Researchers at the University of Wisconsin-Madison have found a new way to accelerate a workhorse instrument that identifies proteins. The high-speed technique could help diagnose cancer sooner and point to new drugs for treating a wide range of conditions.
Proteins are essential building blocks of biology, used in muscle, brain, blood and hormones. If the genes are the blueprints, the proteins patterned on them are the hammers and tongs of life.

Proteins are not only numerous — humans have more than 100,000 varieties — but each one has a complex structure that determines its exact function in the biological realm. Just as tissue from cats and kangaroos can be distinguished by studying the individual ‘letters’ of their genetic codes, protein A can be distinguished from protein B by looking at the amino-acid sub-units that compose all proteins.

The fastest way to count and identify proteins is to use a mass spectrometer, a precise instrument that measures chemical compounds by mass. ‘Mass spec is an essential part of modern biology, and most people use it to look at variations in proteins,’ says Joshua Coon, a professor of chemistry and biomolecular chemistry.

Because mass spectrometers are expensive, and proteins are both numerous and ubiquitous, chemists have recently learned to double up their samples so they can, for example, compare normal tissue to diseased tissue in a single run.

Knowing how the proteins change when good tissue goes bad suggests what has gone wrong.

Now, Coon has doubled-down on the doubling-up process with a technique that has the potential to run as many as 20 samples at once. The new process has already gone to work, says Alexander Hebert, a graduate student who was first author on the new publication.

‘Working with John Denu at the Wisconsin Institute for Discovery, we are looking at mice that lived with or without caloric restriction,’ says Hebert. Caloric restriction is known to increase lifespan in many animals, and scientists are eager to unravel the biochemical pathways that explain this life extension. ‘Some of these mice have lost a certain gene related to metabolism, so we are comparing four types of tissue all at once. We can look at the brain, liver or heart, and ask, how does the abundance of proteins vary?’
Already, Coon and Hebert have performed six simultaneous analyses using the new technique; but it could actually do batches of 20, Coon says.

Key to the original doubling-up process was inserting a ‘tag’ into the amino acids that gives the proteins a slightly different mass. The tags are isotopes — chemically identical atoms that have different masses.

To prepare two samples, one would receive an amino acid containing common isotopes, and the other special, heavier isotopes. The result — proteins that are chemically identical but have different masses — can easily be identified in a mass spectrometer.

The new journal report by Coon and Hebert describes a way to use amino acids built from a broader range of isotopes that would be expected to have identical mass, but do not because some of their mass has been converted to energy to hold the atomic nuclei together. Without this energy, the positively charged proteins would repel each other and the atomic nucleus would be destroyed. The tiny loss of mass due to this conversion to binding energy can be detected in the new, ultra-precise mass spectrometers that are now installed in several labs on campus.

The mass difference in the new technique is more than 1,000 times below the mass differences in the existing doubled-up technique, but it is enough to count and identify proteins from six — and, theoretically, 20 — samples at once. The researchers applied for a patent last fall and assigned the rights to the Wisconsin Alumni Research Foundation. University of Wisconsin-Madison

The Red Cross Blood Transfusion Service of Upper Austria has become the first laboratory in Europe to receive accreditation from the European Federation for Immunogenetics (EFI) for the use of human leukocyte antigen (HLA) tests based on next-generation sequencing with Roche’s GS Junior System. This new method will allow more precise and much more rapid tissue-typing and donor selection for stem cell transplants than has been possible to date. In addition, the HLA testing method previously only used for research will now also be available as a standard routine diagnostic procedure.
“Worldwide, around 50,000 people a year urgently require a stem cell transplant, and the chances of finding an allogeneic stem cell donor are about 1:500,000,” said Thomas Schinecker, Head of Roche Sequencing Solutions. “This accreditation is an example of how the potential of next-generation sequencing can be successfully translated from research into medicine and made widely available to patients in areas of high medical need.”
Underlining the benefits of the new standard method, Dr Christian Gabriel, Medical Director of the Red Cross Blood Transfusion Service of Upper Austria, said: “Standardized laboratory procedures are needed to promote positive therapeutic outcomes for patients. EFI accreditation is an important step, allowing large numbers of patients to benefit from the latest technologies.”

To the list of oncogenic drivers of lung cancer that includes ALK, EGFR, ROS1 and RET, results of a University of Colorado Cancer Center study presented at ASCO 2013 show that mutations in the gene NTRK1 cause a subset of lung cancers.

‘We’re reconceptualising lung cancer as many, related diseases. And we need to learn to identify and treat each individually. We can treat the forms of the disease that depend on ALK and EGFR mutations. We’re getting very close to treating lung cancers that depend on ROS1 and RET. And now we show another oncogenic driver of the disease that begs its own targeted treatment,’ says Robert C. Doebele, MD, PhD, investigator at the CU Cancer Center and assistant professor of medical oncology at the CU School of Medicine.

