Welcome to 2020: DNA Sequencing for Children with Rare Inherited Retinal Diseases.

Meet Pearl. The new DNA-sequencer in PRRL (Pediatric Retinal Research Laboratory)

UPDATE January 2020: 

Just in time for the 4th of July, on July 3rd 2019, we installed and activated an Illumina iSeq-100 DNA sequencing system. Dr. Mitton, Wendy Dailey, Jennifer Felisky, Naomi Haque, Michael Sun, and Ed Guzman from the ERI are all contributing to the setup and eventual ongoing use of this system for high-throughput DNA-sequencing to discover mutations in 8 genes involved in the following rare and inherited conditions:

FEVR (Familial Exudative VitreoRetinopathy), Norrie Disease, and Retinoschisis (RS).

This was done to establish the first DNA-sequencing research service at Oakland University applied to the discovery of genetic disease causing mutations. Our goal is to use these molecular investigations to advance our clinical understanding of these blinding diseases that impacting patients of Associated Retinal Consultants, (Royal Oak Michigan).

Unlike several health care systems in Europe and Canada, DNA sequencing costs are not covered by health insurances in the United States. So for Americans, the access to even DNA-sequencing of genes we already know may be involved in a patient’s disease is simply not available because of the prohibitive costs. As molecular biologists working in vision science, my lab group has developed and then tested a targeted DNA-sequencing panel to analyze many genes related to these rare conditions for only a few hundred dollars per sample. Several developments in technology over the last several years have now made this possible.

  • We use a strategy called “Ampliseq “which is based on PCR (Polymerase Chain Reaction). Using a computational (in silico) design process from Illumina, we designed a large number of PCR primer pairs to copy and amplify all the exon and flanking intronic DNA sequences from 8 genes of interest. These are genes that we either know or think can be involved in these rare pediatric retinal diseases.
  • Each PCR product is about 250 base pairs in length and the total sequence from these 8 genes is over 30,000 base pairs. The primer pairs are mixed in three different pooled combinations (three PCR reactions per patient).
  • Up to 16 patient DNA samples are processed at the same time by PCR amplification at one time. The PCR products are linked to Illumina end sequences, purified, QC tested to make what is called a sequencing library. These are stored frozen until about 32 to 40 samples are ready.
  • The sequencing libraries from, say, 40 patients are mixed together and loaded into a processing cartridge that is then loaded into the iSeq100. The single use cartridge contains all the sequencing microfluidic chemistry reagents, and a sequencing flow cell is also plugged into the cartridge.
  • Using a fluorescent diod-array imaging system, 4.5-Million PCR library products, averaging 250 base pairs each in length, are sequenced simultaneously to generate well over 1.2 GigaBases of DNA sequence. The illumina primers contain a unique 4-basepair sequence for each patient’s sequencing library, so the iSeq100 software then sorts all the sequencing reads into separate folders for each patient using this “DNA barcode”.
  • Each Patient’s DNA sequence fragments are then aligned to a reference Human genome sequence and then any differences in the patient’s DNA sequence from the reference genome is reported to us.
  • We use special bioinformatics software in the cloud and some programming tools called “R” to sort these DNA sequence variants and discover changes that would change or impact the normal proteins encoded by the 8 genes of interest.

To start in late 2019, I am happy to report here that we sequenced the first 35 patients, most who have FEVR. This inherited condition causes the lack of a full and normal retinal vasculature, especially in the periphery of the retina, resulting in a lack of oxygen to the cells of the neural retina. FEVR, Norrie Disease, and Retinoschisis can all have effects that lead to blindness.

The result of the above process is that we now have the ability to test 8 genes for less than $300, and complete the testing in one week starting from a few drops of blood. Previously, only 2 to 3 of these genes in a DNA sample could be sequenced for about $4,500, using different service laboratories, taking months. Not only will this make it possible to sequence more genes for more patients, we can also use this new genetic testing research system to provide advanced training to Oakland University’s undergraduate, graduate and medical school students.

