Gene mutations shed light on ALS Researchers have linked newly discovered gene mutations to some cases of the progressive fatal neurological disease amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease. Shedding light on how ALS destroys the cells and leads to paralysis, the researchers found that mutations in this gene affect the structure and growth of nerve cells. Scientists at the University of Massachusetts Medical School, Worcester, collaborated with international ALS researchers to search for gene mutations in two large families with an inherited form of ALS. The researchers used a technique to decode only the protein-encoding portions of DNA, known as the exome, allowing an efficient yet thorough search of the DNA regions most likely to contain diseasecausing mutations. This deep sequencing of the exome led to the identification of several different mutations in the gene for profilin (PFN1), which were present only in the family members that developed ALS. Further investigations of 272 other familial ALS cases across the world showed that profilin mutations were also found in a small subset (about 1% to 2%) of the familial ALS cases studied. In fly models, disrupting profilin stunted the growth of axons. After identifying the PFN1 mutations in ALS patients, the researchers demonstrated that these mutations inhibited axon growth in laboratorygrown motor neurons as well. They also found that mutant profilin accumulated in clumps in neural cells, as has been seen for other abnormal proteins associated with ALS, Parkinson’s and Alzheimer’s. Neural cells with PFN1 mutations also contained clumps of a protein known as TDP-43. Clumps of abnormal TDP-43 are found in most cases of ALS, further linking profilin to known ALS mechanisms. This research was published in Nature online. Positive steps in cardiac repair therapy Investigators at Zhongshan hospital Fudan University, Shanghai, China have established a novel hyperbranched poly(amidoamine) (hPAMAM) nanoparticle- based hypoxia regulated vascular endothelial growth factor (HRE-VEGF) gene therapy strategy, which is an excellent substitute for the current expensive and uncontrollable VEGF gene delivery system. The discovery provides an economical, feasible and biocompatible gene therapy strategy for cardiac repair. Transplantation of VEGF gene manipulated mesenchymal stem cells (MSCs) has been proposed as a promising therapeutic method for cardiac repair after myocardial infarction. However, the gene delivery system, including the VEGF gene and delivery vehicle, needs to be optimised. On one hand, long-term and uncontrollable VEGF over-expression in vivo has been shown to lead to hemangioma formation instead of functional vessels in animal models. On the other hand, non-viral gene vector can circumvent the limitations of virus, drawbacks of the current non-viral vectors, such as complex synthesis procedure, limited transfection efficiency and high cytotoxicity, still needs to be overcome. Answers for autism’s gender bias Researchers at Emory University School of Medicine have identified five rare mutations in a single gene that appear to increase the chances that a boy will develop an autism spectrum disorder (ASD). Mutations in the AFF2 gene, and other genes like it on the X chromosome, may explain why autism spectrum disorders affect four times as many boys as girls. The mutations in AFF2 appeared in 2.5% of boys examined. Mutations in X chromosome genes only affect boys, who have one X chromosome. Girls have a second copy of the gene that can compensate. Senior author Michael Zwick, assistant professor of human genetics at Emory University School of Medicine, said: “Our data suggest that AFF2 could be one of the major X-linked risk factors for ASDs. The finding bolsters a growing consensus among geneticists that rare variants in many different genes contribute significantly to risk for autism spectrum disorders. The mutations in the AFF2 gene probably do not cause ASDs all by themselves. We do not think that the variants we have identified are monogenic causes of autism. Our data does support the idea that this is an autism susceptibility gene.” In some situations, mutations in a single gene are enough by themselves to lead to a neurodevelopmental disorder with autistic features, such as fragile X syndrome or tuberous sclerosis complex. But these types of mutations are thought to account for a small number of ASD cases. Recent large-scale genetic studies of autism spectrum disorders have identified several rare variants that sharply increase ASD risk. Scientists believe rare variants could explain up to 15% or 20% of ASD cases. However, until now no single variant has been found in more than 1% of ASD cases. Tests showed that in four cases, the affected boys had inherited the riskconferring mutations from their