Metabolic consequences of mitochondrial coenzyme A deficiency in patients with PANK2 mutations

Abstract

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare, inborn error of metabolism characterized by iron accumulation in the basal ganglia and by the presence of dystonia, dysarthria, and retinal degeneration. Mutations in pantothenate kinase 2 (PANK2), the rate-limiting enzyme in mitochondrial coenzyme A biosynthesis, represent the most common genetic cause of this disorder. How mutations in this core metabolic enzyme give rise to such a broad clinical spectrum of pathology remains a mystery. To systematically explore its pathogenesis, we performed global metabolic profiling on plasma from a cohort of 14 genetically defined patients and 18 controls. Notably, lactate is elevated in PKAN patients, suggesting dysfunctional mitochondrial metabolism. As predicted, but never previously reported, pantothenate levels are higher in patients with premature stop mutations in PANK2. Global metabolic profiling and follow-up studies in patient-derived fibroblasts also reveal defects in bile acid conjugation and lipid metabolism, pathways that require coenzyme A. These findings raise a novel therapeutic hypothesis, namely, that dietary fats and bile acid supplements may hold potential as disease-modifying interventions. Our study illustrates the value of metabolic profiling as a tool for systematically exploring the biochemical basis of inherited metabolic diseases.

Figure 1

Plasma metabolites in patients versus healthy controls. The figure depicts the 230 metabolites detected in > 80% of plasma samples. For each metabolite, the significance of fold change (Wilcoxon Rank Sum test) is plotted as a function of fold change in patients versus controls. For each lipid analyte (triacylgylcerols abbreviated as TAG, sphingomyelins as SM, and lysophosphatidylcholines as LPC), the number of carbons and double bonds in the acyl chain(s) are denoted.

Figure 2

Levels of plasma lactate, pyruvate, and alanine in patients and controls measured with global metabolic profiling of non-fasting subjects (top) and using clinical assays on fasting subjects (bottom). Open red circles represent patients with atypical PKAN, filled red circles represent patients with classic PKAN; black filled circles represent controls. Horizontal lines and accompanying values denote mean patient and control levels. For metabolites measured with metabolic profiling, levels are reported as chromatographic peak area (arbitrary units, a.u.). A star (*) denotes significant differences (P value < 0.05; Wilcoxon Rank Sum test for metabolic profiling measurements and two-tailed, unpaired Student’s T Test assuming unequal variance for clinical measurements).

Figure 3

(a) Plasma levels of pantothenate in 18 patient samples and 18 control samples detected with global metabolic profiling, reported as chromatographic peak area (arbitrary units, a.u.). Patients with the Y190X stop mutation are noted with a red triangle, and the one patient taking oral pantothenate supplements is noted with a red square. Open red shapes represent patients with atypical PKAN, filled red shapes represent patients with classic PKAN; black filled circles represent controls.

Figure 4

(a) A simplified schematic of the bile acid biosynthetic pathway. Cofactors are enclosed in rounded rectangles. Arrows (↓↓) denote metabolites detected with metabolic profiling to be significantly lower in patients relative to controls (Wilcoxon Rank Sum P < 0.05). Select enzymes in this pathway, bile acid CoA ligase (BAL) and bile acid-CoA:amino acid N-acyltransferase (BAAT), are shown in italics. (b) A summary of bile acids measured in this study.

Figure 5

Plasma markers of sterol biosynthesis and absorption. Lathosterol and lanosterol (left) in patient and control plasma under non-fasting (top) and fasting conditions (bottom). 7a-hydroxycholesterol and 27-hydroxycholesterol (right) in patient and control plasma under non-fasting (top) and fasting conditions (bottom). Two stars (**) denote significant differences (P values < 0.005); other symbols as in Figure 2.

Figure 6

Cholesterol precursors and fatty acids in fibroblasts. (a) Lathosterol and lanosterol in fibroblasts derived from patients and healthy controls. (b) Palmitic, oleic, myristic and stearic acid levels in fibroblasts derived from patients and healthy controls. Symbols as in Figure 2.

Figure 7

Markers of nutrient absorption. Plant sterols campesterol and sitosterol were measured in plasma derived from patients and controls under non-fasting (top) or fasting conditions (bottom). Symbols as in Figure 2.