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1 Supplementary Information Recurrent PAX3-MAML3 Fusion in Biphenotypic Sinonasal Sarcoma Xiaoke Wang 1,, Krista L. Bledsoe 2,, Rondell P. Graham 1,, Yan W. Asmann 3, David S. Viswanatha 1, Jean E. Lewis 1, Jason T. Lewis 1, Margaret M. Chou 4, Michael J. Yaszemski 5, Jin Jen 6, Jennifer J. Westendorf 5, André M. Oliveira 1, 5 1 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 2 Mayo Graduate School, Rochester, Minnesota 3 Department of Health Sciences Research, Mayo Clinic, Jacksonville, Florida 4 Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia and the Perelman School of Medicine, Philadelphia, Pennsylvania 5 Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota 6 Medical Genome Facility, Center of Individualized Medicine, Mayo Clinic, Rochester, Minnesota These authors contributed equally to this work. Supplementary Figure Supplementary Figure Supplementary Figure Supplementary Figure Supplementary Figure Supplementary Table Supplementary Table Supplementary Table Supplementary Table Supplementary Table

2 Supplementary Figure 1. Cytogenetics and molecular genetic findings for the PAX3-MAML3 fusion. (a) Partial karyogram showing the chromosomal translocation t(2;4)(q35;q31.1). (b) and (c) FISH break apart strategy shows rearrangements of PAX3 and MAML3 loci, respectively (see also Fig. 1b). The percentage of rearranged cells for these loci ranged from 25 to 78% (median, 60%). Endothelial cells, inflammatory cells, sinonasal epithelial cells and other nonneoplastic cells showed no rearrangement of these loci. (d) Representative RT-PCR gel showing the fusion PAX3-MAML3 in three examples of SNS (lanes 1-3). Several other tumors (lanes 4-8: alveolar rhabdomyosarcoma, dermatofibrosarcoma protuberans, melanoma, malignant peripheral nerve sheath tumor and synovial sarcoma, respectively) and normal sinonasal tissue (lane 9) were negative for the fusion product; lanes 10-11: no template; see also Supplementary Table 2). (e) Confirmatory chromatogram showing the PAX3-MAML3 sequence at the fusion breakpoint in case 1. 2

3 Supplementary Figure 2. The PAX3-MAML3 fusion protein localizes to the nucleus and binds DNA. (a) Western blots were performed with anti-pax3 or tubulin antibodies on lysates of HEK293T cells expressing pcdna3 (Co), FLAG-PAX3-MAML3 (P-M) or HA-PAX3. (b) Immunofluorescence images showing nuclear expression of FLAG-PAX3-MAML3 in C2C12 cells. Cells were counterstained with phalloidin to detect cytoplasmic F-actin and with DAPI to detect DNA. (c) Electrophoretic mobility shift assay (EMSA) of FLAG-PAX3-MAML3 binding to double-stranded DNA containing a PAX3 binding element. Lysates from COS-7 cells expressing FLAG-PAX3-MAML3 (P-M) or HA-PAX3 were incubated with a 32 P-labeled PAX3 DNA probe in the presence or absence of 100-fold excess of the wildtype (WT) probe, a mutated (Mut) probe, control IgG, anti-ha or anti-flag. The arrow points to PAX3- MAML3/DNA complexes. The arrowhead indicates PAX3/DNA complexes. The asterisk indicates the free probe. (d) Transient transcription assays demonstrating the effect of PAX3- MAML3 on a Notch reporter, Hes5p-Luc, in C2C12 cells in the presence or absence of the Notch 1 intracellular domain 1 (NICD1). * p < 0.01, ** p < relative to control in unpaired t-tests. 3

4 Supplementary Figure 3. Pathway enrichment analysis. (a) Gene ontology analysis shows that SNS expression profile is enriched in genes involved in several developmental processes, including skeletal system morphogenesis and general organ development (minimum p = 3.39x10-14 for skeletal system morphogenesis, false discovery rate (FDR) = 1.37x10-10 ; maximum p = 1.14x10-10 for organ morphogenesis, FDR = 4.64x10-8 ) (b) Enrichment process network analysis shows that SNS exhibits differential expression of several biologic circuits involved in neural, cardiac, bone and other developmental processes (minimum p = 4.19x10-4 for neural development, FDR = 0.058; maximum p = for olfactory transduction, FDR = 0.427). Enrichment analyses were performed using the MetaCore gene regulatory network database in order to identify networks that are significantly enriched by differentially expressed genes. Modified gene ontology terms are based on the GO database ( 4

