Primary hyperparathyroidism (PHPT) is a common endocrine disorder that is associated with inappropriately elevated parathyroid hormone (PTH) with hypercalcemia and predominantly affects post-menopausal women. The majority of PHPT disease occurs due to single gland parathyroid adenoma (PA; ∼85 %) followed by multi-glandular disease (double PA and hyperplasia), which accounts for ∼15 % of cases, while parathyroid carcinoma (PC) is rare (∼1 %) [1], [2]. PHPT often occurs as a sporadic disease (90 %), however, the familial disease is also reported in 10 % of the total cases in the form of either multiple endocrine neoplasia syndrome 1 (MEN1), MEN2, MEN3, MEN4, hyperparathyroid-jaw tumor (HPT-JT) syndrome, familial isolated hyperparathyroidism (FIHP), or familial hypocalciuric hypercalcemia (FHH) [3]. The prevalence of PHPT in the general population ranges from 0.1 % to 0.8 %, with notable differences between Western and Eastern populations [2]. In Western countries, PHPT typically occurs later in life, with 80–90 % of cases reported during the asymptomatic stage. In contrast, in Eastern countries such as India and China, the majority of the cases are reported in middle age and are often symptomatic, mainly presented with bone disease, renal stone disease and gastrointestinal manifestations [1], [2], [4], [5]. Recent studies have reported an increase in asymptomatic cases from China and India *[6], [7].

PHPT is caused either by abnormal proliferation of parathyroid cells or alteration in calcium inhibition set point control followed by cell proliferation [1], [2]. In sporadic PHPT, tumor cells accumulate genetic and epigenetic alterations (primarily DNA methylation and histone modifications) leading to activation of protooncogenes and loss of tumor suppressor genes. Given the higher frequency of symptomatic PHPT in the East, the molecular pathogenesis may differ, prompting numerous molecular studies from Eastern countries, especially in India. The current review succinctly covers the current knowledge of molecular pathogenesis of sporadic PHPT with focus on findings from symptomatic cases. Additionally, we discuss recent technical advances and their utility in identifying new potential molecules involved in parathyroid tumorigenesis.

MEN1 gene is a ubiquitously expressed gene located at chromosome 11q13 and encodes 610 amino acids protein menin. It interacts with multiple proteins for its diverse functions including transcriptional regulation (differentiation, proliferation, cell cycle, apoptosis), epigenetic regulation, DNA replication, repair, and genome stability [8]. MEN1 gene was initially identified in context of familial PHPT – MEN1 syndrome, have genetic predisposition to parathyroid tumors and other endocrine tumors -pancreatic neuroendocrine tumors and pituitary tumors [9], [10]. Germline-inactivating mutations in MEN1 gene were reported in 90 % of familial cases and ∼45 % of sporadic MEN1 cases. Patients who have parathyroid tumors as a part of MEN1 syndrome generally have multiglandular parathyroid tumors, are younger and have borderline hypercalcemia [11], [12]. In sporadic PA, somatic biallelic inactivating mutations (frameshift, nonsense, missense) and large deletions in MEN1 gene were observed in 30–40 % of cases [13], [14], [15]. In sporadic PC, somatic mutation is reported in ∼15 % of cases [16], [17], however overall mutations germline or somatic mutations are rare in PC [18] A recent study by our group has identified 26 germline variants in MEN 1 gene (including eight pathogenic/likely pathogenic, nine VUS, eight benign, one splice site, and one regulatory variation) in 50 % (42/81) of sporadic PHPT. The in-silico functional analysis found that c.1525 C>A (p.Leu509Met) was associated with multi-glandular parathyroid tumors and c.–35A>T (5′UTR) as the most common variant associated with PHPT recurrence [19]. Patients with germline variants were significantly younger (mean age 30 years) and higher number of patients had bone disease and renal stone disease compared to variant-negative patients. Thus, screening of young symptomatic sporadic PHPT for germline variants in MEN1 gene could link to better diagnosis and management plans.

