Whole Exome Sequencing Identifies Cause of Metabolic Disease

Sequencing a patient's entire genome to discover the source of his or her disease is not routine -- yet. But geneticists are getting close.

A case report, published this week in the American Journal of Human Genetics, shows how researchers can combine a simple blood test with an "executive summary" scan of the genome to diagnose a type of severe metabolic disease.

Researchers at Emory University School of Medicine and Sanford-Burnham Medical Research Institute used "whole-exome sequencing" to find the mutations causing a glycosylation disorder in a boy born in 2004. Mutations in the gene (called DDOST) that is responsible for the boy's disease had not been previously seen in other cases of glycosylation disorders.

Whole-exome sequencing is a cheaper, faster, but still efficient strategy for reading the parts of the genome scientists believe are the most important for diagnosing disease. The report illustrates how whole-exome sequencing, which was first offered commercially for clinical diagnosis in 2011, is entering medical practice. Emory Genetics Laboratory is now gearing up to start offering whole exome sequencing as a clinical diagnostic service.

It is estimated that most disease-causing mutations (around 85 percent) are found within the regions of the genome that encode proteins, the workhorse machinery of the cell. Whole-exome sequencing reads only the parts of the human genome that encode proteins, leaving the other 99 percent of the genome unread.

The boy in the case report was identified by Hudson Freeze, PhD and his colleagues. Freeze is director of the Genetic Disease Program at Sanford-Burnham Medical Research Institute. A team led by Madhuri Hegde, PhD, associate professor of human genetics at Emory University School of Medicine and director of the Emory Genetics Laboratory, identified the gene responsible. Postdoctoral fellow Melanie Jones is the first author of the paper.

"This is part of an ongoing effort to develop diagnostic strategies for congenital disorders of glycosylation," Hegde says. "We have a collaboration with Dr. Freeze to identify new mutations."

Glycosylation is the process of attaching sugar molecules to proteins that appear on the outside of the cell. Defects in glycosylation can be identified through a relatively simple blood test that detects abnormalities in blood proteins. The sugars are important for cells to send signals and stick to each other properly. Patients with inherited defects in glycosylation have a broad spectrum of medical issues, such as developmental delay, digestive and liver problems and blood clotting defects.

The boy in this case report was developmentally delayed and had digestive problems, vision problems, tremors and blood clotting deficiencies. He did not walk until age 3 and cannot use language. The researchers showed that he had inherited a gene deletion from the father and a genetic misspelling from the mother. "Over the years, we've come to know many families and their kids with glycosylation disorders. Here we can tell them their boy is a true 'trail-blazer' for this new disease," Freeze said. "Their smiles -- that's our bonus checks."

The researchers went on to show that introducing the healthy version of the DDOST gene into the patient's cells in the laboratory could restore normal protein glycosylation. Thus, restoring normal function by gene therapy is conceivable, if still experimental. However, restoration of normal glycosylation would be extremely difficult to achieve for most of the existing cells in the body.

The research was supported by the National Institutes of Health and by the Rocket Fund.

Journal Reference:

1. Melanie A. Jones, Bobby G. Ng, Shruti Bhide, Ephrem Chin, Devin Rhodenizer, Ping He, Marie-Estelle Losfeld, Miao He, Kimiyo Raymond, Gerard Berry, Hudson H. Freeze, Madhuri R. Hegde. DDOST Mutations Identified by Whole-Exome Sequencing Are Implicated in Congenital Disorders of Glycosylation. The American Journal of Human Genetics, 2012; DOI: 10.1016/j.ajhg.2011.12.024

Courtesy: ScienceDaily

New 'Biopsy in a Blood Test' to Detect Cancer

Scientists from The Scripps Research Institute, Scripps Health, and collaborating cancer physicians have successfully demonstrated the effectiveness of an advanced blood test for detecting and analyzing circulating tumor cells (CTCs) -- breakaway cells from patients' solid tumors -- from cancer patients. The findings, reported in five new papers, show that the highly sensitive blood analysis provides information that may soon be comparable to that from some types of surgical biopsies.

"It's a next-generation technology," said Scripps Research Associate Professor Peter Kuhn, PhD, senior investigator of the new studies and primary inventor of the high-definition blood test. "It significantly boosts our ability to monitor, predict, and understand cancer progression, including metastasis, which is the major cause of death for cancer patients."

The studies were published February 3, 2012, in the journal Physical Biology.

The new test, called HD-CTC, labels cells in a patient's blood sample in a way that distinguishes possible CTCs from ordinary red and white blood cells. It then uses a digital microscope and an image-processing algorithm to isolate the suspect cells with sizes and shapes ("morphologies") unlike those of healthy cells. Just as in a surgical biopsy, a pathologist can examine the images of the suspected CTCs to eliminate false positives and note their morphologies.

Kuhn emphasizes that this basic setup can be easily modified with different cell-labeling and image-processing techniques.

Five New Studies, Five Steps Forward

To test the new technology, members of the Kuhn lab at Scripps Research teamed up with pathologists and oncologists at Scripps Health in La Jolla, California; UC San Diego Moores Cancer Center at the University of California, San Diego; the Billings Clinic in Billings, Montana; the Division of Medical Oncology at the University of California, San Francisco; the Center for Applied Molecular Medicine at the University of Southern California, in Los Angeles; and the Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital in Amsterdam, the Netherlands.

The five new studies that resulted from the collaboration not only demonstrate the accuracy and effectiveness of the new test for a number of different cancer types, but also begin to explore the utility of the technology for diagnosing and monitoring patients and improving cancer research in the lab. While other tests for CTCs typically use "enrichment" steps in which suspected CTCs are concentrated -- and these methods inadvertently exclude some types of CTCs -- the new studies show HD-CTC works well as a no-cell-left-behind process and enables a more complete analysis.

