Bladder Pain Syndrome

Author's Accepted Manuscript Correlation of Gene Expression with Bladder Capacity in Interstitial Cystitis/Bladder Pain Syndrome Marc Colaco , David S. Koslov , Tristan Keys , Robert J. Evans , Gopal H. Badlani , Karl-Erik Andersson , Stephen J. Walker

PII: DOI: Reference:

S0022-5347(14)03576-9 10.1016/j.juro.2014.05.047 JURO 11485

To appear in: The Journal of Urology Accepted Date: 9 May 2014 Please cite this article as: Colaco M, Koslov DS, Keys T, Evans RJ, Badlani GH, Andersson KE, Walker SJ, Correlation of Gene Expression with Bladder Capacity in Interstitial Cystitis/Bladder Pain Syndrome, The Journal of Urology® (2014), doi: 10.1016/j.juro.2014.05.047. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain. All press releases and the articles they feature are under strict embargo until uncorrected proof of the article becomes available online. We will provide journalists and editors with full-text copies of the articles in question prior to the embargo date so that stories can be adequately researched and written. The standard embargo time is 12:01 AM ET on that date.

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Correlation of Gene Expression with Bladder Capacity in Interstitial Cystitis/Bladder Pain Syndrome

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Marc Colaco1, 2, David S Koslov1, 2, Tristan Keys1, 2, Robert J Evans1, Gopal H Badlani1, KarlErik Andersson2, Stephen J Walker2 1

Department of Urology, Wake Forest School of Medicine-Winston-Salem, NC Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA

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Running Title: Interstitial Cystitis Molecular Expression

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Key Words: interstitial cystitis, genetics, molecular expression

Address for Correspondence:

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Financial Disclosures: None Conflicts of Interest: None Word Count: 2,482

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Marc Colaco, MD MBA Department of Urology Wake Forest School of Medicine Medical Center Boulevard Phone: (336) 716-9601 FAX: (336) 716-5711 E-mail: [email protected]

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ABSTRACT Purpose: Interstitial cystitis and bladder pain syndrome (IC/BPS) are terms used to describe a heterogeneous chronic pelvic and bladder pain disorder. Despite its significant prevalence,

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understanding of the disease’s etiology is poor. We aimed to molecularly characterize IC/BPS and determine if there are clinical factors that correlate with gene expression.

Materials and Methods: Bladder biopsies from female subjects with IC/BPS and female

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controls without signs of the disease were collected and sorted into either normal or low

anesthetized bladder capacity. The samples then underwent RNA extraction and microarray

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assay. The data generated by these assays was analyzed using Qlucore Omics Explorer, GeneSifter© Analysis Edition 4.0 and Ingenuity Pathway Analysis to determine similarity among samples within and between groups as well as to measure differentially expressed transcripts unique to each phenotype.

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Results: A total of 16 subjects were included in this study. Principal component analysis and unsupervised hierarchical clustering showed a clear separation between gene expression in tissues from subjects with low bladder capacity and subjects with normal bladder capacity. Gene

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expression in tissue from IC/BPS patients with normal bladder capacity was not significantly different from IC/BPS-free control subjects. Pair-wise analysis revealed that pathways related to

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inflammatory and immune response were most involved. Conclusions: Microarray analysis provides insight into the potential pathology underlying IC/BPS. In this pilot study low capacity and normal capacity IC/BPS have significantly different molecular characteristics, which may reflect a difference in disease pathophysiology.

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INTRODUCTION Painful bladder syndrome, interstitial cystitis, and bladder pain syndrome (IC/BPS) are all terms used to describe a chronic disease of uncertain etiology that primarily affects women. Patients

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suffer from vague pelvic pain that can be exacerbated by bladder filling, and is often associated with urinary frequency and urgency.1 This is a relatively common entity: IC/BPS is projected to affect approximately 3-8 million women and 1-4 million men in the United States,2,3 and incurs

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as much as $750 million in medical costs per year.1 Yet, despite this burden, the etiology of IC/BPS is still poorly understood.4 Theories include urothelial dysfunction,5–7 mast cell over

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activity, 8,9 neurogenic inflammation,10,11 and allergic or autoimmune response,12 but evidence is scant. Presentation is heterogeneous; while the classic presentation involves the presence of Hunner’s ulcers or glomerulations, neither is a prerequisite for diagnosis. Moreover, very few patients who present without ulcers ever develop such lesions, and as such it is believed that

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ulcerative and non-ulcerative IC may be separate subtypes of the same disorder or distinct entities entirely with similar symptoms.

