Annotation of Gene Alteration in Amniotic Fluid Cell Free RNA Transcriptome via Weighted Gene Co-expression Network Analysis

Annotation of Gene Alteration in Amniotic Fluid Cell Free RNA Transcriptome via Weighted Gene Co-expression Network Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Annotation of Gene Alteration in Amniotic Fluid Cell Free RNA Transcriptome via Weighted Gene Co-expression Network Analysis Shufa Yang, Chenghong Yin, Yan Liu, Taifeng Zhuang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-67755/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Human amniotic fluid (AF) cells are commonly used in prenatal diagnosis. The AF cell-free mRNA (cfRNA) derived from necrotic or apoptotic cells of fetus may provide feasible marker of organ development and fetal malformation. Analysis of gene co-expression network will help to annotate AF cfRNA gene variations in fetal diseases. Methods Datasets of amniotic fluid free RNA were downloaded from the Gene Expression Omnibus database. Co-expressed modules based on normal fetus AF cfRNA transcriptome were established via Weighted Gene Co-expression Network Analysis. The relationship between modules and tissues were set up via tissue-specific gene expression analysis of genes in modules. Differential expressed genes in Down syndrome (DS), Edwards syndrome (ES), and Turner syndrome (TS) were analyzed via linear models implemented in the limma package. Gene Ontology (GO) analysis of modular specific differential expressed genes for these three syndrome fetus was performed separately. Based on relationship of GO terms, GO graph of all enriched GO terms were constructed. The associated enriched GO terms in GO graph were considered as the same subset. Numbers of GO terms of the same subset with diseases and modules were calculated. Results A total of 22 co-expressed modules were constructed. Most of co-expressed module eigengenes showed no correlation with gestation weeks. Probe sets with higher expression values showed significant clustering tendencies. Dominant tissues and modules were different for different modules and tissues respectively through analysis of tissue-specific genes distribution in modules. The total numbers of enriched GO terms in six major modules were related to diseases severity for ES, DS, and TS. Disease-specific modules for ES, DS, and TS were detected in relation to biological processes through GO analysis. Differential expressed genes in disease-specific module were identified for DS, ES, and TS. Conclusions The gene co-expression network for functional classification provides a potential tool for annotating AF cfRNA variations in fetal diseases. Maternal & Fetal Medicine Sexual & Reproductive Medicine amniotic fluid cell-free fetal RNA co-expression network fetal development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cells in amniotic fluid (AF) are commonly used for prenatal diagnosis to detect genetic diseases. AF supernatants contain cell-free mRNA (cfRNA) derived from apoptosis or necrosis of fetal cells [ 1 – 3 ]. The sources of AF cfRNA are not only limited to cells in amniotic fluid[ 4 ]. The AF cfRNA could be influenced by fetal sex [ 2 ], gestation age [ 2 , 5 – 7 ], genetic disorders [ 1 , 8 – 11 ], developmental disorders [ 12 – 15 ], maternal obesity [ 16 ], and preeclampsia [ 17 ]. Therefore analysis of the transcriptome for the AF cfRNA may provide feasible markers for organ development and fetal malformation [ 18 ]. Tissue-specific gene expression analysis and gene set enrichment analysis are the major annotation methods for the AF cfRNA research. Tissue-specific gene analysis is based on public gene expression data in human tissues, such as BioGPS [ 19 ], Genomics Institute of the Novartis Research Foundation [ 20 ], DFLAT [ 21 ], and Gene Atlas [ 20 ]. According to the results of tissue-specific gene expression analysis [ 1 , 2 , 6 , 7 , 14 , 22 , 23 ], the genes derived multiple tissues (brain, liver, kidney, lung, blood, ovary, pancreas, tongue, adrenal gland, thyroid, etc) are detected in AF cfRNA. Enrichment analyses of different expressed genes in abnormal fetus AF cfRNA include Gene Ontology analysis [ 13 , 23 ], Kyoto Encyclopedia of Genes and Genomes pathway analysis [ 17 ], and QIAGEN’s Ingenuity Pathway Analysis (IPA) [ 23 ]. Gene Ontology analysis was the most frequent method used. Disease-associated pathways and -biological processes are enriched via enrichment analysis. Hematologic/immune category functions and gastrointestinal/metabolic functions are significantly enriched in Turner syndrome (TS) [ 10 ]. The neurological disease is identified as the most highly enriched disease pathway in Down syndrome (DS) and Edwards syndrome (ES) [ 1 ]. Embryonic development comprises the dynamic complex processes, including multiple coordinated development and gene co-expression intra-tissues or inter-tissues. The above relationships are not included in tissue-specific gene analysis and gene function enrichment analysis. We assumed that gene co-expression networks in AF cfRNA may reflect these coordinated development and gene co-expression. To verify this hypothesis, AF cfRNA array data from GEO database were analyzed for weighted co-expression networks analysis. Enrichment analysis of different expressed genes and tissue-specific gene analysis were also performed. Materials And Methods Microarray data The following datasets of amniotic fluid cell free RNA (AF cfRNA) were downloaded from Gene Expression Omnibus (GEO) database ( http://www.ncbi.nlm.nih.gov/geo ) [ 24 ]: GSE16176 [ 8 ], GSE46286 [ 5 ], GSE47394 [ 12 ], GSE48521 [ 16 ], GSE25634 [ 9 ], and GSE58435 [ 10 ] (Affymetrix GPL570 platform, Affymetrix Human Genome U133 Plus 2.0 Array). Sample information including sample record (GSM), series record (GSE), clinical information, pregnancy trimester, sex, gestational age, platform accession number (GPL), and fetus karyotype were summarized in Table S1, Table S2, Table S3, and Table S4. Table S1 containing 26 normal samples with gestation age information were used in co-expression networks analysis. Samples in Table S2 (7 DS versus 7 normal fetus), Table S3 (5 ES versus 6 normal fetus), and Table S4 (5 TS versus 5 normal fetus) were used in differential genes analysis for DS, ES and TS groups. Microarray data were preprocessed (background correction, normalization, and summarization) using the Robust Multi-array Average approach [ 25 ] implemented in the oligo package [ 26 ]. Probe sets were converted to gene symbol based on Affymetrix Human Genome U133 Plus 2.0 Array annotation data ("hgu133plus2.db" package). Co-expression net works analysis An unsigned co-expression net works was performed with Weighted Gene Co-expression Network Analysis (WGCNA) package [ 27 ]. Normal second trimester samples (Table S1) with available gestational ages were included in WGCNA analysis. Before WGCNA analysis, raw expression values of probe sets were preprocessed, and the batch effects were removed with ComBat function in the SVA package [ 28 ]. Soft-thresholding power was set to 15, which was calculated by the pickSoftThreshold algorithm. The automatic block-wise network construction and module detection method was employed. Module assignments were made using dynamic tree cutting method by setting mergeCutHeight to 0.2. Module eigengene was calculated via moduleEigengenes algorithm. To generate co-expressed modular genes, probe sets in clustered modules were annotated to genes via "hgu133plus2.db" package. Tissue-specific gene analysis Gene expression data in human tissues were downloaded from tissue-based map of the human proteome [ 29 ] in the Human Protein Altas database ( http://www.proteinatlas.org ). The Human Protein Atlas aims to map all the human proteins in cells, tissues and organs using the integration of various omics technologies. Tissue-specific genes in a specific tissue were defined as expression value in particular tissue higher than cut-off value times the median expression value of all other tissues. Cut-off value was set as 5, 10, 15, 20, 25, and 30 respectively. Tissue-specific gene names were transformed to probe sets for further analysis via "hgu133plus2.db" package. Tissue-specific genes that were not tested by array were discarded. Distribution of Tissue-specific genes (probes) at a different cut-off values in co-expression modules were analyzed via R script. Gene differential expression analysis Before gene differential expression analysis, probe sets expression values were preprocessed (background correction, normalization, and summarization) and log2-transformed. Differential expressed probe sets were analyzed via linear models implemented in the limma [ 30 ] package in Bioconductor [ 31 ]. Differential expressed probe sets (DEPs) were deemed significant based on p -values < 0.05. Differential expressed genes (DEGs) were obtained after transforming differential expressed probe sets into genes via "hgu133plus2.db" package. Gene ontology analysis According to the results of WGCNA analysis, modular distribution of DEPs from ES, DS, and TS groups were analyzed. After transforming probe sets into gene names, modular specific DEGs (DEGs in the certain module) from ES, DS, and TS groups were obtained. Gene ontology analysis of modular specific DEGs from ES, DS, and TS groups was performed separately via the clusterProfiler package [ 32 ]. P -values were adjusted using the Benjamini-Hochberg method. Adjusted p -values < 0.05 were considered statistically significant. Based on the relationship of GO terms (is_a, part_of, regulates, positively_regulates, etc), GO graph of all enriched GO terms from ES, DS and TS groups were constructed. The associated enriched GO terms in the GO graph were considered as a same subset. Numbers GO terms of the same subset concerning diseases and modules were calculated. Statistical Analysis All other data statistical analyses and plots were created using R language (version 3.5.3), ggplot2 (version 3.3.1) and VennDiagram (version 1.6.20). Results Summary of