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Supplementary Data Supplementary Methods Mutation and microdeletion screening by high resolution melting High-throughput mutation screening of DIS3L2 exons 1-16 and HDAC4 was performed by Lightscanner high resolution melting analysis (Idaho Technology, Salt Lake City, UT). Exons 17-21 of DIS3L2 were not sequenced due to an apparent genomic duplication and consequent inability to uniquely amplify these exons. DNA samples were amplified using LightScanner mastermix under the manufacturer’s guidelines (Idaho Technology). After PCR, samples were heated at 0.1°C/s in the Lightscanner instrument and fluorescence was collected from 60 to 95°C. Melting curves were analyzed using LightScanner software (v2.0, Idaho Technology). Microdeletion screening across DIS3L2 was performed on paired normal- tumor samples using Lightscanner Lunaprobe SNP genotyping. Seven SNPs, ~60 Kb apart (rs2679184, rs12988522, rs4973500, rs3100586, rs3116179, rs923333 and rs2633254) were amplified in separate reactions and analyzed as above. Detailed conditions and primer/probe sequences are available on request. Variant amplicons were sequenced as described below. Direct sequencing was also performed for all exons where a common polymorphism might mask detection of a mutation by Lightscanner. Samples with known LOH were analyzed entirely by direct sequencing, since Lightscanner detects altered melting profiles of DNA heteroduplexes and these cannot exist in hemizygous samples. Sequencing of candidate genes Direct sequencing of exons and flanking consensus splice signals was performed for DIS3L2, GIGYF2, NPPC, HDAC4, TWIST2 and miR-562. PCR amplification was performed using HotStarTaq Mastermix and Q solution (Qiagen, Valencia, CA); all conditions and primers are available on request. PCR products were treated with shrimp alkaline phosphatase and exonuclease-I (New England Biolabs, Ipswich, MA) and sequenced using BigDye terminator chemistry on a 3730xl sequencer (Applied Biosystems, Foster City, CA). Sequencing products were analyzed using Mutation Surveyor software (Softgenetics, State College, PA). Bisulfite sequencing of DIS3L2 promoter Bisulfite sequencing of the DIS3L2 promoter CpG island (chr2:232,534,322-232,535,080, hg18 assembly) was performed only on tumors showing LOH across this region. Primers are available on request. DNA (1 μg) was bisulfite converted using the EpiTect Bisulfite kit (Qiagen) according to the manufacturer’s protocol. PCR amplification was carried out using HotStarTaq Mastermix and Q solution (Qiagen, Valencia, CA). The resulting PCR products were cloned using the TOPO TA cloning system (Invitrogen, Carlsbad, CA). Individual clones were miniprepped and sequenced using M13 reverse primer and BigDye terminator chemistry on a 3730xl sequencer (Applied Biosystems, Foster City, CA). The resulting sequences were aligned and analyzed using 4 Peaks (http://mekentosj.com) and MethTools (http://genome.imb-jena.de/methtools/). Supplementary Table 1: Functional annotation from bioinformatics analysis of putative miR-562 targets miR-Target Gene Description Chr Expression in Association with renal cell Gene Kidney* processes/disease (Metacore pathway tool) Symbols ABCA1 ATP-binding cassette, sub-family 9 Overexpressed Renal nephritis A (ABC1), member 1 AGTR1 angiotensin II receptor, type 1 3 Overexpressed Renal atrophy, Renal fibrosis, Renal and urological disease, Renal system process ALOX12 arachidonate 12-lipoxygenase 17 Not expressed Renal cell carcinoma BCAM basal cell adhesion molecule 19 Not expressed Renal and urological system development and (Lutheran blood group) function C3AR1 complement component 3a 12 Under expressed Renal system process receptor 1 CASP1 caspase 1, apoptosis-related 11 Under expressed Renal necrosis, Renal and urological disease cysteine peptidase (interleukin 1, beta, convertase) CCNE1 cyclin E1 19 Under expressed Renal cell carcinoma CHRNB3 cholinergic receptor, nicotinic, 8 Not expressed Renal system process beta 3 COL4A3 collagen, type IV, alpha 3 2 Overexpressed Renal nephritis, Renal and urological disease, (Goodpasture antigen) Renal and urological system development and function CTSL2 cathepsin L2 9 Overexpressed Renal and urological system development and function CUL2 cullin 2 10 Overexpressed Renal cell carcinoma DAB1 disabled homolog 1 (Drosophila) 1 Overexpressed Renal and urological disease EFNB2 ephrin-B2 13 Overexpressed Renal cell carcinoma ESR1 estrogen receptor 1 6 Under expressed Renal nephritis, Renal and urological disease, Renal cell carcinoma EYA1 eyes absent homolog 1 8 Under expressed Renal hypoplasia; overexpressed in Wilms (Drosophila) tumor FGFR1 fibroblast growth factor receptor 8 Under expressed Renal and urological disease 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome) GJA1 