Yeast screens explore the therapeutic potential of chemical rescue.

Anyone who’s worked in a lab knows that sinking feeling of discovering that the temperature of an incubator, carefully set the night before, has crept up high enough to ruin the experiment. While such a mishap usually spells disaster, occasionally, it can lead to an unexpected discovery.

One such revelation was prompted by an uncooperative incubator in the lab of Michael McMurray, a cell biologist at University of Colorado’s Anschutz Medical Campus. McMurray studies the septin family of cytoskeletal proteins, and inside the incubator were plates of yeast with temperature-sensitive septin mutations. The mutant yeast could survive only at mild temperatures, so the incubator was set to a comfortable 27°C.

For this experiment, a chemical called guanidine hydrochloride had been added to some of the plates, to test whether it would stop the mutants from growing at the permissive temperature. When the incubator was found roasting away at more than 30°C, however, all of the yeast should have been dead.

“Amazingly, one of the mutants actually grew,” says McMurray. “The guanidine restored its viability.”

That discovery launched an investigation of how, exactly, guanidine had protected the mutant from normally lethal conditions. In a paper in the September issue of G3: Genes|Genomes|Genetics, Hassell et al. report several mutants that can be rescued by guanidine. They also show that another naturally occurring small molecule can correct an even broader range of mutants.

Guanidine can stand in for a lost arginine

Guanidine’s molecular structure mimics the side chain of the amino acid arginine. Researchers had previously shown that guanidine could restore function to an enzyme that had been mutated to lack an arginine in its active site. But all of this work had been done in vitro. This piqued McMurray’s interest even more. “Arginine is the most commonly mutated amino acid in human disease,” he says. “If guanidine can restore function to arginine mutant proteins, why has no one explored this in living cells?”

McMurray’s team began by testing enzymes in which a single arginine mutation disabled the enzyme enough to cause disease, such as ornithine transcarbamylase (OTC). OTC deficiency is an inherited metabolic disease that leads to a buildup of toxic ammonia in the body. The researchers created yeast with the same OTC arginine mutation that causes the human disease, making the yeast unable to grow without nutritional supplementation. Adding guanidine hydrochloride to the growth media restored some of the lost enzyme function.

“The effect was pretty small,” McMurray says. “It wasn’t a full rescue, but it was something.”

Next, the researchers decided to broaden their investigation. Instead of testing candidate enzymes, they screened hundreds of yeast mutants to see if guanidine restored function to any of them. “We decided to let the cells tell us what would work best,” McMurray says. “That’s when things started to get interesting.”

The screen uncovered 11 new candidates, the most interesting of which was an arginine mutant of actin, another cytoskeletal protein. “It just so happens that arginine is also mutated in human cardiac beta actin, and that mutation causes disease,” McMurray says.

As an ATPase, actin is technically an enzyme, but the arginine mutation is far from the active site, and guanidine isn’t restoring catalytic activity per se. Instead, McMurray says, it’s helping the protein fold into its proper 3D shape. “All proteins have to fold,” McMurray says. Protein folding results from chemical interactions between the side chains of various amino acids. “To rescue the mutant, the guanidine just has to be able to fix what’s missing and restore the folding.”

The idea of rescuing mutants by restoring proper protein folding led them to investigate other chemicals that can influence protein folding. “From a biological perspective, what are other cases in nature in which organisms have to deal with alterations in protein folding?” McMurray says. “Then we thought of sea creatures — sharks and rays.”

Moving beyond guanidine

Because they live in saltwater, sharks maintain high concentrations of urea in their bodies to keep from losing water through osmosis. Urea, however, is toxic to proteins, and causes them to unfold. To counteract the urea, these animals also have high levels of a chemical called trimethylamine oxide (TMAO), which promotes protein folding.

Does the shark’s protein protection trick work in other contexts? To follow up, research assistant Daniel Hassell screened yeast mutants using TMAO. He turned up hundreds of mutants that were rescued by the molecule. The genes and mutant types were all very different from each other, suggesting that TMAO has a more general stabilizing effect rather than specifically replacing a particular amino acid. This broad effect suggests a potential role for the molecule in synthetic biology, as a way to design proteins with an on/off switch system.