gene co-expression network analysis A total of 26 second term normal AF cfRNA chip results (15 males and 11 females) were selected for WGCNA. The mean gestation ages of male and female fetus were 15 and 19 weeks respectively (Table S1). Raw data from each microarray were pre-processed for background correction, normalization and batch correction (Fig 1A). All the 54675 probe sets on the chip were analyzed by WGCNA. For block-wise network construction, a computationally inexpensive and relatively crude clustering method was adopted to classify pre-cluster probe sets into three blocks. A full network analysis was performed in each block separately. A total of 22 distinct probe set modules were generated (Fig 1 B-D). These 22 modules were shown in different colors and module names were labeled as colors, i.e., a color represented a group of co-expressed genes. The size of modules ranged from 20 to 1198 probe sets (Table S6). Probe sets without obvious co-expressions relationship were labeled as grey. After probe sets were converted to gene symbol, co-expressed genes in 22 modules were obtained and summarized in Table S5. Most of the co-expressed modules showed no correlation with gestation weeks Module eigengene (ME) is defined as the first principal component of a given module. For all samples employed in WGCNA analysis, MEs of 22 modules were calculated, and compared to modular expression values of the probe sets. As shown in Fig S1, MEs showed the same tendency with expression values of the probe sets and could be representative for expression values of probe sets in the correspondence modules. The correlation between MEs and gestation age was evaluated by Spearman correlation analysis. As shown in Table S6, most modules showed no significant correlation with gestation age except for green and turquoise modules. Modules were clustered using hierarchical clustering. Green and turquoise modules showing significant correlation with gestation age were classified into different clusters. Modules with similar correlation coefficient were divided into different clusters (Fig 2A). MEs of major modules (turquoise, blue, brown, yellow, green and red) were smoothed by locally weighted regression and were shown in Fig 2B. A downward trend was observed for MEs of green and turquoise modules with the increase of gestation age. No obvious trend was found for MEs of other modules concerning gestation age. Expression of genes in green and turquoise modules reduced with augment of gestation age in second term. High expression probe sets showing significant clustering tendencies Probe sets with higher expression values are more inclined to be used as a marker for fetal organ development. To investigate the expression character of clustered probe sets, mean values and coefficient of variation for all probes in the normal 26 samples employed in WGCNA analysis were calculated. A scatter plot (mean expression value VS coefficient of variation) was drawn to show clustered probe sets distribution in all probe sets, and the clustered probe sets were labeled as modular colors (Fig 3A). Compared to probe sets with low expression values, the higher portions of the probe sets with higher expression values were clustered. Relationship between modules and tissues via dominant tissues and modules To establish the relationship between fetal tissues and modules, the distribution of tissue-specific genes in modules were analyzed. Tissue-specific genes were obtained at a different cut-off value conditions. After tissue-specific genes were converted to probe sets, numbers of tissue-specific genes (probe sets) under different cut-off value (5, 10, 15, 20, 25 and 30) were counted (Fig 3B). The number of tissue-specific genes (probe sets) decreased with higher cut-off value. The distribution of tissue-specific probe sets in all 22 modules, was analyzed and summarized in Table S6. As shown in Fig 3C, tissue-specific probe sets were mainly distributed in turquoise, blue, brown, yellow, green, and red modules, which were called major modules. Considering the difference of gene expression in fetus and adult tissues, the cut-off value was set as 5 to get more gene specific genes. The numbers of tissue-specific probe sets derived from different tissues in major modules were shown in Fig 3D, while cut-off value was set as 5 (detailed data see Table S7). Tissue-specific genes from skeleton, liver, and testis accounted for the largest specific genes in turquoise module. The above three tissues were defined as dominant tissues in turquoise module. Similarly, dominant tissues were calculated in blue module (placenta, skeletal muscle, and testis), brown module (testis, cerebral cortex, and skeletal muscle), yellow module (testis, cerebral cortex, and cerebellum), green module (small intestine, liver, and colon) and red module (esophagus, tongue, and tonsil). Tissue-specific probe sets derived from liver mostly distributed in green and turquoise modules. Green and turquoise modules were defined as dominant modules for liver. Dominant modules for cerebral cortex included yellow and blue modules. Dominant tissues and dominant modules were different for different modules and tissues respectively. Blue, brown, and yellow modules included the largest neural-specific genes (cerebral cortex, cerebellum, basal ganglia, etc). Green modules contained more digest system specific genes. Differential expressed genes in DS, ES, and TS Samples used for DEGs analysis were listed in Table S2 (DS), Table S3 (ES), and Table S4 (TS). For differential expressed probe sets (DEPs) analysis in each group, the expression values of the abnormal fetus were compared to that of normal fetus via a linear regression model. There were 1049, 1507, and 1448 DEPs detected in DS, ES, and TS groups respectively. Volcano map of DS, ES, and TS group were shown in Fig 4A-C. Relationship of DEPs between DS, ES, and TS group were shown in Venn diagrams (Fig 4D). There were 41 common DEPs between DS ES group, 38 common DEPs between DS and TS group, 43 DEPs between ES and TS group. No common DEPs were found in DEPs of DS, ES, and TS. Disease-specific modules via GO analysis Numbers of DEPs of DS, ES, and TS in major clustered modules were counted (Fig 5A). Blue module was the most abundant distributed module in TS and DS groups. However, the brown module had the largest number of DEPs of ES. To analyze the function of clustered genes, DEPs were converted to gene symbols. The modular distribution of DEGs in three groups was summarized in Table S8. Modular specific DEGs, which mean DEGs of every group (DS, ES, and TS) in a certain module, were extracted from Table S8. Modular specific DEGs in major modules (turquoise, blue, brown, yellow, green, and red) were performed functional enrichment analysis (Table S10). The numbers of enriched GO terms were shown in Fig 5B. The yellow, blue, and red modules were specific modules for DS, ES, and TS groups respectively. DEGs of DS in the yellow module included SORBS1 and FFAR4. DEGs of ES in blue module included AKNAD1, SOX9, ZNF395, PID1, PRPF38A, FAM220A, ATP1B1, NME7, CD9, EPC1, and MAML2. DEGs of TS in the red module included S100A8 and IVL. The total number of enriched GO terms in ES, DS, and TS group ranked the first, the second, and the third respectively. In green and turquoise modules, similar numeric distribution of enriched GO term was shown for DS and ES, rather than TS. In green and turquoise modules, a similar number of GO term numbers were enriched in DS and ES group. To get an overall view of all enriched GO terms, the GO graph was established based on the relationship of GO terms. Go maps of all enriched GO terms were list in Table S9. Based on the GO graph, interrelated GO Terms were classified into the same subsets. As shown in Table S10, subsets contained a certain number of enriched GO terms from different modules and abnormal fetus (DE, ES, and TS). A total of 184 subsets were established. The numbers of GO terms in a subset ranged from 1 to 332. The largest nine subsets were summarized in Table 1. Functions of these subsets included basic physiological processes, absorption and transport of nutrients, response to external stimuli, multi-organ (kidney, lung, and heart) development, protein synthesis process, protein catabolic process and proteolysis, thermoregulation, signal transduction of TGF, and bone development. Table 1 Subsets of enriched GO terms ID No Fig 6 Function 1 332 A Basic physiological processes 34 100 B Absorption and transport of nutrients 20 79 C Response to external stimuli 48 60 D Multi-organ (Kidney, lung and heart) development 11 48 E Protein synthesis process 17 38 F Protein catabolic process and proteolysis 38 31 G Thermoregulation 47 15 H Signal transduction of TGF 106 12 I Bone development ID: Subsets id shown in Table S9 subset_id column. No: Number of enriched GO terms in subsets, were shown in Table S9 subset_number column. Fig 6: Modular distribution of enriched GO term subsets in abnormal fetus as shown in Fig 6. Function: summarized function according to in corresponding biological GO term subsets. To analyze the relationship between subset function and disease in different modular condition, enriched GO terms numbers in the nine largest subsets (Table 1) in relation to modules and fetus disease were shown in Fig 6. Besides that yellow, blue, and red modules were specific modules for DS, ES, and TS groups, more specific modules were shown in certain subsets. Green, turquoise, and brown modules were specific for ES group in absorption and transport of nutrients subset. The green module was specific to the DS group in multi-organ (kidney, lung, and heart) development subset, protein synthesis process subset, thermoregulation subset and signal transduction of TGF subset. Discussion AF cfRNA were considered a biomarker source for abnormal fetal development. And the gene expression level in AF cfRNA could be influenced by fetal status (such as fetal sex [ 2 ], gestation age [ 2 , 5 – 7 ], genetic disorders [ 1 , 8 – 