Gene Responsible For Debilitating Blood Cell Disorder Discovered

Now, a team of Boston area researchers has lifted the seemingly impenetrable curtain surrounding the 5q minus syndrome, with the long-awaited discovery of the guilty gene. As described in the January 17 issue of Nature, the gene encodes a ribosomal protein — a protein that helps to build other proteins in practically every cell in the body. Though such a broad-acting gene may seem an improbable culprit for a disorder that strikes only blood cells, the discovery highlights a previously unknown genetic connection to a related disease. And through that connection, some light has at last been shed on the biological missteps that underlie the 5q minus syndrome.

“People have been looking for this gene since the 1970’s,” said first author Benjamin Ebert, an assistant professor at Harvard Medical School and Brigham and Women’s Hospital and a researcher at the Broad Institute working with Cancer Program director Todd Golub and other Broad Institute scientists. Because those efforts were largely fruitless, the researchers felt a different approach was needed, Ebert explained.

Clinically speaking, a defining feature of the 5q minus syndrome is anemia, or a low number of red blood cells. In fact, the anemia can be so severe that patients require frequent blood transfusions to survive. The numbers of other cells in patients’ blood, however, are mostly unchanged. “That very specific anemia without other low blood counts is very unique,” and a signature of the 5q minus syndrome, said Ebert.

To uncover the genetic factors that trigger the defect, Ebert and his colleagues diverged from a well-trodden path in biological research, and questioned a deep-rooted belief about cancer and related syndromes like myelodysplasia. That belief, known as Knudson’s two-hit hypothesis, states that to cause cancer, genes must be disabled with a one-two punch, meaning both copies need to be deleted or otherwise inactivated.

It is well known that in the 5q minus syndrome, patients’ blood cells harbor one mangled copy of chromosome 5, a mere shadow of its normal self with a shortage of some 40 genes. According to Knudson’s hypothesis, that multi-gene deletion reflects the fateful first punch. The other copy of the chromosome, though, appears largely intact and scientists have spent years scouring it to pinpoint tiny mistakes that might represent a second hit. Since none have been found, the second punch — and the culprit gene’s identity — remained in doubt.

That protracted search sparked a question in the minds of Ebert and his colleagues. Is it possible that the 5q minus syndrome defies the two-hit rule? Instead of sustaining blows to both copies of a gene, perhaps just one hit is sufficient to cause the disease. To explore this unconventional idea, the researchers used a powerful technique known as RNA interference, which partially eliminates, or “knocks down”, the activity of specific genes. Like the dimmer on a light switch, it can enable genes to stay switched on, but at only half the typical level.

Working with normal cells grown in the laboratory, the scientists ratcheted down, one by one, the activity of each of the missing genes in the 5q minus syndrome — delivering the laboratory equivalent of the first of Knudson’s two hits. Then, they examined the cells to see if any resembled those seen in patients with the disease.

Out of the 40 genes analyzed, the researchers observed the hallmark signs of the syndrome after the manipulation of just a single gene. And when a working version of that same gene was transferred into abnormal blood cells from 5q minus patients, another striking result emerged: the characteristic defects disappeared. The gene responsible for these seemingly magical feats was RPS14, a component of ribosomes, the fundamental machinery within cells that churns out proteins.

“It seemed like the most unlikely candidate in the world — a ribosomal gene that acts ubiquitously in all cells. At first glance, that’s pretty uninteresting,” said Ebert.

But the finding turned out to be very interesting. Ebert and his colleagues recalled the striking resemblance between the 5q minus syndrome and another disease, an inherited blood disorder called Diamond Blackfan Anemia (DBA) that affects children in the first few months of life. The two diseases share a similar deficit of red blood cells as well as a predisposition to cancer. “If you had to pick a congenital disease that looks like the 5q minus syndrome, there is no question that would be Diamond Blackfan Anemia,” said Ebert.

Yet the researchers soon realized the similarities between the diseases extend beyond outward signs, and reach all the way down to DNA. In DBA, six disease-causing mutations were recently mapped — each of them to a ribosomal gene. The DBA results confirmed the connection between defects in ribosomal genes and anemia, giving the Ebert’s team even greater confidence in their findings for the 5q minus syndrome.

The biological significance of the team’s findings for the 5q minus syndrome became clearer in light of the growing knowledge of red blood cells. In the body, hundreds of billions of red blood cells are made every day, charged with the vital task of ferrying oxygen to tissues. To fulfill this mission, the cells are packed full — like balloons about to burst — with oxygen-carrying molecules called hemoglobin, an amalgam of protein and iron. Logistically, that means that red blood cells must keep the synthesis of both the protein and iron-containing parts of hemoglobin running at full throttle. Moreover, the two halves must be produced at roughly equal amounts. A mismatch can be toxic, and cause the cells to die.

According to Ebert, this emerging picture of red blood cells — miniature hemoglobin factories where production is both exquisitely synchronized and nearly pushed to the limit — helps to explain their sensitivity to even small deficiencies in the cellular machinery, including ribosomal proteins. Genetic alterations in just one copy of a ribosomal gene, as in the 5q minus syndrome, can be enough to shift red blood cells to their tipping point, thereby inciting disease. That sensitivity also helps to explain the tortured past that has plagued research on the 5q minus syndrome. Previous efforts to find the responsible gene came up empty-handed in part because of the two-hit bias. “If you lost both copies of these essential ribosomal genes, there is no way the cell would survive,” said Ebert. “So cells with two hits simply would never be found.”

For Ebert, one of the most exciting implications of his work is that it unites two fields that, for pragmatic reasons, once seemed as immiscible as oil and water. Because the 5q minus syndrome and DBA arise at opposing ends of life’s spectrum, one in the elderly and the other in the very young, adult doctors tend to treat one group and pediatric doctors the other. So, the people who spend their waking hours ruminating about one disease or the other probably have never exchanged ideas — until now.

Another implication of the research is that the RNA interference-based approach used by Ebert and his colleagues can be readily adapted to a variety of other diseases. The tools for systematically knocking down genes across the genome have only recently become available. The RNAi Consortium, a collaboration among life science companies and academic institutions based at the Broad Institute, developed the reagents used in the current study.

Indeed, a broader application of RNA interference may lead to the discovery that other diseases, particularly cancers, stem from subtle, single gene hits, instead of the dual hits of traditional scientific lore. What that might mean for unraveling the genetic basis of those disorders is too early to predict. But with any luck, at least some of the lessons learned will mirror those for the 5q minus syndrome.

Other Broad researchers who contributed to the work include Jocelyn Bosco, Cindy Chang, Jennifer Pretz, David Root, and Pablo Tamayo.

Written by Nicole Davis

Paper cited:

Ebert et al. (2008) Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature doi:10.1038/nature06494