Researchers with the UC Davis MIND Institute and Agilent Laboratories have found that Prader-Willi syndrome — a genetic disorder best known for causing an insatiable appetite that can lead to morbid obesity — is associated with the loss of non-coding RNAs, resulting in the dysregulation of circadian and metabolic genes, accelerated energy expenditure and metabolic differences during sleep.
The research was led by Janine LaSalle, a professor in the UC Davis Department of Medical Microbiology and Immunology who is affiliated with the MIND Institute. It is published online in Human Molecular Genetics.
‘Prader-Willi syndrome children do not sleep as well at night and have daytime sleepiness,’ LaSalle said. ‘Parents have to lock up their pantries because the kids are rummaging for food in the middle of the night, even breaking into their neighbours’ houses to eat.’
The study found that these behaviours are rooted in the loss of a long non-coding RNA that functions to balance energy expenditure in the brain during sleep. The finding could have a profound effect on how clinicians treat children with Prader-Willi, as well as point the way to new, innovative therapies, LaSalle said.
The leading cause of morbid obesity among children in the United States, Prader-Willi involves a complex, and sometimes contradictory, array of symptoms. Shortly after birth children with Prader-Willi experience failure to thrive. Yet after they begin to feed themselves, they have difficulty sleeping and insatiable appetites that lead to obesity if their diets are not carefully monitored.
The current study was conducted in a mouse model of Prader-Willi syndrome. It found that mice engineered with the loss of a long non-coding RNA showed altered energy use and metabolic differences during sleep.
Prader-Willi has been traced to a specific region on chromosome 15 (SNORD116), which produces RNAs that regulate gene expression, rather than coding for proteins. When functioning normally, SNORD116 produces small nucleolar (sno) RNAs and a long non-coding RNA (116HG), as well as a third non-coding RNA implicated in a related disorder, Angelman syndrome. The 116HG long non-coding RNA forms a cloud inside neuronal nuclei that associates with proteins and genes regulating diurnal metabolism in the brain, LaSalle said.
‘We thought the cloud would be activating transcription, but in fact it was doing the opposite,’ she said. ‘Most of the genes were dampened by the cloud. This long non-coding RNA was acting as a decoy, pulling the active transcription factors away from genes and keeping them from being expressed.’
As a result, losing snoRNAs and 116HG causes a chain reaction, eliminating the RNA cloud and allowing circadian and metabolic genes to get turned on during sleep periods, when they should be dampened down. This underlies a complex cycle in which the RNA cloud grew during sleep periods (daytime for nocturnal mice), turning down genes associated with energy use, and receded during waking periods, allowing these genes to be expressed. Mice without the 116HG gene lacked the benefit of this neuronal cloud, causing greater energy expenditure during sleep.
The researchers said that the work provides a clearer picture of why children with Prader-Willi syndrome can’t sleep or feel satiated and may change therapeutic approaches. For example, many such children have been treated with growth hormone because of short stature, but this actually may boost other aspects of the disease.
‘People had thought the kids weren’t sleeping at night because of the sleep apnea caused by obesity,’ said LaSalle. ‘What this study shows is that the diurnal metabolism is central to the disorder, and that the obesity may be as a result of that. If you can work with that, you could improve therapies, for example figuring out the best times to administer medications.’ UC Davis Department of Medical Microbiology and Immunology

