Researchers from ERIBA, Radboud UMC, XJTU, Saarland University, CWI and UMC Utrecht have made a big step towards a better understanding of the human genome. By identifying large DNA variants in 250 Dutch families, the researchers have clarified part of the ‘dark matter’, the great unknown, of the human genome. These new data enable researchers from all over the world to study the DNA variants and use the results to better understand genetic diseases.

Although our knowledge of the human DNA is extensive, it is nowhere near complete. For instance, our knowledge of exactly which changes in our DNA are responsible for a certain disease is often insufficient. This is related to the fact that no two people have exactly the same DNA. Even the DNA molecules of identical twins have differences, which occur during their development and ageing. Some differences ensure that not everybody looks exactly alike, while others determine our susceptibility to particular diseases. Knowledge about the DNA variants can therefore tell us a lot about potential health risks and is a first step towards personalized medicine. Many small variants in the human genome – the whole of genetic information in the cell – have already been documented. Although it is known that larger structural variants play an important role in many hereditary diseases, these variants are also more difficult to detect and are, therefore, much less investigated.

By comparing the DNA of 250 healthy Dutch families with the reference DNA database the researchers were able to identify 1.9 million variants affecting multiple DNA ‘letters’. These variants include large sections of DNA that have disappeared, moved or even appear out of nowhere. When this happens in the middle of a gene that encodes a certain protein, it is likely that the functionality of the gene, and thus the production of the protein, is compromised. However, large structural variants often occur just before or after the coding part of a gene. The effect of this type of variation is hard to predict.

In the paper two occasions are described in which an extra piece of DNA was found just outside the coding region of a gene. In these occasions the variants had a demonstrable effect on the gene regulation. This proves that even structural variants that occur outside the coding regions need to be monitored closely in future DNA screenings. The catalogue of variants provided by this research enables other scientists to predict the occurrence of large structural variants from the known profile of the smaller ones. This technique opens new possibilities for studying the effects of large structural changes in our genomes.

Additionally, the research resulted in the discovery of large parts of DNA that were not included in the genome reference. This ‘extra’ DNA does contain parts that could be involved in the production of proteins. One of the extra pieces of DNA that was described in the paper is a new ‘ZNF’ gene that has previously never been found in humans. Nevertheless it appears to be present in roughly half of the Dutch population. This particular gene is a member of the ZNF gene family that was known from the reference genomes of several species of apes. The new variant will now be added to the human reference database. Authors subsequently showed that this gene is also present in genomes of several other human populations, however its function remains unknown. The fact that these and other pieces of ‘dark matter’ now have been placed on the genetic map enables scientists worldwide to study them and use the results to better understand human genetic diseases.

EurekAlert www.eurekalert.org/pub_releases/2016-10/su-mt100716.php

Coeliac disease is a chronic, immunological disease that is manifested as intolerance to gluten proteins present in wheat, rye and barley. This intolerance leads to an inflammatory reaction in the small intestine that hampers the absorption of nutrients. The only treatment is a strict, life-long, gluten-free diet.

It has been known for some time that coeliac disease develops in people who have a genetic susceptibility, but despite the fact that 40% of the population carry the most decisive risk factor (the HLA-DQ2 and DQ8 polymorphisms), only 1% go on to develop the disease. ‘What we have here is a complex genetic disease in which many polymorphisms play a role, each making a very small contribution to its development,’ explained the UPV/EHU researcher Ainara Castellanos, who has led the work published in Science.

One of the added risk factors is to be found, according to this research, in the so-called ‘junk’ DNA, in other words, in 95% of the DNA. It is the least-known part because, unlike the remaining 5%, it is not involved in synthesising proteins. Nevertheless, light is gradually being shed on its role in the control of the overall functioning of the genome, in other words, it regulates important processes in our organism such as immune response and that is where it might be possible to find the causes of auto-immune diseases such as coeliac disease.

A key gene in the regulating of the inflammatory response observed in coeliac patients has been found in one of the regions of the junk genome: it is the 1nc13. The ribonucleic acid produced by this gene belongs to the family of long, non-coding RNAs or lncRNA and is responsible for maintaining the normal levels of expression of pro-inflammatory genes. In coeliacs, this non-coding RNA is hardly produced at all so the levels of these inflammatory genes are not properly regulated and their expression is increased. But besides being produced in low quantities, the 1nc13 produced by coeliac patients has a variant that alters the way it functions. ‘That way an inflammatory environment is created and the development of the disease is encouraged,’ said Ainara Castellanos.

