Abstract

Objective

   To explore the difference in parathyroid tissue-derived cells between
   male and female PHPT patients.

Methods

   Resected parathyroid tissues were collected from PHPT patients of both
   sexes. Single cells were isolated and sequenced for RNA expression
   profiles. The cell sequencing data were annotated by cell type,
   followed by population analysis, functional analysis, pathway analysis,
   cell communication analysis, differential gene expression analysis, and
   pseudotime trajectory analysis. The subcluster analyses were also
   performed in the parathyroid cells.

Results

   No substantial difference in the cell population, function, or
   communication is found between the two sexes. The interferon-a
   response, oxidative phosphorylation, and reactive oxygen species
   pathways are up-regulated in females than in male patients, mainly
   contributed by fibroblast cells, endothelial cells, parathyroid cells,
   and myeloid cells, which also have significantly more up-regulated
   pathways and cellular interactions than the other three cell types. The
   subcluster analysis of parathyroid cells identified five
   subpopulations: SPARCL1-OC and ISG15-OC are predominant in females,
   while more S100A13-PCC and PTHLH-OC are found in males. The cellular
   functions are also elevated in females compared with males. Cells from
   female patients show a higher expression level of parathyroid hormone
   (PTH) but a lower expression level of parathyroid hormone-like hormone
   (PTHLH). The cell pseudotime trajectory and pathway analyses show that
   the oxyphil cells may be more mature and functionally active than the
   chief cells in both sexes.

Conclusion

   These findings suggest that the sex difference in PHPT may be caused by
   the differentially expressed genes and activated pathways in different
   cell types in the parathyroid tissue. The heterogeneity of parathyroid
   cell subpopulations, especially in oxyphil cells, may be associated
   with the sex differences in PHPT pathogenesis.

   Keywords: primary hyperparathyroidism, ScRNA-seq, sex difference,
   cellular interactions, gene expression

Introduction

   Primary hyperparathyroidism (PHPT) is a common endocrine disorder
   induced by the overproduction of parathyroid hormone (PTH) ([39]1).
   Specifically, the hyperplastic parathyroid glands secrete PTH
   regardless of the blood calcium level, causing excessive calcium to be
   released from bones and kidneys into the blood, leading to
   hypercalcemia and a series of skeletal, renal, and neurological
   complications ([40]1). Typical clinical presentations of PHPT include
   osteoporosis, nephrolithiasis, gastrointestinal disorders, and
   neurological and cognitive impairment ([41]2). The incidence rate of
   PHPT ranges between 0.4 to 82 cases per 100,000 people across different
   races and regions worldwide ([42]2–[43]4). In countries and regions
   where blood calcium is routinely measured, such as the US and western
   Europe, PHPT is often diagnosed early and well managed. Therefore, most
   PHPT patients appear to be asymptomatic ([44]1). In contrast, the
   majority of PHPT patients in Asia, South Africa, and the Middle East
   are symptomatic ([45]4). Despite the regional difference, the incidence
   of PHPT varies significantly by sex: the female-to-male ratio in PHPT
   patients is approximately 3:1 ([46]5). This sexual dimorphism appears
   to be wider in patients above 50 years old ([47]6), i.e., the highest
   prevalence of PHPT is reported in postmenopausal women ([48]7).
   Interestingly, male patients tend to be significantly younger and more
   frequently symptomatic than female patients, although no sex difference
   can be found in the levels of PTH, blood calcium, and urinary calcium.
   Nephrolithiasis and osteoporosis are the dominant PHPT symptoms in men
   and women, respectively ([49]8). Unfortunately, little is known about
   the sex differences in PHPT incidence and clinical presentation. While
   estrogens might be potentially associated with PTH secretion, no direct
   evidence has been discovered ([50]9). The role of sex in PHPT
   pathogenesis and outcome remains unclear, and therefore, personalized
   and sex-specific treatment of PHPT is still impossible to be
   implemented.

