Scientists at the Stowers Institute for Medical Research have reported a detailed description of how function-impairing mutations in polr1c and polr1d genes cause Treacher Collins syndrome (TCS), a rare congenital craniofacial development disorder that affects an estimated 1 in 50,000 live births.
Collectively the results of the study reveal that a unifying cellular and biochemical mechanism underlies the etiology and pathogenesis of TCS and its possible prevention, irrespective of the causative gene mutation.

Loss-of-function mutations in three human genes, TCOF1, POLR1C and POLR1D, have been implicated in TCS and are thought to be responsible for about 90 percent of the diagnoses of this congenital craniofacial condition.
The clinical manifestations of TCS include facial anomalies such as small jaws and cleft palate, hearing loss, and respiratory problems. Patients with TCS typically undergo multiple surgeries, but rarely are they fully corrective. By uncovering a mechanism of action common to all three genes, Stowers scientists have advanced scientific understanding of TCS etiology and pathogenesis and identified possible new avenues for preventing or treating the birth defect. This latest study from the laboratory of Stowers Investigator Paul Trainor, Ph.D., focused on Polr1c and Polr1d, whose roles as a genetic cause of TCS were revealed in a 2011 study of a small group of patients who had been diagnosed with TCS but who did not have the TCOF1 mutation. Unlike POLR1C and POLR1D, TCOF1 has been long recognized as a causative gene in TCS and as a result has been more extensively investigated.
“Before we began the study, nothing was known about the role of Polr1c and Polr1d in craniofacial development,” said Kristin Watt, Ph.D., lead author of the PLoS Genetics paper and postdoctoral scientist in the Trainor Lab. “Using zebrafish as our animal model, we set out to explore the functional roles of polr1c and polr1d during embryogenesis and more specifically in craniofacial development.”
Trainor, Watt and their collaborators compared the results of their findings on polr1c and polr1d with their and other labs’ previous research results on Tcof1. In all three loss-of-function models, the researchers found that the chain of cellular events that led to the TCS phenotype of abnormal craniofacial development originated in ribosomes, the cellular components that translate messenger RNA into proteins. Like the Tcof1 gene, polr1c and polr1d mutations were found to perturb ribosome biogenesis, or production of ribosomes, which affects the generation and survival of progenitor neural crest cells, the precursors of craniofacial bone, cartilage and connective tissue.
In animal models of all three causative genes, the scientists determined that deficient ribosome biogenesis triggered a p53-dependent cell death mechanism in progenitor neural crest cells. As a result of the activation of the p53 gene, developing embryos no longer made the quantity of neural crest cells needed to properly form the craniofacial skeleton.
However, in the polr1c and polr1d models as in the Tcof1 models, Stowers scientists found that by experimentally blocking p53 activation, they could restore the neural crest cell population and thereby rescue the animal models’ cranioskeletal cartilage.
Despite the rescue effect, Trainor said that he does not view the “guardian of the genome,” as the p53 gene is often called due to its ability to suppress cancer, as the basis of a potential therapy to prevent or reduce TCS during embryonic development. The p53 gene’s association with cancer makes inhibiting its function too risky, he said.
A less risky and perhaps more effective target for the prevention or treatment of TCS could be enhancing ribosomes, Trainor said, because the loss-of-function mutations in all three causative genes involve ribosome RNA (rRNA) transcription. Polr1c and Polr1d, for example, are subunits of RNA polymerases I and III that are essential for ribosome biogenesis.
“Rather than blocking p53, a better approach may be to try to prevent TCS by treating the problem in ribosome biogenesis that triggers the activation of p53 and the loss of neural crest cells,” said Trainor.
In their research with zebrafish embryos, Trainor and collaborators also determined that polr1c and polr1d are spatiotemporally and dynamically expressed, particularly during craniofacial development. Furthermore, zebrafish embryos with the polr1c and polr1d loss-of-function mutations develop abnormalities in craniofacial cartilage development that mimic the clinical manifestations of TCS in patients. Trainor said that he and his fellow researchers were surprised that mutations in polr1c and polr1d as well as Tcof1 specifically affected craniofacial development, because ribosome biogenesis occurs in every cell of the body. The mutation of a gene that is part of the ribosome complex would be expected to be detrimental to each of these cells, he said. However, in the zebrafish models, the mutation appears to primarily affect progenitor neural crest cells. Trainor said that he and his team theorize that progenitor neural crest cells may be particularly sensitive to deficiencies in ribosome biogenesis during embryogenesis.
Thus, the study revealed new animal models for TCS: zebrafish with polr1c and polr1d loss-of-function mutations. Moreover, the existence of a common mechanism of action may simplify the research, particularly the search for a therapy to prevent or treat TCS. Because of the similarities among the three causative genes, “we may be able to develop creative ways of preventing TCS that will prove effective in at-risk individuals who have one of the gene mutations,” said Trainor, who has investigated the molecular origins and development of TCS and related craniofacial developmental disorders for 10 years.

