Background
Thoracic ossification of the posterior longitudinal ligament (T-OPLL) is characterized by pathological heterotopic ossification of this region. T-OPLL is a rare but has a high disability rate disease, which results in intractable myelopathy and radiculopathy, the prevalence of T-OPLL in individuals of Japanese ethnicity is 1.6–1.9%, and the mean age of onset is > 40 years old [
1,
2]. T-OPLL has a marked ethnic predilection, as this disease occurs more frequently in Japanese and Chinese individuals. Although the pathogenesis of T-OPLL remains unclear and linked to several factors, including mechanical stress, genetic, and metabolic which probably contribute to the etiology of OPLL, most studies suggest that cervical-OPLL (C-OPLL) is a “genetic” disease [
3‐
12]. Our previous whole-genome sequencing and candidate gene-association studies demonstrated that the presence of the rs201153092A single nucleotide polymorphism (SNP) in the
COL6A1 gene is potentially associated with T-OPLL susceptibility [
13,
14]. Therefore, we hypothesize that
COL6A1 might be involved in the formation of OPLL of the thoracic spine.
COL6A1 is a crucial component of the extracellular matrix and involved in membranous or endochondral ossification [
15]. Although the
COL6A1 has been identified as potentially pathogenic loci for C-OPLL, the mutations reported in previous studies were located in the promoter regions or intronic regions of the
COL6A1 gene and lack relevant functional validation. The rs201153092A site mutation is located in the exonic region of the
COL6A1 gene. Mutation in the exonic region can affect the expression of the protein by affecting the amino acid sequence composition.
The present study aimed to determine whether the rs201153092A site mutation causes abnormal expression of the COL6A1 gene in patients with T-OPLL among a Han Chinese population and to determine whether COL6A1 is involved in the pathogenicity of T-OPLL.
Genotyping
EDTA-anticoagulated peripheral blood samples were obtained from all C-OPLL patients for DNA extraction. Genomic DNA samples were extracted from peripheral leukocytes with the standard procedure using a Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA). The polymerase chain reaction (PCR) fragments were submitted for Sanger sequencing at the Beijing Genomics Institute, and the forward and reverse sequence reads were assembled and analyzed in DNA Star version 7.1. Details of the primer sequences are listed in Table
2. The primer was used for PCR as described previously [
17]. PCR was performed with 20 ng genomic DNA per 15-μl reaction mixture, containing 0.2 μM of each primer, 200 μM of deoxyribonucleotides, 50 mM KCl, 10 mM Tris HCl (pH 8.3), 1.5 mM MgCl2, and 0.5 units of Taq DNA polymerase in a DNA Gradient PCR machine (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions were as follows: initial denaturation at 95 °C for 10 min; followed by 35 cycles of 95 °C for 30 s, annealing at an assay-specific temperature (48 to 65 °C) for 45 s, and elongation at 72 °C for 45 s; and a final terminal elongation step at 72 °C for 5 min. The PCR products were analyzed by direct sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with POP-7™ Polymer in a 3730XL DNA Analyzer with Sequencing Analysis Software version 5.2 (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Table 2Details of the rs201153092 in COL6A1 and the associated primers
COL6A1 | Forward 5′-TGAAAGGGTGAGTGTCCAA-3′ Reverse 5′-GTGCCCAGTCCACTAAAGAG-3′ |
Statistical analysis
All statistical analyses were performed using SPSS v17.0 software (SPSS, Inc., Chicago, IL, USA). Descriptive data for continuous variables are presented as the mean ± standard deviations. The Student’s t test was used to compare the means between 2 groups. The differences in T-OPLL subtypes between patients with or without COL6A1 gene mutation were applied using one-way analysis of variance with post hoc Fisher’s test. The genotypic and allelic distributions were obtained using the χ2 test. P ≤ 0.05 was considered to be statistically significant.
