Research on an investigational DNA methylation test for colorectal cancer demonstrated that the only clinical variable that influenced test results was age, according to findings presented by researchers from the Mayo Clinic in Rochester, USA, at the AACR Annual Meeting 2012, held from March 31 – April 4. The group at the Mayo Clinic, in collaboration with Exact Sciences, developed the multimarker molecular stool test, which is highly sensitive to the critical cancer screening targets of early-stage cancers and precancerous adenomas. The researchers examined common patient variables, including age, gender, race, alcohol consumption, tobacco use, body mass and medication use in 500 patients undergoing screening colonoscopy or polyp follow-up. Patients had a normal colonoscopy in the last three years.With the exception of age, none of the variables influenced test results, nor did family history of colorectal cancer or polyps or personal history of polyps. Researchers have now selected the two markers least affected by age for further test development and validation based on these study results to try and minimise false positives and avoid unnecessary colonoscopies.

A new genetic mutation that causes familial amyotrophic lateral sclerosis (ALS), a fatal neurological disorder also known as Lou Gehrig’s Disease, has been identified by a team of scientists led by researchers at the University of Massachusetts Medical School (UMMS). Mutations to the profilin (PFN1) gene, which is essential to the growth and development of nerve cell axons, is estimated to account for one to two percent of inherited ALS cases. The finding points to defects in a neuron’s cytoskeleton structure as a potential common feature among diverse ALS genes.
‘This discovery identifies what may possibly be a common biological mechanism involved across familial ALS cases regardless of genetics,’ said John Landers, PhD, associate professor of neurology and senior author of the study. ‘We know of at least three other ALS genes, in addition to PFN1, that adversely impact axon growth. If indeed, this is part of the disease’s mechanism, then it might also be a potential target for therapeutics.’
Robert Brown, MD, DPhil, a co-author on the study and chair of neurology at UMass Medical School, said ‘Dr. Landers has done great work in defining this new pathway for motor neuron death. We are delighted to have identified the defects in families from the U.S., Israel and France that we have been investigating for several years. Our finding is particularly exciting because it may provide new insights into ALS treatment targets.’
ALS is a progressive, neurodegenerative disorder affecting the motor neurons in the central nervous system. As motor neurons die, the brain’s ability to send signals to the body’s muscles is compromised. This leads to loss of voluntary muscle movement, paralysis and eventually respiratory failure. The cause of most cases of ALS is not known. Approximately 10 percent of cases are inherited. Though investigators at UMass Medical School and elsewhere have identified several genes shown to cause inherited or familial ALS, almost 50 percent of these cases have an unknown genetic cause.
The current study details the discovery of the PFN1 gene mutation among two large ALS families. Both families were negative for known ALS-causing mutations and displayed familial relationships that suggested a dominant inheritance mode for the disease. For each family, two affected members with maximum genetic distance were selected for deep DNA sequencing. To identify an ALS-causing mutation, genetic variations between the family members were identified and screened against known databases of human genetic variation, such as the 1000 Genomes Project. This narrowed down the resulting number of candidate, ALS-causing mutations to two within the first family and three within the second. Interestingly, both families contained different mutations within the same gene – PFN1, the likely causative mutation. With additional screening, the team documented that in a total of 274 families sequenced, seven contained a mutation to the PFN1 gene, establishing it as a likely cause for ALS.
While it is not certain how the PFN1 mutation causes ALS, the cellular functions it controls within the motor neurons are responsible for regulation of a number of activities, including the growth and development of the axon, the slender projection through which neurons transmit electrical impulses to neighboring cells, such as muscle. When introduced into motor neuron cells, normal PFN1 protein was found diffused throughout the cytoplasm. Conversely, the mutant PFN1 observed in ALS patients was found to collect in dense aggregates, keeping it from functioning properly. Motor neurons producing mutated PFN1 showed markedly shorter axon outgrowth.
‘The discovery that mutant PFN1 interferes with axon outgrowth was very exciting to us,’ said Claudia Fallini, PhD, a postdoctoral researcher at Emory University School of Medicine who collaborated with the UMass Medical School authors to investigate PFN1’s functions in cultured motor neurons. ‘It suggests that alterations in actin dynamics may be an important mechanism at the basis of motor neuron degeneration.’
‘In healthy neurons, PFN1 acts almost like a railroad tie for fibrous filaments called actin, which make up the axon’ said Landers. ‘PFN1 helps bind these filaments to each other, promoting outgrowth of the axon. Without properly functioning PFN1 these filaments can’t come together. Here we show that mutant PFN1 may contribute to ALS pathogeneses by accumulating in these aggregates and altering the actin dynamics in a way that inhibits axon outgrowth.’ EurekAlert