mothers. One boy had a “de novo” (not coming from the parents) mutation. Compared with X-linked genes in unaffected people, mutations in AFF2 were five times more abundant in the boys with ASDs. The AFF2 gene had already been identified as responsible for a rare inherited form of intellectual disability with autistic features. This effect is seen when the AFF2 gene is deleted or silenced completely. AFF2 has some similarity to FMR1, the gene responsible for fragile X syndrome. Like FMR1, it can be silenced by a triplet repeat. In these cases, the presence of the triplet repeat (three genetic bases repeated dozens of times) triggers a change in chromosomal structure that prevents the gene from being turned on. In contrast, the mutations Zwick’s team found are more subtle, slightly changing the sequence of the protein AFF2 encodes. Little is known about the precise function of the AFF2 protein. A related gene in fruit flies called lilliputian also appears to regulate the development of neurons. Zwick says one of his laboratory’s projects is to learn more about the function of the AFF2 gene, and to probe how the mutations identified by his team affect the function. His team is also working on gauging the extent to which other genes on the X chromosome contribute to autism risk. doi: 10.1093/hmg/dds267 New gene-editing tool reduces investigation time Development of a new way to make a powerful tool for altering gene sequences should greatly increase the ability of researchers to knock out, or otherwise alter, the expression of any gene they are studying. The method allows investigators to quickly create a large number of TALENs (transcription activator-like effector nucleases), enzymes that target specific DNA sequences and have several advantages over zinc-finger nucleases (ZFNs), which have become a critical tool for investigating gene function and potential gene therapy applications. J. Keith Joung, associate chief for Research in the Massachusetts General Hospital (MGH) Department of Pathology and co-senior author of the report, said: “I believe that TALENs and the ability to make them in high throughput, which this new technology allows, could literally change the way much of biology is practiced by enabling rapid and simple targeted knockout of any gene of interest by any researcher.” TALENs take advantage of TAL effectors, proteins naturally secreted by plant bacteria that are able to recognise specific base pairs of DNA. A string of the appropriate TAL effectors can be designed to recognise and bind to any desired DNA sequence. TALENs are created by attaching a nuclease, an enzyme that snips through both DNA strands at the desired location, allowing the introduction of new genetic material. TALENs are able to target longer gene sequences than is possible with ZFNs and are significantly easier to construct. But until now there has been no inexpensive, publicly available method of rapidly generating a large number of TALENs. The method developed by Joung and his colleagues, called the FLASH (fast ligation- based automatable solid-phase highthroughput) system, assembles DNA fragments encoding a TALEN on a magnetic bead held in place by an external magnet, allowing automated construction by a liquid-handling robot of DNA. The system encodes as many as 96 TALENs in a single day at a cost of around US$75 per TALEN. Joung’s team also developed a manual version of FLASH that would allow labs without access to robotic equipment to construct up to 24 TALEN sequences a day. In their test of the system in human cells, the investigators found that FLASHassembled TALENs were able to successfully induce breaks in 84 of 96 targeted genes known to be involved in cancer or in epigenetic regulation. Jeffry D. Sander, co-senior author of the FLASH report and a fellow in Joung’s laboratory, said: “The ability to make a TALEN for any DNA sequence with a high probability of success changes the way we think about gene-altering technology because now the question isn’t whether you can target your gene of interest but rather which genes do you want to target and alter.” The research team also found that the longer a TALEN was, the less likely it was to have toxic effects on a cell, which they suspect may indicate that shorter TALENs have a greater probability of binding to and altering unintended gene sites. Joung notes that this supports the importance of designing longer TALENs for future research and potential therapeutic applications. One-step test for RASopathies A new gene test will greatly improve the speed and clarity of diagnosis for a complex range of genetic disorders known as RASopathies. The new test has been developed by molecular diagnostic testing company NewGene in collaboration with the South West Thames Regional Genetics Service at St George’s Healthcare NHS Trust in London, the