| Aims: The prevalence of diabetes varies between ethnic groups,with about 14.7%of the Han Chinese compared to 4.3% of the Tibetan population.In China,the prevalence of diabetes in Tibetans is relatively low,however,there is a lack of research on the genomics of Tibetan T2 DM patients.Therefore,further exploration of whether the difference in T2 DM prevalence between Tibetans and Han Chinese is genomically related may elucidate the pathogenesis of T2 DM from a genetic perspective.METHODS: In this study,we conducted a clinical study and integrative analysis of RNA-seq and DNA methylation for the Han and Tibetan T2 DM populations.120 Han and Tibetan T2 DM patients were included and analyzed for differences in general condition,laboratory indices,Traditional Chinese Medicine(TCM)patterns and TCM constitution.A paired design was used to enroll 12 male Han and Tibetan T2 DM patients with yin deficiency.RRBS and Poly(A)sequencing were used to analyze the DNA methylation and transcriptome of peripheral white blood cells of Han and Tibetan T2 DM patients with yin deficiency,to screen for differentially methylated regions and differentially expressed genes between the two ethnic groups,and to enrich candidate genes for functional validation and mechanism of action analysis.RESULTS: A comparison of clinical data between 120 Han and Tibetan T2 DM patients showed that:(i)the Tibetan group had a higher BMI(25.87±3.20 vs 24.07±4.75,P=0.017),Hb(147.73±12.79 vs 134.52±18.05,P<0.001),Hb A1c(10.31±2.44 vs8.89±2.14,P=0.001),LDL(3.11±0.82 vs 2.58±0.97,P=0.002),ALT(33.05±20.86 vs.24.83±13.87,P=0.014),GGT(55.38±50.37 vs.26.98±18.88,P<0.001),e GFR(131.79±40.90 vs.96.62± 28.06,P<0.001)were higher than those in the Han group,while HDL(0.98±0.20 vs 1.10±0.31,P=0.014)and BUN(5.10±1.45 vs 6.25±2.37,P=0.002)were lower than those in the Han group;(ii)the distribution of TCM constitutions in the Tibetan group was Yin deficiency(31.7%),Qi deficiency(18.3%),Dampness-heat(11.7%),Peace quality(8.3%),special quality(8.3%),Yang deficiency(6.7%),Phlegm-dampness(6.7%),Blood stasis(5%),and Qi stagnation(3.3%);the distribution of the Han group was Yin deficiency(35%),Qi deficiency(15%),Yang deficiency(13.3%),Peace quality(11.7%),Dampness-heat(8.3%),Phlegm-dampness(6.7%),Blood stasis(5%),Special quality(3.3%),and Qi Stagnation(1.6%);(iii)The distribution of TCM syndrome types in the Tibetan group from high to low is Blood stasis in collaterals syndrome(36.7%),Qi-yin deficiency syndrome(33.3%),Yin deficiency and heat syndrome(15%),and Dampness-heat trapping spleen syndrome(11.6)%),Yin-yang deficiency and water retention with blood dysfunction(3.3%);the distribution of TCM syndrome types in the Han group from high to low is Qi-yin deficiency syndrome(41.7%),Blood stasis in collaterals syndrome(20%),and Yin deficiency and heat syndrome(16.7%),Dampness-heat trapping spleen syndrome(16.7%),Yin-yang deficiency and water retention with blood dysfunction(5%).The results of comparing 40 of these Han and Tibetan patients with T2 DM of yin deficiency quality showed that:(i)the Tibetan group had BMI(25.86±3.20 vs 23.40±2.42,P<0.001),Hb A1c(10.22±2.04 vs 8.76±1.55,P=0.014),FBG(10.71±2.07 vs8.60±3.09,P=0.016),GGT(66.53±65.46 vs 28.52±19.55,P=0.015)and e GFR(147.74±41.86 vs 94.20±26.93,P<0.001)were higher than those in the Han group,while TBA(4.11±2.68 vs 6.35± 3.71,P=0.036)and BUN(5.11±1.25 vs 6.16±1.89,P=0.047)were lower than those in the Han group.Comparison of 12 Han and Tibetan male patients with yin deficiency T2 DM showed that:(i)DNA methylation analysis of peripheral blood showed that compared with Han,the Tibetan group had 7558differentially-methylated regions(DMRs),7102 hypermethylated regions,involving1253 genes.Differentially methylated genes mainly enriched in 19 GO terms(P<0.05)as well as 20 KEGG signaling pathways(P<0.05);(ii)Poly(A)sequencing showed that there were 75 Differentially-expressed genes(DEGs)between the two groups,with 57up-regulated genes and 18 down-regulated genes.GO enrichment analysis showed that up-regulated genes were mainly enriched in 30 GO terms and down-regulated genes were in 26 GO terms;KEGG enrichment analysis showed that the up-regulated genes were mainly involved in 30 signaling pathways,while the down-regulated genes were mainly involved in 3 signaling pathways;(iii)Differentially-expressed m RNA analysis showed that 83 genes were up-regulated while only 12 genes were down-regulated;differentially-expressed nc RNA analysis showed that 14 genes were up-regulated while4 genes were down-regulated;(iv)Statistical analysis of the expression levels of new transcript regions revealed a total of 3927 new transcripts;(v)Protein interaction network analysis showed that ALB,HNF4 A and CDH17 may have protein interaction between the Han and Tibetan groups;(vi)Genome-wide methylation and transcriptome The joint analysis showed that there were 25 genes with both differentially methylated regions and differentially expressed genes,of which 10 genes were hypermethylated at high transcriptional levels in Tibetans and 15 genes were hypermethylated at low transcriptional levels in Tibetans;GO and KEGG enrichment analysis was performed on individual genes,and the differentially methylated and differentially expressed genes were associated with 20 GO terms and with 6 KEGG signaling pathways.Conclusions:(i) The obesity index and glycemic control of Tibetan T2 DM patients were slightly less favorable than those of the Han group.(ii)Both Tibetan and Han Chinese T2 DM patients had the most Yin deficiency constitution,with Qi deficiency ranking second constitution,followed by Dampness-heat constitution in Tibetans,and Yang deficiency constitution in Han Chinese.(iii)Gene expression differences existed between Han and Tibetan T2 DM male patients,and the main signaling pathways of DEGs were associated to lipid metabolism,insulin secretion and utilization,including HNF4 A,FABP1,CD248,ERBB3,APOB,PAX8-AS1 genes,and PI3K-Akt,c AMP,Wnt,Hippo signaling pathways.These genes can be further validated as candidates in the Han and Tibetan populations,combining the signaling pathways of action to explore the different mechanisms underlying the development of T2 DM in the Han and Tibetan populations. |
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