The group, in collaboration with Pasi A. Jänne, MD, PhD from the Dana-Farber Cancer Institute, started with lung cancer tumour samples from 36 ‘pan-negative’ patients, meaning that no other driver oncogene had been identified. So if not EGFR, ALK and the like, what was driving the cancer? Doebele and colleagues took the question to Foundation Medicine (Cambridge, MA), which used targeted, next-gen sequencing to analyse the samples for possible mutations in a couple hundred potential oncogenes identified as drivers of other cancers. NTRK1 had been identified as a driver of thyroid cancer and so was included in the panel (though drug development had stalled due in part to the relative rarity of the thyroid disease). Sure enough, next-gen sequencing identified NTRK1 gene fusions as the potential driver in two of these samples.

Doebele and colleagues took the lead back to the CU labs, where Marileila Varella Garcia, PhD, developed a specific test for NTRK1 fusions based on fluorescence in situ hybridisation (FISH), similar to what is used for ALK, ROS1 and RET fusions. This allowed the group to validate the finding of NRTK1 as a novel oncogene in these patient samples.

But the study went a step beyond identifying the oncogene. Doebele describes the relative ease with which genes that are improperly activated can be silenced – ‘whether a drug is already is in clinical trials, or already approved for another cancer, or just sitting on the pharma shelf somewhere, many drugs exist that turn off these candidate genes,’ Doebele says.

In this case, Doebele describes ‘walking up the street to Array BioPharma (Boulder, CO), who happened to have several compounds specific for this gene.’

The group showed that mutated NRTK1 genes in cells treated with drug candidates ARRY-772, -523, and -470 and others was effectively turned off.

‘This is still preclinical work,’ Doebele says, ‘but it’s the first – and maybe even second and third! – important steps toward picking off another subset of lung cancer with a treatment targeted to the disease’s specific genetic weaknesses.’ University of Colorado Cancer Center

A pair of studies tells the tale of how a neuroscientist at Mayo Clinic in Florida helped to discover the first African-American family to have inherited the rare movement disorder dystonia, which causes repetitive muscle contractions and twisting, resulting in abnormal posture. The research may improve diagnosis of this neurological condition in a population not known to suffer from it.
In the first study, Mayo Clinic’s Zbigniew Wszolek, M.D., and a team of neuroscientists from other institutions in the U.S. described three generations of an African-American family in Georgia who had dystonia. The team excluded mutations in genes previously associated with dystonia. The study was the first description of an African-American family with late-onset primary dystonia.
In the second study, Dr. Wszolek was part of an international team of researchers led by Mark LeDoux, M.D., Ph.D., a neurologist and neurogeneticist from the University of Tennessee Health Science Center in Memphis. The investigators identified the specific genetic abnormality seen in the African-American family and in several other white families. In the African-American family, the mutation produced a protein in which one amino acid was substituted for another.
While this isn’t the only gene anomaly linked to dystonia, it is the first found in an African-American family. All other genes found to be linked to this disorder were discovered in families of other ethnic origins.
The findings may improve diagnosis and treatment of dystonia in African-Americans, says Dr. Wszolek, who has been a driving force behind international research efforts to uncover genes that play a role in neurological disorders. Mayo Clinic

Researchers from the RIKEN Center for Integrative Medical Sciences in Japan have identified the first gene to be associated with adolescent idiopathic scoliosis (also called AIS) across Asian and Caucasian populations. The gene is involved in the growth and development of the spine during childhood.
AIS is the most common pediatric skeletal disease, affecting approximately 2% of school-age children. The causes of scoliosis remain largely unknown and brace treatment and surgery are the only treatment options. However, many clinical and genetic studies suggest a contribution of genetic factors.
To understand the causes and development of scoliosis, Dr Ikuyo Kou, Dr Shiro Ikegawa and their team have tried to identify genes that are associated with a susceptibility to develop the condition.
By studying the genome of 1,819 Japanese individuals suffering from scoliosis and comparing it to 25,939 Japanese individuals, the team identified a gene associated with a susceptibility to develop scoliosis on chromosome 6. The association was replicated in Han Chinese and Caucasian populations.
The researchers show that the susceptibility gene, GPR126, is highly expressed in cartilage and that suppression of this gene leads to delayed growth and bone tissue formation in the developing spine. GPR126 is also known to play a role in human height and trunk length.
‘Our finding suggest the interesting possibility that GPR126 may affect both AIS susceptibility and height through abnormal spinal development and growth,’ explain the authors.
‘Further functional studies are necessary to elucidate how alterations in GPR126 increase the risk of AIS in humans,’ they conclude. Medical News Today