Untargeted 1H NMR-based metabolomics and high-pressure liquid chromatography analysis reveal sexual dimorphism in the serum metabolic profiles of Parkinson’s disease patients harbouring rare genetic variants

Background Recent evidence indicates that sex and genetic status significantly influence serum metabolic profiles in idiopathic Parkinson’s disease (iPD) and in patients carrying at least a mutation in GBA1, LRRK2, TMEM175, PARK2, PINK1, or PARK7 genes. However, Parkinson’s disease patients (PD) may also carry rare genetic variants of uncertain significance, whose effects on systemic metabolism have yet to be studied.

We combined untargeted 1H-NMR-based metabolomics to characterize serum metabolome profiles, with high-performance liquid chromatography (HPLC) to quantify D- and L-amino acids involved in neurotransmission, energy homeostasis, and oxidative stress. The study included 212 clinically and genetically well-characterized PD patients—121 with iPD and 91 with at least one rare genetic variant (rvPD) in PD-linked genes—and 140 sex-matched healthy controls (HCs). The same cohort was also investigated by targeted-association analysis of genes encoding key enzymes involved in glycine and serine metabolism, including SRR, DAO, DAOA, SHMT1, SHMT2, PHGDH, AMT, GCSH, GLDC, and N-methyl-D-aspartate receptor (NMDAR) subunits such as GRIN1, GRIN2A, and GRIN2B. Data were replicated in independent case-control datasets.

Notably, integrating untargeted NMR-based analysis, demographic and genetic information, our findings revealed substantial differences in serum metabolome profiles between the rvPD and HCs groups across sexes. Significantly, male patients showed more extensive metabolic disruption in amino acid metabolism, glutathione biosynthesis, and energy-related pathways. In contrast, female patients showed fewer metabolic changes, mainly affecting metabolites implicated in lipid-related pathways. Intriguingly, multivariate analysis did not distinguish serum metabolome profiles between individuals with iPD and rvPD in either sex, indicating shared biochemical abnormalities across patients with different genotypes. Noteworthy, consistent with NMR results, HPLC analysis substantiated a significant reduction in L-glutamate levels in male, but not female, rvPD patients compared to sex-matched HCs. Furthermore, we found that in male patients with rvPD, serum levels of L-serine, glycine, L-aspartate, and L-glutamate significantly correlated with MDS-UPDRS III scores. In the same way, L-serine and L-glutamine were associated with a worsening of motor symptoms in female patients. Finally, in line with prominent dysregulation of glycine-serine metabolism, association analyses identified SHMT1, SHMT2, and GCSH as potential genetic modifiers in sex-stratified rvPD.

In conclusion, we found that sex differences affect serum metabolomic changes and the relationships between amino acid levels and clinical characteristics in PD patients with rare variants. Trial registration The study protocols have been registered in clinicaltrial.gov with the numbers NCT02403765 (Release Date: 04/01/2015), NCT04620980 (Release Date: 11/03/2020), NCT05721911 (Release Date: 30/01/2023). Competing Interest Statement The authors have declared no competing interest. Funding Statement This study was partially funded by Italian Ministry of University and Research (PRIN 2022 COD. 2022XF7YYL to AU and PRIN 2022 COD. 2022W3RKLJ to TE). The work of A.U., T.N. and T.E. was supported by NEXTGENERATIONEU (NGEU) and funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) A Multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022). The work of T.E. was supported by Next Generation EU - PNRR M6C2 Investimento 2.1 valorizzazione e potenziamento della ricerca biomedica del SSN grant n. PNRR-MAD-2022-12375960 and grant n. PNRR-MCNT2-2023-12377375. TE was also supported by Ministry of Health, Ricerca Corrente. The study of TE was partially funded by Ministry of Enterprises and Made in Italy (MIMIT) project Neurotechno n. F/180029/01/X43. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All procedures involving human participants were approved by the Institutional Review Board of the IRCCS Neuromed Italy. The study protocols N_9/2015, N_19/2020, N_4/2023 have been registered in clinicaltrial.gov with the numbers NCT02403765 (Release Date: 04/01/2015), NCT04620980 (Release Date: 11/03/2020), NCT05721911 (Release Date: 30/01/2023). Clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all participants. The research was carried out following the recommendations set out in the Global Code of Conduct for Research in Resource-Poor Settings. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability Metabolomics data have been deposited to the EMBL-EBI MetaboLights database (https://www.ebi.ac.uk/metabolights/) with the identifier MTBLS12746