By Katherine Lawless on August 16, 20192019-08-16
“Mitochondria are essential for life. Mitochondrial diseases interfere with the body’s ability to make energy and affect more than 2 million people in the world” - Mito-FIND team.
Mitochondrial diseases, as a group, are the most common inborn errors of metabolism. The mitochondria play an essential role in generating energy for life through oxidative phosphorylation (OXPHOS) and maintain many other cellular processes. Children can present with neurological, heart or liver disease, growth and developmental delay and can have a reduced life expectancy.
Adults can have muscle weakness, visual difficulties, hearing loss, stroke and digestive problems. But since mitochondria are needed almost everywhere in the body, we continue to learn that there are a huge variety of symptoms that can be due to mitochondrial disease.
The project’s beginnings
Dr. Aneal Khan, Professor of Medical Genetics and Pediatrics at the University of Calgary, has devoted his life’s work to understanding and finding ways to treat mitochondrial diseases. His team saw that methods to study common diseases, like high blood pressure and diabetes, were not going to work for rare diseases that affect the mitochondria2,.
“To find a diagnosis, patients had to go through minor surgeries to get a muscle biopsy, lumbar puncture or brain MRI. These tests are distressing to young children and they would need an anesthetic to do the test.”
In 2014, with help from a grant from MITO-CANADA, Dr. Khan and his team started the MITO-FIND project, which stands for mitochondrial functional and integrative next generation diagnostics, with a goal to use next generation sequencing (NGS) to create a faster and easier path to getting a diagnosis.
According to Dr. Khan, in comparison to a process that takes invasive samples, generates results in months or years, with a diagnosis rate of under 20%, MITO-FIND (using Next Generation Technology) can now generate whole exome sequencing (WES) results on up to 48 patients per week in as little as 26 hours at an average 40x coverage.
MITO-FIND has improved the overall rate of diagnosis to about 35% using their exome (nuclear DNA and cell free mitochondrial DNA) platform without using any needles or anesthetic. MITO-FIND also uses an up-to-date process to analyze the results with the average analysis lasting less than 3 hours per case. So a patient seen in clinic on Monday can have results by Friday which provides previously unmatched speeds in a clinic setting to help solve cases.
When needed, Mito-FIND can also add traditional methods of testing - muscle biopsy, lumbar puncture and brain MRI - in combination with NGS with a single anesthetic procedure to provide insight into the diagnosis. For example, if NGS finds an extremely rare disease, the gene result should be compared to tissue biopsy.
“Mito-FIND’s objective was to establish the fastest, least invasive, and least expensive method of diagnosing mitochondrial disease using NGS to identify the causative gene(s) and test whether gene changes cause an abnormality in mitochondrial function. We believe we now have the fastest, most comprehensive strategy.” – The Mito-FIND team.1
What they hope to solve
We have two types of DNA or two genomes in our bodies – nuclear DNA and mitochondrial DNA (mtDNA). Methods to test mtDNA have been more difficult and have received less attention. Dr. Khan helped develop a new method (patent pending) to specifically target cell-free mtDNA in plasma that is demonstrating to be as effective as mtDNA from a muscle biopsy.
In addition, Dr. Khan and his team helped to develop a mitochondrial OXPHOS analysis on skin fibroblasts, called hybrid clear/blue native polyacrylamide gel electrophoresis (BCN-PAGE) that also demonstrates to be as effective as samples from muscle biopsies.
These methods, as reported by MITO-FIND, produce a higher diagnostic yield on mitochondrial-optimized NGS testing using non-invasive sampling than a typical NGS (exome/genome) or even traditional invasive tissue sampling.
Dr. Khan hopes that most patients will no longer need a muscle biopsy to make a diagnosis of mitochondrial disease.
Not satisfied with making a diagnosis faster and easier on patients, Dr. Khan and his team are putting efforts into find a cure. They have developed protocols using muscle jumping power to measure muscle function in patients with different types of muscle disease and have tested vibration therapy to help improve mobility in patients with neuromuscular disease,.