‘This study confirms the importance of the regions of the genome previously regarded as ‘junk’ in the development of common complaints such as coeliac disease and opens up the door to a new possibility for diagnosis. Right now, we are interested in finding out whether the low levels of this RNA are an early feature of coeliac disease (and of other immune diseases), which could be used as a diagnostic tool before its onset,’ explained the UPV/EHU’s lecturer in Genetics José Ramón Bilbao, another of the authors of the work. University of the Basque Country

The formation of large numbers of polyps in the colon has a high probability of developing into colon cancer, if left untreated. The large-scale appearance of polyps is often due to a hereditary cause; in this case the disease can occur in multiple family members. Under the leadership of human geneticists of the University Hospital Bonn, a team of researchers discovered genetic changes in the MSH3 gene in patients and identified a new rare form of hereditary colon cancer.

Colon polyps form like mushroom-shaped growths from the mucosa and are several millimetres to several centimetres in size. They are benign and generally do not cause any symptoms – however, they can turn into malignant tumours (colon cancer). Physicians refer to the development of a large number of polyps in the colon as ‘polyposis.’ Scientists have already discovered several genes associated with a polyposis. ‘However, about one-third of families affected by the disease do not have any abnormalities in these genes,’ says Prof. Dr. Stefan Aretz, head of the working group at the Institute of Human Genetics at the University of Bonn Hospital. Therefore, there would have to be even more genes involved in the formation of polyps in the colon.

Together with pathologists from the University Hospital Bonn, scientists from the Yale University School of Medicine in New Haven (USA), and the Frankfurt University Hospital, the team working with Prof. Aretz investigated the genetic material (DNA) of more than 100 polyposis patients using blood samples. In each patient, all of the about 20,000 protein-coding genes known were simultaneously examined. In this process, the scientists filtered the rare, possibly relevant genetic changes out of the gigantic quantity of data, like the proverbial needle in a haystack. In two patients, genetic changes (mutations) were discovered in the MSH3 gene on chromosome 5.

Proof of causes is like a trial based on circumstantial evidence

‘The challenge is proving the causal connection between the mutations in this gene and the disease,’ says Prof. Aretz. The process is similar to that of a trial based on circumstantial evidence. Family members also play a role here: The siblings with the disease have to have these same MSH3 mutations as the patient who was first examined, but not the healthy relatives. That was the case. In addition, the scientists investigated the consequences for patients resulting from the loss of function of the MSH3 gene. ‘It involves a gene for the repair of the genetic material,’ reports Dr. Ronja Adam, one of the two lead authors from Prof. Aretz’s team. ‘The mutations cause the MSH3 protein to not be formed.’ Since the protein is missing in the cell nucleus of the patient´s tissues, there is an accumulation of genetic defects. The mutations which are not repaired then predispose to the more frequent occurrence of polyps in the colon.

The newly discovered type of polyposis, in contrast to many other forms of hereditary colon cancer, is not inherited dominantly, but instead recessively. ‘This means that siblings have a 25 percent chance of developing the disease; however, the parents and children of affected persons only have a very low risk of developing the disease,’ explains Dr. Isabel Spier from the Institute of Human Genetics, who was also very involved in the study.

The annual colonoscopy is the most effective cancer screening method for polyposis patients. As a result, the development of colon cancer can be effectively prevented. By investigating the MSH3 gene, a clear diagnosis can be made prospectively in some other, previously unexplained polyposis cases. Afterwards, healthy persons at risk in the family can be tested for the mutations. ‘Only proven carriers would need to take part in the intensive surveillance program,’ says the human geneticist. In addition, science would gain new insights into the development and biological foundations of tumours through the identification of mutations in the MSH3 gene. Prof. Aretz: ‘The knowledge about molecular mechanisms which lead to cancer is also a precondition for the development of new targeted drugs.’

University of Bonn www.uni-bonn.de/Press-releases/discovery-of-a-novel-gene-for-hereditary-colon-cancer

Researchers at Brigham and Women’s Hospital have found two new potential drug targets for treating arterial diseases such as atherosclerosis. By using proteomics to screen a vast number of molecules, the researchers identified PARP9 and PARP14 – two members of the PARP family of proteins – as regulators of macrophage activation, which has been linked to arterial disease by systems biology.

Though the mechanisms that activate macrophages, a type of digestive white blood cell that targets foreign cells, remain incompletely understood, previous research shows that macrophages play an important role in the development of atherosclerosis and its thrombotic complications. Masanori Aikawa, MD, PhD, director of the Center for Interdisciplinary Cardiovascular Sciences (CICS) at the Brigham, his research fellow Hiroshi Iwata, MD, PhD, and colleagues studied atherosclerosis on the protein-level to determine which molecules were most involved in the regulation of macrophages.

Once Aikawa and his colleagues narrowed down their search to these two proteins, they silenced each gene in cultured macrophages and found that tamping down PARP14 increased macrophage activation while tamping down PARP9 had the opposite effect.