Structural analysis revealed that the menin protein lacks a DNA-binding pocket but instead interacts with transcription regulatory machinery components to control transcription. Studies have also explored MEN1's epigenetic role, as menin can interact with MLL1/2 complex which is involved in H3K4me3 histone methylation. However, H3K4me3 levels remain unaffected in MEN1-related PA [16], [20]. Additionally, parathyroid-specific Men1 knockout mice develop parathyroid hyperplasia with hypercalcemia, establishing MEN1 as a tumor suppressor gene in sporadic PHPT [21], [22], [23].

Cyclin D1, a cell cycle regulator, involved in G1 to S phase transition, has an oncogenic role in molecular pathogenesis of sporadic PHPT. It was initially identified in sporadic PAs involved in pericentromeric inversions with upstream PTH promoter at chromosome 11q13 and named accordingly as parathyroid adenoma 1 (PRAD1) [3], [24]. Extracellular signals activate cyclin D1, and at G1-S phase transition binds to the cyclin-dependent kinases - CDK4 and CDK6. Cyclin D1- CDK4/6 complex in turn phosphorylates retinoblastoma protein (RB), resulting in its dissociation from E2F family transcription factors, which then activate downstream molecules [25]. Overexpression of cyclin D1 in parathyroid cells accelerates progression from G1 to S phase, thereby causing excessive cell proliferation and tumor formation. Although cyclin D1 overexpression both at gene and protein levels has been reported in 20–40 % of sporadic PC and approximately 70 % in sporadic PC from Western countries [26], [27]. However, the rearrangement of CCND1 gene with PTH promoter occurs only in 5–8 % of those with overexpressed cyclin D1 cases [28], [29], suggesting the still unexplored mechanism associated with cyclin D1 overexpression such as activation via upstream signalling molecule, protein structure stability, and alternative splicing.

Study on transgenic mice model with CCND1-PTH rearrangement have shown parathyroid specific cyclin D1 overexpression led to a hyperparathyroid state and confirmed the role of cyclin D1 as a driver of abnormal parathyroid cell proliferation [30]. Recent studies have also reported increased CCND1 copy number (70 %) in sporadic PC compared to sporadic PA (20 %) [31], [32]. Data from symptomatic sporadic PA showed overexpression in 85 % of cases with a strong correlation with adenoma weight and trending with higher PTH secretions. This molecular dissimilarity between the West and East strongly supported the clinical severity of disease in developing countries [33].

Cell cycle regulators are comprised of cyclin-dependent kinases (CDKs) that bind with cyclins and activate G1-S phase activation and CDK inhibitors (CDKIs) that regulate the CDK-Cyclin D1 activity and inhibit the cell cycle progression. Inactivation of CDKIs as well as activation of CDK4/CDK6 can also potentiate the excessive cell proliferation activity in sporadic parathyroid tumors. Our study has shown the over-expression of CDK4, CCND1, E2F1 (a downstream transcription factor in cell cycle mechanism) genes [34]. Both CDKN1 and CDKN2 family of inhibitors act as tumor suppressor molecules and appear to have a role in parathyroid tumorigenesis [35]. Germline mutations in CDKN1B, which encodes the p27 protein, are mainly associated with MEN4 syndrome [36], [37], whereas somatic mutations in CDKN1B are rarely reported in sporadic PHPT [21]. Germline or somatic mutations in CDKN2C, a member of the CDKN2 family of inhibitors that encodes p18 is also rare in sporadic PA [38].

Studies have supported the role of epigenetic mechanism in CDKN2A (p16) and CDKNB (p15) genes in sporadic PA *[39], [40]. Western data primarily from asymptomatic PHPT suggested the infrequent promoter hypermethylation in CDKN2A but not in CDKN2B associated with reduced gene expression [41], *[42]. Our study showed the reduced gene expression of CDKN2A, and CDKN2B and inversely associated with CCND1 expression in symptomatic sporadic PA. DNA promoter hypermethylation was observed in ∼ 50 % of cases with reduced expression. So, CDKN2B has an equivalent role as CDKN2A in symptomatic sporadic PA [39]. Overall genetic alterations are rare in CDKIs and epigenetic alterations in CDKIs are also regulated to upstream signaling are not drivers of parathyroid tumorigenesis.