Discussion
Although the exact cause of the T-OPLL disease is still unclear, it is generally believed that genetic factors have an important role in the development of the disease. Some scholars believe that the accumulation of harmful missense mutations in the human genome creates a genetic basis for various complex diseases [
18]. Disease progression is also affected by the gene expression in peripheral blood cells, with several genes reported to exhibit higher expression in the peripheral blood of OPLL patients compared with healthy controls [
12,
19].
In this study, peripheral blood of T-OPLL patients with or without rs201153092A mutation was collected and analyzed to assess the role of the mutated gene locus. The results of ELISA and qPCR analysis demonstrated that the expression of COL6A1 was significantly higher in the peripheral blood of T-OPLL patients carrying the rs201153092A mutation compared with patients without the mutation. WB were performed to determine COL6A1 expression in patients with T-OPLL, and the results demonstrated that the expression of COL6A1 protein was significantly higher in the T-OPLL patients with the mutation compared with those with the wild-type. Therefore, the rs201153092A site mutation may lead to overexpression of the COL6A1. We also showed that there was no difference in the OPLL classification and JOA score between T-OPLL patients with and without the rs201153092A mutation. The possible association between the rs201153092A site mutation in COL6A1 and the severity of the T-OPLL phenotypes requires larger scale studies in the future.
The rs201153092 site of the COL6A1 gene is located in the exonic region. The sequence of COL6A1 mRNA exhibits no difference between the wild-type and mutant. Thus, RT-qPCR and the Western blot analysis cannot distinguish between the wild-type and mutant COL6A1. The mutation in the exonic region enhances the expression level of the COL6A1 gene mRNA which further translates more COL6A1 protein.
COL6 is a remarkable component of the extracellular matrix of many tissues including muscle, tendon, and cartilage [
20]. Disorders caused by mutations of
COL6A1 genes mainly affect ligaments and muscles; mutations of the
COL6A1 polypeptide chains are causative of a broad spectrum of diseases in humans, including Ullrich congenital muscular dystrophy and C-OPLL [
21]. Tanaka et al. [
15] identified that molecular variants of the extracellular proteins may be implicated in the ectopic ossification observed in OPLL, and
COL6A1 gene may lead to increased bone mass.
COL6A1 gene which promotes bone formation is regulated by various pathways. Izu et al. [
22] demonstrated that cell-cell interactions play an essential role in bone formation;
COL6A1 regulates bone formation through establishing communication cell networks at bone-forming sites. Cheng et al. [
23] indicated that
COL6A1 gene exerts its biological functions through the Akt/PI3K pathway. However, the mechanism by which
COL6A1 gene facilitates T-OPLL remains unclear and we will try to explore it in the future and request additional experiments for this reason.
The results of this study demonstrated that the expression of COL6A1 in T-OPLL patients carrying an rs201153092A mutation was significantly higher in peripheral blood and tissues than in patients without the mutation. It is suggested that the rs201153092A site mutation can lead to overexpression of COL6A1 and play a role in the development of T-OPLL. To the best of our knowledge, there are currently no reports available on the rs201153092A locus related to C-OPLL; to determine whether the rs201153092A site mutation is also associated with cervical-OPLL or only associated with T-OPLL, we performed a case-control association study. And the result showed that this locus was not related to cervical-OPLL. However, this study lacks in-depth research and discussion on the mechanism by which COL6A1 facilitates T-OPLL. Besides, due to the prevalence of T-OPLL, the disease is very rare; this study, T-OPLL patient sample size is small. Further studies with more participants of other ethnicities are needed to confirm these positive findings.
Conclusions
In conclusion, the findings of this study suggest that the rs201153092A site mutation can lead to overexpression of COL6A1 and is a potential pathogenic mutation associated with T-OPLL. The results provide a potential basis for the pathogenesis of T-OPLL and the pathogenic role of COL6A1 in T-OPLL disease.
Further genetic studies with more participants are required to validate these findings and in appropriate model systems.
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