Hoskins Investigates Mutations in Protein Linked to Blood Disorders

Photo of Aaron Hoskins
Professor Aaron Hoskins.

Aaron Hoskins, professor in the Department of Biochemistry, is on the hunt for how gene mutations contribute to a serious blood disorder known as myelodysplastic syndrome, or MDS. The disorder, which is often linked to mutations in the DDX41 gene, affects blood cells and can result in anemia, bone marrow failure, infection, or leukemia. Currently, the only effective cure for MDS is bone marrow transplantation.

Hoskins will investigate how mutations in the DDX41 gene, and the resulting changes to the DDX41 protein the gene produces, impact cellular biochemical function. His research is possible thanks to new funding from EvansMDS — an initiative from the Edward P. Evans Foundation to support research related to MDS treatment — as well as access to new collaborations and technologies.

The DDX41 protein is a member of the DEAD-box protein family, which are essential to RNA processing, function, and regulation. One of the processes most commonly disrupted in patients with MDS is RNA splicing — an essential step in gene expression for all eukaryotes and the primary line of inquiry for the Hoskins Lab.

“It turns out,” says Hoskins, “that if you were to take all the processes in the body that contribute to this disease, the one that really stands out is RNA splicing. The disease is highly correlated with mutations to the splicing machinery.”

Still, detailed knowledge about which mutations interrupt RNA splicing (and how) have remained elusive.

Some forms of MDS, as well as acute myeloid leukemia (AML), arise when a person has two mutated copies of the DDX41 gene, resulting in a malfunctioning protein. Mutations to the DDX41 gene can be inherited, but it is also possible for the gene’s DNA to mutate over the course of one’s life, resulting in a somatic mutation (a mutation that is not inherited). While a mutation which impacts only a single allele of the DDX41 gene is benign, this mutation, when it occurs in a person who has already inherited a mutated copy of the allele, can result in blood diseases and cancers. For reasons not yet understood, people who inherit a mutated copy of the DDX41 gene are predisposed to developing a somatic mutation in the other allele. Thus, disease often occurs when a patient has inherited one DDX41 allele containing a mutation and acquires a second mutated copy later in life.

“For the DDX41 protein, there’s not just one mutation of interest or one mutation hotspot,” says Hoskins. Although it is likely that many mutations to the DDX41 gene are nonpathogenic, “there are hundreds more mutations to identify, and you can’t necessarily predict how each mutation is going to affect the function of the protein.”

Until now, researchers have focused on gene mutations found in MDS patients who have undergone genetic testing. Little is known about how many other mutations may lead to MDS or the diversity of ways that these mutations may result in illness. A deeper understanding of the downstream effects of these mutations may be a significant step toward developing directed treatments — especially important because the few therapeutic options available for patients with MDS are aggressive and harsh.

“We’re at the stage where we need to really understand basic biochemistry about the structure and function of the protein, which can pave the way for new therapeutic treatments,” explains Hoskins.

Unfortunately, current technology only allows scientists to decipher the functional impacts of just a few mutations at a time. At that rate, it could take many years to fully explore changes to protein function on the suite of mutations possible on the DDX41 gene.

Hoskins hopes for significant progress in his research on MDS thanks to recent advances in microfluidics technology developed at Stanford University. The new technology may offer opportunities for scientists to test the impact of thousands of mutations at once, removing a significant barrier to the research of Hoskins and others looking to establish a catalog of the biochemical characterizations of pathogenic mutations.

Hoskins first learned about the new technology through an article in Science describing how it was used by Stanford University researchers to better understand how thousands of different mutation impact an enzyme’s activity and structure using assays and quantitative analyses. He wondered if the same tool could be used to examine DDX41 protein function and the impact of pathogenic mutations.

Aaron Hoskins visiting the Fordyce Lab at Stanford University.
From left to right: Aaron Hoskins with Fordyce Lab researchers Albert Lee, Jennifer Ortiz Cárdenas, and Karl Krauth.

Wanting to learn more, Hoskins visited Stanford University in 2022 to see the technology for himself in the lab of its innovator, Polly Fordyce. He soon saw potential for a collaboration.

“Now, we can study the effects of mutations at every single amino acid in the protein and predict the consequences of mutations not yet discovered,” says Hoskins. “Before, this would have taken an entire team of researchers years. With the technology developed in the Fordyce Lab, we can do it in a matter of weeks.”

Fordyce, a professor in Stanford University’s Schools of Engineering and Medicine and 2023 recipient of a National Institutes of Health Director’s High-Risk, High-Reward Pioneer Grant, also sees her technology as an opportunity for scientists to explore the interconnections between mutations and protein function like never before.

“The goal is to predict not only changes in form resulting from mutations, but also changes in function,” explains Fordyce. “We take experiments that have typically taken place in test tubes, flasks, and petri dishes — experiments where a very experienced researcher with a lot of good luck could look at maybe five mutations in a week — and do thousands of them in parallel within microfluidic devices. We can do experiments cheaper and faster, while maintaining the quality of the information provided.”

With the newly developed technology, researchers are able to assess the function of large numbers of mutations and the resulting proteins using only the parts of the cell required for protein production. Through this method, scientists can omit steps required to grow cell cultures for each mutation. Then, using assays (a technique used to test for enzyme activity and inhibition, among other things), researchers assess the function of thousands of protein variants simultaneously.

The grant from Evans MDS, which will provide $600,000 over 3 years, opens the door for Hoskins and Fordyce to collaborate in addressing critical questions about MDS: which mutations are associated with the disease and how do those mutations disrupt cells’ biochemical function?

“We are hoping to do really precise quantitative measurements of important biochemical parameters, like how well the protein binds to substrates or inhibitors,” says Hoskins. “Our primary goal is to understand the biochemical basis for the function of this protein and how it goes wrong in human diseases.”

As the collaborators embark on this new line of investigation, Hoskins sees exciting possibilities for interrogating additional protein mutations known to impact human health.

“The protein we’re studying is part of a larger class of proteins called DEAD-box proteins, and they’re involved in essentially every step of gene expression in humans,” says Hoskins. “This protein is just the beginning. We can learn so much about other systems involved in gene expression using similar approaches. We can just keep going.”

Written by Renata Solan.