WNT β-catenin signaling pathway molecules are associated with cell proliferation, angiogenesis, differentiation and genetic alterations in molecules are frequently reported in many cancers [43]. However, somatic mutation initially thought to be a major contributor is rarely reported in sporadic PA (<1 %). The low-density lipoprotein receptor-related protein 5 (LRP5), a WNT co-receptor appears to be truncated in 86 % of sporadic PHPT [41], [44]. Additionally, there is a report of LRP 5 mutation found exclusively presented with MEN1 frameshift mutation in a patient, making it difficult to study its functional role [21]. Studies have reported the role of WNT signaling inhibitors like APC and RASSF1A. APC causes familial adenomatous polyposis coli and is named based on disease. Reduced expression of APC and promoter region hypermethylation have been reported in sporadic PHPT[45], [46]. RASSF1A promoter region was also frequently hypermethylated insporadic PA with reduced expression both in symptomatic and asymptomatic cases equally *[39], [47], [48]. So, the role of β-catenin and WNT signaling pathway molecules in sporadic PHPT still needs to be established.

The CASR gene consists of two promoters, the P1 promoter containing a TATA box and a CAAT box, and the P2 promoter is GC rich that result in alternative transcripts into exon 1 A and exon 1B [49]. The two promoter regions of CASR suggest tissue-specific regulation and alternatively, spliced mRNA transcripts and contain vitamin D response elements (VDREs) that allow 1,25-dihydroxy vitamin D3, the active form of vitamin D, to activate CASR expression [50]. In PHPT, CASR expression was reduced in both sporadic PA and PC [51], *[52]. Reduced CASR leads to insensitivity of CASR receptor, and alterations in calcium-set point which in turns require higher PTH secretion for extracellular calcium regulation, however mechanistic linked still not established. In animal studies, using cinacalcet as a positive allosteric modulator of the CASR, could control hyperplasia state in parathyroid, and was reversed upon stopping the treatment in mice with chronic kidney disease [53], [54]. Our group has reported that reduced CASR expression at both the gene and protein levels is affected by DNA methylation at promoter 2 and histone 3 trimethylation at lysine 9 (H3K9me3) in symptomatic PA [52]. A recent study also reported significantly lower CASR expression in symptomatic compared to asymptomatic PHPT from the same centre [51]. These findings propose CASR as a potential contributor to symptomatic sporadic parathyroid tumors and link the specific molecular signatures with clinical phenotypes.

The vitamin D receptor (VDR) is a nuclear receptor that instigates PTH secretion and parathyroid cell proliferation by regulating calcium and phosphate homeostasis through calcitriol (vitamin D) [55]. Due to the high prevalence of vitamin D deficiency in developing countries such as India, severe symptomatic PHPT cases are frequently observed [56]. This occurs because insufficient vitamin D disrupts normal feedback mechanisms, leading to uncontrolled PTH release and abnormal parathyroid cell proliferation. Studies have shown the reduced expression of VDR both at gene and protein levels [57], *[58], Dysregulation of VDR expression, as evidenced by a marked overrepresentation of polymorphic VDR alleles a, b, and T, is considered significant in sporadic PHPT [59]. Reduced VDR expression has been associated with increased PTH levels and disrupted calcium regulation in parathyroid tumors. A recent study showed a significant downregulation of VDR at both the gene protein levels in severe PHPT [51]. Genomic and transcriptomic studies, along with epigenetic modification analyses, have provided deep insights into VDR expression in PHPT [40], [60]. A recent data has indicated that inhibition of EZH2-mediated H3K27me3 restores VDR expression in the in-vitro PTHC-1 continuous cell line (unpublished data). This could be possible because the VDRE is present on the EZH2 gene by which VDR expression and functions could be regulated by EZH2-mediated histone modifications [61], [62]. Future research should prioritize the exploration of histone modifications regulated VDR-linked molecular events in sporadic PHPT.