Massachusetts General Hospital (MGH) researchers and their colleagues have used digital versions of a standard molecular biology tool to detect a common tumour-associated mutation in the cerebrospinal fluid (CSF) of patients with brain tumours. In their report the investigators describe using advanced forms of the gene-amplification technology polymerase chain reaction (PCR) to analyse bits of RNA carried in membrane-covered sacs called extracellular vesicles for the presence of a tumour-associated mutation in a gene called IDH1.
‘Reliable detection of tumour-associated mutations in cerebrospinal fluid with digital PCR would provide a biomarker for monitoring and tracking tumours without invasive neurosurgery,’ says Xandra Breakefield, PhD, of the MGH Molecular Neurogenetics Unit, corresponding author of the paper. ‘Knowing the IDH1 mutation status of these tumours could help guide treatment decisions, since a number of companies are developing drugs that specifically target that mutant enzyme.’
Both normal and tumour cells regularly release extracellular vesicles, which contain segments of RNA, DNA or proteins and can be found in blood, CSF and other body fluids. A 2008 study from the MGH team was able to identify a relatively large tumour-associated mutation in extracellular vesicles from the blood of brain tumour patients, but most current diagnostic technologies that analyse CSF do not capture molecular or genetic information from central nervous system tumours.
In addition, explains Leonora Balaj, PhD, of MGH Neurology, co-lead author of the current report, ‘Tumour-specific EVs make up only a small percentage of the total number of EVs found in either blood or cerebrospinal fluid, so finding rare, single-nucleotide mutations in a sample of blood or CSF is very challenging. These digital PCR techniques allow the amplification of such hard-to-find molecules, dramatically improving the ability to identify tumour-specific changes without the need for biopsy.’
The current study used two forms of digital PCR – BEAMing and Droplet Digital PCR – to analyse extracellular vesicles in the blood and CSF of brain tumour patients and healthy controls for the presence of a single-nucleotide IDH1 mutation known to be associated with several types of cancer. Both forms of PCR were able to detect both the presence and abundance of mutant IDH1 in the CSF of 5 of the 8 patients known to have IDH1-mutant tumours. Two of the three mutation-positive tumours that had false negative results were low grade and the third was quite small, suggesting a need for future studies of more samples to determine how the grade and size of the tumours affect the ability to detect mutations. The failure to detect tumour-associated mutations in blood samples with this technology may indicate that CSF is a better source for extracellular vesicles from brain tumours.
The ability to non-invasively determine the genetic makeup of brain tumours could have a significant effect on patient care, explains study co-author Fred Hochberg, MD, MGH Neurology. ‘The current approach for patients who may have a brain tumour is first to have a brain scan and then a biopsy to determine whether a growth is malignant. Patients may have a second operation to remove the tumour prior to beginning radiation therapy and chemotherapy, but none of these treatments are targeted to the specific molecular nature of the tumour.
‘Having this sort of molecular diagnostic assay – whether in spinal fluid or blood – would allow us to immediately initiate treatment that is personalised for that patient without the need for surgical biopsy,’ he adds. ‘For some patients, the treatment could shrink a tumour before surgical removal, for others it may control tumour growth to the point that surgery is not necessary, which in addition to keeping patients from undergoing an unnecessary procedure, could save costs. We still have a long way to go to improve survival of these malignancies, so every improvement we can make is valuable.’ Massachusetts General Hospital

A Chinese research team composed of Shenzhen Second People’s Hospital, BGI and other institutes reports their latest study on bladder cancer genomics. The discoveries were made using whole-genome and exome sequencing technologies and provide evidence that genetic alterations affecting the sister chromatid cohesion and segregation (SCCS) process may be involved in bladder tumorigenesis and open a new way for the treatment of bladder cancer.

Transitional cell carcinoma (TCC) is the most common type of bladder cancer diagnosed, accounting for 90% of all bladder malignancies in North America, South America, Europe, and Asia. It’s reported that there were an estimated 386,300 new bladder cancer cases and 150,200 deaths in 2008 alone. And the number was up to 170,000 deaths in 2010. Until now, there has been no complete genomic data available for developing new therapeutic approaches to combat bladder cancer.

To have a deeper understanding of the genetic basis underlying TCC, Chinese scientists conducted exome sequencing on the tumour and matched peripheral blood samples from 99 TCC patients, and identified 1,023 somatic substitutions and 67 indels respectively. They performed whole genome sequencing (WGS) to detect copy number alterations (CNAs) and obtained 4-fold mean haploid coverage for each sample.
After evaluating the genetic alterations or variants, researchers found frequent alterations in two genes, STAG2 and ESPL1, which are associated with the sister chromatid cohesion and segregation (SCCS) process. Among them, STAG2 was particularly notable as to harbour a greater number of non-synonymous mutations and a higher ratio of non-synonymous to synonymous mutations. Their study indicated that chromosomal instability and aneuploidy had an influence on bladder cancer, and provided evidence that bladder cancer became the first type of cancer with major genetic lesions in SCCS genes.

Furthermore, researchers detected a recurrent fusion involving two other SCCS-associated genes, FGFR3 and TACC3, by transcriptome sequencing of 42 DNA-sequenced tumors. They suggested that FGFR3/TACC3 is related with bladder tumorigenesis, and the high expression of TACC3 was mediated by transcriptional regulatory elements in the promoter of the fusion partner, FGFR3, not the amplification of TACC3. BGI

Researchers from Boston University School of Medicine (BUSM) and George Washington University (GWU) have developed a method to rapidly identify pathogenic species and strains causing illnesses, such as pneumonia, that could help lead to earlier detection of disease outbreaks and pinpoint effective treatments more quickly.
Emerging sequencing technologies have revolutionised the collection of genomic data for bioforensics, biosurveillance and for use in clinical settings. However, new approaches are being developed to analyse these large volumes of genetic data. Principal investigator Evan Johnson, PhD, assistant professor of medicine at BUSM, and Keith Crandall, PhD, director of the Computational Biology Institute at GWU, have created a statistical framework called Pathoscope to identify pathogenic genetic sequences from infected tissue samples.

