By Ben Butkus
In the past six months, three separate groups comprising scientists from academia and industry have published research papers demonstrating the potential of rapid and inexpensive PCR-based molecular tests for fragile X syndrome.
The tests' developers are hopeful that the flurry of positive research results may have finally lowered the barrier to implementing population-wide screening for the syndrome in the form of mandated newborn screening, elective screening of mutation-carrying parents, or both.
Such screening programs, potential recommendations for which are currently being debated by professional organizations such as the American College of Medical Genetics and the American Congress of Obstetricians and Gynecologists, would instantly open up a sizable market for companies with a horse in the race, which include Quest Diagnostics, Celera Genomics, Abbott, and Asuragen.
It remains to be seen, however, whether a market for fragile X population screening will develop any time soon, since mandating newborn screening for the disease may be highly dependent on the development of a novel therapeutic; and carrier screening will likely continue to rely on a case-by-case cost-benefit analysis, according to ACMG Executive Director Michael Watson.
"Fragile X, I would guess, in absence of a treatment that would make it appropriate for newborn screening, is more likely a carrier screening type of assay," Watson told PCR Insider this week.
Genetic testing for fragile X in young, symptomatic children or for pre-mutation allele carriers has been available for almost two decades. One of the most commonly used methods is PCR amplification of CGG repeat regions in the FMR1 gene followed by Southern blot of the genomic DNA.
Although this method is relatively effective, it is both expensive and time-consuming, and not conducive to large-scale population screening.
"It's a very manual technique, and it's one thing to do a few thousand a year, it's another thing to do a million a year," Charles Strom, medical director of the Genetic Testing Center at the Quest Diagnostics Nichols Institute, and co-developer of one of the new fragile X PCR tests, told PCR Insider this week.
"Everybody in the field knew that … if all of a sudden we said, 'Hey, it's time for population-based carrier screening,' there just aren't enough molecular genetic technologists in the world to be able to do it," Strom said. "And Southern blots don't always work, and they have to be repeated, so you might be talking two to four weeks to get a result."
What diagnostic labs and companies have been seeking — "the holy grail of genetics testing," Strom said — is a PCR test that was easy, rapid, inexpensive and, most importantly, sensitive and specific enough to conduct large-scale screening and identify potential carriers or patients that could then be reflexed to traditional Southern blot follow-on testing for confirmation.
In the past two years, at least three separate research groups have developed such a test — largely due the development of a PCR method dubbed triplet repeat primed PCR, in which one primer is anchored outside a trinucleotide repeat region, while the other overlaps the repeat and an adjacent unique sequence.
The technique was originally developed in the 1990s as a method for detecting large CAG repeat expansions associated with Huntington's and related diseases, but researchers Paul Hagerman and Randi Hagerman of the University of California-Davis' MIND Institute have in recent years successfully adapted it to detect fragile X CGG repeat expansions.
Leading the way is Quest Diagnostics, which in February introduced its XSense test, a laboratory-developed triplet-primed PCR test that also uses capillary electrophoresis detection to qualitatively assess expanded CGG repeats.
Other recent test developers include Asuragen, which has developed a research-use only PCR kit for fragile X that uses the triple repeat primed PCR with proprietary gene-specific primers, amplification buffers for CG-rich templates, and PCR cycling conditions; and the ARUP Institute for Clinical and Experimental Pathology at the University of Utah, which used analyte-specific reagents made by Celera and distributed by Abbott to develop its own triple repeat primed/capillary electrophoresis test (PCR Insider 3/4/2010 and 5/6/2010).
"A few different places made the breakthroughs basically within a month of each other, and that's exciting because it lends credibility to the field," Strom said. "If just one group figured out how to do it, people might look at it skeptically. But now you have three different groups basically using the same kind of technology, each slightly different, but each saying, 'Eureka, we did it!'"
Of the three, Quest's test has the most impressive data supporting its potential use as a population screening tool. In a research paper published in March in Genetics in Medicine, Strom and colleagues at the Nichols Institute and Quest Diagnostics reported that their test showed 100 percent agreement with Southern blot tests in a panel of 1,275 blood samples.
The test was also capable of detecting so-called "mosaic" patterns, which have been difficult to detect using traditional methods; and was able to detect mutations for fragile X in six of the 1,275 patients tested.
Meantime, in a study published in March in Clinical Chemistry, researchers from Asuragen and the MIND Institute used their test to detect every one of 66 full mutations that were co-detected using Southern blot analysis across 146 clinical samples. Then, in an April study published in the Journal of Molecular Diagnostics, ARUP Institute and Celera showed their test to have 100 percent concordance with PCR/Southern blot analysis in 205 samples.
"So now we have at least three different ways to do something that previously was not possible to do," Strom said. "So the next issue is: Is it time for population-based carrier screening? And the response is that now is the time to begin thinking about it."
Recommendations on the Table
Strom said that he believes organizations such as ACMG "will have some sort of recommendations in the near future for carrier-based screening, because it's such a devastating disease, and the prevalence of carriers is so high," with about one in every 250 people carrying the mutant allele. "And because it's an X-linked disease, you don't need both parents to be carriers," Strom said.
Likewise, the rapid PCR tests may have opened up the possibility of newborn screening, Strom said, since the current average age of diagnosis is around 2.5 years. That idea was echoed in an interview last month with ARUP Institute's Elaine Lyon, who said that newborn screening might be feasible if the cost of testing was reduced by a high volume of reagent sales.
ACMG and ACOG are currently working on recommendations for both population-based carrier and newborn screening.
ACMG's position statement on carrier screening will take into consideration that "it has to be a relatively inexpensive test; it has to be a bad disease; the markers need to be pretty consistently associated with the bad forms of the disease; and that there is probably not a treatment available," ACMG's Watson said.
If ACMG were to recommend carrier screening, it could go a long way toward convincing physicians to recommend such a test to women in the early stages of pregnancy or even couples considering having a child.
But, he added, in carrier screening "people have a choice, and payors may or may not cover the screening," Watson said. "So it depends on what segment of the population can access the test when they have to pay for it themselves."
Newborn screening, on the other hand, would certainly present the larger market opportunity for companies offering tests, especially if it was made mandatory as part of the battery of tests administered to newborns using dried blood spots collected at birth.
But it has its own issues — first and foremost a debate over whether early identification of fragile X in newborns will make any difference in the outcome of the disease, since there are currently no fragile X treatments on the market.
"There is some hope that even earlier infantile intervention can make a little bit more difference," Watson said. "That's kind of the thing on which the argument for newborn screening is currently predicated."
Watson said there is also an argument as to whether the indirect benefits to families of fragile X patients should be considered. Parents may want to know if their first child has fragile X before having another child, but the diagnosis is not currently made until the first child is a few years old.
"So the question becomes do we consider identification and the elimination of the diagnostic odyssey … to be a benefit that would justify newborn screening programs?" Watson said. "That's going to be a long debate, I suspect, because it would be a real departure from benefits that are directly to the infant."
There are also potential fragile X treatments on the horizon being developed by companies such as Novartis. "There are several studies ongoing, and if a significant improvement was recognized by identifying people with fragile X and treating them, presumably younger, then it would completely change the perspective of whether newborn screening was appropriate or not," Watson said.
"If there was a treatment that made a significant difference in outcome, you'd probably end up with newborn screening instead of carrier screening," he added.