Group Testing Approach for Trinucleotide Repeat Expansion Disorder Screening.

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Fragile X syndrome (FXS) is an X-linked disorder caused by trinucleotide repeat expansions in the FMR1 gene.
• Current FXS testing relies on single-plex PCR assays due to the long, low-complexity nature of pathogenic alleles.
• There is a need for more efficient and cost-effective methods for FXS screening, including newborn screening.

Purpose of the Study:

• To develop and evaluate a group testing experimental design for accurate sizing of expanded FMR1 alleles.
• To assess the performance of a novel nonadaptive group testing scheme, StairCase (SC), for FXS testing.

Main Methods:

• Developed the StairCase (SC) group testing scheme tailored for FXS testing constraints.
• Compared SC performance against existing group testing schemes via simulation.
• Experimentally validated SC using pooled PCR and capillary electrophoresis on 210 Coriell Institute samples across multiple pooling layouts.

Main Results:

• The SC pooled PCR approach achieved perfect classification of samples into clinical categories (normal, intermediate, premutation, full mutation).
• The method accurately identified 90 positive samples among 1800 negatives.
• A batch of 210 samples required only 21 assays, demonstrating high efficiency.

Conclusions:

• Group testing based on the SC scheme is a viable approach for trinucleotide repeat expansion disorder testing.
• SC offers a potential cost reduction of at least 10-fold compared to current single-plex methods.
• This method provides an implementable and cost-effective solution for FXS diagnosis.