Next-generation sequencing to provide precision medicine for rare metabolic disorders

Advances in next-generation-sequencing technology that allow researchers to look at billions of pieces of genetic information are changing the way a disease is diagnosed. Correct identification of changes in the human genetic code responsible for rare metabolic disorders provides scientists and physicians with fact-based guidelines for the treatment.

An international team of scientists from Switzerland Spain, has studied the genetic basis of aromatase deficiency, a rare metabolic disorder that prevents the production of estrogens in humans, according to new research in JCEM (Journal of Clinical Endocrinology and Metabolism). The latest studies on aromatase deficiency in humans come from the group of Amit V. Pandey and Christa E. Flück at the Department for BioMedical Research, University of Bern, and Pediatric Endocrinology, University Children’s Hospital Bern, done in collaboration with Laura Audí at Hospital Vall d’Hebron, Autonomous University of Barcelona, Spain. Scientists found the answers by analysis of the DNA map of patients with aromatase deficiency and comparing those with DNA of the broader human population from different ethnic groups.

“Supplementation with steroids can target multiple different pathways, so we wanted to know which part of genetic code was changed in patients in order to target therapies to the appropriate place,” writes Dr. Amit Pandey, University of Bern and Inselspital, University Hospital Bern, with coauthors. “We knew we had cases of genetic disorders leading to aromatase deficiency, but we needed to find the exact cause of disease, and modern DNA sequencing helped us find it.”

Bern expertise helps solving a special case

Collaborating with genetics specialists at the Vall d’Hebron Hospital in Barcelona (Laura Audi, Núria Camats and Mónica Fernández-Cancio) and Hospital Universitario La Paz, Madrid (Sara Benito-Sanz), Pandey focused on a patient with aromatase deficiency identified by Dr. Juan-Pedro López-Siguero at Hospital Carlos Haya, Universidad de Málaga, Spain. What caused the attention of geneticists in Barcelona and Madrid was the observation that patient has symptoms of aromatase deficiency, but, when the gene for aromatase (CYP19A1) was sequenced, no defects were found.

Through the use of next-generation sequencing technology that simultaneously looks at billions of pieces of genetic code, Spanish scientists identified an error in the gene for cytochrome P450 oxidoreductase (POR). Laboratory of Pediatric Endocrinology in Bern, Switzerland, is a world leader in metabolic disorders caused by mutations in POR

The Swiss and Spanish teams joined forces and set out to find how the aromatase deficiency was being caused by a defect in the POR gene. Amit Pandey has been studying the POR gene for many years and knew that aromatase activity to produce estrogens requires energy supply from POR and had the systems in place to measure the impact of changes in POR on estrogen production. Dr. Shaheena Parween, in the laboratory of Amit Pandey, was able to genetically modify the POR gene to duplicate the defect found in the patient and used E.coli bacteria in the laboratory to produce a copy of POR enzyme matching the genetic sequence of the patient.

Scientists in Bern could show that POR made with the genetic code of the patient had lost most of its ability to provide energy to the aromatase enzyme. Therefore, even with a correct aromatase enzyme, the patient could not produce sufficient estrogens. Learning the exact metabolic step where the aromatase deficiency was originating from, allows the physicians to guide the therapy very precisely and prevent side effects associated with steroid supplementation. The study demonstrated the powerful diagnostic ability of next-generation sequencing technologies.

Supporting research in Africa and India

The Bern team extended their studies by looking at more patients with aromatase deficiency from Africa and India and identified the exact causes of genetic defects responsible for the loss of estrogen production. These findings have been reported in JES (Journal of the Endocrine Society), and recently been discussed in PNAS (Proceedings of the National Academy of Sciences of the United States of America). Collaboration with the scientists in Bern, Switzerland, allowed the use of advanced diagnostic and assay technologies not available in local hospitals, which highlights the role of international collaborations in the diagnosis and therapy of rare metabolic disorders.

Variations of POR common in specific populations

The research team of Pandey and Flück is also studying genetic mutations in POR that cause other diseases, such as congenital adrenal hyperplasia, a common inherited disorder that affects a large number of people each year. Pandey highlights the value of translational medicine in his research. From his previous work, Pandey knew that the POR gene in humans has lots of variations, and some populations carry specific mutations to a greater extent than others.

The Laboratory of Gianfranco Gilardi and Giovanna Di Nardo at the University of Torino, Italy, had studied a common variation of aromatase that was predominant in the south-east Asian population. The scientists in Bern and Turin teamed up to check what if the same individual has an alternative form of both the POR and aromatase genes. By preparing the variations of POR and aromatase proteins based on genetic changes observed in the South-east Asian population, scientists could show that a compounding effect can be found when there is a change in POR and aromatase genes at the same time. These findings have been reported in JSBMB (Journal of Steroid Biochemistry and Molecular Biology).

