Aims/hypothesis The aim of this study was to identify differentially expressed long non-coding RNAs (lncRNAs) and mRNAs in whole blood of people with type 2 diabetes across five different clusters: severe insulin-deficient diabetes (SIDD), severe insulin-resistant diabetes (SIRD), mild obesity-related diabetes (MOD), mild diabetes (MD) and mild diabetes with high HDL-cholesterol (MDH). This was to increase our understanding of different molecular mechanisms underlying the five putative clusters of type 2 diabetes. Methods Participants in the Hoorn Diabetes Care System (DCS) cohort were clustered based on age, BMI, HbA1c, C-peptide and HDL-cholesterol. Whole blood RNA-seq was used to identify differentially expressed lncRNAs and mRNAs in a cluster compared with all others. Differentially expressed genes were validated in the Innovative Medicines Initiative DIabetes REsearCh on patient straTification (IMI DIRECT) study. Expression quantitative trait loci (eQTLs) for differentially expressed RNAs were obtained from a publicly available dataset. To estimate the causal effects of RNAs on traits, a two-sample Mendelian randomisation analysis was performed using public genome-wide association study (GWAS) data. Results Eleven lncRNAs and 175 mRNAs were differentially expressed in the MOD cluster, the lncRNA AL354696.2 was upregulated in the SIDD cluster and GPR15 mRNA was downregulated in the MDH cluster. mRNAs and lncRNAs that were differentially expressed in the MOD cluster were correlated among each other. Six lncRNAs and 120 mRNAs validated in the IMI DIRECT study. Using two-sample Mendelian randomisation, we found 52 mRNAs to have a causal effect on anthropometric traits (n=23) and lipid metabolism traits (n=10). GPR146 showed a causal effect on plasma HDL-cholesterol levels (p = 2×10–15), without evidence for reverse causality. Conclusions/interpretation Multiple lncRNAs and mRNAs were found to be differentially expressed among clusters and particularly in the MOD cluster. mRNAs in the MOD cluster showed a possible causal effect on anthropometric traits, lipid metabolism traits and blood cell fractions. Together, our results show that individuals in the MOD cluster show aberrant RNA expression of genes that have a suggested causal role on multiple diabetes-relevant traits. Graphical abstract

Aims/hypothesis Micro- and macrovascular complications are common among persons with type 2 diabetes. Interest into the potential of circulating small non-coding RNAs (sRNAs) as biomarkers or drivers of the development of diabetic complications is growing. In this study we investigated if circulating sRNAs levels associate with prevalent chronic kidney disease (CKD) in persons with type 2 diabetes. Methods Plasma sRNAs levels were determined using sRNA-seq, allowing detection of miRNAs, snoRNAs, piRNAs, tRNA-fragments and various other sRNA classes, in persons with type 2 diabetes, with CKD (n=69) or without CKD (n=405). Multiple regression analyses were used to test for associations between the sRNAs and primary endpoint CKD. In secondary analyses, we also tested the association with eGFR and albuminuria (UACR). Results In total, twelve sRNA were associated with prevalent CKD (logFC = -0.32 to -1.28, all P≤6.24x10-4). Although miRNAs represent the majority of the sRNAs measured (64%) only four miRNA were significantly associated with prevalent CKD. Interestingly, the majority of the significant sRNA belonged to the snoRNAs (58%). Similar results were observed for eGFR and UACR. Only one of the twelve sRNAs showed its highest expression in kidney whereas several others showed high expression in liver or colon. Conclusions/interpretation Small RNAs present in the circulation associate with CKD in persons with type 2 diabetes. Further studies are warranted to elucidate the biological role of sRNA in diabetic CKD and in particular snoRNAs. High expression of these circulating sRNAs in tissues other than kidney suggest a role in inter-organ communication.