Drs. Jill Johnsen (left) and Doug Fowler: 'This has never been done before to be able to test a lot of (DNA) changes at once. This is the innovative part.'
World Federation of Hemop Nearly 40,000 people globally suffer from Hemophilia B, according to the World Federation of Hemophilia. It is a monogenetic disorder, that is, Hemophilia B is caused by one gene only – the Factor 9 gene – that lacks the protein needed to properly clot blood.
For decades, physicians and researchers have been trying to answer the question, "What are the DNA changes in the gene that cause Hemophilia B?”
UW Medicine’s Jill Johnsen, M.D., is one of those physicians. She is a professor in the UW Medicine Division of Hematology and Oncology, and a faculty member at the Institute for Stem Cell & Regenerative Medicine.
“Answering that question has been has been really hard technically,” she said. “How are we going to interrogate this gene for all the different ways DNA changes might change the protein?”
The rarity of the disorder, one case in 20,000 male births she said, adds to difficulty.
“It’s a rare disorder and often when we do genetic testing, we find a gene change no one has seen before, or a gene change only in seen only a few people,” said Johnsen. “We didn’t have additional evidence, other than identifying a gene that could cause Hemophilia B. These are actionable test results. We really care.”
BBI’s Doug Fowler, Ph.D., and his lab colleagues in the Department of Genome Sciences, also really care. They and Johnsen, together with others at BBI and the UW, have created the first mutational scanning method aimed at secreted human proteins: MultiSTEP.
“Using MultiSTEP, we measured the effect of approximately 10,000 Factor 9 single letter variants on secretion from cells,” Fowler said. “These data have enabled us to more accurately diagnose individuals with Hemophilia B.”
The ideal way to confirm the Hemophilia B gene is to test it in a lab, according to Johnsen. “That’s high level evidence,” she said. “If you can show it under the right conditions, using the right experiments, and all the right controls that you confidently think that change in that particular gene, you can use that evidence in the clinical lab. Then you can act on that information. It is useful for diagnoses. You can use it for family planning, and to test other relatives at risk.”
Their efforts and results are covered in the paper, “Multiplex, multimodal mapping of variant effects in secreted proteins,” which has been accepted for publication later this year.
“MultiSTEP can be combined with diverse antibodies to quantify secretion and post-translational modifications, which until now have been beyond the reach of Multiplexed Assays for Variant Effects…” the paper states. “The ability to characterize the diverse effects of massive numbers of secreted human protein variants on the surface of human cells creates the opportunity to understand variant effects on secreted protein structure, function, and pathogenicity.”
So, how did Fowler, Johnsen, and their colleagues create this new mutational scanning method, MultiSTEP?
“Instead of just expressing the Factor 9 gene and letting it leave the cell, one of our MD-PhD candidates, Nick Popp, who completed his Ph.D. in Doug’s lab, engineered a microscopic tail with a hook at the end that sticks in the membrane of the cell,” Johnsen said. “So, the DNA in the cell has the variant we want to test. It makes the protein and heads to the cell surface – all normal behavior. It leaves the cell, but is now tethered to it, and thereby, remains attached to the cell that made it.”
As a result, researchers are able to examine that protein outside the cell that made it.
“We will be able to tell – with confidence – if this protein is directly related to the DNA change in that cell,” Johnsen said. “This has never been done before to be able to test a lot of changes at once. This is the innovative part.”
That innovation is expected to become a standard for other researchers. The NIH-funded Clinical Genome Resource (ClinGEN), a centralized system regarding the clinical relevance of genes and variants, is expected to incorporate a way to use evidence from Multi-STEP into its rules.
Meanwhile, Johnsen and Fowler are planning to extend MultiSTEP to other secreted proteins involved in blood clotting.
“We hope to provide variant effect data that clinicians can use to interpret related variants,” Fowler said.