The power of genomics for Precision Medicine

Some of the variations in the POR gene are quite common in specific populations, so Pandey advises looking at changes in the POR gene whenever a defect in other genes that are dependent on POR is identified. With the power of whole-genome sequencing at our disposal, it is time to move on from the theories of monogenic disorders as individual patients in the world are not the same in their genetic composition, says Pandey. “All humans have very similar genes, but still may have up to a million or more differences in their genetic code, even between a daughter and her mother, so if we find out exactly what causes a disease, then precise therapies can be used that are tailored for individual patients,” Pandey said. “We are now seeing the power of genomics tailored for use in Precision Medicine, allowing the design of specific treatments for a patient according to their genetic makeup.”

Precision medicine will be a vital component of the NextGen Precision Health Initiative by helping to accelerate medical breakthroughs for both patients in Switzerland and beyond. A center of rare diseases has recently been established at Inselspital, University Hospital Bern, to focus on diagnosis and research on rare metabolic disorders.

These studies were funded by the Swiss National Science Foundation, Novartis Foundation for Medical Biological Research, Switzerland, Burgergemeinde, Bern, Switzerland , Fondo de Investigación Sanitaria, ISCIII, Spain, and CRT Foundation, Italy.

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Light drinking may protect brain function

Light to moderate drinking may preserve brain function in older age, according to a new study from the University of Georgia.

The study examined the link between alcohol consumption and changes in cognitive function over time among middle-aged and older adults in the U.S.

“We know there are some older people who believe that drinking a little wine everyday could maintain a good cognitive condition,” said lead author Ruiyuan Zhang, a doctoral student at UGA’s College of Public Health.

“We wanted to know if drinking a small amount of alcohol actually correlates with a good cognitive function, or is it just a kind of survivor bias.”

Regular, moderate alcohol consumption has been shown to promote heart health and some research points to a similar protective benefit for brain health. However, many of these studies were not designed to isolate the effects of alcohol on cognition or did not measure effects over time.

Zhang and his team developed a way to track cognition performance over 10 years using participant data from the nationally representative Health and Retirement Study.

During the study, a total of 19,887 participants completed surveys every two years about their health and lifestyle, including questions on drinking habits. Light to moderate drinking is defined as fewer than eight drinks per week for women and 15 drinks or fewer per week among men.

These participants also had their cognitive function measured in a series of tests looking at their overall mental status, word recall and vocabulary. Their test results were combined to form a total cognitive score.

Zhang and his colleagues looked at how participants performed on these cognitive tests over the course of the study and categorized their performance as high or low trajectories, meaning their cognitive function remained high over time or began to decline.

Compared to nondrinkers, they found that those who had a drink or two a day tended to perform better on cognitive tests over time.

Even when other important factors known to impact cognition such as age, smoking or education level were controlled for, they saw a pattern of light drinking associated with high cognitive trajectories.

The optimal amount of drinks per week was between 10 and 14 drinks. But that doesn’t mean those who drink less should start indulging more, says Zhang.

“It is hard to say this effect is causal,” he said. “So, if some people don’t drink alcoholic beverages, this study does not encourage them to drink to prevent cognitive function decline.”

Also of note, the association was stronger among white participants versus African American participants, which is significant, said Zhang, and prompts further exploration into the mechanisms of alcohol’s effect on cognition.

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Materials provided by University of Georgia. Original written by Lauren Baggett. Note: Content may be edited for style and length.

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New eye drops may prevent a common cause of blindness

Researchers at Columbia University Irving Medical Center have developed eye drops that could prevent vision loss after retinal vein occlusion, a major cause of blindness for millions of adults worldwide.

A study, in mice, suggests that the experimental therapy — which targets a common cause of neurodegeneration and vascular leakage in the eye — could have broader therapeutic effects than existing drugs.

The study was published in Nature Communications.

What is Retinal Vein Occlusion?

Retinal vein occlusion occurs when a major vein that drains blood from the retina is blocked, usually due to a blood clot. As a result, blood and other fluids leak into the retina, damaging specialized light-sensing neurons called photoreceptors.

Standard treatment for the condition currently relies on drugs that reduce fluid leakage from blood vessels and abnormal blood vessel growth. But there are significant drawbacks. These therapies require repeated injections directly into the eye, and for the patients who brave this daunting prospect, the treatment ultimately fails to prevent vision loss in the majority of cases.

The new treatment targets an enzyme called caspase-9, says Carol M. Troy, MD, PhD, professor of pathology & cell biology and of neurology in the Taub Institute for Research on Alzheimer’s Disease and the Aging Brain at Columbia University Vagelos College of Physicians and Surgeons, who led the studies. Under normal conditions, caspase-9 is believed to be primarily involved in programmed cell death, a tightly regulated mechanism for naturally eliminating damaged or excess cells.

