Changes in the Cerebrospinal Fluid and Plasma Lipidome in Patients with Rett Syndrome

doi:10.3390/metabo12040291

. 2022 Mar 25;12(4):291.

doi: 10.3390/metabo12040291.

Changes in the Cerebrospinal Fluid and Plasma Lipidome in Patients with Rett Syndrome

Affiliations

¹ Department of Paediatrics and Adolescent Medicine, Division of General Paediatrics, University Childrens' Hospital Graz, Medical University of Graz, 8036 Graz, Austria.
² Institute of Molecular Biosciences, NAWI Graz, University of Graz, 8010 Graz, Austria.
³ Core Facility Mass Spectrometry, Medical University of Graz, 8010 Graz, Austria.
⁴ Neurologic Clinic and Policlinic, Departments of Medicine, Biomedicine, and Clinical Research, University Hospital Basel, University of Basel, 4031 Basel, Switzerland.
⁵ Department of Child Neurology, University Children's Hospital Zurich, 8032 Zurich, Switzerland.
⁶ Department of Pediatric Neurology, Klinikum Kassel, 34125 Kassel, Germany.
⁷ Clinic for Paediatrics I, Inherited Metabolic Disorders, Medical, University of Innsbruck, 6020 Innsbruck, Austria.

PMID: 35448478
PMCID: PMC9026385
DOI: 10.3390/metabo12040291

Changes in the Cerebrospinal Fluid and Plasma Lipidome in Patients with Rett Syndrome

Martina Zandl-Lang et al. Metabolites. 2022.

. 2022 Mar 25;12(4):291.

doi: 10.3390/metabo12040291.

Authors

Affiliations

¹ Department of Paediatrics and Adolescent Medicine, Division of General Paediatrics, University Childrens' Hospital Graz, Medical University of Graz, 8036 Graz, Austria.
² Institute of Molecular Biosciences, NAWI Graz, University of Graz, 8010 Graz, Austria.
³ Core Facility Mass Spectrometry, Medical University of Graz, 8010 Graz, Austria.
⁴ Neurologic Clinic and Policlinic, Departments of Medicine, Biomedicine, and Clinical Research, University Hospital Basel, University of Basel, 4031 Basel, Switzerland.
⁵ Department of Child Neurology, University Children's Hospital Zurich, 8032 Zurich, Switzerland.
⁶ Department of Pediatric Neurology, Klinikum Kassel, 34125 Kassel, Germany.
⁷ Clinic for Paediatrics I, Inherited Metabolic Disorders, Medical, University of Innsbruck, 6020 Innsbruck, Austria.

PMID: 35448478
PMCID: PMC9026385
DOI: 10.3390/metabo12040291

Abstract

Rett syndrome (RTT) is defined as a rare disease caused by mutations of the methyl-CpG binding protein 2 (MECP2). It is one of the most common causes of genetic mental retardation in girls, characterized by normal early psychomotor development, followed by severe neurologic regression. Hitherto, RTT lacks a specific biomarker, but altered lipid homeostasis has been found in RTT model mice as well as in RTT patients. We performed LC-MS/MS lipidomics analysis to investigate the cerebrospinal fluid (CSF) and plasma composition of patients with RTT for biochemical variations compared to healthy controls. In all seven RTT patients, we found decreased CSF cholesterol levels compared to age-matched controls (n = 13), whereas plasma cholesterol levels were within the normal range in all 13 RTT patients compared to 18 controls. Levels of phospholipid (PL) and sphingomyelin (SM) species were decreased in CSF of RTT patients, whereas the lipidomics profile of plasma samples was unaltered in RTT patients compared to healthy controls. This study shows that the CSF lipidomics profile is altered in RTT, which is the basis for future (functional) studies to validate selected lipid species as CSF biomarkers for RTT.