5 Supplementary Figure 4. Representative qpcr and immunohistochemistry studies confirm differential gene expression in SNS. (a) and (b) High expression of NTRK3 and MYOD1 were observed in most examples of SNS. (c) MLANA and MITF (not shown) were not expressed in SNS. (d) NTRK3 showed a strong membranous pattern of expression in virtually all SNS (24/25). No staining was observed in the sinonasal epithelium. (e) MYOD1 nuclear expression was focal in a few SNS tumors (4/25); arrow indicating an example cell with focal MYOD1 expression. (f) Melan A and MITF (not shown) were universally negative in all SNS tested (n=22). qpcr and/or immunohistochemistry also confirmed the overexpression of PLUNC, PCP4, GDF7, PIK3C2G, ALX1 and PAX3, and downregulation of HOXB7 (data not shown; see also Supplementary Table 1; additional information about the phenotype of SNS can be found in ref. 1) (RE, relative expression; SNS, sinonasal sarcoma (n=5); NST, normal sinonasal tissue (n=4); ARMS, alveolar rhabdomyosarcoma (n=3); DFSP, dermatofibrosarcoma protuberans (n=3); MPNST, malignant peripheral nerve sheath tumor (n=3); SS, synovial sarcoma (n=3). *** and ** indicate Kruskal- Wallis test p < 0.01 and p < 0.05, respectively, and post hoc Dunett s test p < 0.05; * indicates Wilcoxon-Mann-Whitney test p < 0.05; error bars represent mean ± SD). 5

6 Supplementary Figure 5. Differential expression of myogenin between SNS and ARMS. (a) Biphenotypic sinonsal sarcoma (SNS) is a monomorphic spindle cell neoplasm that exclusively occurs in the head and neck region of middle aged individuals, most commonly females (HE x100). (b) ARMS is a pediatric small round cell tumor that shows a characteristic pseudoalveolar growth pattern (HE x100). (c-d) qpcr and immunohistochemical analysis show that while all ARMS (n = 11) express high levels of MYOG (myogenin) mrna (c) and protein (d, inset), SNS (n = 11) is consistently negative for this marker (c/d); * indicates Wilcoxon-Mann-Whitney test p < ; error bars represent mean ± SD). 6

7 Supplementary Table 1. Summary of Clinical Features, Immunophenotype and Molecular Findings in Biphenotypic Sinonasal Sarcoma Case Sex Age Location PAX3-MAML3 PAX3 MAML3 Phenotype Profile # (RT-PCR) (FISH) (FISH) PAX3 S100 Actin NTRK3 MyoD1 Myogenin Desmin Melan A LR LR (mo) OS (mo) 1^ Δ F 47 ethmoid/cribriform F+ - + F+ - F+ - Y ^ M 74 ethmoid/orbit F 42 nasal/ethmoid N * F 47 nasal/ethmoid F 47 ethmoid/nasal cavity F ** F 57 ethmoid F F 24 ethmoid/sphenoid F Y M 39 nasal cavity F N ** F 26 nasal cavity F N F 85 sphenoid Y F 45 nasal cavity F+ + + F N F 51 nasal cavity F+ - N * F 32 nasal cavity F N ** F 47 nasal cavity F ** F 46 ethmoid N F 58 frontal/ethmoid F F+ - Y F 56 ethmoid F F 52 frontal/ethmoid F N ** M 76 nasal cavity ND ND ND ND ND N F 49 nasal cavity N *** M 46 nasal cavity/ethmoid/frontal F N * F 38 nasal cavity F+ F Y M 56 ethmoid ND ND ND - ND F 66 ethmoid F M 61 nasal cavity ND + ND ND ND ND , positive; -, negative; Y, yes; N, not observed; LR, local recurrence; OS, overall survival; mo, months; ND, not done due to lack of material; F, focal positivity (<30% of total tumor area). * Findings suggestive of a cryptic MAML3 locus rearrangement (cases 4, 13 and 22). ** Findings suggestive of alternate PAX3 fusion genes. *** Findings suggestive of an alternate MAML3 fusion gene. ^ Index case for cytogenetics. Karyotypes: 46,XX,t(2;4)(q35;q31.1)[22]/46,XX[7] (case 1) and 46,XX,t(2;4)(q35;q31.1)[cp20]/46,XX[3] (case 2). Index case used for transcriptome sequencing. Δ An alternate splicing variant of PAX3-MAML3 was found in this case (GenBank accession no. KF727277). #, immunohistochemical analysis for S100, actin (muscle specific actin and smooth muscle actin) and desmin were described in ref.1; all tumors were universally positive for ALX1 and negative for MITF, SILV, HMB45 and HES1. 7