However, in studies of mice, the Troy lab discovered that when blood vessels are injured by retinal vein occlusion, the caspase-9 becomes uncontrollably activated, triggering processes that can damage the retina.

Eye Drops Prevent Retinal Injury

The Troy lab found that a highly selective caspase-9 inhibitor, delivered in the form of eye drops, improved a variety of clinical measures of retinal function in a mouse model of the condition. Most importantly, the treatment reduced swelling, improved blood flow, and decreased neuronal damage in the retina.

“We believe these eye drops may offer several advantages over existing therapies,” says Troy. “Patients could administer the drug themselves and wouldn’t have to get a series of injections. Also, our eye drops target a different pathway of retinal injury and thus may help patients who do not respond to the current therapy.”

Next Steps

The researchers are preparing to test the eye drops in people with retinal vein occlusion during a phase I clinical trial.

Moving forward, the Troy lab will also study whether caspase-9 inhibitors can be used to treat other vascular injuries caused by overactivation of the enzyme, including diabetic macular edema (another common cause of blindness) and stroke.

“Vascular dysfunction is at the heart of many chronic neurological and retinal disorders, because high energy demands in the brain and eye render these tissues exceptionally vulnerable to disruption in blood supply,” says the study’s first author, Maria Avrutsky, PhD, postdoctoral research scientist in pathology & cell biology at Columbia University Vagelos College of Physicians and Surgeons.

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Promising treatment to slow kidney disease doesn’t prove out in clinical trial

Historically, half or more of people with type 1 diabetes develop kidney disease, which frequently progresses to kidney failure requiring hemodialysis or a kidney transplant for survival. The high rate of this diabetic complication has dropped slightly in recent years, with the advent of better ways to control blood glucose (sugar) levels and improved blood pressure drugs, “but diabetic kidney disease is still a huge problem,” says Alessandro Doria, MD, PhD, MPH, Senior Investigator in Joslin Diabetes Center’s Section on Genetics and Epidemiology.

Progression of kidney disease in type 1 diabetes is correlated with increased amounts of a compound in the blood called uric acid. Hoping that a drug that reduces these uric acid levels would slow the disease, Doria and his colleagues launched a multi-institution randomized clinical trial that enrolled 530 participants with type 1 diabetes and early-to-moderate kidney disease.

Results of the Preventing Early Renal Loss in Diabetes (PERL) study were just published in the New England Journal of Medicine (NEJM), the leading clinical research journal. Unfortunately, this study did not show the desired clinical benefits. “This is not the result that we wanted,” says Doria, “but it does give a very clear answer to an important scientific question.”

A second trial by Australian researchers on patients with a variety of chronic kidney diseases, some with diabetes, published alongside the PERL study in the NEJM, found similar results.

The PERL trial grew out of several studies that followed a cohort of people with type 1 diabetes, including one in which Doria partnered with Andrzej Krolewski, MD, PhD, head of the Section on Genetics and Epidemiology. In a 2011 paper, the Joslin scientists demonstrated that in this cohort, people with higher levels of uric acid in their blood were more likely to display a high rate of kidney function loss. Two other research groups in Denver, Colorado and Copenhagen, Denmark obtained similar results.

“This was an actionable discovery, because allopurinol, a drug that’s been on the market since the 1960s, can easily reduce uric acid,” says Doria, who is also a professor of medicine at Harvard Medical School.

Allopurinol is prescribed for gout, an inflammatory condition caused by excess uric acid, he explains. It’s an inexpensive generic drug with known side effects that can largely be avoided. Additionally, allopurinol produced apparent benefits in much smaller clinical trials among people with chronic kidney disease, a minority of whom had diabetes.

Doria teamed up with S. Michael Mauer, MD, of the University of Minnesota Medical School to design and carry out a clinical trial with support from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and JDRF. The PERL consortium eventually grew to 16 sites.

Participants in the three-year, placebo-controlled and double-blinded trial received the current standard of care, including a renin-angiotensin system inhibitor — an existing type of drug shown in the 1990s to slow kidney damage, albeit incompletely.

The key measurement of kidney function for PERL was glomerular filtration rate (GFR), a measure of how much blood is filtered every minute by the kidneys. GFR drops as kidney disease progresses.

Over the three years of the study, levels of uric acid dropped about 35% on average among people given allopurinol compared to those who weren’t. “But despite this very nice reduction in uric acid, we could not see any effect on GFR,” Doria says.

He and his colleagues will continue to follow participants through their medical records and through national databases that track people who eventually progress to dialysis or kidney transplants.

Researchers at Joslin and other institutions continue to examine other potential routes to guard against kidney disease among people with type 1 diabetes.

