Most 'silent' genetic mutations are harmful, not neutral -- a finding with broad implications

In the early 1960s, University of Michigan alumnus Marshall Nirenberg and a few other scientists deciphered the genetic code of life, determining the rules by which information in DNA molecules is translated into proteins, the working parts of living cells.

They identified three-letter units in DNA sequences, known as codons, that specify each of the 20 amino acids that make up proteins, work for which Nirenberg later shared a Nobel Prize with two others.

Occasionally, single-letter misspellings in the genetic code, known as point mutations, occur. Point mutations that alter the resulting protein sequences are called nonsynonymous mutations, while those that do not alter protein sequences are called silent or synonymous mutations.

Between one-quarter and one-third of point mutations in protein-coding DNA sequences are synonymous. Ever since the genetic code was cracked, those mutations have generally been assumed to be neutral, or nearly so.

But in a study scheduled for online publication June 8 in the journal Nature that involved the genetic manipulation of yeast cells in the laboratory, University of Michigan biologists show that most synonymous mutations are strongly harmful.

The strong nonneutrality of most synonymous mutations -- if found to be true for other genes and in other organisms -- would have major implications for the study of human disease mechanisms, population and conservation biology, and evolutionary biology, according to the study authors.

"Since the genetic code was solved in the 1960s, synonymous mutations have been generally thought to be benign. We now show that this belief is false," said study senior author Jianzhi "George" Zhang, the Marshall W. Nirenberg Collegiate Professor in the U-M Department of Ecology and Evolutionary Biology.

"Because many biological conclusions rely on the presumption that synonymous mutations are neutral, its invalidation has broad implications. For example, synonymous mutations are generally ignored in the study of disease-causing mutations, but they might be an underappreciated and common mechanism."

In the past decade, anecdotal evidence has suggested that some synonymous mutations are nonneutral. Zhang and his colleagues wanted to know if such cases are the exception or the rule.

They chose to address this question in budding yeast (Saccharomyces cerevisiae) because the organism's short generation time (about 80 minutes) and small size allowed them to measure the effects of a large number of synonymous mutations relatively quickly, precisely and conveniently.

They used CRISPR/Cas9 genome editing to construct more than 8,000 mutant yeast strains, each carrying a synonymous, nonsynonymous or nonsense mutation in one of 21 genes the researchers targeted.

Then they quantified the "fitness" of each mutant strain by measuring how quickly it reproduced relative to the nonmutant strain. Darwinian fitness, simply put, refers to the number of offspring an individual has. In this case, measuring the reproductive rates of the yeast strains showed whether the mutations were beneficial, harmful or neutral.

To their surprise, the researchers found that 75.9% of synonymous mutations were significantly deleterious, while 1.3% were significantly beneficial.

"The previous anecdotes of nonneutral synonymous mutations turned out to be the tip of the iceberg," said study lead author Xukang Shen, a graduate student research assistant in Zhang's lab.

"We also studied the mechanisms through which synonymous mutations affect fitness and found that at least one reason is that both synonymous and nonsynonymous mutations alter the gene-expression level, and the extent of this expression effect predicts the fitness effect."

Zhang said the researchers knew beforehand, based on the anecdotal reports, that some synonymous mutations would likely turn out to be nonneutral.

"But we were shocked by the large number of such mutations," he said. "Our results imply that synonymous mutations are nearly as important as nonsynonymous mutations in causing disease and call for strengthened effort in predicting and identifying pathogenic synonymous mutations."

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Genetic mutation may identify women with difficulty producing breast milk

Leading health care organizations recommend exclusive breastfeeding for six months after birth, yet some mothers report stopping due to a perceived lack of milk supply. Penn State College of Medicine researchers found in a recent study that women who stopped breastfeeding because they believed they had inadequate milk supply -- a condition called perceived inadequate milk supply (PIMS) -- are more likely to have a specific mutation in a gene found in mammary tissue. These women were also more likely to have babies who gained less weight. The researchers said that screening for this mutation, when combined with maternal characteristics like age and body mass index, could be useful in identifying mothers at risk for stopping breastfeeding prematurely due to a perceived lack of milk supply.

"The World Health Organization, the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists recommend exclusive breastfeeding for at least six months because it provides developing infants with optimum nutrition and is associated with improved health outcomes," said Dr. Steven Hicks, lead researcher and pediatrician at Penn State Health Children's Hospital. "While 83% of women initiate breastfeeding, only a reported 57% continue to six months. Socioeconomic and environmental factors may contribute to early cessation, but milk supply is also an often-cited reason. Identifying women who are more likely to have low milk supply could help get them resources to continue breastfeeding such as lactation consultation services."

