PMC6499182_01
Pedigree and Sanger sequencing chromatogram of c.163G > T (p.Asp55Tyr) mutation in NOG. (A) Family pedigree. Black arrow indicates the proband, II-4. P, represents pregnancy, III-3. (B) The mutation c.163G > T in NOG is identified in II-4 (proband), I-2 and II-2 (red arrows). Blue arrow indicates wild type in III-2. The reference sequence NM_005450 of NOG is used.
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F1
In the studied family, a 43-year-old gravida at 11 weeks of gestation (the proband, II-4), her mother (I-2) and sibling (II-1) were presented with abnormal fusions of joints (Figure 1A). Figure S1). Furthermore, Sanger sequencing analysis identified p.Asp55Tyr in all the three affected family members (I-2, II-1, and II-4) while it was absent in the unaffected member (III-2) (Figure 1B).
Photographic features of hands and feet in the affected individuals. Abnormal joint osseous fusion in fingers and difficulties in making fists were seen in all of patients II-4 (a,b), I-2 (e,f) and II-1 (j,k). For patient II-4, the motion of the elbow joint is normal (c), and a little short phalange of second toe is noticed in both her feet (d). Radiographs of the hands reveal that patient I-2 (g) and II-1 (l) have proximal symphalangism in hands. Radiographs of right feet in anteroposterior and oblique view show that patient I-2 (h,i) presents proximal symphalangism in toes and tarsal coalitions, while patient II-1 (m,n) has both proximal and distal symphalangism in toes and tarsal coalition.
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F2
In the initial observation on the fingers of the proband (II-4), it was noticed that both thumbs were normal (Figure 2a); however, finger 2-5 lacked creases on flexor and extensor surfaces of the interphalangeal joints (Figure 2a), and was not able to flex (Figure 2b). Further examination on elbow joints demonstrated that she had normal abilities to move them and was able to touch her shoulder with hands (Figure 2c). On checking both feet of the proband (Figure 2d), we found abnormalities similar to the fingers: (i) both thumbs were normal; (ii) both toe 2-4 lacked creases on flexor and extensor surfaces of interphalangeal joints; and (iii) the symptomatic toes were not able to flex. Furthermore, a little short phalange of second toe was noticed in both feet (Figure 2d). For patient I-2, proximal interphalangeal joint osseous fusion of left finger 4-5 and right finger 4-5 were observed (Figure 2e-g), but tarsal-carpal coalition was not found. Foot radiographs revealed that patient I-2 had proximal interphalangeal joint osseous fusion of left toe 3-5 and right toe 2-4 (Figure 2h), and she had bilateral talonavicular coalition and talocalcaneal coalition of feet (Figure 2h,i). For patient II-1, both observation and radiographs showed proximal symphalangism of left fingers 3-5 and right fingers 4-5 (Figure 2i-k). Proximal symphalangism was evident in right toe 3-4, while distal interphalangeal joint osseous fusions were observed on left toe 2-5 (Figure 2m,n). Furthermore, fusion of multiple tarsal bones was found in patient II-1 (Figure 2m,n), that is, bilateral talonavicular coalition, bilateral talocalcaneal coalition, coalition of the cuboid bone and the third cuneiform bone on left side as well as coalition of the third cuneiform bone and the third metatarsal bone on the left side.
Functional in silico docking analysis of the p.Asp55Tyr mutation in the NOG gene. (A) The disease-associated mutational hotspots on noggin were mapped to show that p.Asp55Tyr is located within the type II receptor binding region. Known mutations are colored green and Asp55 is in red on the Coulombic surface of the protein. Three regions that were previously established are circled. Snapshots of noggin from different point views were taken by rotating the protein 90  at a time. (B) Molecular surface view of noggin is derived from a crystal structure (1M4U). The binding ligand, BMP-14, was removed for clear visualization of noggin. The BMP-interacting clip is colored blue. The location of the mutated residue, Asp55Tyr, is colored red. The type II receptor binding site, where Asp55 resides, is enlarged to show its special arrangement. Among all known mutations, only four residues are hydrogen-bonded (lines colored orange) via side chains to form a network: Glu48, Asp55, Arg167, and Arg204 (the EDRR-tetrad). Asp55 preferentially interacts with Arg167. All residues known to be mutated in diseases are colored green. Detected hydrogen bonds are indicated by lines in orange. (C)
In silico generated conformation of the EYRR-tetrad that harbors the p.Asp55Tyr mutation. All three hydrogen bonds are abolished between residue 55 and Arg167. D55Y, Asp55Tyr; C215, Cys215; W217, Trp217; I220, Ile220; D55, Asp55; R167, Arg167; R204, Arg204; E48, Glu48; P50, Pro50; Y55, Tyr55; E48, Glu48.
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F3
To better elucidate the mechanism by which the loss of function occurs, we mapped the identified p.Asp55Tyr (red) and all mutations previously reported in literature (green) to the structure (Figure 3A). The disease-associated mutations are concentrated in three regions as previously reported: type I and type II receptor binding domains and the dimerization domain (Figure 3A, in circles). The aspartate residue (Asp55) is located at a distance from the BMP binding-site called the type I receptor-binding clip (Figure 3B left panel, colored blue) and hence, it is unlikely that this mutation directly affects noggin's binding affinity to type I site of BMP. Closer look at this mutational hotspot reveals that the aspartate side chain strongly interacts with the positively charged Arg167 residue by hydrogen bonding (Figure 3B right panel). Tyr167 interacts with Glu48 with one hydrogen bond (Figure 3B). These four amino acid residues, two being positively and two being negatively charged, form a tight cluster and are very likely to hold the elbow-shaped BMP-interacting arm in position. All the three hydrogen bonds that were visualized between the aspartic acid and tyrosine 167 were abolished (Figure 3C); this indicated that p.Asp55Tyr was indeed a destabilizing mutation of the local protein folding.