COLGALT1 is a Prognostic Biomarker in Clear Renal Cell Carcinoma Correlated with Immune Inltrates: A Study Based on TCGA Data

Background: COLGALT1, as one gene enriched in metabolic pathways, which may be related to the tumorigenesis and progression. We aim to explore the potential value of COLGALT1 in clear cell renal cell carcinoma (ccRCC) through the study. Methods: We searched The Cancer Genome Atlas (TCGA) database to collecte ccRCC patients’ information including clinicopathologic parameters and COLGALT1 gene expression. We also validiated the COLGALT1 mRNA expression by qRT-PCR. Then, We evaluated the relationship between COLGALT1 and overall survival (OS) by the Cox regression analyses. Gene Set Enrichment Analysis (GSEA) was utilized to compare between tissues with different COLGALT1 expression levels. Microsatellite Instability (MSI), Tumor Mutational Burden (TMB) and Neoantigen were evaluated through different tools. By TIMER, correlations between COLGALT1 and immune cell inltrations were analyzed. The ESTIMATE algorithm was used to calculate the estimate, stromal and immune scores for ccRCC. Finally, CIBERSORT was carried out to explore the connection between the COLGALT1 and the tumor immune microenvironment. Results: Signicant gene expression of COLGALT1 was identied between normal and ccRCC tissues. Multivariate analysis indicated that high expression of COLGALT1 was linked to poor OS (P = 0e+00). GSEA results demonstrated that high COLGALT1 expression was associated with metabolic pathways. COLGALT1 was identied to be one independent prognostic factor through the univariate and multivariate Cox regression analyses. One nomogram was integrated including both the clinicopathologic variables and COLGALT1 expression to provide a quantitative approach to clinicians for predicting prognostic risk. Futher more, we nd out some genes which are signicantly correlated with COLGALT1. Besides, MSI and TMB showed strong correlations with COLGALT1 in ccRCC. Also, the correlations between COLGALT1 with immune inltrations were found in ccRCC. Finally, immune microenvironment including immune checkpoint molecules, immune cells and mismatch repair protein were proved to be linked to COLGALT1 in ccRCC. Conclusions: Our results revealed that COLGALT1 could act as a favorable prognostic factor for ccRCC. Besides, this study also provided one method to determine the immune inltration of patients and some signal pathways which are potential regulated by COLGALT1 in ccRCC.


Introduction
Renal cell carcinoma (RCC) is one of the ten most common cancers in both males and female around the world [1]. Clear cell renal cell carcinoma (ccRCC), as the predominant histology of renal cell carcinoma, accounts for 75% of all cases and is the main lethal type, the overall 5-year survival rate of RCC is 74% [2]. Nowadays, the overall prognosis of ccRCC is still poor especially for those who were diagnosed with advanced stages although the 5-year survival rates have been improved [3].
As we all know, Tumor stage (TNM) is one of the important prognostic factors for patients with malignant tumor [4]. According to these reports, the 5-year recurrence free-survival of patients with stage I over 92%, while for those patients with stage II and III is only 40% [5,6]. The UISS, one authoritative organization has been developed which classify patients into low-risk, medium-risk, and high-risk prognostic groups based on the clinicopathologic parameters [7]. Although, the cancer prognosis has improved signi cantly over the years through the development of some targeted drugs, none of these therapies is curative [8].
Thus, the early diagnosis and treatment of ccRCC is vital and imperative. Nowadays, some tools have been explorered to analyse the association between clinical outcomes and different gene signatures. We plan to discover novel biomarkers for early screening of ccRCC through the cancer database and these methods.
The tumor microenvironment (TME) is a mixture of chemokines, uids, extracellular matrix molecules, stromal cells, numerous cytokines and immune cells. The molecules and cells of the TME are in a dynamic process, re ecting the evolutionary characteristics of cancer, tumor growth and metastasis [9].
Tumour-in ltrating immune cells (TIICs) including immune cells that migrate from the brink to tumour tissues and exert a negative or positive effect on the growth of cells [10]. Recently, the in ltration of immune cells into different tumors has made signi cant progress, but the function of these immune cells in immune defense and tumor initiation or tolerance still needs further exploration.
In this study, we discovered the gene COLGALT1 enriched in metabolic pathways by means of gene set enrichment analysis (GSEA). As is known to us, the occurrence and development of tumors are closely related to metabolic pathways. Hence, we want to explore the prognostic value of COLGALT1 in ccRCC based on TCGA database. Also, we evaluate the connection between the COLGALT1 and the TIICs.
Through the further analysis, we demonstrated that the COLGALT1 could play one signi cant role as a favorable prognostic and therapeutic factor for ccRCC.

