A targeted search combined with today's rapid DNA sequencing technology leads researchers to a genetic culprit for a rare disease.
Senior-Loken syndrome is a rare condition that dramatically impacts the lives of those it afflicts. Children with the disorder require dialysis or a kidney transplant by the time they reach adolescence. At about the same time, their retinas deteriorate, leaving them blind. Now, an international research team has used powerful gene-sequencing technology to identify a new genetic culprit that can singlehandedly cause these disparate symptoms.
The newly identified gene mutation, like nine others found previously, causes defects in signaling from the hair-like structures called cilia that protrude from most cells. To find it, researchers identified segments of the genome they suspected might be linked to the disease, then sequenced those and searched for places at which they differ between people with and without the disorder. Their targeted approach dramatically reduced the time needed to identify the new gene because the scientists did not sift through the complete set of 23,000 genes in the human genome.
Exon capture is incredibly powerful in single-gene disorders like the ones we’re studying. But we knew we’d get many, many changes. So we tried to a priori restrict the regions we’re looking at for the disease mutation.
The research, conducted by a large, multi-institutional team led by Howard Hughes Medical Institute investigator Friedhelm Hildebrandt at the University of Michigan, was published online in Nature Genetics on September 12, 2010.
Senior-Loken syndrome causes 10 to 15 percent of the cases of childhood genetic kidney diseases known as nephronophthisis, a group of illnesses affecting approximately 1 in 50,000 births. Although nine genetic mutations have already been linked to the disorder, together they are responsible for less than half of the cases, leaving the majority of cases unexplained—and additional genetic culprits difficult to identify.
Five years ago, Hildebrandt's group and others began to realize that the common thread among the genes that had so far been associated with Senior-Loken syndrome—as well as genes for other inherited kidney disease, such as Bardet-Biedl syndrome—was their involvement with cilia function. Cilia are commonly thought of as hair-like structures that wave rhythmically to clear contaminants from the airways or to move an egg through the fallopian tubes, but most cells in the body also possess a more rigid projection called a primary cilium. These cilia are cells’ antennae, and help sense the presence of chemicals, hormones, or photons of light. The varied roles of cilia explain why other organs are often affected in cystic kidney disease, including the retina, brain, and liver, Hildebrandt said. “The involvement of cilia was an absolutely surprising common denominator,” he recalled.
“In the 1,200 children with nephronophthisis, we can only explain about 50 percent, so we know there are dozens of other single-gene causes. Finding them will allow us to piece together the signaling machinery of ciliary function,” he said. Ultimately, he and others hope that by identifying the many genes that cause cystic kidney disease they will move closer to understanding disease mechanisms and ultimately find treatments.
In the new study, Hildebrandt’s team set out to hunt for new causative genes in patients with Senior-Loken who did not carry one of the nine mutations already associated with the syndrome.
Recent advances in DNA sequencing technology have made sequencing a person’s entire genome less costly than it would have been just five years ago. Using the technology, researchers can generate a sequence that they can then use to identify regions of the patient's genome that differ from a reference sequence of DNA from an individual who does not have Senior-Loken syndrome. But even if the analysis is limited to the one percent of DNA that actually encodes proteins—the exons—thousands of genetic letters are expected to differ between the patient’s sequence and the reference sequence. For a disease like Senior-Loken syndrome, which is thought to be caused by a single mutation, only one of these differences will be meaningful, Hildebrandt said.
That’s a lot of genetic variation to sift through, so Hildebrandt and his team decided to narrow their search. “Exon capture is incredibly powerful in single-gene disorders like the ones we’re studying,” he said. “But we knew we’d get many, many changes. So we tried to a priori restrict the regions we’re looking at for the disease mutation.”
Based on what Hildebrandt and his colleagues already knew about Senior-Loken syndrome, they chose to concentrate only on the 828 genes that contribute to cilia function. Normal variations within just those genes’ exons would still make for a daunting search for the causative mutation, Hildebrandt said, so they restricted their search further using a technique called homozygosity mapping.
This approach was possible because the team studied two siblings with Senior-Loken whose parents were related to one another. They concentrated their analysis on exons that were identical—that is, homologous—between the siblings, indicating they had been inherited from an ancestor common to their mother and father. The disease-causing gene would almost certainly be in these homologous regions, they reasoned. This allowed them to reduce the DNA sequence in their analysis another 80-fold. “That is probably, technically, the biggest novelty in the paper,” Hildebrandt said.
By combining these filtering techniques, plus eliminating any mutations they knew would be innocuous, the team was able to reduce the total number of exons to be evaluated more than 2,700-fold. Within four homozygous segments on the siblings’ genome, they uncovered a homozygous mutation on a gene called SDCCAG8, which produces a protein found at the base of cilia. Mutations in SDCCAG8 were responsible for Senior-Loken syndrome in 10 families in their study.
Once the Senior-Loken gene was discovered, the team confirmed in laboratory experiments that it supports the function of cilia. They eliminated the gene in zebrafish, and found that the affected fish developed kidney cysts. Because diseases linked to cilia function affect how a cell orients itself, the group also eliminated the gene in a line of kidney epithelial cells and watched how they grew. This changed the behavior of the cells, which, when grown in a gel matrix, usually arrange themselves into hollow spheres. Without SDCCAG8, the cells clumped, failing to make hollow spheres.
“The power of studying these rare diseases is to identify disease mechanisms where a single gene out 23,000 is sufficient in itself to cause disease,” Hildebrandt said. “There are probably hundreds of genes in children with kidney disease that are not known yet. In kidney disease in general, there is surprisingly little known about the disease causes. We only know a few parts of the ciliary machinery to be able to understand how the disease comes about. I think exon capture will really speed this up immensely.”