Catecholamine metabolites affected by the copper-dependent enzyme dopamine-beta-hydroxylase provide sensitive biomarkers for early diagnosis of menkes disease and viral-mediated ATP7A gene therapy

Abstract

Menkes disease is a lethal X-linked recessive disorder of copper metabolism caused by mutations in ATP7A, a copper-transporting ATPase with diverse and important biological functions. Partial deficiency of dopamine-beta-hydroxylase is a biochemical hallmark of this illness due to the normal role of ATP7A in delivery of copper as an enzymatic cofactor. We exploited this fact to develop a diagnostic test for Menkes disease, which proved highly sensitive and specific. The assay has enabled early identification of affected patients, leading to enhanced survival and improved neurodevelopment after early copper treatment, including some completely normal outcomes. In preclinical efforts to develop improved therapies for patients with non-copper-responsive ATP7A mutations, we used brain-directed adeno-associated viral gene therapy to rescue a murine model of the disease. Statistically significant improvement in brain catechol ratios correlated with enhanced survival, and cerebrospinal fluid catechols represent candidate surrogate markers of treatment effect in a future gene therapy clinical trial.

Keywords: Adeno-associated virus; Catechol ratios; Copper transport; Dopamine-beta-hydroxylase; Gene therapy; Menkes disease; Newborn screening.

Figure 11.1

Overview of cellular copper metabolism. In neuronal cells, ATP7A is required for maturation of DBH and PAM. ATOX1, copper transport protein ATOX1; ATP7A, copper-transporting ATPase 1; ATP7B, copper-transporting ATPase 2; CCS, copper chaperone for SOD1; CCO, cytochrome c oxidase; COX11, cytochrome c oxidase assembly protein Cox11; Cox17, cytochrome c oxidase copper chaperone; CP, ceruloplasmin; CTR, copper transporter 1; DBH, dopamine-β-hydroxylase; DMT1, divalent metal transporter 1; PAM, peptidylglycine α-amidating monooxygenase; Sco1, protein SCO1 homologue; SOD1, superoxide dismutase. Reproduced from Kaler (2011), with permission.

Figure 11.2

The catecholamine biosynthetic pathway. Tyrosine hydroxylation represents the rate-limiting step.

Figure 11.3

Chromatographic pattern of catechol standards that have undergone batch alumina extraction. A solvent front precedes individual peaks. HPLC peaks typically start out sharp and tall and become smaller and wider as retention time increases.

Figure 11.4

Predicted effects of reduced DBH activity. Proximal metabolites are expected to accumulate and distal metabolites are predicted to be reduced, relative to normal.

Figure 11.5

A distinctively abnormal plasma catechol pattern exists in Menkes disease. (A) Normal catechol pattern in a newborn at risk for Menkes disease. (B) Abnormal catechol pattern in a newborn at risk for Menkes, who later was shown to have this condition based on independent clinical and molecular findings. Note the low peaks for DHPG and NE and high peaks for DOPA, DA, and DOPAC.

Figure 11.6

Summary of the NIH experience in prospective testing of at-risk newborns for Menkes disease. Elevated plasma catechol ratios are invariably present in subjects later shown to be affected. Early diagnosis enabled institution of experimental therapy from a young age (<1 month) resulting in improved overall survival and neurodevelopmental outcome.

Figure 11.7

Biochemical effects of brain-directed treatment in the mottled-brindled (mo-br) model of Menkes disease. (A) Brain copper levels at 12 days of age, by treatment category. Only AAV5 + copper (Cu) combination-treated mo-br mice showed significantly higher copper levels in comparison to untreated mo-br mice (P<0.03). (B) Brain catechol ratios at 12 days of age, by treatment category. Only AAV5+Cu combination-treated mo-br mice showed significantly lower ratios, indicative of improved dopamine-beta-hydroxylase activity (P<0.01). (C) Brain cytochrome c oxidase activity at 12 days of age, by treatment category. All treatment groups showed significantly increased activity compared to untreated mo-Br mice. (D) In contrast, the activity of Cu/Zn superoxide dismutase was ~80% of normal and treatment did not significantly enhance activity. Reproduced from Donsante et al. (2011), with permission.