Patient samples
We obtain the data including the raw RNA-Sequencing reads and corresponding clinical informationn of kidney renal clear cell carcinoma patients for a total of 539 ccRCC and 72 normal samples in the TCGA database. In this current study, overall survival (OS) was the primary outcome. We use the Bioconductor packages in an R statistical environment to process and normalize the RNA expression levels of the samples. Finally, we conduct further analysis using these samples gene expression data with the Clear cell renal cell carcinoma and the clinical information.
Identifying genes of differential expression All data from the TCGA were analyzed using the R software (https://www.r-project.org/). Through the R/Bioconductor package of edgeR, the different expression of mRNA in Clear cell renal cell carcinoma was calculated [11]. The cut-off standard DEGs was de ned as |log2fold-change (log2FC)| > 2 and adjusted P-value (P) < 0.05. And we determine the COLGALT1 met the criteria.Then we compare gene expression data between adjacent non-tumor tissue and tumor tissue. Also, compare the COLGALT1 expression in different cancer clinical stages.

Cell culture
Clear cell renal cell carcinoma cell line Caki-1 and Human Kidney-2 cell (Hk-2) were purchased from the Shanghai Enzyme-linked Biotechnology Co.,Ltd. (Shanghai, China). Clear cell renal cell carcinoma cell line were maintained in DMEM (Gibco, USA) ,which contained 10% fetal bovine serum (FBS, HyClone, USA) and 1% penicillin/streptomycin at 37 °C with 5% CO2. Hk-2 were cultured in DF-12 medium (Gibco, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin. Functional and pathway enrichment Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis [12] is an encyclopedia of genes and genomes. We performed it for the selected gene to nd potential pathways using the database for annotation, visualization and Integrated discovery (http://www.kegg.jp/ or http://www.genome.jp/kegg/). Nominal P-value (P) less than 0.05 and FDR less than 25% was considered statistically signi cant.

Relationships between COLGALT1 and adjacent genes
We plan to evaluate the relationships between the COLGALT1 and adjacent genes. The Ensemble Genome Browser was carried out to identify these genes. We downloaded its expression level from TCGA and expressed it as RPKM value. We then perform differential expression analysis of the adjacent genes. Pearson's correlation coe cient was used to analyze the potential relationships between the COLGALT1 and adjacent genes. All the above statistical analyses were performed using SPSS software [13].
Evaluation of independent prognostic index (Riskscore) Univariate Cox and multivariate Cox regression analysis were employed in the TCGA dataset on the prognostic gene signature and clinicopathological parameters to identify the impact of COLGALT1 as one independent factor correlated with OS. The package of "survivalROC" in R was carried out to complete ROC and AUC for the evaluation of the prognostic ability of RS. Moreover, by using the R "rms" package, we conduct one nomogram-based model to visualize the relationship between survival rates and individual predictors .

The evaluation of Microsatellite Instability(MSI), Tumor Mutational Burden(TMB) and Neoantigen
The MISA (http://pgrc.ipk-gatersleben.de/misa/misa.html) was applied to identify all the autosomal microsatellite tracts containing ve or more repeating subunits 1-5 bp in length. Detailed calculations were performed as mentioned before [14]. The total number of somatic nonsynonymous mutations (NSM) which was determined by comparing sequence data from tumor tissues and matched samples was used to de ne the Tumor mutation burden through previously described method [15]. We used seq2 HLA, version 2.2, 17 with default settings to generate 4-digit typing for various tumors in TCGA. Then, pvac-seq, version 4.0.8,18 was ran to generate Neoantigen on each sample [16,17].

Correlation analysis between COLGALT1 with tumor microenvironment
The purity-adjusted Spearman correlations between COLGALT1 and the six different kinds of immune cell in ltrations were analyzed by the TIMER. The ESTIMATE algorithm was utilized for the normalized expression matrix to caculate the estimate,stromal and immune scores for ccRCC [18].

