Patent Application
20150080235
Type 1 diabetes is caused by the destruction of pancreatic β cells due to an autoimmune process, which occurs over years. By the time clinical symptoms appear, the mass of β cells is reduced by at least 70-80% (Cnop, M. et al., (2005) Diabetes December; 54 Suppl. 2:S97-107).
A nested PCR diagnostic for diabetes has been developed that detects the relative amount of circulating un-methylated β cell insulin DNA as a result of cell death (Akirav, E. M. et. al., (2011) Proc. Natl. Acad. Sci. 108: 19018-19023). However, nested PCR produces biases and artifacts.
Accordingly new methods for diagnosing and monitoring type 1 diabetes are needed.
Featured herein are novel primers, probes and assays for diagnosing and monitoring type 1 diabetes.
The assays are highly sensitive and may be performed in a multiplexed fashion to diagnose diabetes before the onset of clinical symptoms and provide clinicians with a tool to decide for whom and when immune therapy might be useful.
Described herein are specific primers and probes, which can be used to measure the relative amounts of methylated and un-methylated insulin DNA in serum obtained from a human subject. The un-methylated form of the gene expresses functional insulin, while the methylated form does not express protein. Since β cells are the only significant source of insulin gene expression, the assay is able to measure epigenetically circulating un-methylated insulin DNA as a marker for β cell death. Methylated insulin DNA is used for normalizing the varying levels of DNA between individual specimens.
The nucleotide sequence of the unmethylated insulin DNA detection probe is: ATTTAAGATTTGTTGGGAGGTAGAG (SEQ ID NO: 1) and the nucleotide sequence of the methylated insulin DNA detection probe is: ATTTAAGATTCGTCGGGAGGTAGAG (SEQ ID NO: 2). One of skill in the art could alter the probes by deleting, replacing of altering one or more nucleotides without substantially changing probe function.
The probes target a region of the human insulin gene with 2 CpG sites (i.e. regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide). When DNA is treated with bisulfite, the un-methylated CpG is converted to TG (thymine, guanine), while a methylated CpG site is protected from this change. The two probes described herein target either a methylated or un-methylated insulin gene.
Use of these probes significantly reduces the background noise thereby increasing the noise to signal ratio.
The forward and reverse human insulin gene primers used in the examples are GTGGTTTATATTTGGTGGA (SEQ ID NO: 5) and ATTAACTCACCCTACAAATC (SEQ ID NO: 6). However, one of skill in the art may vary these sequences, e.g., by deleting, substituting or altering certain nucleotides without substantially impacting primer function.
The probes and primers described herein may be contacted with isolated and bisulfite-treated genomic DNA from an appropriate sample (e.g. serum, islet cells or peripheral blood mononuclear cells (PBMCs)). The reaction mixture may be loaded into a droplet generator. The droplets may be deposited on a plate and transferred into a polymerase chain reactor for amplification. The plate may then be transferred to a droplet reader for analysis of the data.
Use of the described probes and primers in a sensitive diagnostic assay, such as Droplet Digital Polymerase Chain Reaction (ddPCR), results in a highly specific and sensitive assay. In particular, as shown in the following examples, fewer than 13 copies were detected in a 25 ul PCR reaction with the target gene. In addition, in a recovery assay, where 5000 copies of L2_M plasmid representing bisulfite-treated methylated human insulin gene DNA were spiked with fewer than 10 copies of L2_UM plasmid representing bisulfite-treated un-methylated target DNA, L2_UM DNA was successfully detected.
As the results reported in the following example show, use of the described probes, primers and assays can be used to determine insulin DNA methylation status at low concentrations of DNA.
These primers and probes alone or in conjunction with instructions for use may be prepared as a kit for diagnosing or monitoring type 1 diabetes.
Table 1 lists additional probes and primers for amplification of methylation sensitive sites. Each assay has one set of probes, which can be used with 1 to 7 different sets of primers. Some probes will require modifications to the probes themselves to increase the annealing temperature and specificity such as—minor groove binder (MGB) modification, locked nucleic acid modification (LNA) and or Zen modification.
