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Clinical Chemistry 54: 964-972, 2008. First published April 10, 2008; 10.1373/clinchem.2007.096503
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(Clinical Chemistry. 2008;54:964-972.)
© 2008 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Single-Step Scalable-Throughput Molecular Screening for Huntington Disease

Clara R. L. Teo1, Wen Wang2,3, Hai Yang Law4, Caroline G. Lee5 and Samuel S. Chong1,2,3,a

1 Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 2 Children’s Medical Institute, National University Hospital, Singapore; 3 Preimplantation Genetic Diagnosis Center, National University Hospital, Singapore; 4 Department of Pediatric Medicine, KK Women’s and Children’s Hospital, Singapore; 5 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

aAddress correspondence to this author at: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Level 4, NUH, 5 Lower Kent Ridge Rd., Singapore 119074, Singapore. Fax 65-6779-7486; e-mail paecs{at}nus.edu.sg.

Background: Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG trinucleotide repeat in exon 1 of the HTT (huntingtin) gene and typically has an adult onset. Molecular diagnosis and screening for HD currently involve separate amplification and detection steps.

Methods: We evaluated a novel, rapid microplate-based screening method for HD that combines the amplification and detection procedures in a single-step, closed-tube format. We carried out both the PCR for the HTT CAG-repeat region and the subsequent automated melting-curve analysis of the amplicon in the same wells on the plate. To establish cutoff melting temperatures (Tms) for each allelic class, we used a panel of reference DNA samples of known CAG-repeat sizes that represent a range of HTT alleles [normal (≤26 repeats), intermediate (27–35 repeats), reduced penetrance expanded (36–39 repeats), and fully penetrant expanded (≥40 repeats)]. We also measured well-to-well variation in Tm across the thermal block and validated cutoff Tms with DNA samples from 5 different populations. We also conducted a blinded validation analysis of clinical samples from an additional 40 HD-affected and 30 unaffected individuals.

Results: We observed a strong correlation between CAG-repeat size and amplicon Tm among the reference DNA samples. Use of the Tm cutoffs we established revealed that 5 samples from unaffected individuals had been misclassified as affected (1.1% false-positive rate). All samples from HD-affected and unaffected individuals were correctly identified in the blinded analysis.

Conclusions: This simple and scalable homogeneous assay may serve as a convenient, rapid, and accurate screen to detect the presence of pathologic expanded HD alleles in symptomatic patients.






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