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Currently there is no absolute histological or pathological marker for IC/BPS. IC/BPS is a

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clinical diagnosis of exclusion, and while workup only requires confirmed negative urinalysis and urine culture as well as a thorough history and physical examination, patients often undergo

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prolonged evaluations with invasive testing such as cystoscopy, urodynamics, and biopsy to rule out confounding processes before a presumed diagnosis of IC/BPS is made. The purpose of this pilot study was to examine gene expression in bladder tissue from female IC/BPS patients with varying clinical presentations. We examined several clinically relevant parameters as delineators of molecular variation including anesthetized bladder capacity (capacity measured under anesthesia at 100 mL H2O pressure), symptomology using validated

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questionnaires, mastocytosis, presence or absence of glomerulations, duration of symptoms, and presence or absence of ulcerations. Although other researchers have attempted to molecularly characterize ulcerative,14 and non-ulcerative disease,15 to our knowledge these findings have

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never previously been correlated with clinical presentation.

MATERIALS AND METHOD

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Subjects

Permission to conduct this study was obtained from the Wake Forest University Health Sciences

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Institutional Review Board. Experimental subjects were prospectively enrolled from the population of female patients between the ages 18 and 80, who presented for evaluation of IC/BPS with no history of other bladder pathology. Control subjects were drawn from the population of female patients who presented to the same urology clinic for urological evaluation

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requiring biopsy unrelated to IC/BPS.

Clinical Evaluation and Subject Grouping

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Prior to cystoscopy all subjects underwent assessment including history and physical examination. The clinical diagnosis of IC/BPS was based upon the most current American

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Urological Association guidelines definition: “An unpleasant sensation (pain, pressure, or discomfort) perceived to be related to the urinary bladder, associated with lower urinary tract symptoms for more than six weeks duration, in the absence of infection or other identifiable causes”.16 Patients were also asked to complete the Interstitial Cystitis Symptom Index (ICSI), the Interstitial Cystitis Problem Index (ICPI), and the Pelvic Pain and Urinary/Frequency Patient

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Symptom Scale (PUF) preoperatively. These instruments have all been shown to be valid measurements of IC/BPS symptomology and provide a baseline to track response to therapy.17,18

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Tissue Procurement

Experimental tissue was collected either during cystoscopy or at surgery (for patients who were undergoing cystectomy for end stage disease) under general anesthesia. Cystoscopy patients first

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underwent hydrodistention at 100 mL of H20 for a period of 5 minutes. Following

hydrodistention, the bladder was emptied and the degree of glomerulations was assessed based

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upon the Interstitial Cystitis Database (ICDB) criteria of none, mild, moderate, or severe.19 All cystoscopies and glomerulation grading was done by a single physician. Study biopsies were taken post hydrodistention from the posterior bladder wall using a cold-cup technique with a portion of each biopsy specimen was sent for normal clinical analysis by the Wake Forest

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Medical Center pathology department (following standard hospital protocol). The remaining sample was immediately submerged in 200 µL of RNAlater® and stored at -20oC until use. For patients undergoing cystectomy, an amount of tissue similar to that which would be collected at

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biopsy was harvested from the posterior bladder through a single scalpel incision and submerged in 200 µL of RNAlater® and stored at -20oC in a similar fashion. Bladder capacity data and

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cystoscopic findings for these subjects were retrieved from the patient’s last cystoscopy recording in the medical record and included in this analysis. Control tissue was likewise collected during cystoscopy. As these patients did not have any clinical indication for the performance of hydrodistention this procedure was not done.