TTTGTGTTAGGT (SEQ ID NO: 78)
TTTGCGTTAGGT (SEQ ID NO: 79)
TAGGGAGGATGTGGTTGGGT (SEQ ID NO:
TAGGGAGGACGTGGTTGGGT (SEQ ID NO:
TTGTTGGTGTTGTTGG (SEQ ID NO: 98)
TTGTTGGCGTTGTTGG (SEQ ID NO: 99)
TTAGTTGTAGTT (SEQ ID NO: 114)
TTAGTCGTAGTT (SEQ ID NO: 115)
TTGTGTGGTTTA (SEQ ID NO: 124)
TTGTGCGGTTTA (SEQ ID NO: 125)
AGTGTGTGGGGAA (SEQ ID NO: 134)
AGTGTGCGGGGAA (SEQ ID NO: 135)
AGATTTGTTGGGAG (SEQ ID NO: 146)
AGATTCGTCGGGAG (SEQ ID NO: 147)
TTGGTTGTTTTT (SEQ ID NO: 158)
TTGGTCGTTTTT (SEQ ID NO: 159)
TTTGGTGTTTTT (SEQ ID NO: 170)
TTTGGCGTTTTT (SEQ ID NO: 171)
TGGTTATGTTTTAA (SEQ ID NO: 184)
TGGTTACGTTTTAA (SEQ ID NO: 185)
TGAGGATGGTGTTG (SEQ ID NO: 194)
TGAGGACGGTGTTG (SEQ ID NO: 195)
TGGTTTTGGTAGTT (SEQ ID NO: 201)
TGGTTTCGGTAGTT (SEQ ID NO: 201)
AGTTTTGAGATA (SEQ ID NO: 210)
AGTTTCGAGATA (SEQ ID NO: 211)
TGGGTATGTTTTT (SEQ ID NO: 220)
TGGGTACGTTTTT (SEQ ID NO: 221)
TTATTTGTTTTT (SEQ ID NO: 230)
TTATTCGTTTTT (SEQ ID NO: 231)
TGTTTTGTAGTT (SEQ ID NO: 240)
TGTTTCGTAGTT (SEQ ID NO: 241)
ATGATTGTAGA (SEQ ID NO: 250)
ATGATCGTAGA (SEQ ID NO: 251)
TGTTTTATGTTGTTTGT (SEQ ID NO: 254)
TGTTTTACGTTGTTTGT (SEQ ID NO: 255)
TTGGGTGAATAT (SEQ ID NO: 262)
TTGGGCGAATAT (SEQ ID NO: 263)
ATTATGTTTGGAGG (SEQ ID NO: 270)
ATTACGTTCGGAGG (SEQ ID NO: 271)
AGGAGGGTGTGGTTG (SEQ ID NO: 276)
AGGAGGGCGTGGTTG (SEQ ID NO: 277)
TTTTGTTGTTAGG (SEQ ID NO: 282)
TTTTGTCGTTAGG (SEQ ID NO: 283)
TTTTATGGTAG (SEQ ID NO: 294)
TTTTACGGTAG (SEQ ID NO: 295)
TGTGGGTGTTGGG (SEQ ID NO: 306)
TGTGGGCGTTGGG (SEQ ID NO: 307)
TTAGTTTGGTTGG (SEQ ID NO: 318)
TTAGTTCGGTTGG (SEQ ID NO: 319)
AGTGTGATTTA (SEQ ID NO: 330)
AGTGCGATTTA (SEQ ID NO: 331)
TTGGCGGGTAG (SEQ ID NO: 340)
TTGGCGGGTAG (SEQ ID NO: 341)
AGGTGGGTA (SEQ ID NO: 346)
AGGCGGGTA (SEQ ID NO: 347)
TGTTTTGTTGTTGTT (SEQ ID NO: 354)
TGTTTCGTCGTTGTT (SEQ ID NO: 355)
TGTTTTGGAAT (SEQ ID NO: 364)
TGTTTCGGAAT (SEQ ID NO: 365)
TTTTGTGTGGTATGTTTT (SEQ ID NO: 374)
TTTTGCGCGGTATGTTTT (SEQ ID NO: 375)
TTGGGTGGGGGT (SEQ ID NO: 384)
TTGGGCGGGGGT (SEQ ID NO: 385)
ATTAGATGTAGTT (SEQ ID NO: 400)
ATTAGACGTAGTT (SEQ ID NO: 401)
TAGTTTGTAGG (SEQ ID NO: 406)
TAGTTCGTAGG (SEQ ID NO: 407)
ATTTGTTGTTTT (SEQ ID NO: 412)
ATTCGTCGTTTT (SEQ ID NO: 413)
The invention now being generally described, will be more readily understood by reference to the following example, which is included merely for purposes of illustration and is not intended to be limiting.