Aikawa founded CICS and hopes that this hypothesis-generating method can be used to streamline the lengthy process of drug development. Aikawa and CICS are using a more systematic approach which hinges on network analysis; this analysis predicts which pathways are most likely to control their studied effect so that they can prioritize these pathways. Ideally, this process would take a fraction of the time in comparison to searching through each individual pathway unaware of their likelihood of affecting their studied effect.

Aikawa and his colleagues plan to augment these findings to develop targeted therapeutics for atherosclerosis and other diseases.

‘Macrophage activation plays a role in not only vascular disorders but also various inflammatory and autoimmune diseases,’ said Aikawa. ‘These results could provide important information about the mechanisms of these diseases and help to develop much needed new therapeutics.’

EurekAlert www.eurekalert.org/pub_releases/2016-10/bawh-aot102516.php

Researchers at UMass Medical School have identified a new molecular pathway critical for maintaining the smooth muscle tone that allows the passage of materials through the digestive system. This finding, based on studying calcium ion-controlled pathways in mice, may lead to new treatments for a host of digestive disorders ranging from common gastroesophageal reflux disease (GERD), to swallowing disorders, incontinence and pancreatitis.
“We are excited about the potential to target identified genes to treat disorders such as reflux and incontinence,” said Ronghua ZhuGe, PhD, associate professor of microbiology and physiological systems and a senior author of the study. “Knowing how these muscles stay contracted for such long periods of time will allow us to develop potential new treatments for these diseases. The next step is to see whether this molecular mechanism in mice also operates in humans.”

The human body, and those of other mammals, contains a number of ring-shaped structures made of smooth muscle encircling openings in hollow organs such as the intestines and bladder called sphincters. Smooth muscle is involuntarily controlled, unlike the muscles we use to walk, for example, so that we don’t need to consciously move digested food from stomach to small intestine. Dysfunction in the sphincters, either structurally or functionally, can have severe consequences leading to diseases that impair the ability of the muscle to contract or relax. This can lead to achalasia, which makes it difficult to swallow; gastroesophageal reflux disease (GERD), which allows stomach acid to enter the oesophagus or incontinence of the bowels.

“A healthy sphincter opens transiently but remains closed most of the time, maintaining a basal tone. This basal tone requires constant generation of force produced by the contraction of smooth muscle cells that make up the sphincters,” said Dr. ZhuGe. “However, the genetics governing how the sphincter smooth muscle stays contracted for such long periods of time remains unknown.”

Smooth muscle operates by generating force as the muscle motor protein myosin and actin filaments move past each other. This happens after a molecule called the myosin regulatory light chain (MLC) is turned on through a common molecular transformation called phosphorylation. How much phosphorylation takes place is controlled by the relative amounts of two enzymes; calcium dependent MLC kinase (MLCK), which promotes phosphorylation, and calcium independent MLC phosphatase (MLCP), which reverses phosphorylation. Through this process, contraction and relaxation of the muscle is achieved.

To understand the molecular mechanism responsible for the involuntary and continuous contraction of the sphincter muscle, Dr. ZhuGe and colleagues examined the internal anal sphincter that controls bowel continence in mice. They showed that genetic deletion of the MLCP enzyme in the smooth muscle had no effect on the basal tone of the mouse sphincter, but deletion of MLCK essentially abolishes the basal tone and mice become incontinent as a result.

“Although previous biochemical studies suggested that lower MLCP activity may be related to the basal tone of this sphincter, our genetic study indicates this doesn’t seem to be the case. It turns out MLCK is essential for the tone formation,” said ZhuGe. “This prompted us to look for specific calcium signals that regulate MLCK.”

Co-author Lawrence Lifshitz, PhD, associate professor of molecular medicine, said, “Calcium signalling is our favourite subject. We originally hypothesized that localized releases of calcium inside the cell, near target ion channels, might do the trick, as we knew that such releases can regulate the contraction of smooth muscle in blood vessels and airways.”

But experiments showed that these local calcium releases have no direct role in muscle tone. Instead, three types of ion channels act in concert to generate a rise in cytosolic calcium which eventually results in MLCK activation and muscle tone.

To test this hypothesis, ZhuGe’s group teamed up with Minsheng Zhu, PhD, at Nanjing University in China, to generate a line of mice in which one of these channels could be turned off in smooth muscle only. “These mice were a powerful tool for establishing our hypothesis,” said ZhuGe. “They helped us identify the Tmem16a (also called Ano1) gene as a critical component for basal tone formation and fecal continence. When we were able to turn off the TMEM16A channels in these mice, they lost the majority of the basal tone and became incontinent. ”

UMass Medical School www.umassmed.edu/news/news-archives/2016/04/umms-scientists-identify-genes-that-control-smooth-muscle-contraction-in-digestive-system/