| 1. |
- Davids, Mariska, et al.
(författare)
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Simultaneous determination of asymmetric and symmetric dimethylarginine, L-monomethylarginine, L-arginine, and L-homoarginine in biological samples using stable isotope dilution liquid chromatography tandem mass spectrometry
- 2012
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Ingår i: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 900, s. 38-47
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Tidskriftsartikel (refereegranskat)abstract
- Production of the endogenous vasodilator nitric oxide (NO) from L-arginine by NO synthase is modulated by L-homoarginine, L-monomethylargine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Here we report on a stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of these metabolites in plasma, cells and tissues. After addition of the internal standards (D-7-ADMA, D-4-L-homoarginine and C-13(6)-Larginine), analytes were extracted from the samples using Waters Oasis MCX solid phase extraction cartridges. Butylated analytes were separated isocratically on a Waters XTerra MS C18 column (3.5 mu m. 3.9 mm x 100 mm) using 600 mg/L ammonium formate in water - acetonitrile (95.5:4.5, v/v) containing 0.1 vol% formic acid, and subsequently measured on an AB Sciex API 3000 triple quadrupole mass spectrometer. Multiple reaction monitoring in positive mode was used for analyte quantification. Validation was performed in plasma. Calibration lines were linear (r(2) >= 0.9979) and lower limits of quantification in plasma were 0.4 nM for ADMA and SDMA and 0.8 nM for the other analytes. Accuracy (% bias) was <3% except for MMA (<7%), intra-assay precision (expressed as CV) was <3.5%, inter-assay precision <9.6%, and recovery 92.9-103.2% for all analytes. The method showed good correlation (r(2) >= 0.9125) with our previously validated HPLC-fluorescence method for measurement in plasma, and was implemented with good performance for measurement of tissue samples. Application of the method revealed the remarkably fast (i.e. within 60 min) appearance in plasma of stable isotope-labeled ADMA, SDMA, and MMA during infusion of D-3-methyl-1-C-13-methionine in healthy volunteers.
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| 2. |
- Fresard, Laure, et al.
(författare)
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Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts
- 2019
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Ingår i: Nature Medicine. - : NATURE PUBLISHING GROUP. - 1078-8956 .- 1546-170X. ; 25:6, s. 911-919
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Tidskriftsartikel (refereegranskat)abstract
- It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene(1). The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches(2-5). For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases(6-8). This includes muscle biopsies from patients with undiagnosed rare muscle disorders(6,9), and cultured fibroblasts from patients with mitochondrial disorders(7). However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution.
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