[go: up one dir, main page]

WO2015066640A1 - Kit et procédé pour identifier la sensibilité individuelle à la thérapie stéroïde du syndrome néphrotique - Google Patents

Kit et procédé pour identifier la sensibilité individuelle à la thérapie stéroïde du syndrome néphrotique Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
kit
genes
gene
patients
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2014/063736
Other languages
English (en)
Inventor
William E. SMOYER
Shipra Agrawal
Richard F. RANSOM
Saraswathi SUNDARARAJAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nationwide Childrens Hospital Inc
Original Assignee
Nationwide Childrens Hospital Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nationwide Childrens Hospital Inc filed Critical Nationwide Childrens Hospital Inc
Publication of WO2015066640A1 publication Critical patent/WO2015066640A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un kit et un procédé pour identifier un patient pédiatrique qui est résistant à un traitement stéroïdien du syndrome néphrotique.
PCT/US2014/063736 2013-11-01 2014-11-03 Kit et procédé pour identifier la sensibilité individuelle à la thérapie stéroïde du syndrome néphrotique Ceased WO2015066640A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201361898932P 2013-11-01 2013-11-01
US201361898928P 2013-11-01 2013-11-01
US61/898,932 2013-11-01
US61/898,928 2013-11-01

Publications (1)

Publication Number Publication Date
WO2015066640A1 true WO2015066640A1 (fr) 2015-05-07

Family

ID=53005273

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/063736 Ceased WO2015066640A1 (fr) 2013-11-01 2014-11-03 Kit et procédé pour identifier la sensibilité individuelle à la thérapie stéroïde du syndrome néphrotique

Country Status (1)

Country Link
WO (1) WO2015066640A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018094021A1 (fr) * 2016-11-16 2018-05-24 The Research Institute At Nationwide Children's Hospital Résistance aux stéroïdes dans le syndrome néphrotique
CN110923311A (zh) * 2019-12-24 2020-03-27 广州市妇女儿童医疗中心 用于指导肾病综合征cyp3a5非表达型患儿使用他克莫司的多态性位点
CN114836528A (zh) * 2021-12-22 2022-08-02 浙江大学 一种用于儿童激素抵抗型肾病综合征基因诊断试剂盒
US11555073B2 (en) 2018-12-20 2023-01-17 23Andme, Inc. Anti-CD96 antibodies and methods of use thereof
CN117487914A (zh) * 2023-10-27 2024-02-02 广东药科大学 靶向zc3h18/pd-l1信号轴在肿瘤免疫逃逸检测、治疗、预后中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050266484A1 (en) * 2000-01-20 2005-12-01 Institut National De La Sante Et De La Recherche Medicale (Inserm) NPHS2 gene involved in the steroid-resistant nephrotic syndrome, protein encoded by said gene and diagnostic and therapeutic uses
US20100285475A1 (en) * 2007-10-22 2010-11-11 Agency For Science, Technology And Research Fused genes
WO2012118750A2 (fr) * 2011-02-28 2012-09-07 Genentech, Inc. Marqueurs biologiques et procédés de prédiction de réponse à des antagonistes de lymphocytes b

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050266484A1 (en) * 2000-01-20 2005-12-01 Institut National De La Sante Et De La Recherche Medicale (Inserm) NPHS2 gene involved in the steroid-resistant nephrotic syndrome, protein encoded by said gene and diagnostic and therapeutic uses
US20100285475A1 (en) * 2007-10-22 2010-11-11 Agency For Science, Technology And Research Fused genes
WO2012118750A2 (fr) * 2011-02-28 2012-09-07 Genentech, Inc. Marqueurs biologiques et procédés de prédiction de réponse à des antagonistes de lymphocytes b

