Abstract
Molecular diagnosis of Huntington disease (HD) is currently performed by fluorescent repeat-flanking or triplet-primed PCR (TP-PCR) with capillary electrophoresis (CE). However, CE requires multiple post-PCR steps and may result in high cost in high-throughput settings. We previously described a cost-effective single-step molecular screening strategy employing the use of melting curve analysis (MCA). However, because it relies on repeat-flanking PCR, its efficiency in detecting expansion mutations decreases with increasing size of the repeat, which could lead to false-negative results. To address this pitfall, we have developed an improved screening assay coupling TP-PCR, which has been shown in CE-based assays to detect all expanded alleles regardless of size, with MCA in a rapid one-step assay. A companion protocol for rapid size confirmation of expansion-positive samples is also described. The assay was optimized on 30 genotype-known DNAs, and two plasmids pHTT(CAG)26 and pHTT(CAG)33 were used to establish the threshold temperatures (TTs) distinguishing normal from expansion-positive samples. In contrast to repeat-flanking PCR MCA, TP-PCR MCA displayed much higher sensitivity for detecting large expansions. All 30 DNAs generated distinct melt peak Tms which correlated well with each sample’s larger allele. Normal samples were clearly distinguished from affected samples. The companion sizing protocol accurately sized even the largest expanded allele of ~180 CAGs. Blinded analysis of 69 clinical samples enriched for HD demonstrated 100% assay sensitivity and specificity in sample segregation. The assay targets the HTT CAG repeat specifically, tolerates a wide range of input DNA, and works well using DNA from saliva and buccal swab in addition to blood. Therefore, rapid, accurate, reliable, and high-throughput detection/exclusion of HD can be achieved using this one-step screening assay, at less than half the cost of fluorescent PCR with CE.
Highlights
Huntington Disease (HD; OMIM 143100) is an autosomal dominantly inherited progressive neurodegenerative disorder characterized by involuntary movement abnormality, cognitive loss and psychiatric manifestations and affects 5~10 persons per 100,000 in descents of western European [1]
To compare the performance of the triplet-primed PCR (TP-PCR) melting curve analysis (MCA) assay with the published repeat-flanking PCR MCA assay, one normal and three HD-affected samples were analyzed in parallel using the previously published repeat-flanking PCR MCA method [32] and the new TP-PCR MCA method
The normal sample GM07175 produced a single melt peak with a Tm of 85.75 ̊C, which is lower than the 87.35 ̊C TT of the 26 CAG repeat control plasmid pHTT(CAG)26
Summary
Huntington Disease (HD; OMIM 143100) is an autosomal dominantly inherited progressive neurodegenerative disorder characterized by involuntary movement abnormality, cognitive loss and psychiatric manifestations and affects 5~10 persons per 100,000 in descents of western European [1]. The disease causing mutation is a CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (HTT) gene located on chromosome 4p16.3 [2, 3]. HTT alleles are classified based on the number of CAG repeats: normal ( 26 CAGs), intermediate (27–35 CAGs), HDcausing with reduced-penetrance (36–39 CAGs), and HD-causing with full-penetrance The age of on-set is inversely correlated with the number of CAG repeats. The clinical symptoms of HD progress gradually, usually with cognitive and psychiatric manifestations appearing first followed by movement abnormalities, and death [6, 7]
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