A new test from Washington University’s Genomic Pathology Services will help physicians quickly zero in on genetic mutations that may be contributing to kidney disease.
Many kidney disorders are difficult to diagnose. To address this problem, scientists and clinicians have developed a diagnostic test that identifies genetic changes linked to inherited kidney disorders. This testing is now available nationwide through Genomic Pathology Services (GPS) at Washington University School of Medicine in St. Louis.

“For many kidney diseases, diagnosis can be an odyssey in which you sequence one gene after another over a long period of time to learn what’s going wrong and what the best options are for treatment,” said GPS chief medical officer and Washington University pathologist Jonathan Heusel, MD, PhD. “It makes more sense to screen all the possible contributing genes at once with a single test and consider options for treatment.”
To make this possible, the GPS team developed the test with kidney disease specialists, including Joseph Gaut, MD, PhD, a renal pathologist.

The test employs next-generation sequencing technology to decode genes associated with kidney disease. Using software developed at the university, clinical genomics specialists analyse and interpret the observed genetic alterations to identify disease-related genetic changes, or variants. They then must determine whether a given variant poses clinical risks based on available medical knowledge.

“The variants have to be evaluated on a case-by-case basis, which can be time-consuming and labour-intensive,” Heusel said.

GPS continues to update the kidney test as new links between kidney problems and DNA are identified.

“We stay abreast of the literature, and as new genes become clinically meaningful, we will incorporate those into the test,” said Catherine Cottrell, PhD, medical director for GPS.

The kidney test will check for:
•          Alport syndrome, which is characterized by progressive loss of kidney function, hearing loss and eye abnormalities;
•          Nephrotic syndrome, which includes symptoms such as protein in the urine, low blood-protein levels, high levels of cholesterol and triglycerides, and swelling;
•          Metabolic disorders associated with renal disease and including other systemic abnormalities such as diabetes, amyloidosis and others;
•          Complement (immune system) defects related to kidney disease, including atypical hemolytic uremic syndrome. Washington University

By combing through the DNA of more than 100,000 people, researchers at Broad Institute, Massachusetts General Hospital, and elsewhere have identified rare, protective genetic mutations that lower the levels of LDL cholesterol — the so-called “bad” cholesterol — in the blood. The researchers’ findings reveal that these naturally occurring mutations also reduce a person’s risk of coronary heart disease by about 50 percent. Remarkably, the mutations disrupt a gene called Niemann-Pick C1-Like 1 (NPC1L1) — the molecular target of the FDA-approved drug ezetimibe, often used as a treatment for high LDL.

“Protective mutations like the one we’ve just identified for heart disease are a treasure trove for understanding human biology,” said Sekar Kathiresan, a senior author of the study, Broad associate member, and director of preventive cardiology at Massachusetts General Hospital. “They can teach us about the underlying causes of disease and point to important drug targets.”

Over the past several years, evidence has been mounting that certain loss-of-function mutations — mutations that reduce or completely eliminate a gene’s ability to work — can, at the same time, protect against disease. With this latest discovery, the list now stands at four genes that have been found to offer protective effects against either heart or metabolic disease. (The genes PCSK9, AP0C3, and now NPC1L1 have been found to protect against heart disease, and SLC30A8 has been shown to protect against type 2 diabetes.)

The scientific community is interested in these protective mutations not only because of what they can reveal about the biological basis of disease, but also for their ability to suggest potential paths toward new therapeutics. From a pharmaceutical perspective, it is much more feasible to develop a drug that disables, rather than activates, a gene.

Kathiresan’s long-standing interest in the genetics of blood cholesterol and heart disease first led him to uncover rare mutations in the NPC1L1 gene in just a handful of patients. He wondered if other patients carried similar mutations, so he set off on a massive hunt.

With the combined expertise of Broad Institute’s Genomics Platform, led by Stacey Gabriel, and major support from the National Human Genome Research Institute, Kathiresan and his colleagues sequenced the exomes (the protein-coding portions of the genome) of over 20,000 people of European, African, or South Asian ancestry. They discovered 15 distinct mutations in NPC1L1, all of which serve to inactivate or dampen gene activity. Roughly 1 in 650 people carries one of these inactivating NPC1L1 mutations.

“When it comes to rare variant studies, there is simply no substitute for extremely large sample sizes,” said co-author Gabriel, director of Broad Institute’s Genomics Platform. “This has become crystal clear through our work on NPC1L1 as well as several other similar projects here at the Broad. We now know the right path to get statistically robust results, and that’s the path we are on.”

