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

A team of researchers from the Hospital Universitario La Paz Research Institute (IdiPAZ, Madrid), LifeSequencing S.L. (Valencia), Era7 Bioinformatics (Madrid) and Roche Spain (Barcelona) announced on March 14^th the sequencing of the whole genome of three antibiotic resistant strains of Klebsiella pneunomiae isolated from a recent outbreak in a Spanish hospital. The sequence data, generated using Roche’s 454 GS FLX+ System, is some of the first for this particular bacterial species, providing new insights into how antibiotic resistance evolves within this microorganism and can lead to hospital outbreaks.

Klebsiella pneumoniae is a bacterium frequently found in the mouth and gut of healthy humans. In most instances, it does not lead to disease but it can mutate opportunistically and cause diverse types of infections. The bacterium also has a significant capacity to acquire antibiotic resistance. Three closely related isolates of a pathogenic strain of K. pneumonia with increasing degrees of antibiotic resistance were obtained in the Microbiology department at Hospital Universitario La Paz and sequenced at LifeSequencing in Valencia, Spain using the long read GS FLX+ System, developed by 454 Life Sciences, a Roche Company. The sequencing data was assembled using the GS De Novo Assembler software and functional annotation was performed to identify the relevant genes codified in the three genomes with BG7, the optimized system developed by Era 7 Bioinformatics, providing rich functional annotation of 454 Sequencing data.

The researchers found that the three bacteria strains showed an increasing resistance pattern to a wide range of the antibiotics most commonly used at the hospital. Comparison of the genomes will give insights regarding how antibiotic resistance evolves within K. pneumonia and will aid in efforts to reduce the increasing prevalence of antibiotic resistance worldwide. In addition, the comparison of these genomes with other previously studied bacteria will help to understand how a microorganism that is part of our normal microbiome can become a dangerous pathogen.

“Fast and affordable sequencing of pathogenic bacteria is a huge qualitative and quantitative advance that is radically changing the way researchers and clinicians view the infectious disease process,” said Dr. Jesús Mingorance lead researcher at the Hospital Universitario La Paz. The GS FLX+ and GS Junior Systems from Roche are aiding in these pathogen detection and bacterial comparative genomics efforts worldwide.
_{For life science research only. Not for use in diagnostic procedures.}

The Caterpillar got down off the mushroom … remarking as it went, ‘One side will make you grow taller, and the other side will make you grow shorter.’

—Lewis Carroll, ‘Alice’s Adventures in Wonderland’

UCLA geneticists have identified the mutation responsible for IMAGe syndrome, a rare disorder that stunts infants’ growth. The twist? The mutation occurs on the same gene that causes Beckwith–Wiedemann syndrome, which makes cells grow too fast, leading to very large children.

The UCLA findings could lead to new ways of blocking the rapid cell division that allows tumours to grow unchecked. The discovery also offers a new tool for diagnosing children with IMAGe syndrome, which until now has been difficult to identify accurately.

The discovery holds special significance for principal investigator Dr. Eric Vilain, a professor of human genetics, paediatrics and urology at the David Geffen School of Medicine at UCLA.

Nearly 20 years ago, as a medical resident in his native France, Vilain cared for two boys, ages 3 and 6, who were dramatically short for their ages. Though unrelated, the children shared a mysterious malady marked by minimal foetal development, stunted bone growth, sluggish adrenal glands, and undersized organs and genitals.

‘I never found a reason to explain these patients’ unusual set of symptoms,’ said Vilain, who also directs the UCLA Institute for Society and Genetics. ‘I’ve been searching for the cause of their disease since 1993.’

When Vilain joined UCLA as a genetics fellow, the two cases continued to intrigue him. His UCLA mentor at the time, geneticist Dr. Edward McCabe, recalled a similar case from his previous post at Baylor College of Medicine. The two of them obtained blood samples from the three cases and analysed the patients’ DNA for mutations in suspect genes but uncovered nothing.

Vilain and McCabe approached the Journal of Clinical Endocrinology and Metabolism and in 1999 published the first description of the syndrome, which they dubbed IMAGe, an acronym of sorts for the condition’s symptoms: intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia and genital anomalies.

Over the next decade, about 20 cases were reported around the world. But the cause of IMAGe syndrome remained a mystery.

Help arrived unexpectedly last year, when Vilain received an email from Argentinian physician Dr. Ignacio Bergada, who had unearthed the 1999 journal article. He told Vilain about a large family he was treating in which eight members suffered the same symptoms described in the study. All of the family members agreed to send their DNA samples to UCLA for study.

Vilain realized that he had stumbled across the scientific equivalent of winning the lottery. He assembled a team of UCLA researchers to partner with Bergada and London endocrinologist Dr. John Achermann.

‘At last, we had enough samples to help us zero in on the gene responsible for the syndrome,’ Vilain said. ‘Sequencing technology had also advanced in sophistication over the past two decades, allowing us to quickly analyse the entire family’s DNA samples.’

Vilain’s team performed a linkage study, which identifies disease-related genetic markers passed down from one generation to another. The results steered Vilain to a huge swath of Chromosome 11.

The UCLA Clinical Genomics Center performed next-generation sequencing, a powerful new technique that enabled the scientists to scour the enormous area in just two weeks and tease out a slender stretch that held the culprit mutation. The team also uncovered the same mutation in the original three cases described by Vilain and McCabe in 1999.

‘We discovered a mutation in a tiny sliver of the chromosome that appeared in every family member affected by IMAGe syndrome,’ Vilain said. ‘This was a big step forward. Now we can use gene sequencing as a tool to screen for the disease and diagnose children early enough for them to benefit from medical intervention.

‘We were a little surprised, because the mutation was located on a famous gene recognised for causing Beckwith–Wiedemann syndrome,’ he added. ‘The two diseases are polar opposites of each other.’

Children born with Beckwith–Wiedemann syndrome — named for the two doctors who discovered it — grow very large, with big adrenal glands, elongated bones and oversized internal organs. Because their cells grow so fast, one in five children with the disorder die of cancer at a young age. The disease appears in approximately one out of 15,000 births.

‘Finding opposite functions in the same gene is a rare biological phenomenon,’ Vilain emphasised. ‘When the mutation appeared in the slim section we identified, the infant developed IMAGe syndrome. If the mutation fell anywhere else in the gene, the child was born with Beckwith–Wiedemann. That’s really quite remarkable.’ UCLA