A functional framework in patient fibroblasts informs ATP7A variant pathogenicity and identifies p.Q990P as a novel...

ATP7A is a P-type ATPase copper transporter and a central component of the intracellular network to maintain copper (Cu) homeostasis, trafficking between the trans-Golgi network (TGN) and the cell periphery in response to intracellular Cu levels. Pathogenic ATP7A variants cause Menkes disease (MNK), occipital horn syndrome (OHS) and X-linked distal hereditary motor neuropathy (HMNX), with the clinical severity inversely related to residual ATP7A function. In MNK and OHS, variants result in loss of ATP7A function due to absent (MNK) or markedly reduced (OHS) protein levels, leading to abolished ATP7A trafficking and/or impaired Cu transport capacity. HMNX-associated variants are thought to retain partial Cu transport activity, although functional data remain limited. To date, three missense variants (p.T994I, p.P1386S, p.A991D) are established causes of HMNX, but the number of ATP7A variants reported in patients with a distal motor neuropathy phenotype is increasing, creating a need for functional assessment of variants of uncertain significance (VUS). We have developed a functional framework for evaluating HMNX-associated ATP7A variants. Using three patient fibroblast lines carrying the confirmed variants, we demonstrate reduced ATP7A TGN localization under low-Cu conditions and impaired capacity of these cell lines to maintain intracellular Cu levels. By applying these assays to five VUS (p.R703H, p.Y760C, p.A768G, p.Q990P, p.M1311V) identified in individuals from unsolved peripheral neuropathy families, we identify p.Q990P as a novel ATP7A variant in a patient with progressive peripheral motor neuropathy. This approach enables comparison of Cu trafficking and handling across ATP7A alleles, providing a functional framework to support diagnostic variant classification.