Network-based metaanalysis identifies HNF4A and PTBP1 as longitudinally dynamic biomarkers for Parkinson’s disease Jose A. Santiago and Judith A. Potashkin1 Cellular and Molecular Pharmacology Department, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved January 7, 2015 (received for review December 11, 2014) Environmental and genetic factors are likely to be involved in the associated with PD. We performed a transcriptomic and net- pathogenesis of Parkinson’s disease (PD), the second most preva- work-based metaanalysis to identify key regulators and poten- lent neurodegenerative diseaseamongtheelderly.Network- tial diagnostic biomarkers. This is a powerful approach to in- based metaanalysis of four independent microarray studies iden- tegrate gene expression data and to gain insight into complex tified the hepatocyte nuclear factor 4 alpha (HNF4A), a transcrip- diseases (9). The utility of network biology to identify biolog- tion factor associated with gluconeogenesis and diabetes, as ically relevant biomarkers for neurodegenerative diseases has a central regulatory hub gene up-regulated in blood of PD been demonstrated recently. In this context, network analyses patients. In parallel, the polypyrimidine tract binding protein 1 identified the amyloid precursor protein (APP) and superoxide SOD2 (PTBP1), involved in the stabilization and mRNA translation of in- dismutase 2 ( ) mRNAs as blood biomarkers of PD (10, 11) PTPN1 sulin, was identified as the most down-regulated gene. Quantita- and protein tyrosine phosphatase 1 ( )mRNAasadi- tive PCR assays revealed that HNF4A and PTBP1 mRNAs were up- agnostic tool for progressive supranuclear palsy (12; reviewed and down-regulated, respectively, in blood of 51 PD patients and in ref. 9). In this study, network-based metaanalysis identified hepato- 45 controls nested in the Diagnostic and Prognostic Biomarkers for HNF4A Parkinson’s Disease. These results were confirmed in blood of 50 cyte nuclear factor 4 alpha ( ) and polypyrimidine tract binding protein 1 (PTBP1), previously implicated in diabetes, as PD patients compared with 46 healthy controls nested in the Har- NEUROSCIENCE vard Biomarker Study. Relative abundance of HNF4A mRNA corre- the most significant up- and down-regulated genes in blood of PD patients. These results were confirmed in blood samples lated with the Hoehn and Yahr stage at baseline, suggesting its from two independent clinical trials. Relative abundance of clinical utility to monitor disease severity. Using both markers, PD HNF4A mRNA correlated with disease severity in PD patients. patients were classified with 90% sensitivity and 80% specificity. Moreover, longitudinal analysis of HNF4A and PTBP1 revealed Longitudinal performance analysis demonstrated that relative HNF4A PTBP1 that their relative abundance changed over time, thus suggesting abundance of and mRNAs significantly decreased their potential use in tracking the clinical course of PD patients. and increased, respectively, in PD patients during the 3-y follow- HNF4A PTBP1 HNF4A PTBP1 Further evaluation of and mRNAs in patients at up period. The inverse regulation of and provides risk for PD is warranted. a molecular rationale for the altered insulin signaling observed in PD patients. The longitudinally dynamic biomarkers identified in Results this study may be useful for monitoring disease-modifying thera- Metaanalysis of Blood Microarrays in PD. To identify a common pies for PD. transcriptional signature in blood of PD patients, four microarray Parkinson’s disease | HNF4A | PTBP1 | network analysis | blood biomarkers Significance ubstantial efforts have been devoted to the development of ’ ’ Development of therapeutic strategies for Parkinson s disease Sdiagnostic strategies for Parkinson s disease (PD). In partic- (PD) is hampered by the lack of reliable biomarkers to identify ular, changes in mRNA from cellular whole blood can facilitate patients at early stages of the disease and track the therapeutic the identification of dysregulated processes and diagnostic bio- effect of potential drugs and neuroprotective agents. Readily markers for PD (1, 2). Several molecular signatures in blood accessible biomarkers capable of providing information about have been identified. For example, 22 unique genes were found disease status are expected to accelerate this progress. We iden- differentially expressed in blood of PD patients compared with tified hepatocyte nuclear factor (HNF4A) and polypyrimidine healthy controls (1). Likewise, specific splice variants in blood tract binding protein 1 (PTBP1) mRNAs as promising blood bio- were associated with PD in samples obtained from two in- markers for