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Australian Team Develops Melt Curve Methylation Assay for Fragile X Detection, Potential Screening

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NEW YORK (GenomeWeb) — Scientists at the Murdoch Childrens Research Institute at the Royal Children's Hospital in Melbourne, Australia, have developed an assay combining the qualitative strengths of high-resolution melt curve analysis and the high-throughput, quantitative real-time PCR standard curve method to accurately quantify DNA methylation in a single assay.

The method, published last week in Clinical Chemistry, is called methylation specific-quantitative melt analysis, or MS-QMA. The study's authors applied it to detect an expanded CpG region associated with Fragile X Syndrome, and to detect between 92 percent and 100 percent of all males and females carrying Fragile X gene full mutations using newborn dried blood spots. The study also showed a strong correlation between level of methylation in venous blood from females ages six to 35 and verbal impairment using the Wechsler IQ test.

In an email with PCR Insider, David Godler, the lead researcher on the study, said this advance "may finally make cost-effective newborn screening for FXS possible." It could also allow for prognostic testing and early interventions.

The new assay can be adapted to detect other methylated nucleic acids. MS-QMA cannot analyze methylation at a single dinucleotide level, but rather provides mean methylation of all CpG sites within the target amplicon. However, this should not be a major issue if the CpG sites within the target amplicon have been previously clinically validated, Godler said.

Fragile X mutations are usually CGG expansion repeats that result in downregulation of transcription and a heterogeneous neurological phenotype, including variable levels of intellectual disability.

Previously, gold standard testing involved Southern blot analysis to determine methylation status of the FMR1 CpG island. However, this method was cumbersome and "the methylation information provided by the standard testing protocol was not clinically informative in females" with respect to prognosis, Godler said.

The new MS-QMA method stems from prior discovery of a novel epigenetic region located on the Fragile X mental retardation 1, or FMR1 gene. This region is at the exon1/intron1 boundary, expanding downstream into FMR1 intron 1, Godler said. The researchers named the region Fragile X Epigenetic Element 2, or FREE2.

"Our discovery of specific methylation in FREE2 suggested that the FMR1 promoter is much larger than previously thought, and that [in addition to] the 5’UTR, the CGG expansion, and FMR1 exon 1, it also included a non-coding portion of FMR1 – the FMR1 intron 1," he said.

In the resulting body of work, which was described in prior studies published in The Journal of Molecular Diagnostics and Clinical Chemistry, Godler's group showed that methylation of FREE2 was significantly associated with fragile X mental retardation protein levels, as well as cognitive impairment in FMR1 full mutation males. It also correlated with impairment in FM females.

The initial studies used Sequenom’s EpiTYPER system to characterize epigenetic status of the FMR1 CpG island and proximal regions at single base pair resolution. "While the EpiTYPER system-based analysis of the FREE2 region proved to be a wonderful research tool, most Fragile X research groups and diagnostic laboratories do not have access to Sequenom’s matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) machine," Godler said.

The MS-QMA method was therefore developed to make FREE2 methylation analysis more accessible.

According to Godler, MS-QMA is a high-throughput approach. It combines the qualitative nature of high-resolution melt analysis, or HRM, and quantitative real-time PCR to accurately quantify methylation in a single assay. "Some of its unique and unexpected features turned out to be a very low detection limit of about 2 percent methylation, and the ability to analyze CpG sites within FREE2 that could not be analyzed using the EpiTYPER based MALDI-TOF MS analysis due to the large size of their fragments," Godler said.

MS-QMA is performed on bisulfite converted DNA with one pair of primers that amplify both methylated and unmethylated sequences with almost equivalent efficiency, Godler said.

In the method, after bisulfite conversion DNA is serially diluted and an intercalating dye is incorporated into the double-stranded PCR products as part of real-time PCR. This is quantified using a relative standard curve method.

The products are then melted. PCR products from methylated sequences melt at a higher temperature than products from the unmethylated sequences, releasing the intercalating dye. "This release is detected as part of standard HRM," Godler said.

"MS-QMA however only utilizes the emitted fluorescence at one temperature point, which is the lowest temperature where all unmethylated products have first completely melted, while the methylated products are still melting," he said.

The fluorescence is then converted to a methylation ratio from an HRM standard curve created by spiking completely unmethylated DNA with completely methylated DNA at different ratios.

The data analysis from real-time PCR and HMR is performed simultaneously by a custom-designed algorithm incorporated into an app, named Q-MAX. After a filtering step, Q-MAX generates a methylation ratio.

Including the bisulfite conversion, the method requires less than 10 hours, compared with 48 to 72 hours for the EpiTYPER technique.

In addition, "the analysis step takes less than one minute, and can potentially be performed on real-time PCR and HRM data from any real-time PCR machine that can be used for standard HRM analysis," Godler said.

The latest Clinical Chemistry study used MeltDoctor HRM reagents and the ViiA 7 Real-Time PCR system from Life Technologies "because of the attractive price of the reagents and availability of this equipment in the laboratory," Godler said. However, he said there is no reason why other real-time PCR systems or HRM reagents would not work equally well. "We also now know that the data generated by the most recent Bio-Rad real-time machines is suitable for Q-MAX based analysis," he said.

In terms of commercialization, Godler believes "the FREE diagnostic technology has the potential to be market-disruptive as the definitive next generation in diagnostics for FXS, autism spectrum disorder, and FXS-related conditions, with important applications in both targeted testing and newborn screening."

The Murdoch Childrens Research Institute has a suite of patent families directed toward the use of FREE2 and other associated regions, as well as a broader patent application covering the MS-QMA technology, Godler said.

"In partnership with Bio Link Australia, a specialist biotechnology business development consultancy, MCRI is pursuing various strategies including licensing for global commercialization of the technology," he said.

At present, commercial rights to the technology are available on a global, exclusive basis, and MCRI would welcome enquires from interested parties, Godler said.

In addition, a number of cross-sectional and longitudinal studies are being conducted by Godler's group and their collaborators. These studies are examining prognostic utility of FREE2 biomarkers for cognitive and behavioral impairments that can potentially be improved through early intervention strategies.

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