Fig. 2: Missense variants in THSD4 lead to haploinsufficiency or impaired assembly of fibrillin-1 microfibrils.

(a) Evaluation of nonsense-mediated messenger RNA (mRNA) decay (NMD) in skin fibroblasts from THSD4 premature termination codon (PTC) variants carriers. Skin fibroblasts were obtained from the proband (II.6) carrying the p.(Leu247*) variant in TAA-1889 and patients (III.3, II.2, and I.1) carrying the p.(Ala468Glnfs*45) variant from family TAA-1839. Fibroblasts from individual I.2 from family TAA-1839 were used as the control. Fibroblasts were grown with or without emetine (an inhibitor of the NMD pathway). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to evaluate the basal level of expression of THSD4 in those conditions. RT-qPCR data are presented as means ± standard error of the mean (n = 6 for variant carriers, n = 3 for control, experiment repeated on two independent cell cultures). Statistical differences were analyzed using a two-tailed Mann–Whitney test (*P < 0.05; **P < 0.01; ***P < 0.001). (b–f) Immunocytochemistry of fibrillin-1 (red) was performed in MG-63 cells transfected with empty vector (b) or vectors encoding wild-type (WT) ADAMTSL6 (c) or the different mutated forms (p.[Arg781Trp] [d], p.[Tyr321Asn] [e] and p.(Gly753Asp) [f]) 5 days after plating. Images are representative of three independent experiments. Scale bar for (a–e), 20 µm. (g) A semiquantitative assay was performed using fibrillin-1 staining normalized to the nuclear surface, in images obtained from the immunocytochemistry analysis 5 days after plating. Results are expressed as means of normalized fibrillin-1 staining ± standard error of the mean of 25 images per condition (WT was analyzed in duplicate). The dashed line indicates the mean level of fibrillin-1 staining normalized to surface of nuclei, obtained with empty vector. Statistical differences were analyzed using a two-tailed Mann–Whitney test (*P < 0.05; ***P < 0.001).