A new subtype of brachydactyly type B caused by point mutations in the bone morphogenetic protein antagonist NOGGIN

Abstract

Brachydactyly type B (BDB) is characterized by terminal deficiency of fingers and toes, which is caused by heterozygous truncating mutations in the receptor tyrosine kinase-like orphan receptor 2 (ROR2) in the majority of patients. In a subset of ROR2-negative patients with BDB, clinically defined by the additional occurrence of proximal symphalangism and carpal synostosis, we identified six different point mutations (P35A, P35S, A36P, E48K, R167G, and P187S) in the bone morphogenetic protein (BMP) antagonist NOGGIN (NOG). In contrast to previously described loss-of-function mutations in NOG, which are known to cause a range of conditions associated with abnormal joint formation but without BDB, the newly identified BDB mutations do not indicate a major loss of function, as suggested by calculation of free-binding energy of the modeled NOG-GDF5 complex and functional analysis of the micromass culture system. Rather, they presumably alter NOG's ability to bind to BMPs and growth-differentiation factors (GDFs) in a subtle way, thus disturbing the intricate balance of BMP signaling. The combined features observed in this phenotypic subtype of BDB argue for a functional connection between BMP and ROR2 signaling and support previous findings of a modulating effect of ROR2 on the BMP-receptor pathway through the formation of a heteromeric complex of the receptors at the cell surface.

Figure 1.

Pedigrees of families with identified NOG mutations, as indicated. Affected persons are indicated by blackened symbols. Symbols with horizontal lines indicate individuals for whom mutation analysis was performed.

Figure 2.

Clinical phenotypes caused by the NOG mutations. In panel A, pictures in each vertical group belong to one patient; corresponding mutations are depicted above. In hands, note variable terminal deficiency of fingers. Terminal deficiency—particularly of phalanges IV and V, with a milder involvement of distal phalanges II and III (“intermediate” BDB in table 1)—are depicted in patients 1, 2, 4, and 6. Severely affected hands with absent distal and middle phalanges of fingers II–V (“severe” BDB in table 1) are shown in patient 3. Hypoplastic but present distal phalanges of fingers (“mild” BDB in table 1) are shown in patient 5. Note proximally set thumbs and additional cutaneous syndactyly in some affected hands. Radiographs show proximal SYM of fingers II–V, present in fingers consisting of at least two phalanges. Fusion of carpal bones is a further typical feature (in patient 1, note the atypically configured carpal bones with fusion of hamate, capitate, trapezoid, and trapezium). Shortened metacarpal bones I can be seen in most affected hands. In feet, toes are similarly affected (patient 6 had surgical removal of toes). B, Magnifications. a, Proximal SYM. b, Proximal and distal SYM, as observed in patient 5. c, Accelerated development of carpal bones and the proximal epiphysis of metacarpal bone I in a 6-mo-old child. Note narrowed distance between hamate and triquetrum, indicating a future fusion. d, Absent flexion creases due to the fused interphalangeal joints. e, Symmetric constriction rings in the middle of both second toes in patient 2, imitating a condition caused by amniotic bands.

Figure 3.

Three-dimensional model of NOG-GDF5 complex highlighting altered NOG amino acids found in patients with BDB2. NOG is depicted in a ribbon structure, with one monomer in dark and the second in light pink. GDF5 is shown as a surface model, with one monomer in dark and the second in light gray. Amino acids of GDF5 that interact with BMP receptor type I are highlighted in dark and light orange, and amino acids that interact with BMP receptor type II are highlighted in dark and light green. The binding of NOG to BMPs/GDFs blocks both receptor-binding sites. NOG mutations identified in patients with BDB2 are indicated. They affect three different structural clusters: (1) the site that covers the type I receptor pocket in BMPs/GDFs (P35 and A36 [red]) (2) the site that covers the type II receptor pocket (E48 and R167 [yellow]), and (3) the NOG-dimerization site (P187 [blue]).

Figure 4.

Secretion of Nog mutant proteins by DF-1 cells and functional analysis in the micromass culture system, with western-blot analysis of conditioned media from DF-1 cells infected with RCASBP-B containing the coding sequence of either Nog WT sequence (WT Nog), BDB-associated mutant sequence (P35A, P35S, A36P, R167G, and P187S), or SYM1/TCC–derived sequence (P35R). As a negative control, DF-1 cells were infected with empty RCASBP-B (control). A, SDS-PAGE under nonreducing conditions and immunodetection with use of anti-Nog antibody. WT Nog is secreted as a disulfide-bonded dimer, as are mutants P35A, P35R, P35S, A36P, and R167G. The mutant P187S is secreted predominantly as a monomer, and only a small amount of disulfide-bonded dimer is secreted, which supports the hypothesis that this amino acid change directly affects the dimerization of Nog. B, SDS-PAGE under reducing conditions and immunodetection with use of anti-Nog antibody. All Nog dimers resolve to monomers. C, WT Nog or indicated mutants, retrovirally overexpressed in micromass cultures from chicken limb buds and grown for 6 d in culture medium. Chondrogenic differentiation of the cultures was quantified by histomorphometric analysis of Alcian blue–stained nodules. WT Nog completely inhibited nodule formation in the micromass cultures. The inhibition of chondrogenesis by WT Nog was set to 100%. In comparison with WT Nog, the analyzed BDB2-associated Nog mutants resulted only in a minor reduction of Nog activity. P35A, A36P, and P187S showed nearly full activity, whereas P35S and R167G showed a reduction of 20%–25%. In contrast, the SYM1/TCC–associated Nog mutant P35R showed an obvious reduction of ∼60% biological activity, indicating a major loss of function. D, Photographs of Alcian blue–stained micromass cultures.