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Development of High-throughput, low-cost targeted DNA-sequencing panels for rare inherited pediatric retinal diseases: First publication on FEVR patients sequenced just published March 2022. In addition to generating new information on multiple protein-altering variant combinations, the results identified the cause of FEVR in individual children from 9 families!
Survey of Multigenic Protein-Altering Variant Frequency in Familial Exudative Vitreo-Retinopathy (FEVR) Patients by Targeted Sequencing of Seven FEVR-Linked Genes
From 2019 into 2020, we developed our custom targeted-sequencing panel using the Illumina DNA-sequencing platform. This method uses 180 simultaneous PCR reactions to amplify all of the exons, intron/exon boundaries, and promoter regions of candidate genes whose DNA sequences may be altered and cause several orphan (rare) inherited retinal pathologies in children. We screen eight genes that are involved in the following diseases: FEVR (Familial Exudative VitreoRetinopathy), Norrie Disease, Persistent Fetal Vascular Syndrom and Retinoschisis. By establishing this shift in testing methods we transition from the testing of one or two genes costing about $2,000 per sample down to just $250 dollars per person to test at least 8 genes. This supports our research into the relationship between disease severity and specific inherited genetic mutations, as well as helping more families with these rare genetic conditions. The scientific goal is to dramatically increase our knowledge of specific gene changes and how they correlate to different disease characteristics, such as clinical appearance and rate of changes in the retina’s structure and function. The humanity goal is to help more families identify the molecular cause of their inherited condition and to give their physicians and genetic counselors more information to manage and eventually treat their progressive blindness. We are one of the first groups to tackle the development of this kind of sequencing panel for orphan retinal disease genes.
We analyze the data, using computational tools, databases, and protocols that I teach to our group, along with our sequencing technologist Wendy Dailey, using the resources of our Pediatric Retinal Research Lab (PRRL), in the Eye Research Institute of Oakland University. We still provide this research DNA-sequencing service to patient families of Associated Retinal Consultants (Michigan) at no cost. We seek donations from the public to the PRRL, or through the Pediatric Retinal Research Foundation and The Carls Foundation, two charitable foundations that kindly fund this program.
Our first paper with this new analysis method concerns FEVR patients (76) and was published in the journal GENES. This study has established that our group of FEVR patients had changes to more than one FEVR-related protein at a rate (30%) much higher than expected in the general world population (3%).
Dr. Mitton is a member of the newly formed Global Eye Genetics Consortium.
US Small Business Technology Transfer. We completed a successful Phase-I STTR feasibility study to make a novel protein therapeutic based upon the Human Norrin protein. “Noregen” (TM) is a modified human recombinant protein for research on treatments to repair and rebuild a damaged retinal vasculature. I was the Primary Investigator who submitted a National Eye Institute (National Institutes of Health) Phase-1 STTR grant (US Small Business Technology Transfer) as the academic partner working with Caeregen Therapeutics (formerly Retinal Solutions LLC). My group designed and successfully demonstrated a biotechnology production method for biologically active Noregen protein in bacterial factories. Noregen (TM) is based on the Human Norrin protein. We completed this Phase-1 study in 2021, despite pandemic disruptions to supply chains, and met all of the Phase-1 milestones including fermentation yield, purity suitable for intraocular injection, and demonstrated the ability to refold this disulfide-bond rich protein into a conformation that binds its receptor (Frizzled-4). and activates the Norrin-wnt signaling pathway in primary Human Retinal Microvascular Endothelial Cells. We also demonstrated that Noregen can improve the regrowth of the retinal vasculature in the mouse Oxygen Induced Retinopathy model, similar to what we have published from studies with Norrin in the last decade. (Dr. Mitton is not an employee of Caeregen and does not have financial interests in this company.)
Based on the successful Phase-1 STTR, Caeregen Therapeutics was able to submit the next stage Phase-2 SBIR (Small Business Innovation Research) grant to the NEI/NIH to continue the development of Noregen to support more pre-clinical research on effectiveness and safety. Caeregen has also obtained the orphan drug status designation for Noregen. My lab group will participate in more preclinical development research of Noregen using the mouse OIR-model and primary Human Retinal Endothelial Cells under this Phase-2 SBIR. This Phase-2 application achieved a very high score in recent peer-review and we are very hopeful for awarding of funding. For our contribution, there will also be training and educational opportunities for Oakland University undergraduate students and one Master’s student in Biological Sciences or BioEngineering.
