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- Title
Functional Testing of Wilson Disease Patient Variants in the ATP Binding Domain and C-Terminus of ATP7B.
- Authors
Pon, Julia; Davies, Lisa; Macintyre, Georgina; Cox, Diane
- Abstract
Wilson disease (WND) is an autosomal recessive disorder characterized by liver and neurological symptoms. WND results from copper accumulation due to mutations in the copper transporting ATPase, ATP7B. The clinical presentation of WND is variable and more than 580 variants have been identified in WND patients. Molecular analyses to distinguish disease-causing mutations from normal variants are essential. To determine whether eleven WND patient missense variants in the ATP binding domain and C-terminus are disease causing mutations, full length ATP7B variant cDNAs were stably transfected into Chinese Hamster Ovary (CHO) cells, and confirmed by ATP7B cDNA sequencing. Insertion location and expression were verified by β-galactosidase assays and RT-PCR. As functional ATP7B protects CHO cells from copper toxicity by exporting copper, cell lines were assayed for copper resistance. G1149A and L1371P conferred no protection from copper. In contrast, A1250G and D1407E exhibited normal levels of protection. P1052L, I1184T, V1262F, P1273Q, L1305P, Y1331S and I1336T showed intermediate copper protection. Similar results were found at a lower ATP7B induction level. Wildtype ATP7B traffics between the Golgi and cytoplasmic vesicles in response to excess copper. Trafficking defects were observed for P1052L, G1149A, I1184T, V1262F, L1350P, I1336T, and L1371P. On western blots, variants with trafficking defects were undetectable, while the other variants appeared similar to wildtype. A1250G and D1407E are suspected non-disease causing variants, while the other nine variants likely contribute to the Wilson disease phenotype. Our identification of amino acids required for copper transport and ATP7B trafficking advances our understanding of ATP7B structure and function.
- Subjects
HEPATOLENTICULAR degeneration; NEUROSCIENCES; ADENOSINE triphosphate; LIVER physiology; BINDING sites; COPPER
- Publication
UBC Medical Journal, 2011, Vol 2, Issue 2, p40
- ISSN
1920-7425
- Publication type
Abstract