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WO2015066640A1 - Kit and method for identifying individual responsiveness to steroid therapy of nephrotic syndrome - Google Patents

Kit and method for identifying individual responsiveness to steroid therapy of nephrotic syndrome Download PDF

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Publication number
WO2015066640A1
WO2015066640A1 PCT/US2014/063736 US2014063736W WO2015066640A1 WO 2015066640 A1 WO2015066640 A1 WO 2015066640A1 US 2014063736 W US2014063736 W US 2014063736W WO 2015066640 A1 WO2015066640 A1 WO 2015066640A1
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WIPO (PCT)
Prior art keywords
kit
genes
gene
patients
patient
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PCT/US2014/063736
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French (fr)
Inventor
William E. SMOYER
Shipra Agrawal
Richard F. RANSOM
Saraswathi SUNDARARAJAN
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Nationwide Childrens Hospital Inc
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Nationwide Childrens Hospital Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Nephrotic Syndrome is among the most common kidney diseases seen in both children and adults.
  • Nephrotic Syndrome (NS) is a condition caused by damage to the kidneys that leads to several symptoms, such as swelling, low blood protein levels, high cholesterol and triglyceride levels, but most notably, the release of excess protein in the urine (i.e., proteinuria).
  • NS is characterized by increased permeability in the capillaries of the glomerulus (i.e., the filtering unit of the kidney) which results in low blood protein levels and high levels of urine proteins, since the proteins freely pass from the blood into the urine.
  • Research over the past few decades has identified the importance of renal podocytes, the filtering cells of kidney, as a site of injury in NS.
  • kidneys affected by NS have small pores in the podocytes that are large enough to permit proteinuria.
  • the high urine protein levels disable the glomulerus from safely filtering blood, and results in symptoms of NS.
  • the primary therapy for NS is steroids, such as oral glucocorticoids (GC), which effectively result in remission for the majority of patients.
  • GC oral glucocorticoids
  • SRNS Steroid Resistant Nephrotic Syndrome
  • SSNS Steroid Sensitive Nephrotic Syndrome
  • glucocorticoids glucocorticoids
  • SRNS SR-induced side effects as well as progression of disease.
  • the kit comprises a plurality of binding reagents.
  • the kit comprises binding reagents , wherein each of the binding reagents specifically binds to a gene product encoded by a gene selected from the group of genes consisting of: RFFL, INPP1, MCM2, CSNK1G1, SIGLEC10, AGPAT6, DCAF8, FAM193A, RSPRY1, KCTD20, BLOC1S3, TOE1, ACAD10, SULF2, RAB8A,
  • TMEM14B TMEM14B
  • MTIF2 MTIF2
  • YBX1 PLSCR1
  • TAF13 TAF13
  • C220RF46 AHSA1, SAT1
  • DDX60L DDX60L
  • ZNF226 ZNF226.
  • the expression of said genes or gene products has been found to be significantly different between patients with Steroid-Resistant Nephrotic Syndrome and patients with Steroid-Sensitive Nephrotic Syndrome.
  • a plurality of reagents is provided wherein the reagents specifically bind to ten or more gene products encoded by genes selected from the group consisting of: RFFL, AGPAT6, SULF2, TMEM219, PRKCSH, STX4, GANAB, ZC3H18, EXOC7, ACINI, rfGAL, SH2D1B, TRMT6, NACC1, ZC3H12D, TOPBP1, GALNT2, CHMP4B, TRAPPC1, SPRYD3, MTMR14, S1PR5, NDUFS1, LGALS9, SUPT5H, JARID2, KLF2, AKIRINl, OCIADl, KIAA1033, UBE2W, LOC220930, ATP11B, and FGFR10P2.
  • the method comprises the following steps: a) assaying marker genes expressed in the patient, wherein the marker genes are selected from the group of genes consisting of: RFFL, INPP1, MCM2, CSNK1G1, SIGLEC10, AGPAT6, DCAF8, FAM193A, RSPRY1, KCTD20, BLOC1S3, TOE1, ACAD10, SULF2, RAB8A, NCRNA00294, ZNF318, CDK4, TMEM219, PRKCSH, SLC9A7, LEOl, STX4, PTPRK, GANAB, C170RF63, ZC3H18, MED 14, TRAPPC5, EXOC7, ACINI, ITGAL, SH2D1B, TRMT6, NACC1, RECQL5, ZC3H12D, PRR5L, UBE2J2, TOPBP1, GALNT2, BTN2A2, GPR108,
  • RNASEK RNASEK
  • PTAR1, PECAM1, KIAA1267, ZNF281, CX3CR1, SH3BGRL3, PITPNA MAPRE2, MGATl, LM02, S100A4, PCMTDl, SNX18, NFKBIZ, C130RF18, CLEC12A, USP15, C40RF3, CAB39, FCGR3A, GABARAPL1, C3AR1, TNFAIP8L2, RAB33B, ELF2, MBOAT2, SEMA4A, SKAP2, RAB8B, PPP1CB, FUT4, EMB, PTGER4, EPS 15, STIM1, GNLY, CNDP2, LONP2, LIN7C, MGC12916, ACTR1A, LST1, MAPKAPK3, GLRX, ARL8B, TBRG1, AFF3, MARK3, GCC1, C20RF29, ZMAT2, LCOR, CDC42, PHIP, RNF103, TNFSFIO, PTP4A
  • the method may further comprise treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non-steroidal therapies of Nephrotic Syndrome.
  • the method of treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non-steroidal therapies of Nephrotic Syndrome comprises administering a drug selected from the group consisting of adrenocorticotropic hormone, Cyclosporine, Tacrolimus, Mycophenolate mofetil, Plasmapheresis, column A immunoabsorbtion, and Rituximab.
  • Figure 1 shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 72-gene set.
  • Figure 2 shows differential clustering of 72 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
  • Figure 4 shows analysis of gene expression ratios of 72 genes using statistical methods.
  • Figure 6 shows arylsulfatase enzyme activity in SRNS and SSNS patient plasma samples.
  • Figure 7B shows differential clustering of 16 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
  • Figure 8A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 54-gene set.
  • Figure 8B shows differential clustering of 54 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
  • Figure 9A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 40-gene set.
  • Figure 10A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 24-gene set.
  • Figure 10B shows differential clustering of 24 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
  • Figure 11A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 78-gene set.
  • Figure 12A shows a volcano plot of the expression ratios of genes following
  • Figure 12B shows differential clustering of 85 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
  • Figure 13A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 76-gene set.
  • Figure 13B shows differential clustering of 76 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment
  • Figure 14A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 71 -gene set.
  • Figure 14B shows differential clustering of 71 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
  • kits are provided.
  • the kit of the present disclosure comprises one or more binding reagents, or a plurality of binding reagents.
  • the total reagents of the kit of the present disclosure may range from about 1 to about 500 reagents.
  • the reagents of the kit may specifically bind to one or more genes or a gene products.
  • the reagents of the kit may comprise, but are not limited to nucleic acids, antibodies, oligonucleotides, peptides and polypeptides, primers, probes, proteins, compounds, transcription factors, enzymes, nucleotides and polynucleotides, labels, tags, amino acids, molecules, receptors, hormones, vitamins, ligands, genes, and gene products.
  • the nucleic acids of the present kit may be RNA or DNA, or synthetic RNA or DNA, for example the DNA may be copy DNA (i.e., cDNA).
  • the average number of reagents of the kit described herein ranges from about
  • nucleic acids of one embodiment of the kit may comprise a set of two primers and a probe (i.e., 3 reagents) for detecting mRNA expressed by said genes.
  • Each set of two primers may include one forward primer (i.e., that amplifies in the 5' to 5' direction) and one reverse primer (i.e., that amplifies in the 3' to 5' direction).
  • the nucleic acids of the kit may only comprise a set of two or more primers (i.e., 2 reagents) for detecting mRNA expressed by said genes.
  • Primers or oligonucleotides and probes of the present invention may be of any length or size, including but not limited to between about 8 to about 75 bases, from about 8 to about 50, from about 10 to about 70 bases, from about 15 to about 65 bases from about 10 to about 50 bases, from about 15 to about 45 bases, from about 15 to about 35 bases, from about 10 to about 50 bases, from about 10 to about 30 bases, from about 10 to about 25 bases, from about 15 to about 30 bases, from about 15 to about 28 bases, from about 15 to about 25 bases, from about 10 to about 25 bases, from about 10 to about 20 bases, from about 10 to about 15 bases, and from about 5 to about 15 bases.
  • Reagents of the present kit may be designed to bind or hybridize to any region of said genes or gene products by methods known to persons of ordinary skill in the art.
  • reagents of nucleic acids may be designed to bind to introns, exons, 5'-UTR, 3'- UTR, and/or promoter regions of said genes or gene products.
  • the coding regions of said genes or said gene products are ideal locations to design binding reagents of the present kit in order to detect gene expression.
  • the nucleic acids of the kit may also comprise only one or more probes (i.e., 1 reagent) for detecting mRNA expressed by said genes or said gene products.
  • the probes of the kit may further comprise a detection marker, wherein the marker may be a label.
  • the label may be a fluorescent label or a non-fluorescent label.
  • a fluorescent label may include, but is not limited to fluorescein (e.g., 6-FAM), tetrachlorofluorescein (e.g., TET), VIC, SYBR Green, etc.
  • the nucleic acids of the present kit have sufficient sequence identity to bind specifically to the target gene product under moderate to stringent conditions.
  • the nucleic acid probe and/or primer of the kit should have, for example, from about 80% to about 100% sequence identity, such as about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100% sequence identity with the target sequence of the gene product.
  • said genes or gene products comprise proteins.
  • the reagents may comprise antibodies for detecting the proteins expressed by said genes or said gene products.
  • reagents of the kit may further include any substance necessary to facilitate the binding of the reagents to said genes or said gene products.
  • reagents of the present disclosure may include enzymes, one or more nucleotides, water, buffers and/or master mixes (i.e., buffers containing additional reagents such as nucleotide and/or polymerase), solutions, solvents, antibodies, labels, ions (e.g., Mg 2+ or Mn 2+ ), oligonucleotides (e.g., primers), probes, salts, stabilizers (e.g., MgCl 2 ), and control samples, and any other substances known in the art that are necessary or preferable for successful binding and amplification of a reagent to a gene or a gene product.
  • enzymes of the present disclosure include any enzyme known to facilitate binding and amplification of nucleic acids, including but not limited to DNA polymerase (i.e., Taq polymerase) or Reverse Transcriptase.
  • Nucleotides of the present invention include one or more deoxynucleoside or deoxynucleotide triphosphates or dntps, such as dATP, dTTP, dCTP, and dGTP.
  • the control sample of the present kit may comprise any gene, tissue, cell, or marker wherein the levels of said genes or gene products do not significantly differ in patients with Steroid-Resistant Nephrotic Syndrome and patients with Steroid-Sensitive Nephrotic Syndrome.
  • the control sample may be a Nephrotic Syndrome patient that is sensitive and/or responsive to steroid treatment.
  • the control sample may be normal kidney tissue.
  • the kit of the present invention may further comprise supplies.
  • supplies of the kit may include any known supplies necessary to facilitate binding of a reagent to a gene or gene product.
  • Exemplary supplies of the kit include any immobilized support that facilitates the binding of a reagent to a gene or gene product, including, but not limited to tubes, plates, chips, and strips.
  • a plate, a tube, a chip, or a strip may comprise from 1 to about 500 wells, spots, or locations for the reagents to bind to the genes or gene products.
  • the supplies of the kit comprise instructional materials.
  • the kit may comprise instructions for the detection and identification of a patient with resistance to steroid treatment of Nephrotic Syndrome, including
  • the reagents of the kit disclosed herein may bind to genes or gene products ranging from about 1 to about 100 genes or gene products.
  • the genes or gene products of the present kit and method may range from about 1 to about 95, from about 1 to about 90, from about 1 to about 85, from about 1 to about 72, from about 1 to about 78, from about 1 to about 76, from about 1 to about 71, from about 1 to about 71, from about 1 to about 54, from about 1 to about 40, from about 1 to about 34, from about 1 to about 24, and from about 1 to about 16, from about 1 to about 12, from about 1 to 4, from about 1 to 3, from about 1 to 2, from about 2 to about 85, from about 2 to about 78, from about 2 to about 76, from about 2 to about 72, from about 2 to about 71, from about 2 to about 54, from about 2 to about 40, from about 2 to about 34, from about 2 to about 24, and from about 2 to about 16, from about 2 to about 12, from about 2 to 3, from about 2 to 4, from about 3 to
  • a kit may include reagents to detect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 20, 24, 34, 40, 50, 54, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 85, 90, 95, or 100 genes or gene products.
  • Reagents of the present kit specifically hybridize or bind to one or more marker genes or gene products of the present disclosure and in doing so, are capable of identifying a patient who is resistant to steroid treatment of Nephrotic Syndrome with an accuracy of 95% or greater. More specifically, said genes or said gene products of the present invention have differential expression among NS patient groups.
  • genes and said gene products differentiate between patients having Steroid-Resistant Nephrotic Syndrome (SRNS) and patients having Steroid-Sensitive Nephrotic Syndrome (SSNS). Accordingly, the genes and gene products of the present kit are also utilized in the method of identifying patients with SRNS and SSNS as described herein.
  • SRNS Steroid-Resistant Nephrotic Syndrome
  • SSNS Steroid-Sensitive Nephrotic Syndrome
  • An exemplary gene or gene product of the presently described kit and method is SULF2.
  • Reagents of the present kit may bind to a gene product of the SULF2 gene.
  • the mRNA sequence used to derive cDNA of the SULF2 gene is as follows:
  • Reagents of the present kit may bind specifically to SEQ ID NO: 7.
  • reagents that bind to a SULF2 gene product may have about 80% to about 100% identity to SEQ ID NO: 7, wherein the sequence identity may be from about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100% with SEQ ID NO: 7.
  • the amino acid sequence of the SULF2 protein is also described herein as SEQ ID NO: 8.
  • Reagents of the present kit may bind specifically to SEQ ID NO: 8.
  • antibody, peptide, and/or polypeptide reagents of the present kit may bind to SEQ ID NO: 8.
  • reagents of the present kit or method may comprise gene products of the genes described herein.
  • the expression of said gene products is significantly different between patients with Steroid-Resistant Nephrotic
  • Reagents that bind to the genes and gene products of the kit described herein may be purchased commercially.
  • persons of ordinary skill in the art may also use publicly available information, for example, the NIH's GENE database, to design, prepare, and synthesize reagents of the present kit to hybridize to the genes or gene products of the kit by methods known to persons of ordinary skill in the art.
  • Reagents of the present invention may specifically bind to gene products of genes selected from the group consisting of the following: RFFL, INPP1, MCM2, SNK1G1, SIGLEC10, AGPAT6, DCAF8, FAM193A, RSPRY1, KCTD20, BLOC1S3, TOE1,
  • a kit comprises reagents for measuring the gene expression of SULF2 (GER(S2/Sl)suLF2 or GER
  • the kit may comprise reagents for detecting one or more of the other discriminator gene products disclosed herein.
  • a method of identifying a patient who is resistant to steroid therapy of Nephrotic Syndrome comprises the steps of a) assaying marker genes products expressed in the patient through the use of reagents that bind to the gene products of the genes identified in the immediate above paragraph, b) analyzing the expression levels of the marker gene products relative to a control sample, wherein the control sample is taken from a patient that has Nephrotic Syndrome and is responsive to steroid treatment, or relative to standard values that have been established based on population data, including for example from patients that have Nephrotic Syndrome that is responsive to steroid treatment, and c) identifying a patient as being resistant to steroid treatment of Nephrotic Syndrome based on the relative expression of said marker genes.
  • the method of the present invention may further comprise treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non- steroidal therapies of Nephrotic Syndrome.
  • the method further comprises a step of measuring the enzymatic activity of SULF2, (S2/S1) SULF2 - It is believed that expression of SULF2 (GER(S2/S1) SULF2 ) and enzyme analysis of SULF2 (S2/S1) SULF2 ) coupled with the other discriminator genes disclosed herein provides an effective means to identify Nephrotic Syndrome patients that will be non-responsive to steroidal treatments.
  • the method comprises treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non-steroidal therapy including for example, administering a drug selected from the group consisting of adrenocorticotropic hormone, Cyclosporine, Tacrolimus, Mycophenolate mofetil,
  • Plasmapheresis column A immunoabsorbtion, and Rituximab.
  • Nephrotic Syndrome as defined herein is a kidney disorder characterized by a group of symptoms in a patient that include, but are not limited to, protein in the urine, low blood protein levels (e.g., albumin), and swelling.
  • the patient of the present disclosure may be an adult patient or a pediatric patient.
  • An exemplary patient of the present invention is a pediatric patient, meaning a patient that is 18 years old or younger.
  • intron refers to any nucleic acid sequence comprised in a gene (or expressed nucleotide sequence of interest) that is transcribed but not translated. Introns include untranslated nucleic acid sequence within an expressed sequence of DNA, as well as a corresponding sequence in RNA molecules transcribed therefrom. Introns may be used in combination with a promoter sequence to enhance translation and/or mRNA stability.
  • 5 '-untranslated region refers to an untranslated segment in the 5' terminus of pre-mRNAs or mature mRNAs.
  • a 5'-UTR typically harbors on its 5' end a 7-methylguanosine cap and is involved in many processes such as splicing, polyadenylation, mRNA export towards the cytoplasm, identification of the 5' end of the mRNA by the translational machinery, and protection of the mRNAs against degradation.
  • 3 '-untranslated region refers to an untranslated segment in a 3' terminus of the pre-mRNAs or mature mRNAs.
  • this region harbors the poly- (A) tail and is known to have many roles in mRNA stability, translation initiation, and mRNA export.
  • polyadenylation signal refers to a nucleic acid sequence present in mRNA transcripts that allows for transcripts, when in the presence of a poly- (A) polymerase, to be polyadenylated on the polyadenylation site, for example, located 10 to 30 bases downstream of the poly-(A) signal.
  • polyadenylation signals are known in the art and are useful for the present invention.
  • isolated refers to a biological component (including a nucleic acid or protein) that has been separated or removed from other biological components in the cell of the organism in which the component naturally occurs (i.e., other chromosomal and extra-chromosomal DNA).
  • a biological component including a nucleic acid or protein
  • polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • purified in reference to nucleic acid molecules does not require absolute purity (such as a homogeneous preparation). Instead, “purified” represents an indication that the sequence is relatively more pure than in its native cellular environment. For example, the “purified” level of nucleic acids may be at least 2-5 fold greater in terms of concentration or gene expression levels as compared to its natural level. Additionally a “purified polypeptide” is used herein to describe a polypeptide which has been separated from other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates.
  • the claimed DNA molecules may be obtained directly from total DNA or from total RNA.
  • cDNA clones and copies are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified, naturally occurring substance (messenger RNA).
  • messenger RNA messenger RNA
  • the construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA).
  • Individual cDNA clones may be purified from the synthetic library by clonal selection of the cells carrying the cDNA library.
  • the process which includes the construction of a cDNA library from mRNA and purification of distinct cDNA clones yields an approximately 10 6 -fold purification of the native message.
  • a DNA sequence may be cloned into a plasmid.
  • a clone is not naturally occurring, but rather is preferably obtained via manipulation of a partially purified, naturally occurring substance, such as a genomic DNA library.
  • purification of at least one order of magnitude preferably two or three orders, and more preferably four or five orders of magnitude, is favored in these techniques.
  • nucleic acid molecules and proteins that have been “purified” include nucleic acid molecules and proteins purified by standard purification methods.
  • the term “purified” also embraces nucleic acids and proteins prepared by recombinant DNA methods in a host cell, as well as chemically- synthesized nucleic acid molecules, proteins, and peptides.
  • recombination has occurred. It also includes a molecule (e.g. , a vector, plasmid, nucleic acid, polypeptide, or a small RNA) that has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration may be performed on the molecule within, or removed from, its natural environment or state.
  • a molecule e.g. , a vector, plasmid, nucleic acid, polypeptide, or a small RNA
  • the alteration may be performed on the molecule within, or removed from, its natural environment or state.
  • the term "expression” refers to the process by which a polynucleotide is transcribed into mRNA (including small RNA molecules) and/or the process by which the transcribed mRNA (also referred to as "transcript") is subsequently translated into peptides, polypeptides, or proteins.
  • Gene expression may be influenced by external signals, for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene may also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules, such as mRNA, or through activation, inactivation,
  • Gene expression may be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, qRT-PCR, qPCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).
  • nucleic acid molecule As used herein, the terms “nucleic acid molecule,” “nucleic acid,” or
  • polynucleotide refers to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms, and mixed polymers thereof.
  • a "nucleotide” may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of either type of nucleotide.
  • a nucleic acid molecule is usually at least ten bases in length, unless otherwise specified. The terms may refer to a molecule of RNA or DNA of indeterminate length. The terms include single- and double-stranded forms of DNA.
  • a nucleic acid molecule may include either or both naturally- occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of ordinary skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally-occurring nucleotides with an analog, intemucleotide modifications (e.g., uncharged linkages, such as, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages, such as, phosphorothioates,
  • uncharged linkages such as, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • charged linkages such as, phosphorothioates
  • nucleic acid molecule also includes any topological conformation, including single- stranded, double- stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.
  • RNA is made by sequential addition of ribonucleotide-5' -triphosphates to the 3' terminus of the growing chain with a requisite elimination of the pyrophosphate.
  • discrete elements e.g., particular nucleotide sequences
  • upstream relative to a further element if they are bonded or would be bonded to the same nucleic acid in the 5' direction from that element.
  • discrete elements may be referred to as being "downstream” relative to a further element if they are or would be bonded to the same nucleic acid in the 3' direction from that element.
  • base position refers to the location of a given base or nucleotide residue within a designated nucleic acid.
  • a designated nucleic acid may be defined by alignment with a reference nucleic acid.
  • hybridization refers to a process where oligonucleotides and their analogs hybridize by hydrogen bonding, which includes Watson- Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary bases.
  • nucleic acid molecules consist of nitrogenous bases that are either pyrimidines, such as cytosine (C), uracil (U), and thymine (T), or purines, such as adenine (A) and guanine (G).
  • oligonucleotide refers to a short nucleic acid polymer. Oligonucleotides may be formed by cleavage of longer nucleic acid segments or by
  • oligonucleotides may bind to a complementary nucleotide sequence, they may be used as probes for detecting DNA or RNA.
  • Oligonucleotides composed of DNA may be used in Polymerase Chain Reaction, a technique for the amplification of small DNA sequences.
  • Polymerase Chain Reaction an oligonucleotide is typically referred to as a "primer" which allows a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.
  • PCR Polymerase Chain Reaction
  • sequence information from the ends of the region of interest or beyond needs to be available, so that oligonucleotide primers may be designed.
