Diagnostic profile of the AmplideX Fragile X Dx and Carrier Screen Kit for diagnosis and screening of fragile X syndrome and other FMR1-related disorders

ABSTRACT

Introduction: In 2009, a novel, CGG repeat primed FMR1 PCR assay was designed with primers flanking the triplet repeat region, as well as a third chimeric primer complementary to the (CGG)n repeat, that was capable of amplifying alleles throughout the repeat range. This assay for the first time allowed consistent detection of large full mutation alleles with PCR, resolution of heterozygosity in females and mapping of AGG interspersions.

Areas Covered: The AmplideX Fragile X Dx and Carrier Screen Kit (Asuragen, Inc.) represents a refined assay that underwent validation with sensitivity analyses for FDA approval. Single-site precision, analytical sensitivity and specificity, limit of detection and diagnostic performance were assessed in comparison to reference methods at three independent sites. Single-site precision across all genotype categories showed 100% agreement at 20 ng input across multiple operators, days, instruments and kit lots. Compared to Southern Blot analysis, the overall percent agreement was over 98% for all expanded alleles.

Expert Opinion: Limitations include no methylation assessment and hard to see full mutation peaks in some mosaic samples, but overall the assay is considered a highly accurate and time-efficient assay for FMR1 allele size determination.

KEYWORDS:

• FMR1
• fragile X syndrome
• FXTAS
• FXPOI
• CGG repeat
• molecular diagnosis

Article highlights

• The AmplideX FMR1 assays have resolved the problem of difficulty detecting large FMR1 premutations and full mutations by PCR.

• These assays have allowed highly accurate detection of full mutation alleles, detection of AGG interspersions, resolution of zygosity for females with one normal allele, and detection of low level mosaicism.

• The AmplideX Fragile X Dx and Carrier Screen Kit showed high reproducibility and good precision, as well as accurate performance across different thermal cyclers and DNA preparations.

• Limits of detection for mosaic alleles was defined and reproducibility for detection of mosaic alleles was good for all situation studied.

• Limitations include the need for careful manual inspection for low level mosaic alleles and the need for assays giving accurate percent methylation for full mutation alleles.

Acknowledgments

The authors acknowledge Dr. Gary Latham from Asuragen, Inc. for help with obtaining Figures and Tables existing at Asuragen from a prior poster presentation and from the analyses for FDA approval of the kit that are contained in the Instructions for Use. All Figures and Tables are used with permission from Asuragen.

Declaration of interest

Asuragen supported the validation assays for FDA approval at the RUMC and UC Davis sites, and the creation of cell lines at RUMC to serve as control and FMR1 mutation standards for the assays. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.