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Combined Analysis of GAD65, miR-375 and Unmethylated Insulin DNA Following Islet Transplantation in T1D Patients.

Aim: several biomarkers were proposed to detect pancreatic beta cell destruction in vivo but were so far not compared for sensitivity and significance.

Methods: we used islet transplantation as model to compare plasma concentrations of miR-375, GAD65 and (un)methylated insulin DNA, measured at subpicomolar sensitivity, to study their discharge kinetics, power for outcome prediction and detection of graft loss during follow-up.

Results: at 60 min post-transplantation, GAD65 and miR-375 consistently showed near-equimolar and correlated increases, proportional to the number of implanted beta cells. GAD65 and miR-375 showed comparable power to predict poor graft outcome at 2 months, with AUC respectively of 0.833 and 0.771 (p=0.53). Using ROC analysis we defined likelihood ratios (LRs) for rationally selected result intervals: in GADA-negative recipients (N=28), GAD65 <4.5 pmol/l (LR=0.15) and >12.2 pmol/l (LR=∞) respectively predicted good and poor outcome. miR-375 could be used in all recipients irrespective of GADA status (N=46), with levels <1.4 pmol/l (LR=0.14) or >7.6 pmol/l (LR=9.53) as dual thresholds. The post-transplant surge of (un)methylated insulin DNA was inconsistent and unrelated to outcome. Combined measurement of these 3 biomarkers was also tested as liquid biopsy for beta cell death during 2 month follow-up: incidental surges of GAD65, miR-375 and (un)methylated insulin DNA, alone or combined, were confidently detected but could not be related to outcome.

Conclusions: GAD65 and miR-375 perform equally well to quantify early graft destruction and to predict graft outcome, outperforming unmethylated insulin DNA.

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