title: A novel methylation PCR that offers standardized determination of FMR1 methylation and CGG repeat length without southern blot analysis.
abstract: 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.
authors.affiliation: Laboratory of Human Genetics, Galliera Hospital, Genoa, Italy. Electronic address: marina.grasso@galliera.it.
authors.forename: Marina
authors.initials: M
authors.lastname: Grasso
authors.affiliation: Laboratory for Diagnostic Genome Analysis, Department of Clinical Genetics, Leiden University Medical Center (LUMC), Leiden, the Netherlands.
authors.forename: Elles M J
authors.initials: EM
authors.lastname: Boon
authors.affiliation: Asuragen, Inc., Austin, Texas.
authors.forename: Stela
authors.initials: S
authors.lastname: Filipovic-Sadic
authors.affiliation: Laboratory for Diagnostic Genome Analysis, Department of Clinical Genetics, Leiden University Medical Center (LUMC), Leiden, the Netherlands.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: van Bunderen
authors.affiliation: Laboratory of Human Genetics, Galliera Hospital, Genoa, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Gennaro
authors.affiliation: Asuragen, Inc., Austin, Texas.
authors.forename: Ru
authors.initials: R
authors.lastname: Cao
authors.affiliation: Asuragen, Inc., Austin, Texas.
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Latham
authors.affiliation: Asuragen, Inc., Austin, Texas.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Hadd
authors.affiliation: Laboratory of Human Genetics, Galliera Hospital, Genoa, Italy.
authors.forename: Domenico A
authors.initials: DA
authors.lastname: Coviello
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000032:analysis / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D018911:Trinucleotide Repeats
chemical_list: D004247:DNA; D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human