  • PCR primers will be identical or similar in sequence to opposite strands of the nucleic acid template to be amplified.
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material.
  • PCR may be used to amplify specific RNA sequences or DNA sequences from total genomic DNA and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc.
  • the term "primer” refers to an oligonucleotide capable of acting as a point of initiation of synthesis along a complementary strand when conditions are suitable for synthesis of a primer extension product.
  • the synthesizing conditions include the presence of four different deoxyribonucleotide triphosphates (i.e., A,T,G, and C) and at least one polymerization-inducing agent or enzyme such as Reverse Transcriptase or DNA polymerase. These reagents are typically present in a suitable buffer that may include constituents which are co-factors or which affect conditions, such as pH and the like at various suitable temperatures.
  • a primer is preferably a single strand sequence, such that amplification efficiency is optimized, but double stranded sequences may be utilized.
  • the term "probe” refers to an oligonucleotide or polynucleotide sequence that hybridizes to a target sequence.
  • qRT-PCR quantitative Real Time Polymerase Chain Reaction or qRT-PCR, such as the TaqMan ® or TaqMan ® -style assay procedure (e.g., SYBR Green)
  • the probe hybridizes to a portion of the target situated between the annealing site of the two primers.
  • a probe includes about eight nucleotides, about ten nucleotides, about fifteen nucleotides, about twenty nucleotides, about thirty nucleotides, about forty nucleotides, or about fifty nucleotides, or about sixty nucleotides, or about seventy nucleotide, or about eighty nucleotides, or about eighty- five nucleotides. In some embodiments, a probe includes from about eight nucleotides to about fifteen nucleotides.
  • a probe may further include a detectable label, such as, a radioactive label, a biotinylated label, a fluorophore (e.g., Texas-Red ® , fluorescein isothiocyanate, etc.,).
  • the detectable label may be covalently attached directly to the probe oligonucleotide, such that the label is located at the 5' end or 3' end of the probe.
  • a probe comprising a fluorophore may also further include a quencher dye (e.g. , Black Hole QuencherTM, Iowa BlackTM, etc.).
  • sequence identity or “identity” may be used interchangeably and refer to nucleic acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity refers to a value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences or amino acid sequences) over a comparison window, wherein the portion of a sequence in the comparison window may comprise additions, substitutions, mismatches, and/or deletions (i.e. , gaps) as compared to a reference sequence in order to obtain optimal alignment of the two sequences.
  • a percentage is calculated by determining the number of positions at which an identical nucleic acid or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.
  • Methods for aligning sequences for comparison are well known.
  • Various bioinformatics or computer programs and alignment algorithms, such as BLAST, ClustalW, and Sequencher, GAP 10, and others are also well known in the art and/or may be used accordingly.
  • NCBI National Center for Biotechnology Information
  • BLASTTM Altschul et al. (1990) J. Mol. Biol. 215:403-10
  • BLASTTM Altschul et al. (1990) J. Mol. Biol. 215:403-10
  • Bethesda, MD National Center for Biotechnology Information
  • Blastn the "Blast 2 sequences" function of the BLASTTM (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.
  • promoter refers to a region of DNA that is generally located upstream of a gene (i.e., towards the 5' end of a gene) and is necessary to initiate and drive transcription of the gene.
  • a promoter may permit proper activation or repression of a gene that it controls.
  • a promoter may contain specific sequences that are recognized by transcription factors. These factors may bind to a promoter DNA sequence, which results in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene.
  • the promoter generally refers to all gene regulatory elements located upstream of the gene, including, 5'-UTR, introns, and leader sequences.
  • transformation encompasses all techniques in which a nucleic acid molecule may be introduced into a cell. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation; lipofection; microinjection, bacterial-mediated transfer; direct DNA uptake; and microprojectile
  • stable transformation refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms.
  • Transient transformation refers to the introduction of a nucleic acid fragment into the nucleus or DNA-containing organelle of a host organism, resulting in gene expression without genetically stable inheritance.
  • transduce refers to a process where a virus transfers nucleic acid into a cell.
  • transgene refers to an exogenous nucleic acid sequence.
  • a transgene is a gene sequence, such as a gene encoding an industrially or pharmaceutically useful compound.
  • a transgene is an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence.
  • a transgene may contain regulatory sequences operably linked to the transgene (e.g. , a promoter, intron, 5'-UTR, or 3'-UTR).
  • a nucleic acid of interest is a transgene.
  • a nucleic acid of interest is an endogenous nucleic acid, wherein additional genomic copies of the endogenous nucleic acid are desired, or a nucleic acid that is in the antisense orientation with respect to the sequence of a target nucleic acid in a host organism.
  • heterologous coding sequence is used to indicate any polynucleotide that codes for, or ultimately codes for, a peptide or protein or its equivalent amino acid sequence, e.g. , an enzyme, that is not normally present in the host organism and may be expressed in the host cell under proper conditions.
  • heterologous coding sequences may include one or additional copies of coding sequences that are not normally present in the host cell, such that the cell is expressing additional copies of a coding sequence that are not normally present in the cells.
  • the heterologous coding sequences may be RNA or any type thereof (e.g. , mRNA), DNA or any type thereof (e.g. , cDNA), or a hybrid of RNA/DNA.
  • Examples of coding sequences include, but are not limited to, full-length transcription units that comprise such features as the coding sequence, introns, promoter regions, 5'-UTR, 3'-UTR, and enhancer regions.
  • Heterologous coding sequences also include the coding portion of the peptide or enzyme (i.e. , the cDNA or mRNA sequence), the coding portion of the full-length transcriptional unit (i.e., the gene comprising introns and exons), "codon optimized” sequences, truncated sequences or other forms of altered sequences that code for the enzyme or code for its equivalent amino acid sequence, provided that the equivalent amino acid sequence produces a functional protein.
  • Such equivalent amino acid sequences may have a deletion of one or more amino acids, with the deletion being N-terminal, C-terminal, or internal. Truncated forms are envisioned as long as they have the catalytic capability indicated herein.
  • control refers to a sample used in an analytical procedure for comparison purposes.
  • a control can be "positive” or “negative”.
  • a positive control such as a sample from a known plant exhibiting the desired expression
  • a negative control such as a sample from a known plant lacking the desired expression.
  • hybridize As used herein, the terms “hybridize,” “bind(s),” “specifically bind(s),” “specifically hybridize(s)” and/or “specifically complementary” are terms that indicate a sufficient degree of complementarity, such that stable and specific binding occurs between the nucleic acid molecule and a target nucleic acid molecule.
  • Hybridization between two nucleic acid molecules involves the formation of an anti-parallel alignment between the nucleic acid sequences of the two nucleic acid molecules. The two molecules are then able to form hydrogen bonds with corresponding bases on the opposite strand to form a duplex molecule that, if it is sufficiently stable, is detectable using methods well known in the art.
  • a nucleic acid molecule need not be 100% complementary to its target sequence to be specifically hybridizable. However, the amount of sequence complementarity that must exist for hybridization to be specific is a function of the hybridization conditions used.
  • stringent conditions encompass conditions under which hybridization will only occur if there is less than 20% mismatch (i.e., at least 80% identity) between the hybridization molecule and a sequence within the target nucleic acid molecule.
  • Stringent conditions include further particular levels of stringency.
  • “moderate stringency” conditions are those under which molecules with more than 20% sequence mismatch will not hybridize; conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize; and conditions of "very high stringency” are those under which sequences with more than 5% mismatch will not hybridize. The following are representative, non-limiting hybridization conditions.
  • High Stringency condition detect sequences that share at least 90% sequence identity, and include, but are not limited to hybridization in 5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 16 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 20 minutes each.
  • 5x SSC buffer wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.
  • 2x SSC buffer wherein the SSC buffer contains a detergent such as SDS, and additional reagents like
  • Moderate Stringency conditions detect sequences that share at least 80% sequence identity, and include, but are not limited to hybridization in 5x-6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65-70 °C for 16-20 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 5-20 minutes each; and wash twice in lx SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 55-70 °C for 30 minutes each.
  • 5x-6x SSC buffer wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.
  • 2x SSC buffer wherein the SSC buffer contains a detergent
  • detectable marker refers to a label capable of detection, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme.
  • detectable markers include, but are not limited to, the following: fluorescent labels (e.g. , FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g. , horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g. , leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • a detectable marker may be attached by spacer arms of various lengths to reduce potential steric hindrance.
  • detecting is used in the broadest sense to include both qualitative and quantitative measurements of a specific molecule, for example, measurements of a specific polypeptide.
  • treating includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
  • treating Nephrotic Syndrome will refer in general to altering reducing and/or alleviating clinical symptoms of said disease.
  • treating Nephrotic Syndrome may comprise, treating the patient with steroids, such as corticosteroids, such as prednisone or prednisolone, or glucocorticoids (GCs).
  • Treatment of Nephrotic Syndrome (NS) with non-steroidal therapy may also occur in patients that are unresponsive or resistant to treatment with steroids.
  • steroids such as corticosteroids, such as prednisone or prednisolone, or glucocorticoids (GCs).
  • Treatment of Nephrotic Syndrome (NS) with non-steroidal therapy may also occur in patients that are unresponsive or resistant to treatment with steroids.
  • adrenocorticotropic hormone (ACTH) Cyclosporine
  • TAC Tacrolimus
  • MMF Mycophenolate mofetil
  • Rituximab are known in the art to be non-steroidal treatment alternatives for patients that are resistant to steroid treatment of NS.
  • Adrenocorticotropic hormone (ACTH) reduces proteinuria in nephrotic patients during treatment focused on lipid-lowering effects. It has been reported that 2 mg/week for 1 year of ACTH was as effective as methylprednisolone pulses and cytotoxic drugs. In addition, ACTH reduced proteinuria in children non- responding to traditional therapy, however, without protection on functional decline.
  • Cyclosporine CYA reduced the relative risk of persistent NS and produced significant benefits in adults.
  • CYA reduced proteinuria in 70-80% of patients with steroid-resistant NS, lasting after drug withdrawal in 40% of patients.
  • the frequency of either complete or partial remission of NS was significantly higher in the group receiving CYA (85% versus 55%), albeit without difference in renal
  • TAC in association with steroids in CYA-resistant Nephrotic Syndrome induced remission in 12/25 adults, with reversible nephrotoxicity in 40% of adult patients.
  • MMF Mycophenolate mofetil
  • an "effective" amount or a "therapeutically effective amount” of a compound refers to a nontoxic but sufficient amount of the compound to provide the desired effect.
  • desired effects of an effective amount of a compound would be the prevention or treatment of Nephrotic Syndrome, as measured, for example, by a decrease of protein in the urine, normal to higher blood protein levels (e.g., albumin) or levels of electrolytes, creatinine, and blood urea nitrogen (BUN), and a decrease in swelling in the body, for example on eyes, face, ankles, or feet.
  • blood protein levels e.g., albumin
  • BUN blood urea nitrogen
  • the amount that is "effective” will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • NS patients of the present invention comprise adults or children, adults and children, but are preferably pediatric patients.
  • Steroid response (e.g., steroid-sensitivity, steroid-resistance, and steroid- dependence) was assessed in patients approximately twelve months after NS disease presentation, such that patients were previously classified as "steroid- sensitive” and "steroid- resistant” according to clinical parameters.
  • Two blood samples were collected from each NS patient, one at presentation (sample SI), so before steroid therapy, and the second typically about 4 weeks to about 8 weeks after the initiation of steroid therapy (sample S2).
  • Total leukocytes were isolated from patient blood from eleven pediatric patients having SSNS and SRNS by standard protocols known in the art and stored at -140°C in frozen PaxGene tubes.
  • the leukocytes in frozen PaxGene tubes were processed to isolate total peripheral blood RNA according to the manufacturer's instructions (PreAnalytiX).
  • RNA samples (SI and S2) taken from the eleven patients were processed for transcriptome-wide RNA sequencing analysis using RNAseq, according to the
  • samples were assessed for gene expression using quantitative Real Time Polymerase Chain Reaction (qRT-PCR) analysis or aryl sulfatase enzyme activity assays. Samples from 38 NS patients were also processed for qRT-PCR and aryl sulfatase enzyme assays only (see Table 1).
  • qRT-PCR quantitative Real Time Polymerase Chain Reaction
  • Total leukocyte RNA was further prepared using Trizol reagent (Life Technologies).
  • the NuGen Ovation RNASeq System (San Carlos, CA) was used to generate cDNA libraries from 10 ng of total RNA per sample (NuGen Technologies, San Carlos, CA).
  • This method uses poly-dT and random hexamer primers for cDNA synthesis, the latter designed to reduce cDNAs from rRNA by at least 90%.
  • the NuGen cDNA library preparation was able to capture and remove RNA lacking poly A tails, including ncRNA,
  • RNAs, etc. since sequence analysis on annotated coding genes only was of interest.
  • a linear amplification step enables RNAseq analysis on small amounts of RNA ranging from about 10 to about 20 ng of total RNA.
  • Samples were processed to produce 8-barcoded, paired-end libraries or 16-barcoded, single-end libraries. Samples were sequenced using the SOLiD
  • Peripheral blood cDNA was produced from patient whole-blood RNA
  • RNAseq for the eleven NS patients (having paired samples SI and S2, from before and after GC treatment, respectively) were aligned to the International Human Genome Sequencing Consortium human reference sequence (Hgl9 ref). This gene alignment was performed using gene annotation from the University of California at Santa Cruz's genome browser and SOLiD Lifescope v2.5.1 (Life Technologies). Samples were aligned in an 8-barcode set with paired-end reads (with 50 bases sequenced in the forward direction and 35 bases for the reverse) or in a 16-barcode set with single-end alignment performed for the 75 bases sequenced in the forward direction.
  • the primary data set consisted of expression values for greater than 20,000 genes, which were reduced to 15,092 genes, 7334 genes, and 3280 genes by the removal of genes with little or no expression in one or several of the patient samples. The remaining genes were evaluated for gene expression. Gene expression values were used to calculate the Gene Expression Ratio (GER) in patients before (SI) and after (S2) treatment with GCs, annotated as the GER(S1/S2) value. Table 1. Pediatric patient clinical data on steroid treatment SULF2 expression and arylsufatase activity values.
  • Gene expression of coding exons was measured in reads per kilobase per million reads, as reported by the wtcounts module of the SOLid Lifescope technology, and was calculated based on reads that aligned to coding regions of genes annotated by RefSeq.
  • the number of sequence fragments generated and mapping percentages had an average of approximately 60% of generated reads for each sample mapping to the genome. Overall, a number from about 1 million to about 200 million fragments may be generated per sample. Preferably, from about 1 million to about 150 million fragments, from about. 2 million to about 135 million fragments, from about 3 million to about 125 million fragments, from about 4 million to about 120 million, or from about 5 million to about 112 million fragments are generated per sample.
  • read alignments were concentrated into peaks at known exons, other regions of the genome such as introns and intergenic space also showed read coverage. Mature mR A reads derived from known exons were included in this study, whereas all other expressed sequences were excluded.
  • GER(S1/S2) values of 7334 genes were initially analyzed by volcano plotting to identify a smaller set of genes that could discriminate between patients with SSNS and SRNS (see Figure 1).
  • i is the mean GER(S1/S2) value for SSNS patients
  • r 2 is the mean GERCS1/S2) value for SRNS patients.
  • Figure 1 shows the difference in ri and r 2 (f'i-r 2 ) for the SSNS and SRNS patient groups, respectively, as plotted on the X-axis.
  • Figure 1 identifies 72 genes (in the upper right and upper left regions of the plot) that were differentially expressed in the leukocytes of both NS patient groups following the treatment with GCs. Names of the 72 genes and their gene symbols or IDs are listed in Table 2, including SULF2. To independently assess the discriminatory potential of NS patients by these 72 genes, the genes were hierarchically grouped in a clusterogram according to their gene expression patterns as expressed by their GER(S1/S2) values. This clustering method calculates the pairwise (Euclidian) distances between all pairs of genes.
  • Figure 2 shows the resulting cluster tree; the left side of the tree is a cluster representing the Set I gene group (genes 1-57) that is distinctly partitioned from the Set II gene cluster (genes 58-72) on the right side of the tree. Cluster analysis of these genes (see top of Figure 2) clearly
  • SRNS and SSNS patient groups were distinguished by characteristic clusters of increased (light) and decreased (dark) GER(S1/S2) values following glucocorticoid (GC) treatment.
  • the GER(S1/S2) values of Figure 2 correlate to gene expression, such that light colors indicate upregulated gene expression and dark colors represent downregulated gene expression. Accordingly, Set I genes tended to exhibit a higher GER(S1/S2) value in SRNS patients, as compared to the SSNS patients where the GER(S1/S2) value for these genes tended to be lower.
  • solute carrier family 2 (facilitated glucose transporter)
  • FIG. 3 shows a scatter diagram where the GER(S1/S2) values of the 72 genes differentially expressed in SRNS and SSNS patients are plotted against each other.
  • Figure 3 demonstrates a clear and linear separation of Set I genes (diamond symbols) and Set II genes (round symbols), and thereby also of the SRNS patients and the SSNS patients.
  • sequencing methods such as RNAseq produce a plethora of information
  • any sequencing method known in the art may be used in the present invention.
  • sequencing methods comprising next generation sequencing, high-throughput, and low-throughput sequencing, and other sequencing methods known in the art may all be used in the present invention.
  • the subsequent sequencing data analysis remains a challenge.
  • SULF2 and its paralog SULF1 are distinguished by both their extracellular location coupled with endo- sulfatase activity that strictly requires a neutral pH, as is found in the extracellular space. Both SULF2 and SULF1 exhibit the same substrate specificity and cannot be distinguished biochemically. In particular, the biochemical assay for aryl sulfatase activity, described herein, cannot differentiate between SULF1 and SULF2.
  • Other sulfatases in the family are located in lysosomes and have an exo-sulfatase activity on proteoglycan chains, or are located in the endoplasmic reticulum and the Golgi apparatus.
  • enzymatic assays at a neutral or slightly alkaline pH that use the extracellular liquids (e.g., plasma) capture only SULF2 and SULFl, and exclude other sulfatases.
  • the pH to exclude sulfatases other than SULFl and SULFl in a biochemical assay may range from about 6 to about 10, from about 6 to about 9, from about 6 to about 8, from about 7 to about 10, from about 7 to about 9, from about 7 to about 8, from about 8 to about 10, from about 8 to about 9, or have a pH of about 8.
  • heparin/heparan sulfate chains thus remodeling the cell's surface which affects a variety of transmembrane signaling processes.
  • SULFl knock-out mice but not SULFl knock-out mice, exhibited an increase of UA2S-GlcNS6S in kidneys, supporting the theory of a sulfatase function in kidneys.
  • negatively charged heparan sulfate proteoglycans, and in particular their degree of sulfation in the basement membrane play a major role in
  • mice with deleted SULFl and SULFl genes developed proteinuria resulting from injuries of the glomerular endothelial cells and podocytes.
  • the present invention is directed to a gene involved in a site of injury of NS disease in a patient.
  • the site of injury in the patient may be a kidney, a glomerulus, a podocyte, or a combination thereof.
  • SULFl and SULFl as a discriminatory indicators for patients with SSNS and SRNS
  • gene expression before (SI) and after (S2) GC treatment was measured by quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and Reverse Transcription PCR (RT-PCR).
  • qRT-PCR Real Time Polymerase Chain Reaction
  • RT-PCR Reverse Transcription PCR
  • Relative SULFl mRNA expression in the NS patient peripheral lymphocytes was measured and compared with expression of SULFl, according to methods known in the art. Expression analysis was performed using 35 cycles of RT-PCR amplification.
  • Figure 5 shows detection of SULFl and SULFl expression among NS patient groups.
  • gene expression of SULFl and SULFl was measured in SSNS and SRNS patient leukocytes.
  • Figure 5A the SULFl expression in patients of both groups was subject to substantial variability.
  • GER(S2/S1) SULF2 can serve as a discriminatory value of gene expression between both SSNS and SRNS patient classes.
  • induction of SULFl activity in response to the GC therapy may be associated with its therapeutic efficacy in NS patients, particularly considering the known role of SULFl in podocytes.
  • RT-PCR conditions were employed using the following primer sequences to amplify the genes of interest:
  • TTCTTGGTCTCCTCCTCCTTGGAC SEQ ID NO: 6 for RPL19.
  • RT-PCR was sufficient to provide robust signals for the expression of both SULFl and the control gene
  • RPL19, SULFl gene expression was undetectable in NS patients, both before and after GC therapy (see Figure 5B).
  • RT-PCR was conducted using cDNA from commercial RNA extracted from whole human kidneys (BioChain, Newark, CA) as a template, and thus, as a SULFl positive control (see Figure 5B).
  • the primer sequences used to amplify the genes of interest are described above.
  • the primer design for RT-PCR using standard cycling conditions resulted in expected cDNA amplicon lengths of 196 bp (SULFl), 198 bp (SULFl), and 220 bp (RPL19).
  • the position of the 200 bp molecular mass marker band is indicated by the bar on the left side of Figure 5B.
  • Figure 5B shows SULF1 gene expression assessed in leukocytes from four representative NS patients, three SSNS patients (patient 9, 16, and 31) and one SRNS patient (patient 21).
  • Gene expression of SULF1 (lanes #1) was undetected in all NS patients, while SULF2 (lanes #2) and the RPL19 control gene were present (lanes #3). According to these data, SULF2 expression was much greater than that of SULF1 in all analyzed NS patient samples.
  • Figures 5A and 5B indicate that SULF1 expression in patient leukocytes is unlikely to contribute to any measured aryl sulfatase activity (see Figure 6).
  • the Sulfatase 2 (SULF2) gene encodes a large extracellular enzyme comprising approximately 870 amino acids with endoglucosamine-6-sulfatase activity. Upon desulfation, extracellular ligands can no longer bind to the cell membrane. This enzyme liberates 6-O-S mainly from disaccharide units (e.g., IdoA2S-GlcNS6S, UA2S-GlcNS6S, UA-GlcNS6S) within S domains of extracellular heparin/heparan sulfate chains.
  • disaccharide units e.g., IdoA2S-GlcNS6S, UA2S-GlcNS6S, UA-GlcNS6S
  • SULF2 remodels the 6-O-sulfation of the cell's surface heparin/heparan chains which has consequences for the modulation of transmembrane signaling processes. For example, the modulation of the interaction of VEGF-160 or FGF-1 with heparin or heparan sulfate, and the promotion of the Wnt/p-catenin signaling pathway.
  • the enzyme SULF2 is synthesized as pre-pro -protein. After cleavage of the signal sequence, the pro-protein is further proteolytically cleaved resulting in a 75 kDa and a 50 kDa fragments that become linked by a disulfide bond to form a mature protein. A portion of the mature protein becomes partially secreted, with the other portion being retained at the cell surface, notably in the lipid rafts. Catalytic activity of SULF2 requires post-translational modifications, including the conversion of a cysteine residue in the catalytic center into C- formylglycine.
