Study 1: Antibodies to post-translationally modified insulin in LADA. Hypothesis/Aims: The general hypothesis is that oxPTM-Ins reactivity could be higher than the reactivity of the native insulin in LADA patients. It was also evaluated whether oxPTM-Ins antibodies can be used as biomarker to differentiate LADA from T2D. Methods: This study included 37 patients with LADA, 33 patients with T2D and 19 healthy controls (HC). OxPTM insulin was generated using ribose, CuCl2, H2O2 and HOCl. Autoreactivity to oxPTM-Ins was detected by ELISA using sera from study participants. Results: Reactivity to .OH-Ins was significantly higher than NT-Ins in LADA (NT-Ins median ± SE absorbance 0.050 ± 0.03; .OH-Ins median ± SE absorbance 0.122 ± 0.02) (p = 0.01). ELISA data showed that antibody binding to oxPTM-Ins was significantly higher in LADA compared to HC (.OH-Ins median ± SE absorbance in LADA vs. HC: 0.12 ± 0.02 vs. 0.046 ± 0.009, p = 0.02; Gly-Ins: 0.054 ± 0.017 vs. 0.002 ± 0.008, p <0.0001). Reactivity to NT-Ins and oxPTM-Ins was similar between LADA and T2D (NT-Ins median ± SE absorbance in LADA 0.05 ± 0.03; NT-Ins median ± SE absorbance in T2D 0.04 ±0.028) (p=0.88). Antibody binding to Gly-Ins was also not different between LADA and T2D (Gly-Ins in LADA 0.054±0.017; Gly-Ins in T2D 0.07±0.019) (p=0.78). .OH-Ins antibodies were not different in LADA and T2D (.OH-Ins in LADA 0.122±0.023; .OH-Ins in T2D 0.095±0.021) (p=0.64). HOCl-Ins binding was also similar in LADA and T2D (HOCl-Ins in LADA 0.093±0.020; HOCl-Ins in T2D 0.05±0.017) (p=0.53). Conclusions: oxPTM-Ins antibodies are not prevalent in LADA compared to T2D. Study 2: Post-translationally modified collagen type I antibodies in Osteoporosis and Diabetes. Hypothesis/Aims: The objective of this study was to test whether auto-reactivity to oxPTM of collagen type I (oxPTM-CI) is involved in osteoporosis in patients with T2D. Methods: This study included 12 patients with osteoporosis (OP) without diabetes, 12 patients with osteoporosis and T2D (OP + T2D) and 13 healthy controls (C). OxPTM-CI was generated using ribose, CuCl2, H2O2 and peroxynitrite (ONOO-). Reactivity to oxPTM-CI was observed by ELISA using sera from study participants. Statistical analysis revealed no significant differences for the .OH-CI antibody levels in OP, OP +T2D and C (.OH-CI median ± SE absorbance in OP 0.084 ± 0.032 vs. OP + T2D 0.097 ± 0.062 p = 0.46; vs. C 0.114 ± 0.026 p = 0.54.). ONOO-CI antibody binding was the lowest detected by the assay for all groups. Moreover, no differences among the three groups were observed (ONOO-CI in OP 0.044 ± 0.009 vs. OP + T2D 0.058 ± 0.033 p = 0.16; vs. C 0.065 ± 0.033p = 0.21). Conclusions: In conclusion, no reactivity against ox-PTM-CI was observed in osteoporosis, regardless the presence of type 2 diabetes. Study 3: Establishing a genetic basis for parathyroid hormone secretion by studies of patients with normocalcaemic forms of hypoparathyroidism and hyperparathyroidism. Hypothesis/Aims: The objective of this study was to investigate if alterations of the CASR, PTH and vitamin D metabolism genes may be responsible for hypoparathyroidism and hyperparathyroidism. Methods: Five groups of subjects (Euparathyroid forms of hypocalcaemia, n = 13; Euparathyroid forms of hypercalcaemia, n = 11; Normocalcaemic forms of hypoparathyroidism, n = 16; Normocalcaemic forms of hyperparathyroidism, n = 10; Healthy control subjects with normal calcaemia and PTH, n = 58) were selected on basis of serum albumin-adjusted calcium and PTH values for DNA sequence analysis of CASR, PTH and Vit D metabolism genes. DNA sequence analyses of the coding-region and exon-intron boundaries of CASR, PTH, GC and CYP24A1 genes were performed. All SNPs were processed by Sanger sequencing and an automated detection system. Departure from the Hardy-Weinberg equilibrium was determined by Chi-squared analysis (X2). Results: CASR rs1801725 (A986S) was more frequent in the low calcium group compared to the control group (p <0.01). Higher frequency of rs1801726 (Q1011E) in the low calcium group compared to controls was observed (p <0.001). CASR Exon 7 SNP rs1801726 was also observed more frequently in hypoparathyroid subjects compared to controls (p <0.0001). CASR rs4678174 SNP genotype frequencies were higher in hypoparathyroid subjects compared to controls (low PTH vs controls p <0.0001). Conclusions: No SNPs were detected in the causative relation between normal calcium levels and hyperparathyroidism or hypoparathyroidism; however, this study confirmed the involvement of the CASR SNPs rs1801725 and rs1801726 in the condition of hypocalcaemia.

