Caroline Seydel Contributor GEN

Bionano Saphyr Spots Inversions and Large Deletions in Genomes

A long-range optical DNA mapping technique has proved its worth in a clinical setting, successfully identifying large structural variants in DMD, the gene responsible for Duchenne muscular dystrophy. The new system creates a physical map of the order and orientation of functional elements, making it easier than ever to find the kind of disruptions that often stymie genetic tests, such as inversions and large deletions.

Typically, clinicians test for DMD mutations using multiplex ligand-dependent probe amplification (MLPA), in which probes bind each of the gene’s 79 exons. This method reliably picks up deletions and duplications, but since the probes will happily bind the exon sequences regardless of which direction they face, inversions are effectively invisible. Similarly, next-generation sequencing can catch single-nucleotide mutations, while large deletions and inversions may elude detection due to the challenge of assembling the sequence fragments.

Bionano Genomics has developed a genetic mapping system that fills in this gap. The method resembles a modern take on restriction mapping: endonucleases introduce single-strand nicks, which are then repaired using nucleotides bearing fluorescent dye molecules. Then, the individual DNA molecules are stretched out in nanotubes, where the pattern of dye molecules incorporated into the genome can be imaged. This pattern can then be compared with a reference sequence. If labeling sites occur too close together, for instance, that could indicate a deletion; too far apart would seem to be an insertion.

“The technology is really cool,” says Robert Weiss, Ph.D., professor of human genetics at the University of Utah, to GEN. “To take a DNA molecule and thread it through a nanochannel—it’s quite a feat.”

The current work, published in Genome Medicine, applies Bionano’s next-generation mapping (NGM) system to patients with DMD. Led by Eric Vilain, M.D., Ph.D., director of the Center for Genetic Medicine at Children’s National Health System, the researchers tested 8 affected individuals and 3 mothers for mutations in the dystrophin gene. The NGM system, called Saphyr, successfully revealed deletions ranging from 45­–250 Kbp in 6 of the patients, one 13 Kbp insertion, and one 5.1 Mbp inversion in patients. In the mothers, Saphyr also successfully identified an insertion and a deletion, unhindered by the presence of a second, normal allele.

While the deletions and the insertion had been identified by existing tests, the patient with the inversion had undergone MLPA, PCR of all 79 DMD exons, and exome sequencing, and none could turn up a pathogenic mutation. The inversion affected 60% of the DMD gene, completely disabling it, yet because no sequence was gained or lost, remained undetectable.

“This paper offers hope to putative DMD patients that have yet to receive a diagnosis,” says Erik Holmlin, Ph.D., CEO of Bionano. “But it’s more about demonstrating the utility in a well-known case, where it’s a pretty high bar to meet up with what existing technologies can do.”

“Compared with chromosomal microarray and MLPA technology, there is little doubt that the NGM technology is superior in identifying inversions and probably balanced translocations,” writes John Christodoulou, Ph.D., chair of genomic medicine at Murdoch Children’s Research Institute, by email. But he cautions that the existing technologies haven’t been supplanted quite yet. Saphyr would likely have trouble picking up smaller deletions, he says, making MLPA a more reliable method for most DMD testing. And only sequencing, not mapping, can perceive single nucleotide variations.

Still, NGM technology has come a long way since Bionano’s founding in 2003, and continues to improve. Saphyr is the company’s second-generation optical mapping system, processing samples 10 times faster than the original system, Irys. “With Irys, it wasn’t really possible to take on these kind of patient cohorts,” says Holmlin. Studies that would have taken years with Irys, he says, can now be completed in mere months. “Saphyr is the embodiment of our technology that is allowing us to get into clinical and translational research studies.”

“The number of molecules they are counting is quite impressive,” says Weiss. The paper reports 70x coverage of the genome from a single Saphyr chip. “I think this is a sign that it’s actually maturing to a level that it’s perhaps ready to enter clinical testing.”

Holmlin hopes that Bionano’s system will help provide answers for the many patients whose disease likely has a genetic cause, but no mutation can be found using existing technologies.

“The use of whole-exome sequencing on a large series of undiagnosed patients results in a diagnostic yield of only about 30%,” says Vilain. “The detection of structural variants for undiagnosed disorders is an avenue that we are pursuing.” An important next step down this avenue will be to characterize the range of normal structural variation found in the genome. As the authors note in the paper,  they found many other structural variants outside the DMD locus. Not all of these will be disease-causing mutations, and recognizing which variants commonly appear in healthy individuals will inform future testing.

“I think there’s a need for caution, and not over-interpreting the pathogenicity of these types of variants,” says Weiss, citing the difficulty of predicting a phenotype from a single genetic change. “Probably the case it helps better is when one has a clinical diagnosis of disease, yet the standard techniques have failed to yield the expected mutation.” Still, he says, “I think the technology has a lot of promise.”

Caroline Seydel is a freelance science writer based in Los Angeles, CA. She has a MS in genetics.

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