Reliable mobile fast tests for COVID-19 Antibodies. While all research labs were shut down for a few months, late March to mid-June 2020, for all but COVID19-related research, I turned my biochemical skills to a project to help evaluate one version of a serum COVID-19 antibody test that can use a single droplet (10 microliters) of blood from a finger-poke and which gives a reliable result in 15 minutes. Also without using any powered equipment. Cheap, mobile reliable tests like this can be used in almost any location. The problem from January to March 2020 was that many unreliable kits for such tests were rolled out mostly by firms looking to cash in on public fear. The main problem with those test kits was that they either 1) fail to detect antibodies to COVID19 when people have them, or 2) they say you are positive for antibodies to COVID19 BUT you are not.
Using finger-tip blood for tests of this type is possible, and in fact, there are FDA and EU-approved tests that use this format (lateral flow test) for AIDS and for Legionnaires Disease. However, those tests have been around for some time and were of course tested to confirm their reliability. So I got into with a manufacturer of antibody-based research reagents when I heard they were turning their expertise to making a reliable family of tests for COVID19 antibodies in blood. This US company has made reliable reagents I have used in my own research lab for years, and their reagents had worked where others had failed to deliver. Working with one and now three OU students (Nahrain Putris (M4, OUWB, Meron Tarekegn (M4, OUWB), Kaylee Gwyn (OU, senior), we started a research study to evaluate this particular test system and have had the study approved by our Biosafety Committee and our IRB (Institutional Review Board). After testing of the first 30 volunteers, we could determine that the test does NOT suffer from false positives, and it does detect antibodies to COVID19 in persons who have also had positive antibody tests from the standard clinical arm-drawn blood test that is FDA approved. We also have recently started to test persons after one or two doses of an FDA-approved vaccine to COVID19 and we can clearly detect the appearance of antibodies to the COVID19 spike protein in volunteers who have had the Pfizer vaccine after one or two doses. This investigation continues.
We continue to recruit volunteers for a finger-poke based testing. Our test subjects may be persons who:
1.- have had a standard clinical serum antibody test (positive or negative) or
2. – persons who have had COVID19 and are now recovered from the illness or
3. – persons who have had at least one dose of an FDA-approved vaccine for Covid19.
COVID19 Serum Antibody Test Research Project – at Oakland University.
Photoreceptor-Maturation Gene-Activation Database: Genome-wide Map of RNA-Polymerase-II Binding
One of the most useful research resources for researchers interested in genes activated in mammalian photoreceptors in vivo is our mapping of RNA-Polymerase-II (Pol-II) in most genes of the mouse genome. I have programmed a server-link so the research community can compare the presence of Pol-II on photoreceptor genes at age P2 (immature photoreceptors) and age P25 (mature functional photoreceptors) without the need for using any proprietary software. These tracks will load for you in the Genome Browser at UCSC. (Once you get there, find the “Configure” button below the graphical window and set the label area width to 26 characters and text size to 14.) Remember to ZOOM OUT 1.5x, so you see the promoter region of your gene of interest. Substantial amounts of Pol-II may be found throughout the gene as well. Distal (far upstream) and proximal (near the transcription start site) regions of a promoter may have Pol-II peaks because those regions are brought together physically in the promoter activation complex.
Use this link to go there now, and find your gene of interest:
Mouse Retina (Photoreceptor) Gene Activation Database
This will load the data for you in a new browser window and begins with the Rhodopsin gene shown. Orange bars show regions of the DNA that had RNA-Polymerase-II bound to them above background controls. There are also “Peak” tracks that indicate the points where the levels of bound polymerase were maximum. An increase in peak value between age P2 and P25, >1.8 fold ratio, indicates a significant expression increase in vivo as rod-photoreceptors matured. So look for your gene. If you would like high-resolution maps of the binding peaks in any gene to use for your own research or publications, please contact me (firstname.lastname@example.org). I provide this information, no charge, to other retina researchers. If you have questions, email me.
If you are a student at Oakland University, you can complete your 4995 independent research experiences in ERI laboratories. We also mentor Honors College thesis students and more recently have added Medical School Capstone Research students, and Engineering Biology Capstone students. If you are interested in learning how to do science in our laboratories, please use the form link for student applicants found in the main menu if you are on a large screen, or click here:
Undergrad Research – Mitton Lab &PRRL
Current Lab and Lab Alumni News:
May 2022. Our lab group presented five accepted abstracts to the ARVO 2022 meeting (Association for Research in Vision and Ophthalmology), in Denver, CO. Four have to do with our pediatric patient DNA sequencing and include eight OUWB medical students as co-authors. Our latest publication from this sequencing effort was just made public at the journal GENES. Brandon Metcalf, OUWB medical student (M4) who has worked on VEGF research with our group has matched to pathology this year and will take up advanced training nearby at Beaumont.
