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 September 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 all contributed 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 computer design process from Illumina, we designed a large number of PCR primer pairs to copy and amplify 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 DNA base pairs long and the total sequence from these 8 genes is over 30,000 base pairs.
  • 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 40 to 50 samples are ready.
  • The sequencing libraries from 40-50 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.

We have run four iSeq-100 cartridges as of Sept 2020, I am happy to report here that we have successfully sequenced over 90 persons from our Families who have FEVR, Norrie Disease and Retinoschisis. FEVR and Norrie DIsease results in an incomplete retinal vasculature, especially in the periphery of the retina, resulting in a lack of oxygen to the cells. Retinoschisis results in a delicate retina that can suffer layer-separation and retinal detachment due to the functional loss of the Retinoschisis protein, a protein forms an essential structural glue to hold retinal layers together. 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 about $250 per person, and complete the testing in one week starting from a few drops of blood. Previously, only 2 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.

While we are in a pandemic in 2020 we have not stopped work on analysis of our sequencing data for patients and we continue to get ready to sequence more persons with these orphan (rare) retinal conditions. Additional undergraduate students and medical students from Oakland University and MSU are getting their feet wet in genetic analysis over the 2020/2021 year, including Zaid Yahya, Tim Page Jr., Amanda Petrelli Cicerone, and Esi Benedict (MSU).

We also help to raise the funds for DNA sequencing so that no Family has to pay for any DNA sequencing we do. To that end we are doing our 2nd annual Walk for Vision 2020, virtual even, to raise funds for the Pediatric Retinal Research Foundation who have supported the start up of our new sequencing research service for these rare diseases. http://prrf.blueskysweet.com/

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