The clinical data of a single patient with hypocalciuric hypercalcemia were retrospectively analyzed before and after radiofrequency ablation. Informed consent was obtained from the patient and his parents before genomic testing. The study was approved by the ethics committee of Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang province, China and was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Genomic DNA was extracted from the peripheral blood using an Invitrogen genomic DNA extraction kit (Invitrogen, Carlsbad, CA, USA). Standard polymerase chain reaction was carried out to amplify the CaSR gene in a 25-µL reaction mixture containing 1 µL DNA, targeted primers, 0.5 µL dNTPs, and 0.25 µL Taq polymerase (Thermo Fisher Scientific, Waltham, MA, USA; catalogue no.10342020), using the ABI Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The amplified products were analyzed by gel electrophoresis, purified, and sequenced using a 3730XL DNA Sequencer (Applied Biosystems). Sequence analysis results were analyzed using Variant Reporter software v1.0.

Ablation was performed by an experienced ultrasound interventional doctor using a VIVA RF Generator (VRS01, STARmed, Gyeonggi-do, South Korea). The patient was kept in the supine position with the neck fully exposed, and the left inferior parathyroid gland was explored and located. After routine disinfection, a liquid-isolating zone was set up by injecting 10mL 2% lidocaine diluted in normal saline (1:1) into the space between the left inferior parathyroid gland and the surrounding tissue under real-time ultrasound guidance. The radiofrequency needle was inserted into the parathyroid gland, and ablation was started at the power 35 W. Until the high echo completely covered the left inferior parathyroid gland, the radiofrequency needle was pulled out after completing the routine radiofrequency therapy procedure. The left superior parathyroid gland was treated in the same manner. Three days later, the patient had no obvious discomfort such as hoarseness, and radiofrequency ablation of the right inferior parathyroid gland was performed.

Overlap extension was performed on the human wild-type (WT) CaSR expression vector pcDNA3.1-CaSR-WT (Synbio Technologies, Suzhou, China) to introduce point mutations in the constructs pcDNA3.1-CaSR D99N (G→A at 295 Exon3) and pcDNA3.1-CaSR W718X. The W718X mutation is a nonsense mutation that changes nucleotide 2153 in the coding region from G to A. This mutation results in a change from a tryptophan-coding codon to a stop codon, leading to the early termination of protein synthesis, thereby generating a protein lacking the last four transmembrane domains and intracellular tail. All expression plasmids were sequenced directly with an ABI 3730XL Genetic Analyzer (Applied Biosystems).

HEK-293 cells were cultured in Dulbecco’s Modified Eagle’s Medium (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum, in a 37°C incubator with 5% CO₂. Routinely, cultured cells in the log phase were seeded into six-well plates at a density of 5 × 10⁵ cells/well and cultured for 24 h. A total of 5 μg of plasmid and 3.75 μL of Lipofectamine 3000 (Invitrogen) were added to 200 μL of Opti-MEM (GIBCO). The solution was mixed and added to the corresponding cell-culture wells and incubated for 48 h for transfection. Cells transfected with the corresponding plasmids were divided into four groups: blank (empty plasmid), CaSR-WT, CaSR-D99N, and CaSR-W718X.

Immunofluorescence was used to detect the expression of WT and mutated CaSRs (D99N and W718X) in transfected cells. The transfected HEK-293 cells were transferred into 12-well plates (Corning, Inc., Corning, NY, USA) containing sterile glass coverslips. After approximately 48 h, the cells were immobilized in 4% paraformaldehyde (Sigma, St. Louis, MO, USA) and treated with phosphate buffered saline (PBS, GIBCO) containing 0.2% Triton X-100 (Sigma) for permeabilization. After being incubated with 5% bovine serum albumin, the cells were again incubated over night with mouse monoclonal antibody against human CaSR aa200-300 (Abcam, Cambridge, UK; ab19347) at a dilution of 1:100. The cells were then washed three times for 5 min each and then incubated for 1 h at room temperature with anti-mouse IgG antibody conjugated to Alexa Fluor 488 secondary antibody (Abcam; ab150077). Fluorescence images were obtained using an Olympus FLUOVIEW FV1000 laser confocal microscope (Tokyo, Japan).

After cell transfection, the supernatant was discarded, and the cells were washed twice with PBS before lysing with lysis buffer (20 mM Tris HCl, 150 mM NaCl, 1% NP-40). After centrifugation, the protein concentration in the supernatant was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). An aliquot of protein was denatured by boiling in a 100°C water bath for 5 min. SDS-polyacrylamide gel electrophoresis of the denatured proteins (20 μg protein per well) was performed using an 8% separating gel and 5% stacking gel. The separated proteins were transferred onto a polyvinylidene fluoride membrane, which was blocked before incubating with mouse anti-CaSR ab19347 antibody (Abcam) followed by horseradish peroxidase-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific; Product # 31430). Enhanced chemiluminescent reagent (Thermo Fisher Scientific) was added, and the fluorescent signals were imaged using a Chemiluminescence Fluorescence Image Analyzer (FUJIFILM, Osaka, Japan).

