INTRODUCTION: It is widely accepted that the intervertebral disc disorders, including disc degeneration and herniation, have significant genetic predisposition. Although it has been demonstrated that early-onset lumbar disc herniation in teenagers, or juvenile disc herniation (JDH), is driven by hereditary factors, little is known on its natural history, genetic causatives and molecular pathogenesis. Type X collagen (COL10A1, COLX), an extracellular protein in chondrocyte and cartilage, is essential in the development and maturation of mammalian axial skeleton. Mutations in COL10A1 were may lead to autosomal dominant metaphyseal chondrodysplasia in human. Using exome sequencing on blood samples from JDH patients and their family members, we identified a novel initiator codon mutation in COL10A1 gene. With the help of CRISPR/Cas9 gene editing techniques, we demonstrated that deficits in COL10A1 gene led to defective formation of the lumbar vertebral endplates and degenerative phenotypes of disc in mice.
METHODS: Twenty one pedigrees of JDH patients (age <20 years, Chinese Han population) were studied. On lumbar spine magnetic resonance (MR) images, the shape, concavity and Modic change at the lumbar vertebral endplates were evaluated. DNA was extracted from peripheral blood and exome sequencing was performed for each participant. To examine the pathogenicity of the identified COL10A1 mutation (c.2[exon2]T>C, p.Met1Thr, M1T), we overexpressed EGFP- and mCherry-tagged human COL10A1 cDNA in HEK293A cell and detected the subcellular localization of mutant and wild-type (WT) COL10A1 protein using confocal microscope. Via CRISPR/Cas9 system, we generated a Col10a1 gene knock-out (KO) C57L/B6 mouse line. Micro-CT scan and histological study of the juvenile (postnatal-day 21) KO mice were conducted to examine the lumbar discs and endplates.
RESULTS: Upon a pedigree with 4 symptomatic members, we identified a novel initiator codon mutation (c.2(exon2)T>C, p.Met1Thr, heterozygous) in COL10A1 gene. The mutation carriers share similar morphological changes at the lower lumbar spine (L3-S1), namely multiple deformed endplates and herniated discs. In vitro assay revealed that the COLXM1T mRNA was translated into a truncated fragment without the signal peptide, and therefore failed to be processed through endoplasmic reticulum to form a functional homotrimer. We successfully established a Col10a1 gene knock-out (KO) C57L/B6 mouse line. In the juvenile KO mice, Micro-CT revealed defective and irregular-shaped endplates at the thoracolumbar vertebrae, as well as decreased quality of the vertebral cortical bone and trabeculae. Histological study of the KO juvenile mice revealed a thinned hypertrophic zone in the defective endplates, with disrupted chondrocyte columnization and delayed ossification, along with mal-arranged annulus fibrosus in the deformed discs. In brief, the KO mice recapitulated endplate defects and degenerative phenotype of the disc observed in human patients.
DISCUSSION: As a land marker for the hypertrophic chondrocytes, COL10A1 plays a key role in the osteoprogenitor lineage and endochondral ossification. Deficits in COL10A1 gene led to developmental endplate defects and subsequent early-onset disc degeneration. We proposed that COL10A1-associated disc degeneration may be a novel subtype of disc degeneration and herniation. In addition, this work established a novel genetic mouse model of disc degeneration for future investigation.