CIBERSORT and assessment of TIICs
CIBERSORT is a deconvolution algorithm that was reported to predict the fractions of multiple cell types based on gene expression [19,20]. Acoording to the standardized gene expression data, the cellular composition of complex tissues can be estimated which indicated the abundances of speci c cell types [21,22]. For this study, the gene composition of each cell was determined through caculating the expression level of each gene in immune checkpoint molecules, immune cells and mismatch repair protein respectively.

Statistical analysis
All statistical data analysis and gures were carried out on SPSS 24.0 (IBM, Chicago, USA), R 3.3.1 (https://www.r-project.org/) and GraphPad Prism 6.0 (San Diego, CA, USA). Pearson's correlation method was used to analyse the correlation between two different genes. Wilcoxon signed rank test and logistic regression were applied to estimate the association between clinicopathologic parameters and COLGALT1. The survival predictive performance of COLGALT1 and risk score (RS) were carried out by using Kaplan-Meier curve and log-rank test. The relationship between variables and OS were evaluated by Univariate and multivariate Cox regression analyses. The rms package of R software was used to create the nomogram. The package of "survivalROC" in R was carried out to complete ROC and AUC for the evaluation of the prognostic ability of RS. Protein-protein interaction (PPI) network was established by STRING and visualized by Cytoscape software. All statistical results with P < 0.05 were considered statistically signi cant .

Result
The expression of COLGALT1 in ccRCC We found that COLGALT1 is highly expressed in carcinoma tissues, including Clear cell renal cell carcinoma through comparing the expression of normal tissue with carcinoma tissue from the TCGA datesets (P < 0.01, Fig. 1A). We obtain the GOLGAT1 expression level of the normal tissues (n = 72) and the malignant tissues (ccRCC, n = 539) from the TCGA datesets (Fig. 1B). It shows that the expression of COLGALT1 of the ccRCC tissue is higher compared to normal tissue (P < 0.001). We compare the COLGALT1 expression in Clear cell renal cell carcinoma tissue with the para-cancerous tissue in 72 pairs of samples(P = 1.097e-21, Fig. 1C). The results from the cell lines also indicated that COLGALT1 mRNA was increased in ccRCC through qRT-PCR (Fig. 1D).
By using the GOLGAT1 expression as the cut-off point, we classi ed the patients with ccRCC into high and low risk group. The survival curve evaluated for ccRCC patients in the low-risk group and those in the high-risk group were obviously different indicating that patients with high-COLGALT1 expression had a shorter overall survival time than patients with low-COLGALT1 expression (P < 0.001, Fig. 2A). The AUC of a time-dependent ROC curve was used to evaluate the prognostic capacity of the COLGALT1 signature and the AUC of gene biomarker prognostic model was 0.707, see Fig. 2B. The result indicated that the ability of the survival prediction for ccRCC patients was acceptable. The immunohistochemical pictures from the HPA database (http://www.proteinatlas.org/) indicates that COLGALT1 is highly expressed in tumor tissues compared to normal tissues ( Fig. 2C-D).