   Single-cell RNA sequencing (scRNA-seq) is an emerging technology that
   enables the profiling of the transcriptomes of a heterogeneous cell
   population ([51]10). Compared with the traditional bulk RNA-seq
   techniques, scRNA-seq allows the discovery of rare cells in a complex
   cell population, as well as the regulatory relationships between genes
   ([52]11). Due to these unique merits, scRNA-seq has been widely applied
   in diverse fields, such as cancer, neurodevelopment and
   neurodegeneration, and hematological diseases ([53]12–[54]14). The
   parathyroid tissue has been found to be composed of three types of
   cells, including chief cells, oxyphil cells, and lymphocytes, with
   different relative abundance, genetic, and functional behaviors. Such
   heterogeneity may lead to alternate etiologies of PHPT, which may
   further translate into various clinical presentations and sex
   differences ([55]15). Therefore, it might be worth investigating
   whether the parathyroid cells exhibit a heterogeneous transcriptome,
   and how it is associated with PHPT incidence, symptoms, and sex
   differences.

   In this study, we aim to reveal the sex differences of PHPT through the
   scRNA-seq of dissected parathyroid tissues from PHPT patients. By
   annotating seven distinct cell categories, we analyzed their
   proportion, functions, pathways, communication, subclusters, and
   differentially expressed genes. We discover that the proportions of
   mast cells and B cells differ significantly in male and female PHPT
   patients. We also find in the sub-cell type analysis that PTHLH-OC and
   SPARCL-OC are the prominent oxyphil cells in males and females,
   respectively. Sex differences are also observed in the cell function,
   pathway, and communication analyses. Our research discovers new
   evidence for elucidating the sex difference of PHPT from the
   perspective of parathyroid cell heterogeneity, and is thus expected to
   provide insights into the sex-specific PHPT treatment.

Materials and methods

Patients and specimen

   A total of 4 patients (2 males and 2 females) who underwent parathyroid
   resection surgery at Beijing Jishuitan Hospital were recruited in our
   study between January 2020 and January 2022. Signed informed consent
   forms were obtained from all subjects before the study. The resected
   parathyroid tissue samples were collected from all patients during the
   surgery and stored at -80 ˚C. This study was reviewed and approved by
   the Institutional Review Board of Beijing Jishuitan Hospital (Reg. No.
   201905–01).

ScRNA-seq of the parathyroid tissue

   The single-cell suspensions were prepared from the resected parathyroid
   tissue as previously described. The scRNA-seq libraries were
   constructed using the Chromium Single Cell 3’ Reagent Kit v2 under the
   manufacturer’s instructions. Firstly, cells in the suspension were
   loaded onto a Chromium Single Cell Controller (10X Genomics, USA) to
   prepare the Gel Bead-in-Emulsions (GEM), in which the poly-adenylated
   mRNA in each cell was reverse-transcripted into cDNA encoded with a
   16-bp unique sequence that varied between different cells. Next, a
   recovery agent was added to disrupt the emulsion. The cDNA was cleaned
   up with DynaBeads Myone Silane Beads (ThermoFisher Scientific, US),
   amplified by PCR, fragmented, end-paired, A-tailed, and ligated with an
   index adapter following the manufacturer’s instructions. The cDNA
   libraries were sequenced with a HiSeq X platform (Illumina, USA) into
   150-bp paired-end read data.

ScRNA-seq data analysis

   The scRNA-seq data were analyzed following a previously described
   workflow ([56]16). Data loading and quality control were performed with
   the R package Seurat (v 4.0.1) ([57]17). The data of cells that met any
   of the following conditions were discarded before the analysis ([58]1):
   The total unique molecular identifier (UMI) count was below 1,200
   ([59]2); The total number of genes was below 300 ([60]3); The
   mitochondrial gene proportion was more than 20%. Based on these
   criteria, 16,474 cells were selected for further analysis, with 5,726
   and 10,748 cells from male and female patients, respectively. Data
   integration was performed using the R package Harmony (Version 0.1)
   with default parameters ([61]18). A total of 2,000 high variable genes
   were screened. The dimension reduction was performed using the top 30
   principal components (PCs). For optimal cell clustering, the resolution
   for visualization was set to 0.2.

