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1 Supplementary Figures Figure S1. Urodynamic recording of BOO-induced LUTD patients. (A) DO group. BOO patients with increased detrusor pressure and reduced urine flow during pressure flow in combination with involuntary detrusor contractions during filling phase (phasic and/or terminal). (B) BO group. BOO patients without involuntary detrusor contractions during filling phase (phasic and/or terminal). (C) UA group. Patients where detrusor contractions could not be demonstrated during urodynamic study (underactive or acontractile detrusor). Shown are representative recordings from each patients group.
2 Figure S2. Upstream regulator analysis of DO, BO and UA groups based on top 30 elements in mrna-based and mirna target-based pathways. Upstream regulator analysis networks illustrating the interaction of top 30 pathway elements (mrnas) in mrna datasets or expressed mirna targets datasets (mirna targets) of patients groups. (A) mrna dataset of DO patients. JUN with 12 targets is the most interactive element. (B) mrna dataset of BO patients. TNF with 21 targets in our mrna dataset was the most interactive element. (C) mrna dataset of UA patients. TNF with 11 targets was the most interactive element. (D) Expressed mirna targets dataset of DO patients. KLF with 5 targets is the most interactive element. (E) Expressed mirna targets dataset of BO patients. FOS with 16 targets in our mrna dataset is the most interactive element. (F) Expressed mirna targets dataset of UA patients. KRAS with 7 targets was the most interactive element.
3 Figure S3. Upstream regulator analysis of all BOO patients based on top 30 elements in mrna-based and expressed mirna target-based pathways. (A) mrna dataset of all BOO patients. TNF was the most effective element. (B) Expressed mirna targets dataset of abundant significant mirnas in all BOO patients. JUN is the top upstream element.
4 Sex Age Volume desire to void Bladder Capacity Pdet Qmax Pdet max Qmax Retention Retention / Capacity Remarks Supplementary Tables Table ST1. Urodynamic evaluation of patients with BOO-induced LUTD Sample DO #1 M DO #2 M DO #3 M DO #4 M DO #5 M DO #6 M BO #1 M BO #2 M BO #3 M BO #4 M BO #5 M BO #6 M UA #1 M acontractile UA #2 M hypocontratile UA #3 M acontractile UA #4 M acontractile UA #5 M hypocontractile UA #6 M acontractile Groups (n = 6 patients per group): DO = BOO patients with urodynamically determined bladder overactivity, BO = BOO patients without bladder overactivity, UA = underactive bladder Abbreviations: Pdet max = maximum detrusor pressure, Pdet Qmax = detrusor pressure at maximum flow, Qmax = maximum flow, Residual = post-void residual.
5 Table ST2. mirnas and their targets encoding contractile and fibrotic proteins CNN1 CTGF HBEGF HGF JUNB MYC RND1 SNAI1 TGFB2 TGFBR1 TGIF2 VEGFA DO 152-3p 26b-5p -- 26b-5p p p 486-3p 374a-5p BO 10a-5p 133a-3p 26b-5p 145-5p 26b-5p 145-5p 133a-3p 145-5p 490-3p 133a-3p 145-5p 490-3p 497-5p 490-3p 497-5p UA 15b-5p 26b-5p let-7b-5p 203a 199a-3p 203a 26b-5p let-7b- 5p 203a let-7b-5p 320a 199a-3p a
6 Table ST3. Test result variables: prediction probability Markers AUC 1 Sensitivity Specificity Significance 1 Lower Bound 3 NRXN BMP UPK1A hsa-mir-103a-3p hsa-mir-10a-3p hsa-mir-199a-3p Upper Bound 3 1 AUC: Area Under the curve 2 Null hypothesis: true area = (95% confidence interval (CI)
7 Table ST4. Urodynamic parameters of the patients included in the blinded study Diagnosis Capacity (ml) Pdet Qmax (cmh2o) Residual (ml) Qmax (ml/s) BCI Number of contraction during filling BO / / / / /-9 0 DO 458+/ / / / / /-0.4 UA / / / / /-4 0 Samples, processed in the blinded study, originated from BOO patients belonging to the following groups: DO = BOO patients with urodynamically determined bladder overactivity (n=13), BO = BOO patients without bladder overactivity (n=7), UA = underactive bladder patients (n=20). Shown are the group average values ± SEM. Abbreviations: Pdet max = maximum detrusor pressure, Pdet Qmax = detrusor pressure at maximum flow, Qmax = maximum flow, Residual = post-void residual, BCI = bladder contractility index (Pdet Qmax + 5 x Qmax).
