Translation reinitiation at the ATP7A locus associated with favorable early treatment outcomes in classical Menkes disease
Background: Menkes disease (MD) is a neurodegenerative disorder of copper (Cu) transport caused by mutations in a Cu-transporting ATPase (ATP7A). Based on downstream sequence, we previously hypothesized (Nature Genetics 13:21-22) that translation reinitiation may be one mechanism of residual ATP7A activity that could explain successful early treatment outcomes in patients with nonsense mutations in the amino-terminal region of ATP7A.
Purpose: To evaluate the relationship between molecular defects and responsiveness to early copper histidine treatment in two infants with classical Menkes disease caused by different amino-terminal nonsense mutations.
Design/Methods: We conducted a clinical trial of early copper histidine therapy (250 mcg sc b.i.d. to age 1yr, then q.d. to 3yrs) in 16 infants with MD. The trial was approved by the NINDS IRB and the NICHD intramural Data Safety Monitoring Committee. Mean age on study entry was 11.8 d (range: 5-30 d). No infant had neurological symptoms on entry; diagnosis was by plasma neurochemical analysis with molecular confirmation. Primary outcome parameter was neurodevelopment at age 3 years. We identified and characterized the patients' mutations, by sequencing, immunohistochemistry/confocal microscopy with antibodies to ATP7A, and expression of mutant alleles in a yeast complementation assay.
Results: Four patients had good to excellent outcomes and walked independently at or before 15 mos of age. In two responders with aminoterminal nonsense mutations (Q197X, R201X), we detected ATP7A in cultured fibroblasts by confocal microscopy using a carboxy-terminus antibody, indicating translation reinitiation downstream of the premature stop codons. In yeast transfection experiments, we documented that the Q197X, R201X mutant alleles each functionally complement a Cu transport deletion strain (CCC2), restoring growth at approximately 10% of normal.
Conclusions: Translation reinitiation downstream of 5' nonsense mutations generates ATP7A molecules with fewer Cu binding sites. The Q197X and R201X alleles produce some ATP7A capable of Cu transport such that, in combination with early (<10 days after birth) Cu injection treatment, very favorable neurological outcomes are possible.