Pseudotime trajectory analysis

   For pseudotime trajectory analysis, R packages Monocle3 (Version 1.2.9)
   and Monocle (Version 2.18.0) were utilized separately with their
   default parameters ([62]19). The root point was determined with the
   graph learning approach.

Cell communication analysis

   The scRNA-seq data were processed by the R package CellChat (Version
   1.4.0) to investigate cell communication and interaction. From the
   CellChatDB database, we selected a total of 1,939 verified molecular
   interactions for cell communication analysis ([63]20).

Functional enrichment analysis

   We used the R package ClusterProfiler (Version 4.0) to perform the
   functional enrichment analysis through the Gene Ontology (GO) and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) databases ([64]21). We scored
   the gene signatures using the following R packages: UCell (Version
   1.3), singscore (Version 1.2.2), AUCell (Version 1.12.0), GSVA (Version
   1.38.2), and irGSEA (Version 1.1.3) ([65]22–[66]25).

Statistical analysis

   We performed all statistical analyses with the R software (Version
   4.0.5). The scatter plots were generated by the R package ggstatsplot
   (version 0.7.1). P-values below 0.05 were considered statistically
   significant in this study.

Results

Cellular contribution analysis

   We manually annotated the parathyroid cells into seven major cell
   types, including parathyroid cells, fibroblast cells, T cells,
   endothelial cells, myeloid cells, mast cells, and B cells. The
   clustering results are visualized with Umap, as shown in [67]Figure 1A
   . [68]Figure 1B depicts the expression profiles of the characteristic
   markers of each cell type, agreeing well with the clearly separated
   cell populations in [69]Figure 1A . [70]Figure 1C compares the cell
   proportions in male and female patients. It can be observed that female
   patients have a higher proportion of mast cells but a lower proportion
   of B cells compared with male patients, while there is no significant
   difference in other cell types. [71]Figure 1D shows the expression
   profiles of top genes across cell types. Interestingly, in addition to
   the five markers used for the annotation of parathyroid cells (GCM2,
   KL, CASR, VDR, LRP2, and PTH), we find that CHGA, RARRES2, PVALB, and
   CISD1 are also highly expressed, suggesting their potential as specific
   markers for parathyroid cells. [72]Figure 1E displays the localizations
   of cells expressing typical markers on the Umap, demonstrating the high
   annotation accuracy in detail.

Figure 1.

   [73]Figure 1
   [74]Open in a new tab

   Summary of cell composition in PHPT patient samples. (A) Umap
   visualization of the seven manually annotated cell populations. (B) Dot
   plot of representative cell markers of each cell type. (C) Cell
   proportion comparison between male and female patient samples. (D)
   Heatmap of top five markers of each cell type. (E) Feature plot of
   representative cell markers of each annotated cell type.

Cell functional state and pathway analysis

   We explored the cell functions based on the pathway analysis.
   [75]Figure 2A shows the numbers and proportions of
   differentially-expressed genes as evaluated by four algorithms,
   including AUCell, Ucell, singscore, and ssgsea. The results of these
   algorithms are inconsistent when comparing the male and female groups:
   AUCell and Ucell find more up-regulated pathways in males than in
   females, while singscore and ssgsea show an opposite trend. This may
   indicate that the cellular functions do not differ significantly
   between the two sexes. However, these algorithms show similar trends
   when the cells are grouped by cell type ([76] Figure 2B ). Parathyroid
   cells, endothelial cells, fibroblast cells, and myeloid cells have
   higher numbers of up-regulated pathways compared with other cell types.
   These results may suggest that these four types of cells are the
   principal functional cells in PHPT development.

