A total of 54 patients with PHP1 (28 males and 26 females), 127 patients with PHPT (28 males and 99 females), and 65 healthy volunteers (33 males and 32 females) were enrolled in this study. The molecular analysis showed that 16 patients with PHP1 had GNAS mutation with the subtype of PHP1A. The remaining individuals had methylation alterations in one or more GNAS TSS:DMRs; therefore, these 38 individuals were patients with PHP1B.

The distribution of gender, age, biochemical indexes, and eGFR are shown in Table 1. Age and gender ratios were significantly different between the PHP1 and PHPT groups (for both indexes, p < 0.001). The gender ratio between PHP1 and normal control groups was similar as the participants in the normal control group were recruited in a gender-matching pattern (p = 0.809), whereas a significant difference was found in the age distribution between the two groups (p < 0.001). All the participants in our study possessed a normal renal function of eGFR ≥ 60.00 mL/(min·1.73 m2). Patients with PHP1 had higher levels of serum Cr and calculated eGFR than patients with PHPT (p = 0.028 and p < 0.001, respectively). Compared with the normal control group, patients with PHP1 had a significantly higher level of serum Cr and eGFR value (p = 0.033 and p < 0.001, respectively), but the calculated eGFR levels in patients with PHPT were similar to those of the normal control group, even though the Cr level was lower in the PHPT group (p = 0.072 and p < 0.001, respectively, not shown in Table).

The results of iPTH and PTH(1–84) levels are shown in Table 2 and Fig. 1.

Box plot of PTH levels under two analyses of the samples of the 3 cohorts. Hollow squares in each diagram indicate the level of iPTH, while the ones filled with black spots indicate the level of PTH(1–84)

The iPTH and PTH(1–84) levels of patients with PHP1 were significantly lower than that of patients with PHPT (p = 0.030 and p = 0.027 respectively, not shown in the Table). As expected, the iPTH and PTH(1–84) levels of both PHP1 and PHPT were significantly higher than the normal control group. The simple linear regression indicated a strong linear relationship between iPTH and PTH (1–84) in the PHP1, PHPT, and normal control groups (r2 were 0.9661, 0.7733, and 0.9575, respectively; p < 0.001 for all three groups). (Regression equations are marked in Fig. 2).

Linear regression and Bland–Altman plots of iPTH and PTH(1–84) in PHP1, PHPT, and normal control groups. Percent Bias(%): ∆PTH/mean of iPTH and PTH(1–84). In each square, 2 figures of PHP1, PHPT, and normal control, respectively, are shown. A, C, E linear regression figures of PHP1, PHPT, and normal control, respectively, along with regression equations. The horizontal axis indicates the measured iPTH level, while the vertical axis indicates the measured PTH(1–84) level. R2 for linear regression were 0.9661, 0.7733, and 0.9575 for PHP1, PHPT, and normal control group, respectively. B, D, F Bland–Altman plots of Percent Bias (vertical axis) over Mean of iPTH and PTH(1–84) (horizontal axis) of PHP1, PHPT, and normal control, respectively. Mean is indicated with a solid line, while the Mean ± 1.96 SD are shown in the dashed line in the Figure

Serum iPTH levels were significantly higher than PTH(1–84) levels in all the three different groups (p < 0.001 for all), and the medium calculated ∆PTH% were 24.3%, 27.9%, and 15.0% for PHP1, PHPT, and the normal control group, respectively. Even though ∆PTH% and 3rd/2nd ratio of PHP1 and PHPT were similar, the indexes of PHP1 and PHPT groups were significantly higher than the normal control group. Notably, a total of nine cases of PHPT patients were present with a 3rd/2nd ratio over 1.0, which indicated a higher level of PTH (1–84) than iPTH in these nine patients.

