Mitochondrial mutations and rearrangements as a diagnostic tool for the detection of sun exposure, prostate cancer and other cancers

Abstract

Mitochondrial DNA deletions useful for the detection of cancers and sun exposure are provided. In particular, methods and kits for detecting mitochondrial DNA deletions for the early detection, diagnosis and progression of prostate cancer, sun exposure and non-melonoma skin cancer are provided.

What is claimed is: 
     
         1 . A method of detecting a deletion spanning approximately nucleotides 547 to 4443 in the minor arc of the human mtDNA genome, wherein said deletion is associated with skin cancer and/or UV exposure, comprising:
 (a) providing a biological sample;   (b) extracting mtDNA from the biological sample; and   (c) detecting the presence of the deletion in the mtDNA.   
     
     
         2 . The method of  claim 1 , wherein the biological sample is a skin sample from a subject. 
     
     
         3 . The method of  claim 1 , wherein the biological sample is a tissue culture sample. 
     
     
         4 . The method of  claim 3 , further comprising exposing the tissue culture sample to at least one sub-lethal dose of ultraviolet radiation (UVR) prior to the step of extracting mtDNA from the tissue culture sample. 
     
     
         5 . The method of  claim 4 , wherein the biological sample is exposed to a series of repetitive sub-lethal doses of UVR. 
     
     
         6 . The method of  claim 5 , wherein the series comprises exposing the biological sample to daily doses of UVR. 
     
     
         7 . The method of  claim 4 , wherein the UVR is from a solar-simulated UVR source. 
     
     
         8 . The method of  claim 4 , wherein the UVR comprises UVA, UVB, or UVA/UVB. 
     
     
         9 . The method of  claim 1 , wherein the method is use to detect skin cancer. 
     
     
         10 . The method of  claim 1 , wherein the deletion is approximately 3895 bp. 
     
     
         11 . The method of  claim 1 , wherein the deletion comprises a span of mtDNA from approximately mtTF1 binding site in the D-loop to tRNA methionine. 
     
     
         12 . The method of  claim 1 , wherein the deletion comprises substantially a 12s rRNA gene, a 16s rRNA gene, a ND1 gene and promoters for transcription of H and L strands. 
     
     
         13 . The method of  claim 1 , wherein the step of detecting the presence of the deletion in the mtDNA comprises amplification of the mtDNA. 
     
     
         14 . The method of  claim 1 , wherein the step of detecting the presence of the deletion in the mtDNA comprises PCR analysis selected from: deletion specific PCR analysis, radioactive PCR analysis, quantitative PCR, real-time PCR (RT-PCR) analysis, or RT-PCR using recombinant, thermostable, Taq DNA polymerase. 
     
     
         15 . The method of  claim 1 , wherein the step of detecting the presence of the deletion in the mtDNA comprises PCR analysis using a primer that bridges the mtDNA junction formed by the deletion spanning approximately nucleotides 547 to 4443. 
     
     
         16 . The method of  claim 14 , wherein a PCR primer that bridges the mtDNA junction formed by the deletion spanning approximately nucleotides 547 to 4443 is used for the PCR analysis. 
     
     
         17 . The method of  claim 14 , wherein the PCR analysis is quantitative PCR analysis comprising use of a primer that bridges the mtDNA junction formed by the deletion spanning approximately nucleotides 547 to 4443. 
     
     
         18 . The method of  claim 14 , wherein the deletion is detected using a primer having a sequence corresponding to SEQ ID NO:145, SEQ ID NO:146, or SEQ ID NO:153. 
     
     
         19 . The method of  claim 2 , wherein the biological sample is from non-lesional skin. 
     
     
         20 . The method of  claim 2 , wherein the biological sample is from a dermis or an epidermis layer of the subject. 
     
     
         21 . The method of  claim 1 , wherein the biological sample comprises occasionally sun exposed tissue; usually sun exposed tissue; or rarely sun exposed tissue. 
     
     
         22 . The method of  claim 21 , further comprising a step of comparing the presence of the deletion in the biological sample with a reference sample. 
     
     
         23 . The method of  claim 22 , wherein the reference sample is: rarely sun exposed skin tissue; occasionally sun exposed skin tissue; usually sun exposed skin tissue; or a blood sample. 
     
