NEW YORK (360Dx) – A team of investigators at the University Hospital of Wales has, in collaboration with PerkinElmer, developed an automated immunoassay that could be used to screen newborns for a biomarker linked to Duchenne muscular dystrophy.
The developers maintain that the new assay would address an unmet market need, and pilot studies are now underway in the US and China, following a successful evaluation of the test in the UK, detailed recently in the journal Clinical Chemistry.
DMD is the most common lethal X-linked neuromuscular disorder diagnosed in childhood. The UK's National Health Service estimates that about 100 children — primarily boys — are born with the disorder in Britain every year, while the US Centers for Disease Control and Prevention reports that between 400 and 600 are born with DMD annually, a prevalence rate of about 15 in every 100,000 people.
Laboratories have used blood spot creatine kinase (CK) enzyme assays to screen newborns for DMD in the past. These tests measure the enzyme activity for several isoenzyme forms of CK, but are seen by some as nonspecific because they quantify total CK enzyme activity in blood, according to Stuart Moat, a consultant biochemist and director of the Wales Newborn Screening Laboratory at the University of Hospital of Wales in Cardiff, UK.
"The enzyme assays are also nonstandardized and various sources of enzymes have been used as calibrators — bovine, rabbit, and human — and all produced different screening cutoffs or action values," Moat said of conventional approaches. He also said that a lack of stable reagents for the traditional blood-spot CK enzyme test has made it difficult to automate, limiting its adoption for widespread screening.
Moat is also affiliated with Cardiff University School of Medicine, an institution that also supported his work, which developed out of a Wales newborn screening program for DMD, which ran from 1990 to 2011. Moat became director of the Wales Newborn Screening Lab in 2008. Based on Moat's experience with the traditional enzyme activity assay, his laboratory developed another assay focused specifically on CK-MM, a creatine kinase protein found in the damaged skeletal muscle of children with DMD, with the belief that CK-MM would be a more stable marker.
After developing a chemiluminescence-based assay around the marker, Moat said that his lab decided to collaborate with PerkinElmer, citing the Waltham, Massachusetts-based company's activity in immunoassay development as well as its portfolio of instruments and assay kits for neonatal screening.
According to Linh Hoang, vice president of neonatal screening at PerkinElmer, after Moat's lab showed proof of concept for using an immunoassay using CK-MM and chemiluminisence for detecting Duchenne from dried blood spot samples, PerkinElmer further refined the CK-MM immunoassay concept into a standardized CK-MM immunoassay using time-resolved fluorescence.
"Currently there are no commercial [or] standardized IVD assays available for screening newborns for Duchenne muscular dystrophy using creatine kinase as a marker from dried blood spots," Hoang said of PerkinElmer's interest in the assay.
He reiterated Moat's critique of conventional approaches, citing a lack of specificity associated with looking at CK activity from blood instead of muscle, reagent stability issues, and a high background, resulting in inaccurate readouts from normal samples.
Working with Moat's lab, Hoang said that PerkinElmer developed a solid phase, two-site fluoroimmunometric assay based on a direct sandwich technique, in which the calibrators, controls, and test samples are dried blood spots on paper suitable for blood collection.
Sample disks are punched into the assay wells and processed, and the fluorescence in each well is then measured, with the fluorescence of each sample proportional to the concentration of CK-MM in the sample. The assay is run on PerkinElmer's Genetic Screening Processor (GSP) instrument, a fully automated, high-throughput analyzer that can be used for quantitative or qualitative measurement of neonatal dried blood spot samples in a 96-well microplate format.
Moat noted that the assay when used on the GSP is capable of testing 2,500 samples, standards, and controls in just under 13 hours, in his words "permitting high-throughput screening for DMD for the first time." He noted the assay is standardized, allowing the transferability of screening cutoffs from lab to lab, or country to country.
PerkinElmer is now offering the assay for research use only, Hoang said, but evaluation of the assay for IVD use is underway.
Moat is the lead author on a study carried out with partners at PerkinElmer and Cardiff University that sought to evaluate the assay's performance. They determined, based on a variety of metrics, that the test can be used to reliably quantify the CK-MM protein in blood spots. "The development of this CK-MM assay on a commercial immunoassay analyzer would enable standardized and high-throughput newborn blood spot screening of DMD," the authors concluded.
"The CK-MM immunoassay offers greater specificity over the existing enzyme activity tests as it detects the skeletal muscle form of CK, CK-MM, and it is the CK-MM form that is found predominantly in skeletal muscle and is significantly elevated in patients with muscular dystrophy," said Moat of the results.
However, despite this success, he said his lab will not be using the assay routinely, because the UK National Screening committee does not yet recommend newborn screening in the UK. On its website, last updated in November, the committee cited concerns about the reliability of conventional tests, as well as questions as to when therapy and treatment should be implemented, and if screening improves prognosis.
According to Hoang, a pilot study of the assay is currently underway in China at a "large hospital" that he declined to name. He said that PerkinElmer and its partners intend to collect data on the prevalence of DMD in newborns in China, generate a follow-up and treatment protocol for those DMD patients identified early, and to"study the suitability and viability of the CK-MM assay concept for screening for DMD in newborn babies."
Meantime, a separate study is taking place in Wisconsin that is aimed at studying CK-MM reference ranges in de-identified newborn screening samples collected at different time points after birth.
Though the disease is not curable, an early diagnosis can help families seek out therapy and medications to support those affected. Corticosteroid treatment, for instance, is recommended to improve muscle strength and function in those diagnosed with DMD.
Moreover, gene therapies are also in development. In February, Exonics Therapeutics, a new firm exploring the use of CRISPR/Cas9 to create new treatments for DMD, received a $5 million grant from CureDuchenne Ventures. In January, Parent Project Muscular Dystrophy awarded $250,000 to University of Texas Southwestern Medical Center to develop a CRISPR/Cas9-based treatment for DMD.
Moat said that he is aware of the development of new molecular therapies for treating DMD, and that their availability could lead to increased screening.
"One of the main reasons that many countries do not screen for DMD is that there is no treatment for this condition," Moat said. In September, the FDA, in fact, approved a drug from Sarepta Therapeutics for some mutations that cause DMD. However, the drug is controversial, and there was intense disagreement within the agency whether the treatment, called Exondys 51 (eteplirsen), meaningfully impacts patients and should have been approved. Moreover, it targets mutations that are implicated in only a relatively small share of DMD cases.
"The fact that there are treatments on the horizon will have a significant impact upon the decision-making process on whether to screen for DMD in the future," Moat said.