Figure 2.

   [77]Figure 2
   [78]Open in a new tab

   Functional analysis of PHPT patient samples. (A) Bar plot of the count
   and proportion of significant regulation pathways based on four
   different algorithms by sex. (B) Bar plot of the count and proportion
   of significant regulation pathways based on four different algorithms
   by cell type. (C-K) Pathway analysis of three key PHPT-related pathway,
   including interferon (C–E), oxidative phosphorylation (F–H), and
   reactive oxygen species (I–K). Violin plots compare the UCell scores of
   these pathways between the two sexes (C, F, I). Feature plots based on
   Umap show the pathway score distribution across all cell types (D, G,
   J). Ridge maps show the pathway scores of each cell type (E, H, K).

   We then sought to identify the pathways differentially regulated
   between males and females. As shown in [79]Figures 2C, F, I , the
   interferon-a response, oxidative phosphorylation, and reactive oxygen
   species pathways are up-regulated in female than in male patients.
   Mapping these pathways to cells shows that these differences may be
   attributed to certain cell types ([80] Figures 2D, E, G, H, J, K ).
   Specifically, the interferon-a response pathways may be attributed to
   myeloid cells and endothelial cells, whereas the oxidative
   phosphorylation and reactive oxygen species pathways may be attributed
   to parathyroid cells. Fibroblast cells may also have contributed to the
   reactive oxygen species pathway upregulation. Taken together, these
   results suggest that these four types of cells may serve as the key
   regulatory cells in PHPT pathogenesis and play essential roles in the
   sex differences of PHPT.

Cell communication analysis

   The results of the cell communication analysis are shown in
   [81]Figure 3 , showing a similar trend to the pathway analysis. As
   shown in [82]Figure 3A , the interactions between parathyroid cells,
   endothelial cells, fibroblast cells, and myeloid cells are predominant
   compared with those between other cell types. However, there is no
   significant difference between male and female patients in either the
   number of interactions or the interaction strength ([83] Figures 3B, C
   ). By visualizing the differential interaction strength by cell type
   between sexes ([84] Figure 3D ), i.e., subtracting the interaction
   strength of males from that of females, we find that the interactions
   related to fibroblast cells are dominant compared with all interactions
   involving other cell types. This is further confirmed in [85]Figure 3E
   , as the number of interactions and interaction strength from
   fibroblast cells far outnumber those from all other cell types in
   females than in males. [86]Figure 3F shows the top five up-regulated
   pathways related to cell communication, among which the FGF pathway is
   identified as the most activated pathway. As shown in [87]Figure 3G ,
   fibroblast cells and endothelial cells are the major cell types that
   send regulatory signals through the FGF pathway to themselves, as well
   as to the parathyroid cells. In [88]Figure 3H , we show in detail the
   sender-receiver interactions between FGFs and FGFRs in different cell
   types and sexes. It can be observed that the FGF7-FGFR1 and FGF7-FGFR2
   communications from fibroblast cells to parathyroid cells show higher
   probabilities in females than in males, which might be associated with
   the sex differences.

Figure 3.

   [89]Figure 3
   [90]Open in a new tab

   Cell communication analysis of PHPT patient samples. (A) String plot of
   the aggregated cell-cell communication network, showing the number of
   interactions between cell types. (B) Comparison of the number of
   interactions between male and female patients.ns=not statistically
   significant (unpaired t-test). (C) Comparison of the interaction
   strength between male and female patients. ,ns=not statistically
   significant (unpaired t-test). (D) Differential interaction strength
   comparison between male and female patients. (E) Heatmap of interaction
   numbers and strength in different cell types. (F) Pathway enrichment
   analysis of genes differentially expressed in two sexes. (G) FGF
   signaling pathway network of all cell types. (H) Enriched
   receptor-ligand pairs between parathyroid cells and other cell types in
   the FGF pathway.