Previous research has linked maternal genetics with nutrients in breast milk, but few studies have explored how genetics may relate to supply. The researchers studied 18 genes highly expressed in mammary, or milk-producing, tissue in women. They looked for mutations in those genes to see whether mutations were associated with mothers' perceived milk supply.

The study team followed 88 women between 19 and 42 years old for the first year of their baby's life. The mothers completed surveys about their infant's feeding habits at one, four, six and twelve months of age that asked questions about perceived milk supply, whether women supplemented their child's diet with formula and reasons why they did so. Decreased or low milk production, signs of allergies from breastfeeding and other personal reasons such as work, day care or time constraints were included as possible reasons for why women began to supplement with formula. Mothers also provided a DNA sample by having saliva collected.

Using responses from the surveys, the researchers classified the mothers as having either PIMS or perceived adequate milk supply (PAMS). They found that the 45 mothers with PIMS were more likely to breastfeed for shorter periods, report lower milk supply and have infants who were not gaining adequate weight.

The researchers analyzed the mothers' DNA samples and looked for mutations among 18 genes that are involved in the secretion of breast milk. Although modifications in 10 of the genes studied were found among some women, the team found that only one, a variant in the milk fat globule EGF and factor V/VIII domain containing gene (MFGE8), occurred more frequently in women with PIMS. Those without the mutation were more likely to have adequate milk supply and report a longer duration of breastfeeding.

Using statistical modeling, the researchers found that maternal characteristics like age, previous breastfeeding duration and body mass index alone could not differentiate between mothers with PIMS and PAMS. However, when adding in MFGE8 mutation status into the model, it strongly predicted which women reported adequate or inadequate milk supplies. The researchers published their results in the journal Breastfeeding Medicine.

"Identifying risk of PIMS at the outset of breastfeeding could provide opportunities for early, targeted interventions such as guidance from a trained lactation support professional," Hicks said. He noted that current assessment of PIMS is guided by subjective reports and that counseling may help identify foods and medications that help or hinder milk production.

Hicks said that the study's findings will need to be validated in a larger study that includes more mothers. He also said that more research is needed to uncover the biological processes that determine how this particular gene affects milk supply in moms in order to better understand its association with PIMS status.

"Moms with this mutation still produce milk, even if it may be less than women without the mutation, but challenges like poor diet, hydration or sleep could be enough to hinder the supply that they do have," Hicks said. "Screening for this variant and combining that with maternal reports and characteristics could help identify moms and babies that may need additional support."

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Scientists discover early signs of frontotemporal dementia in personalized cerebral organoids

Frontotemporal dementias are a group of fatal and debilitating brain disorders for which there are no cures. In an article published July 26 in Cell, Mount Sinai researchers describe how they were able to recreate much of the damage seen in a widely studied form of the disease by growing special types of cerebral organoids in petri dishes. This form of the disease is caused by a genetic mutation in tau, a protein that is a hallmark of Alzheimer's disease and other dementias. By studying these organoids, the scientists discovered how the mutated tau protein may trigger the death of a specific class of neurons known to be vulnerable in frontotemporal dementia. They also showed that they could prevent the death of these neurons by treating the organoids with an experimental drug, originally designed to combat Crohn's disease.

"Frontotemporal dementia is a devastating disease for patients and their loved ones. Understanding the causes of dementia can be difficult, as most of the damage to the brain occurs well before any symptoms appear. It's like trying to unravel the events that created a crime scene. In this study, we were able to model many aspects of the pathology seen in the brains of patients who carry the V337M mutation in tau," said Alison M. Goate, DPhil, Director, Ronald M. Loeb Center for Alzheimer's Disease at Mount Sinai, and a senior author of the study. "Our results identified several very early transcriptomic and proteomic changes that lead to the formation of tau pathology and neuronal death. Our goal is to help researchers develop novel treatments against frontotemporal dementias and prevent the suffering experienced by patients and their families."

Frontotemporal dementia is a rare form of dementia that usually begins between ages 40 and 60. It affects the front and side (temporal) areas of the brain, leading to behavior changes and difficulty with speaking and thinking.

The study was led by Kathryn Bowles, PhD, an instructor in Dr. Goate's lab at Mount Sinai. Working with scientists at the Neural Stem Cell Institute (NSCI) in Rensselaer, New York, Washington University in St. Louis, Missouri, Massachusetts General Hospital in Boston, and the University of Southern California, Los Angeles, the researchers created thousands of cerebral organoids from induced pluripotent stem cells (iPSCs).

Induced pluripotent stem cells are created by genetically and chemically reprogramming a person's skin or blood cells into newborn stem cells, which have the potential to become any cell in the body. From these stem cells, the NSCI created thousands of tiny, 3D cerebral organoids, which mimic the early growth and development of the cerebral cortex for intensive study by collaborating scientific groups.