  • SULF2 (and SULF1) has an arylsulfatase activity that was used for enzymatic measurements since activity of all other sulfatases are suppressed at this pH.
  • a modified arylsulfatase assay was employed to measure SULF2 enzyme activity (Uchimura et al. 2006).
  • Patient plasma was clarified by centrifugation at 15,000 x g for 30 min at 4°C, and 10 ⁇ of supernatant (excluding any of the upper, lipid-rich layer) was combined with stock solutions to a final concentration of 50 mM HEPES-KOH, pH 8.0, 10 mM lead acetate, and 10 mM 4-methylumbelliferyl sulfate in a total volume of 50 ⁇ . After incubation at 37 °C for 2 h, 40 ⁇ of this mixture was combined with 200 ⁇ of 0.5 M
  • a one-sided Shapiro-Wilk test indicated that the collected GER(S2/S 1)SULF 2 (see Figure 5) and aryl sulfatase activity (S2/S l)(see Figure 6) data exhibit standard normal distribution, with a significance level (p value) of 0.05.
  • Statistical significance of the differences between the SSNS and SRNS patient groups for both the GER(S2/S 1)SULF 2 and the aryl sulfatase activities (S2/S 1) was determined by the unpaired Student's i-test.
  • Figure 5 shows the SULF2 activity ratio, (S2/S1) SULF 2, is also induced in SSNS patient and therefore, it can also serve to discriminate between SRNS and SSNS patient classes.
  • the eleven marker genes identified in Figure 5 may be included with SULF2 to comprise one embodiment of the kit or the method described herein for diagnostic analyses. More specifically, a 12-gene set embodiment of the kit may be prepared and used for diagnostic analyses of NS in patients. The genes, gene symbols, and gene IDs of the 12-gene set are listed in Table 3.
  • the eleven other identified marker genes are involved in diverse cellular processes, including endocytosis (RFFL), vesicle transport (STX4), DNA replication (TOPBP1), lysosphingolipid signaling (S1PR5), transcription (KLF2), cytoskeletal functions (NCRNA00081), glucose transport (SLC2A1), formation of the glycosylphosphatidyl-inositol anchors of cell surface proteins (PIGB), proteins of the tRNA metabolism (TRMT6), unknown functions (FAM40A), and in the development of T-cell lymphoma (FGFRl OP2), or following an in-frame fusion with the FGFRl gene (see Table 3).
  • RFFL endocytosis
  • STX4 DNA replication
  • S1PR5 lysosphingolipid signaling
  • KLF2 transcription
  • NCRNA00081 cytoskeletal functions
  • SLC2A1 glucose transport
  • PIGB glycosylphosphatidyl-inositol anchors of cell
  • another embodiment of the present inventions comprises a particular set of 34 genes that accurately classified the two NS patient groups.
  • the 34 genes of this set are indicated by closed symbols in Figure 3 for both SRNS and SSNS patient groups. Additionally, the 34 genes, gene symbols, and gene IDs are listed in bold amongst the 72 gene-set in Table 2.
  • a gene expression cassette comprising primers and probes to these 34 genes was able to differentiate amongst SRNS and SSNS patients with over a 95% accuracy.
  • FIG. 7A shows the volcano plot that filtered the initial data set down to 991 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 16 gene that significantly differentiated between SRNS and SSNS patients.
  • Figure 7B shows the heat map
  • Figure 8A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 54 gene that significantly differentiated between SRNS and SSNS patients.
  • Figure 8B shows the heat map
  • Table 5 shows the gene, gene symbol, rank, and gene ID comprised in the 54-gene set.
  • H2AFY H2A histone family member Y 9555
  • ZC3HAV1 zinc finger CCCH-type, antiviral 1 56829
  • GLT1D1 glycosyltransferase 1 domain containing 1 144423
  • AHA1 activator of heat shock 90kDa protein ATPase
  • FIG. 9A shows the volcano plot that filtered the initial data set based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 40 gene that significantly differentiated between SRNS and SSNS patients.
  • Figure 9B shows the heat map clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 40 genes. The genes, gene symbols, and gene IDs of the 40-gene set are listed in Table 6.
  • FIG. 10A shows the volcano plot that filtered the initial data set based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 24 gene that significantly differentiated between SRNS and SSNS patients.
  • Figure 10B shows the heat map clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 24 genes. The genes, gene symbols, and gene IDs of the 24-gene set are listed in Table 7. Table 7. 24-Gene Set with differential ex ression in SRNS vs. SSNS atients (Fi ure 10).
  • 78 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 8).
  • Figure 11 A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 78 gene that significantly differentiated between SRNS and SSNS patients.
  • Figure 1 IB shows the heat map
  • RNASEK ribonuclease RNase K 440400
  • MAPKAPK3 mitogen-activated protein kinase-activated protein kinase 3 7867
  • TNFSF10 tumor necrosis factor (ligand) superfamily member 10 8743
  • PTP4A1 protein tyrosine phosphatase type IVA member 1 7803
  • FIG. 12A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 85 gene that significantly differentiated between SRNS and SSNS patients.
  • Figure 12B shows the heat map
  • RNASEK ribonuclease RNase K 440400
  • MAPRE2 microtubule-associated protein RP/EB family, member 2 10982
  • COTL1 coactosin-like F-actin binding protein 1 23406
  • SEC11A SEC11 homolog A (S. cerevisiae) 23478
  • GIMAP5 GTPase IMAP family member 5 55340
  • HLA-B major histocompatibility complex class 1, B 3106
  • PIP4K2A phosphatidylinositol-5-phosphate 4-kinase, type II, alpha 5305
  • 76 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 10).
  • Figure 13A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 76 gene that significantly differentiated between SRNS and SSNS patients.
  • Figure 13B shows the heat map
  • MYL12A myosin, light chain 12A, regulatory, non-sarcomeric 10627
  • TAF7 RNA polymerase II TATA box binding protein (TBP)-
  • HIATL1 hippocampus abundant transcript-like 1 84641
  • 71 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 11).
  • Figure 14A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 71 gene that significantly differentiated between SRNS and SSNS patients.
  • Figure 14B shows the heat map
  • PARP9 poly (ADP-ribose) polymerase family member 9 83666
  • EIF2S3 eukaryotic translation initiation factor 2, subunit 3 gamma, 52kDa 1968
  • EIF2AK1 eukaryotic translation initiation factor 2-alpha kinase 1 27102
  • AHA1 activator of heat shock 90kDa protein ATPase homolog 1

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Abstract

Provided are a kit and a method for identifying a pediatric patient that is resistant to steroid treatment of Nephrotic Syndrome.

Description

KIT AND METHOD FOR IDENTIFYING INDIVIDUAL RESPONSIVENESS TO STEROID THERAPY OF NEPHROTIC SYNDROME
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is an International Application claiming priority under 35
U.S.C. § 119(e) to U.S. Provisional Application Nos. 61/898,928 and 61/898,932, each filed on November 1, 2013, and the contents of which are both incorporated herein by reference.
GOVERNMENT LICENSE RIGHTS
This invention was made with government support under NIH R01
DK075533, NIH U01 GM092655, and NIH R01 DK095059-01A1 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases. The government has certain rights in the invention.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 14 KB ACII (Text) file named "232343_SEQList_ST25" created on October 31, 2014. BACKGROUND
Nephrotic Syndrome is among the most common kidney diseases seen in both children and adults. Nephrotic Syndrome (NS) is a condition caused by damage to the kidneys that leads to several symptoms, such as swelling, low blood protein levels, high cholesterol and triglyceride levels, but most notably, the release of excess protein in the urine (i.e., proteinuria). NS is characterized by increased permeability in the capillaries of the glomerulus (i.e., the filtering unit of the kidney) which results in low blood protein levels and high levels of urine proteins, since the proteins freely pass from the blood into the urine. Research over the past few decades has identified the importance of renal podocytes, the filtering cells of kidney, as a site of injury in NS. For example, kidneys affected by NS have small pores in the podocytes that are large enough to permit proteinuria. The high urine protein levels disable the glomulerus from safely filtering blood, and results in symptoms of NS. The primary therapy for NS is steroids, such as oral glucocorticoids (GC), which effectively result in remission for the majority of patients. However, approximately 20% of patients either present with, or develop, steroid resistance later in their course of treatment; these patients are classified as having Steroid Resistant Nephrotic Syndrome (SRNS). NS patients who show efficacy with steroid treatment are often diagnosed as having Steroid Sensitive Nephrotic Syndrome (SSNS).
The known anti-inflammatory and immunosuppressive effects of glucocorticoids (GCs) have served as indirect evidence that their mechanism of action in NS involves inhibition of soluble mediators released by T lymphocytes. In particular, several podocyte proteins have been identified as playing crucial roles in the development of NS, and a number of rare mutations in these proteins have been identified as causative of the disease or for development of resistance to GC treatment. However, the majority of NS patients present with unknown underlying disease etiology. Similarly, the molecular basis for clinical resistance to GC therapy remains largely unclear.
Interestingly, children with SRNS fail to enter remission after prolonged GC steroid treatment, and are at high risk for steroid-induced side effects as well as progression of disease. Approximately 10-30% of SRNS are due to mutations in genes specifically expressed in the filtering cells of kidney, the podocytes, while the etiology of the other approximate 80% of cases is unknown. Identification and development of diagnostic tools to identify SRNS at the time of initial clinical presentation of NS could prevent unnecessary exposure to the toxic effects of steroids and improve the treatment of patients with Nephrotic Syndrome.
To design individualized therapies for NS patients, a better understanding of both the complex responses to glucocorticoids (GCs) and the factors that determine susceptibility of the disease and resistance to GC is necessary. Unbiased genome-wide, transcriptome-wide, and proteome-wide systems biology approaches have been identified as key tools to help understand complex diseases, including various forms of primary glomerulopathies. Transcriptome-wide sequencing of RNA enables the assessment of the protein-coding potential of all genes of interest expressed in a tissue. For example, RNAseq is a suitable screening approach for identifying the molecular basis of diseases with unknown etiology, including acquired forms of NS.
Identification of the type of NS (steroid sensitive vs. steroid resistant) is crucial to determine the best treatment regimen for NS patients. In particular, the ability to provide patients genetic tests to identify resistant cases of NS. The present invention is able to provide reliable testing to identify patients who have or will develop steroid resistant NS, and thus enable the avoidance of patient exposure to potentially ineffective or adverse
medications (i.e., steroids).
Described herein are a kit and a method for identifying a patient that is resistant to steroid treatment of Nephrotic Syndrome (NS). The kit and the method comprise reagents for detecting the expression of one or more genes or gene products to identify, diagnose, and/or treat NS in patients with a lack of responsiveness to steroids, such as glucocorticoids (GCs). Further described are gene biomarkers that can differentiate between a patient having Steroid Resistant Nephrotic Syndrome (SRNS) and a patient having Steroid Sensitive Nephrotic Syndrome (SSNS). Specifically, the present invention describes a kit or a method to identify steroid resistance in a patient with NS, wherein the patient is a pediatric patient.
SUMMARY
Disclosed herein are a kit and a method for identifying a patient with resistance to steroid therapy of Nephrotic Syndrome. In an exemplary embodiment of the kit and the method, the patient is a pediatric patient. In an embodiment of the kit, the kit comprises instructional materials.
In one embodiment, the kit comprises a plurality of binding reagents. In one embodiment of the kit, the kit comprises binding reagents , wherein each of the binding reagents specifically binds to a gene product encoded by a gene selected from the group of genes consisting of: RFFL, INPP1, MCM2, CSNK1G1, SIGLEC10, AGPAT6, DCAF8, FAM193A, RSPRY1, KCTD20, BLOC1S3, TOE1, ACAD10, SULF2, RAB8A,
NCRNA00294, ZNF318, CDK4, TMEM219, PRKCSH, SLC9A7, LEOl, STX4, PTPRK, GANAB, C170RF63, ZC3H18, MED 14, TRAPPC5, EXOC7, ACINI, ITGAL, SH2D1B, TRMT6, NACC1, RECQL5, ZC3H12D, PRR5L, UBE2J2, TOPBP1, GALNT2, BTN2A2, GPR108, CHMP4B, ALDOA, TRAPPC1, SPRYD3, MTMR14, S1PR5, FAM134C,
ALDOC, TCP1, CLCN3, NDUFS1, LGALS9, SUPT5H, JARID2, FAM40A, KLF2, AKIRIN1, ZNF782, OCIAD1, KPNA1, KIAA1033, UBE2W, LOC220930, NCRNA00081, ATP11B, SLC2A1, PIGB, SEPHS2, FGFR10P2, GLIPR2, ARHGEF2, S100A8, H2AFY, SELL, TRIM22, ZC3HAV1, MY05A, ZNF641, SRSF4, ZBTB34, NCOA4, ANTXR2, DDX3X, CHST11, PRNP, SLC35B4, GM2A, EZH1, LOC100289230, MYLIP, PCMTD2, ARHGAP15, AKAP10, ARHGAP19, B3GNT2, SYNJ1, MBD5, ZNF266, TMEM87B, KCTD10, ZNF587, SLC30A6, RABGAP1, PL-5283, RCSD1, ANXA7, KIAA0754, TMEM87A, ZNF187, GLT1D1, TGIF2-C20ORF24, TOR1B, ZNF41, AHSA2, TAOK3, CPl lO, GRPELl, LOC283070, KIAA0141, ZMATl, ZNF398, SPATA13, HGSNAT, MCC, PDHA1, COPB2, ADIPOR1, LILRB2, FBX07, C20ORF108, NFE2, LIN7A, SNCA, ODC1, HBA2, TIFA, HEMGN, BAG1, DCAF12, SIRPB1, RPGR, AQP9, STRADB, SNAP29, PADI2, SLC25A5, PPIB, C90RF78, FAM46C, PROK2, MKRN1, MPZL3, FAM104A, THOC5, GSPT1, SCARNA3, IGSF6, ARL11, HBA1, CD59, EIF2AK1, PFKFB3, PRDX5, RAB4A, CDK6, FCGR2A, GNAI2, GNB1, STX7, ARHGDIB,
RNASEK, PTAR1, PECAM1, KIAA1267, ZNF281, CX3CR1, SH3BGRL3, PITPNA, MAPRE2, MGATl, LM02, S100A4, PCMTDl, SNX18, NFKBIZ, C130RF18, CLEC12A, USP15, C40RF3, CAB39, FCGR3A, GABARAPL1, C3AR1, TNFAIP8L2, RAB33B, ELF2, MBOAT2, SEMA4A, SKAP2, RAB8B, PPP1CB, FUT4, EMB, PTGER4, EPS 15, STIM1, GNLY, CNDP2, LONP2, LIN7C, MGC12916, ACTR1A, LST1, MAPKAPK3, GLRX, ARL8B, TBRG1, AFF3, MARK3, GCC1, C20RF29, ZMAT2, LCOR, CDC42, PHIP, RNF103, TNFSFIO, PTP4A1, RUNX3, GOSRl, KIAA0922, OSBP, NUP214, CCNI, ERP29, LAMP1, LASP1, ARCN1, FUS, VPS24, MARCH8, MBNL3, MAX, BNIP3L, PIK3CG, PRKCB, HLA-E, STAT5A, CYTH4, GRB2, MXI1, GPSM3, C220RF13, ARF3, SLC25A37, TPM3, S100A6, CFL1, YTHDF3, C190RF22, ATP6V0D1, CLIC1, PIM1, STAT5B, TRIM58, UBE2H, ATP5B, NHP2L1, C160RF72, EPB41, BSG, GNS, PSAP, ARPC1B, BLCAP, ARHGAP30, ACTB, WDR26, CCDC69, PLEKH02, PTPRE, COTL1, PPT1, CREB5, RPS23, MSN, DAZAP2, SEC11A, GIMAP5, EIF4G2, IL6R, GIMAP4, TPD52L2, TCP11L2, HLA-B, AIF1, UBE2J1, ATF7, MEF2A, PIP4K2A, RGS2, GIMAP6, SETD2, NR2C2, WAC, TSPAN14, IL6ST, STK17B, NCRNA00282, FAM120B, WASF2, WNK1, SH3BP5, PAFAH1B1, RBM38, PPP1R15B, MLL5, BTG1, PIK3R5, SIAH2, PCGF3, PIK3IP1, WAPAL, CD53, XRCC5, ZNF638, RBL2, NUFIP2, OAZ1, CNST,
MYL12A, TAF7, TNIPl, FAM117A, PCFl l, KLHL6, HIATLl, SERTAD2, RBM5, CHD7, JAK3, GNG2, S1PR1, MYD88, IKZFl, UBE2G1, ELF1, ENSA, AQP3, IRF9, SESN3, DCAF6, FCER1G, ZNF776, INSIG2, RPS9, SNHG12, ZNF791, CD96, HSP90B1, UBAP2, MARCKS, ZFX, SNX3, DYNC1LI1, STAT6, MME, TNFSF13B, MSH6, PIK3AP1, CEACAM1, CD180, CHPT1, BCL6, C20RF24, ADM, IGBP1, DNAJC2, KDM6A,
C40RF14, SOD2, PARP9, TUBA 1 A, ZFP62, SEPT6, UBE2L6, ZNF121, EIF2S3, IL7R, SNORA45, ANXA3, JAZF1, SNORD89, PEBP1, CCNDBP1, CCNT1, ASNSD1,
TMEM14B, MTIF2, YBX1, PLSCR1, TAF13, C220RF46, AHSA1, SAT1, DDX60L, and ZNF226. As disclosed herein, the expression of said genes or gene products has been found to be significantly different between patients with Steroid-Resistant Nephrotic Syndrome and patients with Steroid-Sensitive Nephrotic Syndrome.
In another embodiment of the kit, the reagents of the kit specifically bind to gene products comprising mRNA, cDNA, or proteins of the gene. In an exemplary embodiment of the kit, the reagents comprise nucleic acids, and the nucleic acids may be DNA or RNA, including for example, wherein the DNA is cDNA. The nucleic acids of the kit may also comprise a set of two primers and optionally a probe for detecting mRNA expressed by said genes. The nucleic acids of the kit may comprise a set of two or more primers for detecting mRNA expressed by said genes. The nucleic acids of the kit may also comprise one or more probes for detecting mRNA expressed by said genes. In an
embodiment of the kit, the probes and primers may further comprise a detectable marker, wherein the marker is a fluorescent label or a non-fluorescent label. In another embodiment, the reagents comprise antibodies for detecting the proteins expressed by said genes.
In one embodiment of the kit, a reagent is provided that specifically binds or hybridizes to a SULF2 gene or gene product. In such an embodiment, the reagents may specifically hybridize to SEQ ID NO: 7. In a further embodiment of the kit, each of the reagents specifically binds to a gene product selected from the group of genes consisting of: RFFL, INPP1, MCM2, CSNK1G1, SIGLEC10, AGPAT6, DCAF8, FAM193A, RSPRY1, KCTD20, BLOC1S3, TOE1, ACAD10, SULF2, RAB8A, NCRNA00294, ZNF318, CDK4, TMEM219, PRKCSH, SLC9A7, LEOl, STX4, PTPRK, GANAB, C170RF63, ZC3H18, MED 14, TRAPPC5, EXOC7, ACINI, ITGAL, SH2D1B, TRMT6, NACCl, RECQL5, ZC3H12D, PRR5L, UBE2J2, TOPBP1, GALNT2, BTN2A2, GPR108, CHMP4B, ALDOA, TRAPPC1, SPRYD3, MTMR14, S1PR5, FAM134C, ALDOC, TCP1, CLCN3, NDUFS1, LGALS9, SUPT5H, JARID2, FAM40A, KLF2, AKIRIN1, ZNF782, OCIAD1, KPNA1,
KIAA1033, UBE2W, LOC220930, NCRNA00081, ATP11B, SLC2A1, PIGB, SEPHS2, and FGFR10P2. In another embodiment of the kit of the present disclosure, binding reagents are provided, wherein each of the reagents specifically binds to a gene product encoded by a gene selected from the group of genes consisting of: RFFL, AGPAT6, SULF2, TMEM219, PRKCSH, STX4, GANAB, ZC3H18, EXOC7, ACINI, ITGAL, SH2D1B, TRMT6, NACCl, ZC3H12D, TOPBP1, GALNT2, CHMP4B, TRAPPC1, SPRYD3, MTMR14, S1PR5, NDUFS1, LGALS9, SUPT5H, JARID2, KLF2, AKIRIN1, OCIAD1, KIAA1033, UBE2W, LOC220930, ATP1 IB, and FGFR10P2. In a further embodiment of the kit, unique binding reagents are provided, wherein each of the reagents specifically binds a gene product selected from the group of genes consisting of: RFFL, SULF2, TOPBP1, SIPR5, FAM40A, STX4, KLF2, NCRNA00081, SLC2A1, TRMT6, PIGB, and FGFR10P2.
In another embodiment of the kit, a plurality of reagents is provided wherein the reagents specifically bind to ten or more gene products encoded by genes selected from the group consisting of: RFFL, AGPAT6, SULF2, TMEM219, PRKCSH, STX4, GANAB, ZC3H18, EXOC7, ACINI, rfGAL, SH2D1B, TRMT6, NACC1, ZC3H12D, TOPBP1, GALNT2, CHMP4B, TRAPPC1, SPRYD3, MTMR14, S1PR5, NDUFS1, LGALS9, SUPT5H, JARID2, KLF2, AKIRINl, OCIADl, KIAA1033, UBE2W, LOC220930, ATP11B, and FGFR10P2. In a further embodiment of the kit a plurality of reagents is provided wherein the reagents specifically bind to ten or more gene products encoded by genes selected from the group consisting of: RFFL, SULF2, TOPBP1, SIPR5, FAM40A, STX4, KLF2, NCRNA00081, SLC2A1, TRMT6, PIGB, and FGFR10P2.
