Metabolic syndrome and the plasma proteome: from association to causation.

BACKGROUND: The metabolic syndrome (MetS), defined by the simultaneous clustering of cardio-metabolic risk factors, is a significant worldwide public health burden with an estimated 25% prevalence worldwide. The pathogenesis of MetS is not entirely clear and the use of molecular level data could help uncover common pathogenic pathways behind the observed clustering. METHODS: Using a highly multiplexed aptamer-based affinity proteomics platform, we examined associations between plasma proteins and prevalent and incident MetS in the KORA cohort (n = 998) and replicated our results for prevalent MetS in the HUNT3 study (n = 923). We applied logistic regression models adjusted for age, sex, smoking status, and physical activity. We used the bootstrap ranking algorithm of least absolute shrinkage and selection operator (LASSO) to select a predictive model from the incident MetS associated proteins and used area under the curve (AUC) to assess its performance. Finally, we investigated the causal effect of the replicated proteins on MetS using two-sample Mendelian randomization. RESULTS: Prevalent MetS was associated with 116 proteins, of which 53 replicated in HUNT. These included previously reported proteins like leptin, and new proteins like NTR domain-containing protein 2 and endoplasmic reticulum protein 29. Incident MetS was associated with 14 proteins in KORA, of which 13 overlap the prevalent MetS associated proteins with soluble advanced glycosylation end product-specific receptor (sRAGE) being unique to incident MetS. The LASSO selected an eight-protein predictive model with an (AUC = 0.75; 95% CI = 0.71-0.79) in KORA. Mendelian randomization suggested causal effects of three proteins on MetS, namely apolipoprotein E2 (APOE2) (Wald-Ratio = - 0.12, Wald-p = 3.63e-13), apolipoprotein B (APOB) (Wald-Ratio = - 0.09, Wald-p = 2.54e-04) and proto-oncogene tyrosine-protein kinase receptor (RET) (Wald-Ratio = 0.10, Wald-p = 5.40e-04). CONCLUSIONS: Our findings offer new insights into the plasma proteome underlying MetS and identify new protein associations. We reveal possible casual effects of APOE2, APOB and RET on MetS. Our results highlight protein candidates that could potentially serve as targets for prevention and therapy.

Macrocalcitonin Is a Novel Pitfall in the Routine of Serum Calcitonin Immunoassay.

CONTEXT: Calcitonin (CT) is a sensitive marker of medullary thyroid carcinoma (MTC) and is used for primary diagnosis and follow-up after thyroidectomy. However, persistently elevated CT is observed even after complete surgical removal without evidence of a recurrent or persistent tumor. OBJECTIVE: To investigate the presence of assay interference in the serum CT of MTC patients who are apparently without a structural disease. PATIENTS AND METHODS: We studied three index MTC cases for CT assay interference and 14 patients with metastatic MTC. The CT level was measured using an immunofluorometric assay. Screening for assay interference was performed by determination of CT levels before and after serum treatment with polyethylene glycol. Additionally, samples were analyzed by chromatography on ultra-performance liquid chromatography and protein A-Sepharose. RESULTS: Patients with biochemical and structural disease showed CT mean recovery of 84.1% after polyethylene glycol treatment, whereas patients suspected of interference showed recovery from 2-7%. The elution profile on UPLC showed that the immunometric CT from these three patients behaved like a high molecular mass aggregate (>300 kDa). Additionally, when these samples were applied to the protein A-Sepharose, CT immunoreactivity was retained on the column and was only released after lowering the pH. CONCLUSIONS: For the first time, our results show the presence of a novel pitfall in the CT immunoassay: "macrocalcitonin." Its etiology, frequency, and meaning remain to be defined, but its recognition is of interest and can help clinicians avoid unnecessary diagnostic investigations and treatment during the follow-up of MTC.

Skewed mutational spectrum of RET proto-oncogene Exon10 in Iranian patients with medullary thyroid carcinoma.