Subgroup comparisons were also performed between the 16 patients with PHP1A and 38 patients with PHP1B. PHP1A and PHP1B groups showed similar iPTH and PTH (1–84) levels [medium iPTH level were 103.6 pg/mL and 74.6 pg/mL, and medium PTH (1–84) level was 77.2 pg/mL and 54.3 pg/mL, p = 0.495 and 0.437, respectively]. The calculated ∆PTH and ∆PTH% did not show any significant difference between the two subgroups (medium ∆PTH was 21.7 pg/mL and 19.4 pg/mL, and average ∆PTH% were 24.7% and 24.3%, p = 0.684 and 0.886, respectively). The average 3rd/2nd ratios were 0.75 and 0.76 for patients with PHP1A and PHP1B, respectively, which showed no significant difference (p = 0.887). Both iPTH/ULN and PTH (1–84)/ULN values were similar between the two subgroups (p = 0.495 and 0.437, respectively). Together, the two subgroups presented with similar levels of iPTH, PTH (1–84), ∆PTH, ∆PTH%, and 3rd/2nd ratio, iPTH/ULN, and PTH(1–84)/ULN compared with each other and with the entire PHP1 group, respectively (all p > 0.05, data not shown in Table).

Among the patients, 49 out of 54 patients with PHP1 and 89 out of 127 patients with PHPT had their serum sample stored under −80 °C for a median of 379 days (317.5, 473.5) and 90 days (72, 385), respectively. The medium level of iPTH at the time of serum collection was 129.2 pg/mL and 145.0 pg/mL for the two samples, respectively, which suggested a distinct declination of iPTH in both groups during storage. The calculated decay rate was 38.2 ± 18.2% in the PHP1 group and 39.8 ± 19.8% in the PHP1 group. The levels of iPTH at the time of sample collection and the decay rate did not differ significantly between the PHP1 and PHPT groups (p value were 0.091 and 0.631 respectively). Moreover, no significant differences were observed in the comparisons of ∆PTH%, 3rd/2nd ratio, iPTH/ULN, and PTH (1–84)/ULN between the stored sample and fresh samples (data not shown).

The results of the conformity test between PTH (1–84) and iPTH for evaluating PTH concentrations in PHP1 and PHPT patients are shown in Table 3.

Among patients with PHP1, 27 of them (50.0%) presented with increased PTH levels under both iPTH and PTH (1–84) assays, whereas 21 patients (38.9%) had normal PTH levels under both assays (Table 3A). The Kappa conformity test showed that in patients with PHP1, the Kappa value of PTH (1–84) assay and iPTH assay were 0.778 (p < 0.001). The number of patients with PHPT with increased PTH levels under both assays was 85 (66.9%), whereas 14 patients (11.0%) had a PTH level that was within the normal range of both assays (Table 3B). In patients with PHPT, the Kappa value of the two methods was 0.395 (p < 0.001), which indicated relatively poor conformity.

The conformity test was also performed in different subtypes of PHP1 (Table 4). In the 16 patients with PHP1A, the two assays showed good conformity with a Kappa value of 0.738 (p = 0.002, Table 4A), whereas the Kappa value of the test in the 38 patients with PHP1B was 0.791 (p < 0.001, Table 4B). Both conformity tests indicated that iPTH and PTH(1–84) agreed well with each other in evaluating PTH concentration in patients with PHP1.

For further conformity analysis, Bland–Altman plot was prepared. In PHP1 patients, the Bland–Altman plot showed that 96.3% of the data points were within Mean ± 1.96 SD (Fig. 2B). The mean of percent bias of PHP1 group was 26.1%, and the range of Mean ± 1.96 SD of percent bias was 2.2%–50.0% (Table 5). The proportion of data points within Mean ± 1.96 SD was 93.7% and 98.5% in the PHPT group and the normal control group, respectively (Fig. 2D, F). The mean of percent bias of these two groups were 31.2% and 17.0%, respectively, and the range of Mean ± 1.96 SD were −14.3%–76.6% and 6.7%–27.2%, respectively (Table 5).