Genome-wide association and functional studies identify the DOT1L gene to be involved in cartilage thickness and hip osteoarthritis

Abstract

Hip osteoarthritis (HOA) is one of the most disabling and common joint disorders with a large genetic component that is, however, still ill-defined. To date, genome-wide association studies (GWAS) in osteoarthritis (OA) and specifically in HOA have yielded only few loci, which is partly explained by heterogeneity in the OA definition. Therefore, we here focused on radiographically measured joint-space width (JSW), a proxy for cartilage thickness and an important underlying intermediate trait for HOA. In a GWAS of 6,523 individuals on hip-JSW, we identified the G allele of rs12982744 on chromosome 19p13.3 to be associated with a 5% larger JSW (P = 4.8 × 10(-10)). The association was replicated in 4,442 individuals from three United Kingdom cohorts with an overall meta-analysis P value of 1.1 × 10(-11). The SNP was also strongly associated with a 12% reduced risk for HOA (P = 1 × 10(-4)). The SNP is located in the DOT1L gene, which is an evolutionarily conserved histone methyltransferase, recently identified as a potentially dedicated enzyme for Wnt target-gene activation in leukemia. Immunohistochemical staining of the DOT1L protein in mouse limbs supports a role for DOT1L in chondrogenic differentiation and adult articular cartilage. DOT1L is also expressed in OA articular chondrocytes. Silencing of Dot1l inhibited chondrogenesis in vitro. Dot1l knockdown reduces proteoglycan and collagen content, and mineralization during chondrogenesis. In the ATDC5 chondrogenesis model system, DOT1L interacts with TCF and Wnt signaling. These data are a further step to better understand the role of Wnt-signaling during chondrogenesis and cartilage homeostasis. DOT1L may represent a therapeutic target for OA.

Fig. 1.

(A) Association results by chromosome. The −log P values for each of the 2.5 million tests performed as part of the genome-wide association of minimal joint space (MJS) of the hip. The black solid horizontal line corresponds to P value threshold of 5 × 10⁻⁸ (GWS). (B) Regional association plot for the locus of JSW (19 p31.3). SNPs are plotted by position in a 400-kb window against association with mJSW (−log10 P). The purple diamond highlights the most significant SNP in discovery analysis. Blue peaks indicate recombination rates. The SNPs surrounding the most significant SNP are color coded to reflect their LD with this SNP (from pair-wise r² values from the HapMap CEU). Genes, exons and the direction of transcription from the University of California at Santa Cruz genome browser are depicted underneath the plot.

Fig. 2.

Forest plots for rs12982744. Black squares represent effect estimate and 95% CI for each study, and the red diamond is a summary effect estimates. mJSW measurements units are in millimeters.

Fig. 3.

Risk for HOA. Values represent OR and 95% CI.

Fig. 4.

Functional analysis of Dot1l during chondrogenesis. Stable ATDC5 clones were established using either the control noninterfering pGIPZ or the pGIPZ-shmiRNA directed against mouse Dot1l. Three different antibiotic-resistant clones were selected. Knockdown efficiency was assessed by quantitative RT-PCR. Stably-transfected ATDC5 clones were cultured as micromasses as described previously (14, 15). Each condition was performed in triplicate. Total RNA from was isolated after 1, 7, 14, or 21 d. Data presented are representative of the three independent clonal colonies. Results are expressed as the mean ± SD of three independent replicates. Comparisons were made by ANOVA, followed by Fisher’s t post hoc test. Statistically significant differences vs. day 1 are indicated as *P < 0.05, and vs. control-transfected cells as ^#P < 0.05. (A) Dot1l knock-down reduces proteoglycan and collagen content, and mineralization during chondrogenesis. Stainings were performed on ATDC5 micromass cultures stably transfected with either control or Dot1l shmiRNA producing vector, over 21 d. AB, Alcian blue; AR, Alizarin red; SO, Safranin O; SR, Sirius red. (B) Dot1l knock-down reduces mRNA expression of markers of chondrogenesis. mRNA levels were normalized to S29 (reference gene) (n = 3). Quantitative real-time PCR conditions and primers are available upon request. (C) Dot1l knock-down affects Wnt signaling during chondrogenesis. mRNA levels of Wnt target genes Tcf1 and osteocalcin were normalized to S29 (reference gene) (n = 3). (D) DOT1L interacts with the Wnt signaling pathway TCF4. Coimmunoprecipitation of DOT1L and TCF4 using 100 μg of total proteins (input) from micromass cultures (at day 7) of either control or Dot1l knocked-down cells. Proteins were isolated from ATDC5 micromasses. Coimmunoprecipitations were performed and 20 μL of elution fraction was probed after protein binding on either mock (donkey anti-goat IgG) or TCF4 column-immobilized antibody. (E) DOT1L is expressed during joint development and in mature articular cartilage of mice. Immunohistochemistry on paraffin-embedded EDTA decalcified adult knee sections and nondecalcified embryonal sections, was performed with rabbit anti-Dot1L antibody (5 μg/mL). After overnight incubation of the sections at 4 °C, 1:100 peroxidase goat anti-rabbit IgG was applied and peroxidase activity was determined using DAB. In the developing limb (embryonic day 15.5), expression was detected in resting (R), proliferating (P), prehypertrophic (PH), and hypertrophic (H) chondrocytes, as well as in the mesenchyme surrounding the bones (M). Immunohistochemistry also detected expression in articular cartilage chondrocytes in healthy mice knee (age 9 wk). IgG as a negative control is also shown.