The AM esters of the fluorescent probesFluo-3 (Invitrogen; F23915) and FuraRed (Invitrogen; F3021) were separately diluted with Ca²⁺-free extracellular solution (135 mM NaCl, 2 mM KCl, 2 mM MgCl₂, 10 mM HEPES, 22.2mM glucose; pH 7.4) to a working concentration of 5 μM and 15 μM, respectively. After 24 h of cell subculture, the medium was discarded, and the cells were washed with PBS 3 times. The Fluo-3 and FuraRed working solutions were separately added to the cells, which were then incubated at 37°C for 40 min. The fluorescent probe was discarded and replaced with new Ca²⁺-free extracellular solution. The cells were incubated at 37°C for 20 min to ensure that the AM ester was fully hydrolyzed. Cells were examined using an Olympus FLUOVIEW FV1000 laser confocal microscope, and cells exhibiting good adherence, extended morphology, and bright fluorescence intensity were selected. The scanning conditions were set at an excitation wavelength of 488 nm, and emission fluorescence at 530/30nm (Fluo-3) and 610/20nm (Fura Red) was recorded. One image was collected every 7 s for approximately 25 min. After the fluorescence intensity curve stabilized, NPS R-568 (final concentrations 10 μM, Sigma) was added to each group of cells; after the curve stabilized again, ATP (final concentrations 100 μM, Sigma) was added, and the experiment was continued until the curve had stabilized again. The ratios of the relative peak fluorescence values after NPS R-568 and ATP addition were analyzed.

Intracellular cAMP levels were determined in the different cell groups after dimethyl sulfoxide (control) or PTH (30 nM, Sigma) was added to the transfected cells and incubation for 6 h. A cAMP ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) was used according to the manufacturer’s protocol.

Statistical analysis was performed with SPSS software v14.0 (SPSS, Inc., Chicago, IL, USA). Values are reported as the mean ± standard error of the mean. A t-test was used to compare differences in intracellular Ca²⁺ concentrations and cAMP levels. P < 0.05 was considered to indicate statistically significant results.

The patient was a 40-year-old male. A physical examination seven years prior revealed elevated blood calcium but no apparent discomfort. His blood calcium remained high in several subsequent follow-ups, with the highest level reaching 4.01 mmol/L. He had a history of hypertension, type 2 diabetes mellitus, and fatty liver disease. The control of blood pressure (via amlodipine tablets) and blood glucose (via diet) was acceptable. The patient’s paternal and maternal grandmothers were half-sisters. The patient’s mother had mild hypocalciuric hypercalcemia (calcium in 2.8 mmol/L), and any other family history of parathyroid disease or a medication history of thiazide diuretics, lithium preparations, and calcium-containing antacids was denied. The patient’s height and weight were 165 cm and 75 kg, respectively. No physical anomalies were found upon examination.

The lab results included high blood calcium, low urine calcium, and abnormal PTH levels ( Table 1 ). No abnormalities were observed upon parathyroid ultrasound, and no tumors were detected on emission computed tomography or positron emission tomography–computed tomography (CT) of the parathyroid glands. 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.

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 ).

In a preliminary diagnosis, it was concluded that the patient’s hypercalcemia was caused by an inactivating CaSR mutation based on the clinical lab results and sequence analysis of hypercalcemia-related genes. However, the patient did not respond to treatments with diuretics, calcitonin, or bisphosphonates. Cinacalcet was subsequently administered with initiated dose of 25mg per day, and the patient’s blood calcium levels decreased but was still above normal range. The patient followed a low-calcium diet, drank around 3 L of water daily, and gradually increased cinacalcet dose to 100mg per day. For nearly 4 years, the patient’s blood calcium was maintained at 3.0–3.3 mmol/L and PTH at 70 pg/mL. Owing to the heavy financial burden and poor effectiveness of the calcium-lowering treatments, the patient was admitted to our department in July 2019 for further treatment.

Parathyroid ultrasound displayed echoes from three parathyroid-like structures, although the ultrasound could not confirm the right inferior parathyroid gland ( Figures 3A – C ). No hyperparathyroidism was identified on parathyroid imaging. The decision to perform radiofrequency ablation was arrived at after multidisciplinary discussions, complete examination, and ruling out contraindications. Ultrasound-guided radiofrequency ablation of the left inferior and bilateral superior parathyroid glands was performed during several sessions between August and September 2019. 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 µg per day) was administered based on the blood calcium levels. During follow-up examinations after surgery until April 2020, the patient’s blood calcium was maintained at 2.0–2.3 mmol/L, and the blood PTH was maintained at 9–10 pg/mL ( Table 1 ).

After transfection, the expression of 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 ). Western blotting revealed no expression of CaSR protein in the blank group, whereas the WT and D99N groups showed a 130-kDa protein corresponding to the known molecular weight of the complete CaSR protein. The W718X group expressed a prematurely terminated, smaller protein fragment of 80 kDa ( Figure 4B ).

The Fluo-3 and Fura Red double labeled fluorescent probe was used to determine intracellular Ca²⁺ concentration, which was represented by the change in Fluo-3/Fura Red ratio (Fluo-3 Ratio). We found that the intracellular Ca²⁺ concentration of the blank and W718X groups did not change significantly after adding 10 μM 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 μM). 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).

PTH increases cAMP levels in renal tubular cells to enhance Ca²⁺ reabsorption. We next added PTH to each group of cells to explore the effects of different CaSR gene mutations on this process by which PTH promotes calcium absorption. As shown in 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). These results suggest that the inactivation mutation of D99N CaSR weakens the antagonizing function toward the effect of PTH.