Highly-sensitive blood test may improve FXS prognosis

Assay can measure trace FMR1 mRNA levels in fragile X patients

Margarida Maia, PhD avatar

by Margarida Maia, PhD |

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Researchers have developed a highly-sensitive test that can reliably detect trace activity levels of FMR1 — the faulty gene in fragile X syndrome (FXS) — in the blood of patients who’d been thought to have none by current methods.

This was done through the measurement of FMR1’s messenger RNA (mRNA), the intermediate molecule derived from DNA that guides protein production.

Importantly, even trace levels of FMR1 mRNA levels resulted in the production of FMRP, the protein encoded by the gene, and higher mRNA levels were associated with better intellectual ability.

These findings, confirming the biological importance of even trace FMR1 mRNA levels, may partly explain the high symptom variability observed across FXS patients, the researchers noted.

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“In the future, this assay could be an important part of the molecular diagnosis of FXS, may be useful in predicting clinical outcomes, and help developing personalized and improved therapeutic strategies,” the researchers wrote.

The findings were detailed in the study “A sensitive and reproducible qRT-PCR assay detects physiological relevant trace levels of FMR1 mRNA in individuals with Fragile X syndrome,” published in Scientific Reports.

Fragile X is a genetic condition that results in a range of developmental problems including learning difficulties and cognitive impairment. Symptoms can vary widely from one patient to another, and often are more severe in males.

The disease is caused by a mutation in the FMR1 gene called a CGG trinucleotide expansion, where three nucleotides (the building blocks of DNA) get repeated an excessive number of times.

The abnormal presence of 55 to 200 CGG repeats is classified as a premutation. When the number of CGG repeats goes above 200 — called a full mutation — FMR1 is silenced (turned off) and makes little or no FMRP protein, which is needed for the brain to develop normally.

 Full-mutation, fully methylated

About 60% of patients with a full mutation are thought to also have a fully methylated FMR1 gene. Methylation is a chemical modification that turns off a gene. When this happens, patients are said to have a full-mutation, fully methylated (FM–FM) FMR1 gene and are expected to have zero levels of FMR1 mRNA, and ultimately no FMRP protein.

A few years back, however, researchers reported that some FM–FM patients have low FMR1 mRNA levels, which could explain — at least in part — why symptoms can vary widely.

But because measuring very low FMR1 mRNA levels can be difficult with current techniques used in diagnostic testing for FXS, a team of researchers at the Cincinnati Children’s Hospital Medical Center in Ohio set out to develop a new assay that is more sensitive.

There’s a “need for better molecular assays for FXS diagnosis,” the researchers wrote.

They optimized a method called quantitative real-time polymerase chain reaction (qRT-PCR) to reliably detect tiny amounts of FMR1 mRNA. qRT-PCR can make many copies of a piece of DNA from a matching piece of mRNA, and the number of DNA copies help tell how much of that specific mRNA is present in cells.

The optimized qRT-PCR assay was run on 148 blood samples from 138 people who either developed typically (26 males, six females), had FXS (55 males, 22 females), or were premutation carriers (two males, 27 females). Participants’ ages ranged from 1.2 years to 67.6 years.

Samples from 19 participants were excluded from the analyses due to insufficient assay quality or insufficient RNA concentration.

Men vs. women

As expected, people with fragile X had significantly lower FMR1 mRNA levels than premutation carriers and typically developing controls. Among FX patients, men had significantly lower FMR1 mRNA levels than women.

Moreover, FMR1 mRNA levels were particularly low in FM–FM male patients, compared with those with a full mutation or full methylation alone. However, no significant differences were seen between these groups among females.

These findings highlight that “low levels of FMR1 mRNA can be reliably detected” even in FM–FM patients, the researchers wrote, meaning that “in a subgroup of individuals meeting the current molecular diagnostic criteria of FXS, the FMR1 gene is not completely silenced.”

The team then compared FMR1 mRNA levels in blood samples with those of the FMRP protein measured on dried blood spots from 120 of the participants. A dried blood spot is a blood sample that’s blotted and dried on filter paper.

They found that the higher the FMR1 mRNA levels, the higher the FMRP protein levels. This association between mRNA and protein levels also was observed in 30 FM–FM patients who had an available dried blood spot sample.

Higher FMR1 mRNA levels also were associated with higher intelligence quotient (IQ) scores, which provide an estimate of intellectual ability. However, this link only reached statistical significance for females, not males.

Further experiments showed that FMR1 mRNA levels explained “over one-third of variance in IQ in individuals with FXS,” the team wrote, meaning that even trace FMR1 mRNA levels “can be physiologically relevant” and may contribute to symptom variability.

“Future studies are needed to understand these relationships and leverage our findings to improve clinical care,” the researchers concluded.