Pedigree and segregation analysis in families of childhood glaucoma patients who carried more than one variant in MMP-related genes. In families PCG219 and PCG291, only proband DNA samples were available for the study. Black and grey symbols indicate childhood glaucoma and primary open-angle glaucoma, respectively. +: wildtype allele.

Localization of ADAMTSL4 protein in adult human ocular tissues. Fluorescent immunohistochemistry was performed on 3 μm histological sections. The sections were incubated with a primary antibody, rabbit anti-ADAMTSL4 (1:250) (MBS716409, Quimigen, Nanterre, France), followed by incubation with a green-labeled secondary antibody (Cy2-conjugate donkey anti-rabbit) (1:1000). In the resulting images, ADAMTSL4 immunoreactivity, DAPI nuclear staining and tissue autofluorescence are represented by green, blue and red signals, respectively. As a negative control, sections were incubated only with the secondary antibody (H–N). Confocal micrographs were captured to visualize the localization of ADAMTSL4 in different ocular tissues, including the lens (A), corneal epithelium (B), retina (C), corneal endothelium (D), iris (E), ciliary processes (F), and trabecular meshwork (G). The scale bars in panels (B–G) correspond to 50 μm, while in panel (A), it corresponds to 25 μm. CEN (corneal endothelium), CEP (corneal epithelium), CS (corneal stroma), GCL (ganglion cell layer), IF (iris fibroblasts), INL (inner nuclear layer), IPL (inner plexiform layer), IPE (iris pigment epithelium), IS (iris stroma), ISM (iris sphincter muscle), LC (lens capsule), LEP (lens epithelium), NPCE (non-pigmented epithelium), ONL (outer nuclear layer), OPL (outer plexiform layer), PCE (pigmented epithelium), PHL (photoreceptor layer), S (stroma), TM (trabecular meshwork). *: non-specific fluorescence.

Localization of ADAMTSL4 protein in zebrafish eye larvae (6 dpf) by confocal fluorescence immunohistochemistry. Fluorescent immunohistochemistry was performed on 10 μm histological sections of zebrafish eyes from wild type (+/+) (A) and adamtsl4 KO (−/−) (B) specimens. The sections were incubated with a rabbit anti-ADAMTSL4 primary antibody (MBS716409, Quimigen) at a dilution of 1:250, followed by a Cy2-conjugated donkey anti-rabbit secondary antibody at a dilution of 1:1000. As a negative control (C), a section was incubated only with the secondary antibody. In the resulting images, ADAMTSL4 immunoreactivity is represented by green signals, DAPI nuclear staining by blue signals, and tissue autofluorescence by red signals. Scale bars in the panels indicate 50 μm. The images are representative of the observed results in three zebrafish of each genotype. C: cornea. GCL: ganglion cell layer. INL: inner nuclear layer. IPL: inner plexiform layer. LEP: lens epithelium. ONL: outer nuclear layer. OPL: outer plexiform layer. PHL: photoreceptor layer. POM: periocular mesenchyme.

Evaluation of the functional effect of four rare ADAMTSL4 variants identified in childhood glaucoma patients, in HEK-293T cells. Subcellular distribution of the wild type protein (A) and variants R98W (E), S719L (I), R774W (M), and R1083H (Q) in HEK293T Cells (green signals). The cells were transfected with cDNA constructs encoding the different variants, fused to GFP at their C-terminal as a reporter protein and analyzed 48 h after transfection. (U) Non-transfected cells were used as a negative control. (B,F,J,N,R,V) Immunocytochemistry was performed to detect PDI, a marker of ER stress (orange signals). (C,G,K,O,S,W) DAPI nuclear staining (blue signals). (D,H,L,P,T,X) Merged signals. The scale bars in the panels correspond to 20 μm. The white arrowheads indicate increased PDI signal colocalized with the granular expression pattern of the R98W and R774W variants. The inserts in panels (E,M) show granular deposits of recombinant protein, and dotted squares indicate the magnified areas. The images are representative of five fields per variant. Additional representative photographs are shown in Figure S3. R98W: p.Arg98Trp. S719L: p.Ser719Leu. R774W: p.Arg774Trp. R1083H: p.Arg1083His.

Western blot analysis of ER stress associated with the expression in HEK-293T cells of four rare ADAMTSL4 variants identified in childhood glaucoma patients. HEK-293T cells were transfected with various cDNA constructs as explained in the legend of Figure 3. Forty-eight hours after transfection, cells lysates were prepared and analyzed by Western immunoblot. PDI, beta-actin (protein loading control), and NPTII (plasmid expression control) immunosignals were quantified by densitometry. PDI levels were normalized using beta-actin and NPTII. We conducted two independent transfections per cDNA construct, each performed in triplicate. *: p < 0.05. R98W: p.Arg98Trp. S719L: p.Ser719Leu. R774W: p.Arg774Trp. R1083H: p.Arg1083His.

Evaluation of the functional effect of four rare ADAMTSL4 variants identified in childhood glaucoma patients, by heterologous expression in zebrafish. One-cell zebrafish embryos were microinjected with pcDNA3.1-hADAMTSL4-GFP cDNA constructs encoding the four variants (R98W, n = 374; S719L, n = 381; R774W, n = 477; and R1083H, n = 485) and the wild type protein (n = 1013). (A) Embryo lethality was quantified at 24 h after microinjection and (B–S) the phenotypes and recombinant protein expression were evaluated in larvae by bright field and fluorescence microscopy (3 dpf), respectively. The images are representative of the larvae expressing the recombinant proteins. Additional representative larvae are shown in Figure S4. (T–V) pcDNA3.1-GFP was microinjected as a control (n = 784). *: p < 0.05; ***: p < 0.001. R98W: p.Arg98Trp. S719L: p.Ser719Leu. R774W: p.Arg774Trp. R1083H: p.Arg1083His.

Functional interaction between zebrafish adamtsl4 and cpamd8 genes. (A–D) To obtain double heterozygotes (adamtsl4/+ and cpamd8/+), zebrafish progenitors with heterozygous mutations in both adamtsl4 and cpamd8 genes were crossed. The progeny were genotyped, and the ocular anterior segment was evaluated in adult specimens (2 months) using brightfield microscopy. Sibling wild type (+/+) and single heterozygous (+/−) animals were used as controls. The photographs are representative of the eyes observed. (E,F) The volumes of the anterior segment and the lens within the anterior chamber were calculated as indicated in Methods section. These calculations assumed that the anterior chamber and lens can be represented as spherical caps. In panel (A), the base (r) and height (h) of the spherical caps for the anterior chamber and lens are depicted in yellow and red, respectively. These values were normalized to those of wild type eyes. The following number of eyes were used: Wild type: n = 16; adamtsl4 +/and cpamd8 +/−: n = 16; adamtsl4 +/− cpamd8 +/−: n = 158. **: p < 0.01. ***: p < 0.001.