EZH2, a histone methyl transferase enzyme, is part of an evolutionarily conserved Polycomb repressive complex 2 (PRC2) comprised of three molecules EZH2, SUZ12 and EED. EZH2 catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3) on the chromatin surface leading to the transcriptional silencing of genes [63]. Studies have highlighted the role of EZH2 in various cancers including endocrine and non-endocrine diseases involving heart, kidney, pancreas, glioblastoma, ovaries, adrenal, thyroid, etc., but studies are limited [64], [65]. EZH2 is involved in tumor progression by transcriptional silencing of target genes through mono-di-tri methylation of H3K27, a repressive chromatin histone marker. Overexpression and/or mutations inhibit tumor suppressor genes and activate oncogenes, increasing cell proliferation, anti-apoptotic pathways and migration potential in cancers [66]. An in-vivo study utilizing an EZH2 knockout mouse model revealed that EZH2 regulates parathyroid gland development during embryogenesis by regulating the expression of embryonic transcription factors such as Tbx1 [67]. In parathyroid tumors, whole exome sequencing and expression studies identified low frequencies of EZH2 mutation, copy number variations and gene expression were significantly higher in PC cases only [68], [69], [70]. An unpublished study from Asian Indian symptomatic PHPT identified a major contribution of EZH2 in PHPT with high gene and protein nuclear positivity in sporadic PA, highest in sporadic PC and atypical parathyroid tumors, associating significantly with H3K27me3 histone modification and Ki67 proliferation index in the in vitro experiments (unpublished data). These findings suggest that EZH2 is a potential contributor to parathyroid tumors. However, the identification and functional characterization of downstream signaling pathways, and molecules targeted by EZH2 in parathyroid tumor progression need future studies.

MicroRNAs (miRNAs) are short, single-stranded non-coding RNAs, 17–25 nucleotides in size that regulate gene expression at the post-transcriptional stage [71]. MiRNAs can be detected in various body fluids such as saliva, blood, urine by which they can serve as an informative biomarker for disease progression. Deregulation of miRNAs has been identified as candidate regulators in parathyroid tumors by oncogenic and tumor suppression functions. Several studies have identified specific miRNAs with altered expression profiles in parathyroid neoplasms. miR-24–1 expression profiles revealed that its presence is observed only in MEN-1-related PA [72], [73], [74]. Studies reported miR-296 and miR-139 were significantly downregulated, while miR-503 and miR-222 were upregulated in sporadic PC and miR-126 could differentiate sporadic from PC [75], [76]. Serum exosomal miRNA profiling have found miR-27a-5p as a non-invasive biomarker to predict tumor progression [77]. Studies have also observed that miRNAs can modulate hormone synthesis and secretion by regulating CASR expression, as well as the initiation and progression of endocrine tumorigenesis, by modulating several signaling pathways such as the Wnt/β-catenin pathway [71], [73]. The future of miRNA research in parathyroid tumors holds promise for enhancing diagnostic precision and developing targeted therapies. Advancements in high-throughput sequencing and bioinformatics will facilitate the identification of novel miRNAs and regulatory networks involved in tumorigenesis.