Subcluster analysis of parathyroid cells

   We then performed a subcluster analysis of the parathyroid cells and
   identified five subtypes, including two types of parathyroid chief
   cells (S100A13-PCC and NEAT-PCC) and three types of oxyphil cells
   (ISG15-OC, PTHLH-OC, and SPARCL-OC), as shown in [91]Figure 4A . The
   expression levels of subtype-specific markers are shown in
   [92]Figure 4B . An intriguing finding is that the subpopulations of
   parathyroid cells differ significantly between females and males:
   SPARCL1-OC and ISG15-OC are predominant in females (especially
   SPARCL1-OC, which is exclusively found in females), whereas more
   S100A13-PCC and PTHLH-OC are found in males ([93] Figures 4C, D ).
   Functional analysis reveals that the parathyroid cells in females are
   up-regulated compared with those in males, as predicted by all four
   scoring algorithms ([94] Figure 4E ). This may indicate that the female
   parathyroid cells may have a higher level of functional activation than
   males. In the functional analysis by parathyroid cell subtype, we find
   that the oxyphil cells (ISG15-OC, PTHLH-OC, and SPARCL-OC) have more
   significantly up-regulated pathways than the chief cells in PHPT
   pathogenesis ([95]Figure 4F). (S100A13-PCC and NEAT-PCC), suggesting
   that the oxyphil cells may play more important roles than the chief
   cells in PHPT pathogenesis. In addition, we also calculated the Simpson
   index of overall cell proportion and parathyroid cells proportion
   separately. Compared with male, female have a lower heterogeneity of
   overall cells (male, 0.75; female, 0.70) but higher heterogeneity in
   parathyroid cells (male,0.55; female, 0.69).

Figure 4.

   [96]Figure 4
   [97]Open in a new tab

   Subpopulation analysis of parathyroid cells. (A) Umap visualization of
   parathyroid subpopulation (B) Dot plot of representative cell markers
   of each cell subtype. (C) Umap visualization of cells derived from male
   and female patients. (D) Cell subpopulation proportion comparison
   between male and female patients. (E) Bar plot of the count and
   proportion of significant regulation pathways based on four different
   algorithms by sex. (F) Bar plot of the count and proportion of
   significant regulation pathways based on four different algorithms by
   cell type.

Differential gene expression analysis

   By performing the differential gene expression analysis, we identified
   78 differentially expressed genes between male and female parathyroid
   cells, including 41 up-regulated genes and 37 down-regulated genes
   ([98] Figure 5A ). Compared with males, the top up-regulated genes in
   females are FAB5, VIM, and NEFH, while the top down-regulated genes in
   females are CFB, SLC7A7, and PLCG2. Functional analysis reveals that
   these up-regulated genes are associated with interferon-gamma response
   and estrogen transportation. It can be observed from [99]Figure 5B that
   PTH is expressed in all cell subpopulations in both sexes, but elevated
   in females compared with males. In contrast, the expression level of
   parathyroid hormone-like hormone (PTHLH) is increased in the males
   compared with females, as shown in [100]Figure 5C .

Figure 5.

   [101]Figure 5
   [102]Open in a new tab

   Subpopulation analysis of parathyroid cells. (A) Volcano plot and
   functional enrichment analysis of the differential expressed genes
   between female and male patients. Red points represent the
   significantly up-regulated genes in males compared with females
   (adjust-p<0.01, logFC>1). Blue points represented the significantly
   down-regulated genes in males compared with females (adjust-p<0.01,
   logFC<-1). (B, C) Expression levels of PTH and PTHLH in different cell
   subpopulations.