Association between COLGALT1 expression and different clinicopathologic parameters
We compare the COLGALT1 expression in ccRCC patients with different clinical stages. The patient' data with ccRCC obtained from TCGA are divided into different groups according to the clinicopathologic stage. The corresponding COLGALT1 expression is shown in Fig. 3A-D. We discover advanced tumors have higher expression of COLGALT1 than early tumors in the (P < 0.01). However, there is no signi cant difference of COLGALT1 expression between African and Caucasian, male and female .
GSEA identi es a COLGALT1-related signaling pathway Gene Set Enrichment Analysis (GSEA) was carried out to nd out potential signaling pathways which are differentially activated in ccRCC patiens with low and high COLGALT1 expression. We selected the most signi cantly enriched signaling pathways based on the normalized enrichment score (NES) and FDR qval (FDR < 0.01) ( Fig. 4 and Table 2). The result shows that butanoate metabolism, fatty acid metabolism, histidine metabolism, ppar signaling pathway, propanoate metabolism, pyruvate metabolism and tryptophan metabolism are differentially enriched in high COLGALT1 expression phenotype.
Multivariate analysis of OS and establishment of ccRCC prognostic prediction nomogram Univariate Cox and multivariate Cox regression analysis were employed to identify the impact of COLGALT1 as one independent factor correlated with OS ( Table 1). The univariate Cox analysis revealed that the OS of ccRCC patients was signi cantly associated with the COLGALT1 expression, agepathological stage, grade, T stage and M stage (Fig. 5A). Multivariate Cox regression analysis showed ccRCC patients with high COLGALT1 expression were correlated with a poor OS (HR = 1.024; P < 0.01). Moreover, some other clinicopathologic parameters correlated with poor overall survival consist of low grade, advanced stage and old age (Fig. 5B). Therefore, the COLGALT1 expression might be an independent prognostic factor of OS compared with these variables.
Predictive ability of the COLGALT1 expression and clinicopathologic variables was demonstrated by using the ROC curve analysis (Fig. 5C). The AUC of the COLGALT1 expression (AUC = 0.703) was obviously higher than that of age (AUC = 0.660), gender (AUC = 0.497), race (AUC = 0.528), M stage (AUC = 0.680) and lymph nodes status (AUC = 0.459). However, the AUC for COLGALT1 is lower than tumor grade (AUC = 0.709), pathological stage (AUC = 0.779) and T stage (AUC = 0.779). Thus, using the COLGALT1 expression level alone to predict the survival of ccRCC patients is insu cient. We then integrate these clinicopathologic variables and the COLGALT1 expression constructing one nomogram integrating to provide clinicians with an effective approach for predicting prognostic risk (Fig. 5D). We can evaluate 1-, 3-, and 5-year survival probabilities of ccRCC patients by using the nomogram.

Validation of the predictive function of COLGALT1
We nd ten genes (AUH, BMP1, CD276, FDX1, IMPDH1, MICU2, MOCS2, PPP1R18, RBM47 and BRHBDF2) were signi cant correlated with COLGALT1 expression by the Pearson's correlation analysis based on TCGA dataset (P < 0.05) (Fig. 6). Among them, ve genes (AUH, FDX1, MICU2, MOCS2 and RBM47) were negatively correlated with COLGALT1 expression and ve genes(BMP1, CD276, IMPDH1, PPP1R18 and RHBDF2) were possitively correlated with COLGALT1 expression. A PPI network of DEGs was constructed, we can nd the relationship between the COLGALT1 with ten mostly relevant other genes (Fig. 7A). Also, based on the TCGA database, we explore whether COLGALT1 is related to different tumors from three aspects including Microsatellite Instability, Tumor Mutational Burden and Neoantigen. And the COLGALT1 is related to Microsatellite Instability, Tumor Mutational Burden in ccRCC (P < 0.001 , Fig. 7B-D).

Correlations between COLGALT1 with immune in ltrations and methylation in ccRCC
The correlations between the COLGALT1 and the six different immune cell in ltration levels were shown through online analysis with TIMER. And the COLGALT1 was indicated to be positively correlated with the immune in ltrations includind B cell in ltration, CD8 + T cell in ltration, CD4 + T cell in ltration, macrophage in ltration, neutrophil in ltration, and dendritic cell in ltration, respectively (P < 0.01, Fig. 8A). As shown in Fig. 8B, the transcripts per million (TPM) is positively related to the immune score indicating the positive effects of the Somatic copy number (of the COLGALT1) on the immune response in ccRCC.

The connection between the COLGALT1 and the immune microenvironment in ccRCC
We also explored immune microenvironment in tumor tissue based on the TCGA database. As is shown in the Fig. 9A, the COLGALT1 is signi cantly connected with the immune checkpoint molecules like BTLA, CD28, CD40, NRP1 ect in ccRCC. Aslo, we explore the interaction between the immune cells and the COLGALT1 in ccRCC. It showed that the COLGALT1 is linked to relevant immune cells including Actived memory B cell, Actived CD4 T cell, Actived CD8 T cell ect (Fig. 9B). Next, we investigate the relatonship between the COLGALT1 and mismatch repair protein including MLH1, MSH2, MSH6, PMS2 in ccRCC showing potential connection among them (P < 0.001, Fig. 9C).

