Iain Cheeseman and colleagues reveal the underappreciated role of single genes producing multiple proteins in atypical presentations of rare disease, and present case studies of affected patients through a collaboration with Boston Children's Hospital. The findings help to identify genetic mutations that may contribute to rare diseases.
Around 25 million Americans have a rare genetic disease, and many of them struggle with not only a lack of effective treatments, but also a lack of good information about their disease. Clinicians may not know what causes a patients' symptoms, know how their disease will progress, or even have a clear diagnosis. Researchers have looked to the human genome for answers, and many disease-causing genetic mutations have been identified, but as many as 70% of patients still lack a clear genetic explanation.
In a paper published in Molecular Cell on November 7, Whitehead Institute Member Iain Cheeseman, graduate student Jimmy Ly, and colleagues propose that researchers and clinicians may be able to get more information from patients' genomes by looking at them in a different way.
The common wisdom is that each gene codes for one protein. Someone studying whether a patient has a mutation or version of a gene that contributes to their disease will therefore look for mutations that affect the "known" protein product of that gene. However, Cheeseman and others are finding that the majority of genes code for more than one protein. That means that a mutation that may seem insignificant because it does not appear to affect the known protein could nonetheless alter a different protein made by the same gene. Now, Cheeseman and Ly have shown that mutations affecting one or multiple proteins from the same gene can contribute differently to disease.
In their paper, the researchers first share what they have learned about how cells make use of the ability to generate different versions of proteins from the same gene. Then, they examine how mutations that affect these proteins contribute to disease. Through a collaboration with co-author Mark Fleming, the pathologist-in-chief at Boston Children's Hospital, they provide two case studies of patients with atypical presentations of a rare anemia linked to mutations that selectively affect only one of two proteins produced by the gene implicated in the disease.
"We hope this work demonstrates the importance of considering whether a gene of interest makes multiple versions of a protein, and what the role of each version is in health and disease," Ly says. "This information could lead to better understanding of the biology of disease, better diagnostics, and perhaps one day to tailored therapies to treat these diseases."
Rethinking how cells use genes
Cells have several ways to make different versions of a protein, but the variation that Cheeseman and Ly study happens during protein production from genetic code. Cellular machines build each protein according to the instructions within a genetic sequence that begins at a "start codon" and ends at a "stop codon." However, some genetic sequences contain more than one start codon, many that are hiding in plain sight. If the cellular machinery skips the first start codon and detects a second one, it may build a shorter version of the protein. In other cases, the machinery may detect a section that closely resembles a start codon at a point earlier in the sequence than its typical starting place, and build a longer version of the protein.
These events may sound like mistakes: the cell's machinery accidentally creating the wrong version of the correct protein. To the contrary, protein production from these alternate starting places is an important feature of cell biology that exists across species. When Ly traced when certain genes evolved to produce multiple proteins, he found that this is a common, robust process that has been preserved throughout evolutionary history for millions of years.
Ly shows that one function this serves is to send versions of a protein to different parts of the cell. Many proteins contain zip code-like sequences that tell the cell's machinery where to deliver them so the proteins can do their jobs. Ly found many examples in which longer and shorter versions of the same protein contained different zip codes and ended up in different places within the cell.
In particular, Ly found many cases in which one version of a protein ended up in mitochondria, structures that provide energy to cells, while another version ended up elsewhere. Because of the mitochondria's role in the essential process of energy production, mutations to mitochondrial genes are often implicated in disease.
Ly wondered what would happen when a disease-causing mutation eliminates one version of a protein but leaves the other intact, causing the protein to only reach one of its two intended destinations. He looked through a database containing genetic information from people with rare diseases to see if such cases existed, and found that they did. In fact, there may be tens of thousands of such cases. However, without access to the people, Ly had no way of knowing what the consequences of this were in terms of symptoms and severity of disease.
Meanwhile, Cheeseman had begun working with Boston Children's Hospital to foster collaborations between Whitehead Institute and the hospital's researchers and clinicians to accelerate the pathway from research discovery to clinical application. Through these efforts, Cheeseman and Ly met Fleming.
One group of Fleming's patients have a type of anemia called SIFD—Sideroblastic Anemia with B-Cell Immunodeficiency, Periodic Fevers, and Developmental Delay—that is caused by mutations to the TRNT1 gene. TRNT1 is one of the genes Ly had identified as producing a mitochondrial version of its protein and another version that ends up elsewhere: in the nucleus.
Fleming shared anonymized patient data with Ly, and Ly found two cases of interest in the genetic data. Most of the patients had mutations that impaired both versions of the protein, but one patient had a mutation that eliminated only the mitochondrial version of the protein, while another patient had a mutation that eliminated only the nuclear version.
When Ly shared his results, Fleming revealed that both of those patients had very atypical presentations of SIFD, supporting Ly's hypothesis that mutations affecting different versions of a protein would have different consequences. The patient who only had the mitochondrial version was anemic but developmentally normal. The patient missing the mitochondrial version of the protein did not have developmental delays or chronic anemia but did have other immune symptoms, and was not correctly diagnosed until his fifties. There are likely other factors contributing to each patient's exact presentation of the disease, but Ly's work begins to unravel the mystery of their atypical symptoms.
Cheeseman and Ly want to make more clinicians aware of the prevalence of genes coding for more than one protein, so they know to check for mutations affecting any of the protein versions that could contribute to disease. For example, several TRNT1 mutations that only eliminate the shorter version of the protein are not flagged as disease-causing by current assessment tools. Cheeseman lab researchers including Ly and graduate student Matteo Di Bernardo are now developing a new assessment tool for clinicians, called SwissIsoform, that will identify relevant mutations that affect specific protein versions, including mutations that would otherwise be missed.
"Jimmy and Iain's work will globally support genetic disease variant interpretation and help with connecting genetic differences to variation in disease symptoms," Fleming says. "In fact, we have recently identified two other patients with mutations affecting only the mitochondrial versions of two other proteins, who similarly have milder symptoms than patients with mutations that affect both versions."
Long term, the researchers hope that their discoveries could aid in understanding the molecular basis of disease and in developing new gene therapies: once researchers understand what has gone wrong within a cell to cause disease, they are better equipped to devise a solution. More immediately, the researchers hope that their work will make a difference by providing better information to clinicians and people with rare diseases.
"As a basic researcher who doesn't typically interact with patients, there's something very satisfying about knowing that the work you are doing is helping specific people," Cheeseman says. "As my lab transitions to this new focus, I've heard many stories from people trying to navigate a rare disease and just get answers, and that has been really motivating to us, as we work to provide new insights into the disease biology."
