A Haplotype-Based Analysis of the LRP5 Gene in Relation to Osteoporosis Phenotypes in Spanish Postmenopausal Women

Abstract

LRP5 encodes the low-density lipoprotein receptor-related protein 5, a transmembrane protein involved in Wnt signaling. LRP5 is an important regulator of osteoblast growth and differentiation, affecting bone mass in vertebrates. Whether common variations in LRP5 are associated with normal BMD variation or osteoporotic phenotypes is of great relevance. We used a haplotype-based approach to search for common disease-associated variants in LRP5 in a cohort of 964 Spanish postmenopausal women. Twenty-four SNPs were selected, covering the LRP5 region, including the missense changes p.V667M and p.A1330V. The SNPs were genotyped and evaluated for association with BMD at the lumbar spine (LS) or femoral neck (FN) and with osteoporotic fracture, at single SNP and haplotype levels, by regression methods. Association with LS BMD was found for SNP 1, rs312009, located in the 5Ј-flanking region (p ‫ס‬ 0.011, recessive model). SNP 6, rs2508836, in intron 1, was also associated with BMD, both at LS (p ‫ס‬ 0.025, additive model) and FN (p ‫ס‬ 0.031, recessive model). Two polymorphisms were associated with fracture: SNP 11, rs729635, in intron 1, and SNP 15, rs643892, in intron 5 (p ‫ס‬ 0.007 additive model and p ‫ס‬ 0.019 recessive model, respectively). Haplotype analyses did not provide additional information, except for haplotype "GC" of the block located at the 3Јend of the gene. This haplotype spans intron 22 and the 3Ј untranslated region and was associated with FN BMD (p ‫ס‬ 0.029, one copy of the haplotype versus none). In silico analyses showed that SNP 1 (rs312009) lies in a putative RUNX2 binding site. Electro-mobility shift assays confirmed RUNX2 binding to this site.

Functional relevance of the BMD-associated polymorphism rs312009: Novel Involvement of RUNX2 in LRP5 transcriptional regulation

“…(29) In contrast to that indicated by predictive tools (Table 2), the presence of this binding site (BS1) was not conditioned by the presence of the T allele of the rs312009 polymorphism, as shown in gel-shift experiments in Fig. 1A, B.…”

“…(29) RUNX2 is a master regulator in bone biology and targets many important bone genes. Here we assessed the possible functional role of the LRP5 rs312009 polymorphism in the above-mentioned association.…”

“…In this sense, we have previously reported the association of an LRP5 promoter SNP, rs312009, with lumbar spine BMD. (29) A RUNX2 binding element was identified at the polymorphic site by gel-shift experiments, but there were no differences in the binding capacities of the two possible alleles of rs312009. In this study, we analyzed the putative functional involvement of this polymorphism in the transcriptional activity of the LRP5 5' region.…”

“…(29) Twenty and thirty micrograms of protein were resolved by SDS-PAGE (12.5% polyacrylamide), transferred onto nitrocellulose membranes, and analyzed by standard immunostaining using a horseradish peroxidase-conjugated secondary antibody and chemiluminescence detection. The following primary antibodies were used: RUNX2 M-70 at 1:800 dilution (SC-10758x; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and nucleoporine NP62 at 1:4000 dilution (BD Biosciences, San Jose, CA, USA) for the loading control.…”

Wnt receptors, bone mass, and fractures: gene-wide association analysis of LRP5 and LRP6 polymorphisms with replication

“…Although the skeletal impact of common allelic variants is less clear, several polymorphisms of the LRP5 gene have been associated with bone mass and fractures, in candidate gene studies (6,24,25) and in some, but not all, GWASs (26,27).…”