Study Identifies Potential Blood-based Biomarkers, Paths for Treatment

Researchers have identified a set of RNA molecules that are dysregulated in Rett syndrome and may be relevant biomarkers for the disorder.

The study, “Unraveling Molecular Pathways Altered in MeCP2-Related Syndromes, in the Search for New Potential Avenues for Therapy,” was published in the journal Biomedicines.

Rett syndrome is predominantly caused by mutations the gene MECP2, resulting in defective or insufficient levels of MeCP2 protein. A related neurological condition, called MECP2 duplication syndrome (MDS), occurs when there is an extra copy of the MECP2 gene.

Biomarkers are most commonly molecules in the blood that can be taken to give insights about a disease. Identifying biomarkers can aid in making diagnoses and prognoses, and in evaluating the activity of medications.

In the new study, a research team based in Barcelona, Spain, set out to identify new biomarkers of Rett syndrome and MDS.

The researchers analyzed samples from 20 females with Rett syndrome (ages 5 to 33), two males with MDS, one asymptomatic MECP2 duplication carrier female, and 28 healthy females (controls) who did not have any history of learning difficulties or psychiatric problems. Not all samples were included in every analysis due to criteria intended to ensure the results were robust.

Because of the small sample size, the findings for MDS generally failed to reach statistical significance.

The researchers looked at two types of potential RNA-based biomarkers in blood: mRNA and miRNA.

“RNA blood-based biomarkers are easy to measure repeatedly from patients of all ages and health conditions,” the team wrote.

mRNA, short for messenger RNA, is a molecule that carries instructions for making proteins from the nucleus (where those instructions are stored, in the form of DNA) to the cell’s protein-making machinery. As such, measuring mRNA can indicate gene activity — in effect, whether genes are “on” or “off.”

Compared to controls, levels of MECP2 mRNA were abnormally high in MDS samples, as expected given that MDS is characterized by an extra copy of the MECP2 gene. Although less expected, MECP2 mRNA levels were also elevated in Rett syndrome samples. The researchers speculated that this could be due to the cells attempting to compensate for the lack of functional MECP2 protein.

Samples from Rett patients had significantly higher levels of mRNA for the gene THBS3 (previously linked with attention deficits and with mood disorders) than control samples, whereas they had significantly lower mRNA levels for the MeCP2-regulated LIN28A gene. Other analyzed genes were not significantly different between the groups.

Unlike mRNA, miRNA — short for micro RNA — does not encode protein-making instructions. Instead, miRNA are small RNA molecules that play a key biological role in regulating gene activity.

The scientists found that ratios of several different miRNAs were significantly altered in Rett syndrome, compared to controls. The specific miRNA ratios were of the miRNA molecules miR-24-3p, miR-146a-5p, and Let-7a-5p to the molecule miR-132-3p.

To evaluate these potential biomarkers, the investigators calculated the area under the receiver operating characteristic curve (AUC) — a type of statistical test that determines how well a given set of criteria (in this case, levels of the potential biomarkers) can differentiate between two groups. AUC values can range from 0.5 to 1, with higher scores indicating better ability to differentiate.

The biomarkers generally performed well in these tests: for example, the AUC for THBS3 mRNA was 0.84, while the AUC for the Let-7a-5p/miR-132-3p ratio was even higher at 0.938.

Further analyses looked for connections between the various biomarkers and clinical manifestations of Rett syndrome. Certain combinations of miRNA ratios were associated with microcephaly (abnormally small head size) and vasomotor deficits, which can lead to symptoms such as cold feet and hands.

“Our exploratory study identifies potential miRNA biomarkers with clinical relevance, and sets the basis for larger studies leading to the identification of specific miRNA signatures suitable for early [Rett syndrome] screening and/or as quantitative outcome measures in clinical trials,” the researchers wrote.

“Those miRNA pairs [or ratios] are key for differentiation, growth and inflammation, some of the prominent features of [Rett], and could serve as targets for potential treatments,” they added, noting that the clinical relevance of these biomarkers “will require further verification and validation in studies with larger [groups] including samples of a whole range of ages.”