Parathyroids develop from third and fourth endodermal pharyngeal pouches in humans. The transcription factors such as GCM2, PAX1, GATA3, TBX1 and HOX are known to be involved in determining the differentiation of the endodermal pharyngeal pouches [78], [79]. GCM2 interacts with GATA3 and MafB and maintains the PTH expression in parathyroid cells [80]. Besides the known function of these transcription factors (GCM2, PAX1 and GATA3) in parathyroid gland development, their role in parathyroid tumorigenesis has not been extensively explored. Recently it has been shown hypermethylated CpG’s in the GCM2-binding region on the CASR promoter 2 influenced CASR loss in sporadic PA [81], [82]. Likewise, patients with sporadic PA were shown to undergo promoter hypermethylation and increased histone 3 trimethylation at lysine 9 (H3K9me3) of GCM2 concerning the repression of GCM2 [82]. In mouse embryos where PAX1 has been genetically inactivated, it led to reduced GCM2 expression, establishing a clear association between these two transcription factors [83]. The downregulation of PAX1 was detected in a subset of sporadic parathyroid tumors and was associated with both promoter hypermethylation and deacetylation of H3 of lysine 9 (H3K9ac). Using potent epigenetic inhibitors such as DAC (DNA demethylating agent) and BRD4770 (histone demethylating agent) reactivated PAX1 and GCM2 expression in PTHC-1 cells respectively [82], *[84]. GATA3 enables PTH transcription and is key for regulating serum calcium and phosphate homeostasis in cooperation with GCM2 and MafB [85]. In humans, GATA3 haploinsufficiency is linked with hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome [86]. It has been demonstrated to be a sensitive and specific immunohistochemical marker for parathyroid differentiation [87]. Transcription factors GCM2, PAX1 and GATA3, have emerged as attractive putative tissue-specific tumor suppressor genes since they control parathyroid development and are linked to epigenetic aberrations in sporadic PA as well. Further evidence in knowing their functional role in parathyroid tumorigenesis remains to be investigated.

The cell division cycle 73 (CDC73) gene located on chr 11q31.2, also named as HRPT2 is a tumor suppressor gene that encodes parafibromin protein [88]. Parafibromin mainly regulates major cellular processes such as proliferation, differentiation, and apoptosis and acts as a transcriptional modulator of histone modifications and chromatin remodeling. Germline CDC73 mutations are mainly associated with familial PHPT disease - HPT-JT syndrome [21]. Moreover, germline and somatic CDC73 mutations are frequently reported in sporadic PC also, however very rare in sporadic PA. Germline mutations are observed in 20–40 % of sporadic PC with somatic alterations reported in ∼75 % of patients [89], [90]. A study also revealed histone methylation H3K4me and H3K79me in CDC73 genes in sporadic PC [89]. Additionally, loss of heterozygosity at the CDC73 gene was reported in ∼50 % of sporadic PC cases [35]. Negative parafibromin staining is significantly associated with higher tumor volume, and aggressive tumors and acts as a candidate biomarker to differentiate sporadic PC from PA in tumor tissue sections [21], [35].

Next-generation sequencing techniques have the potential to identify driver molecules and lead to better diagnosis and therapeutic approaches in various cancers [91]. NGS analysis by either targeted gene panels, whole exome sequencing (WES) or whole genome sequencing (WGS) identified new somatic variants in sporadic tumors that can be driver molecules of parathyroid tumorigenesis. Recent NGS-based studies have identified activating mutations in PIK3CA and MTOR as well as inactivating mutations in NF1, neurofibromatosis 1 gene, in sporadic PC [92]. NF1 is a regulator of the mTOR pathway, and alterations in these genes could have a possible causative link to PC and need further investigation. Studies identified genetic variants in TP53, NF1, ATM, TSC1, CTNNB1 and PI3KA genes along with CDC73 and MEN1 genes making them as candidate driver molecules for PC [68], [92], [93]. Somatic mutations in PRUNE2, a tumor suppressor gene, are implicated in 18 % of sporadic PC, however, germline variants are rare events [89].

Epigenomics refers to genome-wide epigenetic changes including DNA and histone modifications and chromatin landscape. Epigenetic modifications play a major role in parathyroid tumor pathogenesis. Numerous gene-specific DNA methylation and histone modifications have been identified in different parathyroid tumor phenotypes *[39], [40], *[52], [73], *[84]. Research on both global and gene-specific epigenetic modifications in PHPT is limited. A study measured 5-hydroxymethylcytosine (5hmC) levels within LINE1 and showed higher global hypomethylation sporadic PC compared to PA [48]. Other studies have noted the role of epigenetic de-methylase enzymes TET1/TET2 was extensively reduced in PC, suggesting the reduction of de-methylation events across the genome [94]. However, the mechanism of alteration of de-methylating enzymes remains a futuristic approach. A genome-wide methylation analysis showed low hypermethylation levels in normal tissues, intermediate levels in PA, and higher levels in PCs [42]. Studies have also reported DNA promoter hypermethylation in tumor suppressor genes like WT1, SFRP1, SFRP2, SFRP4, RIZ1, CTTNB1, HOXAC1, POMC1 and HIC1 with APC, and RASSF1A which are already reported in sporadic PHPT *[42], [73]. Advancements in molecular techniques continue to highlight the novel molecular pathology of PHPT. Further, single-cell epigenome profiling in conjunction with genomics is still needed to uncover novel epigenomics mechanisms in sporadic parathyroid tumors.

