A protein has been shown to have a surprising role in regulating the ‘glue’ that holds heart cells together, a finding that may explain how a gene defect could cause sudden cardiac death.

A team led by Oxford University researchers was looking at how a protein, iASPP, might be involved in the growth of tumours. However, serendipitously they found that mice lacking this gene died prematurely of sudden cardiac death. More detailed investigations showed that these mice had an irregular conductance in the right side of the heart, a condition known as arrhythmogenic right ventricular cardiomyopathy (ARVC).

The researchers discovered that iASPP had a previously unknown role in controlling desmosomes – one of the main structures that ‘glue’ individual heart muscle cells (cardiomyocytes) together. The genetic defect was shown to weaken desmosome function at the junctions of heart muscle cells: this affected the structural integrity of the heart, making mice lacking iASPP prone to ARVC.

Further studies of heart tissue from human patients who had died from ARVC showed that some of them have similar defects in desmosomes as in the mice suggesting that the faulty iASPP gene could also be responsible for ARVC deaths in humans. This finding also explains why a previously reported cattle herd with spontaneous iASPP gene deletion died of sudden cardiac death.

‘We set out to investigate how this protein might cause cancer and found by chance that it could play a key role in this rare genetic heart condition,’ said Professor Xin Lu, Director of the Ludwig Institute for Cancer Research at Oxford University, the lead investigator of the report. ‘It took my DPhil student Mario Notari, the lead author of the study, over two years of further detective work, in collaboration with our colleagues in Oxford and London, to show how a single faulty gene can affect the function of desmosomes, one of the main structures that ‘glue’ heart muscle cells together. Our studies suggest that these changes can threaten the structural integrity of the heart and predispose humans and animals to AVRC.’

ARVC is uncommon in humans, affecting around 1 in 2000 people in the UK [1], and is a leading cause of sudden cardiac death, which is estimated to kill around 100,000 people a year in the UK [2]. Whilst approximately 50% of human ARVC cases are related to known genetic defects in desmosomes, the cause of the other 50% of cases still remains unknown. The new study suggests that mutations in the gene encoding the iASPP protein may contribute to the development of ARVC in these previously-unexplained cases. University of Oxford

A team of scientists, led by researchers at The Wistar Institute, has identified a possible explanation for why middle-aged adults were hit especially hard by the H1N1 influenza virus during the 2013-2014 influenza season. The findings offer evidence that a new mutation in H1N1 viruses potentially led to more disease in these individuals. Their study suggests that the surveillance community may need to change how they choose viral strains that go into seasonal influenza vaccines, the researchers say.

“We identified a mutation in recent H1N1 strains that allows viruses to avoid immune responses that are present in a large number of middle-aged adults,” said Scott Hensley, Ph.D., a member of Wistar’s Vaccine Center and an assistant professor in the Translational Tumour Immunology program of Wistar’s Cancer Center.

Historically, children and the elderly are most susceptible to the severe effects of the influenza viruses, largely because they have weaker immune systems.  However, during the 2013-2014 physicians saw an unusually high level of disease due to H1N1 viruses in middle-aged adults—those who should have been able to resist the viral assault. Although H1N1 viruses recently acquired several mutations in the haemagglutinin (HA) glycoprotein, standard serological tests used by surveillance laboratories indicate that these mutations do not change the viruses’ antigenic properties.

However, Wistar researchers have shown that, in fact, one of these mutations is located in a region of HA that allows viruses to avoid antibody responses elicited in some middle-aged adults. Specifically, they found that 42 percent of individuals born between 1965 and 1979 possess antibodies that recognize the region of HA that is now mutated. The Wistar researchers suggest that new viral strains that are antigenically matched in this region should be included in future influenza vaccines.

“Our immune systems are imprinted the first time that we are exposed to influenza virus,” Hensley said. “Our data suggest that previous influenza exposures that took place in the 1970s and 1980s influence how middle-aged people respond to the current H1N1 vaccine.”

The researchers noted that significant antigenic changes of influenza viruses are mainly determined using anti-sera isolated from ferrets recovering from primary influenza infections. However humans are typically re-infected with antigenically distinct influenza strains throughout their life.  Therefore, antibodies that are used for surveillance purposes might not be fully reflective of human immunity. Wistar Institute

Damage to DNA is an issue for all cells, particularly in cancer, where the mechanisms that repair damage typically fail. The same agents that damage DNA also damage its sister molecule messenger RNA (mRNA), which ferries transcripts of the genes to the tens of thousands of ribosomes in each cell. But little attention has been paid to this damage.

 “There may be cases where messenger RNA is just as important as DNA,” said Carrie Simms, PhD, a postdoctoral associate in Zaher’s lab. “Clearly oxidative damage to RNA is somehow involved in neurodegenerative diseases, such as Alzheimer’s and ALS. It’s not necessarily causing the disease; it may just be some sort of by-product; but it’s in the mix.”