8 Supplementary Table 2. Clinico-Pathologic Features and Molecular Profile of Control Tumors and Tissues Used in this Study Histologic Diagnosis Age Sex Location Molecular Profile PM(-) GEP qpcr IHC 1 Adrenal cortical carcinoma 43 F Adrenal - * 2 Alveolar rhabdomyosarcoma-01 7 F Forearm PAX3-FOXO1 * * * * 3 Alveolar rhabdomyosarcoma F Periorbital PAX3-FOXO1 * * * * 4 Alveolar rhabdomyosarcoma-03 1 M Thigh PAX3-FOXO1 * * * * 5 Alveolar rhabdomyosarcoma-04 9 F Lung, met. PAX3-FOXO1 * * * 6 Alveolar rhabdomyosarcoma-05 5 M Abdominal wall PAX3-FOXO1 * * * 7 Alveolar rhabdomyosarcoma M Neck PAX3-FOXO1 * * * 8 Alveolar rhabdomyosarcoma-07 5 F Thigh PAX3-FOXO1 * * * 9 Alveolar rhabdomyosarcoma-08 7 M Sinus PAX3-FOXO1 * * * 10 Alveolar rhabdomyosarcoma M Thigh PAX3-FOXO1 * * * 11 Alveolar rhabdomyosarcoma-10 7 F Pelvic area PAX3-FOXO1 * * * 12 Alveolar rhabdomyosarcoma M Forearm PAX3-FOXO1 * * 13 Alveolar rhabdomyosarcoma M Neck, met. PAX3-FOXO1 * * 14 Alveolar rhabdomyosarcoma F Neck PAX3-FOXO1 * * 15 Alveolar rhabdomyosarcoma F Neck PAX3-FOXO1 * * 16 Alveolar rhabdomyosarcoma F Thigh PAX3-FOXO1 * * 17 Alveolar rhabdomyosarcoma-16 9 F Sinus PAX3-FOXO1 * * 18 Alveolar rhabdomyosarcoma-17 9 M Perirenal PAX3-FOXO1 * * 19 Alveolar rhabdomyosarcoma F Inguinal area PAX3-FOXO1 * * 20 Alveolar rhabdomyosarcoma-19 7 F Calf PAX7-FOXO1 * * 21 Alveolar rhabdomyosarcoma M Groin PAX3-FOXO1 * * 22 Alveolar rhabdomyosarcoma M Neck PAX3-FOXO1 * * 23 Alveolar soft part sarcoma 35 F Pelvis ASPSCR1-TFE3 * 24 Angioleiomyoma 45 M Hand - * * 25 Angiomatoid fibrous histiocytoma 22 M Arm EWSR1-CREB1 * * 26 Breast adenocarcinoma, lobular type 48 F Breast - * 27 Chronic myelogenous leukemia cell line K BCR-ABL * 28 Colon adenocarcinoma 83 M Colon, ascending - * 29 Colon adenocarcinoma 4 F Colon; FAP - * 30 Dedifferentiated liposarcoma 85 M Retroperitoneum - * * 31 Dermatofibroma/Benign fibrous histiocytoma F Arm - * * * * 32 Dermatofibroma/Benign fibrous histiocytoma F Arm - * * * * 33 Dermatofibroma/Benign fibrous histiocytoma F Shoulder - * * * * 8