Dr. Khan’s company, M.A.G.I.C. Clinic Ltd., is also planning to sponsor the development of a clinical trial to use mesenchymal stem cells (MSCs) as a treatment for patients with mtDNA related mitochondrial disease.
DNA collection challenges
Always striving to improve the patient experience, the MITO-FIND team set out to find an alternative diagnosis method for the most fragile patients – newborn infants or patients in intensive care who typically experience complications when disturbed for different tests.
In 2015, Dr. Khan’s team switched from cytobrushes to Oragene•DNA assisted collection saliva kits and ORAcollect•DNA oral sponge kits. The impact of these non-invasive DNA collection methods has further reduced the discomfort and challenges in obtaining high quality DNA samples, says Dr. Khan.
These products use absorbent sponges to sample along the inside of the cheek and all samples collected to date by MITO-FIND have had a high DNA yield for exomes, and most recently, a 40x genome sequencing depth.
“Babies mouths are often dry and dehydrated, and obtaining DNA through a needle poke is distressing as everyone knows. Thus blood and whole saliva samples were not practical. With the older method of using a cytobrush, we would get high bacterial contamination, frequently failing to produce adequate sequencing results. The quality of DNA and ease-of-use of ORAcollect has resulted in using these products as our preferred method of DNA collection. With samples collected in under 30 seconds, we obtain at least 40 ng of DNA per sponge sample which we found suitable to run not only an exome, but a full genome which includes both the nuclear and mitochondrial DNA in one test.” – Marina Kerr, MITO-FIND.
The added value of ORAcollect•DNA oral sponge kits is a superior yield of mtDNA compared to whole saliva or whole blood; furthermore, on the MITO-FIND optimized mitochondrial workflow, they have a better chance of finding results for both nuclear and mitochondrial DNA diseases.
Oragene•DNA and ORAcollect•DNA can be collected in an office or even by parents or patients at home and simply put in the mailbox and sent to the MITO-FIND lab without complex shipping. The kits maintain DNA stability at ambient temperature and do not require freezing or expensive shipping methods.
“Many patients in rural areas can’t get to a phlebotomist or hospital so we send the ORAcollect•DNA kits to them directly for self-collection at home. This was a game changer for Mito-FIND and improved access to DNA testing in Canada.” – Marina Kerr, MITO-FIND.
Next steps downstream
DNA sequencing is the first step. Then the big challenge is interpreting the data. In 2018, Dr. Khan partnered with his colleagues and together they founded a company called Discovery DNA to process their samples. With experience of sequencing more than 200 exomes and multiple whole genome results, Discovery DNA has customized the analysis of DNA variants for mitochondrial diseases.
Dr. Khan and his colleagues continue to work to provide a rapid and reliable method of diagnosing mitochondrial and other rare diseases anywhere.
If you are interested in trying Oragene•DNA whole saliva kits and/or ORAcollect•DNA oral sponge kits for your own research project, click on the free kits request button or email firstname.lastname@example.org.
 Parikh S et al. Diagnosis and management of mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society. Gen Med. 17(9): 689-701 (2015).
 McFarland R. Turnbull D.M. Batteries not included: diagnosis and management of mitochondrial disease. J internal Med. (2009). Doi: 10.1111/j.1365-2796.2008.02066.x
 Newell et al. Plasma-derived cell-free mitochondrial DNA: A novel non-invasive methodology to identify mitochondrial DNA haplogroups in humans. Molecular Genetics and Metabolism. 125 (4): 332-337 (2018).
 Newell et al. Hybrid gel electrophoresis using skin fibroblasts to aid in diagnosing mitochondrial disease. Neurol Genet Jun 2019, 5 (3) e336.
 Newell C et al. Side alternating vibration training in patients with mitochondrial disease: a pilot study. Archives Physiotherapy. 7:10 (2017).
 Newell C et al. Peak Jump Power Reflects the Degree of Ambulatory ability in Patients with Mitochondrial and Other Rare Diseases. JIMD. (2016). Doi: 10.1007/8904_2016_583