Assessment of the association between genetic factors regulating thyroid function and microvascular complications in diabetes: A two-sample Mendelian randomization study in the European population.

Background: Observational studies have identified a possible link between thyroid function and diabetic microangiopathy, specifically in diabetic kidney disease (DKD) and diabetic retinopathy (DR). However, it is unclear whether this association reflects a causal relationship. Objective: To assess the potential direct effect of thyroid characteristics on DKD and DR based on Mendelian randomization (MR). Methods: We conducted an MR study using genetic variants as an instrument associated with thyroid function to examine the causal effects on DKD and DR. The study included the analysis of 4 exposure factors associated with thyroid hormone regulation and 5 outcomes. Genomewide significant variants were used as instruments for standardized freethyroxine (FT4) and thyroid-stimulating hormone (TSH) levels within the reference range, standardized free triiodothyronine (FT3):FT4 ratio, and standardized thyroid peroxidase antibody (TPOAB) levels. The primary outcomes were DKD and DR events, and secondary outcomes were estimated glomerular filtration rate (eGFR), urinary albumin-to-creatinine ratio (ACR) in diabetes, and proliferative diabetic retinopathy (PDR). Satisfying the 3 MR core assumptions, the inverse-variance weighted technique was used as the primary analysis, and sensitivity analysis was performed using MR-Egger, weighted median, and MR pleiotropy residual sum and outlier techniques. Results: All outcome and exposure instruments were selected from publicly available GWAS data conducted in European populations. In inverse-variance weighted random-effects MR, gene-based TSH with in the reference range was associated with DKD (OR 1.44; 95%CI 1.04, 2.41; P = 0.033) and eGFR (ß: -0.031; 95%CI: -0.063, -0.001; P = 0.047). Gene-based increased FT3:FT4 ratio, decreased FT4 with in the reference range were associated with increased ACR with inverse-variance weighted random-effects ß of 0.178 (95%CI: 0.004, 0.353; P = 0.046) and -0.078 (95%CI: -0.142, -0.014; P = 0.017), respectively, and robust to tests of horizontal pleiotropy. However, all thyroid hormone instruments were not associated with DR and PDR at the genetic level. Conclusion: In diabetic patients, an elevated TSH within the reference range was linked to a greater risk of DKD and decreased eGFR. Similarly, decreased FT4 and an increased FT3:FT4 ratio within the reference range were associated with increased ACR in diabetic patients. However, gene-based thyroid hormones were not associated with DR, indicating a possible pathway involving the thyroid-islet-renal axis. However, larger population studies are needed to further validate this conclusion.

Alteration of N-Glycan Profiles in Diabetic Retinopathy.

PURPOSE: To investigate the alteration of vitreal N-glycans in patients with proliferative diabetic retinopathy (PDR). METHODS: Plasma and vitreous samples were collected from 17 patients (10 females and 7 males) with PDR (PDR group) and 17 nondiabetic patients (8 females and 9 males) with epiretinal membrane (ERM) and idiopathic macular hole (MH) (non-diabetes mellitus [DM] group). Profiles of N-glycans were analyzed by a glycoblotting-based high-throughput protocol that we recently developed. Human retinal microvascular endothelial cells (HRMECs) were cultivated with culture media containing either low glucose (5 mM) or high glucose (25 mM), and expression levels of sialyltransferases were analyzed by real-time PCR and ELISA. RESULTS: Amount of N-glycans in the vitreous fluid of the PDR group was significantly higher than that of the non-DM group (495.5 ± 37.4 vs. 142.7 ± 30.8 pmol/100 µg protein, P < 0.005), whereas there was no significant difference in the plasma samples between the PDR and the non-DM group. In addition, profile analysis showed that N-glycans with sialic acids increased in the vitreous of the PDR group (328.4 ± 25.8 pmol/100 µg protein) compared to the non-DM group (92.1 ± 21.2 pmol/100 µg protein, P < 0.0005). Expression levels of sialyltransferases ST3GAL1 and ST3GAL4 were upregulated in the HRMECs after high-glucose stimulation. Consistent with the real-time PCR data, high-glucose stimulation elevated the protein levels of ST3GAL1 (117.4 ± 14.9 pg/mg, P < 0.01) and ST3GAL4 (6.1 ± 0.9 pg/mg, P < 0.05) in the HRMECs compared with the cells cultured with low-glucose culture media (ST3GAL1, 64.4 ± 5.8 pg/mg; ST3GAL4, 3.8 ± 0.3 pg/mg). CONCLUSIONS: Our data demonstrate distinct changes in the N-glycan profile and an increase in sialylated N-glycans in eyes with PDR.

Comparisons of microRNA expression profiles in vitreous humor between eyes with macular hole and eyes with proliferative diabetic retinopathy.

