title: Evaluation of the human fragile X mental retardation 1 polymerase chain reaction reagents to amplify the FMR1 gene: testing in a clinical diagnostic laboratory.
abstract: Fragile X syndrome (FXS) is caused by the absence of a functional fragile X mental retardation protein (FMRP). In most cases, the molecular mutation is an expansion and consequent methylation of the CGG trinucleotide repeat in the 5' end of the FMR1 gene. Polymerase chain reaction (PCR)-based assays that overcome the limitations of amplifying >100-150 CGG repeats have been designed. One such product, Human FMR1 PCR Reagents, can detect expanded mutation alleles without determining methylation status. We used this assay to amplify 70 clinical samples previously tested in three clinical laboratories, including 28 full mutation alleles, 17 premutation alleles, 6 gray zone alleles, and 21 normal samples (51 normal alleles including 5 homozygous females). The results were concordant with previously reported results. All full and premutation alleles were identifiable: repeat sizes are not assigned when the CGG repeat number is >200 and all full and premutation alleles were scored in the same category using this assay. All normal and gray zone alleles were within 0-1 repeat of their previously reported allele sizes. This method identified a mosaic premutation/full mutation pattern in 12/21 samples previously identified as full mutation only and in 5/7 samples previously reported as mosaic premutation/full mutation. These results demonstrate that this assay provides comparable results to the combination of PCR/Southern blot methodologies. Additional issues such as technologist time, reagent costs, turnaround times, and sample requirements are comparable to the PCR/Southern blotting assays currently utilized; however, methylation status cannot be determined using this assay. It is likely that PCR-only based assays will eventually replace previous methods for FXS and that Southern blotting or another methylation assay will only be utilized when determination of methylation status is necessary. This type of assay may also be utilized for other nucleotide expansion disorders.
authors: University Laboratories-Molecular Genetics Diagnostic Laboratory, Detroit Medical Center, Detroit, Michigan 48201, USA. fnahhas@dmc.org
authors: Fatimah A
authors: FA
authors: Nahhas
authors: Thomas J
authors: TJ
authors: Monroe
authors: Thomas W
authors: TW
authors: Prior
authors: Patricia I
authors: PI
authors: Botma
authors: Jin
authors: J
authors: Fang
authors: Pamela J
authors: PJ
authors: Snyder
authors: Sandi L
authors: SL
authors: Talbott
authors: Gerald L
authors: GL
authors: Feldman
mesh_terms: D000483:Alleles; D015139:Blotting, Southern; D019411:Clinical Laboratory Techniques / Q000379:methods*; D019175:DNA Methylation; D019075:Electrophoresis, Capillary / Q000379:methods; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000175:diagnosis / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D019680:Trinucleotide Repeat Expansion / Q000235:genetics
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; D007202:Indicators and Reagents; D011933:Reagent Kits, Diagnostic; C497683:FMR1 protein, human