A 30-year-old woman with a history of upper respiratory infections had no idea she carried an immunodeficiency disorder – until her 6-year-old son was diagnosed with the same illness.
After learning she has common variable immunodeficiency (CVID), a disorder characterised by recurrent infections, such as pneumonia, and decreased antibodies, the woman, her husband, their three children and parents joined a multidisciplinary University of Utah study and researchers identified a novel gene mutation that caused the disease in the mom and two of her children. The researchers discovered that a mutation in the NFKB2 gene impairs a protein from functioning properly, which interferes with the body’s ability to make antibodies and fight infection. The children’s father did not have the mutation, nor did a third sibling or the woman’s parents.
Another 35 people with CVID were tested for the gene mutation, and one other unrelated person was found to have it. His father wasn’t tested, but no one else in his family immediate family had the mutation, so the researchers don’t know whether he could have inherited the disorder from his father or developed the gene mutation sporadically.
CVID typically doesn’t present with symptoms until adulthood and it’s not uncommon for someone to reach their 20s, 30s or beyond before being diagnosed, according to Karin Chen, M.D., co-first author of the study published Thursday, Oct. 17, 2013, in the American Journal of Human Genetics online. Identifying the NFKB2 mutation will make it easier to recognise and treat the disorder, particularly after a test developed in conjunction with the study by ARUP Laboratories becomes available as early as next May.
‘If we can screen patients for genetic mutations, we can identify disease complications associated with that gene, start looking for them and treating them sooner,’ says Chen, instructor of pediatric immunology at the University’s School of Medicine.
There’s no cure for CVID, but it can be treated with monthly infusions of antibodies at a cost of $5,000 to $10,000 per treatment.
Identifying the gene mutation and developing the test for it took approximately two years, a fast turnaround made possible because of the multidisciplinary research that the University of Utah Health Sciences encourages and is known for doing. The study involved researchers from the U School of Medicine’s Departments of Pediatrics, Pathology, Human Genetics and Program in Molecular Medicine and ARUP, which is a University-owned, nationwide testing laboratory.
Emily M. Coonrod, Ph.D., a research scientist with the ARUP Institute for Clinical and Experimental Pathology, is co-first author with Chen. Karl V. Voelkerding, M.D., also of the Institute for Clinical and Experimental Pathology and a U professor of pathology, is the senior author.
CVID probably is underdiagnosed, making it hard to know how common it is. But the disorder is estimated to occur in one in 10,000 people to one in 50,000 people, meaning it is one of more common types of immunodeficiency disorders, according to Chen. University physicians currently treat about 150 CVID patients in the Intermountain Region. Historically, CVID has been diagnosed clinically by doctors who are aware of the symptoms and then have individuals tested for low levels of antibodies.
No mutation had been identified in NFKB2 before this study. But Attila Kumánovics, M.D., assistant professor of pathology and co-author on the study, had perused the medical literature and found that a mouse model had been developed that carried a similar mutation in the NFKB2 gene and also had immunodeficiency. That was a key development, according to Voelkerding. ‘This meant that the finding in our patients could be correlated to literature.’ University of Utah Health Care

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

An international team of scientists—including researchers at GENYO, the Centre for Genomics and Oncological Research (Pfizer-University of Granada- Andalusian Regional Government)—has described a molecular mechanism that facilitates the defence of the human genome against ‘bombarding’ by mobile DNA sequences. Abnormalities in the mechanism could be responsible for some symptoms of DiGeorge syndrome, a rare disease. The research could in the future help develop new therapies against the disease, which is caused by the microdeletion of a small part of chromosome 22.

The study describes a sophisticated mechanism that enables all of our cells to control the uncontrolled movement of mobile DNA in our genomes. In patients with DiGeorge syndrome, the cells present abnormalities in the control mechanism. Currently, the research team are trying to generate stem cells that ‘suffer’ from the disease from cells donated by patients who have it—which would enable them to clarify the molecular base of this complex pathology.

DiGeorge syndrome, also known as deletion 22q11.2, is the most common genetic disease caused by a chromosome microdeletion in humans. It has an estimated prevalence of 1 in 4000 births and symptoms vary greatly. Typically, these affect the heart and immune system, as well as presenting as learning difficulties, mental retardation and psychiatric disorders.