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Patient Grouping Following evaluation of cystoscopy results, patients were grouped for molecular analysis as follows:

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1. Group 1 (Normal Capacity): Subjects with normal anesthetized bladder capacity (defined as ≥ 400 mL on hydrodistention)

2. Group 2 (Low Capacity): Subjects with low anesthetized bladder capacity (defined as

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< 400 mL on hydrodistention)

3. Group 3 (Control): Subjects with no history of IC/BPS.

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The delineation of bladder capacity <400 ml as ‘low’ was chosen because patients with bladder capacity below 400 ml have shown in the past to have significantly better results following surgical treatment for IC/BPS (cystectomy), suggesting that it may represent an alternate pathology from normal capacity disease or a more advanced disease state.20 Secondary analysis

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was conducted using patient subjective symptom scores, degree of glomerulations, and tissue mast cell counts as delineating factors.

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Sample Processing

Biopsy tissue was homogenized by sonication and total RNA was extracted using RNeasy

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Minelute Plus columns (includes on-column DNase digestion) and reagents (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA was eluted from the columns in nuclease-free water and RNA quantity and purity was determined spectrophotometrically on a Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE) by measuring absorbance at 260/280nm (mean for all samples= 2.09 ± 0.02) and RNA quality determined using an Agilent Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA; mean RIN= 9.4 ±

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0.24 for all samples). Total RNA was shipped on dry ice to a microarray core facility (City of Hope Functional Genomics Core, Duarte, CA) for processing where labeled cDNA, generated from total RNA, was assayed on SurePrint Human Gene Expression v2 microarrays (Agilent

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Technologies) containing 60-mers for 50,599 biological features. Data were extracted from scanned images using Agilent’s Feature Extraction Software (Agilent Technologies).

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Data Processing for Gene Expression and Gene Ontology/Pathway Analysis

Following normalization of the raw data, principle component analysis (PCA) and unsupervised

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hierarchical clustering were performed (Qlucore Omics Explorer) to determine similarity among samples within and between groups. Further statistical analyses to measure differentially expressed transcripts (DETs) unique to each phenotype were then performed by applying Student’s t-test (@fold change ≥ 1.5; p ≤ 0.05) for the pair-wise comparison (GeneSifter©

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Analysis Edition 4.0). The list of DETs generated from the pair-wise comparison (e.g. low bladder capacity versus normal bladder capacity) was imported into Ingenuity Pathway Analysis software for gene ontology and pathway analysis. All data is available through Gene

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RESULTS

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Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/).

Patient Analysis

16 subjects were included in this pilot study: 13 IC/BPS patients and 3 controls. Clinical and demographic data of cases and controls can be found in Table 1. Using bladder capacity as the differentiator, nine of the IC/BPS patients had normal anesthetized capacity and four had low anesthetized capacity. On secondary analysis, four patients (3 with low capacity and 1 with

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normal capacity) had undergone cystectomy while the remaining nine had only undergone cystoscopy. Two of the low capacity patients demonstrated Hunner’s ulcers on cystoscopy, while none were seen in the normal capacity cohort. Finally, the patients who demonstrated

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ulcers also demonstrated histopathological denudation of the urothelium, while this was not reported in any other sample.

For control samples, bladder biopsies were obtained from two patients undergoing

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surveillance for bladder cancer that were ultimately negative and one patient suffering from overactive bladder (OAB). Although this patient had elevated PUF and O’Leary Sant scores, this

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was due to high scores on questions regarding frequency and urgency rather than the pain that is characteristic of IC/BPS.

Principal Component Analysis (PCA) and Hierarchical Clustering Based on Bladder

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Capacity

Using ‘capacity’ as the measure, analysis of variance (ANOVA) was performed between the three groups (control, normal capacity, and low capacity) to yield the top 52 differentially

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expressed genes (p = 4.5E-6; FDR = 0.0028). Figure 1 shows the results of this analysis. In the PCA (Figure 1A), the four low capacity subjects were a significant distance from normal

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capacity and control patients, with “capacity” being responsible for 92% of the variation between the samples. Figure 1B displays these results in a heat map of the 52 most significantly differentially expressed genes. Differential gene expression within tissue from subjects with low capacity shows significant up regulation of transcripts compared to gene expression within tissue from normal capacity and control patient samples. Gene expression in tissues from patients with normal bladder capacity is more similar to control tissues than to the low capacity

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samples, but these samples do fall into two distinct clusters demonstrating different degrees of lowered expression or down regulation of target genes that is not sharply delineated

other than bladder capacity, resulted in significant sample separation.