"Induced pluripotent stem cells are powerful tools. They allow researchers to study each patient's personalized disease in a petri dish," said Sally Temple, PhD, Scientific Director of the NSCI and a senior author of the study. "In this study we were able to take this idea to the next level. By combining iPSC-organoid technology with high-throughput, single cell gene activity analysis, we were able to get a better look at what might be going on in a patient's brain at early stages of disease development, even before symptoms emerge."

In this study, the researchers examined the growth and development of organoids derived from the stem cells of three patients, all of whom carried the V337M mutation in tau. They then compared their results with those observed in "isogenic," control organoids. The controls were derived from patient stem cells in which the disease-causing mutation was genetically corrected.

After six months of growth, signs of neurodegeneration were seen in the organoids. Most notably, the patient-derived organoids had fewer excitatory neurons than those derived from the control cells, demonstrating that the tau mutation was sufficient to cause higher levels of cell death of this specific class of neurons. Excitatory neurons usually fire in response to the neurochemical glutamate and are known to die at abnormally high levels in frontotemporal dementia. The patient-derived organoids also had higher levels of harmful versions of tau protein and elevated levels of inflammation.

"Excitatory neuron cell death, tau protein deposits, and inflammation are classic hallmarks of the kind of damage seen in many forms of frontotemporal dementia," said Dr. Bowles. "What we wanted to know next was: what are the cellular and molecular processes that occur before the appearance of these disease hallmarks?"

The researchers found clues by examining two- and four-month-old organoids.

For instance, two-month-old mutant organoids appeared to be undergoing elevated levels of cellular stress, whereas four-month-old ones developed problems with autophagy, or the recycling of proteins. The results also suggested that during these early months the excitatory neurons matured faster in the mutant organoids than in the controls.

Other experiments suggested that many of these changes may have been the by-product of a complex interaction between mutant tau, excitatory neuronal genes, and ELAVL4, a protein that controls gene activity by binding to ribonucleic acid (RNA) molecules.

"Our results suggest that the V337M mutant tau sets off a vicious cycle in the brain that puts excitatory neurons under great stress. It hastens the production of new proteins needed for maturation but prevents disposal of the proteins that are being replaced," Dr. Bowles said.

Further experiments supported this idea. For example, excitatory neurons in mutant organoids were less likely to survive in the presence of toxic levels of glutamate than those in control organoids. The researchers then found that this could be prevented by apilimod, an experimental drug designed to alter a cell's protein recycling system. In other words, the researchers saw no difference in levels of glutamate-induced cell death between mutant and control organoids when they treated samples with apilimod.

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Major advance enables study of genetic mutations in any tissue

For the first time, scientists are able to study changes in the DNA of any human tissue, following the resolution of long-standing technical challenges by scientists at the Wellcome Sanger Institute. The new method, called nanorate sequencing (NanoSeq), makes it possible to study how genetic changes occur in human tissues with unprecedented accuracy.

The study, published today (28 April) in Nature, represents a major advance for research into cancer and ageing. Using NanoSeq to study samples of blood, colon, brain and muscle, the research also challenges the idea that cell division is the main mechanism driving genetic changes. The new method is also expected to allow researchers to study the effect of carcinogens on healthy cells, and to do so more easily and on a much larger scale than has been possible up until now.

The tissues in our body are composed of dividing and non-dividing cells. Stem cells renew themselves throughout our lifetimes and are responsible for supplying non-dividing cells to keep the body running. The vast majority of cells in our bodies are non-dividing or divide only rarely. They include granulocytes in our blood, which are produced in the billions every day and live for a very short time, or neurons in our brain, which live for much longer.

Genetic changes, known as somatic mutations, occur in our cells as we age. This is a natural process, with cells acquiring around 15-40 mutations per year. Most of these mutations will be harmless, but some of them can start a cell on the path to cancer.

Since the advent of genome sequencing in the late twentieth century, cancer researchers have been able to better understand the formation of cancers and how to treat them by studying somatic mutations in tumour DNA. In recent years, new technologies have also enabled scientists to study mutations in stem cells taken from healthy tissue.

But until now, genome sequencing has not been accurate enough to study new mutations in non-dividing cells, meaning that somatic mutation in the vast majority of our cells has been impossible to observe accurately.

In this new study, researchers at the Wellcome Sanger Institute sought to refine an advanced sequencing method called duplex sequencing1. The team searched for errors in duplex sequence data and realised that they were concentrated at the ends of DNA fragments, and had other features suggesting flaws in the process used to prepare DNA for sequencing.

They then implemented improvements to the DNA preparation process, such as using specific enzymes to cut DNA more cleanly, as well as improved bioinformatics methods. Over the course of four years, accuracy was improved until they achieved fewer than five errors per billion letters of DNA.