9 34 Dermatofibrosarcoma protuberans F Chest wall - * * 35 Dermatofibrosarcoma protuberans M Upper back - * * 36 Dermatofibrosarcoma protuberans F Chest wall - * * 37 Dermatofibrosarcoma protuberans M Chin - * * 38 Dermatofibrosarcoma protuberans F Lower back COL1A1-PDGFB * * 39 Dermatofibrosarcoma protuberans M Thigh - * * 40 Dermatofibrosarcoma protuberans M Abdominal wall COL1A1-PDGFB * * 41 Desmoplastic small round cell tumor M Abdomen EWSR1-WT1 * * * * 42 Desmoplastic small round cell tumor M Abdomen EWSR1-WT1 * * * * 43 Desmoplastic small round cell tumor-03 8 M Abdomen EWSR1-WT1 * * * * 44 Embryonal rhabdomyosarcoma F Axilla - * * 45 Embryonal rhabdomyosarcoma-02 7 M Epididymum - * * 46 Embryonal rhabdomyosarcoma-03 5 M Infratemporal - * * 47 Embryonal rhabdomyosarcoma-04 7 M Bladder - * * 48 Embryonal rhabdomyosarcoma F Skull base - * * 49 Embryonal rhabdomyosarcoma M Paratesticular - * * 50 Embryonal rhabdomyosarcoma F Cheek - * * 51 Embryonal rhabdomyosarcoma F Maxilla - * * 52 Embryonal rhabdomyosarcoma-09 5 F Skull base - * * 53 Embryonal rhabdomyosarcoma-10 8 M Shoulder - * * 54 Embryonal rhabdomyosarcoma M Paratesticular - * * 55 Embryonal rhabdomyosarcoma-12 1 M Prostate - * * 56 Embryonal rhabdomyosarcoma-13 6 M Bladder - * * 57 Endometrial stromal sarcoma 34 F Uterus - * * 58 Ewing sarcoma M Leg EWSR1-FLI1 * * * * 59 Ewing sarcoma M Paraspinal EWSR1-FLI1 * * * * 60 Ewing sarcoma F Abdomen EWSR1-FLI1 * * * * 61 Ewing sarcoma F Neck EWSR1-FLI1 * 62 Ewing sarcoma F Pelvis EWSR1-FLI1 * * 63 Ewing sarcoma M Abdomen EWSR1-ERG * * 64 Ewing sarcoma F Sinus EWSR1-FLI1 * 65 Fibroma of tendon sheath M Hand - * * * * 66 Fibroma of tendon sheath F Hand - * * * * 67 Fibroma of tendon sheath M Hand - * * * * 68 Fibrosarcoma, adult-type M Groin - * * 69 Fibrosarcoma, adult-type F Mandible - * 9

10 70 Gastrointestinal stromal tumor F Stomach KIT exon 11 mut. * * * * 71 Gastrointestinal stromal tumor F Stomach KIT exon 11 mut. * * * * 72 Gastrointestinal stromal tumor F Stomach KIT exon 11 mut. * * * * 73 Infantile fibrosarcoma 8w M Thigh ETV6-NTRK3 * * 74 Leiomyosarcoma F Uterus - * * 75 Leiomyosarcoma M Leg - * * 76 Leiomyosarcoma M Leg * * 77 Malignant peripheral nerve sheath tumor F Thigh - * * * * 78 Malignant peripheral nerve sheath tumor F Leg - * * * * 79 Malignant peripheral nerve sheath tumor M Neck; NF1 - * * * * 80 Malignant peripheral nerve sheath tumor M Pelvis - * * 81 Malignant peripheral nerve sheath tumor F Groin - * * 82 Malignant peripheral nerve sheath tumor F Chest wall - * * 83 Malignant peripheral nerve sheath tumor M Neck - * * 84 Malignant peripheral nerve sheath tumor F Chest wall - * * 85 Malignant peripheral nerve sheath tumor M Shoulder - * * 86 Melanoma F Inguinal area, met. - * * * * 87 Melanoma M Chest wall, met. - * * * * 88 Melanoma M Arm - * * * * 89 Melanoma F Arm - * 90 Melanoma M Abdominal wall, met. - * 91 Melanoma M Groin, met. - * 92 Melanoma M Neck - * 93 Melanoma M Mesentery, met. - * 94 Mucoepidermoid carcinoma F Parotid gland CRCT1-MAML2 * * 95 Mucoepidermoid carcinoma M Parotid gland CRCT1-MAML2 * 96 Myofibroblastic sarcoma 51 M Hypopharynx - * * 97 Myofibroma 34 M Chest wall - * * 98 Myxofibrosarcoma 70 M Chest wall - * * 99 Neurofibroma-1 31 F Sciatic nerve; NF1 - * * * * 100 Neurofibroma-2 62 M Chest wall - * * * * 101 Neurofibroma-3 57 F Chest wall - * * * * 102 Normal cardiac skeletal muscle F - - * * 103 Normal cardiac skeletal muscle M - - * 104 Normal parathyroid gland 70 F - - * 105 Normal sinonasal tissue M R - * * * 10