Further disclosed herein is a method of identifying a patient who is resistant to steroid therapy of Nephrotic Syndrome. The method comprises the following steps: a) assaying marker genes expressed in the patient, wherein the marker genes are selected from the group of genes consisting of: RFFL, INPP1, MCM2, CSNK1G1, SIGLEC10, AGPAT6, DCAF8, FAM193A, RSPRY1, KCTD20, BLOC1S3, TOE1, ACAD10, SULF2, RAB8A, NCRNA00294, ZNF318, CDK4, TMEM219, PRKCSH, SLC9A7, LEOl, STX4, PTPRK, GANAB, C170RF63, ZC3H18, MED 14, TRAPPC5, EXOC7, ACINI, ITGAL, SH2D1B, TRMT6, NACC1, RECQL5, ZC3H12D, PRR5L, UBE2J2, TOPBP1, GALNT2, BTN2A2, GPR108, CHMP4B, ALDOA, TRAPPC1, SPRYD3, MTMR14, S1PR5, FAM134C, ALDOC, TCP1, CLCN3, NDUFS1, LGALS9, SUPT5H, JARID2, FAM40A, KLF2, AKIRINl, ZNF782, OCIADl, KPNAl, KIAA1033, UBE2W, LOC220930, NCRNA00081, ATP1 IB, SLC2A1, PIGB, SEPHS2, FGFR10P2, GLIPR2, ARHGEF2, S100A8, H2AFY, SELL, TRIM22, ZC3HAV1, MY05A, ZNF641, SRSF4, ZBTB34, NCOA4, ANTXR2, DDX3X, CHST11, PRNP, SLC35B4, GM2A, EZH1, LOC100289230, MYLIP, PCMTD2, ARHGAP15, AKAP10, ARHGAP19, B3GNT2, SYNJ1, MBD5, ZNF266, TMEM87B, KCTD10, ZNF587, SLC30A6, RABGAP1, PL-5283, RCSD1, ANXA7, KIAA0754, TMEM87A, ZNF187, GLT1D1, TGIF2-C20ORF24, TOR1B, ZNF41, AHSA2, TAOK3,
CPl lO, GRPELl, LOC283070, KIAA0141, ZMATl, ZNF398, SPATA13, HGSNAT, MCC, PDHA1, COPB2, ADIPOR1, LILRB2, FBX07, C20ORF108, NFE2, LIN7A, SNCA, ODC1, HBA2, TIFA, HEMGN, BAG1, DCAF12, SIRPB1, RPGR, AQP9, STRADB, SNAP29, PADI2, SLC25A5, PPIB, C90RF78, FAM46C, PROK2, MKRN1, MPZL3, FAM104A, THOC5, GSPT1, SCARNA3, IGSF6, ARL11, HBA1, CD59, EIF2AK1, PFKFB3, PRDX5, RAB4A, CDK6, FCGR2A, GNAI2, GNB1, STX7, ARHGDIB,
RNASEK, PTAR1, PECAM1, KIAA1267, ZNF281, CX3CR1, SH3BGRL3, PITPNA, MAPRE2, MGATl, LM02, S100A4, PCMTDl, SNX18, NFKBIZ, C130RF18, CLEC12A, USP15, C40RF3, CAB39, FCGR3A, GABARAPL1, C3AR1, TNFAIP8L2, RAB33B, ELF2, MBOAT2, SEMA4A, SKAP2, RAB8B, PPP1CB, FUT4, EMB, PTGER4, EPS 15, STIM1, GNLY, CNDP2, LONP2, LIN7C, MGC12916, ACTR1A, LST1, MAPKAPK3, GLRX, ARL8B, TBRG1, AFF3, MARK3, GCC1, C20RF29, ZMAT2, LCOR, CDC42, PHIP, RNF103, TNFSFIO, PTP4A1, RUNX3, GOSRl, KIAA0922, OSBP, NUP214, CCNI, ERP29, LAMP1, LASP1, ARCN1, FUS, VPS24, MARCH8, MBNL3, MAX, BNIP3L, PIK3CG, PRKCB, HLA-E, STAT5A, CYTH4, GRB2, MXI1, GPSM3, C220RF13, ARF3, SLC25A37, TPM3, S100A6, CFL1, YTHDF3, C190RF22, ATP6V0D1, CLIC1, PIM1, STAT5B, TRIM58, UBE2H, ATP5B, NHP2L1, C160RF72, EPB41, BSG, GNS, PSAP, ARPC1B, BLCAP, ARHGAP30, ACTB, WDR26, CCDC69, PLEKH02, PTPRE, COTL1, PPT1, CREB5, RPS23, MSN, DAZAP2, SEC11A, GIMAP5, EIF4G2, IL6R, GIMAP4, TPD52L2, TCP11L2, HLA-B, AIF1, UBE2J1, ATF7, MEF2A, PIP4K2A, RGS2, GIMAP6, SETD2, NR2C2, WAC, TSPAN14, IL6ST, STK17B, NCRNA00282, FAM120B, WASF2, WNK1, SH3BP5, PAFAH1B1, RBM38, PPP1R15B, MLL5, BTG1, PIK3R5, SIAH2, PCGF3, PIK3IP1, WAPAL, CD53, XRCC5, ZNF638, RBL2, NUFIP2, OAZ1, CNST,
MYL12A, TAF7, TNIPl, FAM117A, PCFl l, KLHL6, HIATLl, SERTAD2, RBM5, CHD7, JAK3, GNG2, S1PR1, MYD88, IKZFl, UBE2G1, ELF1, ENSA, AQP3, IRF9, SESN3, DCAF6, FCERIG, ZNF776, INSIG2, RPS9, SNHG12, ZNF791, CD96, HSP90B1, UBAP2, MARCKS, ZFX, SNX3, DYNC1LI1, STAT6, MME, TNFSF13B, MSH6, PIK3AP1, CEACAM1, CD180, CHPT1, BCL6, C20RF24, ADM, IGBP1, DNAJC2, KDM6A,
C40RF14, SOD2, PARP9, TUBA 1 A, ZFP62, SEPT6, UBE2L6, ZNF121, EIF2S3, IL7R, SNORA45, ANXA3, JAZF1, SNORD89, PEBP1, CCNDBP1, CCNT1, ASNSD1,
TMEM14B, MTIF2, YBX1, PLSCR1, TAF13, C220RF46, AHSA1, SAT1, DDX60L, and ZNF226, b) analyzing the expression levels of the marker genes relative to a control sample, wherein the control sample is taken from a patient that has Nephrotic Syndrome and is responsive to steroid treatment, and c) identifying a patient as being resistant to steroid treatment of Nephrotic Syndrome based on the relative expression of said marker genes compared between samples retrieved from one or more Nephrotic Syndrome patients responsive to steroid treatment, and one or more patient resistant to steroid treatment of Nephrotic Syndrome. The method may further comprise treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non-steroidal therapies of Nephrotic Syndrome. The method of treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non-steroidal therapies of Nephrotic Syndrome comprises administering a drug selected from the group consisting of adrenocorticotropic hormone, Cyclosporine, Tacrolimus, Mycophenolate mofetil, Plasmapheresis, column A immunoabsorbtion, and Rituximab. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 72-gene set.
Figure 2 shows differential clustering of 72 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
Figure 3 shows linear separation of Gene Expression Ratios (GER) of 72 genes in SRNS and SSNS patient groups following GC treatment.
Figure 4 shows analysis of gene expression ratios of 72 genes using statistical methods.
Figure 5A shows SULF2 Gene Expression Ratios (GER) in patient samples of SRNS and SSNS patients.
Figure 5B shows SULF1 gene expression in patient samples of SRNS and
SSNS patients.
Figure 6 shows arylsulfatase enzyme activity in SRNS and SSNS patient plasma samples.
Figure 7A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 16-gene set.
Figure 7B shows differential clustering of 16 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
Figure 8A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 54-gene set. Figure 8B shows differential clustering of 54 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
Figure 9A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 40-gene set.
Figure 9B shows differential clustering of 40 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment
Figure 10A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 24-gene set.
Figure 10B shows differential clustering of 24 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
Figure 11A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 78-gene set.
Figure 11B shows differential clustering of 78 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
Figure 12A shows a volcano plot of the expression ratios of genes following
GC treatment of eleven patients having SSNS or SRNS resulting in a 85-gene set.
Figure 12B shows differential clustering of 85 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
Figure 13A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 76-gene set.
Figure 13B shows differential clustering of 76 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment
Figure 14A shows a volcano plot of the expression ratios of genes following GC treatment of eleven patients having SSNS or SRNS resulting in a 71 -gene set.
Figure 14B shows differential clustering of 71 genes selected according to their expression ratios in the SRNS and SSNS patient groups before and after GC treatment.
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure is directed to a kit and a method for identifying a patient that is resistant to steroid treatment of Nephrotic Syndrome. In one described embodiment, a kit is provided. The kit of the present disclosure comprises one or more binding reagents, or a plurality of binding reagents. The total reagents of the kit of the present disclosure may range from about 1 to about 500 reagents.
The reagents of the kit may specifically bind to one or more genes or a gene products. For example, the reagents of the kit may comprise, but are not limited to nucleic acids, antibodies, oligonucleotides, peptides and polypeptides, primers, probes, proteins, compounds, transcription factors, enzymes, nucleotides and polynucleotides, labels, tags, amino acids, molecules, receptors, hormones, vitamins, ligands, genes, and gene products. The nucleic acids of the present kit may be RNA or DNA, or synthetic RNA or DNA, for example the DNA may be copy DNA (i.e., cDNA).
The average number of reagents of the kit described herein ranges from about
1 to about 5, from about 1 to about 4, from about 1 to about 3, and from about 1 to about 2 reagents per gene or gene product. For example, nucleic acids of one embodiment of the kit may comprise a set of two primers and a probe (i.e., 3 reagents) for detecting mRNA expressed by said genes. Each set of two primers may include one forward primer (i.e., that amplifies in the 5' to 5' direction) and one reverse primer (i.e., that amplifies in the 3' to 5' direction). In one embodiment, the nucleic acids of the kit may only comprise a set of two or more primers (i.e., 2 reagents) for detecting mRNA expressed by said genes.
Primers or oligonucleotides and probes of the present invention may be of any length or size, including but not limited to between about 8 to about 75 bases, from about 8 to about 50, from about 10 to about 70 bases, from about 15 to about 65 bases from about 10 to about 50 bases, from about 15 to about 45 bases, from about 15 to about 35 bases, from about 10 to about 50 bases, from about 10 to about 30 bases, from about 10 to about 25 bases, from about 15 to about 30 bases, from about 15 to about 28 bases, from about 15 to about 25 bases, from about 10 to about 25 bases, from about 10 to about 20 bases, from about 10 to about 15 bases, and from about 5 to about 15 bases. Reagents of the present kit, particularly, primers and probe reagents, may be designed to bind or hybridize to any region of said genes or gene products by methods known to persons of ordinary skill in the art. For example, reagents of nucleic acids may be designed to bind to introns, exons, 5'-UTR, 3'- UTR, and/or promoter regions of said genes or gene products. In particular, the coding regions of said genes or said gene products are ideal locations to design binding reagents of the present kit in order to detect gene expression.
The nucleic acids of the kit may also comprise only one or more probes (i.e., 1 reagent) for detecting mRNA expressed by said genes or said gene products. The probes of the kit may further comprise a detection marker, wherein the marker may be a label. The label may be a fluorescent label or a non-fluorescent label. For example, a fluorescent label may include, but is not limited to fluorescein (e.g., 6-FAM), tetrachlorofluorescein (e.g., TET), VIC, SYBR Green, etc.
The nucleic acids of the present kit have sufficient sequence identity to bind specifically to the target gene product under moderate to stringent conditions. In particular, the nucleic acid probe and/or primer of the kit should have, for example, from about 80% to about 100% sequence identity, such as about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100% sequence identity with the target sequence of the gene product.
In another embodiment of the kit, said genes or gene products comprise proteins. In such an embodiment, the reagents may comprise antibodies for detecting the proteins expressed by said genes or said gene products.
The reagents of the kit may further include any substance necessary to facilitate the binding of the reagents to said genes or said gene products. For example, reagents of the present disclosure may include enzymes, one or more nucleotides, water, buffers and/or master mixes (i.e., buffers containing additional reagents such as nucleotide and/or polymerase), solutions, solvents, antibodies, labels, ions (e.g., Mg2+ or Mn2+), oligonucleotides (e.g., primers), probes, salts, stabilizers (e.g., MgCl2), and control samples, and any other substances known in the art that are necessary or preferable for successful binding and amplification of a reagent to a gene or a gene product.
For example, enzymes of the present disclosure include any enzyme known to facilitate binding and amplification of nucleic acids, including but not limited to DNA polymerase (i.e., Taq polymerase) or Reverse Transcriptase. Nucleotides of the present invention include one or more deoxynucleoside or deoxynucleotide triphosphates or dntps, such as dATP, dTTP, dCTP, and dGTP. The control sample of the present kit may comprise any gene, tissue, cell, or marker wherein the levels of said genes or gene products do not significantly differ in patients with Steroid-Resistant Nephrotic Syndrome and patients with Steroid-Sensitive Nephrotic Syndrome. In one embodiment, the control sample may be a Nephrotic Syndrome patient that is sensitive and/or responsive to steroid treatment. In another embodiment, the control sample may be normal kidney tissue. In addition to the reagents, the kit of the present invention may further comprise supplies. For example, supplies of the kit may include any known supplies necessary to facilitate binding of a reagent to a gene or gene product. Exemplary supplies of the kit include any immobilized support that facilitates the binding of a reagent to a gene or gene product, including, but not limited to tubes, plates, chips, and strips. For example, a plate, a tube, a chip, or a strip may comprise from 1 to about 500 wells, spots, or locations for the reagents to bind to the genes or gene products.
In one described embodiment, the supplies of the kit comprise instructional materials. For example, the kit may comprise instructions for the detection and identification of a patient with resistance to steroid treatment of Nephrotic Syndrome, including
instructions for use of the reagents of the kit described herein.
The reagents of the kit disclosed herein may bind to genes or gene products ranging from about 1 to about 100 genes or gene products. For example, the genes or gene products of the present kit and method may range from about 1 to about 95, from about 1 to about 90, from about 1 to about 85, from about 1 to about 72, from about 1 to about 78, from about 1 to about 76, from about 1 to about 71, from about 1 to about 71, from about 1 to about 54, from about 1 to about 40, from about 1 to about 34, from about 1 to about 24, and from about 1 to about 16, from about 1 to about 12, from about 1 to 4, from about 1 to 3, from about 1 to 2, from about 2 to about 85, from about 2 to about 78, from about 2 to about 76, from about 2 to about 72, from about 2 to about 71, from about 2 to about 54, from about 2 to about 40, from about 2 to about 34, from about 2 to about 24, and from about 2 to about 16, from about 2 to about 12, from about 2 to 3, from about 2 to 4, from about 3 to about 78, from about 3 to about 76, from about 3 to about 72, from about 3 to about 71, from about 3 to about 54, from about 3 to about 40, from about 3 to about 34, from about 3 to about 24, and from about 3 to about 16, from about 4 to about 85, from about 4 to about 78, from about 4 to about 76, from about 4 to about 72, from about 4 to about 71, from about 4 to about 54, from about 4 to about 40, from about 4 to about 34, from about 4 to about 24, and from about 4 to about 16, from about 4 to about 12, from about 12 to about 85, from about 12 to about 76, from about 12 to about 78, from about 12 to about 72, from about 12 to about 71, from about 12 to about 54, from about 12 to about 40, from about 12 to about 34, from about 12 to about 24, and from about 12 to about 16. More specifically, a kit may include reagents to detect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 20, 24, 34, 40, 50, 54, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 85, 90, 95, or 100 genes or gene products. Reagents of the present kit specifically hybridize or bind to one or more marker genes or gene products of the present disclosure and in doing so, are capable of identifying a patient who is resistant to steroid treatment of Nephrotic Syndrome with an accuracy of 95% or greater. More specifically, said genes or said gene products of the present invention have differential expression among NS patient groups. In particular, expression of said genes and said gene products differentiate between patients having Steroid-Resistant Nephrotic Syndrome (SRNS) and patients having Steroid-Sensitive Nephrotic Syndrome (SSNS). Accordingly, the genes and gene products of the present kit are also utilized in the method of identifying patients with SRNS and SSNS as described herein.
An exemplary gene or gene product of the presently described kit and method is SULF2. Reagents of the present kit may bind to a gene product of the SULF2 gene. The mRNA sequence used to derive cDNA of the SULF2 gene is as follows:
GGGCCATTTCTGGACAACAGCTGCTATTTTCACTTGAGCCGAAGTTAATTTCTCG GGGAGTTCTCGGGCGCGCAC AGGCAGCTCGGTTTGCCCTGCGATTGAGCTGCGG GTCGCGGCCGGCGCCGGCCTCTCCAATGGCAAATGTGTGTGGCTGGAGGCGAGC GCGAGGCTTTCGGCAAAGGCAGTCGAGTGTTTCATTACGAGGGGAGCGCCCGGC CGGGGCTGTCGCACTCCCCGCGGAACATTTGGCTCCCTCCAGCTCCGAGAGAGG AGAAGAAGAAAGCGGAAAAGAGGCAGATTCACGTCGTTTCCAGCCAAGTGGACC TGATCGATGGCCCTCCTGAATTTATCACGATATTTGATTTATTAGCGATGCCCCCT GGTTTGTGTGTTACGCACACACACGTGCACACAAGGCTCTGGCTCGCTTCCCTCC CTCGTTTCCAGCTCCTGGGCGAATCCCACATCTGTTTCAACTCTCCGCCGAGGGC GAGCAGGAGCGAGAGTGTGTCGAATCTGCGAGTGAAGAGGGACGAGGGAAAAG AAACAAAGCCAAGACGCAACTTGAGACTCCCGCATCCCAAAAGAAGCACCAGAT CAGCAAAAAAAGAAGATGGGCCCCCCGAGCCTCGTGCTGTGCTTGCTGTCCGCA ACTGTGTTCTCCCTGCTGGGTGGAAGCTCGGCCTTCCTGTCGCACCACCGCCTGA AAGGCAGGTTTCAGAGGGACCGCAGGAACATCCGCCCCAACATCATCCTGGTGC TGACGGACGACCAGGATGTGGAGCTGGGTTCCATGCAGGTGATGAACAAGACCC GGCGCATCATGGAGCAGGGCGGGGCGCACTTCATCAACGCCTTCGTGACCACAC CCATGTGCTGCCCCTCACGCTCCTCCATCCTCACTGGCAAGTACGTCCACAACCA CAACACCTACACCAACAATGAGAACTGCTCCTCGCCCTCCTGGCAGGCACAGCA CGAGAGCCGCACCTTTGCCGTGTACCTCAATAGCACTGGCTACCGGACAGCTTTC TTCGGGAAGTATCTTAATGAATACAACGGCTCCTACGTGCCACCCGGCTGGAAGG AGTGGGTCGGACTCCTTAAAAACTCCCGCTTTTATAACTACACGCTGTGTCGGAA CGGGGTGAAAGAGAAGCACGGCTCCGACTACTCCAAGGATTACCTCACAGACCT CATCACCAATGACAGCGTGAGCTTCTTCCGCACGTCCAAGAAGATGTACCCGCAC AGGCCAGTCCTCATGGTCATCAGCCATGCAGCCCCCCACGGCCCTGAGGATTCAG CCCCACAATATTCACGCCTCTTCCCAAACGCATCTCAGCACATCACGCCGAGCTA CAACTACGCGCCCAACCCGGACAAACACTGGATCATGCGCTACACGGGGCCCAT GAAGCCCATCCACATGGAATTCACCAACATGCTCCAGCGGAAGCGCTTGCAGAC CCTCATGTCGGTGGACGACTCCATGGAGACGATTTACAACATGCTGGTTGAGACG GGCGAGCTGGACAACACGTACATCGTATACACCGCCGACCACGGTTACCACATC GGCC AGTTTGGCCTGGTGAAAGGGAA ATCCATGCC ATATGAGTTTGACATC AGG GTCCCGTTCTACGTGAGGGGCCCCAACGTGGAAGCCGGCTGTCTGAATCCCCACA TCGTCCTCAACATTGACCTGGCCCCCACCATCCTGGACATTGCAGGCCTGGACAT ACCTGCGGATATGGACGGGAAATCCATCCTCAAGCTGCTGGACACGGAGCGGCC GGTGAATCGGTTTCACTTGAAAAAGAAGATGAGGGTCTGGCGGGACTCCTTCTTG GTGGAGAGAGGCAAGCTGCTACAC AAGAGAGAC AATG ACAAGGTGGACGCCC A GGAGGAGAACTTTCTGCCCAAGTACCAGCGTGTGAAGGACCTGTGTCAGCGTGC TGAGTACCAGACGGCGTGTGAGCAGCTGGGACAGAAGTGGCAGTGTGTGGAGGA CGCCACGGGGAAGCTGAAGCTGCATAAGTGCAAGGGCCCCATGCGGCTGGGCGG CAGCAGAGCCCTCTCCAACCTCGTGCCCAAGTACTACGGGCAGGGCAGCGAGGC CTGCACCTGTGACAGCGGGGACTACAAGCTCAGCCTGGCCGGACGCCGGAAAAA ACTCTTCAAGAAGAAGTACAAGGCCAGCTATGTCCGCAGTCGCTCCATCCGCTCA GTGGCCATCGAGGTGGACGGCAGGGTGTACCACGTAGGCCTGGGTGATGCCGCC CAGCCCCGAAACCTCACCAAGCGGCACTGGCCAGGGGCCCCTGAGGACCAAGAT GACAAGGATGGTGGGGACTTCAGTGGCACTGGAGGCCTTCCCGACTACTCAGCC GCCAACCCCATTAAAGTGACACATCGGTGCTACATCCTAGAGAACGACACAGTC CAGTGTGACCTGGACCTGTACAAGTCCCTGCAGGCCTGGAAAGACCACAAGCTG CACATCGACCACGAGATTGAAACCCTGCAGAACAAAATTAAGAACCTGAGGGAA GTCCGAGGTCACCTGAAGAAAAAGCGGCCAGAAGAATGTGACTGTCACAAAATC AGCTACCACACCCAGCACAAAGGCCGCCTCAAGCACAGAGGCTCCAGTCTGCAT CCTTTCAGGAAGGGCCTGCAAGAGAAGGACAAGGTGTGGCTGTTGCGGGAGCAG AAGCGCAAGAAGAAACTCCGCAAGCTGCTCAAGCGCCTGCAGAACAACGACAC GTGCAGCATGCCAGGCCTCACGTGCTTCACCCACGACAACCAGCACTGGCAGAC GGCGCCTTTCTGGACACTGGGGCCTTTCTGTGCCTGCACCAGCGCCAACAA TAACACGTACTGGTGCATGAGGACCATCAATGAGACTCACAATTTCCTCTTCTGT GAATTTGCAACTGGCTTCCTAGAGTACTTTGATCTCAACACAGACCCCTACCAGC TGATGAATGCAGTGAACACACTGGACAGGGATGTCCTCAACCAGCTACACGTAC AGCTCATGGAGCTGAGGAGCTGCAAGGGTTACAAGCAGTGTAACCCCCGGACTC GAAACATGGACCTGGGACTTAAAGATGGAGGAAGCTATGAGCAATACAGGCAGT TTCAGCGTCGAAAGTGGCCAGAAATGAAGAGACCTTCTTCCAAATCACTGGGAC AACTGTGGGAAGGCTGGGAAGGTTAAGAAACAACAGAGGTGGACCTCCAAAAA CATAGAGGCATCACCTGACTGCACAGGCAATGAAAAACCATGTGGGTGATTTCC AGCAGACCTGTGGTATTGGCCAGGAGGCCTGAGAAAGCAAGCACGCACTCTCAG TC AACATGACAGATTCTGGAGGATA ACC AGCAGGAGC AGAGATAACTTCAGGAA GTCCATTTTTGCCCCTGCTTTTGCTTTGGATTATACCTCACCAGCTGCACAAAATG CATTTTTTCGTATCAAAAAGTCACCACTAACCCTCCCCCAGAAGCTCACAAAGGA AAACGGAGAGAGCGAGCGAGAGAGATTTCCTTGGAAATTTCTCCCAAGGGCGAA AGTCATTGGAATTTTTAAATCATAGGGGAAAAGCAGTCCTGTTCTAAATCCTCTT ATTCTTTTGGTTTGTCAC AAAGAAGGAACTAAGAAGCAGGACAGAGGCAACGTG GAGAGGCTGAAAACAGTGCAGAGACGTTTGACAATGAGTCAGTAGCACAAAAG AGATGACATTTACCTAGCACTATAAACCCTGGTTGCCTCTGAAGAAACTGCCTTC ATTGTATATATGTGACTATTTACATGTAATCAACATGGGAACTTTTAGGGGAACC TAATAAGAAATCCCAATTTTCAGGAGTGGTGGTGTCAATAAACGCTCTGTGGCCA GTGTAAAAGAAAATCCCTCGCAGTTGTGGACATTTCTGTTCCTGTCCAGATACCA TTTCTCCTAGTATTTCTTTGTTATGTCCCAGAACTGATGTTTTTTTTTTAAGGTACT GAAAAGAAATGAAGTTGATGTATGTCCCAAGTTTTGATGAAACTGTATTTGTAAA AAAAATTTTGTAGTTTAAGTATTGTCATACAGTGTTCAAAACCCCAGCCAATGAC CAGCAGTTGGTATGAAGAACCTTTGACATTTTGTAAAAGGCCATTTCTTGGGAAA AAAAAAAA (SEQ ID NO: 7). Reagents of the present kit may bind specifically to SEQ ID NO: 7. In addition, reagents that bind to a SULF2 gene product may have about 80% to about 100% identity to SEQ ID NO: 7, wherein the sequence identity may be from about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100% with SEQ ID NO: 7.