Dr Robert Osborne, an alumnus of the Wellcome Sanger Institute who led the development of the method, said: "Detecting somatic mutations that are only present in one or a few cells is incredibly technically challenging. You have to find a single letter change among tens of millions of DNA letters and previous sequencing methods were simply not accurate enough. Because NanoSeq makes only a few errors per billion DNA letters, we are now able to accurately study somatic mutations in any tissue."

The team took advantage of NanoSeq's improved sensitivity to compare the rates and patterns of mutation in both stem cells and non-dividing cells in several human tissue types.

Surprisingly, analysis of blood cells found a similar number of mutations in slowly dividing stem cells and more rapidly dividing progenitor cells2. This suggested that cell division is not the dominant process causing mutations in blood cells. Analysis of non-dividing neurons and rarely dividing cells from muscle also revealed that mutations accumulate throughout life in cells without cell division, and at a similar pace to cells in the blood.

Dr Federico Abascal, the first author of the paper from the Wellcome Sanger Institute, said: "It is often assumed that cell division is the main factor in the occurrence of somatic mutations, with a greater number of divisions creating a greater number of mutations. But our analysis found that blood cells that had divided many times more than others featured the same rates and patterns of mutation. This changes how we think about mutagenesis and suggests that other biological mechanisms besides cell division are key."

The ability to observe mutation in all cells opens up new avenues of research into cancer and ageing, such as studying the effects of known carcinogens like tobacco or sun exposure, as well as discovering new carcinogens. Such research could greatly improve our understanding of how lifestyles choices and exposures to carcinogens can lead to cancer.

A further benefit of the NanoSeq method is the relative ease with which samples can be collected. Rather than taking biopsies of tissue, cells can be collected non-invasively, such as by scraping the skin or swabbing the throat.

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Breakthrough blood test developed for brain tumors

 Genetic mutations that promote the growth of the most common type of adult brain tumors can be accurately detected and monitored in blood samples using an enhanced form of liquid biopsy developed by researchers at Massachusetts General Hospital (MGH).

Comparing blood samples from patients with gliomas with tumor biopsy tissues from the same patients, Leonora Balaj, PhD, Bob S. Carter, MD, and other MGH investigators in the Department of Neurosurgery found that a novel digital droplet polymerase chain reaction (ddPCR) blood test they pioneered could accurately detect and monitor over time two mutations of the gene TERT. The mutations, labeled C228T and C250T, are known to promote cancer growth and are present in more than 60 percent of all gliomas, and in 80 percent of all high-grade gliomas, the most aggressive and life-threatening type.

Their discovery, which has the potential to substantially improve the diagnosis and monitoring of gliomas, is reported in the journal Clinical Cancer Research.

Gliomas are tumors of glia, central and peripheral nervous system cells that support and protect neurons, the cells that transmit electrical impulses.

Liquid biopsy is a method for detecting cancer by looking for fragments of tumor DNA that circulate in blood. The technique has been shown to be sensitive at detecting the presence of some forms of cancer, but brain tumors have until now posed a formidable barrier.

"Liquid biopsy is particularly challenging in brain tumors because mutant DNA is shed into the bloodstream at much lower level than any other types of tumors," Balaj says.

"By 'supercharging' our ddPCR assay with novel technical improvements, we showed for the first time that the most prevalent mutation in malignant gliomas can be detected in blood, opening a new landscape for detection and monitoring of the tumors," she says.

The researchers first tested the performance of the ddPCR assay in tumor tissue and found that the results were in perfect agreement with the results from an independently performed clinical laboratory assessment of TERT mutations in the tumor specimens.

They then looked at samples of blood plasma matched to patient tumors and found that the ddPCR assay could detect TERT mutations both in samples from MGH as well as from similarly matched plasma and tumor samples from collaborators at other institutions.

The ddPCR assay has an overall sensitivity (ability to detect the presence of a glioma) of 62.5 percent, which is a tenfold improvement over any prior assay for TERT mutations in the blood for brain tumors, compared to the standard of tissue-based detection of TERT mutations.

The test is easy to use, quick, and low cost, and could be performed in most laboratories, Balaj says. Importantly, the test can also be used to follow the course of disease. "We envision the future integration of tests like this one into the clinical care of our patients with brain tumors," says Carter, chief of Neurosurgery and co-director of the MGH Brain Tumor Center. "For example, if a patient has a suspected mass on MRI scanning, we can take a blood sample before the surgery and assess the presence of the tumor signature in the blood, and then use this signature as a baseline to monitor as the patient later receives treatment, both to gauge response to the treatment and gain early insight into any potential recurrence."

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