Despite its disappointing conclusion, “PERL was a textbook example of using epidemiology to find treatment targets, and then designing a study to translate those findings and try to find a new intervention,” Doria says. “In this case, it didn’t work. But this is exactly why we do epidemiological studies, and how our scientific understanding advances.”

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Repeated head impacts associated with later-life depression symptoms, worse cognitive function

Scientists have long believed that a single traumatic brain injury (TBI) earlier in life may contribute to problems with memory, thinking and depression later in life. In most previous studies, however, research failed to examine the possible role of having a history of exposure to repetitive head impacts, including those leading to “subconcussive” injuries, in these later-life problems. In the largest study of its kind, an association has been found in living patients exposed to repetitive head impacts and difficulties with cognitive functioning and depression years or decades later.

Scientists from the Boston University (BU) Alzheimer’s Disease and Chronic Traumatic Encephalopathy (CTE) Centers, the University of California, San Francisco (UCSF), and San Francisco VA Healthcare System teamed up to analyze the records of 13,323 individuals age 40 and older (average age 62) who participate in the internet-based Brain Health Registry. Of those, 725 or 5 percent of participants reported exposure to previous repetitive head impacts through contact sports, abuse or military service. In addition to repetitive head impact history, the scientists also examined the effects of having a TBI with and without loss of consciousness.

Along with self-report questionnaires of repetitive head impact and TBI history, participants completed measures of depressive symptoms and computerized cognitive tests. The findings, published in the journal Neurology, reveal that participants with a history of both repetitive head impacts and TBI reported greater depression symptoms than those who did not have such history. In addition, when repetitive head impacts and TBI were examined separately, a history of repetitive head impacts had the strongest effect on later-life symptoms of depression. The findings were independent of age, sex, racial identity and education level.

“The findings underscore that repetitive hits to the head, such as those from contact sport participation or physical abuse, might be associated with later-life symptoms of depression. It should be made clear that this association is likely to be dependent on the dose or duration of repetitive head impacts and this information was not available for this study,” said Michael Alosco, PhD, associate professor of neurology at BU School of Medicine (BUSM) and co-director of the BU Alzheimer’s Disease Center Clinical Core.

There was a dose-response-like pattern between head trauma and depression symptoms. Specifically, participants without any history of either TBI or repetitive head impacts had the fewest symptoms. While depression symptoms increased when a history of TBI alone was present, depression symptoms were highest for the groups who had a history of both repetitive head impacts and TBI. Indeed, the group that had a history of repetitive head impacts and TBI with loss of consciousness reported the most depressive symptoms.

A similar cumulative effect was seen among those exposed to repetitive head impacts and TBI on tests of memory, learning, processing speed, and reaction time. Participants with a history of repetitive head impacts or TBI had worse performance on some of the tests compared to those without any head trauma history, and those with both a history of repetitive head impacts and TBI with loss of consciousness had worse performance on almost all of these computerized cognitive tests.

“These findings add to the growing knowledge about the long-term neurological consequences of brain trauma,” said Robert Stern, PhD, professor of neurology, neurosurgery and anatomy & neurobiology at BUSM and director of clinical research at the BU CTE Center. “It should be noted that not all people with a history of repetitive hits to the head will develop later-life problems with cognitive functioning and depression. However, results from this study provide further evidence that exposure to repetitive head impacts, such as through the routine play of tackle football, plays an important role in the development in these later-life cognitive and emotional problems,” added Stern, one of the senior authors of the study.

A major limitation of the study is that the researchers did not have access to measurements or estimates of the degree of repetitive impact exposure nor TBI frequency. In October, BU researchers reported a dose-response relationship between the number of years of exposure to tackle football (regardless of the number of concussions) and the presence and severity of the degenerative brain disease chronic traumatic encephalopathy (CTE). In a sample of 266 deceased football players, each year of exposure to tackle football was associated with 30 percent increased odds of having CTE and 17 percent increased odds of having severe CTE. It is unknown if any subjects in this study have CTE or any other neurodegenerative disease.

The research team plans to extend their work through continued collaboration between BU and UCSF investigators utilizing data from the Brain Health Registry. “We are excited to partner with BU on this important study that used the Brain Health Registry to increase our understanding on the long-term effects of repetitive head impacts and TBI,” said Michael Weiner, MD, PI of the Brain Health Registry and professor-in-residence in radiology and biomedical imaging, medicine, psychiatry and neurology at UCSF. “The Brain Health Registry is a novel and exciting resource for both the scientific community and the general public. It allows for large-scale recruitment, screening and study of dementia, and more than 60,000 individuals across the world are enrolled. It offers a way for the general public to track their thinking, memory, mood, and behavior over time, and also serves as a readiness registry for future research and clinical trials of prevention and treatment.”

The research was funded by the National Institutes of Health, grant numbers U01NS093334; K23AG046377; K23NS102399; P30AG013846.

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