Stowers Institute for Medical Research www.stowers.org/media/news/jul-22-2016

Researchers led by the University of Dundee’s Professor Dario Alessi have developed a new method of measuring the activity of disease-causing mutations in the LRRK2 gene, a major cause of inherited Parkinson’s disease.

The team believes this research could help pave the way for future development of a clinical test that could facilitate evaluation of drugs to target this form of the condition.

Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. The most common disease-causing mutation in this gene increases the activity of the LRRK2 protein three-fold, implying this may contribute towards the symptoms of the disease in patients. It also suggests that drugs that reduce the activity of the protein (LRRK2 inhibitors) may help treat patients with this form of inherited Parkinson’s disease.

“It is important to better understand how disruption in LRRK2 biology causes Parkinson’s disease and whether a drug that targeted the LRRK2 enzyme would offer therapeutic benefit,” said Professor Alessi, lead author on the study.

“Current drug treatments only deal with symptoms of the condition, such as tremors, but do not affect the progression of Parkinson’s disease. An important question is whether an LRRK2 therapy might have potential to slow progression of the condition, which no other current therapy is able to do.”

When the LRRK2 protein is active it stops another cellular protein called Rab10 from fulfilling its function in the body. There are many proteins in the Rab family, and a number of them have been shown to be low in number or deactivated in different forms of Parkinson’s disease.

The new method of measuring these was developed by a collaboration of researchers from Dundee, The Michael J. Fox Foundation for Parkinson’s Research, GSK and the University of Hong Kong. It analyses how much of the Rab10 protein has been deactivated – a process where phosphate groups are added to the Rab10 molecules by the LRRK2 protein – as a measure of heightened LRRK2 protein activity.

This new experimental assay is straightforward, requires only small amounts of sample material and is suitable for adapting to analyse large samples. This contrast with current mass spectrometry technology that is more complex and cumbersome and requires larger sample sizes.

While acknowledging that more work is needed, the researchers believe this breakthrough could help with future drug developments for patients with this form of Parkinson’s disease.

Professor Alessi continued, “The prediction is that elevation of LRRK2 activity leads to Parkinson’s disease, and this is now testable using our assay. The expectation is that if a sub-group of patients can be identified with elevated LRRK2 activity, these individuals might benefit most from LRRK2 inhibitors.

University of Dundee www.dundee.ac.uk/news/2016/lab-method-sheds-light-on-how-genetic-mutations-cause-inherited-parkinsons-disease.php

Not only is breast cancer more than one disease, but a single breast cancer tumour can vary within itself, a finding that University of Pittsburgh Cancer Institute (UPCI) researchers discovered has the potential to lead to very different patient treatment plans depending on the tumour sample and diagnostic testing used.

The results demonstrate that tumour sampling techniques used with newly developed “personalized medicine” gene expression profile tests may need to be refined to ensure that the most appropriate tumour sections are selected for testing.

“These tests are a good thing—they’ve done an incredible job identifying women with breast cancers that have a low risk of recurrence who don’t need chemotherapy, saving them from the toxicity and discomfort of unnecessary treatment,” said Adrian V. Lee, Ph.D., professor of pharmacology and chemical biology at UPCI, partner with UPMC CancerCenter. “However, as with any new technology, we need to understand how these tests work, and we’re finding that the sampling process, which involves liquefying tumours, loses information that could be important in determining the best treatment plan for patients with more aggressive tumours.”

Gene expression profiling is an increasingly popular type of test that tells doctors what certain genes are doing in a tissue sample, such as causing the cells to actively divide and multiply. Several tests have been developed in recent years to aid oncologists in developing breast cancer treatment plans. They involve taking a small bit of the tumour—or multiple small bits mixed together—and testing it.

The tests can tell oncologists if the cancer has a low, intermediate or high risk of recurring. The level of risk can help doctors and patients decide whether an aggressive treatment plan involving chemotherapy is beneficial or likely to do more harm than good.

Dr. Lee and his team examined 71 cases of a type of breast cancer called “estrogen-receptor-positive” that was caught early and hadn’t yet spread to other parts of the body. In all cases, the tumour had been removed and samples taken for gene expression profiling. A total of 181 samples were taken from various parts of the tumours, and the researchers measured the expression of 141 different genes from five different types of gene expression profile tests commonly used for breast cancer tumours.

For 25 percent of the patients, their tumours received a different risk of recurrence score depending on which sample was processed.

“This indicates that one part of the tumour is more aggressive than another part. If an oncologist were to know this, he or she would likely recommend a treatment plan tailored to destroy the most aggressive section of the tumour,” said Dr. Lee.

