Integrated analysis and exploration of potential shared gene signatures between carotid atherosclerosis and periodontitis

Background Increasing evidence has suggested an association between carotid atherosclerosis (CAS) and periodontitis (PD); however, the mechanisms have not been fully understood. This study aims to investigate the shared genes and molecular mechanisms underlying the co-pathogenesis of CAS and PD. Methods Gene Expression Omnibus (GEO) datasets GSE100927 and GSE10334 were downloaded, and differentially expressed genes (DEGs) shared by both datasets were identified. The functional enrichment analysis of these overlapping DEGs was then conducted. A protein-protein interaction (PPI) network was created using the STRING database and Cytoscape software, and PPI key genes were identified using the cytoHubba plugin. Then, weighted gene co-expression network analysis (WGCNA) was performed on GSE100927 and GSE10334, and the gene modules most correlated with CAS and PD were identified as key modules. The genes in key modules overlapping with PPI key genes were determined to be the key crosstalk genes. Subsequently, the key crosstalk genes were validated in three independent external datasets (GSE43292 [CAS microarray dataset], GSE16134 [PD microarray dataset], and GSE28829 [CAS microarray dataset]). In addition, the immune cell patterns of PD and CAS were evaluated by single-sample gene set enrichment analysis (ssGSEA), and the correlation of key crosstalk genes with each immune cell was calculated. Finally, we investigated the transcription factors (TFs) that regulate key crosstalk genes using NetworkAnalyst 3.0 platform. Results 355 overlapping DEGs of CAS and PD were identified. Functional enrichment analysis highlighted the vital role of immune and inflammatory pathways in CAS and PD. The PPI network was constructed, and eight PPI key genes were identified by cytoHubba, including CD4, FCGR2A, IL1B, ITGAM, ITGAX, LCK, PTPRC, and TNF. By WGCNA, the turquoise module was identified as the most correlated module with CAS, and the blue module was identified as the most correlated module with PD. Ultimately, ITGAM and LCK were identified as key crosstalk genes as they appeared both in key modules and PPI key genes. Expression levels of ITGAM and LCK were significantly elevated in the case groups of the test datasets (GSE100927 and GSE10334) and validation datasets (GSE43292, GSE16134, and GSE28829). In addition, the expression of multiple immune cells was significantly elevated in PD and CAS compared to controls, and the two key crosstalk genes were both significantly associated with CD4 T cells. Finally, SPI1 was identified as a potential key TF, which regulates the two key crosstalk genes. Conclusion This study identified the key crosstalk genes and TF in PD and CAS, which provides new insights for further studies on the co-morbidity mechanisms of CAS and PD from an immune and inflammatory perspective. Supplementary Information The online version contains supplementary material available at 10.1186/s12920-022-01373-y.

Background
The leading pathological process of cardiovascular disease, atherosclerosis, has become a major cause of disability and mortality throughout the world [1]. The carotid artery is one of the primary early onsets of atherosclerosis in seemingly healthy individuals [2]. There is currently a substantial global burden of carotid atherosclerosis (CAS), with 21% of patients aged 30-79 years suffering from carotid plaque [3]. Furthermore, studies have shown that CAS contributes to new occurrences of ischemic stroke [4,5].
Periodontitis (PD) is a chronic inflammatory disease that affects the supporting tissues around the teeth [6]. Increasing evidence suggests an association between PD and CAS [7,8]. The risk factors for PD, such as smoking, diabetes, and obesity [9], are also associated with atherosclerosis [3]. In addition, PD serves as an independent risk factor for atherosclerotic cardiovascular disease [10]. The majority of studies indicate that systemic inflammation plays a significant role in PD and CAS [11]. Inflammatory mediators (e.g., C-Reactive Protein, matrix metalloproteinases, fibrinogen, and other hemostatic factors) are elevated by PD, thus accelerating the progression of CAS through oxidative stress and inflammatory dysfunction [12]. Further, bacteremia attributed to PD can promote direct targeting of bacteria to the distal carotid artery, thereby triggering CAS [13]. Carotid atheromatous plaques obtained during carotid endarterectomy were found to contain DNA of multiple periodontitisassociated bacteria, the most common of which was Porphyromonas gingivalis, followed by Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Eikenella corrodens, Fusobacterium nucleatum, and Campylobacter rectus [14]. These findings suggest a strong linkage between CAS and PD, while molecular mechanisms and pathological interactions are not fully understood.
In this study, we analyzed the publicly available datasets of the GEO database. First, we identified the differentially expressed genes (DEGs) of CAS and PD in test datasets (GSE100927 [CAS] and GSE10334 [PD]) and obtained overlapping DEGs. Subsequently, we performed the functional enrichment analysis of overlapping DEGs. Next, we construct the PPI network using the STRING database. Then, we identified PPI key genes via the Cytoscape software. Furthermore, we performed weighted gene co-expression network analysis (WGCNA) on GSE100927 and GSE10334, thereby screening the most correlated gene modules with CAS and PD. The intersecting genes between PPI key genes and key modules were defined as key crosstalk genes. These key crosstalk genes were verified in the validation datasets GSE43292 (CAS microarray dataset), GSE16134 (PD microarray dataset), and GSE28829 (CAS microarray dataset). In addition, we evaluated the immune cell patterns of PD and CAS by single-sample gene set enrichment analysis (ssGSEA), and calculated the correlation of key crosstalk genes with each immune cell. Finally, we explored the transcription factors (TFs) regulating the key crosstalk genes and finally obtained TFs that were generally upregulated in CAS and PD. Our study may shed new light on the shared pathogenesis of CAS and PD.

Data source
Expression data of CAS and PD were obtained from the Gene Expression Omnibus (GEO) database (https:// www.ncbi.nlm.nih.gov/geo/) [15]. The search strategy of this study: (1) the gene expression profiles were produced by array; (2) PD dataset samples were obtained from gingival tissue, and CAS dataset samples were obtained from carotid plaque; (3) datasets contain samples of control groups; (4) samples are sourced from Homo sapiens. The dataset GSE100927 is based on the GPL17077 Agilent-039494 SurePrint G3 Human GE v2 8 × 60 K Microarray 039381. It included 29 carotid atherosclerotic lesion samples and 12 normal carotid samples. GSE10334 was generated using GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array, which contained 183 gingival tissue samples affected by PD and 64 unaffected control gingival tissue samples. Details of the validation dataset are described in the "Validation of Key Crosstalk Genes in Independent External Datasets" section. The data we analyzed were publicly available; therefore, no ethics committee approval or informed consent was required.

Differentially expression analysis
Analyses based on R packages were carried out on R software (version 4.1.1; https://cran.r-project.org/). DEGs were identified by the "Limma" R package [16]. First, the were both significantly associated with CD4 T cells. Finally, SPI1 was identified as a potential key TF, which regulates the two key crosstalk genes. data were normalized using the normalizeBetweenArrays function. Subsequently, the probe IDs were transformed into official gene symbols. Then, the log 2 fold change and adjusted P-value of each gene were calculated by the lmFit and eBayes functions. The genes with adjusted P-value < 0.05 and |log 2 fold change| > 0.5 were identified as DEGs. Finally, we defined overlapping DEGs as genes that were simultaneously up-regulated or simultaneously down-regulated in both GSE100927 and GSE10334 datasets. The "ggvenn" R package (https://cran.r-project.org/ web/packages/ggvenn/) was performed to plot the Veen diagram for overlapping DEGs.