This unique approach can accurately discriminate between closely related strains of the same species with little coverage of the pathogenic genome. The method also can determine the complete composition of known pathogenic and benign organisms in a biological sample. No other method can accurately identify multiple species or substrains in such a direct and automatic way. Current methods, such as the standard polymerase chain reaction detection or microscope observation, are often imperfect and time-consuming.
‘Pathoscope is like completing a complex jigsaw puzzle. Instead of manually assembling the puzzle, which can take days or weeks of tedious effort, we use a statistical algorithm that can determine how the picture should look without actually putting it together,’ said Johnson. ‘Our method can characterise a biological sample faster, more accurately and in a more automated fashion than any other approach out there.’

This work will be relevant in a broad range of scenarios. For example, in hospitals, this sequencing method will allow for rapid screening of thousands of infectious pathogens simultaneously, while being sensitive enough to monitor disease outbreaks caused by specific pathogenic strains. Veterinarians can even apply the method in their practices. This research is also applicable outside of clinical settings, allowing officials to quickly identify agents of bioterrorism (e.g. in a tainted letter) and harmful pathogens on hard surfaces, soil, water or in food products.
‘This approach has the ability to drastically change the process for identifying and combating pathogens, whether they’re in a hospital, veterinarian’s office or salmon stream,’ Crandall said. Researchers plan to conduct more studies to further verify the efficacy of their approach, and will soon begin to work with the aquaculture industry, helping fishermen with water-quality surveillance.

Findings show how a genetic mutation in untreated patients is linked to aggressive cancer later in life. It was previously thought that the mutation only occurred in response to therapy.

The research highlights why relapses could occur in some men following hormone therapy. And it could help identify those patients that will develop fatal prostate cancer much earlier for life-extending therapy.

Prostate cancer is the most common cancer in men in the UK, with more than 40,000 new cases diagnosed every year. Treatment options for patients diagnosed with early stage prostate cancer vary from ‘watchful waiting’ to hormone-withdrawal therapy, radiotherapy or surgery.

Additional tests for indicators of aggressive cancer are necessary to help categorise patients so that those with a low-risk of the disease spreading can avoid unnecessary treatment, and those diagnosed with a high-risk can be targeted for more aggressive first line therapy.

Hormone-withdrawal therapy often results in a dramatic remission, however the disease invariably relapses with a resistant form of the cancer. A third of these are due to an increase in copy number of a particular gene called the ‘androgen receptor’. The gene is on the X-Chromosome and so there is normally only one copy of this gene present in men. Prostate cancer thrives on male hormones, and one way that they develop to grow better is to increase the number of copies of the androgen receptor gene. This also enables the cancer to resist therapy.

Lead researchers Dr Jeremy Clark and Prof Colin Cooper from UEA’s school of Biological Sciences carried out the research at the Institute of Cancer Research, London, and at UEA.

Dr Clark said: ‘By the age of 60, the majority of men will have signs of prostate cancer. However, only a small proportion of men will die of the disease. The question is – which of these cancers are dangerous and which are not? Deciding which cancers are going to progress and kill the patient is key to effective patient treatment.’

‘Prostate cancer thrives on male hormones, and cutting the supply of hormones to the cancer is a main avenue of therapy. Prostate cancer only kills the patient when it becomes immune to these therapies. A third of these killer cancers are immune to therapy because they have boosted the number of male hormone receptor (AR) genes in their DNA. This gene boosting, also known as amplification, has been thought to be a response of the tumour to the hormone reduction therapy itself.

‘Our research has shown that an early form of this hormone-gene boosting is present in a number of prostate cancers that have never been treated with hormone reduction therapy. We think that it is these cancers that will grow and kill the patient.

‘This discovery can be used to identify these killer cancers in patients much earlier than is currently possible. Patients could then be selected for more aggressive therapy before the cancer has developed full immunity.’

The research team looked at biomarkers from almost 600 patients prior to hormone-withdrawal therapy. But the method of identification used was labour intensive and time consuming. Developing ways of identifying patients for early therapeutic intervention will be key to implementing this discovery in the clinic. The research team are currently looking at more rapid ways of identifying patients that will develop aggressive cancer. University of East Anglia