DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS

The DR3 «death receptor» plays an important role in the initiation of apoptosis, proliferation, or inflammation. This receptor is shown to be involved in various diseases, including infectious conditions. Different variants of mRNA DR3 are formed as a result of alternative splicing. These variant transcripts encode membrane and soluble forms of the receptor which have different functions. Features of their expression and contribution of individual DR3 variants to the immune pathogenesis of infectious mononucleosis (IM) are poorely understood.

The purpose of this work was to develop, validate and test the techniques of DR3 gene expression assays, as well as to evaluate the DR3 mRNA splice variants by means of real-time RT-PCR and RT-PCR in the IM patients.

The original version of real-time RT-PCR allowed to determine relative amounts of DR3 mRNA, DR3 membrane variants (LARD1a + LARD8), and ratios of mRNAs encoding membrane and soluble forms of the receptor. The technique proved to be specific and sensitive (a semi-quantitative detection limit = 34-35 cycles) when tested in healthy volunteers and patients with acute infectious mononucleosis (AIM). Lower expression levels were shown for two alternative membrane variants of DR3 mRNA (LARD1b and DR3beta) thus regarding these isoforms as minor fractions. The relative levels of total DR3 mRNA expression were decreased in patients with AIM, as compared to healthy volunteers, whereas mRNA expression of membrane receptor variants did not differ between IM and controls.

To determine a qualitative contribution of either LARD1a and LARD8 variants into the expression of membrane forms of DR3, a two-step «nested» version of RT-PCR has been developed. It was shown that, in majority of control and IM samples, both main LARD1a, and alternative LARD8 membrane forms are contributing to mRNA expression of membrane DR3 variants.

The presented methods for evaluation of expression and occurrence of DR3 mRNA variants allow to characterize changes in the expression and function of this receptor associated with infectious mononucleosis. Further investigations are required to test the technique in larger number of samples.

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