Oral Presentation 2019 Hunter Cell Biology Meeting

Functional assays for determining the pathogenicity of ATP7A variants using primary fibroblasts and patient derived induced pluripotent stem cells. (#37)

Gonzalo Perez-Siles 1 2 , Anthony Cutrupi 1 2 , Jakob Kuriakose 3 , Melina Ellis 1 , Reinaldo Issao Takata 4 , Speck-Martins Carlos Eduardo 4 , Horvath Rita 5 , Duff Jennifer 5 , Ginzberg Mira 6 , Baets Jonathan ‎ 7 , Deconinck Tine 7 , Bowns Benjamin 8 , Tandy Susannah 8 , Sabir Ataf 8 , Nicholson Garth 1 2 9 , Sharon La Fontaine 10 , Marina Kennerson 1 2 9
  1. Northcott Neuroscience Laboratory, ANZAC Research Institute, Sydney, NSW, Australia
  2. Sydney Medical School, University of Sydney, Sydney, NSW, Australia
  3. School of Life Sciences, University of Technology Sydney, Sydney, NSW, Australia
  4. Sarah Network Rehabilitation Hospitals, Brasilia, DF, DF, Brazil
  5. MRC Centre for Neuromuscular Diseases and John Walton Muscular Dystrophy Research Centre, Institute of Genetic Medicine, Newcastle, United Kingdom
  6. Pediatric Neuromuscular Unit, , Wolfson Medical Center , Holon, Israel
  7. Neurogenetics Group, Center for Molecular Neurology, Antwerp, VIB, Belgium
  8. West Midlands Regional Genetics Service, Birmingham Women's and Children's Hospital, Edgbaston, Birmingham, United Kingdom
  9. Molecular Medicine Laboratory, Concord Repatriation General Hospital, Sydney, NSW, Australia
  10. Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Burwood, Melbourne, VIC, , Australia

Mutations in the ATP7A gene cause X-linked hereditary distal motor neuropathy (dHMNX). To date, p.T994I and p.P1386S in this copper (Cu) transporting ATPase are the only confirmed mutations causing dHMNX. Next generation sequencing is increasing the rapid detection of variants in genes known to cause disease, however the absence and size of additional families, make it challenging to determine the pathogenic or benign status of these identified variants of unknown significance (VUS). We have recently identified new variants in ATP7A in patients with progressive peripheral neuropathy, suggesting further genetic heterogeneity of dHMNX. Two of these new variants, p.E840V and p.M1311V, are located at highly conserved amino acids within domains of ATP7A that are critical for the catalytic cycle of the copper transporter. We have also identified three additional variants (p.R703H, p.Y760C and p.A768G) that cluster within close proximity of the known dHMNX mutations p.T994I and p.P1386S in the 3D structure of ATP7A, strongly suggesting these variants may cause disease by sharing a common pathomechanism.

Our investigations using patient fibroblasts and an Atp7a conditional knock in mouse model for dHMNX have shown defective retrograde trafficking of mutant ATP7A leading to Cu dysregulation.  We have systematically assessed this phenotype on patient fibroblasts. Our results indicate altered Cu induced trafficking of ATP7A can be used to assess pathogenicity of newly identified variants in ATP7A and establishes an important functional assay for validating the pathogenic status of VUS in those cases where genetic evidence is limited.

To investigate how defective ATP7A trafficking leads to degeneration of motor neurons we have generated a line of induced pluripotent stem cells by re-programming fibroblasts from a dHMNX patient with the ATP7A p.T994I mutation. The patient motor neurons will be an ideal neuronal system to model axonal degeneration and to investigate how defective trafficking of ATP7A leads to motor neuron death in dHMNX and other neurodegenerative diseases in which Cu dysregulation occurs.