The amino acid sequence of the SULF2 protein is also described herein as SEQ ID NO: 8. Reagents of the present kit may bind specifically to SEQ ID NO: 8. For example, antibody, peptide, and/or polypeptide reagents of the present kit may bind to SEQ ID NO: 8.
In addition to the SULF2 gene products, reagents of the present kit or method may comprise gene products of the genes described herein. The expression of said gene products is significantly different between patients with Steroid-Resistant Nephrotic
Syndrome and patients with Steroid-Sensitive Nephrotic Syndrome. The genes or gene products described herein are publicly known; their gene sequences may be accessed at the National Institute of Health (NIH) GENE database at the following web address:
http://www.ncbi.nlm.nih.gov/gene/. Reagents that bind to the genes and gene products of the kit described herein may be purchased commercially. Alternatively, persons of ordinary skill in the art may also use publicly available information, for example, the NIH's GENE database, to design, prepare, and synthesize reagents of the present kit to hybridize to the genes or gene products of the kit by methods known to persons of ordinary skill in the art.
Reagents of the present invention may specifically bind to gene products of genes selected from the group consisting of the following: RFFL, INPP1, MCM2, SNK1G1, SIGLEC10, AGPAT6, DCAF8, FAM193A, RSPRY1, KCTD20, BLOC1S3, TOE1,
ACAD10, SULF2, RAB8A, NCRNA00294, ZNF318, CDK4, TMEM219, PRKCSH, SLC9A7, LEOl, STX4, PTPRK, GANAB, C170RF63, ZC3H18, MED 14, TRAPPC5, EXOC7, ACINI, ITGAL, SH2D1B, TRMT6, NACC1, RECQL5, ZC3H12D, PRR5L, UBE2J2, TOPBP1, GALNT2, BTN2A2, GPR108, CHMP4B, ALDOA, TRAPPC1,
SPRYD3, MTMR14, S1PR5, FAM134C, ALDOC, TCP1, CLCN3, NDUFS1, LGALS9, SUPT5H, JARID2, FAM40A, KLF2, AKIRIN1, ZNF782, OCIAD1, KPNA1, KIAA1033, UBE2W, LOC220930, NCRNA00081, ATP11B, SLC2A1, PIGB, SEPHS2, FGFR10P2, GLIPR2, ARHGEF2, S100A8, H2AFY, SELL, TRIM22, ZC3HAV1, MY05A, ZNF641, SRSF4, ZBTB34, NCOA4, ANTXR2, DDX3X, CHST11, PRNP, SLC35B4, GM2A, EZH1, LOC100289230, MYLIP, PCMTD2, ARHGAP15, AKAP10, ARHGAP19, B3GNT2, SYNJl, MBD5, ZNF266, TMEM87B, KCTDIO, ZNF587, SLC30A6, RABGAPl, PL-5283, RCSD1, ANXA7, KIAA0754, TMEM87A, ZNF187, GLT1D1, TGIF2-C20ORF24, TOR1B, ZNF41, AHSA2, TAOK3, CP110, GRPEL1, LOC283070, KIAA0141, ZMAT1, ZNF398, SPATA13, HGSNAT, MCC, PDHA1, COPB2, ADIPOR1, LILRB2, FBX07, C20ORF108, NFE2, LIN7A, SNCA, ODC1, HBA2, TIFA, HEMGN, BAG1, DCAF12, SIRPB1, RPGR, AQP9, STRADB, SNAP29, PADI2, SLC25A5, PPIB, C90RF78, FAM46C, PROK2, MKRNl, MPZL3, FAM104A, THOC5, GSPTl, SCARNA3, IGSF6, ARLl l, HBAl, CD59, EIF2AK1, PFKFB3, PRDX5, RAB4A, CDK6, FCGR2A, GNAI2, GNB1, STX7,
ARHGDIB, RNASEK, PTAR1, PECAM1, KIAA1267, ZNF281, CX3CR1, SH3BGRL3, PITPNA, MAPRE2, MGAT1, LM02, S100A4, PCMTD1, SNX18, NFKBIZ, C130RF18, CLEC12A, USP15, C40RF3, CAB39, FCGR3A, GABARAPL1, C3AR1, TNFAIP8L2, RAB33B, ELF2, MBOAT2, SEMA4A, SKAP2, RAB8B, PPP1CB, FUT4, EMB, PTGER4, EPS 15, STIM1, GNLY, CNDP2, LONP2, LIN7C, MGC12916, ACTR1A, LST1,
MAPKAPK3, GLRX, ARL8B, TBRG1, AFF3, MARK3, GCC1, C20RF29, ZMAT2, LCOR, CDC42, PHIP, RNF103, TNFSF10, PTP4A1, RUNX3, GOSR1, KIAA0922, OSBP, NUP214, CCNI, ERP29, LAMP1, LASP1, ARCN1, FUS, VPS24, MARCH8, MBNL3, MAX, BNIP3L, PIK3CG, PRKCB, HLA-E, STAT5A, CYTH4, GRB2, MXI1, GPSM3,
C220RF13, ARF3, SLC25A37, TPM3, S100A6, CFL1, YTHDF3, C190RF22, ATP6V0D1, CLIO, PIM1, STAT5B, TRIM58, UBE2H, ATP5B, NHP2L1, C160RF72, EPB41, BSG, GNS, PSAP, ARPC1B, BLCAP, ARHGAP30, ACTB, WDR26, CCDC69, PLEKH02, PTPRE, COTL1, PPT1, CREB5, RPS23, MSN, DAZAP2, SEC11A, GIMAP5, EIF4G2, IL6R, GIMAP4, TPD52L2, TCP11L2, HLA-B, AIF1, UBE2J1, ATF7, MEF2A, PIP4K2A, RGS2, GIMAP6, SETD2, NR2C2, WAC, TSPAN14, IL6ST, STK17B, NCRNA00282, FAM120B, WASF2, WNK1, SH3BP5, PAFAH1B1, RBM38, PPP1R15B, MLL5, BTG1, PIK3R5, SIAH2, PCGF3, PIK3IP1, WAPAL, CD53, XRCC5, ZNF638, RBL2, NUFIP2, OAZ1, CNST, MYL12A, TAF7, TNIP1, FAM117A, PCF11, KLHL6, HIATL1, SERTAD2, RBM5, CHD7, JAK3, GNG2, S1PR1, MYD88, IKZF1, UBE2G1, ELF1, ENSA, AQP3, IRF9, SESN3, DCAF6, FCER1G, ZNF776, INSIG2, RPS9, SNHG12, ZNF791, CD96, HSP90B1, UBAP2, MARCKS, ZFX, SNX3, DYNC1LI1, STAT6, MME, TNFSF13B, MSH6, PIK3AP1, CEACAMl, CD 180, CHPTl, BCL6, C20RF24, ADM, IGBPl, DNAJC2, KDM6A, C40RF14, SOD2, PARP9, TUBAIA, ZFP62, SEPT6, UBE2L6, ZNF121, EIF2S3, IL7R, SNORA45, ANXA3, JAZF1, SNORD89, PEBP1, CCNDBP1, CCNT1, ASNSD1, TMEM14B, MTIF2, YBX1, PLSCR1, TAF13, C220RF46, AHSA1, SAT1, DDX60L, and ZNF226. The gene names, gene symbols, and gene IDs of genes from which gene products may be derived for the present invention are described herein. The gene names, gene symbols, and gene IDs of genes and gene products of the present invention may be used to query the gene sequence information on publicly available databases known to persons of ordinary skill in the art. In accordance with one embodiment, a kit is provided that comprises reagents for measuring the gene expression of SULF2 (GER(S2/Sl)suLF2 or GER
(S1/S2)SULF2) and/or the enzymatic activity of SULF2 ((S2/Sl)suLF2),and optionally, the kit may comprise reagents for detecting one or more of the other discriminator gene products disclosed herein.
A method of identifying a patient who is resistant to steroid therapy of Nephrotic Syndrome is also disclosed herein. The method comprises the steps of a) assaying marker genes products expressed in the patient through the use of reagents that bind to the gene products of the genes identified in the immediate above paragraph, b) analyzing the expression levels of the marker gene products relative to a control sample, wherein the control sample is taken from a patient that has Nephrotic Syndrome and is responsive to steroid treatment, or relative to standard values that have been established based on population data, including for example from patients that have Nephrotic Syndrome that is responsive to steroid treatment, and c) identifying a patient as being resistant to steroid treatment of Nephrotic Syndrome based on the relative expression of said marker genes. As described herein, the method of the present invention may further comprise treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non- steroidal therapies of Nephrotic Syndrome. In accordance with one embodiment, the method further comprises a step of measuring the enzymatic activity of SULF2, (S2/S1)SULF2- It is believed that expression of SULF2 (GER(S2/S1)SULF2) and enzyme analysis of SULF2 (S2/S1)SULF2) coupled with the other discriminator genes disclosed herein provides an effective means to identify Nephrotic Syndrome patients that will be non-responsive to steroidal treatments. In a further embodiment the method comprises treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non-steroidal therapy including for example, administering a drug selected from the group consisting of adrenocorticotropic hormone, Cyclosporine, Tacrolimus, Mycophenolate mofetil,
Plasmapheresis, column A immunoabsorbtion, and Rituximab.
Definitions
The term "about" as used herein means greater or lesser than the value or range of values stated by 10 percent, but is not intended to designate any value or range of values to only this broader definition. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values.
"Nephrotic Syndrome" as defined herein is a kidney disorder characterized by a group of symptoms in a patient that include, but are not limited to, protein in the urine, low blood protein levels (e.g., albumin), and swelling. The patient of the present disclosure may be an adult patient or a pediatric patient. An exemplary patient of the present invention is a pediatric patient, meaning a patient that is 18 years old or younger.
As used herein, the term "intron" refers to any nucleic acid sequence comprised in a gene (or expressed nucleotide sequence of interest) that is transcribed but not translated. Introns include untranslated nucleic acid sequence within an expressed sequence of DNA, as well as a corresponding sequence in RNA molecules transcribed therefrom. Introns may be used in combination with a promoter sequence to enhance translation and/or mRNA stability.
As used herein, the terms "5 '-untranslated region" or "5'-UTR" refers to an untranslated segment in the 5' terminus of pre-mRNAs or mature mRNAs. For example, on mature mRNAs, a 5'-UTR typically harbors on its 5' end a 7-methylguanosine cap and is involved in many processes such as splicing, polyadenylation, mRNA export towards the cytoplasm, identification of the 5' end of the mRNA by the translational machinery, and protection of the mRNAs against degradation.
As used herein, the term "3 '-untranslated region" or "3'-UTR" refers to an untranslated segment in a 3' terminus of the pre-mRNAs or mature mRNAs. For example, on mature mRNAs this region harbors the poly- (A) tail and is known to have many roles in mRNA stability, translation initiation, and mRNA export.
As used herein, the term "polyadenylation signal" refers to a nucleic acid sequence present in mRNA transcripts that allows for transcripts, when in the presence of a poly- (A) polymerase, to be polyadenylated on the polyadenylation site, for example, located 10 to 30 bases downstream of the poly-(A) signal. Many polyadenylation signals are known in the art and are useful for the present invention.
As used herein, the term "isolated" refers to a biological component (including a nucleic acid or protein) that has been separated or removed from other biological components in the cell of the organism in which the component naturally occurs (i.e., other chromosomal and extra-chromosomal DNA). For example, a naturally-occurring
polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
As used herein, the term "purified" in reference to nucleic acid molecules does not require absolute purity (such as a homogeneous preparation). Instead, "purified" represents an indication that the sequence is relatively more pure than in its native cellular environment. For example, the "purified" level of nucleic acids may be at least 2-5 fold greater in terms of concentration or gene expression levels as compared to its natural level. Additionally a "purified polypeptide" is used herein to describe a polypeptide which has been separated from other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates.
The claimed DNA molecules may be obtained directly from total DNA or from total RNA. In addition, cDNA clones and copies are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified, naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA). Individual cDNA clones may be purified from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and purification of distinct cDNA clones yields an approximately 106-fold purification of the native message.
Likewise, a DNA sequence may be cloned into a plasmid. Such a clone is not naturally occurring, but rather is preferably obtained via manipulation of a partially purified, naturally occurring substance, such as a genomic DNA library. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude, is favored in these techniques.
Similarly, purification represents an indication that a chemical or functional change in the component DNA sequence has occurred. Nucleic acid molecules and proteins that have been "purified" include nucleic acid molecules and proteins purified by standard purification methods. The term "purified" also embraces nucleic acids and proteins prepared by recombinant DNA methods in a host cell, as well as chemically- synthesized nucleic acid molecules, proteins, and peptides.
The term "recombinant" means a cell or organism in which genetic
recombination has occurred. It also includes a molecule (e.g. , a vector, plasmid, nucleic acid, polypeptide, or a small RNA) that has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration may be performed on the molecule within, or removed from, its natural environment or state.
As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA (including small RNA molecules) and/or the process by which the transcribed mRNA (also referred to as "transcript") is subsequently translated into peptides, polypeptides, or proteins. Gene expression may be influenced by external signals, for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene may also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules, such as mRNA, or through activation, inactivation,
compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression may be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, qRT-PCR, qPCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).
As used herein, the terms "nucleic acid molecule," "nucleic acid," or
"polynucleotide" (all three terms being synonymous with one another) refer to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms, and mixed polymers thereof. A "nucleotide" may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of either type of nucleotide. A nucleic acid molecule is usually at least ten bases in length, unless otherwise specified. The terms may refer to a molecule of RNA or DNA of indeterminate length. The terms include single- and double-stranded forms of DNA. A nucleic acid molecule may include either or both naturally- occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of ordinary skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally-occurring nucleotides with an analog, intemucleotide modifications (e.g., uncharged linkages, such as, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages, such as, phosphorothioates,
phosphorodithioates, etc.; pendent moieties, such as, peptides; intercalators, such as, acridine, psoralen, etc.; chelators; alkylators; and modified linkages, such as, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single- stranded, double- stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.
Transcription proceeds in a 5' to 3' manner along a DNA strand. This means that RNA is made by sequential addition of ribonucleotide-5' -triphosphates to the 3' terminus of the growing chain with a requisite elimination of the pyrophosphate. In either a linear or circular nucleic acid molecule, discrete elements (e.g., particular nucleotide sequences) may be referred to as being "upstream" relative to a further element if they are bonded or would be bonded to the same nucleic acid in the 5' direction from that element. Similarly, discrete elements may be referred to as being "downstream" relative to a further element if they are or would be bonded to the same nucleic acid in the 3' direction from that element.
As used herein, the term "base position" refers to the location of a given base or nucleotide residue within a designated nucleic acid. A designated nucleic acid may be defined by alignment with a reference nucleic acid.
As used herein, the term "hybridization" refers to a process where oligonucleotides and their analogs hybridize by hydrogen bonding, which includes Watson- Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid molecules consist of nitrogenous bases that are either pyrimidines, such as cytosine (C), uracil (U), and thymine (T), or purines, such as adenine (A) and guanine (G). Nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and bonding of a pyrimidine to a purine is referred to as "base pairing." More specifically, A will form a specific hydrogen bond to T or U, and G will specifically bond to C. "Complementary" refers to the base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.
As used herein, the term "oligonucleotide" refers to a short nucleic acid polymer. Oligonucleotides may be formed by cleavage of longer nucleic acid segments or by
polymerizing individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to several hundred base pairs in length. Because oligonucleotides may bind to a complementary nucleotide sequence, they may be used as probes for detecting DNA or RNA. Oligonucleotides composed of DNA (oligodeoxyribonucleotides) may be used in Polymerase Chain Reaction, a technique for the amplification of small DNA sequences. In Polymerase Chain Reaction, an oligonucleotide is typically referred to as a "primer" which allows a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.
As used herein, the terms "Polymerase Chain Reaction" or "PCR" refer to a procedure or technique in which minute amounts of nucleic acid, RNA, and/or DNA, are amplified. Generally, sequence information from the ends of the region of interest or beyond needs to be available, so that oligonucleotide primers may be designed. PCR primers will be identical or similar in sequence to opposite strands of the nucleic acid template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR may be used to amplify specific RNA sequences or DNA sequences from total genomic DNA and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc.
As used herein, the term "primer" refers to an oligonucleotide capable of acting as a point of initiation of synthesis along a complementary strand when conditions are suitable for synthesis of a primer extension product. The synthesizing conditions include the presence of four different deoxyribonucleotide triphosphates (i.e., A,T,G, and C) and at least one polymerization-inducing agent or enzyme such as Reverse Transcriptase or DNA polymerase. These reagents are typically present in a suitable buffer that may include constituents which are co-factors or which affect conditions, such as pH and the like at various suitable temperatures. A primer is preferably a single strand sequence, such that amplification efficiency is optimized, but double stranded sequences may be utilized.
As used herein, the term "probe" refers to an oligonucleotide or polynucleotide sequence that hybridizes to a target sequence. In particular, in quantitative Real Time Polymerase Chain Reaction or qRT-PCR, such as the TaqMan® or TaqMan®-style assay procedure (e.g., SYBR Green), the probe hybridizes to a portion of the target situated between the annealing site of the two primers. A probe includes about eight nucleotides, about ten nucleotides, about fifteen nucleotides, about twenty nucleotides, about thirty nucleotides, about forty nucleotides, or about fifty nucleotides, or about sixty nucleotides, or about seventy nucleotide, or about eighty nucleotides, or about eighty- five nucleotides. In some embodiments, a probe includes from about eight nucleotides to about fifteen nucleotides.
A probe may further include a detectable label, such as, a radioactive label, a biotinylated label, a fluorophore (e.g., Texas-Red®, fluorescein isothiocyanate, etc.,). The detectable label may be covalently attached directly to the probe oligonucleotide, such that the label is located at the 5' end or 3' end of the probe. A probe comprising a fluorophore may also further include a quencher dye (e.g. , Black Hole Quencher™, Iowa Black™, etc.).
As used herein, the terms "sequence identity" or "identity" may be used interchangeably and refer to nucleic acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
As used herein, the term "percentage of sequence identity" or " percentage of sequence homology" refers to a value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences or amino acid sequences) over a comparison window, wherein the portion of a sequence in the comparison window may comprise additions, substitutions, mismatches, and/or deletions (i.e. , gaps) as compared to a reference sequence in order to obtain optimal alignment of the two sequences. A percentage is calculated by determining the number of positions at which an identical nucleic acid or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Methods for aligning sequences for comparison are well known. Various bioinformatics or computer programs and alignment algorithms, such as BLAST, ClustalW, and Sequencher, GAP 10, and others are also well known in the art and/or may be used accordingly.
For example, The National Center for Biotechnology Information (NCBI) Basic
Local Alignment Search Tool (BLAST™; Altschul et al. (1990) J. Mol. Biol. 215:403-10) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the "help" section for BLAST™. For comparisons of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST™ (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.
As used herein, the term "promoter" refers to a region of DNA that is generally located upstream of a gene (i.e., towards the 5' end of a gene) and is necessary to initiate and drive transcription of the gene. A promoter may permit proper activation or repression of a gene that it controls. A promoter may contain specific sequences that are recognized by transcription factors. These factors may bind to a promoter DNA sequence, which results in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene. The promoter generally refers to all gene regulatory elements located upstream of the gene, including, 5'-UTR, introns, and leader sequences.
As used herein, the term "transformation" encompasses all techniques in which a nucleic acid molecule may be introduced into a cell. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation; lipofection; microinjection, bacterial-mediated transfer; direct DNA uptake; and microprojectile
bombardment. These techniques may be used for both stable transformation and transient transformation of a human cell. "Stable transformation" refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. "Transient transformation" refers to the introduction of a nucleic acid fragment into the nucleus or DNA-containing organelle of a host organism, resulting in gene expression without genetically stable inheritance.
As used herein, the term "transduce" refers to a process where a virus transfers nucleic acid into a cell. As used herein, the term "transgene" refers to an exogenous nucleic acid sequence. In one example, a transgene is a gene sequence, such as a gene encoding an industrially or pharmaceutically useful compound. In yet another example, a transgene is an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. A transgene may contain regulatory sequences operably linked to the transgene (e.g. , a promoter, intron, 5'-UTR, or 3'-UTR). In some embodiments, a nucleic acid of interest is a transgene. However, in other embodiments, a nucleic acid of interest is an endogenous nucleic acid, wherein additional genomic copies of the endogenous nucleic acid are desired, or a nucleic acid that is in the antisense orientation with respect to the sequence of a target nucleic acid in a host organism.
