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Stanford Developing Targeted Sequencing Test for Mitochondrial Disease Using Long Padlock Probes

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By Julia Karow

Researchers at the Stanford Genome Technology Center are developing a targeted sequencing assay for genes encoding mitochondrial proteins, with the ultimate goal to develop a diagnostic test for mitochondrial diseases.

Earlier this month, they published a paper in PNAS describing the assay, which uses long padlock probes to capture 524 mitochondrial genes followed by array-based sequencing.

About two-thirds of patients suffering from a mitochondrial disease don’t receive a molecular diagnosis today, said Greg Enns, an associate professor of pediatrics and director of the biochemical genetics program at the Stanford University School of Medicine. "Currently, it's much more common to have an undiagnosed patient than a diagnosed patient," said Enns, who is a co-author of the paper.

A limited menu of gene tests is currently available to patients. "They come in, we can define things up to a certain point, and then we are stuck. So this type of technology that's comprehensive could be a real game-changer as far as clinical diagnosis and also understanding these disorders," he said.

A genetic diagnosis could help enroll patients in clinical trials. Enns said he is in charge of a trial to test a novel medication to treat mitochondrial disease, but the US Food and Drug Administration requires a molecularly confirmed diagnosis for a patient to be included. "So even if I have a child who I know has mitochondrial disease, maybe had a muscle biopsy, or laboratory studies, or head imaging, or any or all of the above, … but we don't have a molecular answer, that child is not eligible for inclusion in this clinical trial."

Overall, more than 1,200 genes in the nuclear genome encode mitochondrial proteins, whereas the tiny mitochondrial genome itself only encodes 13 proteins.

The Stanford researchers decided to target 524 nuclear-encoded mitochondrial genes, including those involved in "traditional" mitochondrial disorders, such as errors of metabolism, and others with roles in diseases where mitochondria appear to play a role, such as diabetes, cancer, and neurodegeneration. They include genes encoding proteins found in several locations within the cell, such as BRCA1 and TP53.

"Traditionally, 'mitochondrial disease' was a term primarily used for energy deficiency disorders. But we now have strong evidence that [mitochondrial genes] underlie many, many diseases," said Curt Scharfe, a research associate at the Stanford Genome Technology Center and the senior author of the paper.

While some of the genes included in the assay are known to be involved in mitochondrial disease, others were chosen on the basis of a network analysis and are "high probability candidate genes" for mitochondrial disorders.

"Mitochondrial diseases are difficult to study as a group," Scharfe said. The team is hoping that the assay "will help on many fronts to improve mitochondrial disease diagnostics," as well as treatment.

The researchers used about 5,600 long padlock probes to target the more than 5,400 exons of their candidate genes. Each probe is 320 bases long and has sequences at both ends that hybridize to a complementary site in genomic DNA. The gap between them is then filled by DNA polymerase and closed by ligase, and the resulting DNA circles are amplified and analyzed on a resequencing array. Stanford researchers originally described the LPP capture method in a paper three years ago (IS 7/18/2008).

The single-tube capture assay currently takes less than eight hours and requires about 0.5 micrograms of genomic DNA, according to the paper.

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In the article, the researchers described how they used the assay to analyze samples from 63 individuals, including 47 with a mitochondrial disease. They found that the test detected DNA variants with a false positive rate of 1 percent and a false negative rate of 3 percent. Only 144 exons could not be amplified in any sample due to high GC content.

In one sample, they were able to detect not only single-nucleotide variants but also copy number changes at exon-level resolution.

One advantage of the LPP method, compared to hybridization-based capture approaches, is its high specificity, Scharfe explained, which he said is especially important for mitochondrial diseases because the genome contains many pseudogenes of mitochondrial genes that one would not want to capture.

The method is limited by the high upfront cost of producing the long padlock probes, and the need to design and test them individually. Once a probe has been synthesized, though, it can be used in an estimated 40 million capture reactions, Scharfe said, making it "very cost effective." He guessed that it would currently cost between $1 million and $3 million to make an LPP library for the entire exome.

The researchers are now using their assay to study about 60 additional samples from mitochondrial disease patients but don't have any results yet, according to Enns.

Scharfe and his colleagues are currently improving the LPP capture technology by redesigning probes targeting GC-rich exons and by analyzing more samples in parallel to bring the cost down. Statistical analysis of data from more samples will also help them distinguish between sequencing errors and true variants, he said.

In addition, they are moving from arrays to next-gen sequencing technologies. When they started their project in 2006, resequencing arrays were "the best technology available" and "perfectly suitable" to improve the capture method, he said. While they continue to use arrays, they also currently analyze samples on the Illumina HiSeq, which can handle 12 to 15 samples per lane.

To develop a diagnostic test with the highest possible sensitivity and accuracy, it might be necessary to use several different capture methods, and the researchers are now combining commercially available exome capture methods with the LPP technology. "In the long term, it might not just be one but several technologies that have to be integrated and combined," Scharfe said. "It will be important in diagnostics to make sure we can detect each and every variant in each patient sample."

According to Scharfe, it will likely take between two and five years before the assay can be implemented as a diagnostic test. "But whether this is really fulfilling the high standards of medical sequencing in terms of accuracy and sensitivity remains to be determined," he said. It will also be difficult to ascertain whether a certain DNA variant is indeed disease-causing because many different genes can cause the same phenotype, he added.

"That's a challenge, not only in mitochondrial disease but in all of genetics," said Enns. "With all of the different types of sequencing modalities, you find sequence changes in everybody. And trying to determine which ones are important, which ones are disease-causing, and which ones are just parts of a population variant is very important. We need more samples, better information, and better data analysis, and also better translational tools to confirm whether or not a change is truly disease-causing."

A sequencing test "should not really be looked at in isolation," he added. "It's a tool that is certainly very useful and can certainly be exceptionally helpful. However, it's not the end of the story." An important next step for researchers developing gene sequencing tests is thus to collaborate with scientists conducting functional studies, Enns said.


Have topics you'd like to see covered in Clinical Sequencing News? Contact the editor at jkarow [at] genomeweb [.] com.

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