DNA Sequencing for Families with Rare Inherited Retinal Diseases.

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

UPDATE JULY 2019: 

Just in time for the 4th of July, on July 3rd, 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 will establish the first sequencing research service at Oakland University applied to the discovery of genetic diseases. 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 and so we have developed and then tested a targeted 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 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, a few hundred PCR primer pairs were designed to copy and amplify all the exon and flanking intron sequences from 8 genes of interest.
  • Each PCR product is about 250 base pairs in length and the total sequence from these 8 genes is about 27,000 base pairs. A few hundred primer pairs are mixed in three different pooled combinations (three PCR reactions per patient).
  • Up to 16 patient DNA samples are processed 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 36 to 45 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 microscope imaging system, 4.5-Million PCR library products, averaging 250 base pairs each in length, are sequenced simultaneously to generate a total of 1.2 GigaBases of DNA sequence. The illumina primers contain a unique 4-basepair sequence for each sample, so the iSeq100 then sorts all the sequencing reads into separate folders for each sample (Patient).
  • Each Patient’s DNA sequence fragments are aligned to the reference, known, Human genome sequence and then any differences in the Patient’s DNA sequence is determined.
  • We use special bioinformatics software developed at Oxford University (UK) to sort these DNA sequence variants and discover changes that would change or impact the normal proteins encoded by the 8 genes of interest.

The result of the above process is that the current ability to test 2 to 3 of these genes in a DNA sample for about $4,500, using different service laboratories, will now be 8 genes for less than $400 of cost. Not only will this make it possible to sequence more genes for more patients, Dr. Mitton is using this new genetic testing research system to provide advanced training to Oakland University’s undergraduate, graduate and medical school students.

Advertisements

On doing Science well.

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.

Sincerely,

Ken Mitton

Lab News: Summer’s Start 2019

Current Lab Member and Lab Alumni News: May 2019

Dr Ken Mitton and Wendy Dailey attended ARVO 2019 (May) 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.

The 2019 Summer Undergraduate Program in Eye Research has begun, with six Oakland University Undergraduate science students learning about, and contributing to, the NIH and PRRF funded research projects in our group. You can learn more about this program at:

http://www.oakland.edu/ERI

John R. Trevithick, PhD, A Canadian Vision Research Pioneer Leaves Us at Age 82.

It is my sad task to note the passing of John Trevithick, PhD, at age 82, at his home in London Ontario. (2/20/2018)

John is well known to many ARVO (Association for Research in Vision and Ophthalmology) and ISER (International Society for Eye Research) members, since the early 70’s as one of the first researchers who took up the lens as a model for cell differentiation. A pioneer in the explorations of cAMP in N. Crassa, he sought to explore this signaling molecule in mammalian cell differentiation. The lens epithelial to fiber cell transition continues during the entire life of the mammalian lens.

            Along the way John explored cataract mechanisms of course but as a biochemist he always was interested in the fundamental knowledge of cell physiology and biochemistry that could be revealed. His lab was one of the first groups to optimize culture media conditions to permit the study of whole living lenses in tissue culture. This permitted the precise manipulation of external and internal chemistry and was used to correlate research done with diabetic rat models and the RCS model. Interesting effects on the RCS lens posterior opacity caught his attention in this model, where degeneration was occurring in the retina. Along with diabetic cataract models his group explored the contribution of antioxidant biochemistry to degenerative processes in the lens and retina. Some surprising findings, of dramatically slowing cataract formation and other degenerations by supplementing endogenous antioxidants resulted in a couple Nature publications. I joined his lab in 1988 as a PhD student, at the University of Western Ontario.

            John took me, and other students, to many meetings around the world and he spent a lot of care teaching us to repeat work and make sure we published reproducible data. He let us write papers early and we tended to graduate with a few publications already completed. With John, I enjoyed discovering that the diabetic rat lens was iso-osmolar for several weeks by dumping Taurine and free amino acids to perfectly balance the increase in sorbitol. Then this amino acid deficiency led to the loss of GSH, ATP production, ion transport capacity, and finally the loss of fluid regulation. This was a lesson John taught many of us, that if you delve into the biochemistry of even an old model system, you will likely find some new knowledge. Do not assume that a decade of literature on a topic has documented everything important.

            John was Emeritus professor of Biochemistry and had continued also as a professor in Kinesiology, working to explore the effects of exercise on the eye in normal and diabetic rats, working with Tom Dzialoszynski and Earl Nobel. Most recently they spent the last several years on space radiation effects upon the lens. That work, funded by the Canadian Space Agency, involves trips to UBC and the use of their busy cyclotron facility. John, always curious to the very end and finding ways to fund vision research in Canada, which does not have a vision-focused funding category as we have in the USA.