As used herein, the term "heterologous coding sequence" is used to indicate any polynucleotide that codes for, or ultimately codes for, a peptide or protein or its equivalent amino acid sequence, e.g. , an enzyme, that is not normally present in the host organism and may be expressed in the host cell under proper conditions. As such,
"heterologous coding sequences" may include one or additional copies of coding sequences that are not normally present in the host cell, such that the cell is expressing additional copies of a coding sequence that are not normally present in the cells. The heterologous coding sequences may be RNA or any type thereof (e.g. , mRNA), DNA or any type thereof (e.g. , cDNA), or a hybrid of RNA/DNA. Examples of coding sequences include, but are not limited to, full-length transcription units that comprise such features as the coding sequence, introns, promoter regions, 5'-UTR, 3'-UTR, and enhancer regions.
"Heterologous coding sequences" also include the coding portion of the peptide or enzyme (i.e. , the cDNA or mRNA sequence), the coding portion of the full-length transcriptional unit (i.e., the gene comprising introns and exons), "codon optimized" sequences, truncated sequences or other forms of altered sequences that code for the enzyme or code for its equivalent amino acid sequence, provided that the equivalent amino acid sequence produces a functional protein. Such equivalent amino acid sequences may have a deletion of one or more amino acids, with the deletion being N-terminal, C-terminal, or internal. Truncated forms are envisioned as long as they have the catalytic capability indicated herein.
As used herein, the term "control" refers to a sample used in an analytical procedure for comparison purposes. A control can be "positive" or "negative". For example, where the purpose of an analytical procedure is to detect a differentially expressed transcript or polypeptide in cells or tissue, it is generally preferable to include a positive control, such as a sample from a known plant exhibiting the desired expression, and a negative control, such as a sample from a known plant lacking the desired expression.
As used herein, the terms "hybridize," "bind(s)," "specifically bind(s)," "specifically hybridize(s)" and/or "specifically complementary" are terms that indicate a sufficient degree of complementarity, such that stable and specific binding occurs between the nucleic acid molecule and a target nucleic acid molecule. Hybridization between two nucleic acid molecules involves the formation of an anti-parallel alignment between the nucleic acid sequences of the two nucleic acid molecules. The two molecules are then able to form hydrogen bonds with corresponding bases on the opposite strand to form a duplex molecule that, if it is sufficiently stable, is detectable using methods well known in the art. A nucleic acid molecule need not be 100% complementary to its target sequence to be specifically hybridizable. However, the amount of sequence complementarity that must exist for hybridization to be specific is a function of the hybridization conditions used.
As used herein, "stringent conditions" encompass conditions under which hybridization will only occur if there is less than 20% mismatch (i.e., at least 80% identity) between the hybridization molecule and a sequence within the target nucleic acid molecule. "Stringent conditions" include further particular levels of stringency. Thus, as used herein, "moderate stringency" conditions are those under which molecules with more than 20% sequence mismatch will not hybridize; conditions of "high stringency" are those under which sequences with more than 10% mismatch will not hybridize; and conditions of "very high stringency" are those under which sequences with more than 5% mismatch will not hybridize. The following are representative, non-limiting hybridization conditions.
High Stringency condition detect sequences that share at least 90% sequence identity, and include, but are not limited to hybridization in 5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 16 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 20 minutes each.
Moderate Stringency conditions detect sequences that share at least 80% sequence identity, and include, but are not limited to hybridization in 5x-6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65-70 °C for 16-20 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 5-20 minutes each; and wash twice in lx SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 55-70 °C for 30 minutes each.
As used herein, the term "detectable marker" refers to a label capable of detection, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme. Examples of detectable markers include, but are not limited to, the following: fluorescent labels (e.g. , FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g. , horseradish peroxidase, β- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g. , leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In an embodiment, a detectable marker may be attached by spacer arms of various lengths to reduce potential steric hindrance.
As used herein, the term "detecting" is used in the broadest sense to include both qualitative and quantitative measurements of a specific molecule, for example, measurements of a specific polypeptide.
As used herein, the term "treating" includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms. For example, as used herein the term "treating Nephrotic Syndrome" will refer in general to altering reducing and/or alleviating clinical symptoms of said disease. For example, treating Nephrotic Syndrome may comprise, treating the patient with steroids, such as corticosteroids, such as prednisone or prednisolone, or glucocorticoids (GCs). Treatment of Nephrotic Syndrome (NS) with non-steroidal therapy may also occur in patients that are unresponsive or resistant to treatment with steroids. For example, adrenocorticotropic hormone (ACTH), Cyclosporine (CYA), Tacrolimus (TAC),
Mycophenolate mofetil (MMF), Plasmapheresis, Column A immunoabsorbtion, and
Rituximab are known in the art to be non-steroidal treatment alternatives for patients that are resistant to steroid treatment of NS. Adrenocorticotropic hormone (ACTH) reduces proteinuria in nephrotic patients during treatment focused on lipid-lowering effects. It has been reported that 2 mg/week for 1 year of ACTH was as effective as methylprednisolone pulses and cytotoxic drugs. In addition, ACTH reduced proteinuria in children non- responding to traditional therapy, however, without protection on functional decline.
In children with steroid-resistant NS, Cyclosporine CYA reduced the relative risk of persistent NS and produced significant benefits in adults. CYA reduced proteinuria in 70-80% of patients with steroid-resistant NS, lasting after drug withdrawal in 40% of patients. The frequency of either complete or partial remission of NS was significantly higher in the group receiving CYA (85% versus 55%), albeit without difference in renal
deterioration rate. However, the fear of CYA toxicity, which may worsen renal function, still exists and has stimulated the search for alternative treatments.
A study of children with NS, dependent on or resistant to traditional therapies, reported a high remission rate with TAC (81% complete and 13% partial, within 5 months). These benefits were reproduced in small groups of cortico-dependent or -resistant children, with complete remission in 50% of the cases and partial remission in 40% of patients. TAC in association with steroids in CYA-resistant Nephrotic Syndrome induced remission in 12/25 adults, with reversible nephrotoxicity in 40% of adult patients.
Mycophenolate mofetil (MMF) induced remission in 19 children resistant to conventional therapy and reduction of relapses with a 50% steroid-sparing effect. However, 60% of the children relapsed after withdrawal. Similarly, in 9/18 adults with steroid- and CYA-resistant NS, MMF induced a decrease in proteinuria, but 50% of patients relapsed at withdrawal. Removing the hypothetical permeability factor is the rationale for
plasmapheresis or protein A immunoadsorption treatment of recurrent NS after
transplantation. Rituximab treatment of NS patients in a dose of four weekly injections has been reported. The decrease in proteinuria was rapid over the first 6 months and continued over 1 year, without any difference between patients previously treated with traditional four doses (e.g., steroids). Thus, Rituximab is an option for non-steroidal treatment of NS. As used herein an "effective" amount or a "therapeutically effective amount" of a compound refers to a nontoxic but sufficient amount of the compound to provide the desired effect. For example, desired effects of an effective amount of a compound would be the prevention or treatment of Nephrotic Syndrome, as measured, for example, by a decrease of protein in the urine, normal to higher blood protein levels (e.g., albumin) or levels of electrolytes, creatinine, and blood urea nitrogen (BUN), and a decrease in swelling in the body, for example on eyes, face, ankles, or feet. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
Unless otherwise specifically explained, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art that this disclosure belongs. Definitions of common terms in molecular biology maybe found in, for example: Lewin, Genes V, Oxford University Press, 1994; Kendrew et al. (eds.), The
Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994; and Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995.
Patient Samples and Blood Collection
Clinical protocols described herein were approved by an Institutional Review
Board. In accordance with the Declaration of Helsinki, written informed consent was obtained from the parents of all pediatric participants described herein before samples were collected. NS patients of the present invention comprise adults or children, adults and children, but are preferably pediatric patients. Pediatric NS patients aged between 18 months and 18 years were included in this study, and all exhibited 4+ proteinuria (greater than 2,000 mg/dl) and edema (see Table 1). Patients were excluded if they received more than six days of GC therapy prior to beginning the study, or if they received investigational drugs.
Steroid response (e.g., steroid-sensitivity, steroid-resistance, and steroid- dependence) was assessed in patients approximately twelve months after NS disease presentation, such that patients were previously classified as "steroid- sensitive" and "steroid- resistant" according to clinical parameters. Two blood samples were collected from each NS patient, one at presentation (sample SI), so before steroid therapy, and the second typically about 4 weeks to about 8 weeks after the initiation of steroid therapy (sample S2). Each blood sample consisted of 8 mis of peripheral blood drawn into a CPT® tube with sodium citrate anticoagulant (Becton & Dickenson, Franklin Lakes, NJ) and 2.5 mis of peripheral blood drawn into a PaxGene® tube (PreAnalytiX GmbH, Switzerland). Samples in CPT tubes were processed immediately to isolate peripheral blood leukocytes and plasma, or, were shipped overnight at ambient temperature before processing.
Total leukocytes were isolated from patient blood from eleven pediatric patients having SSNS and SRNS by standard protocols known in the art and stored at -140°C in frozen PaxGene tubes. The leukocytes in frozen PaxGene tubes were processed to isolate total peripheral blood RNA according to the manufacturer's instructions (PreAnalytiX).
Paired patient RNA samples (SI and S2) taken from the eleven patients were processed for transcriptome-wide RNA sequencing analysis using RNAseq, according to the
manufacturer's instructions. In addition, the samples were assessed for gene expression using quantitative Real Time Polymerase Chain Reaction (qRT-PCR) analysis or aryl sulfatase enzyme activity assays. Samples from 38 NS patients were also processed for qRT-PCR and aryl sulfatase enzyme assays only (see Table 1).
RNA Analysis and Sequencing of RNA
Total leukocyte RNA was further prepared using Trizol reagent (Life
Technologies, Grand Island, NY) according to the manufacturer's instructions. RNA quality was verified on a 2100 Bioanalyzer (Agilent, Santa Clara, CA), with all samples showing an
RNA integrity number greater than 6.5.
The NuGen Ovation RNASeq System (San Carlos, CA) was used to generate cDNA libraries from 10 ng of total RNA per sample (NuGen Technologies, San Carlos, CA).
This method uses poly-dT and random hexamer primers for cDNA synthesis, the latter designed to reduce cDNAs from rRNA by at least 90%. The NuGen cDNA library preparation was able to capture and remove RNA lacking poly A tails, including ncRNA,
RNAs, etc. since sequence analysis on annotated coding genes only was of interest. A linear amplification step enables RNAseq analysis on small amounts of RNA ranging from about 10 to about 20 ng of total RNA. Samples were processed to produce 8-barcoded, paired-end libraries or 16-barcoded, single-end libraries. Samples were sequenced using the SOLiD
5500 Next-Generation Sequencing instrument (Life Technologies), according to
manufacturer's instructions. Quantitative Real Time Polymerase Chain Reaction
Peripheral blood cDNA was produced from patient whole-blood RNA
(BioChain, Hayward, CA) using reverse transcriptase (High Capacity kit, Life
Technologies). Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) was performed on an iQ5 Real Time PCR Detection System (Bio-Rad, Hercules, CA) using SYBR Green master mix (BioRad). The amplification cycling parameters were 5 min at 95°C, 40 cycles of 15 s at 95°C, and 30 s at 55°C. Melting curves were obtained to ensure single products. Gene expression was measured by the Pfaffl method, with expression of the housekeeping gene RPL19 that encodes the ribosomal protein L19 and is known to not be affected by GCs.
RNA Sequence Analysis
Data generated using RNAseq for the eleven NS patients (having paired samples SI and S2, from before and after GC treatment, respectively) were aligned to the International Human Genome Sequencing Consortium human reference sequence (Hgl9 ref). This gene alignment was performed using gene annotation from the University of California at Santa Cruz's genome browser and SOLiD Lifescope v2.5.1 (Life Technologies). Samples were aligned in an 8-barcode set with paired-end reads (with 50 bases sequenced in the forward direction and 35 bases for the reverse) or in a 16-barcode set with single-end alignment performed for the 75 bases sequenced in the forward direction.
The primary data set consisted of expression values for greater than 20,000 genes, which were reduced to 15,092 genes, 7334 genes, and 3280 genes by the removal of genes with little or no expression in one or several of the patient samples. The remaining genes were evaluated for gene expression. Gene expression values were used to calculate the Gene Expression Ratio (GER) in patients before (SI) and after (S2) treatment with GCs, annotated as the GER(S1/S2) value. Table 1. Pediatric patient clinical data on steroid treatment SULF2 expression and arylsufatase activity values.
Figure imgf000033_0001
Figure imgf000034_0001
Gene Expression and Identification
Gene expression of coding exons was measured in reads per kilobase per million reads, as reported by the wtcounts module of the SOLid Lifescope technology, and was calculated based on reads that aligned to coding regions of genes annotated by RefSeq.
The number of sequence fragments generated and mapping percentages had an average of approximately 60% of generated reads for each sample mapping to the genome. Overall, a number from about 1 million to about 200 million fragments may be generated per sample. Preferably, from about 1 million to about 150 million fragments, from about. 2 million to about 135 million fragments, from about 3 million to about 125 million fragments, from about 4 million to about 120 million, or from about 5 million to about 112 million fragments are generated per sample. Although read alignments were concentrated into peaks at known exons, other regions of the genome such as introns and intergenic space also showed read coverage. Mature mR A reads derived from known exons were included in this study, whereas all other expressed sequences were excluded. These read numbers of the samples collected before and after the GC treatment of the patients were used to calculate the Gene Expression Ratios (GER), yielding a GER(S1/S2) value or a GER(S2/S1) value as specified herein. These gene expression ratios were used for subsequent data analyses.
The GER(S1/S2) values of 7334 genes were initially analyzed by volcano plotting to identify a smaller set of genes that could discriminate between patients with SSNS and SRNS (see Figure 1). For each gene, i is the mean GER(S1/S2) value for SSNS patients and r2 is the mean GERCS1/S2) value for SRNS patients. Figure 1 shows the difference in ri and r2 (f'i-r2) for the SSNS and SRNS patient groups, respectively, as plotted on the X-axis.
Significance of the expression analysis was determined using a standard two- sample unpaired t-test criteria resulting in the( -logj0(p values)) plotted on the Y-axis of Figure 1. The selected cutoff threshold of C or (r1-r2)=0.263 on the X-axis and C2 or p value=0.02 on the Y-axis was implemented. Genes that had an absolute value of the difference between ri and r2 (lri-r2l) greater than the Ci cutoff value (0.263) and a p value less than the C2 cutoff value (0.02) were selected as being differentially expressed in NS patients. Figure 1 identifies 72 genes (in the upper right and upper left regions of the plot) that were differentially expressed in the leukocytes of both NS patient groups following the treatment with GCs. Names of the 72 genes and their gene symbols or IDs are listed in Table 2, including SULF2. To independently assess the discriminatory potential of NS patients by these 72 genes, the genes were hierarchically grouped in a clusterogram according to their gene expression patterns as expressed by their GER(S1/S2) values. This clustering method calculates the pairwise (Euclidian) distances between all pairs of genes. Figure 2 shows the resulting cluster tree; the left side of the tree is a cluster representing the Set I gene group (genes 1-57) that is distinctly partitioned from the Set II gene cluster (genes 58-72) on the right side of the tree. Cluster analysis of these genes (see top of Figure 2) clearly
differentiated between the two NS patient groups, as represented by the two main branches correlating to the SRNS and SSNS patient groups. SRNS and SSNS patient groups were distinguished by characteristic clusters of increased (light) and decreased (dark) GER(S1/S2) values following glucocorticoid (GC) treatment. The GER(S1/S2) values of Figure 2 correlate to gene expression, such that light colors indicate upregulated gene expression and dark colors represent downregulated gene expression. Accordingly, Set I genes tended to exhibit a higher GER(S1/S2) value in SRNS patients, as compared to the SSNS patients where the GER(S1/S2) value for these genes tended to be lower. Accordingly, gene expression of Set I genes was reduced following GC treatment in SRNS patients, whereas expression of Set II genes was induced following GC treatment in SRNS patients. The GER(S1/S2) for SULF2 (GER(S1/S2)SULF2) fell into the Set I gene group (marked by the arrowhead on right side of Figure 2) indicating SULF2 expression was reduced after treatment with GCs.
In contrast, the Set II genes tended to exhibit higher GER(S1/S2) values in SSNS patients as compared to the SRNS patients where the GER(S1/S2) tended to be lower. Set I and Set II totaled 72 genes that collectively showed excellent discriminatory power between eleven SRNS and SSNS patients (see Figure 3). In fact, all patients identified as SRNS or SSNS by the clusterogram method were determined to be correctly classified and in agreement with the clinical diagnosis and observations of the patient on record. Thus, these genes may serve as markers for differentiation of patients with SSNS and SRNS. Table 2.72- ene set with differential ex ression in SRNS vs. SSNS atients (Fi ures 1-4).
Figure imgf000037_0001
39 UBE2J2 ubiquitin-conjugating enzyme E2, J2 118424
40 TOPBP1 topoisomerase (DNA) II binding protein 1 11073
41 GALNT2 polypeptide N-acetylgalactosaminyltransferase 2 2590
42 BTN2A2 butyrophilin, subfamily 2, member A2 10385
43 GP 108 G protein-coupled receptor 108 56927
44 CHMP4B charged multivesicular body protein 4B 128866
45 ALDOA aldolase A, fructose-bisphosphate 226
46 TRAPPC1 trafficking protein particle complex 1 58485
47 SPRYD3 SPRY domain containing 3 84926
48 MTMR14 myotubularin related protein 14 64419
49 S1PR5 sphingosine-l-phosphate receptor 5 53637
50 FAM134C family with sequence similarity 134, member C 162427
51 ALDOC aldolase C, fructose-bisphosphate 230
52 TCP1 t-complex 1 6950
53 CLCN3 chloride channel, voltage-sensitive 3 1182
54 NDUFS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa 4719
55 LGALS9 lectin, galactoside-binding, soluble, 9 3965
56 SUPT5H suppressor of Ty 5 homolog (S. cerevisiae) 6829
57 JARID2 jumonji, AT rich interactive domain 2 3720
58 FAM40A striatin interacting protein 1 85369
59 KLF2 Kruppel-like factor 2 10365
60 AKIRIN1 akirin 1 79647
61 ZNF782 zinc finger protein 782 158431
62 OCIAD1 OCIA domain containing 1 54940
63 KPNA1 karyopherin alpha 1 (importin alpha 5) 3836
64 KIAA1033 KIAA1033 23325
65 UBE2W ubiquitin-conjugating enzyme E2W (putative) 55284
66 LOC220930 ZEB1 antisense RNA 1 220930
67 NCRNA00081 BBSome interacting protein 1 92482
68 ATP11B ATPase, class VI, type 11B 23200
solute carrier family 2 (facilitated glucose transporter),
69 SLC2A1 member 1 6513
70 PIGB phosphatidylinositol glycan anchor biosynthesis, class B 9488
71 SEPHS2 selenophosphate synthetase 2 22928
72 FGFR10P2 FGFR1 oncogene partner 2 26127
In addition, scatter plotting was applied as a statistical tool in order to separate averaged GER(S1/S2) values for both groups of patients with SRNS and SSNS. The difference between the two patient groups was reinforced by the separation of the averaged GER(S1/S2) values of the 72 genes. For example, Figure 3 shows a scatter diagram where the GER(S1/S2) values of the 72 genes differentially expressed in SRNS and SSNS patients are plotted against each other. Figure 3 demonstrates a clear and linear separation of Set I genes (diamond symbols) and Set II genes (round symbols), and thereby also of the SRNS patients and the SSNS patients. These results show correlation and/or association between the independent variable GER(S1/S2) for SRNS and SSNS patients, and its use to classify and/or diagnose NS patient groupings. Consistent with the results shown in Figures 1 and 2, Figure 3 shows the average GER(S1/S2) for SULF2 (GER(S1/S2)SULF2) is approximately 1.0, and thus, falls into the group of Set I genes. These results support the indication that Set I gene expression is highly induced in SSNS patients following GC treatment, as compared to the reduction of Set I genes in SRNS patients. Taken together, genetic research coupled with applied statistical methods identified 72 genes (see Table 2), including SULF2, that can discriminate between pediatric patients with SSNS and SRNS, and thus may serve as markers for GC resistance.
Bioinformatic Gene Filtering
While advanced sequencing methods such as RNAseq produce a plethora of information, any sequencing method known in the art may be used in the present invention. In particular sequencing methods comprising next generation sequencing, high-throughput, and low-throughput sequencing, and other sequencing methods known in the art may all be used in the present invention. However, the subsequent sequencing data analysis remains a challenge.
In addition to conventional statistical methods, various machine learning systems and computer algorithms known in the art have been used in life sciences in the recent decade to process such large volumes of data. These approaches have identified genes or biomarkers that are causative of diseases, useful for prognosis, or predictive of drug efficacy or optimal therapies. Accordingly, statistical and computational methods known in the art were used to process large datasets of the present invention.
Sulfatase 1 and 2 Gene Expression
Among the 17 members of the sulfatase superfamily, SULF2 and its paralog SULF1 are distinguished by both their extracellular location coupled with endo- sulfatase activity that strictly requires a neutral pH, as is found in the extracellular space. Both SULF2 and SULF1 exhibit the same substrate specificity and cannot be distinguished biochemically. In particular, the biochemical assay for aryl sulfatase activity, described herein, cannot differentiate between SULF1 and SULF2. Other sulfatases in the family are located in lysosomes and have an exo-sulfatase activity on proteoglycan chains, or are located in the endoplasmic reticulum and the Golgi apparatus. Thus, enzymatic assays at a neutral or slightly alkaline pH that use the extracellular liquids (e.g., plasma) capture only SULF2 and SULFl, and exclude other sulfatases. For example, the pH to exclude sulfatases other than SULFl and SULFl in a biochemical assay may range from about 6 to about 10, from about 6 to about 9, from about 6 to about 8, from about 7 to about 10, from about 7 to about 9, from about 7 to about 8, from about 8 to about 10, from about 8 to about 9, or have a pH of about 8.
Both the Sulfatase 1 and 2 genes (i.e., SULFl and SULFl) have been reported to play a crucial role in kidney development and function. In mice, SULFl expression was found in the nephron progenitor cells and tubules, and SULFl /SULFl double knock-out mice suffered from kidney hypoplasia. Both enzymes release sulfate mainly from various disaccharide units (e.g. IdoA2S-GlcNS6S, UA2S-GlcNS6S, UA-GlcNS6S) of
heparin/heparan sulfate chains, thus remodeling the cell's surface which affects a variety of transmembrane signaling processes. SULFl knock-out mice, but not SULFl knock-out mice, exhibited an increase of UA2S-GlcNS6S in kidneys, supporting the theory of a sulfatase function in kidneys. For example, negatively charged heparan sulfate proteoglycans, and in particular their degree of sulfation in the basement membrane, play a major role in
glomerular filtration. Consistent with this, mice with deleted SULFl and SULFl genes developed proteinuria resulting from injuries of the glomerular endothelial cells and podocytes.