11 ], and developmental disorders [ 12 – 15 ]). Therefore, AF cfRNA was proposed as a mean to monitoring fetal development in real-time. At present, the tissue expression database (Human protein Atlas [ 29 ], Gene Atlas [ 20 ], and BioGPS [ 19 ]) were mostly based on the adult tissues. The lack of fetal-related tissue expression database hinders the identification of abnormal fetal development. Additionally, AF cfRNAs were multiple organ origins, which increased the interferences for discovering abnormal fetal development biomarkers. Compared to single genes, the advantages of gene sets contains noise and dimension reduction, as well as greater biological interpretability [ 33 ]. A total of 22 co-expressed gene sets (modules) were identified via WGCNA [ 27 ]. As shown in Fig. 3 A, probe sets with high expression values show obvious clustering tendency. Analogous to dimension reduction, expression values of clustered probes can be simplified as module eigengenes. Correlation between gene expression and gestation ages is easier to analyze via module eigengenes. As shown in Fig. 2 B, only genes in green modules showed significant correlation with gestation ages. The above results indicated that correlation between gene expression and gestation ages was not an appropriate way to discover the biomarker of the abnormal fetal development. To explore the tissue origins of genes in AF cfRNA, different gene expression atlas and cut-off values were used to define tissue-specific genes in previous studies [ 1 , 5 , 10 , 34 , 35 ]. However, there was no fully comparison to get confirmed standard. Consequently, a series of cut-off values were used to define tissue-specific genes expression in our research. Based on tissue-specific gene expression, the relationships between modules and tissues were constructed. Genes from different organs showed similar co-expression patterns and were classified as the same module. Inter-organ synergetic development was manifested as a co-ordinated expression of genes intra modules. Due to the complexity of gene expression in the same organs, genes from the same organ showed different co-expression patterns. Accordingly, tissue-specific expressed genes from the same organ were divided into different modules. Thus, synergetic developments inter- and intra- organs were shown as gene co-expression networks intra- and inter- modules via gene tissue-specific analysis and weighted correlation network analysis. DS, ES, and TS were the most frequent chromosomal abnormalities in prenatal diagnosis [ 36 ], which were caused by trisomy of chromosome 21, trisomy of chromosome 18, and sex chromosome aneuploidy. The development of multiple tissues and organs was affected in these three symptoms. ES is the most serious defects and most ES cause spontaneous abortion. The most frequent clinical features of ES consist of neurological findings, growth disturbances, malformations of the skull, face, thorax, abdomen, limbs, genitals, skin, skin annexes, and internal organs [ 37 ]. Cardiac, airway, pulmonary, hearing, growth, hematologic, oncologic, autoimmune, musculoskeletal, and neurodevelopmental disorders are the most frequent clinical symptoms in DS [ 38 ]. Endocrinal, gastrointestinal, hepatic, phenotypic, neurocognitive, and psychosocial disorders [ 39 ] were reported in TS patient. Compared with the normal fetus, the differential expressed genes of DS [ 8 ], ES [ 9 ], and TS [ 10 ] were analyzed. Different from common affected organs or closely related clinical symptoms, different expressed genes from these syndrome share few common differential expressed genes [ 4 ]. To explain this contradictory phenomenon, DEGs from DS, ES, and TS compared to normal fetuses were extracted. Similar to Zwemer’s report, few common misregulated genes were detected in three groups [ 4 ] (Fig. 4 D). Interestingly, the rank of the total number of enriched GO terms in ES, DS, and TS group was correlated with disease severity (Fig. 5 B). Biological functions of enriched GO terms were simplified via classifying interrelated GO terms into the same subsets. A subset contains function-related GO terms performing associated embryonic development process. In most subsets, the number of enriched GO terms was positively related with disease severity (Fig. 6 D). Another tendency, shown in the modular distribution of GO term subsets was modular heterogeneity, which suggested disease-specific modules were detected in functional enrichment analysis of modules related DEPs. Pathogenesis-related different expressed genes were classed into different modules in different diseases. In absorption and transport of nutrients subsets, DEGs in DS and ES were associated with yellow and red modules respectively. Consequently, Preprocessing via co-expression analysis could help constructed the relationship between DEGs and disease phenotype. Large numbers of GO terms were enriched in most modules with a small amount of DEGs, as shown in Fig. 5 B. To explain this phenomenon, DEGs in disease-specific modules were extracted. SORBS1 and FFAR4 were two of DEGs from the yellow module in the DS group. SORBS1 was associated with insulin signaling pathway [ 40 ], glucose homeostasis [ 41 , 42 ], cancer growth and migration [ 43 , 44 ], etc. FFAR4 have similar functions including glucose and fatty acid metabolism [ 45 , 46 ], glucose-dependent insulinotropic polypeptide secretion [ 47 ], cell cancelation[ 48 , 49 ], etc. This is accounted for enriched results of yellow modules in DS group. In subsets of a basic physiological process, absorption and transport of nutrients, response to external stimuli, protein synthesis process and thermoregulation were enriched. Taken together, genes without co-expression relationships were filtered via WGCNA. The remaining co-expressed genes were easier to be enriched via functional enrichment analysis. Conclusion Co-expression modules based on samples from the whole second gestation (from 15 to 22 weeks) provided gene classification in this study. Via tissue-specific genes analysis, the relationships between modules and tissues were established. Disease-specific modules can help to annotate AF cfRNA variation in fetal diseases. Pre-grouping via co-expression before functional enrichment analysis could help to build a relationship between DEGs and disease phenotype. With the accumulation of datasets of AF cfRNAs, gestation week specific co-expression modules could be constructed, in which more genes in AF cfRNA would be included. With the finer co-expression modules for different gestation age, comprehensive knowledge for fetal coordinated development and gene co-expression will be elucidated. Abbreviations AF: Amniotic fluid; cfRNA: cell-free mRNA; DS: Down syndrome; ES: Edwards syndrome; TS: Turner syndrome; ME: Module eigengene; WGCNA: Weighted Gene Co-expression Network Analysis; DEPs: Differential expressed probe sets; DEGs: Differential expressed genes. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors have no conflicts of interest to report. Funding This research was founded by the National Key Research and Development Program of China (No: 2016YFC1000101) Authors’ contributions SYang, CYin, TZhang designed the project, SYang and YLiu performed statistical analysis, SYang wrote the manuscript. All authors reviewed and approved the final manuscript. 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Int J Oncol. 2020;56(5):1140–51. Sultan G, Zubair S, Tayubi IA, Dahms HU, Madar IH. Towards the early detection of ductal carcinoma (a common type of breast cancer) using biomarkers linked to the PPAR(gamma) signaling pathway. Bioinformation. 2019;15(11):799–805. Codoner-Alejos A, Carrasco-Luna J, Codoner-Franch P. The rs11187533 C > T Variant of the FFAR4 Gene Is Associated with Lower Levels of Fasting Glucose and Decreases in Markers of Liver Injury in Children with Obesity. Ann Nutr Metab. 2020;76(2):122–8. Kimura I, Ichimura A, Ohue-Kitano R, Igarashi M. Free Fatty Acid Receptors in Health and Disease. Physiol Rev. 2020;100(1):171–210. Reimann F, Diakogiannaki E, Moss CE, Gribble FM. Cellular mechanisms governing glucose-dependent insulinotropic polypeptide secretion. Peptides. 2020;125:170206. Zhou LZ, Cui YX, Wang WZ, Wu J, Sun Z, Ma SY. FFAR4 promotes cell proliferation and migration and servers as a potential biomarker for clinicopathological characteristics and prognosis in laryngocarcinoma. Eur Rev Med Pharmacol Sci. 2019;23(17):7438–44. Chu X, Zhou Q, Xu Y, Jiang J, Li Q, Zhou Q, Wu Q, Jin M, Wang H, Gu Y, et al. Aberrant fatty acid profile and FFAR4 signaling confer endocrine resistance in breast cancer. J Exp Clin Cancer Res. 2019;38(1):100. Supplementary Files TableS1.csv Table S1. Normal samples used in Co-expression networks analysis. Sample information including sample record (GSM), series record (GSE), clinical information, trimester, sex, gestational age, Platform accession number (GPL), and fetus karyotype. TableS2.csv Table S2. Samples used in Gene differential expression analysis for DS. Columns are the same as Table S1. TableS3.csv Table S3. Samples used in Gene differential expression analysis for ES. Columns are the same as Table S1. TableS4.csv Table S4. Samples used in Gene differential expression analysis for TS. Columns are the same as Table S1. TableS5.csv Table S5. Co-expressed genes (probe sets) in all modules. Columns include gene symbol (SYMBOL), ENTREZID, module name (module), gene name and probe sets ID in chips (PROBEID). TableS6.csv Table S6. General character of all modules. Numbers of co-expressed probe sets (module_probe_num), tissue-specific probe sets at different cut-off value (cutoff_5, cutoff_10, cutoff_15, cutoff_20, cutoff_25 and cutoff_30) were included. Correlation coefficient (me_cor_week) and P value (me_cor_week_pvalue) between module eigengenes (MEs) and gestation weeks were listed. TableS7.csv Table S7. Numbers of tissue-specific probe sets derived from different tissues in major modules as cut-off value =5. Tissues (tissue) source and systems (system) source of