REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 48440-567001US_ST25.TXT, created on Apr. 7, 2016, 7,485 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference.

BACKGROUND OF THE DISCLOSURE

Beta cell dysfunction and death is, in part, due to autoimmunity against beta cells, which contributes to the rising blood glucose level in patients with type 1 diabetes (T1D). There is a correlation between autoantibody positivity and loss of beta cell function. However, in order to identify individuals not only at risk but with actual ongoing disease prior to loss of metabolic control, a direct measure of beta cell death is needed. Provided herein are solutions to these and other problems in the art.

BRIEF SUMMARY OF THE DISCLOSURE

The current disclosure provides, inter alia, a quantitative methylation specific nested primer based polymerase chain reaction (qMSP) assay, to identify individuals not only at risk of T1D, but with actual ongoing disease prior to loss of metabolic control. The compositions, methods, and kits provided in this disclosure allow, for example, functional screening of at risk individuals, monitoring of ongoing disease, as well as in evaluating treatment methods including islet cell transplantation. The subject matter provided herein further relates to, inter alia, compositions, methods, and kits detecting unmethylated deoxyribonucleic acid (DNA) in blood of a subject. The disclosure further provides, inter alia, compositions, methods, and kits for detecting unmethylation of isolated DNA from blood of a subject at a CpG site in an insulin gene promoter located at −19 bp, −69 bp, −135 bp, −206 bp, or −357 bp relative to the transcription start site. In embodiments the insulin gene promoter is on DNA from beta cells and isolated from blood of a subject.

The present disclosure provides a method of detecting unmethylated promoter DNA in insulin gene. The method involves modifying DNA isolated from whole blood, plasma, serum, or a tissue sample of a subject to produce a modified DNA. In the method of the present disclosure, DNA is contacted with bisulfite for modifying unmethylated cytosine at a CpG site in the DNA to uridine. The DNA is modified by deamination of cytidine (or nucleoside cytosine) to uridine (or nucleoside uracil). The modified DNA is amplified by using quantitative methylation-specific polymerase chain reaction (qMSP) with methylation specific primers. The method provides detecting unmethylation at a CpG site.

In another aspect, the present disclosure provides nucleotides of SEQ ID NO: 1, 2, 3, and 4. The disclosure further provides nucleic acid comprising SEQ ID NO: 1, 2, 3, or 4, hybridized to a complementary DNA sequence, where the complementary DNA sequence is modified to have uridine.

In a further aspect, kits are provided, in which a first nucleic acid and a second nucleic acid each independently including sequences of SEQ ID NO: 1, 2, 3, 4, 5, or 6, are included. In the kit, the first and second nucleic acids do not simultaneously include the same sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In another aspect, the present disclosure provides a kit including nucleic acids of SEQ ID NO: 1, 2, 3, 4, 5, and 6.

In another aspect, the disclosure provides detecting unmethylated insulin gene promoter in blood of a subject as a measure of beta cell death and a prognostic indicator of autoimmunity resulting in type 1 diabetes (T1D).

In another aspect, the disclosure provides a method for treating an autoimmunity against insulin producing beta cells in a subject having unmethylated CpG at −19 bp, −69 bp, −135 bp, −206 bp, and/or −357 bp relative to the transcription start site of an insulin gene promoter, determined by the method as set forth in this disclosure. In embodiments, the insulin gene promoter is that of DNA from beta cells.

In another aspect, the disclosure provides a system including: at least one processor; and at least one memory including program code which when executed by the one memory provides operations that includes collecting DNA data associated with a subject; contacting said isolated DNA with bisulfite for modifying unmethylated cytosine at a CpG site in said DNA to uridine detecting unmethylation of DNA at said CpG site in an insulin gene promoter located at −19 bp, −69 bp, −135 bp, −206 bp, or −357 bp relative to the transcription start site; and providing, via a user interface, a prognosis and/or diagnosis for the subject based at least in part on detected unmethylated DNA.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Unless noted to the contrary, all publications, references, patents and/or patent applications reference herein are hereby incorporated by reference in their entirety for all purposes.