For its part, guanidine is already FDA-approved as a treatment for an inherited autoimmune disorder called Lambert-Eaton myasthenic syndrome. McMurray remains curious about whether it has the potential to treat other genetic diseases.

“That would be my ultimate hope, that someone would be inspired by our work to try it in an animal model or the clinic,” McMurray says.

CITATION

Chemical rescue of mutant proteins in living Saccharomyces cerevisiae cells by naturally occurring small molecules
Daniel S Hassell, Marc G Steingeisser, Ashley S Denney, Courtney R Johnson, Michael A McMurray
G3 Genes|Genomes|Genetics 2021; jkab252
https://doi.org/10.1093/g3journal/jkab252

A new fruit fly model of Leigh syndrome reveals the importance of mtDNA variation.

Inherited mitochondrial disorders pose a perplexing problem to researchers and clinicians: people with the same condition can have vastly different clinical manifestations, even if they share the same mutation. For example, a neurodegenerative disorder called Leigh syndrome, which can be caused by many different mutations in genes encoding mitochondrial proteins, is usually first noticed in infancy and causes death within a couple years. Some affected individuals, however, don’t experience any apparent problems related to the mutations until they’re teens or adults.

Researchers Carin Loewen and Barry Ganetzky recently reported in GENETICS that they identified a fly strain exhibiting a Leigh syndrome-like phenotype, including mitochondrial abnormalities, decreased lifespan, and neurodegeneration. They found that the flies had a mutation in the gene ND23, an ortholog of a human gene (NDUFS8) that is mutated in some cases of Leigh syndrome. The gene encodes a core subunit of mitochondrial complex 1, part of the cellular machinery that makes energy.

In characterizing the ND23-mutant flies, Loewen and Ganetzky found that a maternally inherited factor mediated the severity of the Leigh syndrome-like phenotype. The pair hypothesized that the factor was a variant encoded in the mitochondrial genome because mitochondria are inherited only from the mother. In support of this hypothesis, they showed that the onset of the Leigh syndrome-like phenotype could be modified by mitochondria from different fly strains. By sequencing all the protein- and tRNA-coding genes in the mitochondrial genomes of these various strains, Loewen and Ganetzky determined that the mitochondrial genomes of strains in which the ND23-mutant phenotype was delayed shared polymorphisms that were not seen in the mitochondrial genomes of strains with earlier disease onset. None of these polymorphisms had any effect on flies in the absence of the ND23 nuclear mutation, showing how otherwise benign mitochondrial backgrounds can interact with mutations in nuclear genes to mediate the severity of mitochondrial disorders.

Not only did this work provide evidence for one explanation of the tremendous phenotypic variation observed in mitochondrial disorders, it also introduced a new model system for studying Leigh syndrome and its phenotypic variability. Although the prognosis varies, it is uniformly poor, and there is no cure—so further research on Leigh syndrome is desperately needed.

CITATION:
Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a Drosophila Model of Leigh Syndrome
Carin A. Loewen, Barry Ganetzky
Genetics 2018 208: 1535-1552; https://doi.org/10.1534/genetics.118.300818
http://www.genetics.org/content/208/4/1535

The “hyperspin” long-range enhancer deletion recapitulates disease phenotypes.

In recent years, improvements in genetic testing have made it much easier to discover the causes of rare genetic diseases, but sequence data can also present new puzzles. Take split hand/-foot malformation-1 syndrome (SHFM1), which causes limb deformities, such as joined fingers, and sometimes deafness. Candidate culprits were mutations in the genes DLX5 or DLX6, since defects in either gene cause SHFM1-like malformations in mice. But few people with SHFM1 carry the equivalent mutations.