Gene Expression and Pathway Analysis

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between IC/BPS and control. Additional analyses revealed that no other delineating factor,

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Pairwise analysis between the low and normal bladder capacity groups, performed at a fold change of ≥ 1.5 and adj p ≤ 0.05, with Benjamini and Hochberg false discovery rate (FDR)

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adjustment, yielded several thousand (2060) differentially expressed transcripts (DETs) between these two groups. Surprisingly, there were only 193 DETs between the control and low capacity groups (there were 2030 DETs before FDR correction); even less, 50 DETs, between the control and normal capacity groups. The top 50 DETs between low and normal capacity bladder tissues

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(based on fold change) can be found in Table 2. The majority of up regulated transcripts were found to be involved in inflammatory cell signaling, with the greatest fold change differences seen in the expression of chemokine ligands 18,13, and 19. An example of one important

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pathway containing a significant number of up regulated genes in the low capacity group is the Leukocyte Transendothelial Migration pathway (p = 3.15E-6; Figure 2). One of the most down

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regulated transcripts was Uroplakin 1A, a component of the highly specialized biomembrane of urothelial cells that may play a role in epithelial physiology and permeability. An example of one important pathway that contained a highly significant number of down regulated genes from this DET list is the Tight Junction pathway (Figure 3). “The transmembrane proteins [of tight junctions] mediate cell adhesion and are thought to constitute the intramembrane and paracellular diffusion barriers” (excerpted from the KEGG database; http://www.kegg.jp/).

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When the list of 2060 DETs was analyzed in Ingenuity Pathway Analysis (IPA) the most significantly altered disease and biofunctions categories were inflammatory response (p = 2.11E52 to 3.65E-9) and immunological disease (p = 8.41E-49 to 3.6E-9), lending further credibility to

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the suggestion that low bladder capacity represents a pathologic condition.

DISCUSSION

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In this pilot study we sought to assess the feasibility of subcategorizing patients with IC/BPS into clinically relevant groups that could reflect distinct underlying/associated molecular

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phenotypes. Our results indicate that there is a highly significant difference between low capacity patients and both normal capacity patients and controls (Figure 1A) that appears to center around the up regulation of transcripts involved in the inflammatory and immune cellular signaling systems. Down-regulation of transcripts responsible for proteins within the

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cellular membrane complex also appears prevalent within the low capacity samples and although denuding could partially account for this finding it was only reported in two of the low capacity samples (those patients with Hunner’s ulcers) and as such should can not

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fully explain the differential expression between groupings. While differences between ulcerative and non-ulcerative disease have been well documented in other studies,14,15,21 this

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study is the first to document genetic variation based solely on capacity. Finally, glomerulations, mastocytosis, age, duration of symptoms and subjective pain scores do not appear to have any measurable correlation with molecular variation between tissues. Unlike the differences in gene expression observed between low capacity and normal capacity tissues, control tissue showed a high degree of similarity to normal bladder capacity IC/BPS tissue. While we did observe two distinct clusters consisting of normal capacity

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patients and controls, differential expression between these clusters is less pronouced than between “normal capacity” and “low capacity” groups as a whole, and they were not delineated based on presence or absence of disease (Figure 1B). This finding has been

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previously reported: in a recent study of gene expression analysis from urine sentiment Blalock et al,15 were unable to differentiate between the molecular profiles of tissue from patients with non-ulcerative IC/BPS and controls. These differences in genetic expression may explain why

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clinical trials for IC/BPS are so variable in effectiveness and have such a high proponent of nonresponders: the underlying etiology may differ. Further research must be done in order to

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examine the possibility of further stratification.

