Detection and quantification of donor cell-free dna in the circulation of organ transplant recipients

Abstract

This invention provides methods, compositions, and kits relating to detecting donor cell-free DNA in the circulation of an organ transplant recipient for the early identification of transplant rejection

What is claimed is: 
     
         1 . A method of detecting a SNP from a donor to monitor transplant status of a recipient receiving tissue from said donor, the method comprising:
 (a) identifying that a SNP having a minor allele frequency of 0.20 or greater as homozygous in the recipient;   (b) amplifying cell-free (cfDNA) from a serum or plasma sample obtained from the recipient at least 5 days after transplantation of material from donor to generate a cf library;   (c) performing a digital PCR reaction for the SNPs identified in (a) to detect the presence of the alternative allele for one or more of the SNPs, and   (d) selecting a SNP where the alternative SNP is present in the cf library to monitor transplant status of the patient.   
     
     
         2 . The method of  claim 1 , wherein the SNP has a minor allele frequency of 0.40 or greater. 
     
     
         3 . The method of  claim 1 , wherein step (a) is performed using DNA isolated from peripheral blood leukocytes obtained from the patient. 
     
     
         4 . The method of  claim 1 , wherein step (a) is performed using the cfDNA library of step (b). 
     
     
         5 . The method of any one of  claims 1  to  4 , wherein the SNP having a minor allege frequency of 0.20 or greater is a SNP set forth in Table 1. 
     
     
         6 . The method of any one of  claims 1  to  5 , wherein the SNP selected in (d) is homozygous in the donor. 
     
     
         7 . A method of detecting a SNP from a donor to monitor transplant status of a recipient receiving tissue from said donor, the method comprising:
 (a) identifying that a SNP having a minor allele frequency of 0.20 or greater as homozygous in the recipient;   (b) amplifying cell-free (cfDNA) from a blood sample obtained from the recipient 24 hrs or less after transplantation of graft material from donor to generate a cf library;   (c) identifying a SNP having a minor allele frequency of 0.20 or greater as homozygous in the donor using the cf library of step (b);   (d) performing a digital PCR reaction for the SNPs identified in (a) to detect the presence of the alternative allele for one or more of the SNPs in the donor, and   (e) selecting a SNP where the alternative SNP is homozygous in the donor.   
     
     
         8 . The method of  claim 7 , wherein the SNP has a minor allele frequency of 0.40 or greater. 
     
     
         9 . The method of  claim 7  or  8 , wherein step (a) is performed using DNA isolated from peripheral blood leukocytes obtained from the patient. 
     
     
         10 . The method of  claim 7 ,  8 , or  9 , wherein the SNP having a minor allele frequency of 0.20 or greater is a SNP set forth in Table 1. 
     
     
         11 . A method of detecting a SNP from a donor to monitor transplant status of a recipient receiving tissue from said donor DNA, the method comprising:
 (a) identifying at least one SNP having a minor allele frequency of 0.20 or greater as homozygous in a recipient using a DNA sample from the recipient from a source that is free of donor DNA from the patient;   (b) identifying at least one SNP having a minor allele frequency of 0.20 or greater as homozygous in a recipient using a DNA sample obtained from donor cells or tissue; and   (c) selecting a SNP that is homozygous in the recipient for which the donor has the alternative allele.   
     
     
         12 . The method of  claim 11 , wherein the donor is homozygous for the alternative allele. 
     
     
         13 . The method of  claim 11  or  12 , wherein the DNA sample from the recipient from the source that is free of donor DNA is obtained from a PBL sample from the recipient. 
     
     
         14 . The method of any one of  claims 11  to  13 , wherein the SNP has a minor allele frequency of 0.40 or greater. 
     
     
         15 . The method of any one of  claims 11  to  13 , wherein step (a) comprises identifying the SNP alleles in the recipient for at least 5, 10, 15, 20, 25, 30, 35, or 40 of the SNPs shown in Table 1; and step (b) comprises identifying the SNP alleles in the donor for at least 5, 10, 15, 20, 25, 30, 35, or 40 of the SNPs shown in Table 1. 
     
     
         16 . The method of any one of  claims 1  to  15 , further comprising obtaining a cfDNA sample from the recipient following transplantation of donor tissue; and
 quantifying the level of the donor SNP allele in the cfDNA sample. 
 
     
     
         17 . The method of  claim 16 , wherein the quantifying step comprises determining copy number of the donor SNP allele in the cfDNA sample. 
     
     
         18 . The method of  claim 16 , wherein the quantifying step comprises determining the percentage of the donor SNP allele in the cfDNA sample. 
     
     
         19 . The method of any one of  claims 16 ,  17 , or  18 , wherein the transplanted material is a marginal organ. 
     
     
         20 . The method of any one of  claims 16 ,  17 , or  18 , wherein the cfDNA sample is from a blood sample obtained ten days or longer following transplant. 
     
     
         21 . The method of any one of  claims 16 ,  17 , or  18 , wherein the cfDNA sample is from a blood sample obtained a year or longer following transplant. 
     
     
         22 . The method of  claim 20  or  21 , further comprising adjusting an administration schedule or dosage or of an immunosuppressive drug. 
     
     
         23 . The method of any one of  claims 16 ,  17 ,  18 , or  19 , wherein the cfDNA sample is from a blood sample obtained within seven days of transplant. 
     
     
         24 . The method of any one of  claims 16  to  23 , wherein the donor tissue is a liver. 
     
     
         25 . The method of any one of  claims 16  to  23 , wherein the donor tissue is a heart. 
     
     
         26 . The method of any one of  claims 16  to  23 , wherein the donor tissue is a kidney. 
     
     
         27 . The method of any one of  claims 16  to  21 , further comprising detecting donor-specific antibodies in the blood of the recipient. 
     
     
         28 . Use of a method of  claim 23  for detecting reperfusion damage to donor tissue in a transplant recipient. 
     
     
         29 . Use of a method of  claim 24  for detecting damage to donor liver tissue by a hepatitis virus in a transplant recipient 
     
     
         30 . The use of a method of any one of  claims 16 ,  17 , or  18  for determining the minimal effective dose of an immunosuppressive drug. 
     
     
         31 . The use of a method of any one of  claims 16  to  26  for detecting transplant damage from donor-specific antibodies. 
     
     
         32 . The use of a method of any one of  claims 16  to  26  for detecting a chronic transplant injury. 
     
     
         33 . Use of a method of  claim 26  for detecting chronic graft nephropathy. 
     
     
         34 . Use of a method of  claim 25 , for detecting graft vasculopathy. 
     
     
         35 . A method of monitoring rejection of a transplant in a transplant recipient, the method comprising:
 obtaining a cfDNA sample from the transplant recipient; and   quantifying the amount of a donor SNP allele for a SNP selected in any one of  claims 1  to  15 .   
     
     
         36 . The method of  claim 35 , wherein the transplant is a marginal organ. 
     
     
         37 . The method of  claim 35  or  36 , wherein the transplant is monitored to detect reperfusion damage. 
     
     
         38 . The method of  claim 35 , wherein the transplant is monitored to determine the lowest effective dose of an immunosuppressive drug. 
     
     
         39 . The method of  claim 35 , wherein the transplant is monitored to detect damage to the transplant from donor-specific antibodies. 
     
     
         40 . The method of any one of  claims 35  to  40 , wherein the transplant is a liver, kidney, or heart.