In a recent mouse study, Johnson et al. found new clues to the mystery. The team examined deaf mice with a naturally occurring mutation they named hyperspin, which causes rapid circling behavior typical of mice with inner ear defects. They found that the hyperspin mice had inner ear malformations that recapitulate those seen in Dlx5-knockout mice—and the same features are sometimes part of the SHFM1 phenotype. The new mutation lies in the gene Slc25a13 and consists of a large deletion that spans introns as well as the exons that encode the majority of the SLC25A13 protein.

Despite the fact that hyperspin disrupts so much of the Slc25a13’s coding sequence, the researchers found that nonfunctional SLC25A13 protein wasn’t to blame for the mice’s phenotype. Completely knocking out Slc25a13 yielded mice with normal hearing and no indication of the hyperspin inner ear abnormalities. Instead, there were signs that a noncoding sequence in Slc25a13 regulates the expression of Dlx5, which is 660 kilobases away from the hyperspin deletion. Mouse embryos homozygous for the hyperspin mutation had strongly reduced expression of Dlx5 in the otic vesicle, which could have caused the inner ear defects they observed.

Unlike Dlx5-knockout mice, hyperspin mice can survive to adulthood, providing researchers a new model system for studying how a lack of Dlx5 affects inner ear function across the lifespan. This work also illustrates how pursuing unexpected occurrences, like this chance mutation in a lab mouse, can open new avenues of research.

CITATION:

Deletion of a Long-Range Dlx5 Enhancer Disrupts Inner Ear Development in Mice
Kenneth R. Johnson, Leona H. Gagnon, Cong Tian, Chantal M. Longo-Guess, Benjamin E. Low, Michael V. Wiles, Amy E. Kiernan
Genetics 2018 208: 1165-1179; https://doi.org/10.1534/genetics.117.300447
http://www.genetics.org/content/208/3/1165

Rhinocladiella mackenziei is a fungus that infects the human brain. It is the most common cause of neurological fungal infections in arid regions of the Middle East, and it is fatal in 70% of cases. However, little is understood about this lethal pathogen—not even its natural habitat.

To learn more about the biology of R. mackenziei, Moreno et al. turned to its genome. They resequenced the genome of two strains isolated from patients and compared them to known sequences from R. mackenziei, as well as other related fungi.

These comparisons gave clues about the natural lifestyle of the fungus. For example, R. mackenziei carries genes similar to fungi from habitats polluted by aromatic hydrocarbons, such as those found in gasoline. This suggests that R. mackenziei might flourish in oil-contaminated desert soil, where these genes would give it a competitive advantage over organisms that are unable to thrive in such a harsh environment.

The authors also identified a number of secreted virulence factors which could permit R. mackenziei to more easily establish itself in the brains of infected humans. The genomes harbor a wide array of genes involved in metabolism of diverse substrates, as well as nitrogen and iron uptake. This metabolic adaptability means that R. mackenziei probably isn’t a true pathogen; a pathogen would have lost some of these pathways because it could rely on its host for nutrients. Rather, this desert fungus is equipped to survive a number of harsh conditions, so its ability to infect human brains is most likely opportunistic.

CITATION

Genomic Understanding of an Infectious Brain Disease from the Desert

The Genetics Society of America (GSA) is pleased to announce that Philip Hieter is the recipient of the 2018 George W. Beadle Award, bestowed in honor of his outstanding contributions to the genetics research community. Hieter is Professor of Medical Genetics in the Michael Smith Laboratories at the University of British Columbia.

Philip Hieter

Geneticists across the model organism and human genetics communities recognize Hieter for his dedication to uniting human biologists with those who work on model organisms such as mice, fruit flies, worms, and yeast. The resulting collaborations are crucial to advancing our knowledge of biology, including human health and disease; connecting model organism researchers and human biologists with one another speeds progress for both groups, facilitates mechanistic understanding of disease gene functions, and helps uncover novel disease mechanisms and candidate therapeutic targets.

