Osteoporosis is a multifactorial disease influenced by numerous environmental and genetic factors. On the basis of bone physiology, candidate genes for the development of osteoporosis are being identified. In bone remodelling, Wnt signalling pathway has an important role, influencing osteoblastogenesis and osteoclastogenesis. The key proteins in Wnt pathway are LRP5, LRP6, SOST, β-catenin and Dickkopf-1. The RANKL/RANK/OPG system plays a crucial role in osteoclast biology. The genes, that code for key proteins in the Wnt signalling pathway and in the RANKL/RANK/OPG system, could also be candidate genes for the development of osteoporosis. In the thesis, these genes were studied and functionally evaluated for their influence on the development of osteoporsis in pre- and postmenopausal women as well as in elderly men.
In postmenopausal women, we first evaluated the role of the polymorphisms BsmI, FokI and Cdx2 in VDR, A1330V in LRP5, I1062V in LRP6, -1397_-1396insGGA in SOST and K3N in OPG, that have already been studied in osteoporosis. In healthy postmenopausal women, a significant association was found for A1330V in LRP5 and bone mineral density in total hip (BMD-th) and lumbar spine (BMD-ls) (p=0.010 and 0.033). In osteoporotic postmenopausal women, a significant association was established for BMD-ls with BsmI and Cdx2 in the VDR gene (p=0.015 and p=0.047), as well as with K3N in the OPG gene (p=0.021). Other polymorphisms were not associated with BMD. None of the studied polymorphisms were associated with biochemical markers of bone turnover. The mutations E232K in the DKK1 gene, and D32Y, G34V and N287S in the CTNNB1 gene were not polymorphic and therefore not associated with BMD and biochemical markers of bone turnover.
Secondly, we searched for new polymorphisms in the promoter of the TNFSF11 gene, which codes for the key protein of osteoclastogenesis RANKL and could influence its expression. Using sequencing method, the polymorphisms -290C>T, -643C>T, -693G>C and -1594G>A were identified and associated with BMD. In 115 postmenopausal women, polymorphism -290C>T showed significant association with femoral neck BMD (BMDfn) (p=0.022). In 43 non-osteoporotic postmenopausal women, the polymorphism - 693G>C was associated with the statistically significant loss in BMD-th and BMD-fn after 1 year (p=0.011 and p=0.037). No other association of the identified polymorphisms with BMD have been established. In the next step, the identified polymorphisms in the TNFSF11 gene promoter were involved in the haplotype and functional analysis, and further evaluated in a larger group of 404 postmenopausal women. Using haplotype analysis, two most common haplotypes were inferred: CCG and TTC (occuring in 44.3 % and 49.3 % of women, respectively). Amplified fragments of the TNFSF11 gene promoter, containing CCG or TTC haplotype, were cloned into pGL3-basic reporter plasmid, which was co-transfected with pRL-TK plasmid into HEK293 cells. Dual Luciferase Reporter Assay was performed. Reporter gene analysis showed significantly higher luciferase activity in CCG than in TTC haplotype (p=0.018). In osteoporotic postmenopausal women, both showed association with BMD-ls (p=0.005 and 0.007 for TTC and CCG, respectively), whereas in femoral neck there was no association with BMD. Association with BMD-ls was established in -290C>T, - 643C>T and -693G>C (p values: 0.001, 0.041 and 0.013). Association with BMD-fn was shown in -693G>C (p=0.049). No association was found between genotype subgroups of TNFSF11 gene promoter polymorphisms and biochemical markers of bone turnover. In postmenopausal women, the combined influence of TNFSF11, VDR and OPG genes, as well as LRP5, LRP6 and SOST genes on BMD was evaluated. The combination of
polymorphisms -290C>T in the TNFSF11 gene and K3N in the OPG gene have a significant influence on BMD-th (p=0,041). In non-osteoporotic women, the combination K3N(OPG)-Cdx2(VDR) was associated with BMD-ls, BMD-th and BMD-fn, and the combination -290C>T(TNFSF11)-K3N(OPG) with BMD-fn. In the premenopause, only K3N polymorphism was associated with BMD in the hip area, whereas in the TNFSF11, VDR, LRP5, LRP6 and SOST gene polymorphisms no association with BMD was found. It is assumed, that genetic factors influence the rate of
BMD changes after 35 years of age. Therefore, the BMD changes were studied after 1 and 3 years. The data show, that the BMD changes after 1 year are lower than the least significant change and after 3 years, significant association with BMD was only found in K3N polymorphism. In premenopausal women, no association was found between genotype subgroups and biochemical markers of bone turnover. To study phamacogenetics of osteoporosis, the impact of polymorphisms in the TNFSF11, VDR, LRP5, LRP6, SOST and OPG genes on the efficacy of treatment was studied in women, treated with alendronate, and in those, treated with raloxifene. After one year of treatment, neither for alendronate nor for raloxifene a significant influence of genotype on the changes in BMD or biochemical markers has been shown. In elderly men, the following associations were shown: in A1330V in the LRP5 gene with BMD-fn (p=0.007), for -1397_-1396insGGA in the SOST gene with BMD-k and BMD-ls (p=0.022 and p=0.046), and for FokI in the VDR gene with BMD-fn and BMD-ls (p=0.040 and p=0.036).
We have shown that different genetic factors influence BMD in respect of age and gender. The next step in the clarification of the biological function is the functional evaluation of the identified polymorphisms in the TNFSF11 gene promoter that could play a role in the regulation of BMD. In the pharmacogenetics study of osteoporosis, no influence of the genes under study on the efficacy of alendronate or raloxifene was shown. In the future, farmacogenetics of osteoporosis will focus on other genes, involved in the development of osteoporosis or pharmacokinetics of the drug.
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