11 106 Normal sinonasal tissue F - - * * 107 Normal sinonasal tissue M - - * * * 108 Normal sinonasal tissue M - - * * * 109 Normal sinonasal tissue M L - * * 110 Normal sinonasal tissue M R - * * * 111 Normal sinonasal tissue M - - * * 112 Normal sinonasal tissue M - - * * * 113 Normal sinonasal tissue F - - * * 114 Normal sinonasal tissue M - - * * * 115 Normal sinonasal tissue M - - * * * 116 Normal sinonasal tissue M - - * * 117 Normal sinonasal tissue M L - * * * 118 Normal skeletal muscle M - - * * * 119 Normal skeletal muscle F - - * * * 120 Osteosarcoma 3 M Femur - * * 121 Pleomorphic adenoma 55 M Submandibular gland - * * 122 Prostatic stromal sarcoma 54 M Prostate - * * 123 Schwannoma F Spine, T7 - * * * * 124 Schwannoma F Sciatic nerve - * * * * 125 Schwannoma M Neck - * * * * 126 Spindle cell rhabdomyosarcoma 1 M Chest wall - * * 127 Synovial sarcoma F Forearm SS18-SSX1 * * * * 128 Synovial sarcoma M Finger SS18-SSX1 * * * * 129 Synovial sarcoma M Thigh SS18-SSX2 * * * * 130 Synovial sarcoma M Abdomen SS18-SSX1 * * 131 Synovial sarcoma M Lung, met. SS18-SSX1 * * 132 Synovial sarcoma F Lung, met. SS18-SSX1 * 133 Synovial sarcoma F Hand SS18-SSX1 * * 134 Synovial sarcoma F Chest wall SS18-SSX2 * * 135 Synovial sarcoma F Shoulder SS18-SSX2 * 136 Synovial sarcoma F Forearm SS18+ FISH * * 137 Synovial sarcoma M Arm SS18-SSX1 * * 138 Synovial sarcoma M Thigh SS18-SSX2 * * 139 Synovial sarcoma F Thigh SS18-SSX1 * * 140 Synovial sarcoma F Abdominal wall SS18-SSX2 * * 141 Synovial sarcoma F Abdominal wall SS18-SSX1 * * 11

12 142 Synovial sarcoma F Pleura SS18-SSX1 * * 143 Undifferentiated small round cell sarcoma-01 7 M Ankle - * * 144 Undifferentiated small round cell sarcoma F Liver - * * 145 Undifferentiated small round cell sarcoma-03 10m M Mediastinum - * * FAP, familial adenomatous polyposis; NF1, neurofibromatosis type I; PM(-), negative for PAX3-MAML3; GEP, gene expression profling. IHC, immunohistochemistry. 12

13 Supplementary Table 3. Top 150 Overexpressed and Downregulated Genes in Biphenotypic Sinonasal Sarcoma Upregulated Genes Downregulated Genes Gene p-value Ratio Gene p-value Ratio 1 PLUNC 4.39E HOXB7 1.04E CXCL E KRT C20orf E HOXD CAPN E HOXB5 9.85E PIGR 2.75E PMP XAGE2B 1.72E PRG4 2.46E SERPINB3 1.39E PLA2G2A 3.52E PCP4 1.79E GABRA C13orf E PCOLCE CTAG1B 1.55E CDH GDF7 8.63E ADH1B GALNT E NKAIN TEKT1 7.53E CIDEC SCGB1A1 1.67E C1orf COL6A6 5.35E TNMD BMP5 3.90E MLANA PIK3C2G 7.34E NPTX ALX1 6.84E ANK2 1.58E RSPO1 1.83E HOXA4 2.12E GABBR2 7.17E LAMA2 1.66E MUC E HOXA2 2.74E CYP2F1 2.88E HOXB3 3.05E SV2C 9.73E MEOX2 9.19E C20orf E LCE6A CDH8 9.01E FLG LCN2 4.99E COMP C1orf E GRIN3A C11orf E TUBB FAM3D 3.20E SIM

14 30 ERN2 1.46E STMN TCEAL USP9Y ADH7 8.56E TOX AGXT 4.62E IL17B DBX1 2.11E HOXA9 3.81E LYPD2 9.38E PKIA 6.01E SCGB2A POU3F SEC14L3 1.27E JARID1D MMP E TK GDF E CYorf15A ASCL1 6.03E NCAM ALX SGCG FGFR2 1.12E BHLHE E CCDC E BCHE 3.33E C4orf E HOXB DNAI1 4.08E LOR NWD1 3.93E CXorf MME 4.24E PRL C10orf E CASP SLPI 5.90E ODZ CAPZA3 2.03E SCRG GALNTL6 4.25E CADM GRP 8.90E KRT CCDC E ISG E KRT E PTTG3P PSCA 4.86E C5orf CLDN3 1.32E GFRA FAM5C 1.16E NPTX TTR 1.42E CYP4F SPRR LOC SLC18A2 9.60E TMEFF CEACAM COL9A GREM LOC ATRNL1 1.83E CDH

