Cerebrospinal fluid metabolomics in autistic regression reveals dysregulation of sphingolipids and decreased β-hydroxybutyrate

Jingya Yan¹, Velda X Han², Hannah F Jones³, Timothy A Couttas⁴, Beverly Jieu⁵, F Markus Leweke⁵, Jennifer Lee⁶, Catherine Loi⁶, Richard Webster⁷, Kavitha Kothur⁸, Manoj P Menezes⁹, Jayne Antony⁷, Tejaswi Kandula¹⁰, Michael Cardamone¹⁰, Shrujna Patel¹¹, Sushil Bandodkar¹², Russell C Dale¹³

Affiliations

¹ Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; School of Mathematical and Physical Sciences, Faculty of Science, University of Technology Sydney, NSW, Australia; Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia.
² Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore; Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
³ Starship Hospital, Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, New Zealand.
⁴ Neuroscience Research Australia, Randwick, NSW, Australia; Brain and Mind Centre, The University of Sydney, NSW, Australia.
⁵ Brain and Mind Centre, The University of Sydney, NSW, Australia.
⁶ Department of Endocrinology, The Children's Hospital at Westmead, NSW, Australia.
⁷ TY Nelson Department of Neurology and Neurosurgery, The Children's Hospital at Westmead, The University of Sydney, Westmead, New South Wales, Australia.
⁸ Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; TY Nelson Department of Neurology and Neurosurgery, The Children's Hospital at Westmead, The University of Sydney, Westmead, New South Wales, Australia.
⁹ Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; TY Nelson Department of Neurology and Neurosurgery, The Children's Hospital at Westmead, The University of Sydney, Westmead, New South Wales, Australia.
¹⁰ Department of Neurology, Sydney Children's Hospital Network, Sydney, NSW, Australia.
¹¹ Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia.
¹² Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; Department of Biochemistry, The Children's Hospital at Westmead, NSW, Australia.
¹³ Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia. Electronic address: russell.dale@health.nsw.gov.au.

PMID: 40138886
PMCID: PMC11986237
DOI: 10.1016/j.ebiom.2025.105664

Cerebrospinal fluid metabolomics in autistic regression reveals dysregulation of sphingolipids and decreased β-hydroxybutyrate

Jingya Yan et al. EBioMedicine. 2025 Apr.

. 2025 Apr:114:105664.

doi: 10.1016/j.ebiom.2025.105664. Epub 2025 Mar 25.

Authors

Affiliations

¹ Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; School of Mathematical and Physical Sciences, Faculty of Science, University of Technology Sydney, NSW, Australia; Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia.
² Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore; Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
³ Starship Hospital, Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, New Zealand.
⁴ Neuroscience Research Australia, Randwick, NSW, Australia; Brain and Mind Centre, The University of Sydney, NSW, Australia.
⁵ Brain and Mind Centre, The University of Sydney, NSW, Australia.
⁶ Department of Endocrinology, The Children's Hospital at Westmead, NSW, Australia.
⁷ TY Nelson Department of Neurology and Neurosurgery, The Children's Hospital at Westmead, The University of Sydney, Westmead, New South Wales, Australia.
⁸ Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; TY Nelson Department of Neurology and Neurosurgery, The Children's Hospital at Westmead, The University of Sydney, Westmead, New South Wales, Australia.
⁹ Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; TY Nelson Department of Neurology and Neurosurgery, The Children's Hospital at Westmead, The University of Sydney, Westmead, New South Wales, Australia.
¹⁰ Department of Neurology, Sydney Children's Hospital Network, Sydney, NSW, Australia.
¹¹ Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia.
¹² Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; Department of Biochemistry, The Children's Hospital at Westmead, NSW, Australia.
¹³ Kids Neuroscience Centre, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia; Clinical School, The Children's Hospital at Westmead, Faculty of Medicine and Health, University of Sydney, NSW, Australia. Electronic address: russell.dale@health.nsw.gov.au.

PMID: 40138886
PMCID: PMC11986237
DOI: 10.1016/j.ebiom.2025.105664

Abstract

Background: Autism is highly heritable, however actionable genetic findings are only found in a minority of patients. Many people with autism suffer loss of neurodevelopmental skills, known as autistic regression. The cause of regression is poorly understood, and the diagnostic and therapeutic pathways are lacking.

Methods: We used untargeted metabolomics using a UPLC-Q-Exactive-HFx Mass Spectrometry to examine cerebrospinal fluid (CSF) from twenty-two patients with autistic regression compared to sixteen controls with neurodevelopmental disorders (but not autistic regression) and thirty-four controls with other neurological disease (headache, encephalitis, epilepsy). The twenty-two patients with autistic regression consisted of two groups: early (infantile) autistic regression <2 years of age (n = 8), and later regression of skills >4 years of age, often in the context of pre-existing developmental concerns (n = 14). Metabolites of interest were then quantified and validated using targeted assays.