Because the patients in this study were all caught early, their risk of recurrence was low to begin with, and there weren’t enough recurrences to make a meaningful determination on whether they would have done better if more samples were tested from their tumours.

“It would be valuable to repeat this study with a much larger group of breast cancer patients and follow them over time so that we could definitively determine if the way sampling is done with these tests is, indeed, resulting in patients getting cancer recurrences that wouldn’t have happened if the sampling process was changed,” said Dr. Lee.

University of Pittsburgh Cancer Institute

www.upmc.com/media/NewsReleases/2016/Pages/lee-tumorhetero-gep-cancerresearch.aspx

In contrast to the general belief that the airways of an infant are sterile until after birth, University of Alabama at Birmingham researchers and colleagues have found that the infant airway is already colonised with bacteria or bacterial DNA when a baby is born — and this is true for infants born as early as 24 weeks gestation.

How microbes get into the airways and the purpose of this pre-birth colonisation are still unclear, but the pattern of colonisation appears to have an important link to later severe neonatal lung disease.

An early microbial imbalance, or dysbiosis, is predictive for the development of bronchopulmonary dysplasia, or BPD, a chronic lung disease of prematurity. The extremely low birth-weight, or ELBW, infants in this study had an average birth weight of 1 pound, 8 ounces. Researchers found that the ELBW infants who went on to develop life-threatening BPD showed abnormal microbial colonisation patterns at birth, as compared to pre-term infants who did not get BPD.

“Right at birth, your respiratory microbiome can possibly predict your risk for BPD,” said Charitharth Vivek Lal, M.D., assistant professor in the UAB Pediatrics Division of Neonatology and the lead investigator of this study.
Extremely premature infants are at risk for BPD, which is the most common lung pathology of these tiny infants and a significant cause of morbidity, mortality and health care expenditures. Adults and children who had BPD as infants have lungs that failed to develop properly and are more prone to worse lung function, asthma, lung infections and pulmonary hypertension.

The researchers also looked at the airway microbiomes of 18 ELBW infants with established BPD and found that their microbiomes had a decreased diversity of types of microbes, and the pattern was very different from those of ELBW infants shortly after birth or full-term infants at birth.

As to specific groups of microbes, the phylum Proteobacteria, which includes bacteria like E. coli, appeared to be involved in BPD pathology, and the genus Lactobaccillus, part of the phylum Firmicutes, appeared to be involved in disease protection.

Lal and colleagues found decreased Lactobacillus abundance in the airway microbiomes of 10 infants born to mothers who had chorioamnionitis — an infection of the membranes of the placenta and an independent risk factor for BPD — as well as decreased Lactobacillus abundance at birth in the airways of the BPD-predisposed, ELBW infants, as compared to BPD-resistant infants. Research elsewhere has suggested a beneficial role for Lactobacillus against airway diseases and for lung development.

“I predict that researchers will study the use of respiratory probiotics, and the role of the gut-lung microbiome axis in the future,” Lal said.

For five ELBW infants who later developed BPD, the researchers collected periodic airway microbiome samples from birth through 9 weeks and saw extremely similar patterns of change in the microbiomes over time.

As for the source of the microbes, Lal and colleagues wrote, “As it is commonly believed that colonization of neonates originates in the birth canal, we were surprised to find that the airway microbiome of vaginally delivered and caesarean section-delivered neonates were similar, which suggests that the microbial DNA in the airways is probably transplacentally derived, consistent with reports that the placenta has a rich microbiome.”

The researchers speculate that this transmission of bacteria or bacterial DNA to the in-utero infant could be via blood or amniotic fluid.

University of Alabama at Birmingham www.uab.edu/news/innovation/item/7505-discovery-of-infants-airway-microbiomes-may-help-predict-lung-disease

Ribonucleic acid (RNA) binding fluorescent probes have been powerful and important analytical tools for the study of RNA structures and functions.

A research group led by Professor Seiichi Nishizawa at Tohoku University’s Graduate School of Science has reported a new RNA probe that binds to double-stranded RNA (dsRNA) in a sequence-specific manner.

The probe has a weak response to mismatch-containing dsRNA sequences, thus enabling sequence-selective fluorescence sensing of dsRNA at the single-base pair resolution. It also shows a preference for binding with dsRNA over dsDNA, which is an important selective process for future applications in a cellular environment where RNA and DNA co-exist.

In contrast to the conventional analytical method which is limited to single-stranded regions of RNA, the new analytical method allows for fluorescent sensing of target dsRNA structure and sequence for the first time.

It is expected that the probe will open up new possibilities for analysing the functions of dsRNA-containing structures, which are closely related to various biological phenomena and diseases.

Tohoku University www.tohoku.ac.jp/en/press/fluorescence_detection_of_rna.html