Keywords: LC-MS; Rett syndrome; biomarker; lipidomics; metabolomics; rare diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
Age distribution and quality control. Demographic information and median age on the study subjects ((A) for CSF; (B) for plasma) (C) Phosphatidylethanolamine (PE) 24:0 as extraction control for sample preparation and UHPLC-MS/MS analysis. Standard deviation (SD) for CSF is 14.7% and for plasma samples 10.1%. (D,E) Overview of all CSF (C) and plasma (D) samples analyzed by MS shown as boxplot of all lipids and as sum of the total ion current to exclude inconsistencies in sample preparation and detection. Dots display single lipid species outliers of the whole lipidome.

**Figure 2**
Supervised multivariate statistical analysis of the CSF (A,C) and plasma (B,D). (A,B) Orthogonal partial least square-discriminant analysis (OPLS-DA) plots were generated using the lipidome data set obtained from UHPLC-MS/MS analysis and the program R with the lipidr package. (C,D) Loading plot of CSF (C) and plasma (D) samples obtained from RTT patients compared to healthy controls. Scattered dots represent various lipid species that were identified as influential variables in the discriminant analysis. The top 10 variables are listed in the right panel. CE (Cholesterol Ester), Chol (Cholesterol), DG (Diacylglycerol), PC (Phosphatidylcholine), LPE (Lyso-Phosphatidylethanolamine), PS (Phosphatidylserine), SM (Sphingomyelin), TG (Triacylglycerol).

**Figure 3**
Univariate analysis of lipid classes analyzed from CSF and plasma samples of RTT patients compared to healthy controls. Lipidome data obtained from UHPLC-MS/MS were analyzed using R and the lipidr package. Data is shown as log² of total area count. Box plots represent lipid classes detected in the CSF (A) and plasma (B) of RTT patients and controls and are calculated as sum of lipid species detected in one lipid class. CE (Cholesterol Ester), Cer (Ceramide), Chol (Cholesterol), DG (Diacylglycerol), HexCer (Hexosylceramide), PC (Phosphatidylcholine), PE (Phosphatidyl-ethanolamine), PI (Phosphatidylinositol), PS (Phosphatidylserine), LPC (Lyso-PC), LPE (Lyso-PE), LPI (Lyso-PI), PC-O (Ether-linked PC), PE-P (Plasmalogens of PE), SM (Sphingomyelin), TG (Tri-acylglycerol); *—FDR adjusted p < 0.05 by Wilcoxon rank-sum test, **—FDR adjusted p < 0.01 by Wilcoxon rank-sum test, ns-not significant.

**Figure 4**
Significant changed lipid species detected in CSF of RTT patients compared to healthy controls. Data is displayed as log₂ of total area count. (A) TG (Triacylglycerol), (B) Sphingolipids including SM (Sphingomyelin) and Cer (Ceramide), (C) Sterols including Chol (Cholesterol) and CE (Cholesterol Ester), (D,E) Phospholipids including LPC (Lyso- Phosphatidylcholine), LPE (Lyso-Phosphatidylserine), PC-P (Ether-linked PC), PE-P (Plasmalogens of PE), PC (Phosphatidylcholine) and PE (Phosphatidylethanolamine); *—FDR adjusted p < 0.05 by Wilcoxon rank-sum test , **—FDR adjusted p < 0.01 by Wilcoxon rank-sum test, ns-not significant.

**Figure 5**
Selection of variable lipids by combined UVA and MVA approaches. (A,B) Comparison between the UVA (purple) and MVA (green) selection for CSF (A) and plasma (B) data sets. Lipids are colored according to their selection by one of the approaches or both (red). The pFDR = 0.05 (respectively, Variable Projection of Importance (VIP) = 1) threshold are displayed as a vertical (respectively, horizontal) line. Plot was created using R and the ropls package (C) VIP score plot of 20 most important lipid species identified in the CSF of RTT patients compared to controls. (D) Receiver Operating Characteristic (ROC) curve for CSF dataset, with the Area under the curve (AUC) value. The ROC curve was created from 74 significantly altered lipid species detected by both, UVA and MVA using the R packages mleval, caret and ggplot.

