Research News

Cleveland Clinic researchers have discovered a new root cause of disease while studying hypomyelinating leukodystrophy-15, a reminder of how studying rare diseases can unearth new information on human health.

Hypomyelinating leukodystrophies (HLDs) are a group of neurodevelopmental disorders that prevent brain cells called neurons from developing properly. HLDs are characterized by developmental delay, reduced muscle strength, defective gait and intellectual disability.

While studying patients diagnosed with a unique form of HLD linked to an essential gene, Cleveland Clinic's Paul Fox, PhD and his lab made a discovery - the patients' symptoms were caused by a genetic mutation that disrupted folding of a single RNA.

Starting with siblings

About five years ago, a pediatric geneticist at the Hospital for Sick Children in Toronto reached out to Dr. Fox for help. Grace Yoon, MD had two patients - siblings whom she first saw when they were four and six years old - who were presenting with a severe neurologic disorder.

Dr. Yoon asked Dr. Fox if he could help research their specific HLD disorder. Both siblings had mutations in the gene that encodes EPRS1, which is essential for creating other proteins and healthy cells. Dr. Yoon knew that this EPRS1 variant likely caused her patients' condition but sought additional information that could connect the variant to the disorder.

Dr. Fox, an expert in this gene and its protein product, was glad to help in any way he could. By analyzing patient cells from Dr. Yoon, Dr. Fox and first author Debjit Khan, PhD, found that the patients had much lower levels of EPRS1 protein than their healthy family members.

How folding affects RNA

Messenger RNA, or mRNA, translates the instructions in our DNA to generate proteins - the machinery of all cells. If there is an error in the mRNA's instructions to build a protein, it can have significant consequences on one or more of the body's systems. Dr. Fox's team found the variant causes an irregular folding of the mRNA product of the EPRS1 gene, which carries the instructions to make the EPRS1 tRNA synthetase, also called glutamyl-prolyl tRNA synthetase. tRNA synthetases are ancient proteins essential in carrying out instructions from genes to correctly create other proteins.

Dr. Fox's team determined that the mRNA is missing a specific modification, known to scientists as m6A methylation, that deformed the folding of the mRNA, in turn preventing the binding of important regulatory proteins, known as "readers".

"Researchers are familiar with several ways that mRNA modifications can be impaired," explains Dr. Fox. "So, we were surprised to see that, even though these pathways in the patients' cells remained intact, their mRNA still wasn't being properly modified because the defective folding prevented the binding of other proteins. This type of defect has not been seen before."

Without the correct modification and binding proteins, the mRNA can't escape the cell nucleus where it is made and can't start the process of providing instructions for proteins to form. Improper mRNA modification, says Dr. Fox, can prevent our bodies from making the proteins they need to function and can ultimately lead to disease.

A previously unknown "ripple effect"

Dr. Fox, Dr. Khan and their team describe the full effects of the variant in a paper published in Nature Communications. Because its mRNA wasn't folded correctly, the EPRS1 protein couldn't be made efficiently. The low EPRS1 protein amount, in turn, could not carry out its functions that ensure other proteins in our bodies are properly made. Each of these problems built upon the last and resulted in these patients developing a neurological disorder with myriad symptoms manifesting in delayed and disturbed mobility and intellectual development.

Now, the research team has evidence that other diseases may also be traced back to minor changes in the mRNA molecules' folding, affecting modifications.

"This is a finding that kicks off our investigation into a novel class of mutations or variants," Dr. Khan says. "We hope that one outcome of this study is more screening and testing is done in the clinical setting, especially for neurodevelopmental disorders, as this study proves there is so much more to learn."

Drs. Fox and Khan plan to continue researching defective RNA folding and its consequences to determine whether it's applicable to other genetic defects and diseases.

"We have a list of hundreds of gene variants that could potentially be linked to diseases caused by a similar defect in mRNA folding," Dr. Fox explains. "There's nothing unique about this type of defect that limits it to rare diseases, so we may find a similar mechanism in a more common disease where tens of thousands of people have this class of genetic RNA-folding defect. It could also relate to disorders beyond neurological, like cancers or heart disease. We're eager to start investigating these possibilities."

Hope for future patients

In the lab, Dr. Fox's team used RNA technology to rescue the RNA's shape so it could be properly modified, fundamentally fixing the process. Investigators are optimistic about the potential to develop treatments that successfully target this form of genetic variant directly within the mRNA. Successful methods included introducing a new piece of RNA that forces the EPRS1 mRNA to fold correctly, or forcing the modification itself using CRISPR. Investigators plan to continue studying methods that could lead to a treatment for diseases linked to this genetic variant.

Reflecting on the journey of this study, Dr. Fox says working with a clinician like Dr. Yoon was integral to making his lab's RNA discovery.

"It was true collaboration: she provided us with the foundational data we needed to pursue this study - the sequencing that showed the gene defect, imaging of the patients' brains and the cell lines," he says. "I'm so grateful she reached out to me so that I could utilize the resources of my lab to investigate the nuances of this rare disorder."