Thyroid cancer is the most common endocrine malignant tumor. Medullary thyroid carcinoma (MTC) is an aggressive tumor arising from calcitonin-producing parafollicular cells. MTC has autosomal dominant inheritance and accounts for 5-10 % of all thyroid cancers. It occurs in hereditary (25 %, hMTC) and sporadic (75 %, sMTC) forms. Gain-of-function mutations in the REarranged during transfection (RET) proto-oncogene have been identified in 98 % of hMTC and 50 % of sMTC. The aim of this investigation was to identify mutation(s) in the much conserved RET exon10 in Iranian MTC patients. We started screening patients with MTC for RET in 2001. This study included 347 individuals (154 with sMTC, 38 with FMTC, 8 with multiple endocrine neoplasia type 2A [MEN2A], 3 with MEN2B, and 3 with pheochromocytoma; 207 index cases and 140 relatives). Germline mutation screening of RET exon10 was performed with PCR-DNA sequencing. A total of 14 missense mutations (10 mutations in men and 4 in women) were identified in cysteine codons 611, 618, and 620 (exon10) in 11 patients and three first-degree relatives as follows: four C611Y (three with FMTC and one relative), one C618R (FMTC), one C618S (sMTC), one C620G (sMTC), four C620R (one with FMTC and three with sMTC), and three C620F (one with FMTC and two relatives). In the present study, six different mutations were identified in exon10 of RET in 14 patients with sMTC and FMTC that were restricted to codons 611, 618, and 620, but not in codon 609. This data showed a skewed pattern of RET exon10 mutation compared to other populations. No mutation was found for MEN2A, MEN2B, and pheochromocytoma in exon10 in this population. In the most common mutations in exon10, the FMTC and sMTC patients were C611Y and C620R, respectively.

Allele-specific expression at the RET locus in blood and gut tissue of individuals carrying risk alleles for Hirschsprung disease.

RET common variants are associated with Hirschsprung disease (HSCR; colon aganglionosis), a congenital defect of the enteric nervous system. We analyzed a well-known HSCR-associated RET haplotype that encompasses linked alleles in coding and noncoding/regulatory sequences. This risk haplotype correlates with reduced level of RET expression when compared with the wild-type counterpart. As allele-specific expression (ASE) contributes to phenotypic variability in health and disease, we investigated whether RET ASE could contribute to the overall reduction of RET mRNA detected in carriers. We tested heterozygous neuroblastoma cell lines, ganglionic gut tissues (18 HSCR and 14 non-HSCR individuals) and peripheral blood mononuclear cells (PBMCs; 16 HSCR and 14 non-HSCR individuals). Analysis of the data generated by SNaPshot and Pyrosequencing revealed that the RET risk haplotype is significantly more expressed in gut than in PBMCs (P = 0.0045). No ASE difference was detected between patients and controls, irrespective of the sample type. Comparison of total RET expression levels between gut samples with and without ASE, correlated reduced RET expression with preferential transcription from the RET risk haplotype. Nonrandom RET ASE occurs in ganglionic gut regardless of the disease status. RET ASE should not be excluded as a disease mechanism acting during development.

Papillary thyroid cancer and polymorphic variants in TSHR- and RET-related genes: a nested case-control study within a cohort of U.S. radiologic technologists.

Several variants in the TSHR and RET signaling pathways genes have been reported to be related to cancer risk. We hypothesized that polymorphic variants in these genes are associated with the risk of papillary thyroid cancer. A nested case-control study was conducted within the U.S. Radiologic Technologists cohort. Eligible validated papillary thyroid cancer cases (n = 167) and frequency-matched (by sex and birth year) controls (n = 491) donated blood for analysis. There were no statistically significant associations between papillary thyroid cancer and 10 selected polymorphic variants in analyses of men and women combined. A borderline significant increasing risk was found for RET G691S (P(trend) = 0.05) and was especially pronounced among young women. For women under 38 years (the median age at diagnosis), the odds ratios were 2.1 (95% confidence interval, 1.2-3.7) for those heterozygous for the RET G691S polymorphism and 3.7 (95% confidence interval, 1.1-11.8) for those who were homozygous (P(trend) = 0.001). Our data provide limited evidence that TSHR- and RET-related genes are related to papillary thyroid cancer risk.