2.1 Study population

This hospital-based case-control study was conducted in the Department of Neurology, All India Institute of Medical Sciences (AIIMS), New Delhi, India in collaboration with CSIR-Institute of Genomics and Integrative Biology (IGIB), New Delhi, India from 2018 to 2020. Patients aged between 18 and 50 years of either sex having IIH as per the modified Dandy Criteria [9] were recruited prospectively in the study. A total of 13 IIH patients were included in the study. Patients were excluded if pregnant, associated malignancy, chronic medical conditions, and not given consent to the study.

A control group comprising 20 individuals was recruited for the study. The control CSF were collected from non-head injury trauma cases and degenerative joint disease patients undergoing spinal anesthesia. Prior written informed consent was taken from all the subjects recruited in the study before CSF collection. The study was approved by the Local Institutional Ethics Committee of AIIMS, New Delhi (Ref. No.: IEC-371/06.07.2018).

2.2 Clinical evaluation

All the patients included in the study underwent detailed clinical examination which included demographic data, clinical symptoms, general medical examination, and detailed eye examination. Body mass index (BMI) more than 25 was considered overweight and was compared with the non-overweight subjects in the study. Eye examination included visual acuity and papilledema assessment. Additionally, other cranial nerve involvements and detailed neurologic examinations were also performed. Included patients had to undergo neuroimaging in the form of magnetic resonance imaging (MRI) brain along with MR venography.

2.3 CSF collection

The included patients underwent lumbar puncture for the CSF examination. Opening pressure was measured in all the patients and CSF was examined for total cells, glucose, protein, bacterial, fungal, and cytopathological examination to rule out an alternate diagnosis. The collected CSF was stored in −80°C with proper labeling until further analysis. No damage to the CSF specimen was assured during collection, cold chain maintenance, transportation, and storage.

2.4 Sample preparation

The proteins from CSF samples collected in the cryo-vials were diluted with 1× phosphate buffer saline (PBS) and precipitated in pre-chilled acetone overnight (12 h) at −20°C. The samples were then centrifuged at 15,000 × g for 15 min at 4°C and supernatants were discarded. The CSF protein pellet was resuspended in 0.1 M Tris-HCl with 8 M urea, pH 8.5.

2.5 Reduction, alkylation, and trypsin digestion

Twenty micrograms of protein from each sample was taken in fresh microcentrifuge tubes and diluted 10 times to bring urea concentration below 1 M before proceeding with reduction, alkylation, and trypsin digestion. 300 µg protein pooled from all the cases and controls were reduced with 25 mM of Dithiothreitol (DTT) for 30 min at 56°C, followed by alkylation using 55 mM of iodoacetamide (IAA) at room temperature (in the dark) for 20 min. These samples were then subjected to trypsin digestion in an enzyme-to-substrate ratio of 1:10 (trypsin: protein) for 16 h at 37°C. The tryptic peptides were vacuum dried in vacuum concentrator.

2.6 SWATH-based untargeted proteomics

2.7 Bioinformatics and statistical analyses

A combined database search was performed for .wiff format raw files generated in DDA mode against UniProtKB human FASTA database (Swissprot and TrEMBL; 152,774 entries) using ProteinPilot Software 5.0.1 (SCIEX). The group output file from ProteinPilot served as the spectral ion library for SWATH analysis. Protein identification in ProteinPilot software was performed using the default settings. Cysteine alkylation through iodoacetamide was set as fixed modification, maximum two missed cleavages were allowed, biological modification was activated in the “ID focus,” search effort was set to “Thorough ID,” detected protein threshold [Unused ProtScore (conf)] was set to 0.05 (10.0%) and false discovery analysis was activated. A 1% global false discovery rate (FDR) at the protein level, excluding shared peptides was considered for import in SWATH 2.0 microapp of PeakView 2.2 software (SCIEX). SWATH run files were added and retention time alignment was performed using peptides from abundant proteins. For SWATH analysis, sequence unique peptides were used for quantification. The processing settings for peak extraction were: maximum of 10 peptides per protein, 5 transitions per peptide, > 95% peptide confidence threshold, 1% peptide FDR. XIC extraction window was set to 55 min, with 75 ppm XIC Width. The protein area was exported in the form of MarkerView (.mrkw) file. In MarkerView 1.2.1 (SCIEX), data normalization was performed using total area sum normalization. There were no missing values in the dataset.