In 1997, when few genome sequences were available, Hieter helped create XREFdb, a public database that linked the functional annotations of genes studied in model organisms with the phenotypic annotations on the human and mouse genetic maps. This resource provided cross-species candidate genes for mammalian phenotypes, including human diseases, and stimulated interactions between basic scientists working on various organisms and the medical genetics community. He has also founded and co-led several multidisciplinary meetings that bridged the gap between biologists working on humans and those working on model organisms. Hieter and Jeannie Lee, a professor at Harvard Medical School and the Massachusetts General Hospital (and 2018 GSA President), were co-chairs of 2016’s Allied Genetics Conference, which brought together over 3,000 attendees from seven different genetic research communities to exchange ideas and findings.

As the 2012 GSA President, Hieter continued to foster closer relationships among different groups of life scientists. “As president of the GSA, Phil had a strong focus on bridging the many separate communities of the Society as well as increasing the interactions of the GSA community with members of the human genetics community,” says Stanley Fields, professor at the University of Washington and 2016 GSA President.

To help biological insights reach patients, Hieter co-founded, in 2014, the Canadian Rare Diseases: Models and Mechanisms National Network, a consortium that connects clinician scientists identifying gene mutations in patients that cause rare diseases to basic scientists analyzing the corresponding genes in model organisms. This network funds pilot studies to expedite collaboration between the two groups, conduct model organism-based functional studies of disease gene variants, and develop new therapeutic strategies using model organisms.

In addition to having connected research communities, Hieter and his lab have made many significant contributions to our understanding of chromosome biology, including the dissection of yeast centromeres and the identification of genes involved in genome stability. Their contributions to the yeast community include physical mapping methods, synthetic lethality screen approaches for identifying cross-species candidate genes as potential cancer drug targets, and a widely used set of vectors and yeast host strains that have been instrumental in work that has led to countless discoveries in recent decades.

The George W. Beadle Award was created by GSA to honor the memory of George W. Beadle (1903–1989), the 1946 GSA President. Beadle and his colleague Edward L. Tatum were awarded the Nobel Prize for Physiology or Medicine in 1958 for work that linked genetics to biochemistry, providing a major part of the foundation for the field of molecular biology. In addition to being a GSA President, Beadle served society in several leadership roles—for instance, as chairman of the National Academy of Sciences Committee on the Biological Effects of Atomic Radiation—and demonstrated a strong commitment to science outreach and education.

The Prize will be presented to Hieter at the 2018 Yeast Genetics Meeting, a GSA Conference to be held August 20–26 at Stanford University.

The prolonged and severe seizures suffered by those with pyridoxine-dependent epilepsy (PDE) can lead to brain dysfunction and death if not treated. Standard antiepileptic drugs are typically ineffective for people with this rare genetic disorder—instead, they need high doses of vitamin B6 in the form of pyridoxine or pyridoxal 5′-phosphate. But even with this supplementation, people with PDE often have lingering problems, and over 75% experience neurodevelopmental delays.

Although more than sixty years have passed since PDE was first described, and treatment remains inadequate for resolving all the condition’s comorbidities, little is known about the pathophysiology of PDE. This is partially because no animal model has been available to study PDE—until now. In this month’s issue of GENETICS, Pena et al. report that they have generated zebrafish with mutations in the same gene that causes PDE when mutated in humans. This gene, ALDH7A1, encodes an enzyme called antiquitin, which is important for the breakdown of the amino acid lysine.

The researchers used CRISPR/Cas9-based gene editing to alter the fish version of the gene, rendering it nonfunctional. They found that fish carrying two damaged copies of the gene develop recurrent seizures at an early age, just like humans with PDE do. Without intervention with vitamin B6, the fish’s seizures result in death. The fish also have the same biochemical abnormalities as humans with PDE do, including a buildup of toxic lysine byproducts. The researchers also found several other previously unknown biochemical abnormalities in the fish’s brains, which may help understand the disorder.

The fact that these fish recapitulate human PDE so well—at both the biochemical and organismal levels—will make them a valuable model for further studying the condition. Not only are zebrafish commonly used model organisms with a multitude of tools available for manipulating their genetics, they’re also often used in epilepsy research with many established protocols for studying the topic, making them an ideal choice for investigating this challenging disorder.

