Next-generation sequencing and stem cell technology have helped scientists identify a mutation that causes retinitis pigmentosa.

Retinitis pigmentosa causes progressive vision loss for one in 4,000 people in the United States. It is not a single disease, however. Rather, it is a group of related conditions thought to be caused by mutations in more than 100 different genes. Fewer than half of those genes have been identified. A new approach, combining next-generation sequencing with stem cell research, has identified a new mutation that causes retinitis pigmentosa, and provides a strategy to tease out additional disease-causing genes.

Many of the mutations that cause retinitis pigmentosa (RP) occur only rarely, and pinning them down has proven remarkably difficult. Edwin Stone, the Howard Hughes Medical Institute investigator who led the new study, says it’s important to continue the search. “If there are 100 different genes that cause RP and we’ve already got half of them, you might ask, ‘Why do you need to find another one?’ You need another one if it’s yours,” he says. “If your disease-causing gene hasn’t been found yet, most of the prior discoveries won’t help you very much.”

We were able to study the disease mechanism in this actual person, the person in whom the mutation was discovered.

Edwin M. Stone

In research published online the week of August 8, 2011, in Proceedings of the National Academy of Sciences, Stone’s team scanned the protein-coding DNA from a patient with a form of retinitis pigmentosa (RP) not caused by any of the mutations so far linked to the condition. After identifying a candidate mutation, they investigated its effects on the eye by creating stem cells from the patient’s skin, which they directed to develop into retinal cells in the lab. Though they began their study with a single patient, an analysis of DNA from a much larger group of individuals with RP turned up the newly identified the mutation in 20 additional people, or approximately one percent of the samples.

The process of identifying the new RP-causing mutation began when the team sequenced the patient’s exome—the portion of DNA that encodes proteins. Although the exome represents only about one percent of a person’s genome, it is where disease-causing mutations are most often found. Their next task was to weed through the resulting data to try to find the disease-causing gene. The difficulty is not just the size of the human genome -- six billion nucleotides -- it is the amount of normal genetic variation, which amounts to around six million differences from person to person. Locating the responsible mutation using the genome of just one affected individual is a challenging task, one that requires clever algorithms, a huge amount of computing power, and a healthy dose of luck says Stone, who worked with long-time collaborator and HHMI investigator Val Sheffield to do just that.

Stone and Sheffield, both at the University of Iowa, Department of Ophthalmology and Visual Sciences, discovered that there was a mutation in both copies of the patient’s MAK (male germ-cell associated kinase) gene. Expression of MAK had so far been associated with sperm cell development; the gene had never been shown to cause human vision loss. But sperm cells have one thing in common with the photoreceptor cells of the human retina: they both contain cilia, thin cellular extensions that act as sensory organelles. Defective cilia in photoreceptor cells can cause the type of vision loss associated with RP, and the researchers began to suspect this might be the case for the patient in their study.

To determine whether the mutation could be a plausible cause of disease, the researchers used donor tissue from human eyes to look for MAK proteins in the retina. Indeed, they found the protein in the inner segments of photoreceptor cells. “This was persuasive that MAK could cause RP, since it is present at the scene of the crime,” says Rob Mullins, a cell biologist researcher at the University of Iowa who participated in the research.

Budd Tucker, a stem cell scientist at the University of Iowa, also brought his expertise to the project, using a skin biopsy from the same RP patient to create a line of induced pluripotent stem cells, which can be prompted to differentiate into almost any tissue type. He coaxed the stem cells to develop into retinal cells, so that the researchers could see how the MAK mutation impacted the eye. “You can’t often biopsy someone’s retina to study the mechanism of an inherited disease,” Tucker says. “Even though sequencing identified a MAK mutation in this person, we couldn’t possibly tell exactly what it was doing to that person’s retina. Now we can.”

The genetic analysis indicated that the patient’s MAK gene had an inserted piece of extra DNA, but it was not entirely clear whether this would affect MAK’s function in the eye. The stem cell experiments provided an answer. The retinal cells derived from the patient with RP were unable to make MAK proteins at all. Retinal cells derived from a healthy patient, as well as those derived from a patient with a different genetic form of RP, had no such difficulties. “We were able to study the disease mechanism in this actual person, the person in whom the mutation was discovered,” Stone says. “This research unites next-generation sequencing with induced pluripotent stem cell technology.”

It was an experiment 20 years in the making. Once Stone and his colleagues had confirmed the mutation in the original patient, they went back to blood samples they’d collected from 1,800 patients with RP over the past two decades. They tested those samples for the MAK mutation and found 20 additional patients who shared the mutation, all of them—including the original patient—of Jewish ancestry.

Scrutinizing the exome of an individual with disease, someone with no affected family members, and wading through the noise to find the true mutation is an incredibly difficult task. In fact, the individual patient whose exome led to the discovery of the MAK mutation also harbored genes with two other known RP-causing mutations, neither of which alone cause disease. Those mutations were eventually ruled out as the cause for his condition, because each occurred on just one chromosome, and both copies of a gene must be mutated to cause recessive diseases like RP, but they complicated the search for the true culprit.

Following this success, the researchers now plan to apply the same combination of techniques to try to identify as many new RP genes as they can. Because there are so many potential RP-causing genes, many of them may be involved in less than one percent of patients with the condition. The ability to use iPSC technology to provide mechanistic confirmation of a mutation’s involvement in disease will be very helpful in identifying these rare causes of rare diseases, Stone says.

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