8 Table ST5. Patients in blinded study, decoding based on the three-mrna and three-mirna signatures and urodynamic diagnosis Code Decode Diagnosis BOO1 BOO1_UA UA BOO2 BOO2_UA UA BOO3 BOO3_UA UA BOO4 BOO4_UA UA BOO5 BOO5_DO DO BOO6 BOO6_DO DO BOO7 BOO7_DO DO BOO8 BOO8_BO BO BOO9 BOO9_BO BO BOO10 BOO10_UA UA BOO11 BOO11_UA UA BOO13 BOO13_UA UA BOO45 BOO45_BO BO BOO44 BOO44_BO BO BOO17 BOO17_UA UA BOO18 BOO18_UA UA BOO19 BOO19_UA UA BOO20 BOO20_UA UA BOO21 BOO21_DO DO BOO22 BOO22_DO DO BOO23 BOO23_DO DO BOO24 BOO24_DO DO BOO25 BOO25_UA UA BOO26 BOO26_UA UA BOO27 BOO27_UA UA BOO28 BOO28_UA UA BOO29 BOO29_UA UA BOO30 BOO30_UA UA BOO31 BOO31_DO DO BOO32 BOO32_DO DO BOO33 BOO33_BO BO BOO34 BOO34_BO BO BOO37 BOO37_DO DO BOO38 BOO38_DO DO BOO39 BOO39_DO DO BOO40 BOO40_DO DO BOO41 BOO41_UA UA BOO42 BOO42_UA UA
9 Table ST6. Top 10 canonical pathways implicated in BOO patients groups Pathways predicted based on mrna dataset Pathways predicted based on expressed mirna targets Canonical Pathway Name z-score Canonical Pathway Name -log(pvalue) -log(pvalue) z-score DO ERK5 Signaling Protein Kinase A Signaling PI3K/AKT Signaling Sphingomyelin Metabolism 3.13 NaN Protein Kinase A Signaling RhoA Signaling 2.42 NaN Colorectal Cancer Metastasis Signaling PI3K/AKT Signaling 2.4 NaN Cholecystokinin/Gastrin-mediated Signaling GADD45 Signaling 2.36 NaN Corticotropin Releasing Hormone Signaling Pyridoxal 5'-phosphate Salvage Pathway 2.34 NaN TGF-β Signaling Cardiac β-adrenergic Signaling Neuregulin Signaling D-myo-inositol-5-phosphate Metabolism 2.14 NaN IGF-1 Signaling phosphoinositide Degradation 2.13 NaN Wnt/β-catenin Signaling VDR/RXR Activation 2.1 NaN BO Dendritic Cell Maturation ILK Signaling IL-6 Signaling IL-6 Signaling Leukocyte Extravasation Signaling PPAR Signaling Production of Nitric Oxide and Reactive Oxygen Species in Macrophages Acute Phase Response Signaling CD28 Signaling in T Helper Cells IL-8 Signaling OX40 Signaling Pathway HMGB1 Signaling HMGB1 Signaling Toll-like Receptor Signaling PKCθ Signaling in T Lymphocytes ERK5 Signaling ILK Signaling CD40 Signaling MIF Regulation of Innate Immunity IGF-1 Signaling UA CD28 Signaling in T Helper Cells p53 Signaling B Cell Receptor Signaling Glioblastoma Multiforme Signaling icos-icosl Signaling in T Helper Cells B Cell Receptor Signaling Role of NFAT in Regulation of the Immune Response ERK5 Signaling Dendritic Cell Maturation IGF-1 Signaling PKCθ Signaling in T Lymphocytes Estrogen-mediated S-phase Entry Tec Kinase Signaling Tec Kinase Signaling Toll-like Receptor Signaling PI3K Signaling in B Lymphocytes PI3K Signaling in B Lymphocytes IL-6 Signaling IL-6 Signaling Cardiac Hypertrophy Signaling Table is sorted based on -log(p-value) of individual pathways, calculated using the right-tailed Fisher Exact Test. Positive z-score indicates activation and negative z-score represents inhibition of a pathway. Input - differentially expressed mrnas and expressed mirna targets of abundant regulated mirnas (abs. fold change 1.5, p-value <0.05 and adjusted p-value< 0.15).