CITATION:

Pena, I.; Roussel, Y.; Daniel, K.; Mongeon, K.; Johnstone, D.; Weinschutz Mendes, H.; Bosma, M.; Saxena, V.; Lepage, N.; Chakraborty, P.; Dyment, D.; van Karnebeek, C.; Verhoeven-Duif, N.; Vu Bui, T.; Boycott, K.; Ekker, M.; MacKenzie, A. Pyridoxine-Dependent Epilepsy in Zebrafish Caused by Aldh7a1 Deficiency.
GENETICS, 207(4), 1501-1518.
DOI: 10.1534/genetics.117.300137
http://www.genetics.org/content/207/4/1501

For many of the roughly 300 million people around the world with rare diseases, the road to diagnosis can be long, painful, expensive, and disheartening. Around eighty percent of very infrequently seen undiagnosed diseases are estimated to have a genetic basis, but even with modern DNA sequencing techniques, the causes are often unclear. In these cases, clinicians and their basic scientist collaborators are increasingly turning to laboratory models like fruit flies and zebrafish to help diagnose disease—and gain clues about how to treat it.

The teamwork between clinicians and model organism researchers goes both ways: clinicians can find candidate genes in patients to test in model organisms, or basic scientists can identify candidate disease genes through research on their organism of choice. In a review appearing in the September issue of GENETICS, Wangler et al. describe numerous tools clinicians and basic scientists have at hand to help them work together on puzzling rare diseases.

One such tool is GeneMatcher, a website that connects researchers who may be separately investigating the same genes. Using GeneMatcher, clinicians can find potential collaborators working on model organisms.

Another mechanism that connects clinicians with model organism researchers is the Canadian Rare Diseases Models and Mechanisms Network (RDMM). Via the RDMM, a clinician can submit a proposal to work with a model organism researcher—or vice versa. Uniquely, they can also use the tool to apply for quick-turnaround grants to fund their investigations of potential disease-causing variants.

Patients themselves can also contribute to this research. People with rare diseases that have resisted diagnosis by any other means can apply to the Undiagnosed Diseases Program (UDP) to spur investigations of their conditions. Not only have patients been diagnosed using the UDP’s combination of detailed clinical investigation and genetic analysis, but new disease genes have also been discovered. For example, mutations in the gene NT5E were found to cause a rare arterial calcification disorder—and as an unexpected bonus, this finding hinted that adenosine metabolism might be linked to more common vascular disorders as well.

The UDP has now been expanded into the Undiagnosed Diseases Network (UDN), a decentralized program involving researchers at several institutions. Using the UDN, a patient is first screened to see if their disease matches a known genetic condition after an extensive phenotypic work-up and sequencing of the whole genome or exome. If not, clinical findings and candidate genetic variants are sent to the Model Organisms Screening Center (MOSC). The MOSC starts by searching databases of known information about the candidate variants to determine which are worth testing in model organisms. The MOSC then looks for other individuals with similar clinical presentations and possible genetic causes.

Once the list of candidate genes is narrowed down, the MOSC researchers design experiments in flies or zebrafish to acquire more knowledge. The MOSC teams aim to match the variant in the human patient’s gene in the model organism. The goal is to learn more about the function of the gene, to determine whether the gene variant found in the patient is the likely cause of the disease, and to understand how the variant may cause problems.

Wangler et al. conclude by endorsing the continued support of these tools by government agencies such as the National Institutes of Health. Only with this financial backing, they say, will crucial improvements in the diagnosis and treatment of rare genetic diseases be possible. And since our understanding of rare diseases often drives discoveries about more common diseases, this research could even have more far-reaching impacts.

CITATION:

Wangler, M.; Yamamoto, S.; Chao, H.; Posey, J.; Westerfield, M.; Postlethwait, J.; Members of the Undiagnosed Diseases Network (UDN); Hieter, P.; Boycott, K.; Campeau, P.; Bellen, H. Model Organisms Facilitate Rare Disease Diagnosis and Therapeutic Research.
GENETICS, 207(1), 9-27.
DOI: 10.1534/genetics.117.203067
http://www.genetics.org/content/207/1/9

A Canadian network focused on rare diseases is playing matchmaker between clinicians and model organism researchers.

