Tests that can distinguish whether HIV-positive people are infected with a drug-resistant strain or a non-resistant strain allow patients to get the most effective treatment as quickly as possible. A team of Brown University researchers describes a new method that works faster and more sensitively in lab testing than the current standard technologies.

The main advance enabling that improved performance is that the system operates directly on the virus’ more readily available RNA rather than requiring extra, potentially error-prone steps to examine DNA derived from RNA. In a single tube, the system can first combine two engineered probes (ligation) if a mutation is present and then make many copies of those combined probes (amplification) for detection.

“LRA (ligation on RNA amplification) uniquely optimizes two enzymatic reactions — RNA-based ligation, and quantitative PCR (polymerase chain reaction) amplification — into a single system,” said Anubhav Tripathi, professor of engineering at Brown and corresponding author on the paper. “Each HIV contains about 10,000 nucleotides, or building blocks, in its genetic material, and a drop of blood from a patient with resistant HIV can contain thousands to millions of copies of HIV. To find that one virus, out of thousands to millions, which is mutated at just a single nucleotide is like finding a needle in a haystack.”

The experiments reported in the paper show that the LRA test was sensitive enough to find a commonly sought K103N mutation in concentrations as low as one mutant per 10,000 strands of “normal” viral RNA. The LRA detection worked within two hours, while alternative technologies such as ASPCR or pyrosequencing, can take as long as eight.

LRA works by sending in many copies of a pair of short engineered probes of genetic material to complement the RNA in the HIV sample. Under optimized conditions, those pairs that perfectly match the target HIV RNA containing a mutation that causes drug resistance can rapidly become fused together, or ligated, by an enzyme. If there is a single nucleotide difference, the pair won’t fuse.

The fusing of the engineered genetic probes is designed to happen at room temperature. After a short period, the LRA system then heats the slightly alkaline solution, which shuts off the fusing reaction but turns on the amplification (copying) of fused pairs. That allows the LRA system to produce a strong signal of fused pairs, if there are any. All this happens in a single step, without any need to change solution. Brown University

Combining the investigative tools of genetics, transcriptomics, epigenetics and metabolomics, a Duke Medicine research team has identified a new molecular pathway involved in heart attacks and death from heart disease.

The researchers found that stress on a component of cells called the endoplasmic reticulum (ER) is associated with risk of future heart events, and it can be detected in bits of molecular detritus circulating in the blood.

“ER stress has long been linked to Type 1 diabetes and Parkinson’s disease, among others, but this is the first indication that it is also playing a role in common heart attacks and death from heart disease,” said senior author Svati H. Shah, M.D., associate professor of medicine and faculty at the Molecular Physiology Institute at Duke. “It’s also exciting that we are able to measure this ER stress in a small drop of blood, providing a potential way to intercede and lower the risk of a major cardiovascular event.”

Even after mapping the human genome and finding genetic traits associated with cardiovascular disease, the mechanisms underlying the inherited susceptibility to this disease have not been fully understood. Shah said the Duke team’s research approach — using a variety of analytical methods measuring over a million data points in 3,700 patients — enabled them to fill in some of the missing steps leading to cardiovascular disease, which is often inherited.

“With genetics, everyone is lumped together if they share a trait,” Shah said. “But everyone knows if you have two people with the same trait, but one is overweight, smokes and has a bad lifestyle, that person has a different pathway that led to heart disease than someone who is normal weight, doesn’t smoke, eats right and exercises.”

The Duke team focused on the intermediates between the genes and the disease pathway. This involved metabolomics — an analysis of the metabolites, or trace chemicals, left behind as the by-products from cellular processes.

Among a group of 3,700 patients referred for cardiac catheterization in the CATHGEN study, Shah and colleagues performed a genome-wide analysis of specific metabolite levels that had previously been identified as predictors of cardiovascular disease.

In their earlier work, the researchers had flagged these metabolites as markers for cardiovascular disease, but had not known how they were generated or what the underlying biological pathways were. The current study resolved that question, finding that these genes were directly linked to ER stress, which occurs when the endoplasmic reticulum organelle becomes overworked in its job managing excess and damaged cellular proteins.

Shah and colleagues then took an epigenetics and transcriptomics approach to determine what the differences were between patients with high or low levels of metabolites. Once again, the ER stress pathway came up as a key component.

“Using this multi-platform ‘omics’ approach, we identified these novel genetic variants associated with metabolite levels and with cardiovascular disease itself,” Shah said. “We don’t believe that the metabolites themselves are causing heart attacks — they might just be by-products of a dysregulated process that people are genetically susceptible to — but that’s something we need to study further.” Duke Medicine

The first patient was a mystery.

Arriving at Duke six years ago at the age of three, the youngster had mild developmental delays and physical characteristics that included a large body and large head circumference. A genetic analysis showed mutation of a specific gene, known as ASXL2, which had never been singled out as causing disease.

The youngster’s doctor, Vandana Shashi, a professor of paediatrics for the Division of Medical Genetics at Duke University School of Medicine, told his parents their son likely had a rare and yet-unidentified disease. And she promised to remain vigilant if any other cases popped up in the medical literature that might provide additional clues.

After none turned up, Shashi set out to see if the mystery case might be solved, instead, using the tools of the Undiagnosed Diseases Network (UDN) at the National Institutes of Health, which links Duke and six other medical teaching sites around the country. The participating centres pool information and innovations about diseases that are so rare they often stump the broader medical community.

Within just six weeks — connected to other UDN research labs and an international database of genes and disease characteristics called GeneMatcher — Shashi had a remarkable trove: Five additional children, all with the same physical features and the ASXL2 gene mutation.