If any members of the vision research community have a memory of John Trevithick they would like to share, please email to me at 

Mitton@oakland.edu

Tom and I are planning to write a short memorial paper about John and his contributions to vision science in Canada and internationally. Many of us owe our thanks to John for the time he devoted to ARVO, ISER, vision science and teaching the next generation of scientists.

A mostly complete list of John Trevithick’s publications in PubMed are found here:

John Trevithick Papers in PubMed

On the pathway to having to teach your own kids: Teacher Shortages in all States as Americans treat teachers so bad.

From twitter @kpmitton:

 #Education in #TrumpVos era. 

Americans treat teachers so bad, soon they will have to teach their own kids! 
#Teachershortage in 50 states. 

Some states are lowering certification requirements so you do not even need a college degree.

#OKLAHOMA’s TEACHER OF THE YEAR leaves state! 

Read more about Student Suicide Investigation; and DeVos Proposes New Student Loan Rules  to make it harder for students to get money back when defrauded by for profit colleges.

  Http://n.pr/2ETJ0Fr

Being A Girl: A Brief Personal History of Violence

A very good read. A very important read.

The Belle Jar

1.

I am six. My babysitter’s son, who is five but a whole head taller than me, likes to show me his penis. He does it when his mother isn’t looking. One time when I tell him not to, he holds me down and puts penis on my arm. I bite his shoulder, hard. He starts crying, pulls up his pants and runs upstairs to tell his mother that I bit him. I’m too embarrassed to tell anyone about the penis part, so they all just think I bit him for no reason.

I get in trouble first at the babysitter’s house, then later at home.

The next time the babysitter’s son tries to show me his penis, I don’t fight back because I don’t want to get in trouble.

One day I tell the babysitter what her son does, she tells me that he’s just a little boy, he doesn’t know…

View original post 1,528 more words

Hurricane Andrew: Working in a Category 5 Storm

THis is a great article from the National Hurricane Center blog, also hosted here on WordPress. This story dating from Hurricane Andrew, tells the story so well of what the NHC provides and why even the NHC itself needed a modernized hurricane proof center to keep those hard working forecasters safe when the world is being shook hard.

NHC

Source: Hurricane Andrew: Working in a Category 5 Storm

Our Lab’s latest publication: Norrin treatment testing for survival of optic nerve cells.

Norrin treatment improves ganglion cell survival in an oxygen-induced retinopathy model of retinal ischemia.  In Experimental Eye Research (2017), accepted, in press.

RGCellLivingEye

 

Highlights
• Norrin treatment accelerates recovery of the mouse OIR model from ischemic insult.
• SD-OCT can compare NFL/GCL (nerve fiber layer/ganglion cell layer) thickness in vivo.
• Norrin treatment counters thinning of the NFL/GCL in the mouse OIR model.
• Norrin treatment increases the surviving population density of RGCs in OIR retinas.

This paper is one of the first to use the in vivo imaging methods of intrinsic fluorescence with a transgenic mouse strain to see individual ganglion cells in the living mouse eye, and to even follow their morphology over a period of many days in the mouse model of oxygen-induced retinopathy. This was done with a Phoenix Research Labs‘ system, in this case the Micron-III version of their imaging system. We used a light filter set recommended by Phoenix to image yellow-fluorescent protein (YFP). Axons and dentrites could be seen on single cells in anesthetized mice. Amazing!

We also employed SD-OCT (Spectral Domain – Optical Coherence Tomography) to capture 3D structural records of the mouse retina and then to measure the changes in thickness of the very thin Nerve Fiber Layer / Ganglion Cell Layer (NFL/GCL).

The ability to use these imaging systems in vivo, which are also used in clinical analysis of the Human retina, enables us to see disease processes as they progress and to use far fewer mice to get the answers to research questions. In this case we were testing the ability of Norrin (Norrie’s Disease Protein) to be used to help avascular regions of retina recover their vasculature more quickly and improve the survival of RGCs (retinal ganglion cells) from the stress of low oxygen. RGCs are the cells that form our optic nerves. Millions of RGCs per eye have axons that extend all the way into connections with our brain. This bundle of a million “wires”, or axons, is the optic nerve.

Our research here and that of other laboratories suggest that Norrin and other agents might have use to maintain a better vasculature in diseases where the blood vessels and capillaries are damaged, such as ROP, Diabetic Retinopathy and AMD.