Reliable and Sensitive Detection of Fragile X (Expanded) Alleles in Clinical Prenatal DNA Samples with a Fast Turnaround Time.

Seneca S, Lissens W, Endels K, Caljon B, Bonduelle M, Keymolen K, Rademaeker MD, Ullmann U, Haentjens P, Berkel KV, Dooren SV.

Source

Center for Medical Genetics, UZ Brussel, Brussels, Belgium; Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Brussels, Belgium.

Abstract

This study evaluated a large set of blinded, previously analyzed prenatal DNA samples with a novel, CGG triplet-repeat primed (TP)-PCR assay (Amplidex FMR1 PCR Kit; Asuragen, Austin, TX). This cohort of 67 fetal DNAs contained 18 full mutations (270 to 1100 repeats, including 1 mosaic), 12 premutations (59 to 150 repeats), 9 intermediate mutations (54 to 58 repeats), and 28 normal samples (17 to 50 repeats, including 3 homozygous female samples). TP-PCR accurately identified FMR1 genotypes, ranging from normal to full- mutation alleles, with a 100% specificity (95% CI, 85.0% to 100%) and a 97.4% sensitivity (95% CI, 84.9% to 99.9%) in comparison with Southern blot analysis results. Exact sizing was possible for a spectrum of normal, intermediate, and premutation (up to 150 repeats) alleles, but CGG repeat numbers >200 are only identified as full mutations. All homozygous alleles were correctly resolved. The assay is also able to reproducibly detect a 2.5% premutation and a 3% full-mutation mosaicism in a normal male background, but a large premutation in a full male mutation background was masked when the amount of the latter was >5%. Implementation of this TP-PCR will significantly reduce reflex testing using Southern blot analyses. Additional testing with methylation-informative techniques might still be needed for a few cases with (large) premutations or full mutations.
Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.