tissue-specific probe sets were listed in the table. Numbers of tissue-specific probe sets derived from different tissues in major modules (turquoise, blue, brown, yellow, green and red) were calculated in the middle. Total number (total) for all major modules were shown in the end. TableS8.csv Table S8. Co-expressed differential expressed genes in DS, ES, and TS. Gene symbol (SYMBOL), ENTREZID, gene name (GENENAME), and probe sets ID in chips (PROBEID) of all co-expressed differential expressed genes were listed. Module column contain which module the gene were clustered into. DEG_DS, DEG_ES, and DEG_TS contain whether or not this gene is differential expressed in DS, ES, and TS respectively. TableS9.csv Table S9. Go maps of all enriched GO terms. Columns include the first GO term (GO term 1), the second GO term (GO term2), and relation from the first Gene Ontology term to the second GO term (relation). TableS10.csv Table S10. GO analysis of modular specific DEGs. GO term ID (GO id) and GO term name (Term) of all enriched GO terms were listed. Information of enriched GO terms included whether or not this GO term were enriched from DEGs in DS (DEG_DS), ES (DEG_ES) or TS (DEG_TS) and in blue (DEG_blue), brown (DEG_browm), green (DEG_green), red (DEG_red), turquoise (DEG_turquoise), or yellow (DEG_yellow) modules. Subsets’ information of enriched GO terms included subset id (subset_id), the number of GO terms in this subset (subset_number) and functions of this subset (subset_function). Fig1S.Png Fig 1S Relationship between module eigengene and module probes values MEs of all modules were calculated in all samples and shown in lower barplots. Expression values of modular probe sets in samples employed in WGCNA analysis were shown in upper heat maps. MEs could be representative of probe sets expression values in the correspondence modules. Module names were labeled as colors. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-67755","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":2318292,"identity":"83371273-1544-478e-98b6-6908bc9c457c","order_by":0,"name":"Shufa Yang","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital, Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shufa","middleName":"","lastName":"Yang","suffix":""},{"id":2318293,"identity":"a9c3d631-4813-49e5-aa9e-e578692fbea4","order_by":1,"name":"Chenghong Yin","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital, Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenghong","middleName":"","lastName":"Yin","suffix":""},{"id":2318294,"identity":"8a4b95e4-7594-4ca6-b657-26bc3206094b","order_by":2,"name":"Yan Liu","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital,Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Liu","suffix":""},{"id":2318295,"identity":"ada76a73-a141-42c3-9fbd-1689e9217cea","order_by":3,"name":"Taifeng Zhuang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie3PsUoDQRDG8QkDUw1Zyz0I3iusBFId+CoDgasMWKZIcRC4FGKfgOAr+AizLlx16VOkOAlYWXilTYi9cnd2Fvur5w/zAUTRvzRqtD2v2KQn38gyG1AQ3vgtVZOkyOeuqfNByTQwYeb0bpa8la/9Rfq8hsBM7FRnSyEFs3mQ7iUlgn+yE058kR+Ej2Dr/UtngmRUPxzxOEB1EPsOzi66EyIEZUGGalTeiwv9yfdy8KzIVzUSiAxILM3B74qKky2hFc25d0u6DvjZFqtbY03bfp2za7N57E5+fvq38yiKouhXF2YgSLCYKicJAAAAAElFTkSuQmCC","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital, Capital Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Taifeng","middleName":"","lastName":"Zhuang","suffix":""}],"badges":[],"createdAt":"2020-08-28 11:31:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-67755/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-67755/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":2433684,"identity":"8bc246aa-fd8a-4f0c-99e6-1940c4db978f","added_by":"auto","created_at":"2020-09-16 15:08:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56612,"visible":true,"origin":"","legend":"WGCNA in AF cfRNA\n(A) Pre-procession for background correction, normalization and batch correction. A similar mean and standard deviation were obtained via pre-procession. (B-D) Cluster dendrogram and module assignment for modules from WGCNA. Probe sets on chip were classified into three blocks. A full network analysis was performed in each block separately. A total of 22 distinct probe sets modules were generated.\n ","description":"","filename":"OnlineFig1.Png","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/OnlineFig1.Png"},{"id":2433685,"identity":"185d16fd-e54c-4e02-973a-add3807d040a","added_by":"auto","created_at":"2020-09-16 15:08:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24924,"visible":true,"origin":"","legend":"Correlation analysis between modules and gestation weeks\n(A) Modules were classified in different clusters (upper clustering dengrogram). Correlation coefficient between MEs and gestation ages were shown in lower color gradient. Blue color means higher correlation coefficient. Modules with similar correlation coefficient (lower heat map) with gestation age were divided into different clusters. (B) MEs of major modules (turquoise, blue, brown, yellow, green and red) were smoothed by locally weighted regression. There was a downward trend for MEs of green and turquoise modules with the increase of gestation age. No obvious trend was observed for MEs of other modules in relation to gestation age.\n ","description":"","filename":"OnlineFig2.Png","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/OnlineFig2.Png"},{"id":2433686,"identity":"c91fcdda-e3a9-4e94-9f0d-52ff1c248b3c","added_by":"auto","created_at":"2020-09-16 15:08:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":256744,"visible":true,"origin":"","legend":"Expression features of genes in clustered modules\n(A) Distribution of clustered probe sets in all probe sets based on mean expression value and coefficient of variation. Clustered probe sets were labeled as module colors. Probe sets with higher expression values were more likely classified into co-expression modules. (B) Numbers of tissue-specific genes (probes) under different cut-off value. Number of tissue-specific genes (probes) decreased with higher cut-off value. (C) Distribution of tissue-specific probe sets in all modules. Tissue-specific probe sets were mainly distributed in turquoise, blue, brown, yellow, green and red modules. (D) Numbers of tissue-specific probe sets in major modules and tissues. Tissues (shown in X axis) were classified into digest, gland, immune, neural, reproductive, urine and other systems. Dominant tissues and dominant modules were different for different modules and for different tissues respectively.\n ","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/fig3.png"},{"id":2433687,"identity":"c919cff4-702d-4126-b717-78d5338d946b","added_by":"auto","created_at":"2020-09-16 15:08:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":356771,"visible":true,"origin":"","legend":"Differential expressed probe sets analysis in DS, ES and TS\n(A-C) Volcano map of differential expressed probes (DEPs) in DS (A), ES (B) and TS (C). (D) Relationship of DEPs in three groups. We found 1049, 1507 and 1448 different expressed probes (DEPs) in DS, ES and TS respectively. There are 41 common DEPs in DS intersecting ES, 38 common DEPs in DS intersecting TS and 43 DEPs in ES intersecting TS respectively. No common DEPs were found DS, ES and TS.\n ","description":"","filename":"OnlineFig4.Png","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/OnlineFig4.Png"},{"id":2433688,"identity":"0516b536-69de-43c8-a6af-b0fb54a6343d","added_by":"auto","created_at":"2020-09-16 15:08:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":17643,"visible":true,"origin":"","legend":"Functional enrichment analysis of modules related DEPs\n(A) Numbers of DEPs of DS, ES and TS in major clustered modules were counted. Blue module was the most abundant distributed modules in TS and DS group. However, brown module had the largest number of DEPs of ES. (B) Numbers of enriched GO terms for modular DEGs in DS, ES and TS. ES enriched the largest number of GO terms. Yellow, blue and red modules were specific modules for DS, ES and TS modules for GO analysis.\n \n\n ","description":"","filename":"OnlineFig5.Png","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/OnlineFig5.Png"},{"id":2433689,"identity":"1e0d1980-ea06-4523-885d-9828d6202f8b","added_by":"auto","created_at":"2020-09-16 15:08:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":42732,"visible":true,"origin":"","legend":"Modular distribution of GO term subsets in abnormal fetus\n(A) Basic physiological processes. (B) Absorption and transport of nutrients. (C) Response to external stimuli. (D) Multi-organ (Kidney, lung and heart) development. (E) Protein synthesis process. (F) Protein catabolic process and proteolysis. (G) Thermoregulation. (H) Signal transduction of TGF. (I) Bone development. In addition to yellow, blue and red modules were specific modules for DS, ES and TS groups, more specific modules were shown in certain subsets. Green, turquoise and brown module was specific for ES group in absorption and transport of nutrients subset. Green module was specific to DS group in multi-organ (Kidney, lung and heart) development subset, protein synthesis process subset, thermoregulation subset and signal transduction of TGF subset.\n ","description":"","filename":"OnlineFig6.Png","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/OnlineFig6.Png"},{"id":13593730,"identity":"67af368e-1bda-4960-a7e0-30b9625c6c9f","added_by":"auto","created_at":"2021-09-17 05:17:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1021705,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/bc226ea7-5b40-4a02-b469-9bb31f424121.pdf"},{"id":2433691,"identity":"26470a35-1adc-465b-a6d7-eb2c1acdb1b8","added_by":"auto","created_at":"2020-09-16 15:08:57","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2071,"visible":true,"origin":"","legend":"Table S1. Normal samples used in Co-expression networks analysis. Sample information including sample record (GSM), series record (GSE), clinical information, trimester, sex, gestational age, Platform accession number (GPL), and fetus karyotype.","description":"","filename":"TableS1.