Cell pseudotime and communication analysis

   We conducted pseudotime trajectory analysis to investigate the cell
   differentiation process in the parathyroid tissue. As shown in
   [103]Figure 6A , the oxyphil cells are more mature than the chief
   cells, suggesting that the oxyphil cells might serve as the major
   functional cells in the parathyroid and may be more closely related to
   PHPT. Cell communication analysis reveals that oxyphil cells contribute
   more to cell interaction than chief cells in both male and female
   patients ([104] Figures 6B, C ). Compared with females, parathyroid
   cells in males have a similar number but a slightly higher strength of
   cell interaction ([105] Figure 6D ). By performing the differential
   analysis, it can be observed that communications between oxyphil cells
   are the major contributors to the apparent difference in interaction
   strength between sexes ([106] Figures 6E–G ). The analyses of PTH and
   FGF signaling pathway networks ([107] Figures 6H, I ), as well as the
   weight analyses by cell subtype ([108] Figures 6J, K ), suggest that
   PTHLH-OC plays a central role in the communications through both
   pathways and may be the major contributor for PTH and FGF production.

Figure 6.

   [109]Figure 6
   [110]Open in a new tab

   Pseudotime trajectory analysis and communication analysis of
   parathyroid cells. (A) Pseudotime trajectory analysis of parathyroid
   cell developmental stages. The colormap shows the pseudotime trajectory
   (B, C) String plot of the aggregated cell-cell communication network of
   male (B) and female (C) parathyroid cells. (D) Interaction number and
   strength comparison between male and female patients *= p < 0.05, ns,
   not statistically significant (unpaired t-test). (E) Differential
   interaction strength comparison between male and female patients. (F)
   Heatmap of interaction numbers and strength in different cell
   subpopulations. (G) Pathway comparison between male and female patients
   by parathyroid cell subpopulation. (H, I) String plot of PTH (H) and
   FGF (I) signaling pathway networks. (J, K) Heatmap of PTH (J) and FGF
   (K) signaling pathway networks.

Discussion

   In this study, we revealed the sex differences in PHPT patients at the
   transcriptome level using scRNA-seq. Specifically, we investigate the
   cell population composition, cell functional state, and communication
   in resected parathyroid tissues from male and female patients. We
   further identify seven subclusters in parathyroid tissue and analyze
   their differentially expressed genes, pathways, and communications with
   regard to sexes.

   Overall, no substantial difference in the cell population or cell
   communication is found between the two sexes. However, by analyzing the
   contributions by cell type, we show that fibroblast cells, endothelial
   cells, parathyroid cells, and myeloid cells have significantly more
   up-regulated pathways and cellular interactions, especially the
   fibroblast cells and endothelial cells. Furthermore, in the pathway
   analysis, we find that the interferon-a response, oxidative
   phosphorylation, and reactive oxygen species pathways are more
   up-regulated in females than in males. The up-regulated pathways are
   also mainly attributed to the aforementioned four cell types. Whereas
   previous studies mainly focused on the parathyroid cell and lymphocyte
   populations ([111]15), our findings suggest that the fibroblast cells
   and endothelial cells are functionally active in the parathyroid and
   may play essential regulatory roles. These two cell populations may
   also be related to the sex difference between PHPT patients, which
   requires further investigation.

   The cell population composition, functional state, and communication in
   parathyroid tissue play an important role in the pathogenesis of
   primary hyperparathyroidism (PHPT). The clinical significance of
   understanding these cellular characteristics lies in the potential to
   identify new therapeutic targets and improve patient outcomes.
   Moreover, understanding the differences in cellular characteristics
   between male and female patients with PHPT can provide insights into
   the mechanisms underlying the sex differences in disease incidence,
   progression, and response to treatment. This knowledge may enable the
   development of sex-specific diagnostic and therapeutic strategies. In
   summary, the study of cell population composition, functional state,
   and communication is crucial for advancing our understanding of PHPT
   pathogenesis and improving patient care.