Discussion
Clear cell renal cell carcinoma (ccRCC) is the most common histological type of RCC accounting for 90% of kidney neoplasms [23]. We plan to explore new reliable method for survival prediction and treatment of ccRCC. Nowadays, many studies have reported that the prognosis of ccRCC has connections with genetic factors, and some genes may provide method for predicting prognosis and selecting treatments [24][25][26][27][28]. Today, with the development of microarray technology, some potential prognostic and therapeutic targets have been found. We nd CLOGAT1 may has connection with some metabolism pathways and immune in ltrations which have impacts on tumor. Hence, our present study focused on the prognostic role of COLGALT1 in ccRCC.
In this study, we identi ed the COLGALT1 expression between the ccRCC tissues and the adjacent normal tissues from the TCGA database. Based on qRT-PCR, the COLGALT1 mRNA expression was high in ccRCC cell line Caki-1 compared with human kidney cell. These results revealed that COLGALT1 shows obviously higher expression in ccRCC tissues than that in adjacent normal tissues. Furthermore, we explored the association among COLGALT1 expression and patient survival based on the database showing that high expression of COLGALT1 indicated a bad prognosis of ccRCC. Additionally, high COLGALT1 expression was signi cantly associated with better pathologic stage, histological grade, T stage, M stage, and satisfactory survival time. The COLGALT1 expression level was proved to be an independent predictive factor of overall survival for ccRCC patients. In order to explore how COLGALT1 was involved in ccRCC pathogenesis, we carried out GSEA between tissues with different COLGALT1 expression levels and found that some pathways including butanoate metabolism, fatty acid metabolism, histidine metabolism, ppar signaling pathway, propanoate metabolism, pyruvate metabolism and tryptophan metabolism are differentially enriched in COLGALT1 high expression phenotype. We further investigated the relationship of COLGALT1 and related genes based on TCGA dataset and discovered that the ve most possitively relevant genes are BMP1, CD276, IMPDH1, PPP1R18 and RHBDF2. In contrast, the ve most negatively relevant genes are AUH, FDX1, MICU2, MOCS2 and RBM47. Also, we nd the relationship between the COLGALT1 with ten mostly relevant other genes through the PPI network. And the COLGALT1 is related to Microsatellite Instability, Tumor Mutational Burden in ccRCC based on the TCGA databse. And, we explore the potential connection between the COLGALT1 with the tumor microenvironment and immune in ltrations in various tumors. Finally ,we nd the COLGALT1 is signi cantly related to these aspects including immune cell in ltration, DNA methyltransferase, immune checkpoint molecules immune cells and mismatch repair protein among ccRCC.
We search some relevant literature for the progress of the COLGALT1. The galactosyltransferases COLGALT1 and COLGALT2 in the endoplasmic reticulum initiate the glycogen glycosylation reaction.
Mutations in the COLGALT1 gene can cause abnormalities in cerebral small blood vessels and malformations of the nostrils, which are common in type IV collagen de ciency. These advances are focused on muscle and small vessel diseases [29][30][31]. As we all know, alterations in mitochondrial metabolism have been described as one of the major factor of both ageing cells and cancer [32]. This may be one of the reasons that the COLGALT1 are related to ccRCC. However, We still need further study and analysis to explore the possible relationship.
In our study, we discovered that high COLGALT1 expression had association with butanoate metabolism, fatty acid metabolism, histidine metabolism, ppar signaling pathway, propanoate metabolism, pyruvate metabolism and tryptophan metabolism by GSEA. These pathways are crucial biological processes in tumorigenesis and development of tumor. We can nd that these pathways are mainly focused on metabolic pathways. Over these years, many studies have begun to explore the molecular and cellular mechanisms that constitute the interaction between nutrition, amino acid, fat metabolism and effects on cancer [33,34]. Given the research background of metabolism, there are many articles focused on nding potential ways to treat or even cure cancer [35][36][37]. COLGALT1 as one gene enriched in these metabolic pathways, which may play a certain role in tumor metabolism and the body's immune process. However, further experiments are needed to verify it.
