title: An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis.
abstract: (CGG)(n) repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel, single-tube CGG repeat primed FMR1 PCR technology was designed with two gene-specific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG)(n) repeat. This PCR was evaluated with 171 unique DNA samples, including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis, including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore, a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles, was resolved with 100% accuracy. Last, AGG interrupter sequences, which may influence the risk of (CGG)(n) expansion in the children of some carriers, were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result, this PCR provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods.
authors.affiliation: Department of Diagnostic Research and Technology Development, Asuragen, Inc., Austin, Texas 78744, USA.
authors.forename: Liangjing
authors.initials: L
authors.lastname: Chen
authors.forename: Andrew
authors.initials: A
authors.lastname: Hadd
authors.forename: Sachin
authors.initials: S
authors.lastname: Sah
authors.forename: Stela
authors.initials: S
authors.lastname: Filipovic-Sadic
authors.forename: Julie
authors.initials: J
authors.lastname: Krosting
authors.forename: Edward
authors.initials: E
authors.lastname: Sekinger
authors.forename: Ruiqin
authors.initials: R
authors.lastname: Pan
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hagerman
authors.forename: Timothy T
authors.initials: TT
authors.lastname: Stenzel
authors.forename: Flora
authors.initials: F
authors.lastname: Tassone
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Latham
mesh_terms: D020121:5' Untranslated Regions; D000483:Alleles; D015139:Blotting, Southern; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D019075:Electrophoresis, Capillary; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D018911:Trinucleotide Repeats
chemical_list: D020121:5' Untranslated Regions; D017931:DNA Primers