The interleukin-11 receptor variant p.W307R results in craniosynostosis in humans

Abstract

Craniosynostosis is characterized by the premature fusion and ossification of one or more of the sutures of the calvaria, often resulting in abnormal features of the face and the skull. In cases in which growth of the brain supersedes available space within the skull, developmental delay or cognitive impairment can occur. A complex interplay of different cell types and multiple signaling pathways are required for correct craniofacial development. In this study, we report on two siblings with craniosynostosis and a homozygous missense pathogenic variant within the IL11RA gene (c.919 T > C; p.W307R). The patients present with craniosynostosis, exophthalmos, delayed tooth eruption, mild platybasia, and a basilar invagination. The p.W307R variant is located within the arginine-tryptophan-zipper within the D3 domain of the IL-11R, a structural element known to be important for the stability of the cytokine receptor. Expression of IL-11R-W307R in cells shows impaired maturation of the IL-11R, no transport to the cell surface and intracellular retention. Accordingly, cells stably expressing IL-11R-W307R do not respond when stimulated with IL-11, arguing for a loss-of-function mutation. In summary, the IL-11R-W307R variant, reported here for the first time to our knowledge, is most likely the causative variant underlying craniosynostosis in these patients.

Figure 1

A craniosynostosis phenotype in a Pakistani family. (a) An extended consanguineous pedigree with three affected individuals who harbor the c.919 T > C variant. Filled black circles and squares, affected females and males, respectively; unfilled shapes, unaffected; crossed by a line represents a deceased individual. An index diagnosis is indicated by an arrowhead. An asterisk designates the individual on whom WES was performed. Genotypes are indicated below the genetic symbols of each examined member: TT (wild type), TC (heterozygous), and CC (homozygous mutant). (b) Facial views of live affected individuals of the Pakistani CS family (subjects V:3 and V:4 at age 13 and 9 respectively) (c). i and ii: axial and sagittal T2-weighted images of individual V:3 show brachycephaly, mild platybasia and a basilar invagination. iii and iv: axial and sagittal T2 weighted images of individual V:4 reveal trigonocephaly and increased transverse diameter, * indicates Chiari-like malformation with cerebellar tonsils extending below the foramen magnum, platybasia and a degree of basilar invagination. (d) Orthopantomograph of the individual (V:3) show over-retained deciduous teeth and erupting succedaneous permanent teeth. Individual (V:4) had no anomaly on the teeth.

Figure 2

A homozygous missense c.919 T > C/p.W307R variant in IL11RA is present in members of the family. (a) Representative sequence electropherograms of wild type sequence and of c.919 T > C in heterozygous (IV:1) and homozygous (V:3) individuals. (b) Schematic of the human IL11RA gene and protein domain structure. The IL-11R protein comprises an immunoglobulin (Ig)-like, two fibronectin type 3 domains (FN3)-like (D2 and D3), a flexible stalk region, a transmembrane (TM) helix and a short intracellular part. Position of (c.919 T > C, p.W307R) is indicated. Conservation of the residue is determined by multiple sequence alignments of IL11RA across different species using Clustal Omega.

Figure 3

The p.W307R variant is located in the arginine-tryptophan-zipper within the domain D3 of the IL-11R and prevents IL-11R maturation. (a) Structure of the extracellular part (domains D1, D2 and D3) of the IL-11R according to the published crystal structure (pdb accession code 6O4P). Within the D3 domain (enclosed), the amino-acid residues which constitute the arginine-tryptophan-zipper are highlighted in dark blue with the exception of W307, which is shown in dark red. Magnification of the D3 domain is shown in the insets on the right hand site of the total structure, which represent the WT situation (W307, left inset) and the patient variant (R307, right inset). (b) HEK293 cells were transiently transfected with expression plasmids encoding IL-11R WT, IL-11R-W307R or mock control. 48 h later, cells were lysed and expression of the IL-11R variants was analyzed by western blot. GAPDH was visualized on the same membrane to verify the loading of equal protein amounts. One representative experiment out of three experiments with similar outcome is shown.

Figure 4

IL-11R-W307R is not transported to the cell surface, but retained intracellularly. (a) HEK293 cells were transiently transfected with expression plasmids encoding IL-11R WT, IL-11R-W307R or mock control. 48 h later, cells were harvested and the amount of IL-11R was assessed via flow cytometry. Prior to the antibody staining, cells were either left untreated for cell surface staining (“surface”, upper panel) or permeabilized using saponin for intracellular staining (“intracellular”, lower panel). The geometric mean fluorescence intensity (gMFI) of HEK293 cells expressing IL-11R-WT was set to 100, and the gMFI values of the other cells were calculated accordingly. One experiment out of three is shown on the left side, while quantification of three independent experiments (mean ± SEM) is shown on the right side. Statistical analysis was performed using one sample T test between IL-11R WT and IL-11R-W307R-expressing cells: **: p < 0.01; n.s.: not significant. (b) The experiment was performed with Ba/F3-gp130, Ba/F3-gp130-IL-11R-WT and Ba/F3-gp130-IL-11R-W307R as described for panel a. The geometric mean fluorescence intensity (gMFI) of Ba/F3-gp130-IL-11R-WT cells was set to 100, and the gMFI values of the other cells were calculated accordingly. One experiment out of three is shown on the left side, while quantification of three independent experiments (mean ± SEM) is shown on the right side. Statistical analysis was performed using one sample T test between IL-11R-WT and IL-11R-W307R-expressing cells: **: p < 0.01; n.s.: not significant. (c) HeLa cells were transiently transfected with expression plasmids encoding either IL-11R WT or IL-11R-W307R. IL-11R was stained with an anti-myc antibody, and cells were either permeabilized or not before IL-11R staining to visualize total IL-11R expression or just the IL-11R at the cell surface. Cellular membranes were visualized with WGA, cell nuclei with DAPI. Staining of IL-11R/myc is shown in green, WGA in purple and DAPI in blue. Representative images from three independent experiments with similar outcome are shown. Scale bar: 10 µm.

Figure 5

Cells expressing IL-11R-W307R do not respond to IL-11 stimulation. (a) Equal amounts of Ba/F3-gp130-GFP, Ba/F3-gp130-IL-11R-WT and Ba/F3-gp130-IL-11R-W307R cells were serum-starved for 120 min and then stimulated for 15 min with either 10 ng/ml IL-11, 10 ng/ml Hyper-IL-6 (Hy-IL-6) or left unstimulated. Cells were directly boiled in Laemmli buffer afterwards and pSTAT3, total STAT3, IL-11R and GAPDH were visualized via western blot. One experiment out of three with similar outcome is shown. (b) Quantification of the three experiments described in panel a (mean ± SEM). Shown is the signal intensity of phosphorylated STAT3 in relation to to the signal intensity of total STAT3. (c–e) Equal amounts of (c) Ba/F3-gp130-GFP, (d) Ba/F3-gp130-IL-11R-WT and (e) Ba/F3-gp130-IL-11R-W307R cells were incubated with increasing amounts of either Hyper-IL-6 (HY) or IL-11 (0.01–100 ng/ml) or left untreated. 48 h later, cell viability was determined as described in Materials and Methods. Shown is one experiment (n = 3 technical replicates, mean ± SEM) out of three experiments performed with similar outcome.