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS1.csv"},{"id":2433692,"identity":"31f631ba-95b9-4e05-ade6-dfe0700fb8d2","added_by":"auto","created_at":"2020-09-16 15:08:57","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1089,"visible":true,"origin":"","legend":"Table S2. Samples used in Gene differential expression analysis for DS. Columns are the same as Table S1.","description":"","filename":"TableS2.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS2.csv"},{"id":2433693,"identity":"1a78e817-8be7-4cbd-a19d-56ed92d78696","added_by":"auto","created_at":"2020-09-16 15:08:57","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":902,"visible":true,"origin":"","legend":"Table S3. Samples used in Gene differential expression analysis for ES. Columns are the same as Table S1.","description":"","filename":"TableS3.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS3.csv"},{"id":2433694,"identity":"42bfdaa1-6326-4e15-b486-e5f4214e9f84","added_by":"auto","created_at":"2020-09-16 15:08:57","extension":"csv","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":810,"visible":true,"origin":"","legend":"Table S4. Samples used in Gene differential expression analysis for TS. Columns are the same as Table S1.","description":"","filename":"TableS4.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS4.csv"},{"id":2433695,"identity":"c85b4860-d94f-475a-aa16-ece629671f7d","added_by":"auto","created_at":"2020-09-16 15:08:58","extension":"csv","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":273618,"visible":true,"origin":"","legend":"Table S5. Co-expressed genes (probe sets) in all modules. Columns include gene symbol (SYMBOL), ENTREZID, module name (module), gene name and probe sets ID in chips (PROBEID).","description":"","filename":"TableS5.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS5.csv"},{"id":2433696,"identity":"8472bdb2-1d4f-4924-ae13-5b69118338e1","added_by":"auto","created_at":"2020-09-16 15:08:58","extension":"csv","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2418,"visible":true,"origin":"","legend":"Table S6. General character of all modules. Numbers of co-expressed probe sets (module_probe_num), tissue-specific probe sets at different cut-off value (cutoff_5, cutoff_10, cutoff_15, cutoff_20, cutoff_25 and cutoff_30) were included. Correlation coefficient (me_cor_week) and P value (me_cor_week_pvalue) between module eigengenes (MEs) and gestation weeks were listed.","description":"","filename":"TableS6.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS6.csv"},{"id":2433697,"identity":"d6c862c9-8c3a-4922-a889-2d2b445d7cb7","added_by":"auto","created_at":"2020-09-16 15:08:58","extension":"csv","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2281,"visible":true,"origin":"","legend":"Table S7. Numbers of tissue-specific probe sets derived from different tissues in major modules as cut-off value =5. Tissues (tissue) source and systems (system) source of tissue-specific probe sets were listed in the table. Numbers of tissue-specific probe sets derived from different tissues in major modules (turquoise, blue, brown, yellow, green and red) were calculated in the middle. Total number (total) for all major modules were shown in the end.","description":"","filename":"TableS7.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS7.csv"},{"id":2433698,"identity":"ab0844a7-a6f2-4574-83c4-ec53dbf1ed94","added_by":"auto","created_at":"2020-09-16 15:08:58","extension":"csv","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":11651,"visible":true,"origin":"","legend":"Table S8. Co-expressed differential expressed genes in DS, ES, and TS. Gene symbol (SYMBOL), ENTREZID, gene name (GENENAME), and probe sets ID in chips (PROBEID) of all co-expressed differential expressed genes were listed. Module column contain which module the gene were clustered into. DEG_DS, DEG_ES, and DEG_TS contain whether or not this gene is differential expressed in DS, ES, and TS respectively.","description":"","filename":"TableS8.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS8.csv"},{"id":2433699,"identity":"7cd5f120-228c-4e48-84d0-201071d894da","added_by":"auto","created_at":"2020-09-16 15:08:59","extension":"csv","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":122187,"visible":true,"origin":"","legend":"Table S9. Go maps of all enriched GO terms. Columns include the first GO term (GO term 1), the second GO term (GO term2), and relation from the first Gene Ontology term to the second GO term (relation).","description":"","filename":"TableS9.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS9.csv"},{"id":2433700,"identity":"3f517861-227d-41d8-98ea-ba1a7c917000","added_by":"auto","created_at":"2020-09-16 15:08:59","extension":"csv","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":166003,"visible":true,"origin":"","legend":"Table S10. GO analysis of modular specific DEGs. GO term ID (GO id) and GO term name (Term) of all enriched GO terms were listed. Information of enriched GO terms included whether or not this GO term were enriched from DEGs in DS (DEG_DS), ES (DEG_ES) or TS (DEG_TS) and in blue (DEG_blue), brown (DEG_browm), green (DEG_green), red (DEG_red), turquoise (DEG_turquoise), or yellow (DEG_yellow) modules. Subsets’ information of enriched GO terms included subset id (subset_id), the number of GO terms in this subset (subset_number) and functions of this subset (subset_function).","description":"","filename":"TableS10.csv","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/TableS10.csv"},{"id":2433701,"identity":"d44a328a-cc19-4d69-904d-aa1c30e6e616","added_by":"auto","created_at":"2020-09-16 15:08:59","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":128078,"visible":true,"origin":"","legend":"Fig 1S Relationship between module eigengene and module probes values\nMEs of all modules were calculated in all samples and shown in lower barplots. Expression values of modular probe sets in samples employed in WGCNA analysis were shown in upper heat maps. MEs could be representative of probe sets expression values in the correspondence modules. Module names were labeled as colors.\n","description":"","filename":"Fig1S.Png","url":"https://assets-eu.researchsquare.com/files/rs-67755/v1/Fig1S.Png"}],"financialInterests":"","formattedTitle":"\u003cp\u003eAnnotation of Gene Alteration in Amniotic Fluid Cell Free RNA Transcriptome via Weighted Gene Co-expression Network Analysis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eCells in amniotic fluid (AF) are commonly used for prenatal diagnosis to detect genetic diseases. AF supernatants contain cell-free mRNA (cfRNA) derived from apoptosis or necrosis of fetal cells [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The sources of AF cfRNA are not only limited to cells in amniotic fluid[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The AF cfRNA could be influenced by fetal sex [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], gestation age [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], genetic disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], developmental disorders [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], maternal obesity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and preeclampsia [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore analysis of the transcriptome for the AF cfRNA may provide feasible markers for organ development and fetal malformation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTissue-specific gene expression analysis and gene set enrichment analysis are the major annotation methods for the AF cfRNA research. Tissue-specific gene analysis is based on public gene expression data in human tissues, such as BioGPS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], Genomics Institute of the Novartis Research Foundation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], DFLAT [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and Gene Atlas [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. According to the results of tissue-specific gene expression analysis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the genes derived multiple tissues (brain, liver, kidney, lung, blood, ovary, pancreas, tongue, adrenal gland, thyroid, etc) are detected in AF cfRNA.\u003c/p\u003e \u003cp\u003eEnrichment analyses of different expressed genes in abnormal fetus AF cfRNA include Gene Ontology analysis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], Kyoto Encyclopedia of Genes and Genomes pathway analysis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and QIAGEN\u0026rsquo;s Ingenuity Pathway Analysis (IPA) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Gene Ontology analysis was the most frequent method used. Disease-associated pathways and -biological processes are enriched \u003cem\u003evia\u003c/em\u003e enrichment analysis. Hematologic/immune category functions and gastrointestinal/metabolic functions are significantly enriched in Turner syndrome (TS) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The neurological disease is identified as the most highly enriched disease pathway in Down syndrome (DS) and Edwards syndrome (ES) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmbryonic development comprises the dynamic complex processes, including multiple coordinated development and gene co-expression intra-tissues or inter-tissues. The above relationships are not included in tissue-specific gene analysis and gene function enrichment analysis. We assumed that gene co-expression networks in AF cfRNA may reflect these coordinated development and gene co-expression. To verify this hypothesis, AF cfRNA array data from GEO database were analyzed for weighted co-expression networks analysis. Enrichment analysis of different expressed genes and tissue-specific gene analysis were also performed.