   An interesting finding is that PTH is widely expressed in all five
   subtypes of parathyroid cells, with higher expression levels in females
   than in males, whereas the PTHLH levels in males are higher than in
   females. PTH is mainly secreted from the parathyroid tissue, while
   PTHLH can be produced by almost all types of tissue in the body. PTH
   and PTHLH share the same PTH/PTHrP receptor but have different roles:
   PTH primarily regulates the blood calcium level, but PTHLH is involved
   in many other processes such as chondrocyte growth, mammary gland
   branching morphogenesis, and cancer ([112]26). It has been reported
   that PTHLH is predominantly expressed in the oxyphil cells in the
   parathyroid tissue ([113]27), but its potential implications in the sex
   difference of PHPT remain unclear. It would be valuable to compare the
   PTH and PTHLH expression levels in both sexes and further investigate
   their associations with PHPT development and clinical manifestations.

   The subcluster analysis of the parathyroid cells also shows a
   substantial difference between males and females in the oxyphil cells.
   The SPARCL1 oxyphil cells are exclusively found in females, while the
   PTHLH oxyphil cells are the major oxyphil cells in males. Our findings
   suggest that the oxyphil cells may play more important roles than the
   chief cells in PHPT: the cell pseudotime trajectory and pathway
   analyses show that the oxyphil cells may be more mature and
   functionally active than the chief cells; cell communication analysis
   reveals that the oxyphil cells are mainly responsible for cellular
   interactions; the PTH signaling pathway network also suggests that the
   oxyphil cells contribute more than the chief cells. Our result agrees
   with previous studies that more PTH is mainly produced by the oxyphil
   cells, instead of the chief cells ([114]15, [115]28). Also, it has been
   reported that the number of oxyphil cells increases dramatically with
   age and in patients with chronic kidney disease, which often leads to
   secondary hyperparathyroidism ([116]29). Our findings confirm at the
   single-cell level that the oxyphil cells are more essential influencers
   than the chief cells in cellular function and communication. Also, the
   heterogeneity of the oxyphil cell subpopulation may lead to the
   dramatic difference in PHPT incidence across sexes, which needs to be
   confirmed in future studies.

   The study found that there was no significant difference in the overall
   cell population, function, or communication between the two sexes.
   However, several pathways, including the interferon-a response,
   oxidative phosphorylation, and reactive oxygen species pathways, were
   up-regulated in females compared to males, mainly contributed by
   specific cell types. The subcluster analysis of parathyroid cells
   revealed different subpopulations in males and females. The study also
   found differences in the cellular functions and expression levels of
   certain hormones. The study suggests that the sex difference in PHPT
   may be caused by the differentially expressed genes and activated
   pathways in different cell types in the parathyroid tissue,
   particularly in oxyphil cells.

Data availability statement

   The datasets presented in this study can be found in online
   repositories. The names of the repository/repositories and accession
   number(s) can be found in the article/supplementary material.

Ethics statement

   The studies involving human participants were reviewed and approved by
   the Institutional Review Board of Beijing Jishuitan Hospital (Reg. No.
   201905–01). The patients/participants provided their written informed
   consent to participate in this study.

Author contributions

   SL, XC and XJ conceived and coordinated the study. SL, XC and XJ
   designed this study. SL, XC, MG, SC, JZ, XZ, CW, and AC performed and
   analyzed the experiments, wrote the paper. SL, XC, XZ, CW, and AC
   carried out the data collection, data analysis. XJ revised the paper.
   All authors contributed to the article and approved the submitted
   version.

Funding Statement

   This study was supported by National Key R&D Project (2022YFC2407501),
   Beijing JST Research Funding (ZR-202206), Beijing Municipal Health
   Commission (BMHC2019-9) and National Key R&D Program of China
   (2021YFC2501700). The funders had no role in study design, data
   collection and analysis, decision to publish, or preparation of the
   manuscript.

Conflict of interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Publisher’s note

   All claims expressed in this article are solely those of the authors
   and do not necessarily represent those of their affiliated
   organizations, or those of the publisher, the editors and the
   reviewers. Any product that may be evaluated in this article, or claim
   that may be made by its manufacturer, is not guaranteed or endorsed by
   the publisher.

References