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The expression of magnesium sensitive genes as a function of the extracellular magnesium concentration was investigated in a cell culture experiment. At low magnesium condition, cells of JVM-13 and Jurkat lines exhibited high expression of SLC41A1, CNNM2, TRPM7, NIPA1 and N33 genes. It was hypothesized, that with knowledge of the expression rate of these genes, one gains insight about the intracellular magnesium status.
The capability of the CNNM2 gene as a biomarker was tested in a screening of patients diagnosed with diabetes mellitus type II. The expression levels of the gene were compared to the corresponding [Mg]plasma and [Mg2+]intra concentration of leukocytes. Although the [Mg]plasma concentrations of 114 from 115 patients were within normal range, about a quarter of all patients showed a low [Mg2+]intra concentration.
However, an intracellular magnesium deficiency correlated with an increased expression of the CNNM2 gene. The results of the screening showed that a magnesium deficiency of the body is more reliably detected by the relative expression of the gene CNNM2 than by the [Mg]plasma concentration. An evaluation of the [Mg]plasma concentration failed to detect 96% of the patients determined to be deficient by intra-cellular measurement whereas determination by gene CNNM2 expression missed only 14%.
The daily supplementation of 300 mg magnesium over the course of one month normalized both the gene CNNM2 expression and the [Mg2+]intra concentration. The average [Mg2+]intra concentration increased significantly from 0.43 mmol/l to 0.61 mmol/l while the mean of the [Mg]plasma value remained within the normal range.
Determination of the expression of the magnesium sensitive gene CNNM2 enables the identification of patients with an intracellular deficiency and might respond to a purposeful magnesium supplementation. Therefore the CNNM2 gene is a promising biomarker of the magnesium status, especially in diabetes mellitus type II. To optimize the assay for extensive future studies in this field of research, the experiments were reviewed and the design improved according to the MIQE guidelines.
The ideal housekeeping genes for an expression analysis in JVM-13 and Jurkat cells as well in leukocytes were determined using the analysis software „geNorm“.
- Expression rate analysis in JVM-13 cells was best normalized by YWHAZ.
- Expression rate analysis in Jurkat cells was best normalized by TUBA1B.
- Expression rate analysis in leukocytes was best normalized by ACTB.
- B2M was equally suitable as HKG for JVM-13 and Jurkat cells, as well as for human leukocytes.
A comparison of the normalizations by B2M, ACTB and TUBA1B showed no or very small deviations of the GED values of 20 samples. The housekeeping gene TUBA1B used for normalization was a stably expressed gene in JVM-13 and Jurkat cells and human leukocytes. Therefore the calculations of the expression data in this thesis were based on a reliable normalization.