A common variant rs2054564 in ADAMST17 is associated with susceptibility to lumbar spondylosis

Abstract

The molecular pathophysiology underlying lumbar spondylosis development remains unclear. To identify genetic factors associated with lumbar spondylosis, we conducted a genome-wide association study using 83 severe lumbar spondylosis cases and 182 healthy controls and identified 65 candidate disease-associated single nucleotide polymorphisms (SNPs). Replication analysis in 510 case and 911 control subjects from five independent Japanese cohorts identified rs2054564, located in intron 7 of ADAMTS17, as a disease-associated SNP with a genome-wide significance threshold (P = 1.17 × 10^-11, odds ratio = 1.92). This association was significant even after adjustment of age, sex, and body mass index (P = 7.52 × 10^-11). A replication study in a Korean cohort, including 123 case and 319 control subjects, also verified the significant association of this SNP with severe lumbar spondylosis. Immunohistochemistry revealed that fibrillin-1 (FBN1) and ADAMTS17 were co-expressed in the annulus fibrosus of intervertebral discs (IVDs). ADAMTS17 overexpression in MG63 cells promoted extracellular microfibrils biogenesis, suggesting the potential role of ADAMTS17 in IVD function through interaction with fibrillin fibers. Finally, we provided evidence of FBN1 involvement in IVD function by showing that lumbar IVDs in patients with Marfan syndrome, caused by heterozygous FBN1 gene mutation, were significantly more degenerated. We identified a common SNP variant, located in ADAMTS17, associated with susceptibility to lumbar spondylosis and demonstrated the potential role of the ADAMTS17-fibrillin network in IVDs in lumbar spondylosis development.

Figure 1

Summary flowchart of the present study.

Figure 2

Expression levels of ADAMTS17 and FBN1 in the annulus fibrosus and nucleus pulposus in the mouse intervertebral disc. qRT-PCR revealed the expression of Adamts17 and Fbn1 in the annulus fibrosus (AF) of mouse intervertebral disc. High expression of Col1a1 in AF, and high expression of ACAN, Skt, and CD24 in nucleus pulposus (NP) confirmed the proper separated harvest of AF and NP tissues. Eight lumbar IVDs from two mice were collected to obtain enough tissues. All reactions were performed in triplicate.

Figure 3

Immunohistochemistry and immunofluorescence images of ADAMTS17 and FBN1 in mouse and human intervertebral discs. Immunohistochemistry (the upper row) and immunofluorescence (the other rows) of ADAMTS17 and FBN1 in intervertebral disc (IVD) sections demonstrated the colocalized expression of Adamts17 and Fbn1 in annulus fibrosus of mouse (left) and human (right) IVDs. For mouse IVD sections, we used lumbar spinal IVDs obtained from C57/B6 mice of postnatal day 14 (P14). Non-degenerated human IVD samples were obtained from an 18-year-old patient during surgery for idiopathic scoliosis. Scale bar, 100 μm.

Figure 4

Promotion of extracellular microfibril biogenesis by ADAMTS17. (a) Immunocytochemistry of MG63 cells transfected with an expression vector of ADAMTS17 or empty vector (EV) revealed the promotion of extracellular microfibril assembly by exogenous expression of ADAMTS17. Red fibers represent extracellular fibrillin fibers. Nuclei are visualized using Hoechst 33342. Upper and lower rows show representative images of different experiments. (b) qRT-PCR confirmed the robust overexpression of ADAMTS17 in MG63 cells transfected with an expression vector of ADAMTS17. (c) Quantification of the extracellular microfibrils by positive areas confirmed significantly increased microfibril biogenesis by exogenous ADAMTS17 expression. (d) Quantification of the extracellular microfibrils by total signal intensities confirmed significantly increased microfibril biogenesis by exogenous ADAMTS17 expression. For quantification in both (c,d), the four images of ADAMTS17 group and the five images of EV group were automatically quantified using a hybrid cell count application in the BZ-X Analyzer software (KEYENCE, Japan). EV empty vector.

Figure 5

Representative preoperative magnetic resonance imaging (MRI) of the lumbar spine in patients with Marfan syndrome and idiopathic scoliosis. Representative preoperative MRI of the lumbar spine in patients with Marfan syndrome (left) and idiopathic scoliosis (right). Both cases were 14-year-old boys. White arrowheads indicate the degenerated intervertebral discs.