“Under normal conditions only about 1 percent of the cellular mRNAs are oxidized,” Zaher said, “but if you have oxidative stress, for whatever reason, a higher percentage can be damaged.

One of the hallmarks of Alzheimer’s is oxidative stress, and studies have shown that in people with advanced Alzheimer’s, half of the RNA molecules in the neurons may be oxidized.

Zaher, Simms and their colleagues report that when they fed oxidized mRNA to ribosomes, the nanomachines that convert mRNA to protein, the ribosomes jammed and stopped.

A stuck ribosome could be rescued by factors that released it from the mRNA and chewed up the damaged transcipt. But if the factors involved in this quality-control system were absent, damaged mRNA accumulated in the cell, just as it does in Alzheimer’s.

The three cellular processes essential to life—making copies of DNA, copying DNA into mRNA, and translating the mRNA into protein—have been penalized for billions of years by evolution, are astonishingly accurate, because evolution has heavily penalized any sloppiness.

Errors in DNA copying occur only once every billion events. When DNA is transcribed to mRNA, there is a mistake about once every ten thousand events .and when the mRNA is translated to protein, there might be an error once every thousand events.

To test the robustness of translation, the Zaher lab set out to break it, by giving faulty mRNA transcripts to ribosomes. They damaged one letter in a three-letter mRNA coding unit, oxidizing a G (the base guanine), to create what is called 8-oxo-G.

“We chose this oxidized base,” he said,” because we knew that when DNA is copied, an oxidized G causes a mistake. Instead of pairing with a C, as it normally would, 8-oxo-G will pair with an A.”

He thought the ribosome would read the three-letter codon C[8-oxo-G]C not as CGC but rather as CAC and conseqeuntly put the wrong amino acid in the protein chain it was making.

But when 8-oxo-G was added to a soup that contained all the factors needed to translate mRNA into protein, something surprising happened.

“We expected that we might get aberrant proteins,“ Simms said. “But the ribosome didn’t make mistakes.  It just stopped. It couldn’t deal with the mRNA at all”

The scientsts could tell it was stuck because levels of the protein the faulty mRNA encodes plummeted.

To make sure it was the presence rather than the position of the 8-oxo-G that mattered, Simms made mRNAs with the 8-oxo-G in each of the three positions of the three-letter coding unit. Each time the ribosome stalled.

Knowing they had found something interesting, the scientists upped their game. Simms built a longer 300-nucleotide mRNA to use as a probe. And instead of adding the damaged mRNA to a reconstituted bacterial system, she put it in extracts of plant and animal cells.

“We couldn’t look at ribosomes in the extracts,” Simms said, “but we could look at the proteins they made. They made short proteins, exactly the length you’d expect if the ribosome were stopping at the damaged base. “

A single mRNA typically has several ribosomes traveling along it, all simultaneously translating this transcript into protein. When the first ribosome stops, the others pile up behind it.

“You get this small product that is telling you the ribosome cannot go through the 8-oxo-G and then you get even smaller products that are telling you there are multiple ribosomes stuck behind the first ribosome. So the backed up ribosomes make a ladder of peptides,” Zaher said.

“This is a problem,” he said. “Among other things, the ribosome is an expensive machine that the cell has invested a lot of energy in making, and now it’s stuck on an mRNA. You need those ribosomes back.”

Fortunately ribosomes have three quality-control systems that keep watch for errors in the mRNA and rescue the ribosome if spot serious mistakes. One of these systems is “no-go decay.” When ribosomes are stuck and can’t go forward, they recruit factors that come in to pry open the ribosome, chew up the mRNA and add a tag to the defective peptide  that marks it for degradation.”

But no-go decay was originally discovered by throwing artificial roadblocks in the ribosome’s way: mRNAs with large hairpin turns in them that the ribosome could not unwind or plow through.

“Four billion years of evolution has made sure your genome does not have sequences that make hairpins, so these are clearly not the intended targets for no-go decay,” Zaher said.

To find out, the scientists turned to yeast cells. If the yeast’s ribosomes jammed on the oxidized mRNA but were rescued by no-go decay, very little damaged mRNA would accumulate in the cell. This proved to be the case.

Simms then deleted the gene for a factor that releases the ribosome from the mRNA when it jams. In these knockout yeast the level of oxidized mRNA went up. Then she deleted the gene for a factor that is recruited to degrade the mRNA after the ribosome is released, and again the level of oxidized mRNA rose. Without no-go decay, the cells were clearly in trouble.

“The system that translates mRNA into protein is highly conserved, so what’s true for yeast is probably true for people as well,” said Zaher. Washington University in St. Louis.