The disease is characterised by absence of the ‘Microprocessor’ protein complex, which means these patients lack a ‘vigilante’ gene to watch out for repeated sequences and, therefore, are potentially susceptible to being bombarded by these DNA fragments.
Sara R. Heras—co-author of the study and GENYO researcher—explains that all our cells contain ‘Microprocessor’, a protein complex whose known function at the moment is that of generating small regulatory molecules of ribonucleic acid (RNA), known as microRNAs. ‘Our study has shown that this complex also acts as ‘vigilante’ and defends the integrity of the human genome. Hence, these proteins are capable of recognising and fragmenting the repeated DNA sequences that escape previous control mechanisms, thus preventing them from replicating and introducing themselves into the genome’. University of Granada

An overactive signalling pathway is a common cause in cases of pilocytic astrocytoma, the most frequent type of brain cancer in children. This was discovered by a network of scientists co-ordinated by the German Cancer Research Center (as part of the International Cancer Genome Consortium, ICGC). In all 96 cases studied, the researchers found defects in genes involved in a particular pathway. Hence, drugs can be used to help affected children by blocking components of the signalling cascade. The project is funded by the German Cancer Aid (Deutsche Krebshilfe) and the Federal Ministry of Education and Research (BMBF).
Brain cancer is the primary cause of cancer mortality in children. Even in cases when the cancer is cured, young patients suffer from the stress of a treatment that can be harmful to the developing brain. In a search for new target structures that would create more gentle treatments, cancer researchers are systematically analysing all alterations in the genetic material of these tumours. This is the mission of the PedBrain consortium, which was launched in 2010. Led by Professor Stefan Pfister from the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ), the PedBrain researchers have now published the results of the first 96 genome analyses of pilocytic astrocytomas.
Pilocytic astrocytomas are the most common childhood brain tumours. These tumours usually grow very slowly. However, they are often difficult to access by surgery and cannot be completely removed, which means that they can recur. The disease may thus become chronic and have debilitating effects for affected children.

In previous work, teams of researchers led by Professor Dr. Stefan Pfister and Dr. David Jones had already discovered characteristic mutations in a major proportion of pilocytic astrocytomas. All of the changes involved a key cellular signalling pathway known as the MAPK signalling cascade. MAPK is an abbreviation for ‘mitogen-activated protein kinase.’ This signalling pathway comprises a cascade of phosphate group additions (phosphorylation) from one protein to the next – a universal method used by cells to transfer messages to the nucleus. MAPK signalling regulates numerous basic biological processes such as embryonic development and differentiation and the growth and death of cells.

‘A couple of years ago, we had already hypothesised that pilocytic astrocytomas generally arise from a defective activation of MAPK signalling,’ says David Jones, first author of the publication. ‘However, in about one fifth of the cases we had not initially discovered these mutations. In a whole-genome analysis of 96 tumours we have now discovered activating defects in three other genes involved in the MAPK signalling pathway that have not previously been described in astrocytoma.’

‘Aside from MAPK mutations, we do not find any other frequent mutations that could promote cancer growth in the tumours. This is a very clear indication that overactive MAPK signals are necessary for a pilocytic astrocytoma to develop,’ says study director Stefan Pfister. The disease thus is a prototype for rare cancers that are based on defects in a single biological signalling process.

In total, the genomes of pilocytic astrocytomas contain far fewer mutations than are found, for example, in medulloblastomas, a much more malignant pediatric brain tumour. This finding is in accordance with the more benign growth behaviour of astrocytomas. The number of mutations increases with the age of the affected individuals.

About one half of pilocytic astrocytomas develop in the cerebellum, the other 50 percent in various other brain regions. Cerebellar astrocytomas are genetically even more homogenous than other cases of the disease: In 48 out of 49 cases that were studied, the researchers found fusions between the BRAF gene, a central component of the MAPK signalling pathway, and various other fusion partners.

‘The most important conclusion from our results,’ says study director Stefan Pfister, ‘is that targeted agents for all pilocytic astrocytomas are potentially available to block an overactive MAPK signalling cascade at various points. We might thus in the future be able to also help children whose tumours are difficult to access by surgery.’ German Cancer Research Center