title: Group Testing Approach for Trinucleotide Repeat Expansion Disorder Screening.
abstract: Fragile X syndrome (FXS, OMIM #300624) is an X-linked condition caused by trinucleotide repeat expansions in the 5' UTR (untranslated region) of the fragile X mental retardation 1 (FMR1) gene. FXS testing is commonly performed in expanded carrier screening and has been proposed for inclusion in newborn screening. However, because pathogenic alleles are long and have low complexity (>200 CGG repeats), FXS is currently tested by a single-plex electrophoresis-resolved PCR assay rather than multiplexed approaches like next-generation sequencing or mass spectrometry. In this work, we sought an experimental design based on nonadaptive group testing that could accurately and reliably identify the size of abnormally expanded FMR1 alleles of males and females.
  
  
  
  We developed a new group testing scheme named StairCase (SC) that was designed to the constraints of the FXS testing problem, and compared its performance to existing group testing schemes by simulation. We experimentally evaluated SC's performance on 210 samples from the Coriell Institute biorepositories using pooled PCR followed by capillary electrophoresis on 3 replicates of each of 3 pooling layouts differing by the mapping of samples to pools.
  
  
  
  The SC pooled PCR approach demonstrated perfect classification of samples by clinical category (normal, intermediate, premutation, or full mutation) for 90 positives and 1800 negatives, with a batch of 210 samples requiring only 21 assays.
  
  
  
  Group testing based on SC is an implementable approach to trinucleotide repeat expansion disorder testing that offers ≥10-fold reduction in assay costs over current single-plex methods.
authors.affiliation: Counsyl, South San Francisco, CA.
authors.forename: Kristjan Eerik
authors.initials: KE
authors.lastname: Kaseniit
authors.affiliation: Counsyl, South San Francisco, CA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Theilmann
authors.affiliation: Counsyl, South San Francisco, CA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Robertson
authors.affiliation: Counsyl, South San Francisco, CA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Evans
authors.affiliation: Counsyl, South San Francisco, CA. research@counsyl.com.
authors.forename: Imran S
authors.initials: IS
authors.lastname: Haque
mesh_terms: D005260:Female; D005600:Fragile X Syndrome / Q000235:genetics*; D005820:Genetic Testing / Q000191:economics / Q000379:methods*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*