Validation of Fragile X Screening in the Newborn Population Using a Fit-for-Purpose FMR1 PCR Assay System

Area of Science:

• Genetics
• Molecular Biology
• Clinical Diagnostics

Background:

• Newborn screening enables early detection of serious conditions for timely intervention.
• Fragile X syndrome is a monogenic neurodevelopmental disorder requiring effective screening methods.
• Clinical laboratories need validated assays for prospective pilot studies before implementing new screening tests.

Purpose of the Study:

• To assess the feasibility of a high-throughput screening assay for fragile X syndrome in newborns.
• To establish and validate a customized PCR and analysis system for detecting fragile X mental retardation 1 gene (FMR1) repeat expansions.
• To evaluate the assay's analytical performance, including precision, accuracy, sensitivity, and specificity, using dried blood spots (DBSs).

Main Methods:

• Development of a customized, high-throughput PCR and analysis software system.
• Detection of FMR1 gene repeat expansions from DNA eluted from DBSs.
• Characterization of assay precision, accuracy, sensitivity, and specificity across various repeat expansion ranges.
• Testing with deidentified newborn DBS samples to determine assay performance and identify positive cases.

Main Results:

• The assay accurately resolved genotypes within three CGG repeats up to 137 repeats and within six repeats up to 200 repeats.
• Accuracy testing showed concordance with reference results.
• The assay detected full and premutation alleles from low DNA inputs (subnanogram) and mixtures with as little as 1% relative abundance.
• Analysis of 963 DBS samples identified 6 premutation specimens, aligning with known prevalence.

Conclusions:

• The developed assay system demonstrates robust analytical performance for detecting FMR1 repeat expansions.
• The high-throughput capability and accuracy make the assay suitable for integration into newborn screening programs.
• This validated assay supports the presymptomatic identification of fragile X syndrome, facilitating early intervention and improved outcomes for affected infants.