A metabolomic map of Zellweger spectrum disorders reveals novel disease biomarkers

Abstract

Purpose: Peroxisome biogenesis disorders-Zellweger spectrum disorders (PBD-ZSD) are metabolic diseases with multisystem manifestations. Individuals with PBD-ZSD exhibit impaired peroxisomal biochemical functions and have abnormal levels of peroxisomal metabolites, but the broader metabolic impact of peroxisomal dysfunction and the utility of metabolomic methods is unknown.

Methods: We studied 19 individuals with clinically and molecularly characterized PBD-ZSD. We performed both quantitative peroxisomal biochemical diagnostic studies in parallel with untargeted small molecule metabolomic profiling in plasma samples with detection of >650 named compounds.

Results: The cohort represented intermediate to mild PBD-ZSD subjects with peroxisomal biochemical alterations on targeted analysis. Untargeted metabolomic profiling of these samples revealed elevations in pipecolic acid and long-chain lysophosphatidylcholines, as well as an unanticipated reduction in multiple sphingomyelin species. These sphingomyelin reductions observed were consistent across the PBD-ZSD samples and were rare in a population of >1,000 clinical samples. Interestingly, the pattern or "PBD-ZSD metabolome" was more pronounced in younger subjects suggesting studies earlier in life reveal larger biochemical changes.

Conclusion: Untargeted metabolomics is effective in detecting mild to intermediate cases of PBD-ZSD. Surprisingly, dramatic reductions in plasma sphingomyelin are a consistent feature of the PBD-ZSD metabolome. The use of metabolomics in PBD-ZSD can provide insight into novel biomarkers of disease.

Keywords: PBD-ZSD; metabolomics; peroxisome; peroxisome biogenesis disorder; sphingomyelin.

Conflict of interest statement

DISCLOSURE

M.F.W., L.H., M.J.M., V.R.S., Q.S., and S.H.E. are employees of Baylor College of Medicine, which has a partnership with Baylor Genetics and derives revenue from genetic testing. A.D.K. is an employee of Metabolon and, as such, has affiliations with or financial involvement with Metabolon. T.R.D. is an employee of Greenwood Genetics. The other authors declare no conflict of interest.

Figures

Figure 1. Clinical characteristics of study subjects with mild or moderate peroxisome biogenesis disorders–Zellweger spectrum disorders (PBD-ZSD).

(a) A 3 12-year-old female. (b) A 3-year-old male subject. (c) A 2 12-year-old male subject. (d) Spectrum of hearing loss in the subjects; number of subjects is shown on the y-axis. (e) Presence of seizures in the subjects; number of subjects is shown on the y-axis. Three subjects had experienced one seizure only at the time of the study. (f) Schematic model of the process of peroxisomal biogenesis showing the role for PEX1 protein; 18 of 19 subjects had mutations in PEX1. (g) Summary of biochemical pathways implicated by peroxisomal functions (see also Supplementary Figure S1 online). (h) Pipecolic acid levels measured by electron capture negative ion mass fragmentography of the pentafluorobenzyl ester of pipecolic acid, shown in μmol/L. Normal controls shown in light blue, a group of subjects with severe PBD-ZSD (not included in our study and more severe subjects) shown in dark blue, and our study cohort of mild or moderate PBD-ZSD shown in royal blue. ****P < 0.0001, ns indicates not significant. Pipecolic acid was elevated, although variable in the cohort (mean ± SD 54.6 ± 60.7 μmol/L in our cohort versus 1.7 ± 1.1 μmol/L in clinical controls over 5 years). (i) C26:0 levels measured by capillary gas chromatography/mass spectroscopy of pentafluorobenzyl bromide fatty acid esters; μg/ml quantity is shown on y-axis. Normal controls shown in light blue, a group of subjects with severe PBD-ZSD (not included in our study and more severe subjects) shown in dark blue, and our study cohort of mild–intermediate PBD-ZSD shown in royal blue. ****P < 0.0001. C26:0 levels are elevated in the cohort (1.65 ± 0.88 μg/ml versus 0.23 ± 0.09 μg/ml in controls). (j) C26:1 levels measured by capillary gas chromatography/mass spectroscopy of pentafluorobenzyl bromide fatty acid esters, μg/ml quantity is shown on y-axis. Normal controls shown in light blue, a group of individuals with severe PBD-ZSD (not included in our study and more severe subjects) shown in dark blue, and our study cohort of mild or moderate PBD-ZSD shown in royal blue. ****P < 0.0001. C26:1 levels are elevated in the cohort (0.78 ± 0.46 μg/ml versus 0.18 ± 0.09 in controls). (k) C24/C22 fatty acid ratios. Normal controls shown in light blue, a group of individuals with severe PBD-ZSD (not included in our study and more severe subjects) shown in dark blue, and our study cohort of mild or moderate PBD-ZSD shown in royal blue. ****P < 0.0001. C24/C22 (1.46 ± 0.32 versus 0.84 ± 0.10, (l)) fatty acids were also elevated, although not to the degree of severe PBD-ZSD (C26/C22: 0.50 ± 0.16). (l) C26/C22 fatty acid ratios. Normal controls shown in light blue, a group of individuals with severe PBD-ZSD (not included in our study and more severe subjects) shown in dark blue, and our study cohort of mild or moderate PBD-ZSD shown in royal blue. ****P < 0.0001. (m) Pristanic acid levels in μg/ml in the subjects exhibited elevated pristanic acid levels (0.73 ± 0.44 μg/ml). The dashed line (ULN) marks the upper limit of normal. (n) Phytanic acid levels in μg/ml in the subjects who exhibited elevated phytanic acid levels (3.8 ± 2.8). The dashed line (ULN) marks the upper limit of normal.

