Revealing the Mutation Behind a Rare Neurological Disease

ABOVE: Researchers found that spinocerebellar ataxia 4, a rare neurological disease that affects the cerebellum, is caused by an excessive GGC repeat mutation. ©ISTOCK, janulla

Individuals with movement disorders called ataxias live with muscle weakness and have poor balance and coordination. Scientists have identified the genetic culprits of some types of ataxias that arise from genetic mutations, but the causes of many ataxias remain a mystery. Although researchers mapped the cause of spinocerebellar ataxia type 4 (SCA4), a rare type of ataxia, to chromosome 16 back in 1996, the specific mutation responsible eluded scientists for decades—until now.¹

Now, more than two decades later, researchers homed in on a mutation in the gene zinc finger homeobox protein 3 (ZFHX3), a transcriptional regulator that seems to mess with a cell’s ability to perform autophagy.² The findings, published in Nature Genetics, could help clinicians improve diagnoses and develop novel treatments for similar conditions.

In 2007, when Stefan Pulst, a clinical neurologist from the University of Utah, met with a patient who had participated in the initial study, he saw a rare opportunity to identify the specific genetic cause of SCA4. “Since we had identified many ataxia genes, we thought it would be relatively straightforward to find this one as well,” Pulst said. “It actually wasn't—it took until just now to define the actual […] mutation.”

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For decades, conventional sequencing failed to reveal mutations associated with the condition because the region is rich in glycine and cysteine—a region which is particularly difficult to sequence—and contains many duplications and pseudogenes. Instead, Pulst’s team turned to long-read single-strand whole-genome sequencing. When they scanned the genomes of individuals with SCA4 that hailed from the Utah patient’s family tree, one genetic fingerprint caught their attention. It was full of glycine repeats, referred to as GGC expansions, and nestled in the region coding for the gene ZFHX3, which functions as a tumor suppressor. Excessive GGC repeats, a type of nucleotide expansion, cause protein clumps that can be toxic and are usually associated with specific diseases, such as Huntington disease.

When they looked at other families that trace their roots back to Sweden, they found the same mutation, which provided more evidence that it is associated with SCA4.

With the mutation in hand, Pulst’s team tested its impact on cellular function. When the researchers introduced the mutation into fibroblasts or induced pluripotent stem cells, they noticed that the cells showed reduced autophagy compared to normal cells. In contrast, blocking ZFHX3 in the cells with the mutation caused the autophagy markers to rebound. With this mutation, “The self-digestion mechanism of the cell is impaired, and a lot of proteins pile up,” said Pulst. “It creates a trash problem.”

Then, when the researchers looked back into the brain of one patient with cerebellar atrophy, they observed clumps of proteins called inclusions permeated the organ—evidence of a trash problem in the brain. When they stained for the ZFHX3 protein, the inclusions lit up. The inclusions, which weren’t present in tissues from healthy individuals, expressed additional autophagy markers, such as ubiquitin and p62.

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“This may be the first time somebody is providing insights into the mechanism of [SCA4],” said Martin Paucar, a neurologist at the Karolinska Institute who was not involved with the study. The inclusions are strong proof that the disease is a nucleotide expansion disease, Paucar added.

"In a preprint last year, Paucar’s team noted similar pathological abnormalities. In their paper, published this year in the Journal of Internal Medicine, they reported the same mutation in individuals from a family with SCA4 in Sweden.³ Pulst said that SCA4 seems to “fit into the ever-growing class of neurological diseases caused by repeat expansions.”  

The findings may improve diagnosis but also help the field find commonalities between these conditions to develop new therapeutic avenues. However, the researchers do not fully understand how the mutation disrupts the ZHFX3 gene or the mechanisms by which it alters cellular processes to cause neurological dysfunction.

Still, the finding advances the field. “For us, this is milestone,” said Paucar. “We are closing 20 years of work.”

Correction: August 14, 2024. An earlier version of the story stated that Paucar's team validated the findings of Pulst and his team. The statement has been corrected to reflect that both teams found similar pathological abnormalities independent of each other around the same time.