The main limitation of this pilot study is the modest number of participants. As this is a pilot study we only included a total of 16 subjects and we recognize that this sample size does not have the power necessary to reveal subtle molecular differences. Furthermore, expanded

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data may demonstrate that other factors such as age may also have a significant impact on the molecular profile. Nonetheless, other gene expression-based IC/BPS studies have used similar sample sizes with publishable results,14,22 and we believe that this study provides valuable

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information despite this limitation. A second weakness was that some samples were collected at biopsy and others at cystectomy. While we made efforts to harvest similar amounts of tissue

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from the same location we recognize that this difference may introduce unwanted variability. Thirdly, some of the variation we observed may be due to a biological defense mechanism triggered by biopsy. The detachment of tissue during biopsy may be responsible for disruption of uroplakin and tight junction proteins, and the inflammatory response may be in part iatrogenic, this would not explain the differential expressions between subject groups and biopsy has been used to examine inflammation genetics in the past.22 Finally, our controls were not from uniform

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tissue sources and did not involve hydrodistention (as it is not clinically indicated in this population). This disunity did not, however, result in significantly different molecular profiles

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between control samples and thus it is unlikely to have biased our study.

CONCLUSIONS

Gene expression analysis provides insight into the patho-biology underlying IC/BPS. We

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demonstrate that low capacity and normal capacity IC/BPS bladders have significantly different molecular characteristics, and this difference may reflect a fundamental difference in disease

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processes. Meanwhile, patients with normal bladder capacity exhibit similar molecular profiles to control subjects. Given the promising results of this pilot study we are conducting further research into the correlation between molecular and clinical findings and the development of an

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IC/BPS biomarker.

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REFERENCES Payne CK, Joyce GF, Wise M, et al: Interstitial cystitis and painful bladder syndrome. J. Urol. 2007; 177: 2042–2049.

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Anon: Clemens JQ, Joyce GF, Wise M et al. “Interstitial cystitis and painful bladder syndrome. In: Urologic Diseases in America.” Edited by M. S. Litwin and C. S. Saigal. Washington, DC: US Department of Healt and Human Services, Public Health Service, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseaes, 2007.

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Bullones Rodríguez MÁ, Afari N, Buchwald DS, et al: Evidence for overlap between urological and nonurological unexplained clinical conditions. J. Urol. 2013; 189: S66– 74.

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Logadottir Y, Fall M, Kåbjörn-Gustafsson C, et al: Clinical characteristics differ considerably between phenotypes of bladder pain syndrome/interstitial cystitis. Scand. J. Urol. Nephrol. 2012; 46: 365–370.

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Parsons CL: The role of a leaky epithelium and potassium in the generation of bladder symptoms in interstitial cystitis/overactive bladder, urethral syndrome, prostatitis and gynaecological chronic pelvic pain. BJU Int. 2011; 107: 370–375.

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Buffington CA and Woodworth BE: Excretion of fluorescein in the urine of women with interstitial cystitis. J. Urol. 1997; 158: 786–789.

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Shie J-H and Kuo H-C: Higher levels of cell apoptosis and abnormal E-cadherin expression in the urothelium are associated with inflammation in patients with interstitial cystitis/painful bladder syndrome. BJU Int. 2011; 108: E136–141.

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Sant GR, Kempuraj D, Marchand JE, et al: The mast cell in interstitial cystitis: role in pathophysiology and pathogenesis. Urology 2007; 69: 34–40.

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Theoharides TC, Sant GR, el-Mansoury M, et al: Activation of bladder mast cells in interstitial cystitis: a light and electron microscopic study. J. Urol. 1995; 153: 629–636.

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10. Dmitrieva N, Shelton D, Rice AS, et al: The role of nerve growth factor in a model of visceral inflammation. Neuroscience 1997; 78: 449–459. 11. Pang X, Marchand J, Sant GR, et al: Increased number of substance P positive nerve fibres in interstitial cystitis. Br J Urol 1995; 75: 744–750. 12. Lin Y-H, Liu G, Kavran M, et al: Lower urinary tract phenotype of experimental autoimmune cystitis in mouse: a potential animal model for interstitial cystitis. BJU Int. 2008; 102: 1724–1730.

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13. Peeker R and Fall M: Toward a precise definition of interstitial cystitis: further evidence of differences in classic and nonulcer disease. J. Urol. 2002; 167: 2470–2472. 14. Gamper M, Viereck V, Geissbühler V, et al: Gene expression profile of bladder tissue of patients with ulcerative interstitial cystitis. BMC Genomics 2009; 10: 199.

