Received on August 14, 2007
Accepted on February 25, 2008

Molecular Diagnostics and Genetics

Single-Step Scalable-Throughput Molecular Screening for Huntington Disease

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

^* To whom correspondence should be addressed. 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 (T_ms) 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 T_m across the thermal block and validated cutoff T_ms 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 T_m among the reference DNA samples. Use of the T_m 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.