Protein-protein interaction (PPI) network construction
To identify interactions between overlapping DEGs, we constructed a PPI network of the overlapping DEGs using the STRING database (Version: 11.5) (http://stringdb.org/). This database enables researchers to construct a functional association network of uploaded proteins of an organism based on three aspects: prior knowledge, computational interaction predictions and direct lab experiments [25]. We obtained the interaction network of overlapping DEGs with the "minimum required interaction score" parameter set to 0.4. Subsequently, we imported the network into Cytoscape software for visualization [26]. MCODE plugin app was used to filter clusters with high connectivity, thus dividing the PPI network into several clusters (default parameters: Degree Cutoff = 2; Node Score Cutoff = 0.2; K-Core = 2; and Max Depth = 100) [27]. The genes in clusters with scores > 10 were subsequently performed functional enrichment analysis.

Weighted gene co-expression network analysis
The "WGCNA" package in R was applied for weighted gene co-expression network analysis [28]. The optimal values of the weighted parameters of the adjacent functions were obtained using the pickSoftThreshold function and were used as soft thresholds for subsequent network construction. Then, a weighted adjacency matrix was constructed, and a hierarchical clustering based on a topological overlap matrix (TOM) with a dissimilarity measure (1-TOM) was used to construct the relevant gene modules. Finally, the correlation of each module with the disease was calculated, and the module with the highest correlation was defined as the key module. The genes within the key modules were screened for subsequent analysis.

Identification and validation of key crosstalk genes
CytoHubba is a Cytoscape plugin app that can screen hub genes in PPI networks [29]. Genes that generally ranked top 20 in four algorithms (Degree, EPC, MCC, and MNC) were identified as PPI key genes. Subsequently, PPI key genes and key module genes were intersected. The intersections were defined as key crosstalk genes, which were subsequently validated in GSE100927 and GSE10334. We examined the diagnostic effectiveness of the key crosstalk genes with the receiver operating characteristic curves (ROCs) using the "pROC" package in R [30]. In addition, the mRNA expression levels of the key crosstalk genes were compared between case and control groups using an independent t-test, with a P-value of less than 0.05 considered statistically significant. Relative expression levels of the key crosstalk genes in case and control groups were visualized by boxplots through the "ggplot2" R package [31].

Validation of key crosstalk genes in independent external datasets
To improve the confidence, we validate the expression of the key crosstalk genes in the GSE43292, GSE16134, and GSE28829 datasets. GSE43292 was generated using GPL6244 [HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array, including 32 carotid atherosclerotic lesion samples and 32 normal carotid samples. GSE16134 was obtained from GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array, which contained 241 gingival tissue samples affected by PD and 69 unaffected control gingival tissue samples. GSE28829 was generated using GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array, including 16 advanced atherosclerotic plaque samples and 13 early atherosclerotic plaque samples from carotid arteries.

Immune infiltration analysis
The expression levels of immune cells for each sample of GSE10334 and GSE100927 were quantified using the ssGSEA algorithm from the GSVA R package [32]. The gene set of the cell marker for immune cells was derived from a previous study by Charoentong and colleagues [33]. We then calculated the correlation between the expression of key crosstalk genes and the expression of immune cells in the samples of case groups in GSE10334 and GSE100927 using the Spearman method.

Identification of transcription factors (TFs) of key crosstalk genes
First, we predicted TFs of the key crosstalk genes through the NetworkAnalyst 3.0 platform (https://www.networkanalyst.ca/) [34]. Then, TFs interacting with at least two key crosstalk genes were screened. Subsequently, we validated the mRNA expression levels of these TFs in the test set (GSE100927 and GSE10334) and the validation set (GSE43292, GSE16134, and GSE28829) by independent t-tests. Finally, TFs that were commonly upregulated in case groups were identified as potential key TFs in CAS and PD. Figure 1 shows the flow diagram for this study. There were 3552 DEGs identified in GSE100927, of which 1915 were up-regulated, and 1637 were down-regulated ( Fig. 2a and c). In addition, 1371 DEGs were identified in GSE100927, including 837 up-regulated genes and 534 down-regulated genes ( Fig. 2b and d). Through Veen diagrams, we identified 293 overlapping up-regulated DEGs and 62 overlapping down-regulated DEGs between GSE100927 and GSE10334 (Fig. 2e).

Functional enrichment analysis of overlapping DEGs
We conducted functional enrichment analyses of overlapping DEGs in the DAVID database. The GO_BP Fig. 1 The flow diagram for the whole study analysis revealed that the most significantly enriched terms were immune response, inflammatory response, neutrophil chemotaxis, cell adhesion, and T cell differentiation. KEGG analysis showed that the overlapping genes were likely related to chemokine signaling pathway, leukocyte transendothelial migration, rheumatoid arthritis, cytokine-cytokine receptor interaction, and hematopoietic cell lineage. Reactome analysis indicated that overlapping DEGs were significantly enriched in immune system, innate immune system, neutrophil degranulation, adaptive immune system, and cytokine signaling in immune system. Figure S1 illustrates the top 20 terms that were significantly enriched in GO_BP, KEGG pathway, and Reactome pathway.

Construction of PPI networks with overlapping DEGs
A PPI network with 302 nodes and 2844 interaction pairs was constructed from the STRING database. The PPI network was visualized using the Cytoscape software (Fig. 3a). The MCODE plugin app identified two clusters with high connectivity (scores > 10) (Fig. 3b and c). KEGG analysis showed that two clusters were significantly enriched in cytokine-cytokine receptor interaction, chemokine signaling pathway, leukocyte transendothelial migration, osteoclast differentiation, NF-kappa B signaling pathway, rheumatoid arthritis, TNF signaling pathway, complement and coagulation cascades, and IL-17 signaling pathway (Fig. 3d). Reactome analysis revealed that two clusters were mainly involved in immune system, innate immune system, signaling by interleukins, cytokine signaling in immune system, neutrophil degranulation, adaptive immune system, interleukin-4 and interleukin-13 signaling, and extracellular matrix organization (Fig. 3e).

Construction of weighted gene co-expression network and screening of key modules
The top 5000 genes with the highest standard deviation of expression were screened for WGCNA. The value of β = 18 and β = 12 (scale-free R2 = 0.80) were selected as the soft-threshold power of CAS and PD respectively to ensure scale-free networks ( Fig. 4a and b). With min-ModuleSize set to 40, 11 modules were identified in GSE100927, and 15 modules were identified in GSE10334 (Fig. 4c and d). Different colors indicated different modules. Finally, based on Pearson correlation coefficients, heat maps on module-trait relationships were generated to assess the association between each module and the disease. As a result, the turquoise module was most correlated with CAS (0.9, p = 5E-16), containing 2441 genes,  (Fig. 4e and f ). The genes in these two modules were subsequently analyzed to screen for key crosstalk genes.

Identification and validation of key crosstalk genes
CytoHubba plug-in of Cytoscape was used to calculate the top 20 genes based on four algorithms (Degree, EPC, MCC, and MNC) (Fig. 5a, b, c, and d). Table S1 presents the top 20 genes calculated by the four algorithms. The Veen diagram showed that ITGAM and LCK existed both in the top 20 genes of the four algorithms and in the two key modules (Fig. 5e). Therefore, these two genes were identified as key crosstalk genes. According to the ROC curves, LCK and ITGAM were effective for diagnosing CAS and PD (Fig. 5f h). In addition, the mRNA expression levels of the key crosstalk genes were significantly upregulated in the case groups of GSE100927 and GSE10334 (Fig. 5g, 5i).

Validation of the key crosstalk genes in independent external datasets
To further verify the efficacy of the key crosstalk genes. We validated the genes in two additional independent external datasets (GSE43292 and GSE16134). Consistent with the results of the test set, the ROC results showed good diagnostic efficacy in the external dataset ( Fig. 6a and c), and the mRNA expression levels of the key crosstalk genes elevated in case groups of two external datasets (Fig. 6b and d). In addition, in the GSE28829 dataset, ROC results suggested the effectiveness of LCK and ITGAM in discriminating between advanced and early atherosclerotic plaques (Fig. 6e), and the expression of the key crosstalk genes was significantly higher in advanced atherosclerotic plaque samples in comparison to early plaque samples (Fig. 6f ), indicating that LCK and ITGAM are strongly associated with CAS severity.