Figure 2. Metabolomic profile of our study cohort: the peroxisome biogenesis disorders–Zellweger spectrum disorders (PBD-ZSD) metabolome.

(a) Principal components analysis is shown; red squares represent the PBD-ZSD samples, blue circles represent a collection of control samples. The principal components, PC1 and PC2, were generated using all metabolomic data. Percent signs indicate the percent of variation explained by that principal component. (b) Heat map of the PBD-ZSD samples; red shows relative increases, while blue shows reductions. Pathways of the analytes are shown on the left axis (individual analytes in Supplementary Table S4 online). Specific compounds noted to be altered are marked at right. Subjects are labeled according to family designation (see Table 1).

Figure 3. Dramatically altered analytes in the peroxisome biogenesis disorders–Zellweger spectrum disorders (PBD-ZSD) metabolome.

(a) Box plots of analytes that are related to biochemical pathways known to be peroxisomal (see Supplementary Figure S1 online); error bars correspond to 95% confidence interval. (b) Box plots of alterations in sphingomyelins including palmitoyl sphingomyelin (palmitoyl sphingomyelin (d18:1/16:0)), palmitoleoyl sphingomyelin (sphingomyelin (d18:2/16:0, d18:1/16:1)*), oleoyl sphingomyelin (sphingomyelin (d18:1/18:1, d18:2/18:0)), nervonoyl sphingomyelin (sphingomyelin (d18:1/24:1, d18:2/24:0)*), myristoyl sphingomyelin (sphingomyelin (d18:1/14:0, d16:1/16:0)*), euricoyl sphingomyelin (sphingomyelin (d18:1/22:1, d18:2/22:0, d16:1/24:1)*), eicosenoyl sphingomyelin (sphingomyelin (d18:1/20:1, d18:2/20:0)*), and behenoyl sphingomyelin (behenoyl sphingomyelin (d18:1/22:0)*).

Figure 4. Comprehensive metabolomic findings are impacted by age in peroxisome biogenesis disorders–Zellweger spectrum disorders (PBD-ZSD) study cohort.

Figures generated using Cytoscape to delineate biochemical pathways (http://cytoscape.org). (a) Biochemical pathway showing metabolic perturbations in plasma from subjects diagnosed with PBD-ZSD and under 10 years of age at the time of sample collection. Red circles indicate biochemicals with positive z-scores (>2) and blue circles indicate biochemicals with negative z-scores (< − 2). The diameters of the circles indicate the magnitude of the z-score. Pink circles represent biochemicals with z-score of 1.5 ≤ z < 2.0 and light blue circles represent biochemicals with − 2.0 < z ≤ − 1.5. Black circles represent other biochemicals in the pathway detected in the samples but had z-scores of − 1.5 < z < 1.5. (b) Biochemical pathway showing metabolic perturbations in plasma from subjects diagnosed with PBD-ZSD and over 10 years of age at the time of sample collection. Red circles indicate biochemicals with positive z-scores (>2) and blue circles indicate biochemicals with negative z-scores (< − 2). The diameters of the circles indicate the magnitude of the z-score. Pink circles represent biochemicals with z-score of 1.5 ≤ z < 2.0 and light blue circles represent biochemicals with − 2.0 < z ≤ − 1.5. Black circles represent other biochemicals in the pathway detected in the samples but had z-scores of − 1.5 < z < 1.5.