Dominant-negative SOX9 mutations in campomelic dysplasia

Abstract

Campomelic dysplasia (CD) is an autosomal dominant, perinatal lethal skeletal dysplasia characterized by a small chest and short long bones with bowing of the lower extremities. CD is the result of heterozygosity for mutations in the gene encoding the chondrogenesis master regulator, SOX9. Loss-of-function mutations have been identified in most CD cases so it has been assumed that the disease results from haploinsufficiency for SOX9. Here, we identified distal truncating SOX9 mutations in four unrelated CD cases. The mutations all leave the dimerization and DNA-binding domains intact and cultured chondrocytes from three of the four cases synthesized truncated SOX9. Relative to CD resulting from haploinsufficiency, there was decreased transactivation activity toward a major transcriptional target, COL2A1, consistent with the mutations exerting a dominant-negative effect. For one of the cases, the phenotypic consequence was a very severe form of CD, with a pronounced effect on vertebral and limb development. The data identify a novel molecular mechanism of disease in CD in which the truncated protein leads to a distinct and more significant effect on SOX9 function.

Keywords: SOX9; bent bone dysplasia; campomelic dysplasia; dominant negative.

FIGURE 1

Radiological findings of four cases of CD. The four CD cases (b), (c), (e–h) were compared with a case with the classic type of CD (a), (d). Full body X‐rays were available for two of the cases: R83–090 (b) and R14–170 (c). Lower limb radiographs show bending of the tibiae and sometimes the femora (d–h). Cases R83–090, R89‐086, and the classic type case, R96‐084, were studied at 20 weeks of gestational age while cases R09–315 and R14–170 were at 14 weeks of gestational age. CD, campomelic dysplasia

FIGURE 2

The SOX9 mutations resulted in the production of truncated proteins. (a) Diagram of SOX9 showing the different protein domains and the positions of the mutations in five cases: R92–124 (p.S181X), classic type; R09–315 (p.Y319X), R89‐086 (p.Q391X), R83–090 (p.R394X), and R14–170 (p.Q412X). (b) Western blot analysis of SOX9 protein in control and patient chondrocytes. Three different control chondrocyte lines were used and compared with cells from a CD classic type, R92–124 (p.S181X) and three of the four patients with truncating mutations, R09–315 (p.Y319X), R89‐086 (p.Q391X), and R14–170 (p.Q412X). The numbers below reflect densitometric quantification of the upper and lower bands. (c) HEK293T cells were transduced with a COL2A1‐luciferase reporter plasmid (4eCOL2A1‐luc) and then transfected with SOX9^WT or SOX9^Q412X; luciferase levels were measured and compared with basal levels in control cells transfected with an empty vector. (d), (e) Primary chondrocytes from control and cases were transduced with 4eCOL2A1‐luc and luciferase levels were determined without induction (d) and after adding BMP‐2 (e). Luciferase was measured in three independent experiments; the graph shows the average ± SD. (f), (g) Endogenous levels of SOX9 (f) and COL2A1 (g) in primary chondrocytes from control and cases were determined by qPCR. The graph shows the average ± SE of two independent experiments. In all experiments statistical analyses were performed using Student’s t test; asterisks indicate significant differences between samples, *p < .05, ***p < .001. CD, campomelic dysplasia; qPCR, quantitative polymerase chain reaction; SE, standard error; SD, standard deviation

FIGURE 3

Type II collagen quantification in control and CD cartilage. (a) SDS‐PAGE of cyanogen bromide (CB) peptides extracted from cartilage of cases and controls. Shown to the right are the positions of the CB peptides derived from type II and type I collagen, respectively. (b) Quantification of type II collagen from SDS‐PAGE in (a). CD, campomelic dysplasia; SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

FIGURE 4

Histological findings. Picrosirius red staining of long bones in control (a), (g), a case of classic type CD (b), (h), and CD due to truncating SOX9 mutations (c–f), (k), (l). (a–f) Macroscopic details of bending at the growth plate and midshaft levels. Image (b) is a composite of two images due to size restrictions. (g–l) Growth plates show reduction of the hypertrophic region (vertical bars). Control, R96‐084, R89‐086, and R83–090 are tibiae at 20 weeks of gestational age. R09–315 and R14–170 are femurs of 14–15 weeks of gestational age. Bars represent 50 μm. CD, campomelic dysplasia