Researchers Find "Hidden" Mutations by Tracing Protein Isoforms

Approximately 25 million individuals in the United States are affected by a rare genetic disorder, and a significant number of them face challenges not only due to the absence of effective treatments but also because of insufficient information regarding their condition.

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Healthcare providers may be unaware of the underlying causes of a patient's symptoms, may not understand the potential progression of the disease, or may even struggle to provide a definitive diagnosis. Researchers have turned to the human genome in search of solutions, and numerous genetic mutations responsible for diseases have been found; however, as many as 70% of patients still do not have a clear genetic explanation for their condition.

In a study published in Molecular Cell, Iain Cheeseman, a member of the Whitehead Institute, suggests that both researchers and clinicians could potentially gain deeper insights from patients' genomes by examining them from a novel perspective.

The current belief is that each gene is responsible for coding a single protein. Consequently, an individual investigating whether a patient possesses a mutation or variant of a gene that plays a role in their illness will typically search for mutations that influence the "known" protein product of that gene.

Cheeseman and colleagues are discovering that most genes actually code for multiple proteins. This implies that a mutation that may appear trivial because it does not seem to impact the known protein could still modify another protein produced by the same gene. Recently, it has been demonstrated that mutations affecting one or several proteins derived from the same gene can have varying contributions to disease.

The researchers initially present the findings regarding how cells utilize the capacity to produce various forms of proteins from a single gene. The researchers investigate the role of mutations that influence these proteins in the development of disease.

The researchers offer two case studies of patients exhibiting unusual manifestations of a rare anemia associated with mutations that specifically impact only one of the two proteins generated by the gene involved 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. 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.

Jimmy Ly, Graduate Student, Whitehead Institute

Rethinking How Cells Use Genes

Cells possess multiple mechanisms to generate various forms of a protein, yet the variation examined by Cheeseman and Ly occurs during the synthesis of proteins from genetic information. Cellular machinery constructs each protein based on the directives contained within a genetic sequence, which initiates at a 'start codon' and concludes at a 'stop codon.'

Certain genetic sequences feature more than one start codon, many of which are concealed in plain sight. If the cellular machinery bypasses the initial start codon and identifies a subsequent one, it may produce a shorter variant of the protein. Conversely, the machinery might recognize a segment that closely resembles a start codon earlier in the sequence than its usual starting point, resulting in the creation of a longer version of the protein.

These occurrences might appear to be errors: the cellular machinery inadvertently generates an incorrect variant of the appropriate protein. On the contrary, the synthesis of proteins from these alternative starting points is a significant aspect of cellular biology that is present across various species. When Ly investigated the timeline of specific genes evolving to generate multiple proteins, he discovered that this is a widespread and resilient process that has been maintained throughout millions of years of evolutionary history.

Ly demonstrates that one of the functions it serves is to transport various forms of a protein to distinct locations within the cell. Numerous proteins possess sequences akin to zip codes that instruct the cell's machinery on where to send them, enabling the proteins to perform their functions. Ly identified numerous instances where both longer and shorter forms of the same protein had different zip codes and consequently were located in different areas of the cell.

Specifically, Ly discovered many instances where one variant of a protein was found in mitochondria, which are structures responsible for supplying energy to cells, while another variant was located elsewhere. Given the mitochondria's critical role in the vital process of energy production, mutations in mitochondrial genes are frequently associated with disease.

Ly considered the implications of a mutation that causes a disease by eliminating one variant of a protein while preserving the other, resulting in the protein reaching only one of its two designated locations. He examined a database of genetic data from individuals suffering from rare diseases to determine if such instances were present, and discovered that they indeed were. In reality, there could be tens of thousands of these occurrences. Nevertheless, without the ability to consult the individuals directly, Ly was unable to establish the effects of this mutation regarding the symptoms and severity of the disease.

In the meantime, Cheeseman collaborated with researchers and clinicians at Boston Children’s Hospital to expedite the transition from research discovery to clinical application. As a result, he encountered Fleming.

A subset of patients treated by Fleming suffers from a form of anemia known as SIFD, Sideroblastic Anemia with B-Cell Immunodeficiency, Periodic Fevers, and Developmental Delay, resulting from mutations in the TRNT1 gene. TRNT1 is among the genes that Ly identified as generating a mitochondrial variant of its protein, as well as another variant that is located in a different area: the nucleus.

Fleming provided anonymized patient data to Ly, who identified two noteworthy cases within the genetic information. The majority of the patients exhibited mutations that affected both forms of the protein; however, one patient had a mutation that specifically removed only the mitochondrial variant of the protein, whereas another patient had a mutation that solely eliminated the nuclear variant.

When Ly presented the findings, Fleming disclosed that both patients exhibited highly unusual manifestations of SIFD, thereby corroborating Ly's theory that mutations impacting various forms of a protein could yield distinct outcomes. The patient possessing solely the mitochondrial variant was anemic yet developmentally typical.

Conversely, the patient lacking the mitochondrial variant did not experience developmental delays or chronic anemia; however, he did present with other immune-related symptoms and was not accurately diagnosed until he reached his fifties. It is likely that additional factors are influencing the specific presentation of the disease in each patient; yet, Ly's research initiates the process of elucidating the enigma surrounding their atypical symptoms.

Cheeseman and Ly aim to increase clinicians' awareness regarding the prevalence of genes that code for multiple proteins, ensuring they are vigilant in checking for mutations that may impact any of the protein variants potentially linked to disease. For instance, numerous TRNT1 mutations that solely eliminate the shorter variant of the protein are not recognized as disease-causing by existing assessment tools.

Researchers in Cheeseman's lab are currently working on a new assessment tool for clinicians, named SwissIsoform, which will pinpoint relevant mutations affecting specific protein variants, including those mutations that might otherwise go undetected.

Jimmy and Iain’s work will globally support genetic disease variant interpretation and help with connecting genetic differences to variation in disease symptoms. 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.

Mark D. Fleming, Department of Pathology, Boston Children's Hospital

In the long run, the researchers anticipate that their findings may contribute to a deeper understanding of the molecular foundations of diseases and facilitate the development of novel gene therapies. Once researchers comprehend the underlying issues within a cell that lead to disease, they will be better equipped to create effective therapies. More urgently, the researchers aim for their efforts to have a significant impact by offering improved information to healthcare professionals and individuals affected by 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. 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.

Iain Cheeseman, Whitehead Institute