June 2021. The ARVO2021 was an ARVO2021 e-meeting. Now that we have had a year of virtual meetings, classes and seminars, meetings are adapting with online platforms that better support virtual scientific meetings. In summer 2020, I attended the International Society for Stem Cell Research (ISSCR 2020) meeting and the American Society for Cell Biology (ASCB) meetings which were fully virtual. I wanted to see how meeting platforms were evolving. (Ken Mitton). They are good enough that our group did our part to support the ARVO 2021 e-meeting in May 2021, based in San Franscisco, which is the largest vision science meeting in the world every year. Naomi Haque, Amanda Cicerone, Michael Sun, and Nahrain Putris all submitted abstracts in early January to the ARVO2021 meeting their abstracts were accepted. Two abstracts deal with our VEGFA control of gene expression research in primary human retinal endothelial cells (these cells line the interior of blood vessels), cells derived from human retinal tissue donors. Two abstracts concern our orphan (rare) pediatric inherited retinal disease DNA-sequencing testing, which we developed to target 8 genes involved in Norrie Disease, FEVR and Retinoschisis. The testing as we developed it has been funded by the Pediatric Retinal Research Foundation of Michigan, and some of us participated in the second annual Hope For Vision Walk in OCT 2020, to help raise over $30,000 for the foundation. Our latest publication is currently in press in Molecular Vision journal reporting our first research that mapped the full dose-response curves for activation of two key intracellular signalling kinases in primary human retinal endothelial cells, using isoforms of VEGFA165. We also would like to congratulate our OU graduate student Naomi Haque who has gained acceptance to medical school for 2021, and also to Nahrain Putris who will be graduating this spring from the OUWB medical school and just matched to the Ophthalmology Residency program at Beaumont Hospital, Royal Oak, Michigan. Matching doctors this year, are matched to the 2022 start of residency, and will first complete a year of Internship through 2021/22.
All in all, our group has faired just fine throughout the pandemic so far. Research does move slower, as it does for most of the United States at this time, and there is more management time required to work safely and monitor safe working practices. However, we continue to strive to move forward one day at a time and we have managed to keep moving forward thanks to everyone in our group, the ERI and Oakland University.
2020. The ARVO2020 meeting had to occur as a virtual meeting, so our lab group had several abstracts that we let publish in the meeting abstracts. Naomi Haque (Masters student) and Nahrain Putris (OUWB medical student, M3 year) had accepted abstracts on the subjects of PLVAP gene expression and p38MAPK signalling in primary human retinal endothelial cells. Dr Ken Mitton, Wendy Dailey, and Michael Sun (OUWB, M2) also had a third abstract on our new orphan pediatric retinal disease gene sequencing test panel system for patients with Norrie Disease, FEVR, and Retinoschisis. While research was shut down for a few months into the summer of 2020, Dr. Mitton begin a COVID19 antibody test system evaluation with its manufacturer Epigentek. This was to evaluate a 15 minutes blood test for reliability because many other tests had flooded the market that were NOT reliable. Through 2020, with the assistance of two M4 year OUWB students (Nahrain Putris, and Meron Tarekegn), we validated that the Epigentek lateral flow assay does not suffer from false positives and it does detect IgG and IgM antibodies to the COVID19 spike protein. This work continues, as we now will use the test to detect antibodies in persons after vaccination. So far it looks like the test also is useful for monitoring vaccination response after the Pfizer vaccine’s first and second doses. In 2020 we also managed a 4th iSeq-100 run and completed the testing and activation of our protocols to establish our ability to sequence run up to 50 patients at once using our 8-gene panel. About 98 persons were sequenced in our first start up phase and OU honors college student Amanda Cicerone came on board our ERI gene sequencing team.