All CpG sites for the human insulin gene (hg19_knownGene_uc021qcd.1 range=chr11:2181009-2182439) were mapped. The sequence was then transformed to a sequence representing bisulfite-treated DNA, where all Cs are converted to Ts and in the case of a methylated CpG site, the C is protected from conversion to T. The probes were designed to include two CpG sites at nucleotides 21814010 and 21814012 (http://genome.ucsc.edu/cgi-bin/hgGateway, Feb 2009 GRCh37/hgl9) in positions +396 and +399 from the transcription start site.
To increase sensitivity, specificity and to reduce background fluorescence, the probes were designed with an internal Zen quencher (ZEN) in addition to the 3′ Iowa Black FQ quencher (3IABkFQ). The ZEN probes were synthesized by Integrated DNA Technologies.
The synthetic L2_M and L2_UM sequences, which are replicas of bisulfite-treated methylated and un-methylated human insulin gene sequences (region 2181155-2181465 on Chr11), respectively, were cloned into pUC57 plasmids (Genewiz, Inc).
Reaction mixtures of 25 ul volume, comprising 1× Droplet PCR supermix, (BioRad), 900 uM primer mix, 250 uM probe mix, 5 ul of plasmid (copy number ranging from 100,000 to 1 and mixed populations of un-methylated and methylated plasmids) or 4 ul of bisulfite treated genomic DNA from either serum, islet cells or peripheral blood mononuclear cells (PBMCs) was prepared. Twenty μl of the PCR reaction mix was loaded into a separate well of a eight channel droplet generator cartridge, and in a separate corresponding well 70 ul of droplet generation oil (BioRad) was loaded. The cartridge was loaded into a droplet generator (Biorad). Forty ul of the generated droplets were carefully transferred to a 96 well PCR plate and the PCR reaction was run on a thermal cycle with the following protocol: 10 min activation at 95° C. followed by 40 cycles of a two-step amplification protocol of 30 seconds at 94° C. denaturation and 60 seconds at 58° C. for a combined annealing-extension step. A final 10 min at 98° C. inactivation step completed the reaction.
The PCR plate was then transferred to a QX100 droplet reader (Biorad), which automatically reads the droplets from each well of the plate. Analysis of ddPCR data was performed using QuantaSoft Analysis software.
Islet, PBMCs and serum samples from non-diabetic and diabetic patients were used to validate the protocol developed using the plasmids. DNA was isolated using the Qiagen blood and tissue DNA extraction kit. DNA was bisulfite treated using an EZ DNA Methylation kit (Zymo Research, Irvine, Calif.).
Limit of detection assays were performed with plasmids, islets and PBMCs. The plasmid suspensions were made in a series of 10 fold serial dilutions from 100,000 copies to 1 copy. Bisulfite-treated DNA from islets and PBMCs were diluted over a ¼dilution series up to 1/1024 with amounts ranging from 148 ng to 0.1 ng.
In the multiplexed assay, the probe for unmethylated human insulin gene DNA successfully detected 1 copy/μl of plasmid and showed no cross amplification with the plasmid representing methylated DNA. The probe for methylated DNA successfully detected 1 copy/ul per reaction and showed minimal cross amplification only with high numbers of plasmid representing un-methylated insulin gene DNA.
In the recovery assay, where approximately 3000 copies/ul of L2_M plasmid were mixed with 10 copies of the L2_UM plasmid, the ddPCR successfully detected the L2_UM plasmid successfully.
Using human islet tissue and PBMCs, we were able to detect unmethylated insulin DNA in as low as 0.1 ng of DNA. Also, the ratio of methylated insulin DNA to unmethylated insulin DNA did not change over varying concentrations. We then tested the assay with clinical samples, and were able to detect unmethylated DNA in pre-diabetics. Also, the ratio of unmethylated insulin coding DNA to methylated insulin DNA was significantly elevated in pre-diabetic (mean 0.4264±0.04034 N=6) patients compared to non-diabetic human controls (0.2122±0.02449 N=13).
While specific embodiments have been discussed, the above specification is intended to be illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification.
This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/879,347, filed Sep. 18, 2013; the contents of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61879347 | Sep 2013 | US |
