PMC8317969_01
Novel homozygous SCN9A mutation and the clinical phenotypes in the proband with Charcot neuroarthropathy. (A) Family pedigree. The proband is indicated by a black arrow. The shaded box represents a suspect patient who is presented with Charcot neuroarthropathy. The genetic relationship of parents is them being first cousins. (B) DNA sequence electropherograms demonstrating c.4015T > C (p. Cys1339Arg) homozygous mutation in the proband. (C) Burns on the finger by a lit cigarette. (D,E) Arrows of different colors represent swollen and deformed joints. (F) Plain radiographs of the right knee joint showed narrowing of the right knee joint space, blurring of the joint surface, bone destruction, and uneven density. The knee is swollen, distorted, and painless. (G) The three-dimensional CT of the left foot showed that the structures of the left ankle joint, the left tarsometatarsal joint, and the intertarsal joint were disordered, and the left ankle showed varus and subluxation. (H) The postoperative x-ray examination of bilateral knee joints showed that the internal fixation device was in place after the correction of the valgus of the right knee. (I) Conservation of residues Cys1339 among different species.
fnins-15-697167-g001.jpg
PMC8317969_01_fnins-15-697167-g001.jpg
F1
He was the fourth child of parents with normal pregnancy and childbirth (Figure 1A). When he started smoking, he often burned his fingers with lit cigarettes but did not experience pain (Figure 1C). In his 30 s, he developed multiple joint swelling, instability, and deformities caused by repetitive microtrauma and painless fractures (Figures 1D,E). Skin ulcers were noted on the right knee and on the lateral side of the left ankle (Figure 1D). In addition, radiography of the right knee joint showed narrowing of the right knee joint space, blurring of the joint surface, bone destruction, and uneven density (Figure 1F). Three-dimensional CT scan of the left foot demonstrated that the structures of the left ankle joint, left tarsometatarsal joint, and the intertarsal joint were disordered, and the left ankle exhibited varus and subluxation (Figure 1G). Postoperative radiographic examination of the bilateral knee joints revealed that the internal fixation device was in place after correction of the valgus of the right knee, and there were no apparent signs of loosening or fracture (Figure 1H). After filtration of the WES data, we finally identified a missense homozygous mutation (c.T4015C; Cys1339Arg) in exon 21 of SCN9A, which was further confirmed by Sanger sequencing (Figure 1B). Multiple alignments of SCN9A orthologs in other animal species indicated that the amino acid at position 1339 was highly conserved (Figure 1I).
The CDS sequence and protein structure pattern of SCN9A. (A) A variety of mutations in different exons in SCN9A cause CIP. The red circles: stopgain; blue diamonds: frameshift; green inverted triangles: missense; black diamonds: in-frame del/ins; yellow parallelograms: splicing. (B) Schematic representation of Nav1.7, the voltage-gated sodium channel (VGSC) a-subunit encoded by SCN9A, and the locations of the identified mutation. SCN9A encodes a plasma membrane protein, Nav1.7, which consisted of four similar domains with each domain comprising six a-helical transmembrane segments (labeled 1-6). Transmembrane segments 5 and 6 are the pore-lining segments and the voltage sensor is located in transmembrane segment 4 of each domain (depicted by a plus symbol). The red arrows indicate the location of the missense mutation identified in this study. The black dotted line correlates the change of CDS with protein change in SCN9A.
fnins-15-697167-g002.jpg
PMC8317969_01_fnins-15-697167-g002.jpg
F2
Cys1339Arg in the extracellular linker joining S5 and S6 of domain III, outside the end of S5 (Figure 2B).
Biophysical characterization of WT and mutant Nav1.7 channels in HEK293 cells. (A,B) Representative inward current traces were recorded from HEK293 cells expressing WT, and mutant p. Cys1339Arg Nav1.7 channels, respectively. (C) Current-voltage relationship of WT and mutant p. Cys1339Arg channels. Peak current density normalized to membrane capacitance is presented as the mean +- SEM.
fnins-15-697167-g003.jpg
PMC8317969_01_fnins-15-697167-g003.jpg
F3
Representative inward sodium channel traces of WT and p.C1339R are shown in Figures 3A,B. The results showed that the average peak current density of the p.C1339R channels was almost abolished compared with that of the WT channels (-9.3 +- 1.5 pA/pF, N = 10. vs. -119.1 +- 16.0 pA/pF, N = 22, P < 0.05) (Figure 3C).