Over half of children with rare, inherited monogenic diseases lack a molecular diagnosis. There are an estimated 7,000 monogenic diseases possible, and only about half of those have been implicated in human disease. The Rare Diseases: Models & Mechanisms (RDMM) Network is addressing this by giving clinicians easy access to researchers who use model organisms to study their new gene of interest.

There are a few hurdles associated with rare disease gene discovery, not least of which is that the definition of rare disease is one that affects fewer than 200,000 people in the US or fewer than 1 in 2,000 people in the EU. Along with the heterogeneity of the clinical features in many of these diseases, the low number of those affected makes it harder to identify patients that share mutations in the same causative gene. As the cost of diagnostic sequencing continues to fall, however, clinicians can obtain detailed sequence information on their patients. The next step then lies in turning that sequence data into actionable information. That’s where the RDMM Network comes in.

“The ability for clinicians to now rapidly be linked to a model organism researcher is a critical step in bringing the gene discovery back to the family in the clinic where it all started,” says Kym Boycott, RDMM Network leader and GENETICS Associate Editor.

Together with Boycott (University of Ottawa and Children’s Hospital of Eastern Ontario), former GSA President Phil Hieter (University of British Columbia) and Janet Rossant (University of Toronto and Hospital for Sick Children) make up the RDMM Network leadership. Established with funding from the Canadian Institute of Health Research, the RDMM Network joins a group of other Canadian organizations – including FORGE Canada and Care4Rare Canada – already working towards rare disease gene discovery.

When clinicians discover a possible disease gene, they can submit the gene to the Network’s Clinical Advisory Committee. If it’s approved there, it goes on to the Scientific Advisory Committee; their job is to match the disease gene to model organism researchers who have already declared an interest in that gene or pathway. The model organism researchers can then submit a short application, and if it’s successful, the RDMM Network will put the clinician and model researcher in touch with each other and make available a $25,000 seed grant to the model organism researcher so that an immediate collaboration can be started.

“The network represents a model to the world for enhancing collaboration at the basic science/rare disease interface,” says Phil Hieter.

The RDMM Network is an example of the forward-thinking approach needed in biomedical research to make progress on identifying new disease genes and understanding their biological mechanisms. By encouraging collaboration between the bedside and the bench, we can expand our knowledge of rare human diseases – and how to treat them.

Canadian model organism researchers can register their genes of interest here, and Canadian clinician scientists can submit a newly discovered disease gene here.

CITATIONS AND FURTHER READING

Beaulieu C.L., Majewski J., Schwartzentruber J., Samuels M.E., Fernandez B.A., Bernier F.P., Brudno M., Knoppers B., Marcadier J., Dyment D., Adam S., Bulman D.E., Jones S.J.M., Avard D., Ngyuen M.T., Rousseau F., Marshall C., Wintle R.F., Shen Y., Scherer S.W., FORGE Canada Consortium, Friedman J.M., Michaud J.L., Boycott, K.M. 2014. FORGE Canada Consortium: Outcomes of a 2-Year National Rare-Disease Gene-Discovery Project. Am J Hum Genet, 94(6):809-817. doi: 10.1016/j.ajhg.2014.05.003 http://www.cell.com/ajhg/abstract/S0002-9297(14)00223-7

Foley K.E. 2015. Model network: Canadian Program Aims to Generate Models for Rare Disease. Nat Med, 21:1242-1243. doi: 10.1038/nm1115-1242
http://www.nature.com/nm/journal/v21/n11/full/nm1115-1242.html

Hieter P., Boycott K.M. 2014. Understanding Rare Disease Pathogenesis: A Grand Challenge for Model Organisms. Genetics, 198(2):443-445. doi: 10.1534/genetics.114.170217 http://www.genetics.org/content/198/2/443.full