Elsevier

Clinica Chimica Acta

Volume 456, 1 May 2016, Pages 137-143
Clinica Chimica Acta

Validation of a robust PCR-based assay for quantifying fragile X CGG repeats

https://doi.org/10.1016/j.cca.2016.02.027Get rights and content

Highlights

  • This PCR-based method is highly accurate on identifying all Fragile X alleles.

  • All repeat sizes could be sized on a low-cost electrophoresis method—Bioanalyzer.

  • This assay avoids reflex testing by Southern-blot in homozygous normal female.

Abstract

Background

Sizing of FMR1 trinucleotide repeats in the clinical laboratory requires the use of capillary sequencer by PCR, or by a labor intensive measurement using Southern blot method. Our aim was to validate an accurate and robust PCR assay for quantification of CGG repeats.

Methods

We performed an analytical and clinical validation of a new PCR-based method that utilizes a low-cost capillary electrophoresis instrument and the FragilEase™ reagent kit. First, analytical performance was demonstrated on 12 Coriell reference samples comprising normal through full mutations. Subsequently, a cohort of 112 archived clinical DNA samples, enriched for premutation and full mutations, was analyzed.

Results

All samples were amplified successfully. Quantification of repeat numbers was interpreted by the use of standards with known repeats. Twenty-five full-mutation samples were successfully amplified with the largest allele size measured at 1380 repeats. The repeat numbers from the new assay were concordant with those obtained with the reference method. The intra-assay (CV < 2.5%) and inter-assay imprecision was within 1 CGG repeat.

Conclusion

This new PCR-based method is reproducible and capable of identifying all Fragile X alleles. It is an accurate and robust method that facilitates Fragile X testing in a broader spectrum of clinical laboratories.

Introduction

Expansion of the CGG trinucleotide repeat region above 200 repeats in the 5′ untranslated region of the FMR1 gene and resulting transcriptional silencing by methylation is the cause of Fragile X syndrome (FXS) [1]. The repeat size is used to classify and predict risk of FXS and other Fragile X-associated disorders [2], [3]. Normal subjects have 10 to 45 repeats. Individuals with 55 to 200 repeats are classified as premutation. Middle-aged males with premutations may develop Fragile X-associated tremor/ataxia syndrome (FXTAS), a neurodegenerative disorder [4], [5], [6], [7], [8], [9]. Women carrying the premutation have elevated risk of developing premature ovarian failure [10], [11], [12], [13], [14] and infertility and if pregnant, warrant invasive prenatal diagnosis [15]. The Fragile X premutation state has also been associated with cognitive deficits, behavioral problems and autism spectrum disorders [16], [17], [18], [19], [20], [21], [22]. Emerging data has proposed that FMR1 repeat testing should be offered for a widening set of clinical indications including developmental delay or autism in children, female infertility, preconception carrier testing and newborn screening [23], [24], [25], [26], [27], [28], [29]. In addition, since the transmission risk of full mutation increases from zero at less than 55 repeats to approximately 100% at repeat sizes of 90 to 200 repeats [30], the need for an accurate, rapid and robust test that can be implemented more widely with less investment in equipment would be advantageous.

Traditionally, Fragile X CGG repeat alleles testing is performed by FMR1-specific PCR followed by capillary electrophoresis fragment sizing on a genetic analyzer instrument. Typical laboratory developed PCR has been incapable of amplifying this challenging 100% GC content template beyond 100 to 130 repeats [31], [32]. In addition, standard capillary analyzer is only capable of detecting and sizing FMR1 PCR products less than 200 repeats. Therefore, differentiating full mutations with greater than 200 CGG repeats from apparently homozygous normal female samples, and confirming full mutations, have historically required a Southern blot reflex test. More recently PCR-based methods using a primer designed to start strand synthesis from randomized locations within the CGG repeat region have been introduced and validated. These require interpretation of “stutter” electropherograms from the repeat-primer (or chimeric primer) PCR products [32], [33], [34], [35] and also require the use of the expensive capillary analyzer instrument to assess the size of PCR products. The purpose of this study was to demonstrate the analytical and clinical performance of a new PCR-based approach using the FragilEase™ kit and a low-cost electrophoresis sizing method.

Section snippets

Reference sample preparation

Genomic DNA reference samples for use as standards and for evaluating the analytical performance of the assay were obtained from the Coriell Institute for Medical Research (Camden, NJ). Samples were selected for which the FMR1 repeat number had been established in previous studies [31]. All reference samples were diluted to a concentration of 50 ng/μL in TE buffer except where noted and 1 μL was used as the input to each PCR reaction.

PCR amplification

The FMR1 repeat region of each reference sample was amplified

FragilEase™ PCR assay repeatability and accuracy

The results of the repeatability and accuracy study of the 12 reference samples are shown in Table 1. Repeatability, characterized by the CV of each allele's sizing over 6 reactions and measurements, ranged from 0% to 2.5%. Repeats for the female reference samples that differ in size by two repeats or less are not resolved on the Bioanalyzer into separate fragment peaks and therefore are reported as normal samples with one repeat size. Interpretation of results from these samples is covered in

Discussion

Efficient testing of Fragile X syndrome or premutation carriers requires rapid, accurate and precise quantification of the FMR1 trinucleotide repeats. For this reason we have validated the FragilEase™ PCR assay which provides reproducibility, precision and adequate detection range through the largest full mutations. To determine the variation in PCR amplification of the CGG repeat size, twelve previously characterized reference DNA samples were PCR amplified in six separate PCR reactions in the

Conclusion

In this study, we demonstrated that this newly developed Fragile X PCR assay has the performance characteristics required to facilitate rapid and accurate measurement of the cytosine–guanine–guanine (CGG) repeat alleles in low to moderate throughput test settings. Results for this PCR, electrophoresis and analysis were obtained in about 6 h, straightforward to accommodate in an 8-h shift. The compact microfluidic Bioanalyzer instrument is much smaller, less costly and simpler to maintain than an

Disclosure

Some of the investigators in this study are employees of and stockholders in PerkinElmer Health Sciences, Inc.

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