identifying early stage PD patients with high dependent clinical trials (2, 3). In addition, altered expression of diagnostic accuracy. Furthermore, HNF4A was identified as a the vitamin D receptor (VDR) in blood and reduced plasma potential biomarker to monitor disease severity. Longitudinal levels of 25-hydroxy vitamin D3 have been associated with PD analysis demonstrated that HNF4A and PTBP1 are longitudinally (1, 4). Furthermore, plasma levels of the epidermal growth factor dynamic biomarkers that provide insights into the molecular have been associated with cognitive decline in PD (5). mechanisms underlying the altered insulin signaling in PD pa- Environmental stressors and genetic factors are most likely tients and may enable novel therapeutic strategies. involved in the pathogenesis of PD. Among the genetic factors associated with PD, mutations in the gene encoding leucine-rich Author contributions: J.A.S. and J.A.P. conceived and designed the project; J.A.S. con- repeat kinase 2 (LRRK2) are the most common cause of auto- ducted microarray metaanalysis, network analyses, qPCR experiments, and statistical somal dominant PD (6) and a considerable risk factor in idio- analyses; and J.A.S. and J.A.P. wrote the paper. pathic forms of the disease (7, 8). Given the complex interaction The authors declare no conflict of interest. between environmental and genetic factors in sporadic PD, we This article is a PNAS Direct Submission. integrated four independent microarray studies from patients 1To whom correspondence should be addressed. Email: judy.potashkin@rosalindfranklin. harboring a mutation in the LRRK2 gene (G2019S; glycine-to- edu. serine substitution at amino acid 2019), sporadic patients, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. untreated PD patients to identify a universal signature in blood 1073/pnas.1423573112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1423573112 PNAS Early Edition | 1of6 Downloaded by guest on October 4, 2021 studies (Table S1) were analyzed using Integrative Meta-Analysis and mannose metabolism, T-cell receptor-signaling pathway, of Expression Data (INMEX), a web interface for the integrative mammalian target of rapamycin-signaling pathway, type 2 di- metaanalysis (13). The overall metaanalysis workflow used in this abetes mellitus, and colorectal cancer. The most important hub study is shown in Fig. 1A. Metaanalysis using a Fisher’stest gene in terms of network topology measures of betweeness (BC) identified a total of 2,781 genes differentially expressed consis- and degree of centrality (DC) was HNF4A (BC = 2,213; DC = tently across four microarray studies. Among this group, 680 84) (Fig. 2A). genes were up-regulated and 2,101 were down-regulated in In parallel, down-regulated genes in blood of PD patients were PD compared with healthy controls. The thy-1 cell-surface anti- associated with the KEGG pathways, including protein pro- gen (THY1)andHNF4A were the most significant up-regulated cessing in the endoplasmic reticulum (ER), Epstein–Barr virus genes across the four microarray datasets. The complete list of infection, and several types of cancer including prostate, endo- differentially expressed genes is provided in Dataset S1.There metrial, and lung cancer. The most prominent hub gene in terms were 921 gained genes uniquely identified in the metaanalysis of network topology measures was ubiquitin C (UBC) (BC = that show relatively weak but consistent expression across the 495; DC = 1630), and PTBP1 was the most down-regulated gene four datasets. A total of 491 genes were classified as lost genes across the four microarray datasets (Fig. 2B and Dataset S1). (i.e., genes identified as differentially expressed genes in in- dividual datasets but not in the metaanalysis). A Venn diagram of Network-Based Metaanalysis. HNF4A was confirmed as potential metaanalysis results is shown in Fig. 1B, and a heat map visuali- key hub gene in blood of PD by network-based metaanalysis zation of the top 20 genes across the different studies is displayed implemented in NetworkAnalyst (14). The most highly ranked in Fig. 1C. node across the four datasets based on network topology mea- sures was HNF4A (BC = 329; DC = 35) followed by GATA1 Biological and Functional Analysis. To identify the overrepresented (BC = 10.5; DC = 8). The resulting zero-order interaction net- biological processes dysregulated in blood of PD patients, we work contained 76 nodes and 81 edges (Fig. S1). In addition, performed a gene pathway analysis using NetworkAnalyst (14). network-based metaanalysis identified the aberrant expression of Pathway analysis was performed using the set of up- and down- several splicing factors