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicroarray data\u003c/h2\u003e \u003cp\u003eThe following datasets of amniotic fluid cell free RNA (AF cfRNA) were downloaded from Gene Expression Omnibus (GEO) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]: GSE16176 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], GSE46286 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], GSE47394 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], GSE48521 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], GSE25634 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and GSE58435 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] (Affymetrix GPL570 platform, Affymetrix Human Genome U133 Plus 2.0 Array). Sample information including sample record (GSM), series record (GSE), clinical information, pregnancy trimester, sex, gestational age, platform accession number (GPL), and fetus karyotype were summarized in Table S1, Table S2, Table S3, and Table S4. Table S1 containing 26 normal samples with gestation age information were used in co-expression networks analysis. Samples in Table S2 (7 DS versus 7 normal fetus), Table S3 (5 ES versus 6 normal fetus), and Table S4 (5 TS versus 5 normal fetus) were used in differential genes analysis for DS, ES and TS groups. Microarray data were preprocessed (background correction, normalization, and summarization) using the Robust Multi-array Average approach [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] implemented in the oligo package [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Probe sets were converted to gene symbol based on Affymetrix Human Genome U133 Plus 2.0 Array annotation data (\"hgu133plus2.db\" package).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCo-expression net works analysis\u003c/h2\u003e \u003cp\u003eAn unsigned co-expression net works was performed with Weighted Gene Co-expression Network Analysis (WGCNA) package [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Normal second trimester samples (Table S1) with available gestational ages were included in WGCNA analysis. Before WGCNA analysis, raw expression values of probe sets were preprocessed, and the batch effects were removed with ComBat function in the SVA package [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Soft-thresholding power was set to 15, which was calculated by the pickSoftThreshold algorithm. The automatic block-wise network construction and module detection method was employed. Module assignments were made using dynamic tree cutting method by setting mergeCutHeight to 0.2. Module eigengene was calculated via moduleEigengenes algorithm. To generate co-expressed modular genes, probe sets in clustered modules were annotated to genes via \"hgu133plus2.db\" package.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eTissue-specific gene analysis\u003c/h2\u003e \u003cp\u003eGene expression data in human tissues were downloaded from tissue-based map of the human proteome [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] in the Human Protein Altas database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.proteinatlas.org\u003c/span\u003e\u003c/span\u003e). The Human Protein Atlas aims to map all the human proteins in cells, tissues and organs using the integration of various omics technologies. Tissue-specific genes in a specific tissue were defined as expression value in particular tissue higher than cut-off value times the median expression value of all other tissues. Cut-off value was set as 5, 10, 15, 20, 25, and 30 respectively. Tissue-specific gene names were transformed to probe sets for further analysis via \"hgu133plus2.db\" package. Tissue-specific genes that were not tested by array were discarded. Distribution of Tissue-specific genes (probes) at a different cut-off values in co-expression modules were analyzed via R script.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGene differential expression analysis\u003c/h2\u003e \u003cp\u003eBefore gene differential expression analysis, probe sets expression values were preprocessed (background correction, normalization, and summarization) and log2-transformed. Differential expressed probe sets were analyzed via linear models implemented in the limma [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] package in Bioconductor [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Differential expressed probe sets (DEPs) were deemed significant based on \u003cem\u003ep\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Differential expressed genes (DEGs) were obtained after transforming differential expressed probe sets into genes via \"hgu133plus2.db\" package.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGene ontology analysis\u003c/h2\u003e \u003cp\u003eAccording to the results of WGCNA analysis, modular distribution of DEPs from ES, DS, and TS groups were analyzed. After transforming probe sets into gene names, modular specific DEGs (DEGs in the certain module) from ES, DS, and TS groups were obtained. Gene ontology analysis of modular specific DEGs from ES, DS, and TS groups was performed separately via the clusterProfiler package [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \u003cem\u003eP\u003c/em\u003e-values were adjusted using the Benjamini-Hochberg method. Adjusted \u003cem\u003ep\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Based on the relationship of GO terms (is_a, part_of, regulates, positively_regulates, etc), GO graph of all enriched GO terms from ES, DS and TS groups were constructed. The associated enriched GO terms in the GO graph were considered as a same subset. Numbers GO terms of the same subset concerning diseases and modules were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll other data statistical analyses and plots were created using R language (version 3.5.3), ggplot2 (version 3.3.1) and VennDiagram (version 1.6.20).\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003ch2\u003eSummary of gene co-expression network analysis\u003c/h2\u003e\n\u003cp\u003eA total of 26 second term normal AF cfRNA chip results (15 males and 11 females) were selected for WGCNA. The mean gestation ages of male and female fetus were 15 and 19 weeks respectively (Table S1). Raw data from each microarray were pre-processed for background correction, normalization and batch correction (Fig 1A). All the 54675 probe sets on the chip were analyzed by WGCNA. For block-wise network construction, a computationally inexpensive and relatively crude clustering method was adopted to classify pre-cluster probe sets into three blocks. A full network analysis was performed in each block separately. A total of 22 distinct probe set modules were generated (Fig 1 B-D). These 22 modules were shown in different colors and module names were labeled as colors, i.e., a color represented a group of co-expressed genes. The size of modules ranged from 20 to 1198 probe sets (Table S6). Probe sets without obvious co-expressions relationship were labeled as grey. After probe sets were converted to gene symbol, co-expressed genes in 22 modules were obtained and summarized in Table S5.\u003c/p\u003e\n\u003ch2\u003eMost of the co-expressed modules showed no correlation with gestation weeks\u003c/h2\u003e\n\u003cp\u003eModule eigengene (ME) is defined as the first principal component of a given module. For all samples employed in WGCNA analysis, MEs of 22 modules were calculated, and compared to modular expression values of the probe sets. As shown in Fig S1, MEs showed the same tendency with expression values of the probe sets and could be representative for expression values of probe sets in the correspondence modules.\u003c/p\u003e\n\u003cp\u003eThe correlation between MEs and gestation age was evaluated by Spearman correlation analysis. As shown in Table S6, most modules showed no significant correlation with gestation age except for green and turquoise modules. Modules were clustered using hierarchical clustering. Green and turquoise modules showing significant correlation with gestation age were classified into different clusters. Modules with similar correlation coefficient were divided into different clusters (Fig 2A). MEs of major modules (turquoise, blue, brown, yellow, green and red) were smoothed by locally weighted regression and were shown in Fig 2B. A downward trend was observed for MEs of green and turquoise modules with the increase of gestation age. No obvious trend was found for MEs of other modules concerning gestation age. Expression of genes in green and turquoise modules reduced with augment of gestation age in second term.\u003c/p\u003e\n\u003ch2\u003eHigh expression probe sets showing significant clustering tendencies\u003c/h2\u003e\n\u003cp\u003eProbe sets with higher expression values are more inclined to be used as a marker for fetal organ development. To investigate the expression character of clustered probe sets, mean values and coefficient of variation for all probes in the normal 26 samples employed in WGCNA analysis were calculated. A scatter plot (mean expression value VS coefficient of variation) was drawn to show clustered probe sets distribution in all probe sets, and the clustered probe sets were labeled as modular colors (Fig 3A). Compared to probe sets with low expression values, the higher portions of the probe sets with higher expression values were clustered.\u003c/p\u003e\n\u003ch2\u003eRelationship between modules and tissues via dominant tissues and modules\u003c/h2\u003e\n\u003cp\u003eTo establish the relationship between fetal tissues and modules, the distribution of tissue-specific genes in modules were analyzed. Tissue-specific genes were obtained at a different cut-off value conditions. After tissue-specific genes were converted to probe sets, numbers of tissue-specific genes (probe sets) under different cut-off value (5, 10, 15, 20, 25 and 30) were counted (Fig 3B). The number of tissue-specific genes (probe sets) decreased with higher cut-off value. The distribution of tissue-specific probe sets in all 22 modules, was analyzed and summarized in Table S6. As shown in Fig 3C, tissue-specific probe sets were mainly distributed in turquoise, blue, brown, yellow, green, and red modules, which were called major modules.