15 64 KRT HOXA PCSK1N 2.61E FOXD DBC1 4.56E SPRR2B SVOPL 3.57E CDCA KRT8 1.67E SCN7A LRRC E PRUNE C3orf POPDC GATA2 1.17E GABRB NTN5 5.20E TMEM132D ALX1 3.59E PGBD WDR E MOXD1 1.71E PAX1 1.46E FLG FMO1 1.15E LCE2B DNAH2 1.49E SALL CEACAM RXRG LOC E CAMK2N MIR TRPC NTRK3 1.02E ANGPTL SHD SOX6 7.71E ALX3 1.82E TCBA GABRP SPRR2E UBL4B PLSCR SLC27A6 6.00E TYRP XAGE2 4.36E SCGB2A ADD2 3.72E LIN28B SFRP DSC PTH2R 7.60E PNMA MSLN 6.30E NELL AGR3 2.57E RGPD PPP1R1A 5.74E TMEM C20orf E KIF1A FOXA PDZRN PAX3 7.29E OGN AGR2 7.77E CHGA

16 98 CCKAR 4.71E HOXA TCN HOXB SERPINB E SLC7A ENKUR 3.48E LOC POU4F LPPR POU3F3 7.51E BIRC NEUROG WIT RASL10B 7.45E COL20A VTCN PCSK C10orf KRTAP MYOCD DCT EYA KRT PTPRT PBK IL E RDH C1orf PKD1L RSPH1 6.02E HAS GSTA DCX TTLL LCE2A VSTM2A 1.28E HOXA ALDH1A2 1.92E HOXA E SLC6A E LYG C1orf E HOXA EYA PASD ADRA2C 4.04E CSRP MYOD CST NOS IGSF SLC27A2 8.86E KRT MIR E NDST CES4 3.79E FABP3 3.54E SPTB 1.34E TMEM16D C1orf E IL1F ADRA2A 2.57E CHRDL LRRC TOP2A SEC1 2.75E ADIPOQ

17 132 KRT6A PSAT PAX DSG ELF EBF ALDH3A BVES 8.11E USP6 4.71E NOG MSMB 4.78E EDN FOXJ1 6.43E LCE2D CYP4B PCDH ACADL 3.41E ZFY PNLDC LCE1C SH3GL RASGEF1C CHRNA6 3.30E COL25A C20orf E LZTS FGF CDCA CA SERPINA ARSI 1.81E CDCA NOV UBE2T NCRNA E MFAP CYP2B7P SLN Note. Genes in boxes (n=6) had their overexpressions confirmed by qpcr and immunohistochemistry, genes in bold (n=4) had their expressions confirmed by qpcr. 17

18 Supplementary Table 4. Top 500 Differentially Expressed Genes Between Biphenotypic Sinonasal Sarcoma and Alveolar Rhabdomyosarcoma*^ Upregulated Genes in SNS Downregulated Genes in SNS Gene p-value Ratio Gene p-value Ratio 1 PLUNC 7.07E TMEM132D SERPINB3 9.40E FGF C13orf SMYD CTAG1B ELA2A C20orf E DCX GDF7 5.65E APOBEC C11orf NELL SV2C GATA SYT NRTN PIGR ACTA XAGE2B 4.27E ACTC TEKT1 1.71E MTMR7 8.58E CXCL FBN CAPN MYH SLC18A C20orf SELV KBTBD PIK3C2G 5.73E MYO18B ADAM FRMPD RSPO TAGLN CAPZA MIMT1 1.73E NOV POPDC3 9.56E TFAP2C RGR 5.65E MYOCD SRRM SH3GL EDN ENKUR LOC RSPH C1orf E NEGR PGBD5 6.87E GALNT OLIG2 5.73E TTR IL17B

19 30 ALX NGB 3.13E GPR SALL MME CKM SOX PBK CES PRDM ZIC CHRNG 8.05E RSPO DUSP C20orf SLC18A1 7.07E CCKAR TRIM E KNDC CHRND 1.07E PRAME BTBD PAX POU4F SVOPL CADM DLX6AS FGFR4 4.27E FAT4 7.63E LZTS C1orf CDH COL6A MYT1L FAM5C MYOG 1.17E CCDC DES MSLN CAPN6 8.44E PTH2R LOC E MMP2 3.29E UTS2R 3.54E SNORD ZSCAN ALS2CR SMPX C10orf GPR HLA-DRB DLGAP C4orf MYOM GABBR CBLN DNAH12L C10orf CYP2F SBK AOX SYN2 1.80E WDR TCF PCSK1N MYO7B CCDC INA

20 64 CYP4B CSRP DUOXA SOX CH25H FAM64A C1orf ELA3A OR2M KREMEN2 3.82E UBL4B CHGA LOC PKP GDF LHX VIT AURKA NTNG ARPP E C9orf TTN GRP FAM181A KRT6A PLEKHG4B HLA-DRB TMSB15A C20orf MYLK POU3F SYT SERPINB CENPF PTHLH AURKB CPZ PKD2L LOC MYL4 1.71E ELF KIFC CYS ZNF WIF MCM COLEC MYH TBX3 5.24E MIR C4orf MYBPHL RGS MYH CP SEZ C22orf CDCA COL27A MICAL2 8.05E SCNN1A TNNT HPGD MYLK CYP2B7P SRPK DNAH CDC45L

