**14. Conclusions**

*Mineral Deficiencies - Electrolyte Disturbances, Genes, Diet and Disease Interface*

**13. Other genes involved in parathyroid tumorigenesis**

mutation or overexpression to parathyroid cells have been reported.

potential significance of *ZFX* as a parathyroid proto-oncogene.

in humans [102].

any familial form of HPT.

was subsequently recognized by DNA sequence analysis to be a member of the cyclin protein family [101]. The gene was therefore re-named cyclin D1 (*CCND1*). Overexpression of *CCND1* in the parathyroid cells of transgenic mice induces cell proliferation and gives rise to the metabolic abnormalities that characterize HPT

parathyroid tumors [103], overexpression of *CCND1* has been demonstrated in 20–40% of sporadic benign parathyroid tumors and in an even larger percentage of parathyroid carcinomas [104–107]. In parathyroid carcinoma, no somatic chromosomal rearrangements on chromosome 11 involving *CCND1* have been reported. Neither germline activating missense mutations of *CCND1* nor chromosomal translocations or rearrangements involving the *CCND1* locus have been reported in

While activating CCND1 missense mutations have not been observed in sporadic

Recurrent mutations in a subset of genes likely relevant to parathyroid tumori-

Soong and Arnold used WES analysis of DNA extracted from 19 parathyroid adenomas and matching germline DNA to identify somatic mutations in *ZFX*, a putative parathyroid proto-oncogene and member of the Krüppel associated box domain-containing family of zinc finger protein transcription factors [112]. Their observations in the discovery cohort were confirmed by direct sequencing of tumor DNA from an additional validation set comprised of 111 parathyroid adenomas [112]. The mutant *ZFX* alleles detected in parathyroid tumors likely act as oncogenes [113]. Such somatically acquired ZFX mutations in parathyroid tumors may be uncommon, however, since an independent mutational analysis of 23 sporadic parathyroid carcinomas and 57 adenomas failed to identify any pathogenic *ZFX* variants [111]. The development of a transgenic mouse model and/or better characterization of the functional properties of the mutant ZFX protein may clarify the

WES analysis of 22 blood-sporadic parathyroid adenoma tumor pairs from a Chinese patient cohort identified recurrent mutations of *ASXL3* [108]. ASXL3 belongs to a family of vertebrate Additional sex combs (Asx)-like proteins that may function as regulators of transcription. It remains unclear if the somatic missense *ASXL3* mutations identified in the parathyroid adenomas, mutations that affected highly conserved residues, would result in gain- or loss of ASXL3 function [108]. Further studies will be required to confirm this initial observation and to clarify the mechanism by which *ASXL3* mutation might drive parathyroid tumor

genesis have been identified by WES analysis of DNA derived from sporadic parathyroid tumors. Eight out of 193 sporadic parathyroid tumors analyzed by WES demonstrated the Y641N missense mutation in the *EZH2* gene on chromosome 7 that encodes the enhancer of zeste 2 polycomb repressive complex 2 subunit [36]. Analysis by WES of 22 parathyroid tumors derived from a Chinese patient population identified a distinct somatic missense mutation, Y646N, in *EZH2* [108]. Acquired mutations of Y641 and Y646 in *EZH2* were described previously in lymphoid malignancy [109, 110]. Molecular genetic profiling of 80 sporadic parathyroid neoplasms by separate investigators failed to uncover any pathogenic *EZH2* mutations however, suggesting acquired *EZH2* mutation may be uncommon in parathyroid tumors [111]. In the context of lymphoma, *EZH2* is thought to function as a proto-oncogene [109]. To date, no transgenic mouse models restricting *EZH2*

**122**

development.

While inherited forms of HPT represent only a small fraction of cases (<5%), study of the molecular pathophysiology of these uncommon familial syndromes has yielded substantial insight into the genetic etiology of both sporadic and familial parathyroid disease and resulted in the identification of genes such as *MEN1, CDC73, CASR, GNA11, AP2S1, CDKN1B, CCND1*, and *GCM2*. It is highly likely that the mutational gain- or loss-of-function of other, yet unrecognized, genes is able to drive parathyroid neoplasia. For example, the risk in the majority of FIHP kindreds predisposing to the development of parathyroid tumors seems to result from the germline mutation of genes not presently recognized as having a role in parathyroid disease. This follows from the observation that nearly 70% of families initially considered as FIHP in multiple studies that examined for germline *MEN1*, *CASR* and *CDC73/HRPT2* gene mutation, had no recognized syndromic etiology (**Figure 1**) [20, 75–77]. From among those FIHP kindreds who are *MEN1*, *CASR* and *CDC73* mutation-negative, only about 20% are estimated to harbor germline activating mutations in the *GCM2* proto-oncogene [21], which leaves nearly 80% of FIHP kindreds with a currently-undefined genetic basis for their disease (**Figure 1**).

The existence of currently unidentified parathyroid tumor suppressors and oncogenes is also suggested by analysis of parathyroid tumors using techniques such as comparative genomic hybridization (CGH) to identify specific chromosomal regions harboring loss or gain of DNA. Several investigators have documented recurrent loss of DNA at the 1p, 6q, 9p, and 13q chromosomal loci in parathyroid tumors, indicating the potential presence there of novel parathyroid tumor suppressor genes [114–117]. The potential presence of novel oncogenes at chromosomal loci 9q, 16p, 19p, and Xq is suggested by results demonstrating specific chromosomal gain at these loci in benign or malignant parathyroid tumors [114, 116–118].

Next-generation sequencing analysis including WES of parathyroid neoplasms is an auspicious approach for the identification of novel acquired and germline gene variations that predispose to the development of HPT and parathyroid neoplasia. The apparent validation of this line of investigation by the identification of *EXH2* [36], *ZFX* [112], and potentially *ASXL3* [108], as candidate driver genes for parathyroid neoplasia was previously discussed. WES analysis of parathyroid cancerderived DNA has similarly underscored the possible significance of recurrent somatic and germline inactivating mutations of *PRUNE2* in the etiology of parathyroid malignancy [40]. The comprehensive quality and great sensitivity of WES and related next-generation sequencing methodologies should further advance our insight into the genetic basis and endocrine pathophysiology of inherited and sporadic parathyroid neoplasia in the decades ahead.