EKF Diagnostics subsidiary, Selah Genomics, has announced a major, four-way collaboration with Greenville Health System (GHS, South Carolina), DecisionQ Corporation (Virginia), and BD (Becton Dickinson and Company, New Jersey). Expected to last 18 months, the collaboration aims to unite classic clinical annotations with proprietary next generation sequencing (NGS) technology and artificial intelligence-based decision support algorithms in order to improve clinical decision support in the treatment of colon cancer patients. More than eight out of ten US patients are treated in the community, and as many as 60 percent of all patients with solid tumours do not respond to first-line treatment. This results in further treatment cycles, higher cost, elevated toxicity and, perhaps most importantly, lost time. A tool that significantly improves the prognostication for patients by bringing centre of excellence expertise to any clinical setting is therefore highly desirable. Using its PrecisionPath NGS technology, Selah Genomics will first determine the genetic profiles of tumour samples provided by the Institute for Translational Oncology Research, which is part of GHS’s Cancer Institute.  The samples, from colon cancer patients with known outcomes, will be provided with full clinical annotation. DecisionQ will employ its advanced machine-learning platform to integrate genetic profile data with clinical annotations to produce a model that will improve clinical decisions related to the treatment of colon cancer patients. The research project is being funded in part by BD in return for the first opportunity to license the technology should the collaboration be a success. After the initial collaboration, a clinical trial is planned to validate the research and affirm the effectiveness of the new system as a clinical decision support tool for the community-based setting.

www.ekfdiagnostics.com www.selahgenomics.com

Scientists have identified a genetic mutation in about 20 percent of colorectal and endometrial cancers that had been overlooked in recent large, comprehensive gene searches. With this discovery, the altered gene, called RNF43, now ranks as one of the most common mutations in the two cancer types.

The investigators from Dana-Farber Cancer Institute and the Broad Institute of MIT and Harvard said the mutated gene helps control an important cell-signalling pathway, Wnt, that has been implicated in many forms of cancer. They suggest that having a mutation in RNF43 may serve as a biomarker that identifies patients with colorectal and endometrial cancer who could benefit from precision cancer drugs that target the Wnt pathway, although no such drugs have yet been approved.

“Tumours that have this mutation may be telling us that they are dependent on the Wnt signalling pathway, and they will be uniquely sensitive to drugs that inhibit this pathway,” said Charles Fuchs, MD, MPH, an author of the paper and director of the Center for Gastrointestinal Cancer at Dana-Farber. He is also affiliated with Brigham and Women’s Hospital and the Harvard School of Public Health.

In pre-clinical cancer models, tumours with RNF43 mutations have been found to be sensitive to new Wnt pathway inhibitors that are now in clinical trials in humans, according to Marios Giannakis, MD, PhD, who is an attending physician at Dana-Farber and is also a conducting research at the Broad Institute.

The researchers were surprised to find RNF43 mutations in such a significant proportion of colorectal and endometrial cancers because they had not been detected in recent comprehensive searches of tumor DNA conducted by scientists of The Cancer Genome Atlas (TGCA) project.

Authors of the new study believe computer algorithms used by TCGA to parse data from DNA sequencing of tumors may have interpreted the “signal” of the RNF45 mutation as an artifact, and discarded it, much as a legitimate email will sometimes be trapped in a junk filter.

“These mutations occur in repetitive regions of the genome where you often have errors in DNA sequencing, so past algorithms may have been more likely to assume that the RNF43 mutation was an artifact of the sequencing process,” explained Eran Hodis, an MD/PhD student at Harvard Medical School and MIT and also affiliated with the Broad and Dana-Farber. Giannakis and Hodis are co-first authors on the new report.

Other frequently mutated genes in colorectal cancer include APC (75 percent), P53 (50 percent), and KRAS (40 percent).

The new evidence for RNF43 mutations first came from analysis of tumour samples of colorectal cancer that were obtained from two large cohort studies – the Nurses’ Health Study, which has been following 121,000 healthy women since 1976, and the Health Professionals Follow-up Study, which includes 52,000 men enrolled in 1986. About 10 years ago, Fuchs, along with Dana-Farber pathologist Shuji Ogino, MD, PhD, MS, began collecting and studying gastrointestinal tumour samples that had been taken from men and women in the studies who developed cancer. Because these specimens are accompanied by a wealth of data about the patients’ lifestyle, medical history, and other factors, Fuchs calls this collection of tumour samples “a gold mine.”

For the new study, 185 colorectal cancer specimens from this collection were analyzed by whole-exome DNA sequencing at the Broad Institute under the leadership of Levi Garraway, MD, PhD, who is affiliated with Dana-Farber, the Broad, and Brigham and Women’s Hospital, and is corresponding author of the report. The RNF43 mutation was identified in 18.9 percent of the colorectal tumors.

This surprising result prompted the investigators to re-analyse 222 colorectal cancer samples from TCGA project and found the RNF43 mutation in 17.6 percent. The researchers, noting that endometrial cancer is dependent on abnormal Wnt signalling, then re-analysed 248 DNA samples from endometrial cancer that had been previously published by TCGA scientists. They found a strikingly similar proportion – 18.1 percent – of RNF43 mutations in those cancers.

The study authors noted that the discovery of such a significant cancer mutation that hadn’t been picked up in the previous gene hunts shows that carrying out these comprehensive genomic searches continues to have value. Dana-Farber Cancer Institute