10 Table ST7. Top 10 canonical pathways using all BOO patients datasets. Pathways predicted based on mrna dataset Pathways predicted based on expressed mirna targets Canonical Pathway Name z- score Canonical Pathway Name -log(pvalue) -log(pvalue) z- score HMGB1 Signaling HMGB1 Signaling IL-6 Signaling SAPK/JNK Signaling Acute Phase Response Signaling ERK5 Signaling Role of IL-17F in Allergic Inflammatory Airway Diseases TREM1 Signaling Colorectal Cancer Metastasis Signaling Leukocyte Extravasation Signaling Hepatic Fibrosis / Hepatic Stellate Cell Activation Colorectal Cancer Metastasis Signaling 2.90 NaN Bladder Cancer Signaling 2.97 NaN NRF2-mediated Oxidative Stress Response B Cell Receptor Signaling IL-8 Signaling Dendritic Cell Maturation p53 Signaling 2.74 NaN p53 Signaling HIF1α Signaling 2.66 NaN Table is sorted based on -log(p-value) of individual pathways, calculated using the right-tailed Fisher Exact Test. Positive z-score indicates activation and negative z-score represents inhibition of a pathway. Input - differentially expressed mrnas and expressed mirna targets of abundant regulated mirnas (abs. fold change 1.5, p-value <0.05 and adjusted p-value< 0.15).
11 Supplementary figure legends Figure S1. Urodynamic recording of BOO-induced LUTD patients (A) DO group. BOO patients with increased detrusor pressure and reduced urine flow during pressure flow in combination with involuntary detrusor contractions during filling phase (phasic and/or terminal). (B) BO group. BOO patients without involuntary detrusor contractions during filling phase (phasic and/or terminal). (C) UA group. Patients where detrusor contractions could not be demonstrated during urodynamic study (underactive or acontractile detrusor). Shown are representative recordings from each patients group. Figure S2. Upstream regulator analysis of DO, BO and UA groups based on top 30 elements in mrna-based and mirna target-based pathways Upstream regulator analysis networks illustrating the interaction of top 30 pathway elements (mrnas) in mrna datasets or expressed mirna targets datasets (mirna targets) of patients groups. (A) mrna dataset of DO patients. JUN with 12 targets in our mrna dataset was the most interactive element. KRAS with 9 ILA1 with 8 MYC with 7 and MAP3K8 with 5 targets were among top targeting pathway element in DO mrna dataset. (B) mrna dataset of BO patients. TNF with 21 targets in our mrna dataset was the most interactive element. (C) mrna dataset of UA patients. TNF with 11 targets was the most interactive element. TNF and NFKBIA were predicted to be activated. (D) Expressed mirna targets dataset of DO patients. KLF with 5 targets in our mrna dataset was the most interactive element. KLF and CTGF were predicted to be activated. (E) Expressed mirna targets dataset of BO patients. FOS with 16 targets in our mrna dataset was the most interactive element. (F) Expressed mirna targets dataset of UA patients. KRAS with 7 targets was the most interactive element. STAT3, JAK2 and NFKBIA were predicted to be activated. Figure S3. Upstream regulator analysis of all BOO patients based on top 30 elements in mrnabased and expressed mirna target-based pathways (A) mrna dataset of all BOO patients. TNF was the most effective element. (B) Expressed mirna targets dataset of abundant significant mirnas in all BOO patients. JUN is the top upstream element.
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