Findings: Untargeted case-control studies revealed good separation of patients from controls using multivariate analysis. β-hydroxybutyrate was significantly decreased in the CSF of patients with autistic regression, and the findings were validated using a targeted β-hydroxybutyrate assay. The sphingolipid, sphingosine-1-phosphate was significantly elevated in the discovery case-control studies, and sphingolipid metabolism pathways were also significantly dysregulated. We therefore developed a targeted metabolite assay of forty sphingolipids. After FDR correction, 21 of the 40 sphingolipids were significantly dysregulated (p_FDR < 0.05) (Benjamini-Hochberg correction) in autistic regression compared to the neurodevelopmental controls, and 26 of the 40 sphingolipids were significantly dysregulated in autistic regression compared to other neurological controls, with elevated ceramides, hexosylceramides, sphingosines (including sphingosine-1-phosphate), and sulfatides. By contrast, sphingomyelin levels were generally decreased in autistic regression.

Interpretation: Our data shows the potential utility of CSF metabolomics in the context of autistic regression, a clinical syndrome which has historically lacked pathophysiological biomarkers and disease modifying therapies.

Funding: Financial support for the study was granted by Dale NHMRC Investigator grant APP1193648, Petre Foundation, Cerebral Palsy Alliance, and Ainsworth and SCHF Neuroscience grant scheme.

Keywords: Inflammation; Lipidomics; Metabolomics; Neurodevelopmental disorders.

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Conflict of interest statement

Declaration of interests The authors have declared that no conflict of interest exists.

Figures

**Fig. 1**
Summary of statistical analyses of cerebrospinal fluid cohort studies, cohort 1 (left) and cohort 2 (right) using ultra-performance liquid chromatography coupled to high resolution mass spectrometry. (a) Partial least squares discriminant analysis score plots of patients with autistic regression (red dots), control patients (green dots) and quality control samples (blue dots) showing clustering of the respective groups. (b) Pathway analysis of statistically significant metabolites after FDR correction and a power analysis: sphingolipid metabolism is significantly dysregulated in both cohort 1 and 2. (c) Elevation of sphingosine-1-phosphate in the autistic regression group compared to controls showing p = 0.0097 (cohort 1; AUTREG, n = 11; Controls, n = 11) and p = 0.0083 (cohort 2; AUTREG, n = 11; Controls, n = 11) (ANOVA and Fisher’s LSD FDR-adjusted). The response ratio is the ratio of *peak area of metabolite: internal standard peak area*.

**Fig. 2**
β-hydroxybutyrate data. (a) Untargeted metabolomics of patients with autistic regression (AUTREG, n = 11) are compared with controls (n = 11) showing β-hydroxybutyrate is reduced in the autistic regression group compared to controls (p = 0.0004) (ANOVA and Fisher’s LSD FDR-adjusted). The response ratio is the ratio of *peak area of metabolite: internal standard peak area*. (b) Validation of untargeted metabolomics study through quantification of β-hydroxybutyrate using a single analyte LiquiColor assay showing decreased β-hydroxybutyrate in patents with autistic regression (AUTREG, n = 17) and controls (n = 12) (p = 0.03) (Mann Whitney U test). (c) β-hydroxybutyrate pathway. HMGCS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGCL, 3-hydroxy-3-methylglutaryl-CoA lyase; BDH1, β-hydroxybutyrate dehydrogenase; HMG-CoA, 3-hydroxy-beta-methylglutaryl-CoA.

**Fig. 3**
Targeted sphingolipids. (a) Targeted sphingolipids data of autistic regression group (AUTREG) compared to neurodevelopmental control group (NEURODEV CONT.) and ‘other neurological disorder’ control (OTHER NEURO CONT.) group (see Table 1). Representative statistically significant sphingolipids which are elevated in autistic regression are presented, including ceramides (Cer), hexosylceramides (HexCer), sulfatides (ST) and sphingosine 1 phosphate (S1P) (all < 0.05 according to *Benjamini-Hochberg correction FDR-adjusted*, see Supplemental Table S3 for values). By contrast, the sphingomyelin (SM) metabolites were generally decreased in autistic regression. (b) FDR adjusted p values of autistic regression (AUTREG) compared to other neurodevelopmental (NDD) controls and compared to ‘other neurological disorder controls are presented for all forty sphingolipids, with elevated values in red and decreased values in blue. Most sphingolipids were elevated in autistic regression, apart from sphingomyelin metabolites which were often decreased (full values in Supplemental Table S3) (c) Pathways of sphingolipid metabolism. SPL, SIP lyase; SK, sphingosine kinase; CDase, ceramidase; CerS, ceramide synthase; DES, dihydroceramide desaturase; HexCerase, hexosylceramidase; HexCerS, hexosylceramides synthase; HSase, hexosaminidase; CK, ceramide kinase; C1PP, ceramide-1-phosphate phosphatase; SMS, sphingomyelin synthase; SMase, sphingomyelinase.

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References

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Cerebrospinal fluid metabolomics in autistic regression reveals dysregulation of sphingolipids and decreased β-hydroxybutyrate

Affiliations

Cerebrospinal fluid metabolomics in autistic regression reveals dysregulation of sphingolipids and decreased β-hydroxybutyrate

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

MeSH terms

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LinkOut - more resources

Full Text Sources