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Label free quantitative proteomic profiling of serum samples of intellectually disabled young patients revealed dysregulation of complement coagulation and cholesterol cascade systems

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Abstract

Intellectual disability is a heterogeneous disorder, diagnosed using intelligence quotient (IQ) score criteria. Currently, no specific clinical test is available to diagnose the disease and its subgroups due to inadequate understanding of the pathophysiology. Therefore, current study was designed to explore the molecular mechanisms involved in disease perturbation, and to identify potential biomarkers for disease diagnosis and prognosis. A total of 250 participants were enrolled in this study, including 200 intellectually disabled (ID) subjects from the subgroups (mild, moderate, and severe) with age and gender matched healthy controls (n = 50). Initially, IQ testing score and biochemical profile of each subject was generated, followed by label-free quantitative proteomics of subgroups of IQ and healthy control group through nano-LC/MS- mass spectrometry. A total of 310 proteins were identified, among them198 proteins were common among all groups. Statistical analysis (ANOVA) of the subgroups of ID showed 142 differentially expressed proteins, in comparison to healthy control group. From these, 120 proteins were found to be common among all subgroups. The remaining 22 proteins were categorized as exclusive proteins found only in disease subgroups. Furthermore, the hierarchical cluster analysis (HCL) of common significant proteins was also performed, followed by PANTHER protein classification and GO functional enrichment analysis. Results provides that the datasets of differentially expressed proteins, belong to the categories of immune / defense proteins, transfer carrier proteins, apolipoproteins, complement proteins, protease inhibitors, hemoglobin proteins etc., they are known to involvein immune system, and complement and coagulation pathway cascade and cholesterol metabolism pathway. Exclusively expressed 22 proteins were found to be disease stage specific and strong PPI network specifically those that have significant role in platelets activation and degranulation, such as Filamin A (FLNA). Furthermore, to validate the mass spectrometric findings, four highly significant proteins (APOA4, SAP, FLNA, and SERPING) were quantified by ELISA in all the study subjects. AUROC analysis showed a significant association of APOA4 (0.830), FLNA (0.958), SAP (0.754) and SERPING (0.600) with the disease. Apolipoprotein A4 (APOA4) has a significant role in cholesterol transport, and in modulation of glucose and lipid metabolism in the CNS. Similarly, FLNA has a crucial role in the nervous system, especially in the functioning of synaptic network. Therefore, both APOA4, and FLNA proteins represent good potential for candidate biomarkers for the diagnosis and prognosis of the intellectual disability. Overall, serum proteome of ID patients provides valuable information of proteins/pathways that are altered during ID progression.

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Data availability

The complete proteomics dataset used and/or analyzed in the present study can be accessed in the electronic supplementary file ESI. Individual healthy/patient’s samples data will be provided on request from the corresponding author.

Abbreviations

AUC:

Area under the curve

DSM:

Statistical manual of mental disorders

FDR:

False discovery rate

GO:

Gene ontology

HCL:

Hierarchical cluster analysis

ID:

Intellectual disability

IQ:

Intelligence quotient

LC/MS:

Liquid chromatography–mass spectrometry

NS-ID:

Non-syndromic intellectual disability

PANTHER:

Protein analysis through evolutionary relationships

ROC:

Receiver operating characteristics

SECs:

Special education centers

S-ID:

Syndromic intellectual disability

STRING:

Search tool for the retrieval of interacting genes/proteins

TFA:

Trifluoroacetic acid

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Acknowledgements

The Villum Center for Bioanalytical Sciences at University of Southern Denmark (SDU), Odense, Denmark, is acknowledged for access to advanced LC-MSMS instrumentation. National Institute for Biotechnology and Genetic Engineering (NIBGE), and Faisalabad Medical University for contributions in the study cohort recruitment, sampling and analysis.

Funding

All the experimental work was performed by a recurring grant of Dr. Panjwani Center for Molecular Medicine and Drug Research (ICCBS). There was no funding available to support the publication of this research work.

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Authors and Affiliations

  1. Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan

    Soma Vankwani, Munazza Raza Mirza, Muneeza Zafar & Muhammad Iqbal Choudhary

  2. Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, P.O. Box. 577, Faisalabad, Pakistan

    Fazli Rabbi Awan, Muneeza Zafar, Muhammad Wasim & Haq Nawaz Khan

  3. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

    Arkadiusz Nawrocki & Martin Rossel Larsen

  4. Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan

    Muhammad Wasim & Haq Nawaz Khan

  5. Department of Pediatrics, Punjab Medical College, Allied & DHQ Hospitals, Faisalabad Medical University, Faisalabad, Pakistan

    Hina Ayesha

Authors
  1. Soma Vankwani
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  2. Munazza Raza Mirza
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  3. Fazli Rabbi Awan
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Contributions

This study was initially conceived and designed by M.R.M., F.R.A., and M.I.C., Sample collection, study approvals and biochemical assays were taken by F.R.A, M.W., H.A., H.N.K., and M.Z., Sample preparation for mass spectrometry and ELISA assays were performed by S.V., and M.R.M. Mass spectrometric analysis was performed by S.V., M.R.M., A.N., and M.R.L. Data analysis and interpretation by S.V., M.R.M., M.Z., and F.R.A. Manuscript first draft was prepared by S.V. which was further reviewed and edited by all the authors of the manuscript.

Corresponding authors

Correspondence to Munazza Raza Mirza or Fazli Rabbi Awan.

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Ethics approval and consent to participate

This study was approved by Ethical Review Committee of National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, and Independent Ethics Committee (Protocol Ref.: ICCBS/IEC-093-HB/HUS-2023/Protocol/2.0), International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Pakistan. Duly signed informed consents were obtained from parents/guardians of all study participants prior to sample (blood and urine) collection. The study was carried out according to the guidelines of the 1964 Declaration of Helsinki.

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Vankwani, S., Mirza, M.R., Awan, F.R. et al. Label free quantitative proteomic profiling of serum samples of intellectually disabled young patients revealed dysregulation of complement coagulation and cholesterol cascade systems. Metab Brain Dis (2024). https://doi.org/10.1007/s11011-024-01351-6

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