“We can now definitively say this is a newly identified disease,” Shashi said. “With just one case, we could not say the gene mutation was the underlying cause. But with six cases, all with the same ASXL2 mutation, it is definitive.”

The new disease, which still has no name, does have similarities to two other rare genetic disorders arising from related genes. A condition called Bohring-Opitz syndrome is the result of a mutation of the ASXL1 gene, while Bainbridge-Ropers syndrome is caused by a flaw in the ASXL3 gene. Both conditions are also rare, and result in similar, but more severe impairments.

It’s unknown how the ASXL2 genetic mutation arises, but Shashi said identifying the root cause of the children’s condition is a first step, and could help drive new therapies and treatment approaches.

The immediate benefit is to the families of the children, who now have an answer to their most basic question.

“It has been wonderful to be connected to other families who share this genetic condition,” said Teresa Locklear, whose son, Issac, was the first patient to present with the mutation at Duke. “When we started, we hoped we would find other families with children who were older than Isaac, to provide a sort of roadmap for what to expect. But it turns out, Isaac is the oldest and we are the ones sharing our experiences with parents of younger children, and that’s been so rewarding.”

Study co-author Loren del Mar Peña, assistant professor in the Department of Pediatrics at Duke, said reducing isolation for families with a rare disease has tremendous impact.

“These families feel truly alone when their child clearly has a disorder, and yet there is no name for it, and no community of people they can relate to with shared experiences,” Peña said. “This will help them be able to connect with others and compare notes. That’s a huge deal – to know you aren’t the only one and there a five other children out there.”

Duke University corporate.dukehealth.org/news-listing/network-and-gene-tools-help-quickly-identify-new-rare-genetic-disease

Skin cancer is the most common of all cancers, and melanoma, which accounts for 2% of skin cancer cases, is responsible for nearly all skin cancer deaths. Melanoma rates in the U.S. have been rising rapidly over the last 30 years, and although scientists have managed to identify key risk factors, melanoma’s modus operandi has eluded the world of medical research.

A new Tel Aviv University study sheds light on the trigger that causes melanoma cancer cells to transform from non-invasive cells to invasive killer agents, pinpointing the precise place in the process where “traveling” cancer turns lethal. The research was led by Dr. Carmit Levy of the Department of Human Genetics and Biochemistry at TAU’s Sackler School of Medicine and conducted by a team of researchers from TAU, the Technion Institute of Technology, the Sheba Medical Center, the Institut Gustave Roussy and The Hebrew University of Jerusalem.

If melanoma is caught in time, it can be removed and the patient’s life can be saved. But once melanoma invades the bloodstream, turning metastatic, an aggressive treatment must be applied. When and how the transformation into aggressive invasion takes place was a mystery until now.

‘To understand melanoma, I had to obtain a deep understanding about the structure and function of normal skin,’ said Dr. Levy, ‘Melanoma is a cancer that originates in the epidermis, and in its aggressive form it will invade the dermis, a lower layer, where it eventually invades the bloodstream or lymph vessels, causing metastasis in other organs of the body. But before invading the dermis, melanoma cells surprisingly extend upward, then switch directions to invade.

‘It occurred to me that there had to be a trigger in the microenvironment of the skin that made the melanoma cells ‘invasive,” Dr. Levy continued. ‘Using the evolutionary logic of the tumour, why spend the energy going up when you can just use your energy to go down and become malignant?’

After collecting samples of normal skin cells and melanoma cells from patients at hospitals around Israel, the researchers mixed normal and cancerous cells and performed gene analysis expression to study the traveling cancer’s behaviour. They found that, completely independent of any mutation acquisition, the microenvironment alone drove melanoma metastasis.

‘Normal skin cells are not supposed to ‘travel,” said Dr. Levy. ‘We found that when melanoma is situated at the top layer, a trigger sends it down to the dermis and then further down to invade blood vessels. If we could stop it at the top layer, block it from invading the bloodstream, we could stop the progression of the cancer.’

The researchers found that the direct contact of melanoma cells with the remote epidermal layer triggered an invasion via the activation of ‘Notch signalling,’ which turns on a set of genes that promotes changes in melanoma cells, rendering them invasive. According to the study, when a molecule expressed on a cell membrane — a spike on the surface of a cell, called a ligand — comes into contact with a melanoma cell, it triggers the transformation of melanoma into an invasive, lethal agent.

‘When I saw the results, I jumped out of the room and shouted, ‘We got it!” Dr. Levy said. ‘Now that we know the triggers of melanoma transformation and the kind of signalling that leads to that transformation, we know what to block. The trick was to solve the mystery, and we did. There are many drugs in existence that can block the Notch signalling responsible for that transformation. Maybe, in the future, people will be able to rub some substance on their skin as a prevention measure.’ Tel Aviv University

Siemens Healthineers will expand the company’s existing manufacturing operations in Shanghai, China to include a new in vitro diagnostics facility. The China manufacturing facility will enable in-country manufacturing capabilities for clinical chemistry and immunoassay reagents. “This investment demonstrates the company’s continued commitment to address the evolving needs in the Chinese market and in healthcare markets across the globe,” said Franz Walt, President, Laboratory Diagnostics, Siemens Healthineers. China is the second largest market for Siemens Healthineers. According to George Chan, President, Greater China, Siemens Healthineers, “The opening of this facility strengthens our ability to support Chinese healthcare reform as we deliver better outcomes at a lower cost to our customers.” The company expects to employ hundreds of additional employees once the project is completed.

www.healthcare.siemens.com