Titled “Abnormally Methylated FMR1 in Absence of a Detectable Full Mutation in a U.S.A Patient Cohort Referred for Fragile X Testing,” the study was published in the journal Scientific Reports.
Fragile X, the most frequent single genetic cause of autism, is the result of the expansion of CGG repeats in the FMR1 gene. C stands for cytosine and G for guanine, which are two of the four building blocks of DNA.
These CGG repeats disrupt FMRP protein production via an epigenetic modification — changes in gene expression but not in the gene itself. This modification adds methyl chemical groups that work as “switch off” signals.
“Full” mutation carriers, those usually diagnosed with fragile X, typically have more than 200 CGG repeats. Those with premutations — 55 to 200 CGG repeats — do not have fragile X syndrome but may have related disorders. In addition, women carrying a premutation have a higher risk of having a child with fragile X.
Despite advances in diagnostic tools and genetic tests, FXS and autism spectrum disorders are usually detected in children in the U.S. only around three years of age, according to the Centers for Disease Control and Prevention. The lack of clinically distinct symptoms at a young age contributes to this delayed diagnosis.
Such delays both preclude prompt medical care and make parents unaware of the risk of having another child with fragile X.
“The impact of delayed diagnosis is significant and potentially preventable not only to the families but also for our health system,” David Godler, PhD, professor at the Murdoch Children’s Research Institute (MCRI) and the study’s lead author, said in a press release.
To help with “more accurate and timely diagnosis,” the team developed a one-step test called Methylation Specific Quantitative Melt Analysis (MS-QMA). The test quantifies the number of methyl groups in the FMR1 gene.
Patients currently are only tested for methyl groups after first determining the size of CGG repeats. This is because this particular test is too expensive to be used as a first strategy for suspected cases of fragile X. However, its limited use means that not all patients may be correctly diagnosed.
The team at MCRI and Lineagen used DNA samples from 316 participants — 218 male and 98 female — in the U.S. and Australia, who either did not have FMR1 mutations or were full-mutation carriers, as confirmed by standard testing. On clinical examination, those without gene alterations revealed intellectual disability with or without autism.
DNA was collected between five days after birth to age 51, with 97 cases involving patients younger than 18. Controls included samples from the general population and children with disorders affecting intellectual development, such as Klinefelter syndrome.
Among the 189 males without gene variants on standard testing, the MS-QMA assay identified 10 cases of abnormal methylation in FMR1. Such cases occurred in 4% of participants with normal CGG size, in 6% of what researchers called the grey zone — specifically, CGG repeats ranging from 45–54 — and in 12% of premutation samples. All of these cases were confirmed with Lineagen’s standard methylation test.
In contrast, no abnormal methylation pattern was detected in 89 males from the general population.
Among 43 females with a normal-sized FMR1 gene copy, the MS-QMA test identified three with abnormal methylation levels. Two of these had no repeat expansion on standard tests while the remaining patient had a full mutation in a small proportion of cells. Researchers noted that this female had the highest level of methylation (48%) but the smallest (yet normal, 24 CGG repeats) size of FMR1.
In three of 96 females from the general population with normal-sized FMR1, the number of methyl groups also was slightly above the maximum value seen in controls.
“We also identified, for the first time, smaller more common FMR1 alleles that are not usually tested for methylation (a tell-tale sign of Fragile X), that had abnormal methylation signatures in a significant number of affected patients,” Godler said.
“These signatures may compromise function of the FMR1 gene, and potentially lead to Fragile X like clinical features, and is an active area of research for my group,” he added.
Setting up the methylation threshold as 37% represented 99.1% sensitivity and 100% specificity with the MS-QMA test.
“We are pleased to have collaborated with Associate Professor Golder and his team to identify up to 15 per cent more patients with Fragile X syndrome than is currently revealed through the current testing paradigm,” added Michael Paul, Lineagen’s CEO.
“Lineagen’s mission is to help provide more accurate and precise genetic diagnoses of pediatric neurodevelopmental disorders sooner, so this is a community we must help and a challenge we cannot ignore,” he added.
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