Immune infiltration analysis
Through enrichment analysis, we found that the immune pathway is involved in crosstalk between PD and CAS; therefore, we calculated the content of each immune cell in the case group versus the control group in both datasets using the ssGSEA algorithm. Figure 7a and b indicated that the immune landscape is considerably altered in PD and CAS compared to the control group. Furthermore, correlation analysis showed that two key crosstalk genes (LCK and ITGAM) were both significantly associated with the expression of activated CD 4 T cells (Fig. 7c).

Exploring key transcription factors (TFs) regulating the key crosstalk genes
We explored the potential TFs that may regulate the key crosstalk genes. According to the results generated by NetworkAnalyst 3.0, we screened TFs that can regulate two key crosstalk genes (ITGAM and LCK) and calculated their mRNA expression levels in all datasets included in our study by independent t-tests. Consequently, the expression levels of SPI1 elevated in all case groups in GSE100927, GSE43292, GSE10334, and GSE16134 ( Fig. 8a and d). In addition, in the GSE28829 dataset, the expression level of SPI1 was significantly higher in advanced atherosclerotic plaque samples in comparison to early plaque samples (Fig. 8e). Therefore, SPI1 might be a potential key TF regulating the two key crosstalk genes (ITGAM and LCK) in the pathological process in CAS and PD (Fig. 8f ).

Discussion
In a study by Ning and colleagues, gene expression profiles of peripheral blood mononuclear cells (PBMCs) obtained from patients with PD or CAS were analyzed [35]. Another study by Trindade et al., incorporating bibliometric analysis and the DisGeNET database, identified the dysregulated molecules, including C-reactive protein, interleukin-6 and 1-beta, myeloperoxidase, and matrix metalloproteinase-9, as important mediators of periodontitis and coronary artery disease [36].
The DisGeNET database, which was utilized in these two studies, allows users to obtain genes relevant to particular diseases [37]. However, this approach may filter out some potentially valuable molecules. Therefore, we performed analysis directly using transcriptomic data of PD and CAS and finally identified key crosstalk genes and TFs, as well as their mechanisms leading to PD and CAS co-morbidity from an immune and inflammatory perspective. Boxplots of the expression of each immune cell between CAS and control in the GSE100927 dataset. (c) The expression of two key crosstalk genes was significantly correlated with the expression of activated CD4 T cells in both GSE10334 and GSE100927 datasets. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 By comprehensive analysis of gene expression profiles, LCK and ITGAM were identified as two key crosstalk genes between CAS and PD. LCK is a member of the Src family of protein tyrosine kinases (PTKs). He et al. identified an essential role for LCK in the imbalance of the immune system in periodontitis through bioinformatics analysis [38]. LCK inhibitor attenuates the development of atherosclerosis and promotes plaque stability [39]. In addition, LCK was reported to inhibit heat shock protein 65-mediated reverse cholesterol transport in T cells, which is involved in one of the causes of atherosclerosis [40]. ITGAM encodes the integrin alpha M chain. Integrins expressed in periodontal tissues are involved in regulating cell attachment, maintaining tissue integrity, and mediating cell signaling, gene expression, and cytokine activation [41]. In addition, it was shown that ITGAM was significantly increased in the gingival tissue of the aged nonhuman primate model [42]. A proteomics study has revealed a significant elevation of ITGAM in gingival tissue of chronic periodontitis patients [43]. Integrin signaling has been associated with multiple aspects of atherosclerosis, including the early induction of inflammation and the development of advanced fibrotic plaques [44]. Zhou et al. identified that high ITGAM level was associated with atherosclerotic plaque instability and poor outcomes in ischemic stroke [45]. Mass spectrometry analysis identified integrin Alpha-M in emboli from patients with high LDL [46]. In addition, ITGAM has been identified as one of the crosstalk genes for atherosclerosis and COVID-19 co-morbidity [47]. Therefore, the two upregulated key crosstalk genes (ITGAM and LCK) might be involved in CAS and PD co-morbidity mechanisms.
Enrichment analysis of crosstalk genes indicated that immune pathways and inflammatory pathways participated in the co-morbidity of PD and CAS. In addition, immune cell infiltration analysis revealed significantly different immune patterns in the disease groups of PD and CAS compared to controls. Two key crosstalk genes were highly correlated with activated CD4 T cells. Thus these two key crosstalk genes may contribute to PD and CAS co-morbidities through immune and inflammatory pathways.
The gum tissue of individuals who neglect oral hygiene may be vulnerable to pathogens such as Porphyromonas gingivalis [48]. Macrophages eliminate pathogenic microorganisms and present antigenic peptides to the T cell receptor (TCR) [49]. The critical role of LCK in TCR signaling and its involvement in T cell activation was previously reported [50]. Thus, the upregulated LCK in PD is partially involved in T cell activation, which initiates the adaptive immune response. Subsequently, levels of circulating cytokines (such as IL-1β, TNF-α, IFN-γ, and MCP-1) rise, elevating the global inflammation level and promoting the conversion of macrophages into foam cells at the artery, ultimately leading to plaque formation [51,52]. Furthermore, pathogens that reach the damaged endothelium of the carotid artery as a result of bacteremia brought about by periodontitis can increase LCK in the carotid vessels, which can produce the inflammatory and immune effects mentioned above and exacerbate the CAS process.
Integrin ITGAM/ITGB2 is implicated in various adhesive interactions of monocytes, macrophages, and granulocytes, as well as in mediating the uptake of complement-coated particles and pathogens [53]. Studies have shown that integrin activation can promote smooth muscle cell proliferation and macrophage infiltration, consequently aggravating atherosclerosis progression [54]. A recent study by Zhou et al. identified that ITGAM might promote the growth and progression of abdominal aortic aneurysms by promoting endothelial cell adhesion and the migration of circulating monocytes and macrophages [55]. Therefore, ITGAM activation resulting from alterations in the gingival microenvironment following a pathogenic attack may be partially involved in the local macrophage activation, consequently enhancing the inflammatory response. In addition, the altered local vascular environment of CAS induces ITGAM upregulation, resulting in the differentiation of monocytes to macrophages, promoting macrophage activation and smooth muscle cell migration to the endothelium, further exacerbating the progression of CAS.
Our study revealed that SPI1, encoding the transcription factor PU. 1, is commonly upregulated in PD and CAS, which regulates key crosstalk genes. Despite few studies reporting specific functions of PU.1 in PD and CAS, there is evidence that PU. 1 is a key regulator of cellular communication in the immune system, capable of modulating cytokines and cytokine receptors regulating inflammation [56]. In addition, PU.1 specifically participates in macrophage activation [57]. Thus in the global inflammatory environment of PD and CAS, SPI1 (PU. 1) may simultaneously activate macrophages and regulate LCK and ITGAM levels. Figure 9 demonstrates the potential role of the key crosstalk genes and TF identified by our study in the mechanism of PD and CAS co-morbidity.
Our study identifies key crosstalk genes and TF in PD and CAS from an immune and inflammatory perspective, thus providing new ideas on the co-morbidity mechanisms of PD and CAS. The significance of this study is to remind people especially those with high cardiovascular disease risk factors and those who suffer from CAS to focus on oral hygiene. It should be noted that the conclusions were obtained by combining bioinformatics analysis and the previous relevant study findings, thus more clinical validation is required in the future. In addition, the specific function of the crosstalk genes remains for validation in vivo and in vitro, for instance whether perturbation of the crosstalk genes on APOE −/− periodontitis mice/rats will alter CAS progression.