After defining the mutational landscape of NPC1L1 in the initial study group of 20,000 people, Kathiresan and his colleagues correlated those mutations with LDL levels. The researchers examined the genomes of another 91,000 people and found that those with inactivating mutations in NPC1L1 tended to have lower LDL levels than those without such mutations. The reductions averaged about 12mg/dL, a 10 percent drop that is similar to what is seen in patients receiving ezetimibe therapy.

Individuals who carry inactivating NPC1L1 mutations also have a lower risk of coronary heart disease — roughly half the risk compared to those individuals without those mutations. Broad Institute

AMSBIO reports on the recent publication in Cell by Dr Meritxell Huch, Prof Hans Clevers et al. of ground-breaking research using Cultrex BME2 reduced growth factor (organoid growth matrix) to enable long-term (>1 year) culture of genome-stable bipotent stem cells from adult human liver. These results open up new experimental avenues towards the use of human liver material expanded in vitro as an alternative cell source for disease modelling, toxicology studies, drug testing, regenerative medicine and gene therapy.
Failure in the management of liver diseases can be attributed to the shortage of donor livers as well as to our poor understanding of the mechanisms behind liver pathology. The value of any cultured cell as a disease model or as a source for cell therapy transplantation depends on the fidelity and robustness of its expansion potential as well as its ability to maintain a normal genetic and epigenetic status.
The research by Huch, Clevers et al. shows that primary human bile duct cells can readily be expanded in vitro as bipotent stem cells into 3D liver organoids using AMSBIO’s reduced growth factor basement membrane extract Cultrex BME2 as extracellular matrix (ECM). In this novel culture system, adult liver stem cells maintain their ability of self-renewal, differentiating into functional hepatocyte cells in vitro and generating bona fide hepatocytes upon transplantation. Extensive analysis of the genetic stability of cultured organoids in vitro demonstrates that the expanded cells preserve their genetic integrity over months in culture (agreeing with the authors’ previous observations in a mouse model). Organoids derived from patients with genetic disorders can be used to model liver disease in vitro.
Commenting on this research, Dr. Huch said “We have obtained culture conditions that allow us to long-term expand genetically stable human donor liver cells in organoid culture. One of the clues to this success is the use of ECM that allows the cells to grow in 3D. BME2 has been our ECM of choice for these experiments.”
AMSBIO has been working with the variability of the cellular microenvironment and how it affects the physiological relevance of cell culture. Factors contributing to this variability include: organ specific stromal cells, growth factors, proteoglycan and protein composition, and stiffness or tensile strength of the basement membrane extract or extracellular matrix. Matrices from AMSBIO not only support cells and cell layers, but also play an essential role in tissue organization that affects cell adhesion, migration, proliferation, and differentiation. Cultrex® Basement Membrane Extract (BME) is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumour. The extract gels at 37°C to form a reconstituted basement membrane. Major components of BME include laminin, collagen IV, entactin, and heparan sulfate proteoglycan.  These extracted proteins can be used in multiple applications, under a variety of cell culture conditions, for maintaining growth or promoting differentiation of primary endothelial, epithelial, smooth muscle and stem cells. BME can also be utilized in cell attachment, neurite outgrowth, angiogenesis, in vitro cell invasion and in vivo tumorigenicity assays. The new BME 2 is a proprietary formulation that has a higher tensile strength than similar products such as Matrigel®.
Dr Meritxell Huch is based at the Gurdon Institute, University of Cambridge, UK; and Professor Hans Clevers is Professor and Director of the Hubrecht Institute and President of the Royal Netherlands Academy of Arts and Sciences.

www.amsbio.comhttp://tinyurl.com/ouyktb3

Down syndrome is the most common chromosomal abnormality in humans, involving a third copy of all or part of chromosome 21. In addition to intellectual disability, individuals with Down syndrome have a high risk of congenital heart defects. However, not all people with Down syndrome have them – about half have structurally normal hearts.

Geneticists have been learning about the causes of congenital heart defects by studying people with Down syndrome. The high risk for congenital heart defects in this group provides a tool to identify changes in genes, both on and off chromosome 21, which are involved in abnormal heart development.

Researchers at Emory University School of Medicine, with colleagues at Johns Hopkins University, Oregon Health Science University, and University of Pittsburgh, report results from the largest genetic study of congenital heart defects in individuals with Down syndrome.