May 2019. Dr Ken Mitton and Wendy Dailey attended ARVO 2019 and presented research on VEGFA isoform regulation of primary human retinal endothelial cells. Also, on Norrin regulation of PLVAP expression in the same cell type. Dr. Mitton co-presented a third abstract with Tom Dzialozsynski, Western University (London Ontario) regarding Gensingoside effects on normalization of serum lipids and slowing of cataract formation in diabetic rats. John Trevithick, PhD, Professor of Biochemistry and Kinesiology (Western University), Tom and Ken’s former supervisor and long time friend and colleague was also on the abstract. Dr Trevithick passed away just over a year ago. The 2019 ARVO meeting was the first one ever outside the United States. Vancouver BC. We wanted to make sure that John Trevithick’s research was presented at this first ever ARVO meeting held in Canada. (Dr. Mitton obtained his PhD under John Trevithick at Western University, London Ontario). Without John Trevithick’s influence, the many many Michiganders trained in biosciences in our lab since 2001 would not have had their opportunities here. Life is connections, and at times a series of wonderful accidents. Peter Chen MD, graduated OUWB school of medicine. The second OUWB medical EMBARK program student in the ERI. Michael Sun has joined our group as the 5th EMBARK program student in the ERI, just completing his M1 year in our OUWB program. Michael will be working with us to help establish patient DNA sequencing for rare inherited retinal vascular diseases in our Pediatric Retinal Research Laboratory.
January 2019. OUBW Embark Research medical student Peter Chen (M4), has matched for Ophthalmology Residency at the University of Cincinnati. Peter contributed to our work on VEGFA isoform differences in activation of the AKT pathway.
October 2018. Undergraduate student Megan Moore presented “Differences in the Activation of Human Retinal Endothelial Cell Gene Expression by Isoforms of VEGFA165”. (Megan Moore, Wendy Dailey, Anju Thomas, Ed Guzman, Jennifer Felisky and Kenneth Mitton.) at the Sigma Xi International Undergraduate Student Research Convention. October 27, 2018. San Francisco, CA. Poster.
March 9th, 2018, undergraduate students Jennifer Felisky and Megan Moore both presented talks at the Michigan Academy of Arts, Sciences and Letters hosted at Central Michigan University.
May 2018. OUWB medical student Austen Knapp, MD, graduated in 2018 and matched for Ophthalmology Residency at Cleveland Clinic. Dr. Knapp’s Embark research contributed our data on differences of MAPK (ERK1/2) pathway activation by two isoforms of VEGFA165, included in our presented abstract at ARVO 2018, Honolulu HI, May 2018.
Camryn DeLooff (SUPER program 2013) is currently in the full time MBA program at the internationally renowned business school, the Rotman School of Management, University of Toronto. Quentin Tompkins (SUPER program 2015) has been accepted into several medical schools for fall 2018. Congratulations Quentin. Brandon Metcalf (SUPER program 2014) is now in his M2 year in the OUWB school of medicine. He has also joined our lab again for his medical school capstone research project. Nahrain Putris, another SUPER program alum, has also joined our lab (M2) for VEGFA research.
My Philosophy on doing Science well (Ken Mitton).
As for many biomedical, basic-science research labs, my research flows and changes over time as we make new discoveries that lead us to new questions we form even as we uncover the answers to previous questions. That is the nature of basic science, and it is the way science investigation has always brought the most benefits to people and medicine in particular. While many organizations and countries have attempted to focus research support (funding) into specific diseases, it turns out that the overwhelming majority of high-impact medical discoveries have come from “serendipity”. That is, great useful ideas and tools were discovered to treat diseases simply by exploring how things work.
For example, drugs for controlling high cholesterol were not discovered by deciding to start making drugs for treating high cholesterol. In the course of biochemists investigating how our cells make cholesterol in the first place, chemicals were used to block enzymes to help figure out how cholesterol was made. Some of these chemicals were obviously the idea to become new drugs that could block cholesterol made in the body. Latanoprost, one of the later generation of drugs developed in the ’80s for reducing high intraocular pressure (IOP), was based on the discovery that prostaglandins made by some cells in the eye could increase the aqueous outflow in the eye, and reduce pressure. The basic science was elucidated in animal models. Again, a basic science discovery in the laboratory of physiologist Laszlo Bito at Columbia University was adopted by a Pharma company as the way to make drugs that mimic natural prostaglandins to produce this new class of drugs. As a result, thousands of people around the world have another class of drugs to reduce their intraocular pressure and reduce their risk of vision loss from Glaucoma.
So, you never really know where benefits will arise for biomedicine. That is why many research funding agencies, such as the NIH (USA) and the MRC (UK), understand the importance of funding physiologists and biochemists to explore how things work. In our case, how things work in the eye, and the retina of the eye.