Padlock Probe Assay Rapidly Detects Cardiac Disease Gene Mutations

A recent paper described a novel next generation sequencing (NGS) assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround time, and cost.

By using double-stranded DNA probes (complementary long "padlock" probes or cLPPs), investigators at Stanford University (Palo Alto, CA, USA) reduced the scale of analysis needed to diagnose hereditary cardiovascular abnormalities. The current method involves sequencing thousands of genes, costs USD 1,000 or more per patient, and requires weeks or months to obtain results. The cLPP-based method analyzes only the 88 genes known to carry heart defect mutations, costs about USD 100 per patient, and returns results within three days.

cLPPs are long oligonucleotides of around 100 bases, containing target complementary regions at both their 5′ and 3′ ends. These regions recognize adjacent sequences on the target DNA and between these segments lie universal primer sites and a unique sequence identifier, the so-called ZipCode. Upon hybridization, the ends of the probes get into adjacent position and can be joined by enzymatic ligation. This ligation and the resulting circular molecule can only take place when both end segments recognize their target sequences correctly. Non-circularized probes are removed by exonuclease treatment, while the circularized ones may be amplified by using universal primers. Subsequently, the target-specific products are detected by a universal complementary ZipCode (cZipCode) microarray. cLPPs have been shown to possess good specificity and very high-multiplexing capabilities in genotyping assays.

The investigators reported in the August 11, 2015, online edition of the journal Circulation Research that they had used cLPPs to capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs (miRNA) associated with inherited cardiac abnormalities, congenital heart disease (CHD), and cardiac development. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499.

This new technique could eventually enable doctors to diagnose genetic heart diseases by rapidly scanning the 88 genes known to cause cardiac anomalies. Senior author Dr. Joseph Wu, professor of cardiovascular medicine and radiology at Stanford University, said, “Suppose you have a 60-year-old patient who comes in with heart failure. We do the angiogram and we find he has no history of heart attack or other issues, and yet the heart is not performing well. We also find that several of his family members have similar heart conditions. So if we run the new genetic test and find the man’s illness has a genetic cause, such as dilated cardiomyopathy, we now have both a cause and a diagnosis, and we can initiate treatment right away.”

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