The team found that infants with congenital heart defects, in the context of Down syndrome, were more likely to have rare, large genetic deletions. Those deletions tended to involve genes that affect cilia, cellular structures that are important for signalling and patterning in embryonic development.

These new findings, along with other recent studies, suggest that the risk for congenital heart defects in Down syndrome can come from several genes and environmental factors, in addition to the substantial risk from the extra chromosome 21.

“In Down syndrome, there’s a 50-fold increase in risk for heart defects, which is enormous,” says senior author Michael Zwick, PhD, associate professor of human genetics and paediatrics at Emory. “Studying congenital heart defects in the ‘at risk’ Down syndrome population can make it possible to reveal genes that impact the risk of heart defects in all children, including those with typical number of chromosomes.”

“Understanding the origin of heart disorders in individuals with Down syndrome may reveal aspects of biology that would allow better personalization of their health care, since genetic alterations that affect the heart may also affect other organs, such as the lungs or gut,” Zwick says.

“Our partnership with families who have a child with Down syndrome and our investment in a comprehensive clinical data and biorepository will continue to provide resources to study not only heart defects, but also other Down-syndrome associated medical conditions such as cognitive function, leukaemia,  and dementia,” says co-author Stephanie Sherman, PhD, professor of human genetics at Emory University School of Medicine.

The study included 452 individuals with Down syndrome. 210 had complete atrioventricular septal defects (AVSDs), a serious heart defect that is relatively common among those with Down syndrome (about 20 percent). The remaining 242 had structurally normal hearts. The Emory team used high density microarrays to probe more than 900,000 sites across the human genome to detect structural variation, including deletions or duplications of DNA.

An atrioventricular septal defect means that the central region of the heart separating the atria from the ventricles has failed to form properly. Such defects increase the workload on the heart, and a complete AVSD leads to heart failure: fluid buildup in the lungs and difficulty breathing, requiring surgery in the first year of life.

The team’s results add to evidence for a connection between AVSDs and cilia. Ciliopathies are a class of genetic disorders that include kidney, eye, and neurodevelopmental disorders. Cells in the airways have mobile cilia which sweep mucus and dirt out of the lungs, but almost every cell in the body has a primary (sensory) cilium.

“The finding that ciliome genes may be disrupted in children with Down syndrome and AVSD may indicate differences in life-time care for these individuals,” Zwick says. “This is a suggestive result that needs replication in a larger group.”

To confirm and strengthen the findings, Zwick and his team are currently performing an independent study of individuals with Down syndrome, using whole genome sequencing to further delineate alterations in genes that perturb heart development in children. Emory Health Sciences

Prostate cancer researchers have drawn a molecular portrait that provides the first complete picture of localized, multi-focal disease within the prostate and also unveils a new gene subgroup driving it.

The discoveries are a further step along the road to personalizing prostate cancer medicine say study co-leads, Dr. Robert Bristow, a clinician-scientist at Princess Margaret Cancer Centre, and Dr. Paul Boutros, an investigator at the Ontario Institute for Cancer Research.

‘Our research shows how prostate cancers can vary from one man to another – despite the same pathology under the microscope – as well as how it can vary within one man who may have multiple tumour types in his prostate,’ says Dr. Bristow. He goes on to say, ‘these sub-types may be important to determining the response to surgery or radiotherapy between patients.’

The study involved molecular profiling of 74 patients with Gleason Score 7 index tumours. (Gleason is the classification system used to evaluate aggressiveness in prostate tumours). Of these, whole-genome sequencing was done on 23 multiple tumour specimens from five patients whose prostates were removed at surgery.  By carefully analysing the genetics of each focus of cancer within each prostate, the researchers could assign ‘aggression scores’ to each cancer which revealed that even small cancers can contain aggressive cells capable of altering a patient’s prognosis.

Dr. Boutros, explained that the more detailed analysis clearly identified that two members of the MYC cancer gene family were at play in disease development, and that one of them – ‘C-MYC’ – was the culprit driving aggressive disease. The other one – ‘L-MYC’ – is already known to be implicated in lung and other cancers.

‘This discovery of a new prostate cancer-causing gene gives researchers a new avenue to explore the biology of the disease and improve treatment,’ says Dr. Paul Boutros, a principal investigator at the Ontario Institute for Cancer Research.

‘By showing that mutations in prostate cancer vary spatially in different regions of a tumour, this study will aid in the development of new diagnostic tests that will improve treatment by allowing it to be further personalized.’ University Health Network