Virginia Kimonis, MD
Especially Beautiful Fundraising Update
November 2017

NUBPL is a “rare” mitochondrial disease.  Research activities represent hope for the lovely Spooner girls and others with this rare condition. In fact, the field of clinical genetics represents a promising pathway to new and better treatments for many rare diseases. And, what this means is that every child with NUBPL disease today has a reason to hope. Nothing is more thrilling than to successfully slow or treat a disease.

With the funds we have completed the following studies:

Phase 2

1. The Kimonis Laboratory has studied the skin fibroblasts from the girls for the mitochondrial function at UC Irvine using the Seahorse instrument in collaboration with Dr. Christina Kenney’s lab.
2. We have studied the effect of paraquat a mitochondrial complex 1 poison and hydrogen peroxide to the cells in order to study mitochondrial function under conditions of stress and have found differences between the patients and age matched controls.
3. Moyra Smith and Ambry have completed the reanalysis of the exome data in the two girls to understand the cause of the differences between the two girls. No further differences have been identified.
4. Our collaborator, Janneke Balk, at the University of East Anglia, UK, has shown in her lab that treating yeast cells with peptones improves mitochondrial function. Therefore, we now recommend treating patients with NUBPL disease with a high-protein diet. As a result of our collaboration Dr, Balk has treated the cells and yeast mutants with components of the mitochondrial cocktail to identify specific drugs that may be beneficial. She has identified that coenzyme Q10 treatment has resulted in improvement in the activity of mitochondrial complex 1. She has also introduced the various mutations found in patients in the yeast in order to study the effect on mitochondrial complex 1 in order to conclusively establish that the mutations are the cause of the disease.
5. We have presented several posters and are completing the manuscript for publication on the 5 new patients in 4 families with NUBPL disease. Dr. Kimonis has contacted the exome abs in order to identify other patients and families with NUBPL disease.
6. Peggy Eis/Eli Hatchwell of Population Diagnostics update.

Phase 3 ($ 20,000 raised from the Especially Beautiful event in 2016)

1. Stem cell line has been made with skin fibroblasts from Calynn, Ryann and Patient 3 by Dr. Schwartz, CHOC Children’s as a collaboration ($5,000 requested by Dr. Schwartz for supplies). These stem cells have made into brain neuron cells to study the effect of the mutations in the gene.
2. Bethany Berg, genetic counseling intern for her thesis (attachment) has performed RNA studies to identify the splicing variants resulting from the Spooner mutation. The Patient 3 splicing mutation analysis was not successful.
3. CRISPR mouse model has been created:The animal model we hope will be the best and most authentic model and will allow us to actually study the brain in NUBPL disease.  Mice share 99% of the human genetic makeup, and have proven to be a very useful model of the human disease based on experience in Dr. Kimonis’ lab for the past seven years. The approximate cost to just make a mouse model is $ 10K (see attachment)
   We can perform imaging of the brain in order to study the impact of manipulating the NUBPL gene on the mouse cerebellum, balance and cognitive function.  We then plan to study the effect of a drug panel on the structure and function of the brain in order to identify potential novel treatments that could be tried in children with NUBPL disease.Expenses:
   Stem cell work                                            $5,000
   CRISPR mouse                                           $10,000
   Supplies for  RNA splicing, Seahorse     $3,000
   Balance:                                                        $2,000

Phase 4— (Goal $ 50,000)

1. CRISPR Mouse Research (research costs—supplies, animal housing, personnel, etc.)a. Experiments to be conducted (breeding mice, behavior studies, study of cerebellum, study of the expression of the gene,)b. Test different treatments (drugs) to improve the cells and the structure of the brain of the mice.

   Because the specific Spooner c.311T>C (p.L104P) gene mutation is a severe mutation  it is  possible that introducing this gene mutation will also be lethal in animals,

2. EMMA Mouse project: As a result of further research I have identified a mouse model that is embryonic lethal by E12l. It is expected the mouse will show the typical clinical features such as the cerebellar atrophy. Currently there is almost no information on the brain pathology and function in this disease. These mice can be a useful resource for future mechanistic and therapeutic studies.If the CRISPR mouse is lethal I plan to use technology to only knock out the gene in specific parts brain to study the effect of NUBPL deficiency.

3. iPSC derived neuron cells will be used to study mechanism and the effect of novel treatments by studying gene expression and further mitochondrial studies such as the Seahorse.. Stem cell work is very intensive and expensive however the potential rewards are enormous because the personalized medicine work is done using cells from the girls.
4. We are working with Pat Jennings, UCSD on understanding the chemistry of NUBPL iron clusters.
5. Janneke Balk is continuing to work on mitochondrial complex 1 activity with NUBPL mutations.

• Balk, J. “Discovery of the NUBPL gene and its role in Complex 1 assembly.
• Calvo, S. “High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency.”
• Kevelam, S. “NUBPL mutations in patients with complex I deficiency and a distinct MRI pattern.” Neurology 80 (17): 1577–1583.
• P.S. Eis, B. Schüle, S. Kim, J.W. Langston, V.E. Kimonis, E. Hatchwell. NUBPL mutations link Parkinson’s disease and other movement disorders to recessive Complex I deficiency. Abstract/Program #1403. Presented at the 66th Annual Meeting of The American Society of Human Genetics, October 2016, Vancouver, Canada.
• Sheftel, A. “Human Ind1, an Iron-Sulfur Cluster Assembly Factor for Respiratory Complex I”. Mcb.asm.org. Retrieved 25 April 2015
• Sheftel, A. “Human ind1, an iron-sulfur cluster assembly factor for respiratory complex I”. Mol. Cell. Biol. 29 (22): 6059–6073. PMID 19752196.