The data were Log₂ transformed as significant skewness or kurtosis was present in the peak area values. After Log₂ transformation and confirmation of normality in the data distribution using the Shapiro-Wilk test, continuous variables were represented by mean and standard deviation (SD) or median and interquartile range (IQR) and were analyzed using student the t-test. Dichotomous variables were represented by number, percentage and were analyzed using the Chi-square test. Differentially expressed proteins were identified using a Log₂ fold change cut-off of > 0.58 (corresponding to fold change > 1.5) for upregulation and < −0.58 (corresponding to < 0.67-fold change) for downregulation and a Log₁₀ p-value cut-off of > 1.3 (corresponding to p-value < 0.05). Receiver operating characteristic (ROC) curves were developed and area under the curve (AUC) was calculated for differentially expressed proteins. Protein interaction network analysis of significantly differentially expressed proteins was performed using STRING (version 11; http://string-db.org/) [10] and Cytoscape 3.9.0 software [11]. Functional analysis was performed using g:Profiler (https://biit.cs.ut.ee/gprofiler/gost) [12]. The results were reported in accordance with the STROBE guidelines [13]. Statistical analysis was conducted in R version 3.6.2 using the following packages: “plyr”, “tidyverse”, “gplots”, and “pheatmap”.

4.1 Baseline characteristics of IIH cases and controls

Our study included CSF samples from 33 subjects, consisting of 13 IIH cases and 20 control subjects. The mean age of cases was 27.69 ± 6.94 and controls was 37.35 ± 13.72 (p = 0.03). There were 11 (84.61%) females in cases and six (30%) in controls (p = 0.02). The duration of symptoms ranged from 2 to 28 weeks. The most common symptoms were headache and transient visual obscurations in the IIH group. The visual acuity and neuroimaging findings are given in Table 1. The details of controls are described in Table 2. All the patients recruited as controls in our study were having trauma or any orthopedics surgery and had undergone spinal anesthesia during which CSF was collected for the study after a written valid consent.

4.2 Identification of differentially expressed proteins in IIH cases and controls using SWATH-MS/MS

Total 262 protein groups were identified in ProteinPilot search in 1% global FDR. Through SWATH analysis, we could quantify 232 proteins in the two groups of samples. A total of 37 differentially expressed proteins were identified between the two groups (Figure 1). Out of 37 proteins, 24 proteins were upregulated while 13 proteins were downregulated in IIH cases compared to the control subjects (Table 3). Nineteen out of 37 proteins were upregulated/downregulated by more than two-folds. Complement C1q subcomponent subunit B was found to be six-fold upregulated in IIH cases while Myosin-reactive immunoglobulin kappa chain variable region was downregulated by 11-fold. A heatmap of all the 232 identified proteins is given in Figure 2. The AUC of 37 differentially expressed proteins is given in Table S1.

A subgroup analysis was conducted to identify the differentially expressed proteins between overweight and non-overweight IIH cases. We observed only two proteins (Oligodendrocyte myelin glycoprotein variant and Semaphorin-7A) both upregulated in nine overweight IIH cases compared to four non-overweight IIH cases (Figure S1 and Table S2).

4.3 Protein-protein interactions

The 37 differentially expressed proteins were loaded into the STRING online tool (Search Tool for the Retrieval of Interacting Genes/Proteins 11). The network nodes represent proteins while the edges represent the protein-protein interactions. Out of 37 proteins, 26 were successfully matched to proteins within the STRING database, while 11 were unmatched due to insufficient interaction data. This interaction network was further analyzed using the Cytoscape 3.9.0 software. The interaction network consisted of 26 nodes and 46 edges. The interaction network for 21 proteins was found to be highly connected showing molecular interactions with other proteins while five proteins (NELL2, SOD3, ENPP2, NCAN, and RARRES2) were either not connected or had interactions of low confidence with the entire network. APOE had a strong interaction profile with other proteins with the highest degree of interaction = 11 followed by VTN and GSN having a degree of interaction = 8. LRP1-APOE had the highest protein-protein interaction score of 0.999 followed by APOE-APP (score = 0.998), LRP1-APP (score = 0.992), and ORM1-ORM2 (score = 0.984). The interaction network is depicted in Figure 3.

4.4 Functional analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)

The functional analysis was performed using the g:Profiler online tool. The top 10 highly significant pathways/processes were selected. In the GO enrichment analysis, the most significant molecular functions included sulfur compound binding, heparin binding and glycosaminoglycan binding. Platelet degranulation, secretion by cell, and export from cell were found to be the most significant biological processes, while the cellular components included collagen-containing extracellular matrix, blood microparticles, and extracellular space. The KEGG pathway enrichment analysis observed that the significantly differentially expressed proteins were involved in the complement and coagulation cascade pathway (Figure 4).

4.5 Biological roles of significantly differentially expressed proteins

The proteins that were significantly differentially expressed in IIH cases compared to control subjects were categorized into various biological functions including neuroendocrine and neurosecretion, inflammation, antioxidants, adhesion molecules/apoptosis, and proteins involved in cell proliferation (Table S3).