**11. Multiple endocrine neoplasia type 2A (MEN2A)**

MEN2A is a familial cancer syndrome characterized by a predisposition to the development of medullary thyroid cancer (MTC), pheochromocytoma (typically benign and often bilateral), and primary HPT. In the context of MEN2A, HPT is usually mild and resembles sporadic HPT. HPT in MEN2A is almost always results from benign parathyroid disease. MEN2A is an autosomal dominant disorder that results from germline gain-of-function mutation in the *RET* proto-oncogene at chromosomal location 10q11. *RET* encodes a receptor tyrosine kinase that binds the ligand glial derived neurotrophic factor, together with a glycosylphosphatidylinositol-anchored protein co-receptor Gfra1 [96].

Germline oncogenic mutations of *RET* are associated with three distinct familial endocrine neoplasia syndromes, all associated with MTC: MEN2A, multiple endocrine neoplasia type 2B (MEN2B), and familial medullary thyroid cancer (FMTC). The disease spectrum of typical MEN2B or FMTC does not include parathyroid tumors and HPT. Genotype–phenotype correlations based on particular *RET* mutations are apparent and account for the distinct patterns of disease. Some 95% of MEN2A cases are due to the presence in the germline of nonsynonymous variants affecting the RET receptor's extracellular cysteine-rich domain, namely missense mutations of *RET* codons 609, 611, 618, 620, or 634 [97]. In fact, germline missense alteration of RET residue cysteine-634 accounts for approximately 85% of cases of MEN2A [98].

### **12. Parathyroid tumorigenesis involving the CCND1 oncogene**

The discovery of the *CCND1* (or *PRAD1*, for parathyroid adenomatosis 1) oncogene resulted from the analysis of several large, non-familial, parathyroid adenomas that harbored DNA re-arrangements that involved the PTH gene locus [99–101]. A breakpoint resulting from the pericentromeric inversion of chromosome 11 DNA was identified just upstream of the *CCND1/PRAD1* oncogene [101]. The inversion positioned the PTH gene regulatory region, that is normally located on the short arm of chromosome 11, just upstream of the *CCND1/PRAD1* protooncogene located on 11q [99–101]. The product encoded by the proto-oncogene

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 in humans [102].

While activating CCND1 missense mutations have not been observed in sporadic 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 any familial form of HPT.

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

Recurrent mutations in a subset of genes likely relevant to parathyroid tumorigenesis 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* mutation or overexpression to parathyroid cells have been reported.

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

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 development.

**123**

*Familial Syndromes of Primary Hyperparathyroidism DOI: http://dx.doi.org/10.5772/intechopen.93036*

sporadic parathyroid neoplasia in the decades ahead.

The author declares no conflict of interest.

The author wishes to thank the members of the Metabolic Diseases Branch, NIDDK for many helpful discussions and suggestions. The Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (ZIA DK043012-18) supported this research. The author declares no competing

**Acknowledgements**

financial interests.

**Conflict of interest**

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

**14. Conclusions**