Researchers at National Jewish Health have discovered a novel genetic mechanism of immune deficiency. Magdalena M. Gorska, MD, PhD, and Rafeul Alam, MD, PhD, identified a mutation in Unc119 that causes immunodeficiency known as idiopathic CD4 lymphopenia. Unc119 is a signalling protein that activates and induces T cell proliferation. The mutation impairs Unc119 ability to activate T cells.
‘A better understanding of the molecular mechanisms associated with this mutation will improve diagnosis and pave the way for development of new therapies,’ said Dr. Gorska.
Nearly a decade ago Drs. Alam and Gorska identified Unc119 as a novel activator of SRC-type tyrosine kinases, important regulators of cellular function. Since then, they have published numerous papers where they characterised the function of this protein in various aspects of the immune system.
Idiopathic CD4 lymphopenia is a rare and heterogeneous syndrome defined by low levels of CD4 T cells in the absence of HIV infection, which predisposes patients to infections and malignancies. Recent research by others had linked the syndrome to reduced activation of the SRC-type kinase known as Lck. The latter kinase is involved in T cell development, activation and proliferation.
So, Drs. Alam and Gorska thought Unc119, an activator of Lck, might be involved. They kept an eye out for patients with CD4 lymphopenia coming to National Jewish Health, which specialises in immune-related disorders as well as respiratory and cardiac diseases. They identified three patients with CD4 lymphopenia, then sequenced their Unc119 gene as well as the Unc119 gene in several patients who suffered low CD4 T cell counts as a result of other conditions.
One of the three patients, a 32-year-old woman with a history of recurrent infections, had a missense mutation in her Unc119 gene. The same mutation was not present in other lymphopenia patients nor in any genetic database.
The researchers then performed several studies with the woman’s blood cells, to understand the mutation’s effect. They introduced the mutated gene into normal T cells and examined the outcome.
The mutation prevents Lck activation and its downstream signalling. It also reduces the amount of Lck found near the plasma membrane, where it plays a major role in propagating signals from the T-cell receptor. Proliferation of T cells, which normally occurs on stimulation of T-cell receptors, was profoundly reduced in cells from the patient.
‘Since we originally published our findings earlier this year, we have received inquiries from many physicians with lymphopenia subjects,’ said Dr. Alam. ‘Working with them, we expect to find several more patients with this novel mutation, which should help us better understand its effect, improve diagnosis and possibly find therapies.’
At this point there is no treatment for CD4 lymphopenia caused by this mutation other than close monitoring of the patient and treatment of resulting infections and malignancies. National Jewish Health

As the NHS cervical cancer screening programme (CSP) rolls out high risk human papilloma virus (HR-HPV) testing for the triage of women with borderline or low-grade cervical abnormalities across England, the Roche cobas HPV test has already enabled more than 30 laboratories to be ready to offer an HPV testing service.

In the financial year 2011/2012, local cervical screening programmes have been preparing to present a business case for approval and central support funding (for the first two years) to support the implementation of HPV Triage and Test of Cure.  Only programmes with a minimum annual workload of 35,000 can apply and the HPV assay used must be approved by NHS CSP [1].  Currently, only five commercially available HPV tests have been approved, including the cobas® HPV test on the cobas 4800 instrument [2]. 