Further support for the connection between SULF1ISULF1 and proteinuria was obtained from children with Wilm's tumor. The largely podocyte-specific transcription factor, Wilm's tumor gene 1 (WTl), regulates the expression of SULFl and SULFl in Wilm's tumor. Children with Wilm's tumor exhibited a kidney phenotype similar to that earlier described for in SULFl /SULFl knock-out mice. Thus, SULFl and SULFl expression seems to be crucial for maintaining the glomerular filtration barrier. In particular, the present invention is directed to a gene involved in a site of injury of NS disease in a patient. The site of injury in the patient may be a kidney, a glomerulus, a podocyte, or a combination thereof.
To evaluate the potential use of SULFl and SULFl as a discriminatory indicators for patients with SSNS and SRNS, gene expression before (SI) and after (S2) GC treatment was measured by quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and Reverse Transcription PCR (RT-PCR). Gene expression was measured in cDNA synthesized from peripheral blood leukocyte RNA from a cohort of NS patients (n=28 of SSNS patients and n=14 of SRNS patients). Relative SULFl mRNA expression in the NS patient peripheral lymphocytes was measured and compared with expression of SULFl, according to methods known in the art. Expression analysis was performed using 35 cycles of RT-PCR amplification.
Figure 5 shows detection of SULFl and SULFl expression among NS patient groups. In particular, gene expression of SULFl and SULFl was measured in SSNS and SRNS patient leukocytes. As shown in Figure 5A, the SULFl expression in patients of both groups was subject to substantial variability. However, using an unpaired, two-tailed t-test, the average GER(S2/S1)SULF2 was shown to be significantly greater (p value=0.020l) in samples from SSNS patients (2.69 + 0.37 SEM) than from SRNS patients (1.55 + 0.29 SEM). The dotted line in Figure 5 A marks a GER(S2/S1)SULF2 = 1, which separates induction (above) from reduced (below) SULFl expression following GC treatment (see Figure 5A). These data indicate that the GER(S2/S1)SULF2 can serve as a discriminatory value of gene expression between both SSNS and SRNS patient classes. Further, induction of SULFl activity in response to the GC therapy may be associated with its therapeutic efficacy in NS patients, particularly considering the known role of SULFl in podocytes.
To assess SULFl gene expression in NS patients, RT-PCR conditions were employed using the following primer sequences to amplify the genes of interest:
TGGAACCTGGGATCTTTCTG (SEQ ID NO: 1) and GCTGATTCAAAATGCCTCGT (SEQ ID NO: 2) for SULFl; ACACGTACTGGTGCATGAGG (SEQ ID NO: 3) and
GCTTGTAACCCTTGCAGCTC (SEQ ID NO: 4) for SULFl; and
TGTACCTGAAGGTCAAAGGGAATGTG (SEQ ID NO: 5) and
TTCTTGGTCTCCTCCTCCTTGGAC (SEQ ID NO: 6) for RPL19.
While RT-PCR was sufficient to provide robust signals for the expression of both SULFl and the control gene, RPL19, SULFl gene expression was undetectable in NS patients, both before and after GC therapy (see Figure 5B). In order to confirm the absence of SULFl in the NS patient samples, RT-PCR was conducted using cDNA from commercial RNA extracted from whole human kidneys (BioChain, Newark, CA) as a template, and thus, as a SULFl positive control (see Figure 5B). The primer sequences used to amplify the genes of interest are described above. The primer design for RT-PCR using standard cycling conditions resulted in expected cDNA amplicon lengths of 196 bp (SULFl), 198 bp (SULFl), and 220 bp (RPL19). The position of the 200 bp molecular mass marker band is indicated by the bar on the left side of Figure 5B.
Figure 5B shows SULF1 gene expression assessed in leukocytes from four representative NS patients, three SSNS patients (patient 9, 16, and 31) and one SRNS patient (patient 21). Gene expression of SULF1 (lanes #1) was undetected in all NS patients, while SULF2 (lanes #2) and the RPL19 control gene were present (lanes #3). According to these data, SULF2 expression was much greater than that of SULF1 in all analyzed NS patient samples. Thus, Figures 5A and 5B indicate that SULF1 expression in patient leukocytes is unlikely to contribute to any measured aryl sulfatase activity (see Figure 6). It should be noted, however, that sulfatase activity originating from SULF1 in plasma, but that is secreted by other tissues cannot be excluded. These results indicate that any measured sulfatase activity in NS patients originates from SULF2 only.
Sulfatase 2 Enzyme Activity
The Sulfatase 2 (SULF2) gene encodes a large extracellular enzyme comprising approximately 870 amino acids with endoglucosamine-6-sulfatase activity. Upon desulfation, extracellular ligands can no longer bind to the cell membrane. This enzyme liberates 6-O-S mainly from disaccharide units (e.g., IdoA2S-GlcNS6S, UA2S-GlcNS6S, UA-GlcNS6S) within S domains of extracellular heparin/heparan sulfate chains. Thus, SULF2 remodels the 6-O-sulfation of the cell's surface heparin/heparan chains which has consequences for the modulation of transmembrane signaling processes. For example, the modulation of the interaction of VEGF-160 or FGF-1 with heparin or heparan sulfate, and the promotion of the Wnt/p-catenin signaling pathway.
The enzyme SULF2 is synthesized as pre-pro -protein. After cleavage of the signal sequence, the pro-protein is further proteolytically cleaved resulting in a 75 kDa and a 50 kDa fragments that become linked by a disulfide bond to form a mature protein. A portion of the mature protein becomes partially secreted, with the other portion being retained at the cell surface, notably in the lipid rafts. Catalytic activity of SULF2 requires post-translational modifications, including the conversion of a cysteine residue in the catalytic center into C- formylglycine. Taken together, these findings reveal several layers of regulation of SULF2 activity, implying that expression alone is not necessarily the critical determinant of extracellular SULF2 activity. This regulatory complexity may account for the imperfect correlation between the observed ratios of expression and activity of SULF2 in the patient samples (see Figures 5 and 6).
For example, at pH 8.0, SULF2 (and SULF1) has an arylsulfatase activity that was used for enzymatic measurements since activity of all other sulfatases are suppressed at this pH. A modified arylsulfatase assay was employed to measure SULF2 enzyme activity (Uchimura et al. 2006). Patient plasma was clarified by centrifugation at 15,000 x g for 30 min at 4°C, and 10 μΐ of supernatant (excluding any of the upper, lipid-rich layer) was combined with stock solutions to a final concentration of 50 mM HEPES-KOH, pH 8.0, 10 mM lead acetate, and 10 mM 4-methylumbelliferyl sulfate in a total volume of 50 μΐ. After incubation at 37 °C for 2 h, 40 μΐ of this mixture was combined with 200 μΐ of 0.5 M
Na2C03/NaHC03, pH 10.7, and fluorescence was measured in black- walled clear bottom plates at 460 nm emission with excitation at 360 nm. Plasma that was heat-inactivated for 30 min at 95°C was used as a negative control.
The arylsulfatase activity was measured in clarified plasma samples from 45 patients (n=30 of SSNS patients and n=15 of SRNS patients). The results of arylsulfatase activity are shown in Figure 6 as the ratio (S2/S 1) of the activities in the plasma sample collected from each patient before (S I) and after (S2) treatment with GCs. Consistent with the SULF2 gene expression ratio or GER(S2/S 1)SULF2 data (see Figures 2-4), the arylsulfatase activity ratio (S2/S 1) was shown to be significantly greater (p value=0.0325) in samples from SSNS patients (1.20 + 0.06 SEM) than from SRNS patients (0.96 + 0.10 SEM) using an unpaired, two-tailed t-test. The dotted line marks where arylsulfatase activity ratio = 1, which separates increased (above) from reduced (below) enzyme activity following GC treatment (see Figure 6). Approximately 73.3% of SRNS patients had arylsulfatase activity ratios less than or equal to 1.0 (11 of 15 of patients), while about 70% of SSNS patients had ratios greater than 1.0 (21 of 30 patients).
A one-sided Shapiro-Wilk test indicated that the collected GER(S2/S 1)SULF2 (see Figure 5) and aryl sulfatase activity (S2/S l)(see Figure 6) data exhibit standard normal distribution, with a significance level (p value) of 0.05. Statistical significance of the differences between the SSNS and SRNS patient groups for both the GER(S2/S 1)SULF2 and the aryl sulfatase activities (S2/S 1) was determined by the unpaired Student's i-test.
Probability values were considered significant at a p value<0.05.
These results indicate that the ability of patients to induce SULF2 expression and activity in plasma in response to GC therapy is associated with the therapeutic efficacy of GC in NS patients. For example, in Figure 3, the average gene expression for SULF2 (GER(Sl/S2)suLF2) falls into the group of Set I genes, indicating that SULF2 is highly induced in SSNS patients following GC treatment, as compared to the SRNS patients.
Similarly, Figure 5 shows the SULF2 activity ratio, (S2/S1)SULF2, is also induced in SSNS patient and therefore, it can also serve to discriminate between SRNS and SSNS patient classes.
It is noted that the potency of the SULF2 gene expression value (GER(S2/S1)SULF2) or activity ratios ((S2/S1)SULF2) may be limited due to the high deviation of expression within the patient groups. However, it is believed that expression and enzyme analysis of SULF2 (GER(S2/S1)SULF2 and (S2/S1)SULF2, respectively) coupled with other discriminator genes, may bolster the diagnostic utility of these gene in NS patients.
Additional Marker Genes of NS Patients
23,080 gene transcripts (including non-coding) were studied, and 4018 showed greater than 1.4 fold ratio change between SRNS and SSNS patients. Notable genes included were PTGS2 and CNTNAP3 which are known to be relevant to NS, and FKBP5 and BMPRII, which are associated with steroid sensitivity. 1109 genes were identified with alternative transcript expression, and 1908 SNPs were detected from 1234 genes with AEI >1.5 in >2 samples. The Mann-Whitney U test identified candidates associated with nephropathy or actin polymerization. Normalization of the data set yielded 991 genes, while an imputed data set yielded 3280 genes, and after using statistical methods of Volcano plotting, Clusterogram, and other conventional statistical methods, machine learning systems, or computer algorithms, several sets of genes were identified to differentiate between SRNS and SSNS patient groups at the time of clinical presentation.
For example, in order to identify smaller sets of gene markers, but retain maximum accuracy in classifying SRNS and SSNS patient groups, statistical analyses were performed. These analyses identified several sets of genes able to classify the two patient groups with an accuracy of greater than 95%, with all samples in all runs being correctly classified according to their corresponding clinical observation (except a single SRNS sample in a single run which is not shown). In the present invention, gene sets ranging from 1 to 100 genes or gene products may be utilized. EXAMPLES
Example 1: 12-Gene Set
Statistical and computational algorithms known in the art were applied to all 72 genes identified as differentially expressed in NS patient lymphocytes. A number of genes occurred more often than others. The average numbers of gene occurrences are shown in Figure 4. Specifically, the twelve genes with the most frequent occurrences in the gene runs are found in the area above the dotted line of Figure 4, and are listed in Table 3. The SULF2 gene was among the top twelve genes identified as a NS disease marker. Although other genes appeared more often than SULF2 in the analysis, biochemical analyses of the patient samples were performed specifically on SULF2 since its role in renal podocytes and development of NS was recently reported.
While the SULF2 gene may be used alone as a marker for NS disease, additional markers that may be assessed with SULF2, increase the accuracy and efficiency of diagnostic capabilities. Accordingly, the eleven marker genes identified in Figure 5 (see Table 3) may be included with SULF2 to comprise one embodiment of the kit or the method described herein for diagnostic analyses. More specifically, a 12-gene set embodiment of the kit may be prepared and used for diagnostic analyses of NS in patients. The genes, gene symbols, and gene IDs of the 12-gene set are listed in Table 3.
The eleven other identified marker genes are involved in diverse cellular processes, including endocytosis (RFFL), vesicle transport (STX4), DNA replication (TOPBP1), lysosphingolipid signaling (S1PR5), transcription (KLF2), cytoskeletal functions (NCRNA00081), glucose transport (SLC2A1), formation of the glycosylphosphatidyl-inositol anchors of cell surface proteins (PIGB), proteins of the tRNA metabolism (TRMT6), unknown functions (FAM40A), and in the development of T-cell lymphoma (FGFRl OP2), or following an in-frame fusion with the FGFRl gene (see Table 3). These eleven genes do not share common functions or properties, although most of the above cellular functions are known to be of crucial importance for many tissues and organs, including kidneys. The molecular basis for the discriminatory potency of these genes in steroid resistance of NS remains to be elucidated. However, the present results unexpectedly indicate that these twelve marker genes together have a high discriminatory accuracy for NS patients. Table 3. 12-Gene Set with differential expression in SRNS vs. SSNS patients (Figure 4).
Figure imgf000046_0001
Example 2: 34-Gene Set
Among the results of the statistical analysis on the 72 genes, another embodiment of the present inventions comprises a particular set of 34 genes that accurately classified the two NS patient groups. The 34 genes of this set are indicated by closed symbols in Figure 3 for both SRNS and SSNS patient groups. Additionally, the 34 genes, gene symbols, and gene IDs are listed in bold amongst the 72 gene-set in Table 2. A gene expression cassette comprising primers and probes to these 34 genes was able to differentiate amongst SRNS and SSNS patients with over a 95% accuracy.
Example 3: 16-Gene Set
In another embodiment of the present invention, 16 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 4). Figure 7A shows the volcano plot that filtered the initial data set down to 991 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 16 gene that significantly differentiated between SRNS and SSNS patients. Figure 7B shows the heat map
clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 16 genes. The genes, gene symbols, and gene IDs of the 16-gene set are listed in Table 4. Table 4. 16-Gene Set with differential expression in SRNS vs. SSNS patienl ts (Figure 7).
# GENE SYMBOL GENE NAME GENE ID
1 G LIP 2 GLI pathogenesis-related 2 152007
2 ARHGEF2 Rho/Rac guanine nucleotide exchange factor (G EF) 2 9181
3 S100A8 S100 calcium binding protein A8 6279
4 H2AFY H2A histone family, member Y 9555
5 SELL selectin L 6402
6 TRI M22 tripartite motif containing 22 10346
7 ZC3HAV1 zinc finger CCCH-type, antiviral 1 56829
8 SEPHS2 selenophosphate synthetase 2 22928
9 MY05A myosin VA (heavy chain 12, myoxin) 4644
10 ZN F641 zinc finger protein 641 121274
11 SRSF4 serine/arginine-rich splicing factor 4 6429
12 ZBTB34 zinc finger and BTB domain containing 34 403341
13 NCOA4 nuclear receptor coactivator 4 8031
14 ANTXR2 anthrax toxin receptor 2 118429
15 DDX3X DEAD (Asp-Glu-Ala-Asp) box helicase 3, X-linked 1654
16 CHST11 carbohydrate (chondroitin 4) sulfotransferase 11 50515
Example 4: 54-Gene Set
In another embodiment of the present invention, 54 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 5). Figure 8A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 54 gene that significantly differentiated between SRNS and SSNS patients. Figure 8B shows the heat map
clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 54 genes. Table 5 shows the gene, gene symbol, rank, and gene ID comprised in the 54-gene set.
Figure imgf000047_0001
CHST11 carbohydrate (chondroitin 4) sulfotransferase 11 50515
MYLIP myosin regulatory light chain interacting protein 29116 protein-L-isoaspartate (D-aspartate) O-
PCMTD2 methyltransferase domain containing 2 55251
A HGAP15 Rho GTPase activating protein 15 55843
AKAP10 A kinase (PRKA) anchor protein 10 11216
H2AFY H2A histone family, member Y 9555
ARHGAP19 Rho GTPase activating protein 19 84986
UDP-GlcNAc:betaGal beta-l,3-N-
B3GNT2 acetylglucosaminyltransferase 2 10678
SYNJ1 synaptojanin 1 8867
MBD5 methyl-CpG binding domain protein 5 55777
ZNF266 zinc finger protein 266 10781
SELL selectin L 6402
TMEM87B transmembrane protein 87B 84910 potassium channel tetramerization domain containing
KCTD10 10 83892
ZNF587 zinc finger protein 587 84914
S100A8 S100 calcium binding protein A8 6279
CLCN3 chloride channel, voltage-sensitive 3 1182
SLC30A6 solute carrier family 30 (zinc transporter), member 6 55676
ZC3HAV1 zinc finger CCCH-type, antiviral 1 56829
RABGAP1 RAB GTPase activating protein 1 23637
PL-5283 C7orf73, chromosome 7 open reading frame 73 647087
SEPHS2 selenophosphate synthetase 2 22928
RCSD1 RCSD domain containing 1 92241
ANXA7 annexin A7 310
KIAA0754 uncharacterized protein-coding KIAA0754 643314
TMEM87A transmembrane protein 87A 25963
ZNF187 ZSCAN26, zinc finger and SCAN domain containing 26 7741
GLT1D1 glycosyltransferase 1 domain containing 1 144423
TGIF2- C20ORF24 TGIF2-C20orf24 readthrough 100527943
GLIPR2 GLI pathogenesis-related 2 152007
TRIM22 tripartite motif containing 22 10346
T0R1B torsin family 1, member B (torsin B) 27348
ZNF41 zinc finger protein 41 7592
AHA1, activator of heat shock 90kDa protein ATPase
AHSA2 homolog 2 (yeast) 130872
TA0K3 TAO kinase 3 51347
CP110 centriolar coiled coil protein HOkDa 9738
GRPEL1 GrpE-like 1, mitochondrial (E. coli) 80273
LOC283070 non-coding RNA, uncharacterized LOC283070 283070
KIAA0141 protein-coding KIAA0141 9812 46 NCOA4 nuclear receptor coactivator 4 8031
47 ZMAT1 zinc finger, matrin-type 1 84460
48 ZNF398 zinc finger protein 398 57541
49 SPATA13 spermatogenesis associated 13 221178
50 HGSNAT heparan-alpha-glucosaminide N-acetyltransferase 138050
51 ZBTB34 zinc finger and BTB domain containing 34 403341
52 MCC mutated in colorectal cancers 4163
53 PDHA1 pyruvate dehydrogenase (lipoamide) alpha 1 5160
54 C0PB2 coatomer protein complex, subunit beta 2 (beta prime) 9276
Example 5: 40-Gene Set
In another embodiment of the present invention, 40 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 6). Figure 9A shows the volcano plot that filtered the initial data set based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 40 gene that significantly differentiated between SRNS and SSNS patients. Figure 9B shows the heat map clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 40 genes. The genes, gene symbols, and gene IDs of the 40-gene set are listed in Table 6.
Table 6. 40-Gene Set with differential expression in SRNS vs. SSNS patients (Figure 9).
Figure imgf000049_0001
17 ST ADB STE20-related kinase adaptor beta 55437
18 SNAP29 synaptosomal-associated protein, 29kDa 9342
19 PADI2 peptidyl arginine deiminase, type II 11240 solute carrier family 25 (mitochondrial carrier; adenine nucleotide
20 SLC25A5 translocator), member 5 292
21 PPIB peptidylprolyl isomerase B (cyclophilin B) 5479
22 C9orf78 chromosome 9 open reading frame 78 51759
23 FAM46C family with sequence similarity 46, member C 54855
24 PR0K2 prokineticin 2 60675
25 MKRN1 makorin ring finger protein 1 23608
26 MPZL3 myelin protein zero-like 3 196264
27 FAM104A family with sequence similarity 104, member A 84923
28 TH0C5 THO complex 5 8563
29 GSPT1 Gl to S phase transition 1 2935
30 SCARNA3 small Cajal body-specific RNA 3 677679
31 IGSF6 immunoglobulin superfamily, member 6 10261
32 ARL11 ADP-ribosylation factor-like 11 115761
33 HBA1 hemoglobin, alpha 1 3039
34 CD59 CD59 molecule, complement regulatory protein 966
35 EIF2AK1 eukaryotic translation initiation factor 2-alpha kinase 1 27102
36 PFKFB3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 5209
37 PRDX5 peroxiredoxin 5 25824
38 RAB4A RAB4A, member RAS oncogene family 5867
39 CDK6 cyclin-dependent kinase 6 1021
40 FCGR2A Fc fragment of IgG, low affinity lla, receptor (CD32) 2212
Example 6: 24-Gene Set
In another embodiment of the present invention, 24 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 7). Figure 10A shows the volcano plot that filtered the initial data set based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 24 gene that significantly differentiated between SRNS and SSNS patients. Figure 10B shows the heat map clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 24 genes. The genes, gene symbols, and gene IDs of the 24-gene set are listed in Table 7. Table 7. 24-Gene Set with differential ex ression in SRNS vs. SSNS atients (Fi ure 10).
Figure imgf000051_0001
Example 7: 78-Gene Set
In another embodiment of the present invention, 78 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 8). Figure 11 A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 78 gene that significantly differentiated between SRNS and SSNS patients. Figure 1 IB shows the heat map
clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 78 genes. The genes, gene symbols, and gene IDs of the 78-gene set are listed in Table 8. Table 8.78-Gene Set with differential ex ression in SRNS vs. SSNS atients (Fi ure 11).
Figure imgf000052_0001
RFFL ring finger and FYVE-like domain containing E3 ubiquitin protein ligase 117584
KCTD10 potassium channel tetramerization domain containing 10 83892
DCAF8 DDB1 and CUL4 associated factor 8 50717
ACTR1A ARPl actin-related protein 1 homolog A, centractin alpha (yeast) 10121 guanine nucleotide binding protein (G protein), alpha inhibiting
GNAI2 activity polypeptide 2 2771
ARHGDIB Rho GDP dissociation inhibitor (GDI) beta 397
RNASEK ribonuclease, RNase K 440400
LST1 leukocyte specific transcript 1 7940
GNB1 guanine nucleotide binding protein (G protein), beta polypeptide 1 2782
CSNK1G1 casein kinase 1, gamma 1 53944
S100A4 S100 calcium binding protein A4 6275
MAPKAPK3 mitogen-activated protein kinase-activated protein kinase 3 7867
KCTD20 potassium channel tetramerization domain containing 20 222658
FAM134C family with sequence similarity 134, member C 162427
PTAR1 protein prenyltransferase alpha subunit repeat containing 1 375743
KPNA1 karyopherin alpha 1 (importin alpha 5) 3836
UBE2W ubiquitin-conjugating enzyme E2W (putative) 55284
GLRX glutaredoxin (thioltransferase) 2745
ARL8B ADP-ribosylation factor-like 8B 55207
TBRG1 transforming growth factor beta regulator 1 84897
AFF3 AF4/FMR2 family, member 3 3899
MARK3 MAP/microtubule affinity-regulating kinase 3 4140
NCRNA00081 BBSome interacting protein 1 92482
GCC1 GRIP and coiled-coil domain containing 1 79571
C2orf29 CCR4-NOT transcription complex, subunit 11 55571
ZMAT2 zinc finger, matrin-type 2 153527
LCOR ligand dependent nuclear receptor corepressor 84458
CDC42 cell division cycle 42 998
PHIP pleckstrin homology domain interacting protein 55023
RNF103 ring finger protein 103 7844
AKIRIN1 akirin 1 79647
TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 8743
PTP4A1 protein tyrosine phosphatase type IVA, member 1 7803
RUNX3 runt-related transcription factor 3 864
KIAA1267 KANSL1, KAT8 regulatory NSL complex subunit 1 284058
GOSR1 golgi SNAP receptor complex member 1 9527
KIAA0922 protein-coding KIAA0922 23240
OSBP oxysterol binding protein 5007 integrin, alpha L (antigen CD11A (pl80), lymphocyte function-
ITGAL associated antigen 1; alpha polypeptide) 3683 76 ACINI apoptotic chromatin condensation inducer 1 22985
77 NUP214 nucleoporin 214kDa 8021
78 CCNI cyclin 1 10983
Example 8: 85-Gene Set
In another embodiment of the present invention, 85 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 9). Figure 12A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 85 gene that significantly differentiated between SRNS and SSNS patients. Figure 12B shows the heat map
clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 85 genes. The genes, gene symbols, and gene IDs of the 85-gene set are listed in Table 9.
