PMC:5722821 / 19067-24947 JSONTXT

{"project":"2_test","denotations":[{"id":"29222432-22402134-139351637","span":{"begin":2646,"end":2648},"obj":"22402134"},{"id":"29222432-24418399-139351638","span":{"begin":2649,"end":2651},"obj":"24418399"},{"id":"29222432-22964273-139351639","span":{"begin":2652,"end":2654},"obj":"22964273"},{"id":"29222432-26770981-139351640","span":{"begin":2655,"end":2657},"obj":"26770981"},{"id":"29222432-25687897-139351641","span":{"begin":5029,"end":5031},"obj":"25687897"},{"id":"29222432-18323559-139351642","span":{"begin":5032,"end":5034},"obj":"18323559"},{"id":"29222432-18323559-139351643","span":{"begin":5403,"end":5405},"obj":"18323559"}],"text":"Methods\nThe ethics committee of the First Affiliated Hospital of Xiamen University reviewed and approved the study protocol. In addition, we can confirm that all methods were performed in accordance with the relevant guidelines and regulations.\n\nStudy Patients\nThe present study was carried out from May 2014 to March 2017 in a total of 2199 patients diagnosed with primary hypertension (1422 males and 777 females) aged 31 to 86 years. All patients were of Han Chinese origin, and they were diagnosed and recruited from the First Affiliated Hospital of Xiamen University, Xiamen city, Fujian province, China. Of them, 1252 patients were comorbid with T2DM as the ‘diabetic cases’ and 947 patients were confirmed to have no clinical indication of T2DM as the ‘nondiabetic controls’, who had no personal or family history of diabetes mellitus among first-degree relatives. All patients read and signed informed consent files for the collection and subsequent use of their blood samples.\n\nClinical Diagnosis\nBP was measured twice by trained physicians or nurses when subjects had rested for at least 5 minutes. Primary hypertension is diagnosed with an average systolic BP reading of \u003e140 mmHg or an average diastolic BP reading of \u003e90 mmHg or the use of antihypertensive medications, and importantly secondary causes of hypertension are excluded after an extensive biochemical and clinical examination. Meanwhile, fasting serum glucose was assayed in all primary hypertensive patients, and some of them had received oral glucose tolerance test (OGTT). T2DM is diagnosed if fasting serum glucose ≥7.0 mmol/L or 2-hour OGTT glucose ≥11.1 mmol/L.\n\nLaboratory Assay\nBefore clinical examination, all primary hypertensive patients were invited to answer a structured questionnaire about their medical histories and lifestyles. Meanwhile, body weight and height were measured on site and taken to calculate BMI. Afterwards, a 5-mL venous blood sample was drawn from all patients for further biomedical examination and genetic analysis.\nBesides fasting serum glucose, other biomedical markers including triglyceride, total cholesterol, HDLC, low-density lipoprotein cholesterol (LDLC), apolipoprotein-A, apolipoprotein-B, BUN and creatinine were assayed subsequently at the Laboratory Medicine Centre of The First Affiliated Hospital of Xiamen University.\n\nGenotyping Assay\nSix genomic variants were selected from the RAGE gene based on current literature, including −429T \u003e C (promoter), −374T \u003e A (promoter), the −407th to −345th fragment insertion/deletion (I/D) (promoter), 82Gly \u003e Ser (exon 3), 1704G \u003e T (intron 7) and 2184 A \u003e G (intron 8) variants15,23,33,34. Genomic DNA was prepared from 200 μl of peripheral blood lymphocytes for all samples by aid of the QIAamp DNA Mini kits (Qiagen) at the university laboratory. Based on a case-control study design, genomic DNA samples of 1252 diabetic cases and 947 nondiabetic controls were subjected to the TaqMan genotyping assay (Applied Biosystems, Inc, Foster City, CA, USA). Experimental protocols are available upon request.\n\nStatistical Analyses\nAll baseline data were typed into a computerized database and were independently cross-checked for any data entry mistakes. Comparisons of continuous data between two groups were done by the Student’s t-tests, and that of quantitative data were done by the Person χ2 contingency tables. Each of the RAGE gene 6 variants was tested to see whether the observed genotype frequencies were concordant with the expected Hardy-Weinberg proportions by the Person χ2 or the Fisher’s exact contingency tables. Genotype and allele frequencies of each variant were calculated, and their differences between cases and controls were determined by the Person χ2 contingency tables. Adjusted ORs with 95% CIs were derived from the multivariate Logistic regression analysis to quantify the association magnitude between the RAGE gene and T2DM risk in primary hypertensive patients. Moreover, the frequencies of haplotypes based on 4 significant variants were estimated by using the program Haplo.Stats, version 1.7.7 developed by Sinnwell and Schaid (http://www.mayo.edu/research/labs/statistical-genetics-genetic-epidemiology/software). The omnibus association of derived haplotypes as a whole with each baseline characteristic was also examined by the program Haplo.Stats.\nThe ROC curves were used to assess the relative predictive capability of RAGE genomic variants beyond that of available baseline risk factors. Areas under the ROC curves were compared accordingly. Meanwhile, the Hosmer-Lemeshow test was used to test the goodness-of-fit for risk prediction models.\nFinally, a predictive nomogram was structured based on the significant attributes from the Forward Logistic regression analysis of available baseline risk factors and 6 genomic variants in the RAGE gene. The nomogram can create a simple graphical representation of a statistical predictive model that generates a single numerical estimate of the probability of a clinical event35,36. A nomogram’s predictive accuracy is measured through a concordance index (c-index), which can quantify the level of concordance between predicted probabilities and the actual chance of having the event of interest. Specifically, the c-index denotes the proportion of pairs, with the responders having a higher predicted probability of response than the non-responders36.\nStatistical analyses were performed with the use of Stata software, version 14.1 (http://www.stata.com) and R language, version 3.3.3 (https://www.r-project.org). All reported P values were based on 2-sided tests. Unless otherwise stated, P value of less than 0.05 was reported to be statistically significant.\n\nAvailability of data and materials\nThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. "}