     
         24 . A method for determining cumulative UV exposure or for monitoring long term safety of clinical UV phototherapy regimes or for monitoring a person for sun exposure and associated non-melanoma skin cancer (NMSC), in a subject having mtDNA, the method comprising:
 (a) providing a biological sample from the subject;   (b) extracting mtDNA from the biological sample; and   (c) detecting the presence of a deletion spanning approximately nucleotides 547 to 4443 in the minor arc of the human mtDNA genome.   
     
     
         25 . The method of  claim 24 , wherein the method is for monitoring the subject for sun exposure and associated non-melanoma skin cancer (NMSC), and wherein the method further comprises the step of:
 (d) repeating steps (a) to (c) after an interval of time.   
     
     
         26 . The method of  claim 24 , wherein the deletion is approximately 3895 bp. 
     
     
         27 . The method of  claim 24 , wherein the deletion comprises a span of mtDNA from approximately the mtTF1 binding site in the D-loop to tRNA methionine. 
     
     
         28 . The method of  claim 24 , wherein the deletion comprises substantially a 12s rRNA gene, a 16s rRNA gene, a ND1 gene and promoters for transcription of H and L strands. 
     
     
         29 . The method of  claim 24 , wherein the step of detecting the presence of the deletion in the mtDNA comprises amplification of the mtDNA. 
     
     
         30 . The method of  claim 24 , wherein the step of detecting the presence of the deletion in the mtDNA comprises PCR analysis selected from: deletion specific PCR analysis, radioactive PCR analysis, quantitative PCR, real-time PCR (RT-PCR) analysis, or RT-PCR using recombinant, thermostable, Taq DNA polymerase. 
     
     
         31 . The method of  claim 24 , wherein the step of detecting the presence of the deletion in the mtDNA comprises PCR analysis using a primer that bridges the mtDNA junction formed by the deletion spanning approximately nucleotides 547 to 4443. 
     
     
         32 . The method of  claim 30 , wherein a PCR primer that bridges the mtDNA junction formed by the deletion spanning approximately nucleotides 547 to 4443 is used for the PCR analysis. 
     
     
         33 . The method of  claim 30 , wherein the PCR analysis is quantitative PCR analysis comprising use of a primer that bridges the mtDNA junction formed by the deletion spanning approximately nucleotides 547 to 4443. 
     
     
         34 . The method of  claim 30 , wherein the deletion is detected using a primer having a sequence corresponding to SEQ ID NO:145, SEQ ID NO:146, or SEQ ID NO:153. 
     
     
         35 . Use of an mtDNA deletion spanning approximately nucleotides 547 to 4443 in the minor arc of the human mtDNA genome as a biomarker for UV damage or as a biomarker for sking cancer. 
     
     
         36 . An array for use in detecting skin cancer, UV exposure, UV damage and/or monitoring associated non-melanoma skin cancer (NMSC), the array comprising at least one nucleic acid member, and a solid substrate, wherein the at least one nucleic acid member is associated with a human mtDNA deletion spanning approximately nucleotides 547 to 4443 of the human mtDNA genome, and wherein the at least one nucleic acid member has a unique position on said array and is stably associated with the solid substrate. 
     
     
         37 . A kit for detecting skin cancer, UV exposure, UV damage and/or monitoring associated non-melanoma skin cancer (NMSC), the kit comprising a means for detecting a mtDNA deletion spanning approximately nucleotides 547 to 4443 in the minor arc of the human mtDNA genome, and at least one member selected from: a solid support, means for holding the solid support, a disposable chip, means for holding the disposable chip, a means for extraction of mitochondrial DNA, primers, reagents and instructions. 
     
     
         38 . The kit of  claim 37 , wherein the means for detecting the mtDNA deletion comprises the array of  claim 36 . 
     
     
         39 . An array for use in detecting prostate cancer, the array comprising at least one nucleic acid member, and a solid substrate, wherein the at least one nucleic acid member is associated with a human mtDNA deletion spanning approximately nucleotides 10744 to 14124 of the human mtDNA genome, and wherein the at least one nucleic acid member has a unique position on said array and is stably associated with the solid substrate. 
     
     
         40 . A kit for detecting prostate cancer, the kit comprising a means for detecting a mtDNA deletion spanning approximately nucleotides 10744 to 14124 of the human mtDNA genome, and at least one member selected from: a solid support, means for holding the solid support, a disposable chip, means for holding the disposable chip, a means for extraction of mitochondrial DNA, primers, reagents and instructions. 
     
     
         41 . The kit of  claim 40 , wherein the means for detecting the mtDNA deletion comprises the array of  claim 39 .