Table 9. 85-Gene Set with differential expression in SRNS vs. SSNS patients (Figure
Figure imgf000054_0001
S100A4 S100 calcium binding protein A4 6275
FBX07 F-box protein 7 25793
CYTH4 cytohesin 4 27128
G B2 growth factor receptor-bound protein 2 2885
MXI1 MAX interactor 1, dimerization protein 4601
RNASEK ribonuclease, RNase K 440400
GSPT1 Gl to S phase transition 1 2935
RBM38 RNA binding motif protein 38 55544
GPSM3 G-protein signaling modulator 3 63940
C22orfl3 GUCD1, guanylyl cyclase domain containing 1 83606
ARF3 ADP-ribosylation factor 3 377 solute carrier family 25 (mitochondrial iron transporter), member
SLC25A37 37 51312
LM02 LIM domain only 2 (rhombotin-like 1) 4005
TPM3 tropomyosin 3 7170
DCAF12 DDB1 and CUL4 associated factor 12 25853
S100A6 S100 calcium binding protein A6 6277
CFL1 cofilin 1 (non-muscle) 1072
YTHDF3 YTH domain family, member 3 253943
C19orf22 R3H domain containing 4 91300
ATP6V0D1 ATPase, H+ transporting, lysosomal 38kDa, V0 subunit dl 9114
CLICl chloride intracellular channel 1 1192
PIM1 Pim-1 proto-oncogene, serine/threonine kinase 5292
STAT5B signal transducer and activator of transcription 5B 6777
TRIM58 tripartite motif containing 58 25893
FAM46C family with sequence similarity 46, member C 54855
UBE2H ubiquitin-conjugating enzyme E2H 7328
ATP synthase, H+ transporting, mitochondrial Fl complex, beta
ATP5B polypeptide 506
NHP2L1 NHP2 non-histone chromosome protein 2-like 1 (S. cerevisiae) 4809
PECAM1 platelet/endothelial cell adhesion molecule 1 5175
C16orf72 chromosome 16 open reading frame 72 29035
EPB41 erythrocyte membrane protein band 4.1 2035
BSG basigin (Ok blood group) 682
GNS glucosamine (N-acetyl)-6-sulfatase 2799
PSAP prosaposin 5660
ARPC1B actin related protein 2/3 complex, subunit IB, 41kDa 10095
MAPRE2 microtubule-associated protein, RP/EB family, member 2 10982
BLCAP bladder cancer associated protein 10904
ARHGAP30 Rho GTPase activating protein 30 257106
ACTB actin, beta 60
WDR26 WD repeat domain 26 80232
CCDC69 coiled-coil domain containing 69 26112 PLEKH02 pleckstrin homology domain containing, family 0 member 2 80301
PTP E protein tyrosine phosphatase, receptor type, E 5791
COTL1 coactosin-like F-actin binding protein 1 23406
NCOA4 nuclear receptor coactivator 4 8031
PPT1 palmitoyl-protein thioesterase 1 5538
CREB5 cAMP responsive element binding protein 5 9586
RPS23 ribosomal protein S23 6228
MSN moesin 4478
ARHGEF2 Rho/Rac guanine nucleotide exchange factor (GEF) 2 9181
DAZAP2 DAZ associated protein 2 9802
GLIPR2 GLI pathogenesis-related 2 152007
SEC11A SEC11 homolog A (S. cerevisiae) 23478
GIMAP5 GTPase, IMAP family member 5 55340
EIF4G2 eukaryotic translation initiation factor 4 gamma, 2 1982
IL6R interleukin 6 receptor 3570
GIMAP4 GTPase, IMAP family member 4 55303
TPD52L2 tumor protein D52-like 2 7165
TCP11L2 t-complex 11, testis-specific-like 2 255394
HLA-B major histocompatibility complex, class 1, B 3106
AIF1 allograft inflammatory factor 1 199
UBE2J1 ubiquitin-conjugating enzyme E2, Jl 51465
ATF7 activating transcription factor 7 11016
MEF2A myocyte enhancer factor 2A 4205
PIP4K2A phosphatidylinositol-5-phosphate 4-kinase, type II, alpha 5305
FAM134C family with sequence similarity 134, member C 162427
RGS2 regulator of G-protein signaling 2 5997
GIMAP6 GTPase, IMAP family member 6 474344
Example 9: 76-Gene Set
In another embodiment of the present invention, 76 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 10). Figure 13A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 76 gene that significantly differentiated between SRNS and SSNS patients. Figure 13B shows the heat map
clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 76 genes. The genes, gene symbols, and gene IDs of the 76-gene set are listed in Table 10. Table 10. 76-Gene Set with differential expression in SRNS vs. SSNS patients (Figure
Figure imgf000057_0001
SIAH2 siah E3 ubiquitin protein ligase 2 6478
RCSD1 RCSD domain containing 1 92241
PCGF3 polycomb group ring finger 3 10336
PIK3IP1 phosphoinositide-3-kinase interacting protein 1 113791
EIF4G2 eukaryotic translation initiation factor 4 gamma, 2 1982
WAPAL wings apart-like homolog (Drosophila) 23063
CD53 CD53 molecule 963
SELL selectin L 6402
X-ray repair complementing defective repair in Chinese
XRCC5 hamster cells 5 (double-strand-break rejoining) 7520
ZNF638 zinc finger protein 638 27332
ADIP0R1 adiponectin receptor 1 51094
RBL2 retinoblastoma-like 2 5934 nuclear fragile X mental retardation protein interacting protein
NUFIP2 2 57532
PIM1 Pim-1 proto-oncogene, serine/threonine kinase 5292
OAZ1 ornithine decarboxylase antizyme 1 4946
CNST consortin, connexin sorting protein 163882
TCP11L2 t-complex 11, testis-specific-like 2 255394
MYL12A myosin, light chain 12A, regulatory, non-sarcomeric 10627
TRIM58 tripartite motif containing 58 25893
TAF7 RNA polymerase II, TATA box binding protein (TBP)-
TAF7 associated factor, 55kDa 6879
TNIP1 TNFAIP3 interacting protein 1 10318
FAM117A family with sequence similarity 117, member A 81558
PCF11 PCF11 cleavage and polyadenylation factor subunit 51585
KLHL6 kelch-like family member 6 89857
HIATL1 hippocampus abundant transcript-like 1 84641
SERTAD2 SERTA domain containing 2 9792
RBM5 RNA binding motif protein 5 10181
CHD7 chromodomain helicase DNA binding protein 7 55636
JAK3 Janus kinase 3 3718
GNG2 guanine nucleotide binding protein (G protein), gamma 2 54331
S1PR1 sphingosine-l-phosphate receptor 1 1901
MYD88 myeloid differentiation primary response 88 4615
IKZF1 IKAROS family zinc finger 1 (Ikaros) 10320
WDR26 WD repeat domain 26 80232
UBE2G1 ubiquitin-conjugating enzyme E2G 1 7326
ELF1 E74-like factor 1 (ets domain transcription factor) 1997
ENSA endosulfine alpha 2029 Example 10: 71-Gene Set
In another embodiment of the present invention, 71 genes were identified with the ability to differentiate between SRNS and SSNS patient groups at the time of clinical presentation (see Table 11). Figure 14A shows the volcano plot that filtered the initial data set down to 3280 genes based on normalization. Those genes were statistically analyzed using computational algorithms known in the art which yielded 71 gene that significantly differentiated between SRNS and SSNS patients. Figure 14B shows the heat map
clusterogram differentiating the SRNS and SSNS patients based on the differential expression data of 71 genes. The genes, gene symbols, and gene IDs of the 71-gene set are listed in Table 11.
Table 11. 71-Gene Set with differential expression in SRNS vs. SSNS patients (Figure
Figure imgf000059_0001
carcinoembryonic antigen-related cell adhesion molecule 1 (biliary
CEACAM1 glycoprotein) 634
CD180 CD180 molecule 4064
CHPT1 choline phosphotransferase 1 56994
BCL6 B-cell CLL/lymphoma 6 604
C2orf24 CNPPD1, cyclin Pasl/PHO80 domain containing 1 27013
ADM adrenomedullin 133
IGBP1 immunoglobulin (CD79A) binding protein 1 3476
DNAJC2 DnaJ (Hsp40) homolog, subfamily C, member 2 27000
KDM6A lysine (K)-specific demethylase 6A 7403
P NP prion protein 5621
C4orfl4 NOA1, nitric oxide associated 1 84273
S0D2 superoxide dismutase 2, mitochondrial 6648
HEMGN hemogen 55363
PARP9 poly (ADP-ribose) polymerase family, member 9 83666
TUBA1A tubulin, alpha la 7846
ZFP62 ZFP62 zinc finger protein 643836
C20orfl08 FAM210B, family with sequence similarity 210, member B 116151
SEPT6 septin 6 23157
UBE2L6 ubiquitin-conjugating enzyme E2L 6 9246
ZNF121 zinc finger protein 121 7675
C9orf78 chromosome 9 open reading frame 78 51759
EIF2S3 eukaryotic translation initiation factor 2, subunit 3 gamma, 52kDa 1968
GSPT1 Gl to S phase transition 1 2935
IL7R interleukin 7 receptor 3575
SNORA45 SNORA45B, small nucleolar RNA, H/ACA box 45B 677826
0DC1 ornithine decarboxylase 1 4953
MARK3 MAP/microtubule affinity-regulating kinase 3 4140
OAZ1 ornithine decarboxylase antizyme 1 4946
ANXA3 annexin A3 306
JAZF1 JAZF zinc finger 1 221895
EIF2AK1 eukaryotic translation initiation factor 2-alpha kinase 1 27102
SNORD89 small nucleolar RNA, C/D box 89 692205
PEBP1 phosphatidylethanolamine binding protein 1 5037
CCNDBP1 cyclin D-type binding-protein 1 23582
CCNT1 cyclin Tl 904
SNCA synuclein, alpha (non A4 component of amyloid precursor) 6622
ASNSD1 asparagine synthetase domain containing 1 54529
TMEM14B transmembrane protein 14B 81853
MTIF2 mitochondrial translational initiation factor 2 4528
YBX1 Y box binding protein 1 4904
PLSCR1 phospholipid scramblase 1 5359 TAF13 NA polymerase II, TATA box binding protein (TBP)-
66 TAF13 associated factor, 18kDa 6884
67 C22orf46 chromosome 22 open reading frame 46 79640
AHA1, activator of heat shock 90kDa protein ATPase homolog 1
68 AHSA1 (yeast) 10598
69 SAT1 spermidine/spermine Nl-acetyltransferase 1 6303
70 DDX60L DEAD (Asp-Glu-Ala-Asp) box polypeptide 60-like 91351
71 ZNF226 zinc finger protein 226 7769
It is intended that the scope of the present methods be defined by the following claims. However, it must be understood that this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope. It should be understood by those skilled in the art that various alternatives to the embodiments described herein may be employed in practicing the claims without departing from the spirit and scope as defined in the following claims.

Claims

WHAT IS CLAIMED IS:
1. A kit for identifying a patient with resistance to steroid therapy of Nephrotic syndrome comprising:
a plurality of binding reagents, wherein each of the reagents specifically binds a gene product of a gene selected from the group consisting of:
RFFL, INPP1, MCM2, CSNK1G1, SIGLEC10, AGPAT6, DCAF8, FAM193A, RSPRY1, KCTD20, BLOC1S3, TOE1, ACAD10, SULF2, RAB8A, NCRNA00294, ZNF318, CDK4, TMEM219, PRKCSH, SLC9A7, LEOl, STX4, PTPRK, GANAB, C170RF63, ZC3H18, MED 14, TRAPPC5, EXOC7, ACINI, ITGAL, SH2D1B, TRMT6, NACC1, RECQL5, ZC3H12D, PRR5L, UBE2J2, TOPBP1, GALNT2, BTN2A2, GPR108, CHMP4B, ALDOA, TRAPPC1, SPRYD3, MTMR14, S1PR5, FAM134C, ALDOC, TCP1, CLCN3, NDUFS1, LGALS9, SUPT5H, JARID2, FAM40A, KLF2, AKIRIN1, ZNF782, OCIAD1, KPNA1, KIAA1033, UBE2W, LOC220930, NCRNA00081, ATP11B, SLC2A1, PIGB, SEPHS2, FGFR10P2, GLIPR2, ARHGEF2, S100A8, H2AFY, SELL, TRIM22, ZC3HAV1, MY05A, ZNF641, SRSF4, ZBTB34, NCOA4, ANTXR2, DDX3X, CHST11, PRNP, SLC35B4, GM2A, EZH1, LOC100289230, MYLIP, PCMTD2, ARHGAP15, AKAP10, ARHGAP19, B3GNT2, SYNJ1, MBD5, ZNF266, TMEM87B, KCTD10, ZNF587, SLC30A6,
RABGAP1, PL-5283, RCSD1, ANXA7, KIAA0754, TMEM87A, ZNF187, GLT1D1, TGIF2-C20ORF24, TOR1B, ZNF41, AHSA2, TAOK3, CP110, GRPEL1, LOC283070, KIAA0141, ZMAT1, ZNF398, SPATA13, HGSNAT, MCC, PDHA1, COPB2, ADIPOR1, LILRB2, FBX07, C20ORF108, NFE2, LIN7A, SNCA, ODC1, HBA2, TIFA, HEMGN, BAG1, DCAF12, SIRPB 1, RPGR, AQP9, STRADB, SNAP29, PAD 12, SLC25A5, PPIB, C90RF78, FAM46C, PROK2, MKRNl, MPZL3, FAM104A, THOC5, GSPTl, SCARNA3, IGSF6, ARL11, HBA1, CD59, EIF2AK1, PFKFB3, PRDX5, RAB4A, CDK6, FCGR2A, GNAI2, GNB1, STX7, ARHGDIB, RNASEK, PTAR1, PECAM1, KIAA1267, ZNF281, CX3CR1, SH3BGRL3, PITPNA, MAPRE2, MGAT1, LM02, S100A4, PCMTD1, SNX18, NFKBIZ, C130RF18, CLEC12A, USP15, C40RF3, CAB39, FCGR3A, GABARAPL1, C3AR1, TNFAIP8L2, RAB33B, ELF2, MBOAT2, SEMA4A, SKAP2, RAB8B, PPP1CB, FUT4, EMB, PTGER4, EPS 15, STIM1, GNLY, CNDP2, LONP2, LIN7C, MGC12916, ACTR1A, LST1, MAPKAPK3, GLRX, ARL8B, TBRG1, AFF3, MARK3, GCC1,
C20RF29, ZMAT2, LCOR, CDC42, PHIP, RNF103, TNFSFIO, PTP4A1, RUNX3, GOSRl, KIAA0922, OSBP, NUP214, CCNI, ERP29, LAMP1, LASP1, ARCN1, FUS, VPS24, MARCH8, MBNL3, MAX, BNIP3L, PIK3CG, PRKCB, HLA-E, STAT5A, CYTH4, GRB2, MXI1, GPSM3, C220RF13, ARF3, SLC25A37, TPM3, S100A6, CFL1, YTHDF3,
C190RF22, ATP6V0D1, CLIC1, PIM1, STAT5B, TRIM58, UBE2H, ATP5B, NHP2L1, C160RF72, EPB41, BSG, GNS, PSAP, ARPCIB, BLCAP, ARHGAP30, ACTB, WDR26, CCDC69, PLEKH02, PTPRE, COTL1, PPT1, CREB5, RPS23, MSN, DAZAP2, SEC11A, GIMAP5, EIF4G2, IL6R, GIMAP4, TPD52L2, TCP11L2, HLA-B, AIF1, UBE2J1, ATF7, MEF2A, PIP4K2A, RGS2, GIMAP6, SETD2, NR2C2, WAC, TSPAN14, IL6ST, STK17B, NCRNA00282, FAM120B, WASF2, WNK1, SH3BP5, PAFAH1B1, RBM38, PPP1R15B, MLL5, BTG1, PIK3R5, SIAH2, PCGF3, PIK3IP1, WAPAL, CD53, XRCC5, ZNF638, RBL2, NUFIP2, OAZ1, CNST, MYL12A, TAF7, TNIP1, FAM117A, PCF11, KLHL6, HIATL1, SERTAD2, RBM5, CHD7, JAK3, GNG2, S1PR1, MYD88, IKZFl, UBE2G1, ELFl, ENSA, AQP3, IRF9, SESN3, DCAF6, FCERIG, ZNF776, INSIG2, RPS9, SNHG12, ZNF791, CD96, HSP90B1, UBAP2, MARCKS, ZFX, SNX3, DYNC1LI1, STAT6, MME, TNFSF13B, MSH6, PIK3AP1, CEACAM1, CD180, CHPT1, BCL6, C20RF24, ADM, IGBP1, DNAJC2, KDM6A, C40RF14, SOD2, PARP9, TUBA 1 A, ZFP62, SEPT6, UBE2L6, ZNF121, EIF2S3, IL7R, SNORA45, ANXA3, JAZF1, SNORD89, PEBP1, CCNDBPl, CCNTl, ASNSDl, TMEM14B, MTIF2, YBXl, PLSCRl, TAF13, C220RF46, AHSA1, SAT1, DDX60L, and ZNF226,
wherein the expression of said gene products is significantly different between patients with Steroid-Resistant Nephrotic Syndrome and patients with Steroid- Sensitive Nephrotic Syndrome.
2. The kit of claim 1, wherein the patient is a pediatric patient.
3. The kit of claim 1, wherein the reagents comprise nucleic acids.
4. The kit of claim 1, wherein the gene products comprise mRNA, cDNA, or proteins.
5. The kit of claim 1, further comprising instructional materials.
6. The kit of claim 1, wherein one of the reagents binds to a SULF2 gene or gene product.
7. The kit of claim 1, wherein the reagents are immobilized on a solid support.
8. The kit of claim 3, wherein the nucleic acids are DNA.
9. The kit of claim 3, wherein the nucleic acids are RNA.
10. The kit of claim 3, wherein the nucleic acids comprise a set of two primers and a probe for detecting mRNA expressed by said genes.
11. The kit of claim 3, wherein the nucleic acids comprise a set of two or more primers for detecting mRNA expressed by said genes.
12. The kit of claim 3, wherein the nucleic acids comprise one or more probes for detecting mRNA expressed by said genes.
13. The kit of claim 8, wherein the DNA is cDNA.
14. The kit of claim 12, wherein the probes further comprise a detectable marker.
15. The kit of claim 4, wherein the reagents comprise antibodies for detecting the proteins expressed by said genes.
16. The kit of any one of claims 1-15, wherein one of the reagents specifically binds to SEQ ID NO: 7.
17. The kit of any one of claims 1-16, wherein each of the reagents specifically binds to a gene product selected from the group of genes consisting of: RFFL, INPPl, MCM2, CSNKIGI, SIGLECIO, AGPAT6, DCAF8, FAM193A, RSPRYl, KCTD20, BLOC1S3, TOEl, ACADIO, SULF2, RAB8A, NCRNA00294, ZNF318, CDK4, TMEM219, PRKCSH, SLC9A7, LEOl, STX4, PTPRK, GANAB, C170RF63, ZC3H18, MED 14, TRAPPC5, EXOC7, ACINI, ITGAL, SH2D1B, TRMT6, NACCl, RECQL5, ZC3H12D, PRR5L, UBE2J2, TOPBPl, GALNT2, BTN2A2, GPR108, CHMP4B, ALDOA, TRAPPCl, SPRYD3, MTMR14, S1PR5, FAM134C, ALDOC, TCP1, CLCN3, NDUFS1, LGALS9, SUPT5H, JARID2, FAM40A, KLF2, AKIRIN1, ZNF782, OCIAD1, KPNA1, KIAA1033, UBE2W, LOC220930, NCRNA00081, ATPl lB, SLC2A1, PIGB, SEPHS2, and FGFR10P2.
18. The kit of any one of claims 1-16, wherein each of the reagents specifically binds to a gene product selected from the group of genes consisting of: RFFL, SULF2, TOPBPl, SIPR5, FAM40A, STX4, KLF2, NCRNA00081, SLC2A1, TRMT6, PIGB, and FGFR10P2.
19. The kit of any one of claims 1-17, wherein each of the reagents specifically binds to a gene product selected from the group of genes consisting of: RFFL, AGPAT6, SULF2, TMEM219, PRKCSH, STX4, GANAB, ZC3H18, EXOC7, ACINI, ITGAL, SH2D1B, TRMT6, NACCl, ZC3H12D, TOPBPl, GALNT2, CHMP4B, TRAPPCl, SPRYD3, MTMR14, S1PR5, NDUFS1, LGALS9, SUPT5H, JARID2, KLF2, AKIRIN1, OCIAD1, KIAA1033, UBE2W, LOC220930, ATPl lB, and FGFR10P2.
20. A method of identifying a patient who is resistant to steroid therapy of Nephrotic Syndrome, comprising:
assaying marker gene products expressed in the patient through the use of the kit of claim 1,
analyzing the expression levels of the marker gene products relative to a control, wherein the control represents standard expression values established for samples taken from one or more patients having Nephrotic Syndrome and that is responsive to steroid treatment, and
identifying a patient as being resistant to steroid treatment of Nephrotic Syndrome based on the relative expression of said marker gene products.
21. The method of claim 20, further comprising treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non-steroidal therapies of Nephrotic Syndrome.
22. The method of claim 21, wherein treating the patient identified as being resistant to steroid treatment of Nephrotic Syndrome with non-steroidal therapy comprises administering a drug selected from the group consisting of adrenocorticotropic hormone, Cyclosporine, Tacrolimus, Mycophenolate mofetil, Plasmapheresis, column A immunoabsorbtion, and Rituximab.
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WO2018094021A1 (en) * 2016-11-16 2018-05-24 The Research Institute At Nationwide Children's Hospital Steroid resistance in nephrotic syndrome
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