21 98 SCGB2A CNR1 3.48E CCDC SLN SNORD MYLPF MIR BEST RARG CST MXRA PLCXD CEACAM CXorf XAGE LHX DNAH MYL CYP2A TROAP ZIC PRL IL NEUROD GREM CACNG OR2M TEX KRT MYBPH PSCA FCRLA BMPER TOP2A C1QTNF LIPG GALNTL SNTG ARSI GTSE LEPR 5.24E PTTG3P LMOD LOC IGFL GRM FAM181B PKIA 6.69E NKX INSM1 8.05E SLC14A KCNT AKAP BHLHE SNORA74A RGPD ERN CDH KRT6B MYO CART C20orf FADS CPA5 8.58E TAS1R KRT33B C10orf SGCG

22 132 GSTA CCNB ADD PROX GABRP SMTNL MUC LOC C21orf BUB C1orf TNNC AGR PITX CEACAM CABP7 5.24E VWA3B SIM CHD DTL FOXL CDCA KRT DBH CD C2orf C1orf SCN4A TACSTD KIF1A FAM3D TRIM PANX ASPM C11orf MYLC2PL AMTN MGC LYPD NKAIN FOXG CAMK2B KLK KIF20A ODAM HSPB OPRK DNAH VSTM2A STMN KCNN HRASLS ARMC ISG CYP1A KIF E PNMA CHRDL SEMA6D TNNI S1PR NCAPG FLJ FAM163A SCUBE LOC CD164L BVES 9.09E

23 166 FLJ KRT NTRK KIF MMP LINGO ESPN OTOF NKD SCN3B ENPP2 8.05E EPHA ABCA ARSF C1orf SYT LCN MYT HPSE COL25A FOXJ NKAIN CCL C16orf ADRA1D ESCO SLC6A HOXD SNORA KCNH2 6.06E ALX MYBPC SEC14L UNC45B WNT5A TK SERPINB PNMA KRT HIST1H1B WNT7B PCDH LOC INHBE 6.51E PTPN LBX ZFHX LOC ASCL ENPP STOML SPSB4 5.24E TWIST KIF4A PNLDC ANKRD CNTN LOC E OR2T MIR DYNLRB AIPL1 1.86E DLX KCNF WNK SGOL GLIS SST

24 200 LLGL ACTN DUOX CCNE SLC6A MPO 1.33E EPHA ASB CCL HJURP C GJD4 6.30E SNORA RAPSN 2.92E FOXA COL22A ABCG COMP MIR27B RHEBL C10orf NHLH KRT NKX PAX LRTM ALX LPPR CYTL DRGX LPPR BLM DPYS LOC ADAM C14orf GPX LAMA HOXC MEOX RNF CASQ ANXA8L C13orf PLCH UBE2T CDC42BPG PRKAG C4orf NES C3orf HAMP NUDT9P KIF18A C6orf TMEM132B DNAI PASD LRRC CA LDLRAD KIAA ASTN TFRC GAL3ST HFE NWD FIT1 4.79E

25 234 TC2N IGF2BP ADH ZNF MIR HRNBP SERPINB FANCI CA MIR133B CLDN KLHL LONRF PEG ATRNL PEG3AS 1.51E HORMAD C13orf SNORD BRSK SCGB1A MYL SOX ATPGD OR2M RRP TMPRSS11E KBTBD PTX NEU TTLL STAC TRPM PPM1E MIR125A CENPE CCL INSM WDR KCNJ CCDC85A KIF B3GNT ANK TMEM PIPOX XG C5orf VEGFC C7orf SLPI BIRC PPP1R1A ZNF UGT2A DDN RARB ESRRG LRRC CTNND C11orf SCT 9.61E VWA3A ALK LRRTM N4BP GAGE NPPA

26 268 C21orf HRC MIRLET7I VGLL SHANK KIR3DS GPR SOX PAX CRMP B3GNT C12orf C1QTNF ANKRD34B 8.96E GAGE C1orf B4GALNT MYPN FGL SGCA SLC27A CSMD GRIA SNTG NCRNA NXPH BMP DPP SNORD ELAVL XYLT SLC26A FAM183A TMEM CTTNBP EFCBP ROPN1L CYP27B TSPAN SPINT GATA EPHA TSPAN COL20A ARSE GP SNORD36B RPL3L SNORA OTX TCN C15orf OR2A COL9A AQP CKAP2L C20orf KCNG LRRC4B MLF1IP GDNF TSG COL24A MCM ALDH1A MYBPC NME OIP

