That gene was described in an earlier pilot study by Justice and her co-authors. In the 2013 paper, the team also reported that statins could mimic the protective genetic effect in mice with MECP2 mutation. Other researchers also found cholesterol synthesis was disturbed, but reported that a statin did not have the same effect in Rett model mice with a different genetic background. Results of a small open label clinical trial testing the efficacy and safety of statins in girls with Rett have not been published but were inconclusive, says Monica Coenraads, executive director of the Rett Syndrome Research Trust (RSRT), which funded both Justice studies and the clinical trial.
The third category of mitigating genetic mutations cluster around neuronal signaling or synaptic activity, which other researchers have implicated previously. The mouse Fred Astaire had an additional mutation in this category. Two other categories are transcriptional repression and regulation of DNA activity, which are gene expression pathways that involve MECP2.
In all categories, some symptoms, such as overall health, longevity and limb-clasping behavior, improved in mice with different mutations in some genes. “It’s not like Adrian Bird’s study, where he reintroduced the MECP2 gene, and mice got so much better,” Justice says, referring to a 2007 study by the University of Edinburgh, Scotland, team that first suggested the possibility that Rett Syndrome could be reversed in girls after symptoms had emerged.
“The other unexpected thing we learned from the project was that some mice had two mutations that occurred in the different gene clusters, and those two mutations improved symptoms better than one alone,” Justice says.
It’s too early to make therapeutic predictions on the findings of the mouse genetic screening. In the absence of a therapeutic that replaces the function of MECP2 itself, Justice thinks treatments to modify other pathological pathways may require combination therapies.
In some respects, the notion of inducing more mutations to improve symptoms from another mutation seems counterintuitive. But MECP2 binds extensively to DNA, likely modulating multiple biological pathways.
The technique used in this study, genetic screening for modifier genes, is usually conducted in short-lived fruit flies or round worms to uncover pathways for gene functions, Justice says. But MECP2 is not found in fruit flies or worms. Yet, because the screen is completely unbiased, “animals tell us what’s important,” she says. "Often, we have been totally surprised, then after generating more data, the reason why symptoms improve makes complete sense."
So, she and her colleagues applied modern technology and adapted statistical techniques from human genome-wide studies to minimize the number of mice needed for the study. Although Rett Syndrome primarily affects girls, the researchers used male mice for the screen, because, in contrast to people and female mice, males show symptoms early and consistently.
The genetic screen identified 106 founder mice missing MECP2 but with other mutations that showed improvement in symptoms and longevity. The researchers identified the genes through massively parallel genetic sequencing and verified phenotypes through cross breeding and association analysis.
“The goal of this paper was to report everything we have found up until now,” Justice says. Plenty of work remains, including sequencing a few remaining founders and confirming other modifiers of Rett-like symptoms in other mouse founder lines.
Her lab will be following up on the DNA damage pathway as soon as the coronavirus pandemic restrictions allow, especially genes responsible for repairing double-stranded breaks.