Ten most signi cantly relevant geneswere discovered in this study. Five genes (BMP1, CD276, IMPDH1, PPP1R18 and RHBDF2) were possitively correlated with GOLGALT1 expression. Recently, outstanding research has provided new clues to the function of CD276 (B7H3) in cancer, and determined that CD276 is the key to tumor cell proliferation, migration, invasion, epithelial-mesenchymal transition, cancer stemness, drug resistance and Warburg effect Promoter. [38]. Also, one article reports that RHBDF2 may be linked to the esophageal cancer [39]. On contrary, Five genes (AUH, FDX1, MICU2, MOCS2 and RBM47) were negatively correlated with GOLGALT1 expression. Among them, three genes (AUH, FDX1, MICU2) are related to mitochondrial energy metabolism [40][41][42]. As we mentioned earlier, mitochondria are closely related to tumor progression. This again proves that COLGALT1 may act on tumors through metabolic pathways. Also, we nd the relationship between the COLGALT1 with ten mostly relevant other genes through the PPI network. Further veri cation need to be carried out to con rm the relationship between them.
Immune response were shown to be linked with various tumors importantly. In COAD, a mouse model showed depletion of CD25+, CD4 + regulatory T cells able to enhance the anti-tumor immunity induced by interleukin2 [43]. CD4 + T cells were found to play signi cant role in the progression and metastasis of lung cancer [44]. Tumor-in ltrating naive CD4 + T cells were proved to be conneted with poor survival in bladder cancer [45]. Here, We explorer the connection between the GOLGALT1 and six different immune cells including B cell in ltration, CD8 + T cell in ltration, CD4 + T cell in ltration, macrophage in ltration, neutrophil in ltration, and dendritic cell in ltration, respectively in ccRCC. The COLGALT1 was indicated to be positively correlated with these immune cells (P < 0.01). The methyltransferase including DNMT1, DNMT2, DNMT3a and DNMT3b was positively correlated with the COLGALT1 in different tumors (P < 0.01).
Immune checkpoints provide a general mechanism for various cancers to avoid immune surveillance and play a signi cant role in the immune system. In lung cancer, anti-CTLA-4 and anti-PD-1/PD-L1 blocking antibodies have shown to be successful for treatment. In addition, in lung cancer, there are some recognition markers for early response, such as TCR library, CD4 + / CD8 + T cell pro le, cytokine markers and the expression of immune checkpoint molecules in tumor cells, macrophages or T cells [46,47].
Here,we nd COLGALT1 is related to MSI, TMB in ccRCC. Also, we explored immune microenvironment in tumor tissue through three aspects including immune checkpoint molecules, immune cells and mismatch repair protein respectively by using the CIBERSORT algorithm. The COLGALT1 is signi cantly connected with the immune checkpoint molecules like BTLA, CD28, CD40, NRP1 ect in ccRCC. Aslo, the COLGALT1 is linked to relevant immune cells including Actived memory B cell, Actived CD4 T cell, Actived CD8 T cell ect. The relationship between the mismatch repair protein including MLH1, MSH2, MSH6, PMS2 with the COLGALT1 was investigated showing potential connection among them in ccRCC. These results may offer help in the development of ccRCC treatments.
Our study has some limitations. First, our results come from the TCGA database and generated by bioinformatic analysis. Considering various factors (region, age, gender, race, etc.) and the heterogeneity in the analysis process, the sample size of renal clear cell carcinoma cannot guarantee su cient. Therefore, the results of our study need to be veri ed with enough clinical samples. Also, we need further investigations to validate our results based on ccRCC samples and clinical data. Second, the relationship between COLGALT1 expression with the ccRCC and these signaling pathways is the rst to be reported, and the regulatory mechanism needs to be further investigated. Therefore, further experiments need to be carried out to explore whether progression in ccRCC should be affected by COLGALT1 through metabolic pathways or other possible pathways.

Conclusions
In conclusion, the COLGALT1 could be a reliable prognostic factor for ccRCC according our study.
Moreover, butanoate metabolism, fatty acid metabolism, histidine metabolism, ppar signaling pathway, propanoate metabolism, pyruvate metabolism and tryptophan metabolism might be the primary pathways regulated by COLGALT1. Besides, MSI and TMB showed strong correlations with COLGALT1 in ccRCC. Moreover, the COLGALT1 was indicated to be positively correlated with the immune in ltrations and the methyltransferase. Furthermore, we verify the connetion between the COLGALT1 and the the immune microenvironment including immune checkpoint molecules, immune cells and mismatch repair protein in ccRCC. We still need more evidence and experiments to nd the potential molecular mechanisms of COLGALT in ccRCC.

Availability of data and material
All the data used to support the ndings of this study are included within the article. Please contact author for data requests.

Competing interests
None declared.

Consent for publication
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