Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

Abstract

Bruck syndrome is a rare autosomal recessive disorder characterized by increased bone fragility and joint contractures similar to those in arthrogryposis and is known to be associated with mutations in the FKBP10 (FKBP prolyl isomerase 10) and PLOD2 (Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2) genes. These genes encode endoplasmic reticulum proteins that play an important role in the biosynthesis of type I collagen, which in turn affects the structure and strength of connective tissues and bones in the body. Mutations are associated with disturbances in both the primary collagen chain and its post-translational formation, but the mechanism by which mutations lead to Bruck syndrome phenotypes has not been determined, not only because of the small number of patients who come to the attention of researchers but also because of the lack of disease models. In our work, we investigated the cellular effects of two forms of the wild-type PLOD2 gene, as well as the PLOD2 gene with homozygous mutation c.1885A>G (p.Thr629Ala). The synthesized genetic constructs were transfected into HEK293 cell line and human skin fibroblasts (DF2 line). The localization of PLOD2 protein in cells and the effects caused by the expression of different isoforms-long, short, and long with mutation-were analyzed. In addition, the results of the transcriptome analysis of a patient with Bruck syndrome, in whom this mutation was detected, are presented.

Keywords: Bruck syndrome; PLOD2; cells; mutations; transfection.

Figure 1

Schematic representation of genetic constructs CMV—CMV promoter: (A) wt PLOD2 (LH2a) expression plasmid, (B) wt PLOD2 (LH2b) expression plasmid, and (C) mutant Thr629Ala allele PLOD2 expression plasmid.

Figure 2

Transfection efficiency of a plasmid with a GFP reporter. Flow cytometry analysis of transfection efficacy of HEK293 (left) and DF2 (right) cells in 24 h (A). Visualization of HEK293 (left) and DF2 (right) cells in 48 h using the EVOS FL Auto Imaging System (B). Green – PLOD2 fused to GFP. Scale bar—200 µm.

Figure 3

Confocal microscopy images of HEK293 cells transfected with three different constructs, including GFP-LH2a (B), GFP-LH2b (C), and GFP-LH2b with mutation Thr629Ala (D) and wild-type without constructs (A). In Figure (D), the arrow shows a change in the morphology of the cell with expression of the LH2b (Thr629Ala). The left column (green) shows the images of GFP-tagged protein PLOD2, the middle column (red) shows the images of ER, and the right column shows the overlaid images of the left and middle columns. Scale bar—25 µm.

Figure 4

Visualization of DF2 cells transfected with three different constructs, including GFP-LH2a (A), GFP-LH2b (B), and GFP-LH2b with mutation Thr629Ala (C) using the EVOS FL Auto Imaging System. The middle column (red) shows the images of ER, and the right column shows the overlaid images of the left and middle columns. Scale bar—100 µm.

Figure 5

Cell viability assays of HEK293 (A) and DF2 (B) 24, 48, and 120 h after transfection. Cell adhesion rate in each group of the HEK293 cells at 30, 60, and 90 min after transfection (C,D). Scale bar—1000 µm; mean ± SEM, Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. N = 6 (A,B) and N = 12 (C).

Figure 6

Cell migration assay of DF2. Scale bar—1000 µm. N = 4.

Figure 7

Cell migration assay of HEK293. Scale bar—1000 µm. N = 6.

Figure 8

(A) Volcano plot showing −log10 of adjusted p-value vs. log2FoldChange. Blue dots represent downregulated differentially expressed genes (DEGs) and red dots represent upregulated DEGs. Enrichment analysis of human phenotype of (B) downregulated DEGs and (C) upregulated DEGs.

Figure 9

GO function and pathway enrichment analysis of down- and up-regulated DEGs. (A) BP of down- and (B) up-regulated DEGs, (C) MF of down- and (D) up-regulated DEGs, and (E) CC of down- and (F) up-regulated DEGs.