Sequencing cancer mutations

Using precise information about an individual’s genetic makeup is becoming increasingly routine for developing tailored treatments for breast, lung, colon and other cancers. But techniques used to identify meaningful gene mutations depend on analysing sequences of both normal and mutant DNA in tumour samples, a process that can yield ambiguous results. Now, a team of Johns Hopkins researchers says it has developed an easy-to-use online computer software application that can clear up any confusion faster and cheaper than other methods currently used to do the job.

The free web-based app is called “Pyromaker” and it generates simulated pyrograms, which are readouts from a gene sequencing technique known as pyrosequencing. Most pyrograms correspond precisely to a person’s unique mutation or set of mutations, but some mutations can be more difficult to interpret than others.

Pyrosequencing works on shorter stretches of DNA than does the traditional method, known as “Sanger”, named for Frederick Sanger who invented the process. But pyrosequencing is also more sensitive in registering the presence of mutant DNA in a tumour sample, which is a mix of tumour and normal cells. That sensitivity makes it very useful for tumour sequencing, says James R. Eshleman, a professor in the departments of pathology and oncology at Johns Hopkins, because the mutant genes that drive a tumour’s abnormal growth typically are less prevalent in a tumour sample, compared with normal versions of those genes.

Pyromaker also may be used with a new sequencing technology known as ion semiconductor sequencing, which detects a change in hydrogen ions instead of a light pulse.


Study finds answers about chromosomal link to lymphomas and leukaemia

A new study by scientists from the US National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), part of the National Institutes of Health, resolves longstanding questions about the origin of recurrent chromosomal rearrangements, known as translocations that drive lymphomas and leukaemias in humans. Translocations occur when broken strands of DNA from one chromosome are erroneously joined with those of another chromosome, thus deregulating genetic information and leading to cell transformation. Sometimes chromosomal rearrangements can be beneficial, in that they enable the immune system to respond to a vast number of microorganisms and viruses. However, translocations can result in tumours.

Specific chromosomal translocations driving human cancer have been known since 1960, when two scientists in Philadelphia, Peter Nowell and David Hungerford, first visualised one such lesion in patients suffering from chronic myeloid leukaemia, an aggressive form of cancer in the blood. The origin of such malignant rearrangements, however, has been unclear. At least three theories have been put forward to explain their aetiology: translocations between two genes are driven by how frequently the genes interact in the nucleus of tumour precursor cells; translocating genes undergo DNA damage more frequently than non-translocating genes; and all genes in the genome have about an equal chance of translocating with one another, but certain translocations are particularly selected because they drive cell transformation.

In the new study, NIAMS scientists explored the three theories and found that the frequency of DNA damage was directly proportional to the frequency of translocation. Intriguingly, the researchers found that an enzyme, called AID, damages approximately 150 genes in the B cell genome, thus making them susceptible to translocations. Among the targeted genes, many have been previously shown to be translocated in human cancer. Further study also revealed that, in the absence of AID, gene proximity or interaction frequency was the driving force behind translocations.

The new results not only clarify the origin of tumour-inducing translocations, but they also suggest that finding ways to stop AID could potentially prevent the development of many human cancers.

Gene therapy for Cystic Fibrosis to be tested

UK Cystic Fibrosis Gene Therapy Consortium (GTC) has launched a trial into Cystic Fibrosis (CF), involving 130 CF patients, both adults and children. The trial has been funded by a £3.1million (US$4.97million) grant from the US National Institute for Health Research (NIHR) and the Medical Research Council (MRC) through the Efficacy and Mechanism Evaluation programme. The MRC, through their Developmental Pathway Funding Scheme (DPFS), will also fund a £1.2million study by the GTC aiming to develop a potentially more efficient delivery method for the gene therapy, which could contribute to an even more effective treatment in the future.

The GTC is a group of scientists and clinical teams from Imperial College London, the Universities of Oxford and Edinburgh, Royal Brompton & Harefield NHS Foundation Trust and NHS Lothian who have worked together for the last decade to develop gene therapy for CF.

The cause of CF, mutations in a gene located on chromosome 7, was identified in 1989, opening the door to replacing this faulty gene using gene therapy. Patients will receive the treatment by inhaling molecules of DNA wrapped in fat globules that deliver the replacement gene into the cells in the lung lining. Half the participants will receive the real treatment and half a placebo in a double-blind study. Patients aged 12 and over at Royal Brompton Hospital, London and Western General and Royal Hospital for Sick Children in Edinburgh, will receive one dose a month for one year.

Over 30 patients have each received a single dose of the gene therapy in the Consortium's previous studies, looking at how effective the therapy is at replacing the protein encoded by the defective CF gene. By delivering multiple doses over the course of a year, the researchers aim to determine whether the therapy can improve symptoms for CF patients. The second study, which will be lab based, will investigate a more advanced version of the therapy using a modified virus to carry the replacement gene into the lungs, which could in future lead to a more efficient delivery mechanism.

Professor Eric Alton, the GTC Coordinator, from Imperial College London and consultant physician at Royal Brompton Hospital, said: “Conventional treatments have extended the life expectancy for people with CF. We’re hoping that this therapy will achieve a step change in the treatment of CF that focuses on the basic defect rather than just addressing the symptoms. This trial will assess if giving gene therapy repeatedly for a year will lead to the patients’ lungs getting better. Eventually we hope gene therapy will push CF patients towards a normal life expectancy and improve their quality of life significantly.”

The outcome of the trial will be known in spring 2014 and regular progress reports will be posted on the Consortium's website.

CF Gene Therapy

Newly viral genome could change what we know about virus evolution

A study published in BioMed Central’s Biology Direct journal reports the existence of a previously undetected group of viruses and, more importantly, a new type of viral genome that could have huge implications for theories of viral emergence and evolution.

Viruses are the most abundant organisms on earth, yet little is known about their evolutionary history since they have exceptionally high rates of genetic mutation which are difficult to track. Viral metagenomics, however, is becoming an increasingly useful tool with which to glimpse virus evolution, as it makes available vast amounts of new sequence data for analysis.

Kenneth M. Stedman’s team from Portland State University in Oregon, USA, used a metagenomics approach to investigate virus diversity in Boiling Springs Lake in Lassen Volcanic National Park, USA, an acidic, high temperature lake (ranging from 52-95°C, with a pH of ~2.5) that sustains a purely microbial ecosystem.

Astonishingly, they found a unique viral genome that has never before been reported – a circular, single-stranded DNA virus encoding a major capsid protein seen previously only in RNA viruses. This unusual genome provides proof that integration of an RNA virus into a DNA virus may have occurred between two unrelated virus groups at some point in evolution – something that has not been observed before. Moreover, this suggests that entirely new virus types may emerge via recombination of functional and structural modules between vastly different viruses, using mechanisms that are as-yet unknown.

The team observed that the Boiling Springs Lake RNA-DNA hybrid virus (BSL RDHV) genome is circular, but its size is roughly double that of typical circoviruses, with the ORFs arranged in an uncommon orientation. They compared the BSL RDHV genome to other metagenomic DNA sequences from the Global Ocean Survey, and found strong evidence to conclude that previously undetected BSL RDHV-like viruses could be widespread in the marine environment and are likely to be found in other environments as well.

No mechanism has been proposed to account for the inferred instances of interviral RNA-DNA recombination. doi:10.1186/1745-6150-7-13  


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