Transcriptomics has emerged as an advanced field post-genomic era, supporting genomic and proteomic research to identify genes expressed in specific cells and their association with various disease conditions [95]. Transcriptome analysis provides information on genome transcription, including gene structure, expression regulation, product functions, and genome dynamics. It reveals regulation networks of biological processes and pathways, pinpointing key genes and pathways crucial for disease diagnosis and therapy. To the best of our knowledge till date, three transcriptome-based studies have been conducted in sporadic PHPT patients. The first study showed the phenotypic difference within the sporadic PHPT based on their molecular (gene) signatures identified through RNA sequencing and categorized them into calcium-sensitive and calcium-resistant. Calcium-resistant PHPT cases have low bone mineral density and higher expression of mitochondrial genes (COX7B, CYCS, ATP5G3, ATP5A1 and PCYT2), whereas calcium-sensitive populations have higher membrane trafficking and ECM proteins (HPSE, SLC8A1, MME, SORL1, DDX43, VCAN) [96]. Another study showed that down-expressed hub genes SPCS2, RPL23, RPL26, RPN1, SEC11C, SEC11A, RPS25, and SEC61G in sporadic PA could contribute to parathyroid tumorigenesis [91]. The most recent study showed a stable and conserved gene profile in sporadic PA with only 7 differentially expressed genes (DEGs), whereas sporadic PC has more diverse gene expression pattern with 1110 DEGs. They also showed upregulation of E2F targets, KRAS, TNF-alpha signaling, and epithelial-mesenchymal transition pathways in Sporadic PC compared to PA [97]. However, identifying parathyroid-specific target genes or contributing molecules by manipulating their overexpression or inhibition post-treatment has not been extensively explored in parathyroid tumors.

Functional information of most of the genes resides in the proteome, so proteome analysis could valuable insight into disease. Proteome information can’t directly link to pathogenesis however it acts as a starting point for identifying potential candidate molecules which will later be established as biomarkers after in vivo and in vitro studies. Proteomics studies are in a very nascent stage. Earliest studies used gel-based proteomics approach with the identification of a limited number of differentially expressed proteins (DEPs) and the recent gel-free approaches led to the identification of a large number of DEPs in sporadic PA and PC [98], [99], [100]. Studies led to the identification of the heterogeneous set of DEPs with a few common proteins that might be associated with differences in methodology, clinical heterogeneity (symptomatic vs asymptomatic; organ involvement, level of hypercalcemia and PTH secretion etc.) and ethnicity. Still, there are some interesting findings like dysregulated molecules from MAPK signalling pathway, PI3K-AKT signalling pathway and chromatin modifier proteins. Ubiquitin-carboxyl-terminal hydrolase isozyme L1 (UCHL1), protein S100-A11, annexin A2 (ANXA2), and annexin A4 (ANXA4) were overexpressed in sporadic PA and showed an upward trend in sporadic PC, whereas albumin (ALB) showed a downward trend in sporadic PC from sporadic PA. Heat shock protein 60 kDa (HSPD1) and ATP synthase, subunit D (ATP5H) showed an upward trend in multiglandular disease from sporadic PA, predicting more mitochondrial role in multiglandular disease; however still needs to be validated in large data sets [98], [99], [100], *[101]. Current proteomics studies on sporadic PHPT are mainly focused on the discovery-proteomics of tumor tissues. Studies involving a large number of samples, types (tissue, serum and FFPE), and with organ involvement categorization need to be undertaken leading to better understanding of protein markers of sporadic PHPT.