27 302 BIK PLSCR SNORA CHRNA ZP HMP SNORD83A GPRIN TMEM200B C6orf LTF ASF1B ADH1C GINS COLEC LMNB YSK RIPPLY GULP DEPDC1B ENPP CTRC MIR LRRC14B EFNA MAPK8IP SNORA RGAG UGT1A CACNG LRP TRPM SLC25A KIF2C LOC GMNN FLRT KCNC CNTNAP TOX LOC TRIM FRMPD PLA2G2A EPHX FAM65B GPR109A PBX PITPNC1 2.70E PCOLCE CCDC ABLIM ALOX12P SEMA5B GRIK ST8SIA C15orf NEB C1orf SIRT PTGER GRIN3A MYCBPAP CENPN LRRIQ ADAMTS MIR LRFN

28 336 BNC CBLN HOTAIR GSG PTPN20A PDLIM RNF PSAT MMP SLC29A CDH LCNL YBX POLQ TUBA RBM RADIL NTRK TP53TG CHEK CACNA1C GABRR DKFZP564J CENPA RBMS3 1.73E ST6GAL CYP26A NGFR CLSTN MIR30C MMP FOXA ABHD12B CEP NEK MELK LOC EXO FBLN P2RX WNT9B KIF18B TMEM HOXB ANXA8L SKA SNORD XRCC SPINT FOXM SNORD FRMD SNORA TACC AGAP RECQL MAML SLC1A TMEM C9orf SNORD TTF IQSEC CDC25A FER1L SLITRK AGR FAM89A

29 370 ODZ CPA MAML UBE2C FLJ SATL DMKN NUF VTRNA KCNG SNORA36B NPY MIR ANK PAPPA TMEM63C SORBS EME LY6H PRODH OR14C C2orf FHOD RASD SNORA NRCAM PION CDC C4orf CORO LRRIQ PDE4C EFHC FAM184A NFATC CCNE HDC KCND SLC5A ONECUT LTBP RELN DNAH LHX EPCAM LEFTY DPYD PLA2G4D AGBL DLK KLHDC7B ZSCAN12L NOSTRIN ARC MAPK DOK CACNA1G KCNJ OR2M1P SPOCK CLCNKB LINGO ODF C21orf ATHL HOXA CHL FLVCR

30 404 NNMT C10orf SLC8A GALNT FLJ C12orf C1RL NMU MRVI COBL TCTEX1D MEGF GLT25D GADD45G ATP8B NECAB IRX TNFRSF13C SNORD96A CCDC BOC CELA2B 8.58E SCTR RXRG NR2E RRM C1orf NDST FCER1A SLC38A TCEAL TRIM ALOX12B CDC CCDC85B CDCA CDC42EP RIMS C10orf SLC25A DLG GRM PGPEP CKB CES FNDC TCEAL HHIP PLP APOC PRDM CACNG SLC13A CAMK2N SCARNA HBQ EVPL HOXA BEST GGNBP CDH LOC C10orf TMEM STK ASB LOC ZNF

31 438 NRG GPR SLC7A RNF C6orf POM121L10P CRISPLD SYNPO2L OR2L SLC7A LRRC FAM71E DCLK BLCAP OSAP FLJ DBX CASC SLC16A MAGEA VTRNA NKAIN GALR LYG TBX XIRP BLID ASB WDR MKRN GALIG COX6A CMAH TGM SNORD HMX SNORA5A ZNF AHR C19orf C9orf OMG PDE1B SLC24A SAMD LGI MIR E2F MRC POU3F ALDH3A DHTKD C9orf BYSL ADAD ESPL SRD5A TNNI LOC ZNF RNF DHRS7C TRIM C20orf SPRR PIF SLFN TMCC

32 472 MAS CDK5R TMEM PALM ADCY LHX MICA TTK STAT KIF MAGEA ZIM MOV10L C8orf VIPR CNPY PREX HIST1H3I SNORD EXOC3L MPL GRWD FAM154B TRIP SFRP DAPL LYNX LILRA CCDC UPB LAMA MXD SPTB SEMA3D CTAG1A KRT GNAL MSTN OVOL RACGAP WDR ENO LMTK PLK TTC HAS MS4A8B CCDC FRMD C4orf PIB5PA CD300C CCDC TFAP2D FLT IGSF LRRK CORO Additional Genes of Interest EPHA MYCN NEBL TNNT PRKAR2B FOXO GREM MYOD