Franziska Weber, Sonja Forss-Petter, Johannes Berger, Willi Weber
Center for Brain Research, Medical University of Vienna, Austria
Objectives: X-linked Adrenoleukodystrophy (X-ALD) is a fatal neurodegenerative disorder with inflammatory demyelination of the brain caused by mutations in the ABCD1 gene, a member of the peroxisomal ABCD transporter family. Currently, the only curative therapies are transplantations of either allogeneic hematopoietic cells or genetically corrected autologous CD34+ stem cells, implicating the importance of CD34+ stem cell-derived immune cells in the arrest of brain inflammation. We compared mRNA levels of peroxisomal ABCD transporters between these immune cells of 5 X-ALD patients and 5 controls.
Methods: Immune cells from blood samples were isolated by magnetic activated cell sorting. Quantitative PCR was used to determine mRNA levels of the ABCD transporters. RNA was reverse transcribed into cDNA. A DNA-binding dye, SYBRgreen, intercalates into double-stranded DNA, emitting fluorescence. The fluorescence intensity increases proportionally to the cDNA copies produced during each PCR cycle. The slope of the fluorescence curve mirrors the growth rate of the cDNA copies. The proportionality factor is determined by measuring DNA standards of known copy numbers, allowing cDNA concentrations to be quantified [1]. A linear mixed effect (lme) model was used with the mRNA copies as dependent variable and two predictor variables, a gene factor (HPRT, ABCD1/2/3) and a population factor (X-ALD, control). Using the lme function from the nlme R-package [2] with the treatment option, we obtained mean values of ABCD1/2/3 mRNA normalized to the housekeeping gene HPRT of the control population and their difference to the X-ALD population.
Results: The three ABCD transporters were differentially expressed in CD34+ derived cells of controls: ABCD1 and ABCD2 mRNA were inversely expressed in all cell types, whereas ABCD3 was equally distributed. Monocytes show high levels of ABCD1, the ABCD2 mRNA is barely detectable. In contrast, T cells express high levels of ABCD2 and moderate levels of ABCD1. ABCD2, closest homolog of ABCD1 could be expected to compensate for the ABCD1 deficiency in X-ALD. However, no differences were found in the expression pattern of ABCD2 in X-ALD.
Conclusions: We propose that the beneficial effect of the transplantations may rely on the replacement of those cells lacking sufficient ABCD2 expression in ABCD1 deficiency. Mixed effect modelling was an effective tool to compare mRNA expression between different cells of a target and a control population.
References:
[1] MW Pfaffl (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29(9)
[2] R Core Team (2013) R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. ISBN 3-900051-07-0. http://www.R-project.org/
Reference: PAGE 22 (2013) Abstr 2881 [www.page-meeting.org/?abstract=2881]
Poster: Other Drug/Disease Modelling