New study maps early genetic disruptions in Rett syndrome

To better understand what drives the emergence of symptoms in Rett syndrome, researchers at Baylor College of Medicine and the Duncan Neurological Research Institute (Duncan NRI) at Texas Children's Hospital took a closer look at brain cells in mice modeling Rett syndrome before symptoms appeared. They identified a set of dysfunctional genes and specific cell types that are vulnerable early on to genetic changes. The study appears in Science Advances.

Rett syndrome is a rare genetic neurological disorder that mainly affects girls. Girls with Rett syndrome typically develop normally during infancy, but between 6 and 18 months of age, they begin to lose skills such as speech, intentional movements and social engagement.

Rett syndrome is caused by mutations in a gene called MECP2, which plays a key role in regulating how other genes are turned on and off in brain cells."

Dr. Huda Zoghbi, corresponding author, Distinguished Service Professor at Baylor, director of the Duncan NRI and Howard Hughes Medical Institute investigator

The mutations cause the gene to lose its function, which affects the proper regulation of thousands of other genes. "MECP2 gene is on the X chromosome," said co-first author Dr. Ashley Anderson, postdoctoral associate of molecular and human genetics in the Zoghbi Lab. "Female cells have two X chromosomes, but each cell randomly turns off one of these chromosomes, creating a mosaic cellular environment, where about half of the brain cells use the healthy version of MECP2 (MeCP2-positive cells) and the other half use the mutated version (MeCP2-negative cells). However, males only have one X chromosome, so all cells have a mutant MECP2, leading to more severe disease early in life."

"What makes Rett uniquely challenging to study is that the healthy and mutant cells influence each other in ways we are only beginning to understand," said co-first author Yan Li, graduate student in the Zoghbi lab. "By studying female mice that mirror this mosaic condition, alongside male mice carrying only the mutant copy, we begin to untangle those effects."

The researchers studied the expression pattern of genes or gene activity in cells of the hippocampus, the brain region involved in learning and memory, which is known to be affected early in the disease. A key technical advance in this study was the physical separation of MeCP2-positive and MeCP2-negative cells before studying the cells, allowing the researchers to compare gene activity in mutant and healthy cells from the same mosaic female brain for the first time.

"We applied two molecular techniques to measure which genes are turned on or off in these cells," Li said. "Bulk RNA sequencing showed us gene activity across the whole tissue and single-nucleus RNA sequencing allowed us to analyze gene activity in individual cells. Using both techniques let us see the 'big picture' and zoom in on specific cell types."

In female mice, the overall changes in gene activity looked modest when measured across whole brain tissue. However, when the researchers examined individual cells, a very different picture emerged. "We found that important changes were not evident in bulk measurements because they occurred only in certain cells," Li said. "For instance, cells carrying the Mecp2 mutation showed strong gene disruptions only in specific cell types, which we did not detect when we analyzed a mixture of cells in bulk studies. This shows that in mosaic conditions like Rett syndrome, studying individual cells is essential to fully understand the disease."

"We uncovered 12 genes that were consistently altered at very early stages of the disease and only in the Mecp2 mutant cells," Anderson said. "These genes were either turned up or down in the same way regardless of sex or disease severity. We propose that these genes likely represent an early 'core disease signature.' Many of these genes are involved in communication between brain cells (synapses), suggesting that disruptions in how neurons connect and signal may be one of the earliest steps in Rett syndrome."

While this study found a core disease signature in the Mecp2 negative cells, it also revealed that even healthy cells (with normal Mecp2 gene) are not completely unaffected in females. "We found that some brain cells with normal Mecp2 had changes in gene activity due to the presence of neighboring defective cells," Anderson said. "This shows that cells can be influenced by their environment and helps explain why Rett syndrome can cause widespread brain dysfunction even when many cells are genetically normal."

One surprising discovery was that a type of neuron called trilaminar interneuron, which had not been associated with Rett syndrome before, showed disruptions that were stronger than those of other neuron types when MeCP2 was malfunctioning. These cells connect across multiple layers of the hippocampus, helping coordinate communication within the brain. Further studies are needed to better understand the role of these interneurons in Rett syndrome.

"Understanding these early and cell-specific changes provides markers to monitor efficacy of interventions and also entry points to understand the brain circuits driving Rett features," Zoghbi said. "If scientists can target the earliest molecular disruptions, or protect the most vulnerable cell types, it may be possible to slow or prevent the progression of Rett syndrome. In addition, this work informs studies of other genetic conditions that involve mosaicism or affect specific brain cell populations."

Guantong Qi, Sih-Rong Wu, Jean-Pierre Revelli, Hu Chen and Zhandong Liu, all at Baylor College of Medicine and/or the Duncan NRI, also contributed to this work.