NEW YORK (GenomeWeb) – Methylation microarrays could provide a simpler and easier way to screen for fragile X syndrome, at least as a first-line option in male patients suspected of having the disorder, according to a new study by researchers from the London Health Sciences Center in Ontario.
Because of the breadth of array-based analysis, this new approach could also offer clinicians the ability to simultaneously screen for multiple different disorders that have overlapping symptoms with fragile X by distinguishing unique methylation signatures associated with each.
Bekim Sadikovic, head of molecular genetics at Ontario's Western University and lead author of the new study published online in the Journal of Molecular Diagnostics, told GenomeWeb this week that his hope is that clinical practice will slowly shift from single-analyte testing for epigenetic disorders to broad methylation array-based screening.
In the meantime, the current study provides important evidence toward a first step of establishing this approach for fragile X, he said. He and his colleagues are also working to publish additional validations for several other diseases where the method could gain a foothold.
According to Sadikovic and his coauthors, fragile X molecular diagnosis is currently relatively complex, involving long-range PCR or a combination of PCR and Southern blot analyses.
The disorder is most frequently caused by a more-than-200-repeat expansion of the CGG trinucleotide in the promoter of the gene FMR1 (although it can also be caused by deletion of FMR1), which results in DNA hypermethylation and inhibition of transcription of the gene.
Traditional PCR methods have used primers flanking the CGG repeat regions to identify the approximate number of repeats in each allele. However, this has limitations, most significantly that amplification can fail in very larger repeats, or it can favor the smaller allele and skew the relative ratio of normal to abnormal alleles in female cases or male mosaics.
More recently, newer PCR methods have been developed — both by commercial companies and academic groups — that can reliably detect even long repeats in fragile X.
PCR-based methylation assays can also play a role in diagnosis of the disorder.
Sadikovic and his coauthors hoped with their study to show that a broader analysis approach, using whole-genome methylation microarrays, could work as a first-line screening method for the disease, with an advantage over current options both in cost and ease, and because it opens up the possibility of simultaneously testing for other intellectual disability disorders that are important to rule out as differential diagnoses.
In their study, Sadikovic and his collaborators used Illumina HumanMethylation450 DNA methylation arrays to measure genome-wide and FMR1-specific methylation in 32 males previously diagnosed with fragile X, five females with a full mutation, 22 "premutation" carrier males or females (in which there is a lower than 200x but higher than normal amplification of the trinucleotide), and 300 normal control DNA samples.
According to the authors, the results demonstrated 100 percent sensitivity and specificity for detection of the disorder in male patients, as well as the ability to differentiate patients with mosaicism.
The approach does not work to identify female cases of fragile X because full mutation females may not show differential methylation at the FMR1 locus in peripheral blood, the investigators wrote.
However, Sadikovic explained, because males (usually young boys) are the ones who present with the most severe phenotypes of the disorder, they are the most common to be identified as the first case, or proband, in a family.
"Females often don't have significant symptoms or any symptoms at all, so the idea is that you will pick up a kid with this method, and then you would test other family members using these other methodologies," he said.
Importantly, because the assay utilizes a whole-genome methylation array, it also allows for the ruling out, or detection of other disorders that might be confused for fragile X, most commonly Prader-Willi syndrome or Sotos syndrome, which each have their own unique methylation signatures, Sadikovic said. If these are detected instead of fragile X, there are then other follow-up molecular or cytogenetic tests that might be warranted.
The Ontario investigators said they have also validated their array-based approach for detection of additional imprinting disorders, including Prader-Willi syndrome, Angelman syndrome, Beckwith-Wiedemann syndrome, and Russell-Silver syndrome, although this data has not yet been published.
"We've collected over 1,000 patients with these very rare conditions in collaboration with multiple [other academic] sites," Sadikovic said. "We are assessing technology to see if we can we take one test and use it as a replacement for all these single gene tests."
In addition to that work, the team has also demonstrated unique DNA methylation signatures in patients with other conditions not previously known to be associated with epigenetic changes, including Floating-Harbor syndrome caused by mutation in the SRCAP gene, and X-linked α-thalassemia/mental retardation, caused by a mutation in the Q8 ATRX gene, the authors wrote.
Those have not yet been published, but another study by the group on their discovery of a methylation profile in a cerebellar ataxia, deafness, and narcolepsy syndrome caused by mutations in the DNMT1 gene just appeared in Clinical Epigenetics.
If methylation array-based screening grows in clinical practice and larger cohorts are amassed, the hope is that still other epigenetic disease associations may be discovered, Sadikovic said.
"When arrays were marketed to replace single FISH tests for things like microdeletions, what happened was that over many years of utilization we've now used genome-wide arrays not only to test conditions we knew, but have [identified] close to 100 new microdeletion-implicated syndromes," he argued.
"So now we are hoping to educate the clinical community about doing the same thing with epigenomics."
According to Sadikovic, his group is now working toward implementing the methylation array approach clinically for fragile X, as well as other disorders that the team has not yet published on.
"The work we've done has been really in research basis, but as of last month we purchased a high-scan instrument in our clinical lab and we are working on validating all these different conditions in the clinical lab," he said. "Immediately when that's done we are going to be offering this test in the province, as well as more broadly— nationally."
Broader adoption may take a while, he said. To try to support that, the team is also working on clinical utility/economics research. In one current study, they are looking prospectively at the incremental diagnostic and clinical value added by incorporating methylation array analyses, along with exome sequencing and regular array testing for about 500 patients with broadly defined intellectual disabilities with a suspected genetic cause.
Sadikovic said he's also planning to present on his work at an Illumina-sponsored workshop at the upcoming American Society of Human Genetics meeting in Vancouver.