The next batch of patient tests samples, the first in 2020 will be sequenced this month, February 2020 and we will continue to sequence more and more patients to help us find the underlying causes of these rare genetic blinding diseases in children. Welcome to 2020.

Repairing and Regeneration, Medicine’s Holy Grail and our Technology Transfer Project

Over the last two decades my natural tendency to pursue research projects closer and closer to future clinical impact has taken me from a very focused study of the mechanisms of regulating gene expression in the retina to understanding retinal diseases that cause most new cases of blindness in the United States each year. Those diseases involve damage to the integrity of the blood-retinal-barrier and even the loss of blood microvessels that nourish the neural retina. As you can see, poking around my website, we work on things like Vascular Endothelial Growth Factor and Wnt (“wint”) signaling to understand what keeps endothelial cells of the retinal blood vessels happy and then to understand how these normal system are perturbed to cause harm.

Along the way, I have worked with Dr. Kim Drenser MD PhD and Dr Michael Trese MD, of Associated Retinal Consultants (Michigan) and the Ophthalmology department in the Beaumont Health System. An atypical Wnt-protein called Norrin has emerged as a protein essential for development of a human baby’s neural retinal vasculature. Long before I came to this project area, real human Families with an inherited condition called Norrie Disease had Sons who were missing a full retinal vasculature, similar to very young premature infants who suffer the same physical problem: a lack of blood supply to nourish an oxygen starved retina. While it was a very rare inherited condition, the eventual discovery of the gene (NDP) and its protein (Norrin) made it clear that this protein is essential to form the initial blood vessels of the neural retina.

Research we have done in animal models, and research in other laboratories, have also revealed that Norrin is also required to establish a strong blood-retinal-barrier which is formed by the endothelial cells that line the interior of blood vessels. But, how can we possibly use this information for a future therapeutic tool? One way would be to make a protein based on human Norrin in large enough and pure enough amounts to be used for both more testing in models and hopefully clinical trial use in human subjects one day. The first step is to come up with a biotechnological solution to make the protein and purify it and to fold it in its required natural 3-D shape so that it has the same activity as Norrin protein made naturally in the retina.

The later project is what I have contributed, using my knowledge and experience designing and producing proteins in bacteria (E. Coli strains used for protein production), to the production of a Norrin activity protein. After demonstrating some preliminary data on this process, I wrote a research plan submitted with a local small business firm Retinal Solutions LLC (Michigan) to obtain NIH STTR (Small business technology transfer) funding to initially iron out the process to make the protein pure, and free of toxic impurities, suitable for testing in the mammalian eye. These begin with a Phase-1 STTR, if one is successful at obtaining the funding. STTR grants require a company, like Retinal Solutions LLC, to work with a non-profit academic partner (that would be me and Oakland University) to develop a technology with the goal of benefiting US citizens with future medical treatments, drugs, and devices.

I am pleased to note that as the Primary Investigator on the project, we obtained funding for the one year Phase-1 STTR from the National Eye Institute (NIH), and we started this developmental research this Fall 2019. In this project, the basic scientist like me, works with my clinical colleagues at Retinal Solutions LLC (Dr. Trese, Dr. Drenser) to identify a clinical need and bring science to bare on the problem. While there are no guarantees of success in any drug development, clear scientific evidence for the genetic necessity of Norrin to make retinal blood vessels simply makes Norrin an excellent candidate as a therapeutic tool, a factor, that would be needed to repair and regenerate the retinal microvasculature and its strong blood-retinal-barrier. Even if Norrin is not the solution on its own, it would likely be one of the essential members required for a therapeutic team. As a protein that is already natural in the neural retina, there is also a much reduced likelihood that there would be any toxicity or ill effects. One this is for certain, just thinking about this as a dream and not trying to see if the dream can come true is not something we wish to do.

So wish us lots of luck as we make Norrin in bacteria, refold it, and put it through our testing in the Eye Research Laboratory and the protein purity characteristics testing by Retinal Solutions LLC. If all goes to plan, then the next future step will be the Phase-II STTR to get this Protein therapeutic closer to future use in human clinical trials. That is the dream, one step at a time.

Ken Mitton