Conclusion
In summary, we identified shared DEGs of CAS and PD, performed enrichment analysis, constructed PPI networks, and conducted WGCNA. Eventually, ITGAM and LCK were identified as key crosstalk genes for CAS and PD, and they may be involved in the crosstalk between CAS and PD through immune pathways and inflammatory pathways. In addition, SPI1 was identified as a potential key TF in CAS and PD. This study provides new insights into the co-pathogenesis of CAS and PD, and the present findings need further validation in the future.


s2fieldsofstudy: Biology Medicine

text:
Cervical cancer cell proliferation inhibition by vanillin oxime through HIF‐1α expression inhibition, ERK1/2 and Akt protein down‐regulation

1Department of Obstetrics and Gynecology, Nantong Maternal and Child Health Center, Nantong University, Jiangsu Nantong, 226006 China; 2Department Maternity and Health Care, Maternal and Child Health Care and Planned Birth Service Center of Licheng County, Shanxi Province, Shanxi Licheng 047600, China; 3Department of Mammary Division, Nantong Maternal and Child Health Center, Nantong University, Jiangsu Nantong 226006, China; 4Department of Obstetrics and Gynecology, Chifeng Second Hospital, Neimenggu Chifeng 024000, China

INTRODUCTION
Cervical cancer is a frequently reported cancer of reproductive tract in females and is worldwide 4th most common malignant tumor (Chen et al., 2020). Annually cervical cancer is detected in ~500 000 patients globally and reports have shown increasing incidence among younger women over the past few years (Fitzmaurice et al., 2015;Wei et al., 2012). Surgery along with radiotherapy have shown significant improvement in the cervical cancer management in patients with early-stage of cancer (Sousa et al., 2018;McClung et al., 2019). However, the treatments available hardly effect tumor growth in patients having late-stage cancer and tumor recurrence. Patients with late-stage cervical cancer have very poor prognosis and it is reported that 1-year survival rate of such patients is below 20% (Sousa et al., 2018;McClung et al., 2019). The comprehensive strategies, including surgery used for cervical cancer have failed to produce any breakthrough in the treatment of advanced and recurrent tumors (Su et al., 2019;. Thus, identification of molecular targets and discovery of potent anti-tumor agents is required to improve the health and survival of patients and inhibit tumor development. There are several reactive oxygen species (ROS) in human bodies which play important role in various metabolic regula tory processes like glucose metabolism (Jambunathan et al., 2010;Wang et al., 2018). ROS act as main signalling factors and regulate several processes including proliferation of cells, migration abilities, and cell differentiation by influencing various proteins and lipids (Lu et al., 2018;Diwanji et al., 2018;Sun et al., 2019). In addition, ROS also influence the functioning and structure of the blood vessels (Lu et al., 2018;Diwanji et al., 2018;Sun et al., 2019). Higher content of ROS induces various disorders in the body by damaging various proteins and membranes.
Hypoxia-inducible factor-1α (HIF-1α) has been found to play a prominent role in invasion of cancer cells, their metastatic ability, and proliferation (Zhang et al., 2017;Lai et al., 2018). Reports have demonstrated that HIF-1α expression inhibition by therapeutic agents exhibits antitumor effect in many cancers such as breast and pulmonary cancer cells (Sun et al., 2011;. Vanillin, a phytoconstituent isolated from Vanilla planifolia plant is commonly known as 4-hydroxy-3-methoxybenzaldehyde. Initial screening revealed anti-microbial, inflammation inhibitory and anti-mutagenic activities of vanillin in rodent as well as human models (Walton et al., 2003;King et al., 2008;Srinivasan et al., 2008;Imanishi et al., 1990). Mechanistic study revealed that vanillin inhibits invasion and migration abilities of liver cancer cells by down-regulation of nuclear factor-κB expression and suppression of matrix metalloproteinases (Liang et al., 2009). Moreover, vanillin treatment led to the inhibition of PI3K phosphorylation to suppress angiogenesis of lung cancer cells and formation of lamellipodia (Lirdprapamongkol et al., 2010;Lirdprapamongkol et al., 2005;Lirdprapamongkol et al., 2009). Vanillin treatment of cervical and breast cancer cells is associated with activation of apoptosis pathway and thereby inhibition of tumor growth (Lirdprapamongkol et al., 2010;Lirdprapamongkol et al., 2005;Lirdprapamongkol et al., 2009). Compound obtained by modification of many natural products has shown strong anticancer property exhibiting 81-82% cytotoxicity. Oxime analogs of curcumin have been found to possess strong antiproliferative property against cancer cells compared to the cyclohexanone and tetralone analogs (Qin et al., 2016). Taking into account string anticancer activity of oxime analogs of the natural products the present study investigated vanillin oxime as anti-proliferative agent against cervical cancer cells and elucidated the associated mechanism.

Reagents and chemicals
Vanillin oxime (Cas number S539570; purity 99%) and dimethyl sulfoxide were supplied by the Merck Chemicals. Dimethyl sulfoxide (DMSO) and many other chemicals were provided by the Sigma-Aldrich (St Louis, MO, USA). Stock solution of vanillin oxime was prepared in physiological saline.

Cell lines and culture
Caski cervical cancer cell line and human cervical epithelial cells (HCvEpC) cells were supplied by the Shanghai Bioengineering Co., Ltd., China. The cells were cultured overnight in RPMI-1640 medium mixed with FBS (10%) in an incubator containing CO 2 (5%) at 37ºC. At the time of entering logarithmic growth phase the cells were digested for 3 min on treatment with 0.25% pancreatin at 37ºC.

Proliferation assay
Caski and (HCvEpC) cells were inoculated in 96-well plates at a density of 2×10 4 cells/ well and incubated for 24 h with vanillin oxime at 2, 4, 6, 8 and 10 μM concentrations. Culture solution from the plates was removed at 24 h of incubation and then 10 μl of MTT (5 mg/ml) solution was added to each well. After 4 h of incubation with MTT, 100 μl DMSO was added to solubilize the formazan crystals that formed in each well. Measurement of absorbance was made three times for each plate at 450 nm using a microplate reader (Thermo, BD, USA, VL0LA0D2) for calculation of cell proliferation.

Determination of ROS level
Fluorescent dye namely, 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA; Invitrogen) was used for ROS content detection in Caski cells. The cells were incubated at 1×10 6 cells/ well distribution in 6-well plates for 24 h with 2 to 10 μM concentrations of vanillin oxime at 37ºC. Afterwards cells were PBS washed twice before incubation for 40 min at 37ºC with 20 μM H 2 DCFDA as per the instructions of the supplier. Excitation and emission wavelengths used were 489 and 528 nm, for detection of fluorescence using enzyme labeling instrument (Tecan Group, Ltd.) and for the determination of ROS content, respectively.

Colony formation assay
Caski cells growing in logarithmic phase were distributed at 1 000 cells/ well density in 60-mm culture dishes and subjected to culture at 37ºC. Incubation with vanillin oxime at 2 and 10 μM concentrations was followed by removal of the medium and subsequently medium was replaced after every 3 days. Washing of cells in PBS, fixing for 10 min in methanol (anhydrous) was followed by staining with crystal violet solution (0.1%). Staining was performed for 20 min at room temperature prior to calculation of colonies formed under light microscope (magnification, ×400; Carl Zeiss AG).

Analysis of cell invasion
Matrigel was adjusted to a concentration of 1 mg/ml using precooled medium devoid of the serum at 4ºC. Upper chambers having filters with 8 μm pore-size were coated for 4 h at 37ºC using Matrigel and set for solidification. The cells in groups of 4×10 5 were suspended in 350 μl serum-free medium and then transwell chambers were put into the 24-well culture plate. Mixture of the cells was put into the upper chamber and the medium mixed with 20% FBS was added into the lower chamber. Removal of the chamber and aspiration of the cell culture was followed by PBS washing of the cells and then fixing for 10 min in methanol (anhydrous). The cells were dyed for 10 min with crystal violet (0.1%), washed in water, dried, and then mounted on the coverslips using neutral gum. Cell invasion was detected randomly in five fields using a light microscope (magnification ×200; Carl Zeiss AG).