specialist centre for Noonan Syndrome and associated hereditary disorders in the UK. Noonan Syndrome and related disorders (RASopathies) are autosomal dominant congenital syndromes. These disorders are characterised by facial dysmorphism, a wide spectrum of cardiac disease, postnatal reduced growth, ectodermal and skeletal defects, and variable cognitive deficits. Although some features are more frequently associated with particular syndromes, variable presentations make it difficult to provide a definitive clinical diagnosis. Correctly identifying the disorder is essential to ensure that appropriate care and monitoring is provided and to preclude unnecessary investigations. The research laboratories at St George’s have worked for over 10 years to clone the genes for Noonan Syndrome. Current genetic testing of suspected Noonan cases is carried out on a gradual basis by testing for one disorder, or gene, and then another until a mutation is identified to confirm the diagnosis. This is both time consuming and costly and delays the determination of the best clinical care pathway for patients. The new work carried out by NewGene and the specialists at St George’s has resulted in the development of a new one-step test that utilises Roche 454 sequencing to provide large scale parallel screening of all 12 genes associated with Noonan disorders in one panel. This comprehensive new method will allow a refined diagnosis to be made more quickly than has been possible previously. This approach will also be far cheaper than screening for all available genes. A positive test result will provide a definite diagnosis of the syndrome in question as the mutation spectrum has been well defined and no cases of nonpenetrance have been identified. Importantly, as a positive result will also determine which disorder is applicable, medical interventions appropriate for that specific disorder can be highlighted more quickly. Professor Michael Patton, medical advisor for the Noonan Syndrome Association UK, said: “Since the discovery of new genes in the RAS MAPK pathway it has become increasingly difficult and expensive to undertake full genetic testing for Noonan syndrome, but the development of the next generation sequencing test at NewGene and St George’s hospital is a major advance in the diagnosis of Noonan syndrome”. Noonan spectrum disorders affect around 1 in 1,000 live births. The early identification of a pathogenic mutation will also facilitate informative genetic counselling and confirmation testing in other affected individuals in the family. A definitive diagnosis would allow families early use of patient support groups such as the Noonan Syndrome Association (NSA). New gene identified for type 2 diabetes Stanford University School of Medicine investigators have strongly implicated a novel gene in the triggering of type-2 diabetes. Their experiments in lab mice and in human blood and tissue samples further showed that this gene is not only associated with the disease, as predicted computationally, but is also likely to play a major causal role. In the study, researchers combed through freely accessible public databases storing huge troves of results from thousands of earlier experiments. They identified a gene never before linked to type-2 diabetes. Drugs now used to treat insulin resistance can’t reverse the progression to fullblown type-2 diabetes. In searching for risk-increasing genes over the past 10 years, scientists have used two approaches to hunt them down. One way is to look for variations in genes’ composition, such as deviations in their chemical sequences that correlate with a higher likelihood of contracting a particular condition. However, genes don’t change from one tissue to the next and, with the exception of mutations that accrue gradually over a lifetime in particular cells and can lead to cancer and other conditions, they remain largely unaltered by disease and the ageing process. What does change dynamically, from one tissue or state to another, is what all those genes are doing and how actively each of them is involved in cranking out the starting materials for the many thousands of proteins critical to each cell’s or tissue’s identity and to every organism’s survival. So, a second approach to understanding our genes has been devised. This latter method flags differences in genes’ activity levels. Both types of approaches have generated staggering amounts of data. Now, investigators are starting to reach in and pull out a treasure-trove of potentially valuable information. In this study, the Stanford scientists wanted to know which genes showed especially marked changes in activity. Mining public databases, they located 130 independent gene-activitylevel experiments. By integrating that data, they were able to search for those genes that showed activity-level