Measurement of Differentially Methylated <em>INS</em> DNA Species in Human Serum Samples as a Biomarker of Islet &#946; Cell Death

Abstract

Summary Islet β cell death precedes development of type 1 diabetes, and detecting this process may allow for early therapeutic intervention. Here, we provide a detailed description of how to measure differentially methylated INS DNA species in human serum as a biomarker of β cell death. The death of islet β cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank hyperglycemia, especially in type 1 diabetes. The development of sensitive and reliable biomarkers of β cell death may allow for early therapeutic intervention to prevent or delay the development of diabetes. Recently, several groups including our own have reported that cell-free, differentially methylated DNA encoding preproinsulin (INS) in the circulation is correlated to β cell death in pre-type 1 diabetes and new-onset type 1 diabetes. Here, we present a step-by-step protocol using digital PCR for the measurement of cell-free INS DNA that is differentially methylated at cytosine at position -69 bp (relative to the transcriptional start site). We demonstrate that the assay can distinguish between methylated and unmethylated cytosine at position -69 bp, is linear across several orders of magnitude, provides absolute quantitation of DNA copy numbers, and can be applied to samples of human serum from individuals with new-onset type 1 diabetes and disease-free controls. The protocol described here can be adapted to any DNA species for which detection of differentially methylated cytosines is desired, whether from circulation or from isolated cells and tissues, and can provide absolute quantitation of DNA fragments.

Immune reconstitution in ART treated, but not untreated HIV infection, is associated with abnormal beta cell function

“…One HIV- control participant and one HIV+/ART+ participant had insufficient serum for proinsulin analysis; therefore, these individuals were excluded from group comparisons of PI:C ratios or analysis of correlations with proinsulin values. Serum levels of unmethylated and methylated preproinsulin ( INS ) DNA were quantified as previously described using a droplet digital PCR-based assay that detects differential methylation at the -69 bp site of the INS gene [ 25 ]. 10 samples (4 controls, and 6 HIV+ samples) with poor total DNA recovery (<10 ng/μL) were excluded from INS DNA analysis.…”

Persistent elevations in circulating INS DNA among subjects with longstanding type 1 diabetes

“…Concentrations of unmethylated and methylated INS DNA were assayed in fasting and 90‐minutes serum samples from all subjects. The assay for serum unmethylated and methylated INS DNA using ddPCR has previously been described . Briefly, DNA was extracted from 50 μL of serum using the QiaAmp DNA Blood Mini kit (Qiagen) with 5 μg of poly‐A DNA as carrier.…”

“…The assay for serum unmethylated and methylated INS DNA using ddPCR has previously been described. 20,30 Briefly, DNA was extracted from 50 μL of serum using the QiaAmp DNA Blood Mini kit (Qiagen) with 5 μg of poly-A DNA as carrier. Bisulfite conversion was performed using the EZ DNA Methylation-Lightning Kit (Zymo Research).…”

Elevated unmethylated and methylated insulin DNA are unique markers of A + β + ketosis prone diabetes

“…Sera and DNA were obtained 4–6 months after the index DKA from 45 A+β+ KPD patients and 67 patients randomly selected to represent all other KPD subtypes, once they were without ketosis on stable doses of insulin or oral medications 3, 6 . Other KPD subtypes included A+β− (N=16), A−β− (N=21), and A−β+ (N=30).…”

“…All DNA samples underwent bisulfite conversion before analysis by droplet digital PCR, utilizing a QX200 Droplet Reader and QuantaSoft Software (Bio-Rad). Values were normalized for DNA recovery, back-calculated to the volume of blood used for DNA isolation and reported as concentrations of copies/µL 3, 6 .…”