Single-step scalable-throughput molecular screening for Huntington disease.
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.
Teo
Clara R L
CR
Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Wang
Wen
W
Yang Law
Hai
H
Lee
Caroline G
CG
Chong
Samuel S
SS
D001322:Autoanalysis; D004247:DNA / Q000032:analysis; D019075:Electrophoresis, Capillary; D005838:Genotype; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012016:Reference Values; D015203:Reproducibility of Results; D044366:Transition Temperature; D019680:Trinucleotide Repeat Expansion
C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D004247:DNA