differences in the most experiments. They zeroed in on a single gene, called CD44, whose activity changed substantially in diabetic tissues compared with healthy tissues in 78 of the 130 experiments. The chance of this occurring by fluke was less than one in 10 million-trillion. The uptick in CD44’s activity was especially pronounced in the fat tissue of people with diabetes. Researchers say this is intriguing, because obesity is known to be a strong risk factor for type-2 diabetes. The team obtained two strains of mice (one carrying the gene, the other lacking it) and divided them into two subgroups, which they fed either a normal or a highfat diet, representative of today’s increasingly common human diet. The team studied these mice using tests commonly applied to humans, for example measuring fasting blood sugar and measuring blood sugar after administering sugar or insulin. As anticipated, the mice on normal diets stayed slim and retained good insulin sensitivity. CD44- containing mice on high-fat diets, also as expected, got tubby and developed insulin resistance. But mice lacking the suspect gene never lost their sensitivity to insulin and didn’t become diabetic on high-fat diets, although they became as plump as their CD44-carrying peers. This suggested that knocking out CD44’s function could improve insulin sensitivity, and that blocking CD44 with a drug might turn out to be an interesting new way to treat type-2 diabetes. So the team tested a prototype drug: antibodies that shut down the receptor’s action in CD44-carrying mice fed a high-fat diet. Though these overfed mice didn’t get any thinner, the prototype drug did reduce their blood-sugar levels within a week. Gene therapy for bone renewal Researchers at the Royal College of Surgeons in Ireland (RCSI) have developed a new method of repairing bone using synthetic bone graft substitute material which, combined with gene therapy, can mimic real bone tissue and has potential to regenerate bone in patients who have lost large areas of bone from either disease or trauma. The researchers have developed an innovative scaffold material (made from collagen and nano-sized particles of hydroxyapatite) which acts as a platform to attract the body’s own cells and repair bone in the damaged area using gene therapy. The cells are tricked into overproducing bone producing proteins known as BMPs, encouraging regrowth of healthy bone tissue. This is the first time these in-house synthesised nanoparticles have been used in this way and the method has potential to be applied to regenerate tissues in other parts of the body. Professor Fergal O’Brien, principal investigator, said: “Previously, synthetic bone grafts had proven successful in promoting new bone growth by infusing the scaffold material with bone producing proteins. These proteins are already clinically approved for bone repair in humans but concerns exist that the high doses of protein required in clinical treatments may potentially have negative side effects for the patient such as increasing the risk of cancer. Other existing gene therapies use viral methods which also carry risks. By stimulating the body to produce the bone-producing protein itself, using nonviral methods these negative side effects can be avoided and bone tissue growth is promoted efficiently and safely.” Sequencing technique offers hope for leukemia patients A collaboration of researchers from the University of California, San Francisco, Pacific Biosciences and Mount Sinai School of Medicine has demonstrated that the gene FLT3 is a valid therapeutic target in Acute Myeloid Leukemia, AML, one of the most common types of leukemia. The discovery may help lead to the development of new drugs to treat AML. Andrew Kasarskis, vice chair of the Department of Genetics and Genomic Sciences at Mount Sinai School of Medicine, said: “By sequencing the FLT3 gene in AML patients who have relapsed on therapy targeted against FLT3, we have determined that FLT3 is a valid therapeutic target, and this will certainly help us better understand the physiology of this type of leukemia in order to help us develop new therapies in the future. In addition, sequencing hundreds of single molecules of FLT3 allowed us to see drug resistance mutations at low frequency. This increased ability to see resistance will let us identify the problem of the resistance sooner in a patient’s clinical course and help us take steps to address it. These finding may have great utility for drug development, as we can begin to test drugs, or a combination of drugs, in patients with AML who have relapsed. Furthermore, if we can find out when the drug resistant mutations occur exactly, clinicians may be able to prescribe another drug more quickly.”    


 

                                   
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