Reverse transcription-quantitative PCR (RT-qPCR) analysis
Total RNA was extracted from vanillin oxime treated Caski cells using TRIzol ® reagent (Invitrogen) and for determination of RNA purity and concentration the ultraviolet spectrophotometer (Tecan Group, Ltd.) was employed. The RNA samples were reverse transcribed to cDNA using the EcoDry™ Premix (Takara Biotechnology Co., Ltd.). Conditions used for reverse transcription were 50 min at 42ºC, for inactivation of the reverse transcriptase 5 min at 98ºC and reverse transcription product saving at 4ºC. The BeyoFast™ SYBR-Green quantitative PCR premix was used for detection of HIF-1α expression. Conditions used for thermocycling were: denaturation for 8 min at 94ºC, 38 cycles for 12 sec at 94ºC and for 55 sec at 58ºC. The PCR amplification was followed by plotting of the melting curve to determine HIF-1α expression using the 2-ΔΔ Cq method.

Flow cytometry assay
Caski cells at 2.5×10 5 cells/ml distribution were collected as single-cell suspension and put into 96-well plates. After treatment with vanillin oxime cells were harvested, washed twice with PBS, and then dyed with Annexin V-FITC and PI for 20 min at 37ºC. The cells were subsequently analysed for apoptosis using the flow cytometry (BD FACSCalibur™; BD Biosciences). Apoptosis was analysed using the CellQuest Pro software (version 5.0; BD Biosciences).

Statistical analysis
Data expressed are the mean ± S.D. of triplicate experiments carried out independently. Differences were determined statistically using one-way ANOVA, Bonferroni post hoc test and Unpaired t-test to compare two groups. Data were analysed using the SPSS 20 (IBM Corp.) software. Differences were taken as statistically significant at P<0.05.

Inhibition of Caski cell viability and promotion of ROS level by vanillin oxime
Cervical cancer, a frequently diagnosed gynecological malignancy in females, was responsible for 311 000 deaths in the year 2018 (Arbyn et al., 2020). Worldwide, cervical cancer has an incidence of around 13·1 cases in every 100 000 women and it was diagnosed in ~570 000 women in 2018 (Arbyn et al., 2020). Treatment strategy presently available for cervical cancer consists of surgical resection followed by radiotherapy in combination with chemotherapy (Menderes et al., 2016). Standard treatment that is effective for cervical cancer inhibition is yet to be established and is urgently required to be developed. Chemotherapeutics used against the cervical cancer are mainly confronted with challenges such as tumor recurrence and rapid distant organ metastasis (Geretto et al., 2017). Highly efficient and novel chemotherapeutic agents possessing least/ no side effects need to be developed for the treatment of the cervical cancer effectively (Geretto et al., 2017;Dueñas-González and Campbell, 2016).
In the present study viability changes by vanillin oxime in Caski and (HCvEpC) cells were measured following incubation with 2, 4, 6, 8 and 10 μM concentrations by MTT method (Fig. 1A). Vanillin oxime treatment led to suppression of Caski cell proliferation, and the reduction was concentration dependent. Suppression in Caski cell proliferation by vanillin oxime treatment was maximum at 10 μM concentration and remained constant at 10 μM. Vanillin oxime treatment also caused significant (P<0.05) reduction in Caski cell proliferation at 2, 4 and 6 μM concentrations. In (HCvEpC) cells vanillin oxime treatment could not affect proliferation at the tested (2 to 10 μM) concentrations. Vanillin oxime induced ROS level in Caski cells was detected at 2 to 10 μM concentrations following incubation for 24 h (Fig. 1B). In vanillin oxime treated Caski cells ROS level showed an increase with enhancement in concentration from 2 to 10 μM.

Vanillin oxime inhibits colony formation and cell invasion
Colony formation inhibition by treatment with vanillin oxime in Caski cells was detected by treatment with concentrations of 2 and 10 μM ( Fig. 2A). Vanillin oxime treatment significantly (P<0.0487) lowered the count of colonies in Caski cells compared to the control. In 10 μM vanillin oxime treated cell cultures very few colonies were detected at 24 h compared to the control cultures. Treatment with vanillin oxime also lowered invasive abilities of Caski cells significantly (P<0.05) at 2 and 10 μM concentrations (Fig. 2B). Transwell assay showed that treatment with 10 μM vanillin oxime lowered invasive abilities of Caski cells to greater extent compared to 2 μM concentrations.

Vanillin oxime induces Caski cell apoptosis
A main anti-tumor mechanism for chemotherapeutics involving several targets to inhibit cell proliferation via complicated process is the apoptosis (Lunghi et al., 2008). Anti-tumor effect of some chemotherapeutics have been demonstrated to be associated with the damage induced by ROS formation (Sarkar et al., 2014;Qu et al., 2013). At baseline values ROS induces DNA synthe-  sis, subsequent cell mitosis and tumor cell proliferation (Hassani et al., 2013). Excessive formation of ROS leads to induction of apoptotic signals and causes necrotic cell death (Pollak et al., 2013). In the present study Caski cells after vanillin oxime treatment at 2 to 10 μM concentrations were analysed for apoptosis by flow cytometry at 24 h (Fig. 3). Treatment with vanillin oxime significantly (P<0.05) raised Caski cell apoptotic count in a dose-dependent way. Caski cell apoptotic count reached to 8.76% and 48.65%, on incubation with 2 and 10 μM concentrations of vanillin oxime, respectively.

Vanillin oxime inhibits MMP expression
Vanillin inhibits tumor migration and suppresses invasive abilities of liver cancer cells via targeting NF-κB expression and MMP activities (Liang et al., 2009). Angiogenesis as well as lamellipodia formation in lung cancer cells is inhibited through interfering with PI3K pathway by vanillin treatment (Lirdprapamongkol et al., 2010;Lirdprapamongkol et al., 2005;Lirdprapamongkol et al., 2009). Growth of cancer cells such as breast and cervical carcinoma is inhibited by vanillin through induction of apoptotic signals (Lirdprapamongkol et al., 2010;Lirdprapamongkol et al., 2005;Lirdprapamongkol et al., 2009). In the present study treatment of Caski cells with vanillin oxime at 2 to 10 µM concentrations for 24 h was followed by MMP expression measurement by western blotting (Fig. 4). After treatment with vanillin oxime a prominent reduction in MMP-2 and -9 levels was observed in Caski cells compared to the control cells. The MMP-2 and -9 levels in Caski cells were reduced to minimum level by vanillin oxime treatment at 10 µM concentration at 24 h.

Vanillin oxime inhibits malignancy protein levels
In Caski cells vanillin oxime treatment at 2 to 10 μM concentrations for 24 h was followed by protein expression determination by western blotting (Fig. 5). A prominent reduction in p-ERK1/2 and p-Akt levels was observed in Caski cells after treatment with vanillin oxime at 2 to 10 μM concentrations. The Bcl-2 level in Caski cells also showed a remarkable reduction after vanillin oxime treatment in a dose-dependent way.

Vanillin oxime inhibits HIF-1α expression
Activation of HIF-1α at a rapid rate plays crucial role in proliferation of tumor cells, their metastatic potential and infiltration abilities (Su et al., 2011). It is also involved in development of resistance in tumor cells against chemotherapies and radiotherapy (Su et al., 2011). In the present study treatment with vanillin oxime caused a significant (P<0.05) suppression in HIF-1α mRNA expression at 2 to 10 μM concentrations in Caski cells (Fig. 6A). After increasing the concentration of vanillin oxime from 2 to 10 μM, the reduction in HIF-1α mRNA showed a dose-dependent reduction. HIF-1α protein level also showed a prominent reduction in Caski cells after treatment with vanillin oxime at 2 to 10 μM concentrations (Fig. 6B, C). Suppression in HIF-1α protein level by vanillin oxime treatment at 24 h was maximum at 10 μM concentration in Caski cells.