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15. Blalock EM, Korrect GS, Stromberg AJ, et al: Gene expression analysis of urine sediment: evaluation for potential noninvasive markers of interstitial cystitis/bladder pain syndrome. J. Urol. 2012; 187: 725–732.

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16. Hanno PM, Burks DA, Clemens JQ, et al: AUA Guideline for the Diagnosis and Treatment of Interstitial Cystitis/Bladder Pain Syndrome. The Journal of Urology 2011; 185: 2162–2170.

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17. Parsons CL, Dell J, Stanford EJ, et al: Increased prevalence of interstitial cystitis: previously unrecognized urologic and gynecologic cases identified using a new symptom questionnaire and intravesical potassium sensitivity. Urology 2002; 60: 573–578. 18. Sirinian E, Azevedo K and Payne CK: Correlation between 2 interstitial cystitis symptom instruments. J. Urol. 2005; 173: 835–840.

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19. Tomaszewski JE, Landis JR, Russack V, et al: Biopsy features are associated with primary symptoms in interstitial cystitis: results from the interstitial cystitis database study. Urology 2001; 57: 67–81. 20. Lotenfoe RR, Christie J, Parsons A, et al: Absence of neuropathic pelvic pain and favorable psychological profile in the surgical selection of patients with disabling interstitial cystitis. J. Urol. 1995; 154: 2039–2042.

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21. Ogawa T, Homma T, Igawa Y, et al: CXCR3 binding chemokine and TNFSF14 over expression in bladder urothelium of patients with ulcerative interstitial cystitis. J. Urol. 2010; 183: 1206–1212.

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22. Homma Y, Nomiya A, Tagaya M, et al: Increased mRNA expression of genes involved in pronociceptive inflammatory reactions in bladder tissue of interstitial cystitis. J. Urol. 2013.

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FIGURE AND TABLE LEGENDS

Figure 1. Gene expression in IC/BPS cases and controls. Hierarchical clustering, based on

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bladder capacity was performed on whole genome microarray data. For the principle components analysis (PCA; Panel A) the significance threshold was set at p = 4.5e-6. Each colored circle represents data from one individual (Panel A). The heatmap represents the top 52 differentially

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expressed genes based on bladder capacity (Panel B). Panels A & B: violet = low capacity;

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yellow = normal capacity; blue = controls

Figure 2. Leukocyte Transendothelial Migration pathway. A large number of genes in the low bladder capacity group were overwhelmingly up regulated (compared to normal bladder capacity samples) in this pathway (p = 3.15E-6). Genes that appear in a red box are genes that populate the DET list in the pairwise comparison between low and normal bladder capacity

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samples. Red arrow = down-regulated gene; green arrow = up-regulated gene.

Figure 3. Tight Junction pathway. A significant number of genes in the low bladder capacity

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group were overwhelmingly down regulated (compared to normal bladder capacity samples) in this pathway (p = 0.011). Genes that appear in a red box are genes that populate the DET list in

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the pairwise comparison between low and normal bladder capacity samples. Red arrow = downregulated gene; green arrow = up-regulated gene.

Table 1. Clinical data and demographic information for cases and controls. Demographic and clinical data for each of the 16 subjects. Subject #11 had biopsy performed during cystoscopy then underwent cystectomy at a later date.

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Table 2. Most differentially expressed transcripts. The top 50 differentially expressed transcripts (sorted by direction of change, and then by fold change) in the comparison between

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low capacity samples (N=4) and normal capacity samples (N=9) are listed.