\u003c/p\u003e\n\u003cp\u003eConsidering the difference of gene expression in fetus and adult tissues, the cut-off value was set as 5 to get more gene specific genes. The numbers of tissue-specific probe sets derived from different tissues in major modules were shown in Fig 3D, while cut-off value was set as 5 (detailed data see Table S7). Tissue-specific genes from skeleton, liver, and testis accounted for the largest specific genes in turquoise module. The above three tissues were defined as dominant tissues in turquoise module. Similarly, dominant tissues were calculated in blue module (placenta, skeletal muscle, and testis), brown module (testis, cerebral cortex, and skeletal muscle), yellow module (testis, cerebral cortex, and cerebellum), green module (small intestine, liver, and colon) and red module (esophagus, tongue, and tonsil). Tissue-specific probe sets derived from liver mostly distributed in green and turquoise modules. Green and turquoise modules were defined as dominant modules for liver. Dominant modules for cerebral cortex included yellow and blue modules. Dominant tissues and dominant modules were different for different modules and tissues respectively.\u003c/p\u003e\n\u003cp\u003eBlue, brown, and yellow modules included the largest neural-specific genes (cerebral cortex, cerebellum, basal ganglia, etc). Green modules contained more digest system specific genes.\u003c/p\u003e\n\u003ch2\u003eDifferential expressed genes in DS, ES, and TS\u003c/h2\u003e\n\u003cp\u003eSamples used for DEGs analysis were listed in Table S2 (DS), Table S3 (ES), and Table S4 (TS). For differential expressed probe sets (DEPs) analysis in each group, the expression values of the abnormal fetus were compared to that of normal fetus via a linear regression model. There were 1049, 1507, and 1448 DEPs detected in DS, ES, and TS groups respectively. Volcano map of DS, ES, and TS group were shown in Fig 4A-C. Relationship of DEPs between DS, ES, and TS group were shown in Venn diagrams (Fig 4D). There were 41 common DEPs between DS ES group, 38 common DEPs between DS and TS group, 43 DEPs between ES and TS group. No common DEPs were found in DEPs of DS, ES, and TS.\u003c/p\u003e\n\u003ch2\u003eDisease-specific modules via GO analysis\u003c/h2\u003e\n\u003cp\u003eNumbers of DEPs of DS, ES, and TS in major clustered modules were counted (Fig 5A). Blue module was the most abundant distributed module in TS and DS groups. However, the brown module had the largest number of DEPs of ES. To analyze the function of clustered genes, DEPs were converted to gene symbols. The modular distribution of DEGs in three groups was summarized in Table S8.\u003c/p\u003e\n\u003cp\u003eModular specific DEGs, which mean DEGs of every group (DS, ES, and TS) in a certain module, were extracted from Table S8. Modular specific DEGs in major modules (turquoise, blue, brown, yellow, green, and red) were performed functional enrichment analysis (Table S10). The numbers of enriched GO terms were shown in Fig 5B. The yellow, blue, and red modules were specific modules for DS, ES, and TS groups respectively. DEGs of DS in the yellow module included SORBS1 and FFAR4. DEGs of ES in blue module included AKNAD1, SOX9, ZNF395, PID1, PRPF38A, FAM220A, ATP1B1, NME7, CD9, EPC1, and MAML2. DEGs of TS in the red module included S100A8 and IVL. The total number of enriched GO terms in ES, DS, and TS group ranked the first, the second, and the third respectively. In green and turquoise modules, similar numeric distribution of enriched GO term was shown for DS and ES, rather than TS. In green and turquoise modules, a similar number of GO term numbers were enriched in DS and ES group.\u003c/p\u003e\n\u003cp\u003eTo get an overall view of all enriched GO terms, the GO graph was established based on the relationship of GO terms. Go maps of all enriched GO terms were list in Table S9. Based on the GO graph, interrelated GO Terms were classified into the same subsets. As shown in Table S10, subsets contained a certain number of enriched GO terms from different modules and abnormal fetus (DE, ES, and TS). A total of 184 subsets were established. The numbers of GO terms in a subset ranged from 1 to 332. The largest nine subsets were summarized in Table 1. Functions of these subsets included basic physiological processes, absorption and transport of nutrients, response to external stimuli, multi-organ (kidney, lung, and heart) development, protein synthesis process, protein catabolic process and proteolysis, thermoregulation, signal transduction of TGF, and bone development.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eSubsets of enriched GO terms\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003eID\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eFig 6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eFunction\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e332\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eBasic physiological processes\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eAbsorption and transport of nutrients\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eResponse to external stimuli\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eMulti-organ (Kidney, lung and heart) development\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eProtein synthesis process\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eProtein catabolic process and proteolysis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eThermoregulation\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eSignal transduction of TGF\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e106\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003eI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"362\"\u003e\n\u003cp\u003eBone development\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\" width=\"68\"\u003e\n\u003cp\u003eID: Subsets id shown in Table S9 subset_id column.\u003c/p\u003e\n\u003cp\u003eNo: Number of enriched GO terms in subsets, were shown in Table S9 subset_number column.\u003c/p\u003e\n\u003cp\u003eFig 6: Modular distribution of enriched GO term subsets in abnormal fetus as shown in Fig 6.\u003c/p\u003e\n\u003cp\u003eFunction: summarized function according to in corresponding biological GO term subsets.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTo analyze the relationship between subset function and disease in different modular condition, enriched GO terms numbers in the nine largest subsets (Table 1) in relation to modules and fetus disease were shown in Fig 6. Besides that yellow, blue, and red modules were specific modules for DS, ES, and TS groups, more specific modules were shown in certain subsets. Green, turquoise, and brown modules were specific for ES group in absorption and transport of nutrients subset. The green module was specific to the DS group in multi-organ (kidney, lung, and heart) development subset, protein synthesis process subset, thermoregulation subset and signal transduction of TGF subset.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":" \u003cp\u003eAF cfRNA were considered a biomarker source for abnormal fetal development. And the gene expression level in AF cfRNA could be influenced by fetal status (such as fetal sex [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], gestation age [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], genetic disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and developmental disorders [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]). Therefore, AF cfRNA was proposed as a mean to monitoring fetal development in real-time. At present, the tissue expression database (Human protein Atlas [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], Gene Atlas [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and BioGPS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]) were mostly based on the adult tissues. The lack of fetal-related tissue expression database hinders the identification of abnormal fetal development. Additionally, AF cfRNAs were multiple organ origins, which increased the interferences for discovering abnormal fetal development biomarkers.\u003c/p\u003e \u003cp\u003eCompared to single genes, the advantages of gene sets contains noise and dimension reduction, as well as greater biological interpretability [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A total of 22 co-expressed gene sets (modules) were identified via WGCNA [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, probe sets with high expression values show obvious clustering tendency. Analogous to dimension reduction, expression values of clustered probes can be simplified as module eigengenes. Correlation between gene expression and gestation ages is easier to analyze via module eigengenes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, only genes in green modules showed significant correlation with gestation ages. The above results indicated that correlation between gene expression and gestation ages was not an appropriate way to discover the biomarker of the abnormal fetal development.\u003c/p\u003e \u003cp\u003eTo explore the tissue origins of genes in AF cfRNA, different gene expression atlas and cut-off values were used to define tissue-specific genes in previous studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, there was no fully comparison to get confirmed standard. Consequently, a series of cut-off values were used to define tissue-specific genes expression in our research. Based on tissue-specific gene expression, the relationships between modules and tissues were constructed. Genes from different organs showed similar co-expression patterns and were classified as the same module. Inter-organ synergetic development was manifested as a co-ordinated expression of genes intra modules. Due to the complexity of gene expression in the same organs, genes from the same organ showed different co-expression patterns. Accordingly, tissue-specific expressed genes from the same organ were divided into different modules. Thus, synergetic developments inter- and intra- organs were shown as gene co-expression networks intra- and inter- modules via gene tissue-specific analysis and weighted correlation network analysis.