CONCLUSION
In summary, vanillin oxime inhibits cervical cancer proliferation, invasive abilities, induces apoptotic signalling, and elevates ROS production. Moreover, HIF-1α expression inhibition, ERK1/2 and Akt protein downregulation is induced in cervical cancer cells after treatment with vanillin oxime. Therefore, vanillin oxime may


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PI4K2A deficiency causes innate error in intracellular trafficking with developmental and epileptic‐dyskinetic encephalopathy

Abstract Objective Intracellular signaling networks rely on proper membrane organization to control an array of cellular processes such as metabolism, proliferation, apoptosis, and macroautophagy in eukaryotic cells and organisms. Phosphatidylinositol 4‐phosphate (PI4P) emerged as an essential regulatory lipid within organelle membranes that defines their lipid composition and signaling properties. PI4P is generated by four distinct phosphatidylinositol 4‐kinases (PI4K) in mammalian cells: PI4KA, PI4KB, PI4K2A, PI4K2B. Animal models and human genetic studies suggest vital roles of PI4K enzymes in development and function of various organs, including the nervous system. Bi‐allelic variants in PI4KA were recently associated with neurodevelopmental disorders (NDD), brain malformations, leukodystrophy, primary immunodeficiency, and inflammatory bowel disease. Here, we describe patients from two unrelated consanguineous families with PI4K2A deficiency and functionally explored the pathogenic mechanism. Methods Two patients with PI4K2A deficiency were identified by exome sequencing, presenting with developmental and epileptic‐dyskinetic encephalopathy. Neuroimaging showed corpus callosum dysgenesis, diffuse white matter volume loss, and hypoplastic vermis. In addition to NDD, we observed recurrent infections and death at toddler age. We further explored identified variants with cellular assays. Results This clinical presentation overlaps with what was previously reported in two affected siblings with homozygous nonsense PI4K2A variant. Cellular studies analyzing these human variants confirmed their deleterious effect on PI4K2A activity and, together with the central role of PI4K2A in Rab7‐associated vesicular trafficking, establish a link between late endosome‐lysosome defects and NDD. Interpretation Our study establishes the genotype–phenotype spectrum of PI4K‐associated NDD and highlights several commonalities with other innate errors of intracellular trafficking.

Introduction
Eukaryotic cells compartmentalize their cellular functions using organelles that are defined by membranes with unique lipid and protein composition as well as signaling properties. Transport of proteins, lipids and other soluble molecules either through vesicular or other intracellular transport pathways ensure proper dynamic adaptation of cells to changing environments. Inositol phospholipids (PPIns) represent a small fraction of cellular phospholipids with important roles in the control of cellular processes. 1 While phosphatidylinositol 4-phosphate (PI4P) has long been viewed solely as an immediate precursor of the regulatory lipid, PI(4,5)P 2 , studies in the last 15 years have clearly demonstrated that PI4P is a regulatory lipid on its own right, which functions specifically in the Golgi apparatus and endosomal membranes where it controls vesicular trafficking. 1 PI4P gradients established at membrane contact sites between opposing organelle membranes have also recently emerged as a driving force to support nonvesicular transport of structural lipids against concentration gradients. 2 This paradigm-shifting discovery has placed PI4P in the center of the lipid homeostatic cellular machinery. Mammalian PI4P is generated by four distinct phosphatidylinositol 4-kinases (PI4Ks): PI4KA, PI4KB, PI4K2A, PI4K2B. While PI4KA establishes the PI4P gradient between plasma membrane (PM) and endoplasmic reticulum (ER), other PI4K enzymes generate PI4P gradients between Golgi-ER or endosome-ER interfaces, hence controlling lipid transport within these membrane compartments. Murine studies showed severe myelination defects in knockout of either PI4KA or PI4KB in Schwann cells 3,4 and spinocerebellar degeneration with lack of Pi4k2a. 5 In light of these findings, the common thread between PI4K enzymes is their critical involvement in establishing the lipid landscape of mammalian cells, especially within neuronal development and plasticity.
The wider availability of massively parallel sequencing has aided the identification of such disease-associated genes from intracellular trafficking pathways. Recent studies described 22 affected individuals with bi-allelic variants in the phosphatidylinositol 4-kinase alpha gene (PI4KA, OMIM *600286) causing a spectrum of neurodevelopmental disorder (NDD), brain malformations, contractures, hypomyelinating leukodystrophy, inflammatory bowel disease (IBD), and primary immunodeficiency (PID). [6][7][8] To this date, only two homozygous variants in the phosphatidylinositol 4-kinase type 2 alpha gene (PI4K2A) were reported to be associated with NDD phenotypes. 9,10 The first and most striking report showed homozygous nonsense variants in PI4K2A in two affected siblings presenting with NDD, epilepsy, myoclonus, and akathisia. In a subsequent study, a homozygous missense variant of uncertain significance was suggested to be responsible for some features in a case with NDD and metabolic cutis laxa. In the present study, we describe two patients from unrelated consanguineous families with PI4K2A deficiency characterized by developmental encephalopathy with hyperkinetic movement disorders, and epilepsy. We further examine whether the pathogenic variants found in these two index cases have an effect on intracellular vesicular trafficking.

Clinical and genomic investigations
In this study, we evaluated two independent consanguineous families of Iranian and Lebanese origin ( Fig. 1A and B). The study was approved by the institutional ethics committees of the participating centers and written informed consent was obtained from the families in accordance with the Declaration of Helsinki. Detailed clinical features, family history, EEG recordings and brain magnetic resonance imaging (MRI) studies were carefully reviewed by a group of clinical geneticists, pediatric neurologists, and a neuroradiologist. Clinical and research exome sequencing and dideoxy sequencing segregation analysis were performed in each family according to previously published studies. 11 The patient from family 1 was sequenced with proband-only whole exome sequencing (WES). The patient from family 2 was sequenced by trio WES (patient, mother, father).

Reagents
Stock solutions of all reagents were dissolved in the indicated solvent and stored in small aliquots at À20°C.
Live-cell confocal microscopy

BRET measurements
BRET-based measurements of PI4P levels within Rab7positive membrane compartments of intact cells has been described in detail previously. 14  After the pre-incubation period, the cell-permeable luciferase substrate, coelenterazine h (40 lL, final concentration 5 lmol/L), was added and a 5 min baseline BRET measurement (30 sec/cycle) was recorded. The plates were then quickly unloaded for manual addition of GSK-A1 (30 nmol/L) or a DMSO vehicle control, which were prepared in a 10 lL volume of the modified Krebs-Ringer solution. Measurements were carried out in triplicate wells and repeated as three independent experiments. From each well, the BRET ratio was calculated by dividing the 530 and 485 nm intensities, which were then normalized to the baseline measurement. To facilitate the pooling of data from individual wells and between replicate experiments, the raw BRET ratios were processed by using a simple moving average with a four-cycle interval across the kinetic. For each iRFP-PI4K2A variant or the iRFP-C1 control, the processed BRET ratios obtained from GSK-A1-treated wells were normalized to an internal DMSO vehicle control.

