PMC8795859_01
Upper half of body CT, head CT, and X-ray images of the both hands of the patient. (A, B) Upper half of body CT and head CT scans, respectively. Red arrows both indicate calcification of cerebral falx. (C) Head CT scan. Red arrow indicates calcification of tentorium cerebelli. (D) X-ray of both hands. Red arrow indicates calcium deposition. (E) Abdominal CT scan. Red arrow indicates calcification in the subcapsular region of the posterior segment of the right liver lobe.
fendo-12-743517-g001.jpg
PMC8795859_01_fendo-12-743517-g001.jpg
f1
A cranial CT revealed evident calcification of the falx cerebri and left tentorium cerebelli ( Figures 1A-C ). X-ray images of both hands revealed a change in calcium deposition around the distal ulna ( Figure 1D ). Abdominal CT revealed calcification in the subcapsular region of the posterior segment of the right liver lobe ( Figures 1E ), and B-mode ultrasound of the urinary system showed no evident abnormalities.
CaSR sequencing results in the patient and parents. Sequencing of the CaSR gene revealed that the patient (A) harbored a 295G>A homozygous mutation (D99N), whereas both parents (B, C) were R (A/G) heterozygous at the same position.
fendo-12-743517-g002.jpg
PMC8795859_01_fendo-12-743517-g002.jpg
f2
Gene sequencing results revealed that the patient harbored a G A mutation at position 295 of exon 3 in the CaSR gene that resulted in an A/A homozygous mutation of CaSR D99N ( Figures 2A ). Sequencing of the CaSR gene of both parents revealed that they both were R (A/G) heterozygous at this position ( Figures 2B, C ). At 15 min, there was a transient peak in the intracellular Ca2+ concentration in all four groups in response to ATP addition (100 muM).  Figure 5E  shows that the intracellular Ca2+ oscillations (Fluo-3ratio of NPS R-568/ATP) in the D99N group were significantly lower than those in the WT group (P < 0.05), and higher than those in the blank (P < 0.01) and W718X groups (P < 0.01).
Radiofrequency ablation of the parathyroid glands. Ultrasound images of the left superior (A), left inferior (B), and right superior (C) parathyroid glands before radiofrequency ablation. Ultrasound images (left) and ultrasound contrast images (right) of left superior (D), left inferior (E), and right superior (F) parathyroid glands after radiofrequency ablation. Red arrow in (A) shows that intraoperative saline and lidocaine mixture were used for local anesthesia and isolation of the left superior parathyroid glands and peripheral tissues.
fendo-12-743517-g003.jpg
PMC8795859_01_fendo-12-743517-g003.jpg
f3
Parathyroid ultrasound displayed echoes from three parathyroid-like structures, although the ultrasound could not confirm the right inferior parathyroid gland ( Figures 3A - C ). After ablation, the parathyroid gland showed very low echo and filling defects ( Figures 3D - F ). After the ablation sessions, the patient exhibited hypoparathyroidism, calcitriol (0.25-0.5 microg per day) was administered based on the blood calcium levels.
Analysis of CaSR protein expression. (A) Expression of CaSR in blank, WT, D99N, and W718X HEK-293 cells was analyzed by immunofluorescence confocal microscopy (1000X) following incubation with a monoclonal anti-CaSR antibody. Green fluorescence indicates CaSR and blue fluorescence (DAPI) indicates the cell nucleus. (B) Western blot analysis. Proteins extracted from untransfected HepG2 cells (blank) and HepG2 cells transfected with human wild-type CaSR (WT) or mutant CaSR (D99N and W718X) were subjected to SDS-polyacrylamide gel electrophoresis, western blotting, and visualization using an anti-CaSR monoclonal antibody. GAPDH was used as a loading control.
fendo-12-743517-g004.jpg
PMC8795859_01_fendo-12-743517-g004.jpg
f4
CaSR protein in each group was examined by immunofluorescence confocal microscopy and western blotting ( Figure 4 ). Confocal immunofluorescence showed that CaSR protein was expressed in the cells of each group except the blank group ( Figure 4A ), which is consistent with results of western blotting ( Figure 4B ). The W718X group expressed a prematurely terminated, smaller protein fragment of 80 kDa ( Figure 4B ).
Detection of intracellular calcium concentration using co-loadedFluo-3andFura Redprobes. (A-D) Changes in the ratio of Fluo-3/Fura Red fluorescence in response to consecutive stimulation with 10 muM calcimimetics NPS R-568; peak 'a') and 100 muM ATP (peak 'b') were used as a measure of the intracellular Ca2+ concentration in the blank (A), CaSR-WT (B), CaSR-D99N (C), and CaSR-W718X (D) groups. (E) Relative fluorescence values of NPS R-568/ATP (peak 'a'/peak 'b' in panels A-D) in each group, reflecting the Ca2+ concentration. Data were obtained from12 wells for each cell type and given as means +- S.E. Statistical significance was determined by Student's test. *P < 0.05, **P < 0.01. Three separated tests were performed and both inter- and intra- assay coefficients of variability was less than 10%.
fendo-12-743517-g005.jpg
PMC8795859_01_fendo-12-743517-g005.jpg
f5
We found that the intracellular Ca2+ concentration of the blank and W718X groups did not change significantly after adding 10 muM NPS R-568 at 5 min but increased rapidly and significantly in the WT and D99N groups, however the increase in D99N group was much lower than that in WT group( Figures 5A - D ). At 15 min, there was a transient peak in the intracellular Ca2+ concentration in all four groups in response to ATP addition (100 muM).  Figure 5E  shows that the intracellular Ca2+ oscillations (Fluo-3ratio of NPS R-568/ATP) in the D99N group were significantly lower than those in the WT group (P < 0.05), and higher than those in the blank (P < 0.01) and W718X groups (P < 0.01).
Measurement of cAMP levels in response to PTH. Intracellular cAMP levels in all groups following treatment with dimethyl sulfoxide (control) or 30 nM PTH. *P < 0.05, ns for no significance..
fendo-12-743517-g006.jpg
PMC8795859_01_fendo-12-743517-g006.jpg
f6
Figure 6 , compared with dimethyl sulfoxide, addition of 30 nM PTH increased the intracellular cAMP levels in all groups, with significant differences within the blank group, D99N group, and W718X group (P < 0.05). Furthermore, the cAMP level in the D99N group treated with PTH was significantly greater than that in the WT group treated with PTH (P < 0.05).