\u003c/p\u003e \u003cp\u003eDS, ES, and TS were the most frequent chromosomal abnormalities in prenatal diagnosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which were caused by trisomy of chromosome 21, trisomy of chromosome 18, and sex chromosome aneuploidy. The development of multiple tissues and organs was affected in these three symptoms. ES is the most serious defects and most ES cause spontaneous abortion. The most frequent clinical features of ES consist of neurological findings, growth disturbances, malformations of the skull, face, thorax, abdomen, limbs, genitals, skin, skin annexes, and internal organs [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Cardiac, airway, pulmonary, hearing, growth, hematologic, oncologic, autoimmune, musculoskeletal, and neurodevelopmental disorders are the most frequent clinical symptoms in DS [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Endocrinal, gastrointestinal, hepatic, phenotypic, neurocognitive, and psychosocial disorders [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] were reported in TS patient. Compared with the normal fetus, the differential expressed genes of DS [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], ES [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and TS [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] were analyzed. Different from common affected organs or closely related clinical symptoms, different expressed genes from these syndrome share few common differential expressed genes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo explain this contradictory phenomenon, DEGs from DS, ES, and TS compared to normal fetuses were extracted. Similar to Zwemer\u0026rsquo;s report, few common misregulated genes were detected in three groups [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Interestingly, the rank of the total number of enriched GO terms in ES, DS, and TS group was correlated with disease severity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Biological functions of enriched GO terms were simplified via classifying interrelated GO terms into the same subsets. A subset contains function-related GO terms performing associated embryonic development process. In most subsets, the number of enriched GO terms was positively related with disease severity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Another tendency, shown in the modular distribution of GO term subsets was modular heterogeneity, which suggested disease-specific modules were detected in functional enrichment analysis of modules related DEPs. Pathogenesis-related different expressed genes were classed into different modules in different diseases. In absorption and transport of nutrients subsets, DEGs in DS and ES were associated with yellow and red modules respectively. Consequently, Preprocessing via co-expression analysis could help constructed the relationship between DEGs and disease phenotype.\u003c/p\u003e \u003cp\u003eLarge numbers of GO terms were enriched in most modules with a small amount of DEGs, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. To explain this phenomenon, DEGs in disease-specific modules were extracted. SORBS1 and FFAR4 were two of DEGs from the yellow module in the DS group. SORBS1 was associated with insulin signaling pathway [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], glucose homeostasis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], cancer growth and migration [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], etc. FFAR4 have similar functions including glucose and fatty acid metabolism [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], glucose-dependent insulinotropic polypeptide secretion [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], cell cancelation[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], etc. This is accounted for enriched results of yellow modules in DS group. In subsets of a basic physiological process, absorption and transport of nutrients, response to external stimuli, protein synthesis process and thermoregulation were enriched. Taken together, genes without co-expression relationships were filtered via WGCNA. The remaining co-expressed genes were easier to be enriched via functional enrichment analysis.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eCo-expression modules based on samples from the whole second gestation (from 15 to 22 weeks) provided gene classification in this study. Via tissue-specific genes analysis, the relationships between modules and tissues were established. Disease-specific modules can help to annotate AF cfRNA variation in fetal diseases. Pre-grouping via co-expression before functional enrichment analysis could help to build a relationship between DEGs and disease phenotype. With the accumulation of datasets of AF cfRNAs, gestation week specific co-expression modules could be constructed, in which more genes in AF cfRNA would be included. With the finer co-expression modules for different gestation age, comprehensive knowledge for fetal coordinated development and gene co-expression will be elucidated.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eAF: Amniotic fluid; cfRNA: cell-free mRNA; DS: Down syndrome; ES: Edwards syndrome; TS: Turner syndrome; ME: Module eigengene; WGCNA: Weighted Gene Co-expression Network Analysis; DEPs: Differential expressed probe sets; DEGs: Differential expressed genes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no conflicts of interest to report.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was founded by the National Key Research and Development Program of China (No: 2016YFC1000101)\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eSYang, CYin, TZhang designed the project, SYang and YLiu performed statistical analysis, SYang wrote the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eHui L, Slonim DK, Wick HC, Johnson KL, Koide K, Bianchi DW. Novel neurodevelopmental information revealed in amniotic fluid supernatant transcripts from fetuses with trisomies 18 and 21. Hum Genet. 2012;131(11):1751\u0026ndash;9.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eLarrabee PB, Johnson KL, Peter I, Bianchi DW. Presence of filterable and nonfilterable cell-free mRNA in amniotic fluid. 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J Exp Clin Cancer Res. 2019;38(1):100.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"amniotic fluid, cell-free fetal RNA, co-expression network, fetal development","lastPublishedDoi":"10.21203/rs.3.rs-67755/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-67755/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eHuman amniotic fluid (AF) cells are commonly used in prenatal diagnosis. The AF cell-free mRNA (cfRNA) derived from necrotic or apoptotic cells of fetus may provide feasible marker of organ development and fetal malformation. Analysis of gene co-expression network will help to annotate AF cfRNA gene variations in fetal diseases.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eDatasets of amniotic fluid free RNA were downloaded from the Gene Expression Omnibus database. Co-expressed modules based on normal fetus AF cfRNA transcriptome were established via Weighted Gene Co-expression Network Analysis. The relationship between modules and tissues were set up via tissue-specific gene expression analysis of genes in modules. Differential expressed genes in Down syndrome (DS), Edwards syndrome (ES), and Turner syndrome (TS) were analyzed via linear models implemented in the limma package. Gene Ontology (GO) analysis of modular specific differential expressed genes for these three syndrome fetus was performed separately. Based on relationship of GO terms, GO graph of all enriched GO terms were constructed. The associated enriched GO terms in GO graph were considered as the same subset. Numbers of GO terms of the same subset with diseases and modules were calculated.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA total of 22 co-expressed modules were constructed. Most of co-expressed module eigengenes showed no correlation with gestation weeks. Probe sets with higher expression values showed significant clustering tendencies. Dominant tissues and modules were different for different modules and tissues respectively through analysis of tissue-specific genes distribution in modules. The total numbers of enriched GO terms in six major modules were related to diseases severity for ES, DS, and TS. Disease-specific modules for ES, DS, and TS were detected in relation to biological processes through GO analysis. Differential expressed genes in disease-specific module were identified for DS, ES, and TS.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe gene co-expression network for functional classification provides a potential tool for annotating AF cfRNA variations in fetal diseases.\u003c/p\u003e","manuscriptTitle":"Annotation of Gene Alteration in Amniotic Fluid Cell Free RNA Transcriptome via Weighted Gene Co-expression Network Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-09-16 14:43:55","doi":"10.21203/rs.3.rs-67755/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b56af15f-4cdb-418c-b40b-870ab918364c","owner":[],"postedDate":"September 16th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":526081,"name":"Maternal \u0026 Fetal Medicine"},{"id":526082,"name":"Sexual \u0026 Reproductive Medicine"}],"tags":[],"updatedAt":"2020-12-13T14:03:04+00:00","versionOfRecord":[],"versionCreatedAt":"2020-09-16 14:43:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-67755","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-67755","identity":"rs-67755","version":["v1"]},"buildId":"369fNeqWncA4NS6XSWjrt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}