Results
Clinical and neuroimaging findings Table 1 shows the clinical and neuroimaging findings in our two cases. Patient 1 was a female patient born to 2nd degree consanguineous Iranian parents who was presented to our specialty clinic with severe neurodevelopmental delay, irritability, movement disorders, epilepsy, and failure to thrive at 18 months of age. The patient was born at full term (38 weeks) with a weight at 2200 g (1st percentile) and head circumference at 34 cm (44th percentile) without relevant abnormalities in perinatal adaptation. She never attained considerable milestones in motor, intellectual or speech development. At the age of 16 months, she first showed intractable myoclonic and tonic seizures. Treatment with clobazam for 1.5 years had moderate response while there were only intermittent improvements on treatment with levetiracetam, primidone, vigabatrin, topiramate, and phenobarbital. Neurological examination showed distinct muscular hypotonia and atrophy, intermittent dystonic postures of the upper limbs with clenched fist and thumb adduction, spasticity of limbs and orofacial dyskinesia (see Video S1). EEG showed slow background, bursts of central theta frequency waves, and frontotemporal spike-wave-bursts. Brain MRI showed marked dysgenesis of corpus callosum. We also observed hypoplastic anterior commissure and massa intermedia, diffuse white matter volume loss with ventriculomegaly, widened anterior interhemispheric fissure, slightly rotated thalami, and hypoplastic brainstem predominantly involving the pons, hypoplastic vermis with widening of the foramen of Magendie, hypoplastic superior and middle cerebellar peduncles, and a mega cisterna magna. Standard metabolic tests (MS/MS and organic acids in urine) and karyotype were normal. The patient showed recurrent severe bouts of infection with 16 hospitalizations within 3 years, failure to thrive (height, weight, and head circumference all below 1st percentile at 34 months), and recurrent constipation which might point towards PID. The patient died at 3 years and 10 months of age. Patient 2 is a female patient born to consanguineous Lebanese parents. She had a severe developmental delay, brain malformation, irritability, orofacial dyskinesia, epilepsy, and failure to thrive at 2 years of age. The parents had a previous miscarriage and comparative genomic hybridization of the fetus revealed evidence of 45,XO chromosomes (Turner syndrome). The patient was born at full term (38 weeks) with a weight at 3325 g (65th percentile), length at 48 cm (15th percentile), and head circumference at 35 cm (71st percentile) without relevant abnormalities in perinatal adaptation. At the age of 6 months, she first showed epileptic spasms that were ultimately managed with triple treatment (vigabatrin, topiramate, and lamotrigine). EEG showed diffuse delta rhythm and hypsarrhythmia. While hypoplasia of corpus callosum was already detected in utero, brain MRI at 6 months of age showed dysgenesis of the corpus callosum with markedly hypoplastic and foreshortened body and underdeveloped rostrum, genu, and splenium, secondary radial configuration of the paramedian gyri with absent cingulate gyri, widened anterior interhemispheric fissure, hypoplastic anterior commissure and massa intermedia, agenesis of the septum pellucidum, asymmetrically rotated thalami, diffuse white matter volume loss, hypoplastic brainstem predominantly involving the pons, hypoplastic vermis with widening of the foramen of Magendie, hypoplastic superior and middle cerebellar peduncles, and a mega cisterna magna. The patient had constipation and failure to thrive with feeding difficulties, and no recurrent infections. Standard metabolic screenings showed no abnormalities.

Molecular findings
A loss of function homozygous variant in PI4K2A NM_018425.4:c.925C>T, p.(Arg309Ter) was found in both patients from unrelated families, whereas the parents of each index patient showed heterozygous variants ( Fig. 1A-C). This novel variant is not reported in multiple variant databases (gnomAD, UKbiobank, Centogene Bio/Databank, QSG, BRAVO TOPMed Freeze 8, GME Variome, Iranome) and leads to a protein truncation at amino acid residue 309 within the catalytic kinase domain.

Functional studies of PI4K2A variants
To test the functionality of the mutated PI4K2A proteins, we generated GFP-and iRFP-fused versions of the wildtype and two homozygous variants (Arg275Trp and Arg309Ter) proteins. The Arg309Ter pathogenic variant was found in the patients described above, while the Arg275Trp variant was previously described in a patient with metabolic cutis laxa. 10 Confocal microscopy showed that the GFP-tagged truncated enzyme was fully cytoplasmic showing no association with any internal membranes,    whereas the wild-type protein was associated with the endosomal compartments as shown previously (Fig. 2D). 15,16 There was no obvious difference between the localizations of the wild-type and the Arg275Trp substituted protein (not shown). In previous studies using PI4K2A K/O HEK293-AT1 cells, we found that PI4K2A generates most of the PI4P in Rab7-positive endolysosomal compartments. 14 In those studies, wildtype PI4K2A but not a catalytically inactive protein was able to restore PI4P levels in the Rab7-positive endosomes. We used this system to test the functionality of the mutated forms of PI4K2A. The PI4P content of the Rab7 positive compartment was monitored by BRET that is based on the energy transfer between a Rab7-targeted Venus and the Luciferase-fused PI4P-selecive protein binding module of Legionella (P4M-2x) as described previously 14,17 and shown in the illustration (Fig. 2C). This approach also takes advantage of the use of GSK-A1, a selective inhibitor of PI4KA, the enzyme that generates the PM pool of PI4P. 12 Upon addition of GSK-A1 to the cells, the PM pool of PI4P is slowly diminished causing the Luciferase-fused PI4P binding module to be released from the PM and re-localize to the other PI4P containing membranes including the Rab7 compartment as reflected in the increased BRET signal (Fig. 2E). As shown in Figure 2E, PI4K2A K/O cells show only a modest increase in the BRET signal monitored in the Rab7 compartment after GSK-A1 addition (gray symbols) that has been attributed to the function of the other member of this enzyme family, PI4K2B. 14 Expression of the wildtype iRFP-fused PI4K2A substantially increased the amount of PI4P in the Rab7 compartment (red symbols). A similar increase was found with the Arg275Trp variant (blue symbols). In contrast, the truncated Arg309Ter variant of PI4K2A failed to restore the PI4P levels in the Rab7 compartment.

