| Background: Ossification of the posterior longitudinal ligament (OPLL) ischaracterized by the replacement of ligamentous tissue in the cervical and thoracicspine by new ectopic bone. This condition may lead to paralysis caused by theresulting compression of the spinal cord and nerve roots. With the increasing pace ofsociety, this disease also showed am increasing trend in China. Until now, there is noeffective treatment, so the etiology of OPLL is a research focu. Previous studiesindicated that genetic factors were suggested to be critical for development of OPLL.Genome wide microarray analysis of candidate genes involved in OPLL resulted inthe identification of a novel, clinically relevant gene encoding bone morphogeneticprotein 4 (BMP4) but was defined only by its expression patterns. This gene mutationand relate dysfunction was not considered. Method: The complete genomic BMP4coding DNA from 450 patients with OPLL and 550 matched controls were sequencedand compared. Our research had identified that missense mutation of rs17563 wasassociated with the occurrence of OPLL and increased severity of OPLL. To buildbone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation modelinduced by BMP4 gene, we packed BMP4 gene carrying rs17563 site into thelentiviral vector and transfected on BMSCs. Then the genetically modified cells wereinjected into thigh four tendon of nude rat to establish BMP4 transgenetic ectopicossification animal model further. The history and radiographic analysis on ossification of the transplant site, were carried out by animal in vivo imaging system(FX PRO). To study the BMP4 abnormal expression and induction into the degree ofdifference of the bone and cell signaling pathways involved in the induction ofosteogenesis caused by BMP4 mutation, qRT PCR and Western Blot technologyswere employed to test the protein levels of the BMP4 gene, osteogenic factors ALPand critical proteins of cell signaling pathways: Smad1/5/8, of p38MAPK fromtransfected cells, heterotopic ossification mass of animals and the posteriorlongitudinal ligament of patient from clinical surgery. Result: 1. Association study:We identified 18 SNPs, among which the minor alleles of SNP8 (C>T; P<0.001; OR:1.58), SNP13 (rs17563C>T; P<0.001; OR: 1.76), and SNP14 (rs76335800A>T;P<0.001; OR: 1.68) were associated with OPLL. Logistic regression analysis showedthat the additive model of SNP8 (P<0.001; OR: 3.48), SNP13 (P<0.001; OR: 2.22),and SNP14 (P<0.001; OR: 1.99) retained statistical significance. Linkagedisequilibrium (LD) analysis identified a 3 kbp block of intense LD in BMP4 and onespecific haplotype, TGGGCTT (P<0.001, OR: 2.54), which was associated withOPLL associated risk alleles and increased severity of OPLL, as shown by thedistribution of ossified vertebrae in patients with OPLL (P=0.002). 2. Function study :At the cell level, (rs17563T)/(rs17563C) BMP4 transgenetic cells: BMP4 protein: 3times (p<0.001), osteogenic factors ALP protein: 3.4 times (p<0.001) and cellosteogenic signaling pathway protein: Smad1,5,8: 4,2 times (p<0.001) and p38MAPK:3.6 times (p<0.001). At the animal level: (rs17563T)/(rs17563C) BMP4 induced bonemass: BMP4 protei: 2.3 times (p<0.01), osteogenic factors ALP protein: 2.6 times(p<0.01) and the signaling pathway protein: Smad1,5,8: 3.1 times (p<0.001), p38MAPK:2.72 times (p<0.001), mass volume: 2.5 times. At the clinical surgical tissue level:(rs17563T)/(rs17563C) posterior longitudinal ligament of patient: BMP4 protein: 2.3times (p<0.01), osteogenic factor ALP protein: 2.6 times (p<0.01), the signalingpathway protein: Smad1,5,8: 3.1 times (p<0.001), p38MAPK: 2.72 times (p<0.001) andthe distribution of ossified vertebrae in patients: 2.6 times. Conclusion: In northernChina, the novel mutation of human BMP4 gene could play a important role inpromoting ectopic bone formation and increasing the severity of OPLL through causing itself overexpression and then over stimulating Smad and p38MAPK signalpathways.
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