NEW YORK – Australian researchers have developed a test they think could be added to standard newborn screening protocols to expand the range of genetic disorders tested for at birth.
Scientists at the Murdoch Children's Research Institute at the Royal Children's Hospital in Melbourne have adapted a melt curve methylation test they first published about in 2014 for three chromosome 15 imprinting disorders: Prader-Willi, Angelman, and chromosome 15 duplication, or Dup15q, syndromes.
The assay combines high-resolution melt curve analysis and high-throughput, quantitative real-time PCR to quantify DNA methylation in a specific gene related to the syndromes and was validated in a study published in JAMA Network Open last month. The low-cost method, called methylation specific-quantitative melt analysis, or MS-QMA, was originally laid out in a 2014 Clinical Chemistry paper by the researchers to detect fragile X syndrome, another chromosomal disorder.
The three additional disorders that the test has been adapted for are marked by differences in methylation in the small nuclear ribonucleoprotein polypeptide N gene promoter. The MS-QMA test can measure and quantify those abnormal levels of methylation to detect the diseases.
David Godler, an associate professor at MCRI and the lead author of the study, said that to run the test, the blood spots are placed onto a traditional 96-well plate — 93 unknown samples and three controls — and are put through the extraction protocol, with the DNA then treated with sodium bisulfite and diluted. An intercalating dye is incorporated into the DNA and the rate of incorporation is measured by PCR to quantify the DNA concentration, using a relative standard curve method. The DNA is melted with high-resolution melt reagents, which causes the dye to release and emit fluorescence.
Software developed by the researchers for the test incorporates an algorithm that determines a methylation ratio from the fluorescence emitted, Godler said, adding when the result is above 90 percent, the sample is likely positive for Prader-Willi, while a result of below 12 percent indicates Angelman syndrome. Methylation results between 80 percent and 90 percent were likely to be Dup15q.
The software can also perform quality control checks on the samples, throwing out those samples where the DNA concentration is too low to detect the methylation, he said.
Godler and his colleagues first validated the test with 1,356 retrospective newborn blood spots, including 109 samples from babies with Prader-Willi, 48 with Angelman, and nine with Dup15q, as well as 1,190 control samples. Samples with a positive result for one of the disorders were then tested by droplet digital PCR, rt-PCR, and low-coverage whole-genome sequencing for confirmation. An additional 16,579 samples were then tested.
In the validation set, the MS-QMA test showed 99 percent sensitivity for Prader-Willi, 94 percent sensitivity for Angelman, and 78 percent sensitivity for Dup15q, and 100 percent specificity for all three disorders, according to the JAMA paper.
The goal of this study was to determine whether MS-QMA testing would be feasible as a first-tier step for newborn screening of these disorders on a population scale. Using MS-QMA as a first step would mean only applying expensive whole-genome sequencing to samples with a high likelihood of positivity, Godler said — thereby saving money and time and leading to "less waste."
A "special thing" about MS-QMA, he noted, is that its requirements "in terms of investment in resources and training are minimal in comparison to the standard of care of genomic testing." A user only needs access to an RT-PCR machine and the appropriate reagents, which aren't particularly specialized. The reagents used for MS-QMA are "in line with reagent costs for other conditions that are included in existing newborn screening," he said. According to the JAMA paper, the reagent cost for the test on a single blood sample was less than $4, compared to approximately $10 for genomic testing technologies that require higher quality DNA.
While none of the disorders have a cure, both Prader-Willi and Angelman syndromes have treatments that, when applied earlier, can significantly improve patients' quality of life, Godler said. The availability of those treatments "provides a strong argument" to put those two disorders into newborn screening protocols to ensure patients are diagnosed early and can receive treatment as soon as possible, he added.
However, one limitation of the study, Godler noted, was that because it looked at retrospective samples, the team couldn't go back to the infants who were tested and prove the benefits of identifying the disorder at birth.
In the current standard of care, clinicians have a list of features to look for to guide diagnosis, such as intellectual disability, motor problems, or seizures, but those features overlap with a host of other diseases. To confirm a suspected diagnosis, the standard is either a methylation test or chromosomal microarray, and doctors often end up ordering multiple tests while looking for the right diagnosis, said David Amor, a group leader at MCRI and another author on the paper.
In addition, that standard diagnostic testing is more expensive than the traditional newborn screening conducted on all babies and requires more DNA, he said. By including MS-QMA testing for these disorders in newborn screening protocols, that cost could be reduced and more patients could be diagnosed, he said.
Whole-genome sequencing, another option for diagnosing these disorders, can also be too sensitive for screening purposes, Godler said, since it picks up "stuff you want to know and stuff you don't," leading to an information overload.
To be added to newborn screening panels in Australia, a test must detect a condition that has an available treatment and must show a certain level of effectiveness, Godler said. The MS-QMA test meets those eligibility requirements, and the team plans to apply for it to be added to standard newborn screening panels there later this year, he said. He added that the researchers are publishing "another couple of papers" this year on the test and are looking to further validate the test in 100,000 infants in Australia.
In the US, the process to add a test to the newborn screening panel is "long and complicated," said Theresa Strong, director of research programs at the Foundation for Prader-Willi Research, which helped fund the JAMA study. Newborn screening programs are run through individual states, meaning that a test must be adopted by every state — a big hurdle for any test, considering that a lot of evidence must be provided to show the worth of adding the assay, including cost-benefit analysis, Strong said.
While there is a uniform recommended screening protocol in the US, not all states follow it, and tests must be feasible to run on a large scale to be included in newborn screening panels, she said. But researchers also must show that the system has the resources to support patients when they're diagnosed and that early diagnosis makes a clear difference in a patient's quality of life.
The Australian researchers led by Godler are working with scientists at RTI International and its Early Check program in North Carolina to further validate the MS-QMA test prospectively and ensure the results can be replicated. That work will be used to support an application to the committee that recommends newborn screening tests in the US, the Committee for the Recommended Uniform Screening Panel, she said.
By including the test in newborn screening panels, patients who may not have the money for or access to extensive testing can get screened and treated earlier, since all newborns go through the process, regardless of socioeconomic background, Godler said. It's about "having equitable access for these kids to be tested," he said.