Discussion
In this report we describe two cases with a bi-allelic pathogenic variant resulting in the truncation of the PI4K2A protein. Taking into account the seminal paper reporting two patients from one family, 9 our findings establish that PI4K2A deficiency is a novel cause of developmental encephalopathy with epilepsy and hyperkinetic movement disorders 18 (Fig. 3A). Our results from Figure 2 clearly showed that the truncated PI4K2A protein was completely inactive, which is not surprising given the fact that half of its catalytic domain was missing (Fig. 2B). It is also likely that such truncated protein is unstable and would be degraded in the patients' cells. The genome aggregation database (gnomAD) lists the gene PI4K2A with a very high probability of being intolerant towards loss-of-function (pLoF pLI = 0.8) which strengthens the case that null variants are not tolerated in the healthy population. In contrast, we found that the Arg275Trp substituted enzyme was fully functional in our experimental conditions testing the protein in situ within cells. This variant was previously proposed as a possible disease-causing allele in a patient with metabolic cutis laxa although the patient also carried a homozygous missense variant in the Twinkle mtDNA helicase gene (TWNK). 10 The patient reported in that study also showed lipodystrophy, microcephaly, muscular hypotonia, global developmental delay, choreoathetosis, brain malformations, progeroid facial appearance, and hepatomegaly. All of these symptoms have been documented in previous reports of TWNK deficiency. 10 Importantly, neither of our patients with PI4K2A deficiency showed any signs of cutis laxa or joint hypermobility. The findings from our cellular experiments raise the possibility that the missense variant in PI4K2A with a substitution in Arg275Trp described in that study may not have been the primary cause of the disease, although we cannot rule out that the PI4K2A Arg275Trp variant has subtle functional alterations not revealed by our assays, which also contributed to the clinical manifestation found in that patient.
It is notable that significant overlap exists in pathogenic variants in PI4KA and PI4K2A with phenotypes including severe combined NDD, epilepsy or even epileptic encephalopathy, brain malformation (hypomyelination/atrophy, corpus callosum abnormalities, pontocerebellar hypoplasia 8 ), movement disorders (dystonia, spastic paraplegia), craniofacial dysmorphia, and feeding difficulties with failure to thrive (Fig. 3B). Patients with variants in PI4KA or PI4K2A share common features of severe developmental encephalopathy reflecting diffuse neurological dysfunction. [6][7][8][9] Brain malformation, severe psychomotor retardation, epilepsy, movement disorders, contractures, spasticity and peripheral nervous system involvement have been described in both defects [6][7][8][9] (Fig. 3B). Movement disorders and neuropathy have been well established in patients with monogenic deficiencies in intracellular trafficking. 19 Severe neurological phenotypes with optic atrophy, epileptic encephalopathy or coloboma also presented with PID or IBD in several patients with PI4KA variants (Fig. 3A). 6 It is unclear to what extent immunodeficiency triggers a severe deterioration of the neurological phenotype.
Immunological features in patients with PI4KA variants include T-cell lymphopenia primarily affecting CD8+ T cells, B-and NK cell lymphopenia, aÀ/hypogammaglobulinemia, splenomegaly, and poor antibody responses to previous immunizations. 6  recurrent infections. Of note, she showed a diffuse grade 3A follicular non-Hodgkin lymphoma with a bcl6 translocation. One of our PI4K2A cases had recurrent infections with multiple hospitalizations. However, the patient died before diagnostic work-up for a suspected immunodeficiency. As PID is a recurring feature in the cohorts of published cases with PI4KA variants (n = 8/22), PID may be underreported in cases with PI4K2A variants due to the much lower number of cases. Further studies are required to lay the groundwork for genotype-phenotype correlations in patients with pathogenic variants in PI4K2A. [20][21][22] We observed major clinical overlaps with other disorders within the emerging group of innate errors in intracellular trafficking that is likely explained by the prominent roles of PI4KA and PI4K2A in the control of intracellular trafficking pathways. A recent study revealed an important role of PI4K2A in Rab7-positive compartments supporting the fusion of lysosomes with autophagosomes. 14 Another recent study reported biallelic variants in the vacuolar protein sorting-associated protein 41 homolog gene (VPS41) in patients suffering from progressive neurodevelopmental disease comprising cognitive impairment, cerebellar atrophy/hypoplasia, motor dysfunction with ataxia and dystonia, and nystagmus due to lysosomal deregulation. 23 VPS41 is part of the homotypic fusion and vacuole protein sorting (HOPS) complex which tethers the complex to Rab7positive endosomes and support lysosomal function. Heterozygous variants in the vacuolar protein sortingassociated protein 16 homolog gene (VPS16), another member of the HOPS complex, were also linked to earlyonset progressive dystonia with predominant cervical, bulbar, orofacial, and upper limb involvement. 24 Patients  [6][7][8] Bi-allelic PI4K2A variants on protein level from one previously published study and our study. 9 Missense/indel variants are found above protein illustrations, frameshift/stop variants below protein illustrations. Color coding relates to each symptoms/disorders in each patient that is associated to variant: bright blue neurodevelopmental disorder, pinkmovement disorder, brownbrain malformation, greenimmunodeficiency and/or inflammatory bowel disorder. Created with IBS Biocuckoo. (B) Comparison of phenotypes in PI4KA deficiency (blue), PI4K2A deficiency (red), and phenotypes found in both PI4K-associated disorders (green) shows overlap specifically within the neurological disorders (dystonia, spastic paraplegia, contractures, peripheral neuropathy, epilepsy/epileptic encephalopathy, intellectual disability and/or global developmental delay, hypomyelination, callosal abnormalities, pontocerebellar hypoplasia) as well as craniofacial dysmorphia, failure to thrive, feeding difficulty, and recurrent infections. Created with BioRender.com. Figure 2. Characterization of PI4K2A variants using intact HEK293 cells. (A) Cartoon depiction of the PI4K2A sequence along with variants relevant to this study. The proline-rich (PR; blue) and kinase (orange) domains, along with the palmitoylated cysteine-rich CCPCC motif (CR; yellow), are specifically highlighted. (B) Ribbon representation of the solved PI4K2A structure in complex with adenosine trisphosphate 35 (PDB Accession: 4PLA), with the membrane-oriented surface facing upwards. Please note, as in (A), the truncated C-Lobe of the kinase that is lost in the Arg309Ter variant has been colored green, and the side chain for residue Arg275 is highlighted as a stick representation in blue. (C) Schematic depicting the principle behind BRET-based measurements of PI4P levels within Rab7-positive endosomal compartments. Briefly, treatment of cells with the PI4KA-selective inhibitor, GSK-A1 (30 nmol/L), acutely reduces the PM levels of PI4P, which causes a well-characterized PI4P-sensitive reporter 17 to drop from this compartment and find endosomal pools of PI4P that are made by PI4K isoforms, including PI4K2A, that are insensitive to GSK-A1. For further information on this approach and important controls, please refer to previously published study. 17 (D) Representative images comparing the subcellular localization of the wild-type EGFP-PI4K2A enzyme (left) with the EGFP-PI4K2A-Arg309Ter variants in HEK293-AT1 cells (10 lm scale bars). (E) Kinetics of PI4P levels within the Rab7-positive endosomal compartments of PI4K2A K/O cells expressing iRFP-PI4K2A variants (wild-type, truncated Arg309Ter), or the Arg275Trp substitution) or a vector control (iRFP-C1) in response to treatment with GSK-A1 (30 nmol/L). BRET measurements were made using the sLuc-P4M2x-T2A-mVenus-Rab7 biosensor 14 and are presented as mean values AE SEM from three independent experiments carried out using triplicate wells. PI4P, phosphatidylinositol 4-phosphate; PI4K, phosphatidylinositol 4-kinase. with bi-allelic variants in VPS16 showed a more severe phenotype with similar features to PI4K deficiency, including delayed myelination, brain atrophy, neutropenia, skeletal abnormalities, and dysmorphic features. 25 Defects in Rab7-mediated vesicular trafficking were also found in other neurodegenerative movement disorders, such as in models of Parkinson's disease for the clearance of toxic protein aggregate alpha-synuclein. 26,27 Another example of dysfunctional Rab7-mediated signaling in NDD is found in patients with Vici syndrome and recessive variants in the ectopic P-granules autophagy protein 5 (EPG5) gene, 28 where EPG5 deficiency causes a blockade in autophagosome-lysosome tethering and deficient Rab7-mediated signaling. 29,30 Patients with Vici syndrome also show microcephaly, corpus callosum abnormalities, and various movement disorders such as spastic paraplegia, similarly to our patients with PI4K2A deficiency. This spectrum of movement disorders was also observed in patients with PI4KA deficiency, including dystonia, spastic paraplegia, opisthotonus, ataxia, and myoclonus. The patients described here with pathogenic variants in PI4K2A show spastic paraplegia, orofacial dyskinesia, and intermittent dystonic postures of the upper limbs with clenched fist.
PI4K2A is known to form a tripartite complex with the biogenesis of lysosome-related organelles complex 1 (BLOC-1) and adaptor-related protein complex 3 (AP-3) that is crucial for the endocytic pathway. 31 Pathogenic variants in both BLOC1 or AP-3 complex (AP3B1 and AP3D1) are associated with Hermansky-Pudlak syndrome, in which neurodevelopmental and non-neuronal phenotypes (albinism, PID) result from lysosomal defects. Sorting motif and kinase-active sites in PI4K2A were found to be required for its localization to LAMP-1-positive endosomes 32 and for the biogenesis of lysosomes by directing the sorting of membrane proteins to lysosomes. 33 One patient with a PI4KA variant showed widespread small-pigmented skin macules, 6 however there is no report of patients with a PI4K2A variant and PID or pigmentation abnormalities.
As outlined above, there is a remarkable overlap in the phenotypes of patients with pathogenic variants in PI4KA and PI4K2A, namely brain malformations, epilepsy and movement disorders. Further clinical studies are required to lay the groundwork for natural history of the disease, genotype-phenotype correlations, and specific diagnostics to allow for timely diagnosis, informed management and anticipatory guidance in an underreported and ultra-rare disorder. 34