Method for diagnosing subclinical and clinical acute rejection by analysis of predictive gene sets

Abstract

Disclosed herein are methods for diagnosing acute cellular rejection (ACR) of an allograft by analysis of predictive gene sets and kits for practicing these methods.

What is claimed is: 
     
         1 . A method for identifying a renal allograft recipient at risk of allograft rejection comprising the steps of:
 (a) providing a blood specimen from the renal allograft recipient;   (b) determining the expression levels of a preselected gene set in the blood of the recipient;   (c) comparing the expression levels of the preselected genes with the expression levels the preselected genes in a control; and   (d) determining the recipient is at risk for allograft rejection if the expression level of one or more genes in the gene set in the specimen is altered from the expression level of the same one or more gene set genes in the control.   
     
     
         2 . The method of  claim 1  wherein the alteration comprises an increase and/or decrease in the expression level of one or more of the gene set genes in the specimen compared to the same one or more gene set genes in the control. 
     
     
         3 . The method of  claim 1  wherein the preselected gene set comprises at least 7 genes selected from the group consisting of SPCS3, ZMAT1, ETAA1, ZNF493, CCDC82, NFYB, F13A1, TUBB1, TSC22D1, SENP6, ANXA5, EFTUD2, SENP7, AP1M1, CLK1, MAP1A and C1GALT1C1. 
     
     
         4 . The method of  claim 1  wherein the determining step comprises applying the expression levels determined in the patient's sample to a penalized logistic regression fitting model (log (p(x))/(1−p(x))=β*0+β*1g1+β*igi+ . . . +β*9g9 (where (p(x) is the probability of rejection β*i is penalized coefficiency and gi is the read count of gene i) to determine the probability of allograft rejection. 
     
     
         5 . A method for treating a renal allograft recipient at risk of rejection of the allograft comprising the steps of:
 (a) obtaining a blood specimen from the renal allograft recipient;   (b) isolating mRNA from the blood specimen;   (c) synthesizing cDNA from the miRNA;   (d) determining the expression levels of a signature gene set in said recipient's blood;   (e) diagnosing the allograft recipient as being at high risk for allograft rejection and allograft loss if the expression level of one or more genes in the gene signature set in the allograft recipient's blood specimen is altered compared to the expression level of the same one or more genes in the control blood specimen and treating the recipient identified as being at high risk to prevent allograft rejection or allograft loss.   
     
     
         6 . The method of  claim 5  comprising administering an anti-rejection drug or a high dose steroid to the recipient diagnosed as being high risk for ACR or graft loss. 
     
     
         7 . The method of  claim 2  wherein the gene signature set comprises at least the genes ANXA5, TSC22D1, AP1M1, CLK1, EFTUD2, SENP6, and SENP7. 
     
     
         8 . The method of  claim 2  wherein the gene signature set comprises at least the genes TSC22D1, ANKA5, EFTUD2, AP1M1, MAP1A, C1GALT1C1, SENP6, CLK1 and SENP7. 
     
     
         9 . The method of  claim 2  wherein the gene signature set comprises at least the genes CCDC82, F13A1, TUBB1, TSC22D1, SENP6, ANXA5, EFTUD2, SENP7, AP1M1, CLK1, and C1GALT1C1. 
     
     
         10 . The method of  claim 2  wherein the gene signature set comprises at least the genes SPCS3, ZMAT1, ETAA1, ZNF493, CCDC82, NFYB, F13A1, TUBB1, TSC22D1, SENP6, ANXA5, EFTUD2, SENP7, AP1M1, CLK1, MAP1A and C1GALT1C1. 
     
     
         11 . The method of  claim 5 , wherein the anti-rejection drug is an immunosuppressive or anti-proliferative agent. 
     
     
         12 . The method of  claim 11 , wherein the immunosuppressive agent is a member selected from the group consisting of a calcineurin inhibitor (CNI) (e.g., cyclosporine or tacrolimus), mycophenolate mofetil (MMF), sirolimus, prednisone, Mycophenolate Mofetil, Mycophenolate Sodium and Azathioprine. 
     
     
         13 . The method of  claim 1  wherein the expression levels are determined by a method selected from the group consisting of Nanostring, MiSEQ and quantitative polymerase chain reaction (qPCR). 
     
     
         14 . A kit for identifying renal allograft recipients suffering from subclinical and clinical acute rejection and at risk for allograft loss comprising in one or more separate containers primer pairs for the preselected signature set, SEQ ID NOs: 1-34, buffers, positive and negative controls and instructions for use. 
     
     
         15 . The kit of  claim 14  wherein said signature set comprises at least the genes ANXA5, TSC22D1, AP1M1, CLK1, EFTUD2, SENP6, and SENP7. 
     
     
         16 . The kit of  claim 14  wherein the gene signature set comprises at least the genes TSC22D1, ANKA5, EFTUD2, AP1M1, MAP1A, C1GALT1C1, SENP6, CLK1 and SENP7. 
     
     
         17 . The kit of  claim 14  wherein the gene signature set comprises at least the genes CCDC82, F13A1, TUBB1, TSC22D1, SENP6, ANXA5, EFTUD2, SENP7, AP1M1, CLK1, and C1GALT1C1. 
     
     
         18 . The kit of  claim 14  wherein the gene signature set comprises at least the genes SPCS3, ZMAT1, ETAA1, ZNF493, CCDC82, NFYB, F13A1, TUBB1, TSC22D1, SENP6, ANXA5, EFTUD2, SENP7, AP1M1, CLK1, MAP1A and C1GALT1C1. 
     
     
         19 . The kit of  claim 14  further comprising housekeeping genes and primers for the housekeeping genes. 
     
     
         20 . The method of  claim 5  further comprising calculating the probability score of acute rejection for said patient using the equation:
   log ( p ( x ))/(1− p ( x ))=β*0+β*1 g 1+β* igi+ . . . + β*4 g 4
 
 
       wherein (p(x) is the probability of developing fibrosis, β*i is the penalized coefficiency and gi is the expression value of gene i. 
     
     
         21 . The method of  claim 20  wherein the probability score is determined using a computer based system. 
     
     
         22 . The method of  claim 20  wherein the probability score is used to determine the cut off value. 
     
     
         23 . The method for selecting a renal allograft recipient for treatment to reduce the risk of renal allograft rejection which comprises:
 (a) providing a blood specimen from the renal allograft recipient;   (b) determining the expression levels of a preselected gene set in the specimen;   (c) comparing the expression levels of the preselected gene set genes in the specimen with the expression levels of the preselected gene set genes in a control, and   (d) selecting the recipient for treatment for allograft rejection if the expression level of one or more genes in the gene set in the specimen is altered compared to the expression level of one or more of the gene set genes in the control.   
     
     
         24 . The method of selecting a renal allograft patient for treatment to reduce the risk of renal allograft rejection or allograft loss which comprises:
 comparing the expression level of a preselected gene set obtained from the patient with the expression level of the preselected gene set in a control sample obtained from an allograft recipient that did not suffer allograft rejection, and selecting the patient for treatment for allograft rejection or loss if the expression level of one or more genes in the preselected gene set from the patient is altered compared to the expression level of one or more of the preselected gene set genes in the control.