gap junction protein, alpha 1, 6 Under expressed Renal and urological system development and 43kDa function HES1 hairy and enhancer of split 1, 3 Under expressed Renal cell carcinoma (Drosophila) KCNJ13 potassium inwardly-rectifying 2 Overexpressed Renal tubular transport, inborn errors channel, subfamily J, member 13 LAMA1 laminin, alpha 1 18 Overexpressed Renal degradation MET met proto-oncogene (hepatocyte 7 Overexpressed Renal cell carcinoma; expressed in Wilms tumor growth factor receptor) MUC1 mucin 1, cell surface associated 1 Under expressed Renal cell carcinoma NFAT5 nuclear factor of activated T-cells 16 Overexpressed Renal hydronephrosis 5, tonicity-responsive NFATC3 nuclear factor of activated T- 16 Under expressed Renal hypertrophy cells, cytoplasmic, calcineurin- dependent 3 NFKB1 nuclear factor of kappa light 4 Overexpressed Renal inflammation, Renal cell carcinoma polypeptide gene enhancer in B- cells 1 (p105) NPHS1 nephrosis 1, congenital, Finnish 19 Overexpressed Renal necrosis, Renal and urological disease; type (nephrin) An association of congenital nephrotic syndrome, Finnish type and human NPHS1 occurs (OMIM: 256300) P2RY11 purinergic receptor P2Y, G- 19 Expressed Renal system process protein coupled, 11 PCMT1 protein-L-isoaspartate (D- 6 Under expressed Renal cell carcinoma aspartate) O-methyltransferase PRKD2 protein kinase D2 19 Under expressed Renal cell carcinoma PSEN1 presenilin 1 (Alzheimer disease 14 Under expressed Renal and urological system development and 3) function PTGDS prostaglandin D2 synthase 9 Under expressed Renal and urological disease 21kDa (brain) PTK2 PTK2 protein tyrosine kinase 2 8 Under expressed Renal cell carcinoma RANBP9 RAN binding protein 9 6 Under expressed Renal and urological disease SLC7A9 solute carrier family 7 (cationic 19 Overexpressed Renal aminoaciduria; Renal tubular transport, amino acid transporter, y+ inborn errors system), member 9 TBXA2R thromboxane A2 receptor 19 Overexpressed Renal system process TRPC6 transient receptor potential cation 11 Under expressed Renal and urological disease; An association of channel, subfamily C, member 6 focal segmental glomerulosclerosis and human TRPC6 occurs (OMIM: 603965) UCHL1 ubiquitin carboxyl-terminal 4 Under expressed Renal cell carcinoma esterase L1 (ubiquitin thiolesterase) *Expression information from WebGestalt, (http://bioinfo.vanderbilt.edu/webgestalt/). Expression is based on the data from the CGAP (Cancer Genome Anatomy Project) expressed sequence tag (EST) libraries and over/under-representation of individual genes in the tissue is evaluated by hypergeometric test. Gene Summary (NCBI RefSeq) Gene Ontology Annotations (http://www.geneontology.org/) The membrane-associated protein encoded by this gene is a member of the superfamily ATP binding/ATPase activity/anion transporter of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules activity/nucleotide binding/protein binding/sterol transporter across extra- and intracellular membranes. ABC genes are divided into seven distinct activity/cholesterol metabolism/lipid metabolism/steroid subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a metabolism/transport/integral to plasma member of the ABC1 subfamily. Members of the ABC1 subfamily comprise the only major membrane/membrane ABC subfamily found exclusively in multicellular eukaryotes. With cholesterol as its substrate, this protein functions as a cholesteral efflux pump in the cellular lipid removal pathway. Mutations in this gene have been associated with Tangier's disease and familial high-density lipoprotein deficiency. Angiotensin II is a potent vasopressor hormone and a primary regulator of aldosterone C-X-C chemokine receptor activity/angiotensin type II secretion. It is an important effector controlling blood pressure and volume in the receptor activity/bradykinin receptor activity/receptor cardiovascular system. It acts through at least two types of receptors. This gene encodes activity/rhodopsin-like receptor activity/G-protein signaling, the type 1 receptor which is thought to mediate the major cardiovascular effects of coupled to IP3 second messenger (phospholipase C angiotensin II. This gene may play a role in the generation of reperfusion arrhythmias activating)/circulation following restoration of blood flow to ischemic or infarcted myocardium. Not available arachidonate 12-lipoxygenase activity/hepoxilin-epoxide hydrolase activity/iron ion binding/lipoxygenase activity/metal ion binding/oxidoreductase activity/potassium channel inhibitor activity/anti-apoptosis/arachidonic acid metabolism/c Lutheran blood group glycoprotein is a member of the immunoglobulin superfamily and a transmembrane receptor activity/cell adhesion/signal receptor
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