Also striking is the quality of the images. "The high definition method gives a detailed portrait of these elusive cells that are caught in the act of spreading around the body," said diagnostic pathologist Kelly Bethel, MD, of Scripps Health, Scripps Research, and UC San Diego School of Medicine, who is the senior clinical investigator on Kuhn's team. "It's unprecedented -- we've never been able to see them routinely and in high definition like this before."

In the first study, the research team examined 83 advanced cancer patients using HD-CTC to document the test's sensitivity and accuracy for different cancer types. The scientists found that the test detected five or more CTCs per milliliter of blood in 80 percent of patients with metastatic prostate cancer, 70 percent of those with metastatic breast cancer, 50 percent of those with metastatic pancreatic cancer, and no healthy subjects. The current gold-standard CTC test, known as CellSearch, was notably less sensitive in detecting tumor cells in these samples.

Most patients whose CTC counts surpassed the detection threshold also showed small aggregates of CTCs, which cancer biologists term "microtumor emboli." These are widely suspected to be incipient metastatic tumors, as well as triggers for the blood clots that often kill advanced cancer patients. In the second study, the scientists showed that HD-CTC could detect these aggregates in 43 percent of 71 patients with advanced prostate, lung, pancreas, and breast cancers, and in none of a group of 15 healthy subjects. "This tells us that HD-CTC could be helpful in studying the origins of cancer metastases and related blood clots, and for predicting them, too," Kuhn said.

In the third study, the team used HD-CTC to compare circulating tumor cells from prostate cancer patients with cells from prostate cancer cell lines that researchers often use as convenient models for prostate cancer biology in the lab. The team found significant differences between the two classes of cells, in their cell morphology and in the way they were labeled by HD-CTC's fluorescent tags. "This underscores the need for studying cancer cells from patients, not just model cancer cells that in some ways may be utterly different from the real thing," Kuhn said.

In the fourth study, the researchers performed HD-CTC tests on 28 patients with advanced non-small-cell lung cancer over periods of up to a year. The team was able to detect CTCs in 68 percent of samples, and found that the numbers of detected CTCs tended to go up as other measures showed cancer progression.

In the fifth and final paper of the series, the team used HD-CTC in 78 patients who had just been diagnosed with various stages of non-small-cell lung cancer. "We demonstrated that we could sensitively detect CTCs even in patients with early-stage cancer," Kuhn said.

This result points to the possibility of using the HD-CTC blood test not only to evaluate already-diagnosed cancer, but also to help detect cancer in people who are unaware they have it. "If HD-CTC works on the day after cancer diagnosis, as we've shown, then one can easily imagine that it would work the day before diagnosis, too," Kuhn said.

Kuhn and his colleagues now intend to study the use of HD-CTC as a potential screening test and to develop it further for use in clinical monitoring and cancer research. Kuhn has founded a San Diego-based biotechnology company, Epic Sciences, Inc., to develop HD-CTC commercially for companion diagnostic products in personalized cancer care.

Journal References:

1. Jorge Nieva, MarcoWendel, Madelyn Luttgen, Dena Marrinucci, Lyudmila Bazhenova, Anand Kolatkar, Roger Santala, BrockWhittenberger, James Burke, Melissa Torrey, Kelly Bethel, and Peter Kuhn. High-- imaging of circulating tumor cells and associated cellular events in non-small cell lung cancer patients: a longitudinal analysis. Physical Biology, Feb 3, 2012
2. Dena Marrinucci1, Kelly Bethel, Anand Kolatkar, Madelyn Luttgen, Michael Malchiodi,, Franziska Baehring, Katharina Voigt, Daniel Lazar, Jorge Nieva, Lyudmilda Bazhenova, Andrew H Ko, W Michael Korn, Ethan Schram, Michael Coward, Xing Yang, Thomas Metzner, Rachelle Lamy, Meghana Honnatti, Craig Yoshioka, Joshua Kunken, Yelena Petrova, Devin Sok, David Nelson, and Peter Kuhn. Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers. Physical Biology, Feb 3, 2012
3. Daniel C Lazar, Edward H Cho, Madelyn S Luttgen, Thomas J Metzner, Maria Loressa Uson, Melissa Torrey, Mitchell E Gross, and Peter Kuhn. Cytometric comparisons between circulating tumor cells from prostate cancer patients and the prostate-tumor-derived LNCaP cell line. Physical Biology, Feb 3, 2012
4. Edward H Cho, MarcoWendel, Madelyn Luttgen1, Craig Yoshioka, Dena Marrinucci1, Daniel Lazar, Ethan Schram, Jorge Nieva, Lyudmila Bazhenova, Alison Morgan, Andrew H Ko, W Michael Korn, Anand Kolatkar, Kelly Bethel, and Peter Kuhn. Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors. Physical Biology, Feb 3, 2012
5. Marco Wendel, Lyudmila Bazhenova, Rogier Boshuizen, Anand Kolatkar, Meghana Honnatti, Edward H. Cho, Dena Marrinucci, Ajay Sandhu, Anthony Perricone, Patricia Thistlethwaite, Kelly Bethel, Jorge Nieva, Michel van den Heuvel, and Peter Kuhn. Fluid biopsy for Circulating Tumor Cell identification in Patients with early and late stage Non-Small Cell Lung Cancer; a glimpse into lung cancer biology. Physical Biology, Feb 3, 2012

Courtesy: ScienceDaily