Post-translational modifications of proteins and genetic polymorphisms associated to metabolic diseases / Valentina Greto , 2017 Sep 20. 29. ciclo

Post-translational modifications of proteins and genetic polymorphisms associated to metabolic diseases

2017-09-20

Abstract

Study 1: Antibodies to post-translationally modified insulin in LADA. Hypothesis/Aims: The general hypothesis is that oxPTM-Ins reactivity could be higher than the reactivity of the native insulin in LADA patients. It was also evaluated whether oxPTM-Ins antibodies can be used as biomarker to differentiate LADA from T2D. Methods: This study included 37 patients with LADA, 33 patients with T2D and 19 healthy controls (HC). OxPTM insulin was generated using ribose, CuCl2, H2O2 and HOCl. Autoreactivity to oxPTM-Ins was detected by ELISA using sera from study participants. Results: Reactivity to .OH-Ins was significantly higher than NT-Ins in LADA (NT-Ins median ± SE absorbance 0.050 ± 0.03; .OH-Ins median ± SE absorbance 0.122 ± 0.02) (p = 0.01). ELISA data showed that antibody binding to oxPTM-Ins was significantly higher in LADA compared to HC (.OH-Ins median ± SE absorbance in LADA vs. HC: 0.12 ± 0.02 vs. 0.046 ± 0.009, p = 0.02; Gly-Ins: 0.054 ± 0.017 vs. 0.002 ± 0.008, p <0.0001). Reactivity to NT-Ins and oxPTM-Ins was similar between LADA and T2D (NT-Ins median ± SE absorbance in LADA 0.05 ± 0.03; NT-Ins median ± SE absorbance in T2D 0.04 ±0.028) (p=0.88). Antibody binding to Gly-Ins was also not different between LADA and T2D (Gly-Ins in LADA 0.054±0.017; Gly-Ins in T2D 0.07±0.019) (p=0.78). .OH-Ins antibodies were not different in LADA and T2D (.OH-Ins in LADA 0.122±0.023; .OH-Ins in T2D 0.095±0.021) (p=0.64). HOCl-Ins binding was also similar in LADA and T2D (HOCl-Ins in LADA 0.093±0.020; HOCl-Ins in T2D 0.05±0.017) (p=0.53). Conclusions: oxPTM-Ins antibodies are not prevalent in LADA compared to T2D. Study 2: Post-translationally modified collagen type I antibodies in Osteoporosis and Diabetes. Hypothesis/Aims: The objective of this study was to test whether auto-reactivity to oxPTM of collagen type I (oxPTM-CI) is involved in osteoporosis in patients with T2D. Methods: This study included 12 patients with osteoporosis (OP) without diabetes, 12 patients with osteoporosis and T2D (OP + T2D) and 13 healthy controls (C). OxPTM-CI was generated using ribose, CuCl2, H2O2 and peroxynitrite (ONOO-). Reactivity to oxPTM-CI was observed by ELISA using sera from study participants. Statistical analysis revealed no significant differences for the .OH-CI antibody levels in OP, OP +T2D and C (.OH-CI median ± SE absorbance in OP 0.084 ± 0.032 vs. OP + T2D 0.097 ± 0.062 p = 0.46; vs. C 0.114 ± 0.026 p = 0.54.). ONOO-CI antibody binding was the lowest detected by the assay for all groups. Moreover, no differences among the three groups were observed (ONOO-CI in OP 0.044 ± 0.009 vs. OP + T2D 0.058 ± 0.033 p = 0.16; vs. C 0.065 ± 0.033p = 0.21). Conclusions: In conclusion, no reactivity against ox-PTM-CI was observed in osteoporosis, regardless the presence of type 2 diabetes. Study 3: Establishing a genetic basis for parathyroid hormone secretion by studies of patients with normocalcaemic forms of hypoparathyroidism and hyperparathyroidism. Hypothesis/Aims: The objective of this study was to investigate if alterations of the CASR, PTH and vitamin D metabolism genes may be responsible for hypoparathyroidism and hyperparathyroidism. Methods: Five groups of subjects (Euparathyroid forms of hypocalcaemia, n = 13; Euparathyroid forms of hypercalcaemia, n = 11; Normocalcaemic forms of hypoparathyroidism, n = 16; Normocalcaemic forms of hyperparathyroidism, n = 10; Healthy control subjects with normal calcaemia and PTH, n = 58) were selected on basis of serum albumin-adjusted calcium and PTH values for DNA sequence analysis of CASR, PTH and Vit D metabolism genes. DNA sequence analyses of the coding-region and exon-intron boundaries of CASR, PTH, GC and CYP24A1 genes were performed. All SNPs were processed by Sanger sequencing and an automated detection system. Departure from the Hardy-Weinberg equilibrium was determined by Chi-squared analysis (X2). Results: CASR rs1801725 (A986S) was more frequent in the low calcium group compared to the control group (p <0.01). Higher frequency of rs1801726 (Q1011E) in the low calcium group compared to controls was observed (p <0.001). CASR Exon 7 SNP rs1801726 was also observed more frequently in hypoparathyroid subjects compared to controls (p <0.0001). CASR rs4678174 SNP genotype frequencies were higher in hypoparathyroid subjects compared to controls (low PTH vs controls p <0.0001). Conclusions: No SNPs were detected in the causative relation between normal calcium levels and hyperparathyroidism or hypoparathyroidism; however, this study confirmed the involvement of the CASR SNPs rs1801725 and rs1801726 in the condition of hypocalcaemia.
20-set-2017
ox-PTM; diabetes; SNPs; normocalcaemic hyperparathyroidism
Post-translational modifications of proteins and genetic polymorphisms associated to metabolic diseases / Valentina Greto , 2017 Sep 20. 29. ciclo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12610/68849
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