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GEE, H ET AL.: "ARHGDIA Mutations Cause Nephrotic Syndrome Via Defective RHO GTPase Signaling.", J CLIN INVEST., vol. 123, no. 8, August 2013 (2013-08-01), pages 3243 - 3253 *
MORIMOTO-TOMITA ET AL.: "Cloning And Characterization Of Two Extracellular Heparin-Degrading Endosulfatases In Mice And Humans.", J BIOL CHEM., vol. 277, no. 51, 20 December 2002 (2002-12-20), pages 4917 - 49185 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018094021A1 (fr) * 2016-11-16 2018-05-24 The Research Institute At Nationwide Children's Hospital Résistance aux stéroïdes dans le syndrome néphrotique
US11555073B2 (en) 2018-12-20 2023-01-17 23Andme, Inc. Anti-CD96 antibodies and methods of use thereof
US12365732B2 (en) 2018-12-20 2025-07-22 23Andme, Inc. Anti-CD96 antibodies and methods of use thereof
CN110923311A (zh) * 2019-12-24 2020-03-27 广州市妇女儿童医疗中心 用于指导肾病综合征cyp3a5非表达型患儿使用他克莫司的多态性位点
CN110923311B (zh) * 2019-12-24 2023-04-18 广州市妇女儿童医疗中心 用于指导肾病综合征cyp3a5非表达型患儿使用他克莫司的多态性位点
CN114836528A (zh) * 2021-12-22 2022-08-02 浙江大学 一种用于儿童激素抵抗型肾病综合征基因诊断试剂盒
CN117487914A (zh) * 2023-10-27 2024-02-02 广东药科大学 靶向zc3h18/pd-l1信号轴在肿瘤免疫逃逸检测、治疗、预后中的应用
CN117487914B (zh) * 2023-10-27 2025-01-03 广东药科大学 靶向zc3h18/pd-l1信号轴在肿瘤免疫逃逸检测、治疗、预后中的应用

Similar Documents

Publication Publication Date Title
US9279158B2 (en) Compositions and methods useful for the treatment and diagnosis of inflammatory bowel disease
US20230020356A1 (en) Methods of stratifying and treating a sub-population of inflammatory bowel disease patients
AU2016295347A1 (en) Gene signature for immune therapies in cancer
WO2008073290A1 (fr) Expression du gène tp53 et ses utilisations
CN109837340B (zh) 用于肺癌无创诊断的外周血基因标记物
KR20160057416A (ko) 식도암에 대한 분자적 진단 검사
WO2015066640A1 (fr) Kit et procédé pour identifier la sensibilité individuelle à la thérapie stéroïde du syndrome néphrotique
AU2022203356A1 (en) Methods of detecting prostate cancer
JP7390002B2 (ja) 頭頸部がんの検出を補助する方法
EP3250710B1 (fr) Adn hôte en tant que biomarqueur de la maladie de crohn
ES2959778T3 (es) Método para detectar tuberculosis activa
CN107475440A (zh) 一种血浆外泌体生物标志物在乳腺癌诊断中的应用
WO2014134728A1 (fr) Procédés et gènes pour la normalisation de l'expression génétique
EP2985351B1 (fr) Un arn non-codant circulant pour prognostiquer la mortalité d'un patient avec défaillance rénale
EP3055426B1 (fr) Détection du virus de l'hépatite delta (vhd) pour le diagnostic et le traitement du syndrome de sjögren et du lymphome
TW201514311A (zh) 用於判定胰臟癌預後之方法
Pan Development of diagnostic methods using cell-free nucleic acids
EP3775282A1 (fr) Biomarqueurs pour le traitement du diabète
CN107583052A (zh) miR‑6734‑5p在制备Luminal型乳腺癌诊断工具中的应用
US20230203586A1 (en) Method and system for rna isolation from self-collected and small volume samples
CN106434951B (zh) 大肠癌纺锤体组装检验点检测用试剂体系和试剂盒及应用
CN118256608B (zh) 尿液微米囊泡miRNA标志物及其应用、诊断继发性肾小球疾病的试剂盒
CN111363808B (zh) 一种与阿尔茨海默相关的生物标志物及其应用
KR102202120B1 (ko) 알츠하이머 질환의 진단 또는 치료를 위한 Ｕｂｅ２ｈ의 용도
WO2017031342A1 (fr) Biomarqueur circulant pour le syndrome de brugada

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14858405

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14858405

Country of ref document: EP

Kind code of ref document: A1