PURPOSE: MicroRNAs (miRNAs) are small noncoding RNAs which regulate the activities of target mRNAs. We compared the expression profiles of the miRNAs in the vitreous of eyes with macular hole (MH) to that in eyes with proliferative diabetic retinopathy (PDR). METHODS: Vitreous and whole blood samples were collected from four patients with MH and from four patients with PDR. We assayed for 168 miRNAs in the vitreous and serum samples by the microRNA PCR Panel method. RESULTS: The mean number of miRNAs expressed in the vitreous was 63 (55-69) in eyes with MH and 86 (65-117) in eyes with PDR. The mean number of miRNAs expressed in the serum was 162 (159-167) in the MH patients and 142 (115-160) in the PDR patients. Twenty-six miRNAs were expressed in the vitreous of both MH and PDR eyes. Although there was no significant difference in the levels of 20 of the 26 (73 %) miRNAs expressed in both MH and PDR eyes, six of 26 miRNAs (24 %) (hsa-miR-15a, hsa-miR320a, hsa-miR-320b, hsa-miR-93, hsa-miR-29a, and hsa-miR-423-5p) were expressed significantly more highly in PDR eyes. In addition, the mean fold changes of three miRNAs, hsa-miR-23a, hsa-miR-320a, and hsa-miR-320b, in the vitreous to serum were significantly higher in the PDR group than in the MH group. CONCLUSIONS: The expression of several miRNAs related to angiogenesis and fibrosis was expressed significantly higher in the vitreous of eyes with PDR. Further studies are needed to understand the role played by the miRNAs in the biological function of the eye.

Assessment of 193 candidate genes for retinopathy in African Americans with type 1 diabetes.

OBJECTIVE: To report in African Americans with type 1 diabetes the association of single-nucleotide polymorphisms in 193 candidate genes with diabetic retinopathy (DR) and/or its progression. METHODS: A custom panel of 1536 single-nucleotide polymorphisms located on 193 candidate genes for DR was genotyped in 437 African Americans with type 1 diabetes who participated in the New Jersey 725 study. Clinical evaluations at baseline and follow-up examinations included structured clinical interview, ocular examination, 7-field stereoscopic fundus photographs, and blood pressure measurements. Severity of DR was determined via masked grading of fundus photographs. Biological evaluations included blood and urine assays. RESULTS: Single-nucleotide polymorphisms in 13 candidate genes for DR involved in glucose metabolism, angiogenesis, inflammation, neurotransmission, hypertension, and retinal development were significantly associated with the prevalence of severe DR. Three of these genes were also significantly associated with progression of DR. Adjusting for sex, duration of diabetes, glycosylated hemoglobin, systemic hypertension, and total cholesterol did not alter the results. CONCLUSIONS: Our data support the role of genetic factors to account for severity and/or progression of DR in African Americans with type 1 diabetes and to identify several prime genes that likely contribute to the risk of DR.

Retina-specific mRNA in the assessment of diabetic retinopathy.

In a previous study we demonstrated the presence and diagnostic usefulness of circulating rhodopsin mRNA in the assessment of diabetic retinopathy (DR). In the present study we investigated three further retina-specific markers in blood to determine their suitability as markers of DR. The markers were RPE65, retinoschisin, and melanopsin. Whole blood was collected from diabetic patients and healthy controls into PAXgene Blood RNA tubes and RNA was extracted using the PAXgene Blood RNA System. Quantitative real-time PCR was used to quantify mRNA for RPE65, retinoschisin, and melanopsin. beta-actin mRNA was used for normalization. RPE65, retinoschisin, and beta-actin mRNA were detected in 100% of subjects; melanopsin was not detected in either controls or diabetic patients. Circulating RPE65 mRNA concentration was 63% higher in diabetic patients than in healthy individuals (P= 0.019), whereas retinoschisin showed no change between the two groups. Compared with healthy controls, circulating RPE65 mRNA concentration was higher in diabetics with no retinopathy (30%; P= NS), background DR (93%; P= 0.01), preproliferative DR (20%; P= NS), and proliferative DR (107%; P= 0.004). Compared with diabetics with no retinopathy, levels of RPE65 mRNA were also significantly higher (60%) in the presence of proliferative DR (P= 0.029). In contrast, levels of retinoschisin mRNA were lower in background DR (34%; P= 0.033), preproliferative DR (43%; P= 0.026), and proliferative DR (47%; P= 0.038) compared to that in diabetics without retinopathy. We conclude that not all retina-specific mRNA species are detectable in circulation (e.g., melanopsin). This may be related to differences in expression levels for the individual markers. Both RPE65 and retinoschisin were detectable and demonstrated contrasting trends in diabetics with and without retinopathy. In combination with rhodopsin, RPE65, and retinoschisin, mRNA may offer a useful tool in developing a blood test for DR.

Assessment of mitochondrial gene in proliferative vitreoretinal tissues from patients with familial diabetes mellitus.

Fibrovascular tissues obtained at therapeutic vitrectomy from 22 patients with proliferative diabetic retinopathy were studied for a mutation of tRNALEU(UUR) at nucleotide position (np) 3243 of mitochondrial DNA (mtDNA). We found a mutation of mtDNA in the vitreous sample of one patient, a 44-year-old woman who had maternally inherited, familial, non-insulin-dependent diabetes mellitus of 25-year duration with recurrent vitreous hemorrhages due to proliferative retinopathy. Heteroduplex analysis also detected the mutant-type mtDNA in the vitreous sample of this patient. The abnormality was, however, not observed in the peripheral blood sample from either this patient orr her family members with diabetes mellitus. The other 21 patients were negative for the mutation in vitreous specimens as well as in peripheral blood. Although a firm conclusion cannot be drawn from a single case report, our observations suggest that this case was an example of tissue-specific expression of mtDNA mutation. Further studies of a larger number of patients with familial diabetes mellitus seem justified.