Subject

Age

Duration of Symptoms (years)

Capacity (mL)

O'Leary-Sant

PUF

Glomerulations

Ulceration

Mast Cell Count

Cystectomy

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Normal Capacity 47

2

900

30

26

Mild

No

30

No

2

27

11

1400

30

25

Mild

No

<20

No

3

57

12

1350

16

17

Moderate

No

<18

No

4

32

16

900

26

26

Moderate

No

20

No

5

37

1

1000

28

30

Severe

No

28

No

6

25

4

900

31

27

Severe

No

<20

No

7

24

8

1200

19

22

Severe

No

<20

No

8

23

4

600

33

27

Severe

No

Not Reported

Yes

9

63

38

400

23

15

Mild

No

20

No

26

Severe

No

35

No

30

Moderate

No

60

Yes*

33

Severe

Yes

20

Yes

17

Mild

Yes

66

Yes

57

6

300

32

11

46

11

225

35

12

67

4

175

35

13

65

24

275

25

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14

76

Not Reported

Not Reported

Not Reported

NA

No

Not Reported

No

15

43

600

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Control

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10

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Low Capacity

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1

14

14

NA

No

20

No

16

53

850

25

25

NA

No

Not Reported

No

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0.000178005 7.21E-05 0.000124367 0.000798191 0.001592473 1.06E-05 9.90E-06 0.001628582 0.000647602 0.007804865 0.000187536 0.001736463 0.005486874 0.008956653 0.00503573 0.002652341 0.000454132 0.00027721 0.001075787 0.002516161 0.000579803 0.000690519 0.002556574 0.002240218 0.000866098 0.002090873 0.002228786 0.000236905

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21.23 20.18 20.04 19.12 18.94 18.07 17.98 42.47 39.08 37.69 36.1 35.58 31.89 27.67 26.39 26.2 24.74 22.52 21.32 20.43 20.15 19.93 19.31 19.12 18.61 18.15 18.14 17.89

Chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated) Chemokine (C-X-C motif) ligand 13 Chemokine (C-C motif) ligand 19 T-cell immunoglobulin and mucin domain containing 4 Complement component (3d/Epstein Barr virus) receptor 2 Chromosome 4 open reading frame 7 CD19 molecule T-cell leukemia/lymphoma 1A Spi-B transcription factor (Spi-1/PU.1 related) Ubiquitin D Hypothetical protein MGC29506 Lactotransferrin B lymphoid tyrosine kinase Chemokine (C-C motif) receptor 7 Tumor necrosis factor receptor superfamily, member 17 Chemokine (C-X-C motif) receptor 5 Chemokine (C-X-C motif) ligand 9 Interleukin 6 (interferon, beta 2) Chitinase 3-like 2 Junctional sarcoplasmic reticulum protein 1 Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) Prepronociceptin Interleukin 2 receptor, alpha CD38 molecule Pre-B lymphocyte 3 Membrane-spanning 4-domains, subfamily A, member 1 T-cell leukemia/lymphoma 1B Cholesteryl ester transfer protein, plasma Hypothetical protein FLJ21511 Death associated protein-like 1 Uroplakin 1A Transmembrane protease, serine 11E Envoplakin-like Uroplakin 1B Calcineurin B homologous protein 2 Chromosome 10 open reading frame 99 E74-like factor 5 (ets domain transcription factor) Cytochrome P450, family 4, subfamily F, polypeptide 22 Transcription factor CP2-like 1 Calpain, small subunit 2 Involucrin Fibroblast growth factor receptor 3 E74-like factor 5 (ets domain transcription factor) Ovo-like 1(Drosophila) Dual oxidase 1 BCL2/adenovirus E1B 19kD interacting protein like A_33_P3400708 Zinc finger protein 750 Syntaxin 19

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0.000290325 0.001968637 0.000116235 1.69E-05 0.001771048 0.004181259 0.000736209 0.001311106 0.000997173 1.75E-05 0.002338876 0.004202359 0.003662396 0.000100188 0.002502974 0.000506539 0.000376546 0.010061171 0.002357518 8.24E-05

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Gene Name ACCEPTED MANUSCRIPT

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p-value

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118.52 85.72 69.34 63.77 61.16 45.78 45.67 42.25 33.69 32.74 29.82 27.44 26.12 25.38 24.81 24.6 24.18 23.82 23.59 22.5

Direction

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Fold Ratio

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Key of Definitions of Abbreviations: IC/BPS- Interstitial Cystitis/Bladder Pain Disease

PUF- Pelvic Pain and Urinary/Frequency Patient Symptom Scale ICSI- Interstitial Cystitis Symptom Index

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DET- differentially Expressed Transcript

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ICPI- Interstitial Cystitis Problem Index

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PCA- Principle Component Analysis