
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Male
Biology Polymerase Chain Reaction Sensitivity and Specificity Pathology and Forensic Medicine Cell Line 03 medical and health sciences Fragile X Mental Retardation Protein 0302 clinical medicine Trinucleotide Repeats medicine Humans Allele 030304 developmental biology Southern blot Genetics 0303 health sciences Base Composition Base Sequence Methylation DNA Sequence Analysis DNA Amplicon DNA Methylation medicine.disease Molecular biology FMR1 Fragile X syndrome Blot Restriction enzyme Blotting Southern Technical Advance Fragile X Syndrome Molecular Medicine Female 030217 neurology & neurosurgery |
| Zdroj: | Grasso, M, Boon, E M J, Filipovic-Sadic, S, Van Bunderen, P A, Gennaro, E, Cao, R, Latham, G J, Hadd, A G & Coviello, D A 2014, ' A novel methylation PCR that offers standardized determination of FMR1 methylation and CGG repeat length without southern blot analysis ', Journal of Molecular Diagnostics, vol. 16, no. 1, pp. 23-31 . https://doi.org/10.1016/j.jmoldx.2013.09.004 Journal of Molecular Diagnostics, 16(1), 23-31. Association of Molecular Pathology The Journal of Molecular Diagnostics, 16(1), 23-31 |
| ISSN: | 1525-1578 |
| Popis: | Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%-100%) with an accuracy of 99% (95% CI: 93%-100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories. |
| Databáze: | OpenAIRE |
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