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References

1. Marballi K., MacDonald J.L. Proteomic and transcriptional changes associated with MeCP2 dysfunction reveal nodes for therapeutic intervention in Rett syndrome. Neurochem. Int. 2021;148:105076. doi: 10.1016/j.neuint.2021.105076. - DOI - PMC - PubMed
1. Amir R.E., Van den Veyver I.B., Wan M., Tran C.Q., Francke U., Zoghbi H.Y. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 1999;23:185–188. doi: 10.1038/13810. - DOI - PubMed
1. Vashi N., Justice M.J. Treating Rett syndrome: From mouse models to human therapies. Mamm Genome. 2019;30:90–110. doi: 10.1007/s00335-019-09793-5. - DOI - PMC - PubMed
1. Buchovecky C.M., Turley S.D., Brown H.M., Kyle S.M., McDonald J.G., Liu B., Pieper A.A., Huang W., Katz D.M., Russell D.W., et al. A suppressor screen in Mecp2 mutant mice implicates cholesterol metabolism in Rett syndrome. Nat. Genet. 2013;45:1013–1020. doi: 10.1038/ng.2714. - DOI - PMC - PubMed
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[1] Marballi K., MacDonald J.L. Proteomic and transcriptional changes associated with MeCP2 dysfunction reveal nodes for therapeutic intervention in Rett syndrome. Neurochem. Int. 2021;148:105076. doi: 10.1016/j.neuint.2021.105076. - DOI - PMC - PubMed

[2] Marballi K., MacDonald J.L. Proteomic and transcriptional changes associated with MeCP2 dysfunction reveal nodes for therapeutic intervention in Rett syndrome. Neurochem. Int. 2021;148:105076. doi: 10.1016/j.neuint.2021.105076. - DOI - PMC - PubMed

[3] Amir R.E., Van den Veyver I.B., Wan M., Tran C.Q., Francke U., Zoghbi H.Y. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 1999;23:185–188. doi: 10.1038/13810. - DOI - PubMed

[4] Amir R.E., Van den Veyver I.B., Wan M., Tran C.Q., Francke U., Zoghbi H.Y. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 1999;23:185–188. doi: 10.1038/13810. - DOI - PubMed

[5] Vashi N., Justice M.J. Treating Rett syndrome: From mouse models to human therapies. Mamm Genome. 2019;30:90–110. doi: 10.1007/s00335-019-09793-5. - DOI - PMC - PubMed

[6] Vashi N., Justice M.J. Treating Rett syndrome: From mouse models to human therapies. Mamm Genome. 2019;30:90–110. doi: 10.1007/s00335-019-09793-5. - DOI - PMC - PubMed

[7] Buchovecky C.M., Turley S.D., Brown H.M., Kyle S.M., McDonald J.G., Liu B., Pieper A.A., Huang W., Katz D.M., Russell D.W., et al. A suppressor screen in Mecp2 mutant mice implicates cholesterol metabolism in Rett syndrome. Nat. Genet. 2013;45:1013–1020. doi: 10.1038/ng.2714. - DOI - PMC - PubMed

[8] Buchovecky C.M., Turley S.D., Brown H.M., Kyle S.M., McDonald J.G., Liu B., Pieper A.A., Huang W., Katz D.M., Russell D.W., et al. A suppressor screen in Mecp2 mutant mice implicates cholesterol metabolism in Rett syndrome. Nat. Genet. 2013;45:1013–1020. doi: 10.1038/ng.2714. - DOI - PMC - PubMed

[9] Lyst M.J., Bird A. Rett syndrome: A complex disorder with simple roots. Nat. Rev. Genet. 2015;16:261–275. doi: 10.1038/nrg3897. - DOI - PubMed

[10] Lyst M.J., Bird A. Rett syndrome: A complex disorder with simple roots. Nat. Rev. Genet. 2015;16:261–275. doi: 10.1038/nrg3897. - DOI - PubMed

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Changes in the Cerebrospinal Fluid and Plasma Lipidome in Patients with Rett Syndrome

Affiliations

Changes in the Cerebrospinal Fluid and Plasma Lipidome in Patients with Rett Syndrome

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

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References

Grants and funding

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