Huntington's Disease Research Today is a free monthly online journal that collates and summarizes the latest research about Huntington's Disease, including details on genetics, causes, symptoms, treatment.

Single-step scalable-throughput molecular screening for Huntington disease.

Teo CR, Wang W, Yang Law H, Lee CG, Chong SS

Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

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(m)s) 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 (< or =26 repeats), intermediate (27-35 repeats), reduced penetrance expanded (36-39 repeats), and fully penetrant expanded (> or =40 repeats)]. We also measured well-to-well variation in T(m) across the thermal block and validated cutoff T(m)s 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.

Published 29 May 2008 in Clin Chem, 54(6): 964-72.
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