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New rare disease discovery sheds light on cellular recycling process

Scientists from the NIH’s National Human Genome Research Institute (NHGRI) and Undiagnosed Diseases Program (UDP) have identified a new rare disease that appears to be associated with autophagy (the cell’s housekeeping process). The study, published in npj Genomic Medicine, points to ATG4D gene mutations as the cause for the disorder and could aid research into related conditions such as Alzheimer’s disease.

The ATG4D gene

The team identified two siblings with the rare neurodevelopmental condition and one unrelated child. The condition appeared to affect the children’s motor coordination and speech.

Combined exome and genome sequencing revealed that they all had rare, conserved, bi-allelic mutations in the ATG4D gene, which is associated with autophagy. This process is involved in the breakdown and recycling of damaged proteins and organelles in the cell. Though autophagy is observed across many cells, it is particularly important for neurons.

Unfortunately, very little is known about the ATG4D gene or how it might contribute to the function of healthy neurons. That is, at least, in humans.

Canine connection

The first clues to the function of the ATG4D gene came from a 2015 study on the Italian dog breed Lagotto Romagnolo. Dogs with the gene defect showed abnormal behaviour, atrophy of the cerebellum, and issues with motor coordination and eye movement. However, no connection to humans was initially made.

“Among genetic diseases, we’ve solved many of the lower hanging fruits,” said May Christine Malicdan, M.D., Ph.D., NHGRI staff scientist and senior author of the study. “Now, we’re reaching for the higher fruits — genes like ATG4D that are more difficult to analyse — and we have the genomic and cellular tools to do so.”

Computational analyses had already predicted that the ATG4D mutations would produce dysfunctional proteins. But some genes perform similar functions to ATG4D, so the team weren’t sure if they could potentially be compensating for (or “masking”) this loss of function in a real-world setting. To get a better understanding, they first analysed the function of ATG4D in skin cells. The children’s variants did not cause any abnormalities, but the researchers wondered if this would be true in the brain as well.

“The brain is so complex, and neurons have very specialized functions. To fit those functions, different neurons use different genes, so changes in redundant genes can have major impacts in the brain,” said Malicdan.

To overcome any differences in the genetic background of the children (ie masking of autophagy) the team generated an ATG4D-deficient HeLa cell model. They found that these cells could not perform autophagy, suggesting that ATG4D gene variations are indeed the cause of the children’s symptoms.

Figure 1: Transmission electron microscopy analyses of autophagosome biogenesis in cultured cells from individuals with bi-allelic variants in ATG4D.
a, b Representative TEM images of cultured primary fibroblasts from Control (GM09503) and Individual 1 (a) or lymphoblastoid cell lines from Control 1 (CCL-104), Control 2 (Family 2: I-2, mother of Individuals 2 and 3), Individual 2 (Family 2: II-3), and Individual 3 (Family 2: II-4) (B) treated with vehicle (DMSO) or 100 nM Torin 1 and 100 nM Bafilomycin A1 for 3 h to induce the formation of autophagosomes and to prevent their degradation, respectively (arrowheads). c–f Quantification of autophagosome area (c, d) and size (e, f) from the experiments represented in (a, b).

The future of autophagy

Despite the promise, there’s still a lot of work to be done to validate these findings. The research is ongoing, and the team aims to identify more patients with the condition in the future.

However, the work does open up interesting avenues for further research into related neurological diseases, like Alzheimer’s, where autophagy is known to play a role. “Rare diseases can help us understand biological pathways, so we can better understand how those pathways contribute to other rare and common conditions,” said Malicdan.

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