The fully automated cobas® HPV Test is ideal for screening large numbers of samples for HR-HPV.  Unlike other HPV assays, this is an FDA approved and CE marked test that provides a separate result for the highest risk HPV genotypes (HPV 16 and HPV 18) in addition to a pooled result for all HR-HPV genotypes.  This separate simultaneous detection of HPV 16 and HPV 18 further enhances risk stratification, allowing women who may need more intensive follow up and intervention to be identified.  The cobas® HPV Test is also the only integrated genotyping test to have undergone full clinical validation [3].

It was anticipated that 10-20 laboratories would be NHS CSP approved by 1st April 2012, from a mixture of cytology, microbiology and virology departments [2].  This diversity of disciplines is reflected in the range of laboratories that have adopted the cobas® HPV test.

Successful bids are required to have adequate access to molecular and HPV expertise, and to maintain the operating standard of 14 day turnaround time for cervical screening.  Additional criteria for local screening programmes, set by the Department of Health and NHS CSP, include: having sufficient sustainable colposcopy capacity to cope with initial increased workloads; adopting an external quality assurance programme for HPV testing; having suitable training for local professionals; appointing a pathway manager to oversee all aspects; and having quality assurance and primary care support for the bid.  The full list of criteria is published in the NHS CSP Implementation Guide (2011) [1].

References

1. NHS Cancer Screening Programmes (2011) NHS CSP Good Practice Guide Number 3.  HPV Triage and Test of Cure: Draft Implementation Guide (July, 2011).
2.  The UK Clinical Virology Network (2011) Roll-out of HPV triage in the NHS. 1 December 2011 (updated on 14 Feb 2012)
3. Stoler, MH, Wright TC, Sharma A et al (2011) High risk Human Papillomavirus Testing in Women with ASC-US Cytology. Results from the ATHENA HPV Study. Am J Clin Pathol 135:468-475.

Scientists have successfully sequenced the genome of a baby in the womb without tapping its protective fluid sac. Maternal blood sampled at about 18 weeks into the pregnancy and a paternal saliva specimen contained enough information for the scientists to map the foetus’ DNA. This method was later repeated for another expectant couple closer to the start of their pregnancy. The researchers checked the accuracy of their genetic predictions using umbilical cord blood collected at birth.
Jacob Kitzman and Matthew Snyder, working in the laboratory of Dr. Jay Shendure, associate professor of genome sciences at the University of Washington, led the study. Kitzman is a National Science Foundation Graduate Research Fellow.
Scientists have long known that a pregnant woman’s blood plasma contains cell-free DNA from her developing foetus. Foetal DNA appears in the mother’s plasma a few weeks after conception. It rises during gestation and normally vanishes after the baby arrives. While the concentration varies among individuals, about 10 percent of the cell-free DNA in a pregnant woman’s blood plasma comes from her foetus.
Based on this phenomenon, other research labs are designing maternal blood tests for major aberrations in the foetus’s genetic makeup. The tests are considered a safer substitute for the more invasive sampling of fluid from the uterus, a common procedure in obstetrical practice. These new tests search for just a few genetic disorders or specific congenital abnormalities. For example, a test targeted for Down syndrome would look for evidence of three copies of chromosome 21.
Kitzman explained what distinguishes his team’s latest methods is the ability to assess many and more subtle variations in the foetus’ genome, down to a minute, one-letter change in the DNA code.
‘The improved resolution is like going from being able to see that two books are stuck together to being able to notice one word misspelled on a page,’ said Kitzman.
With technical advances as well as statistical modelling, the research group overcame several obstacles that had stymied previous efforts to determine foetal genomes. With a preponderance of maternal rather than foetal DNA in plasma samples, a major problem was figuring out which genetic variants had passed from mother to child. The scientists applied a recently developed technique to resolve the mother’s haplotypes, which are groups of genetic variations residing on the same chromosome. From these groupings, the researchers could pick out the parts of the baby’s genetic material inherited from each parent with over 98 percent accuracy.
‘It was rewarding to apply biostatistics to help solve this problem,’ said Snyder, who came to genome sciences from the fields of statistics and economics.
Still, he added, there is more work to be done to improve this technique. The researchers pointed to the need for a more robust, scalable, overarching protocol, as well as ways to lower costs and automate and standardise parts of the process. Washington University