CDC73/HRPT2 CpG island hypermethylation and mutation of 5′-untranslated sequence are uncommon mechanisms of silencing parafibromin in parathyroid tumors

in Endocrine-Related Cancer

The tumor suppressor HRPT2/CDC73 is mutated in constitutive DNA from patients with the familial disorder hyperparathyroidism–jaw tumor syndrome and in ∼70% of all parathyroid carcinomas. In a number of HRPT2 mutant tumors however, expression of the encoded protein parafibromin is lost in the absence of a clear second event such as HRPT2 allelic loss or the presence of a second mutation in this tumor suppressor gene. We sought to determine whether hypermethylation of a 713 bp CpG island extending 648 nucleotides upstream of the HRPT2 translational start site and 65 nucleotides into exon 1 might be a mechanism contributing to the loss of expression of parafibromin in parathyroid tumors. Furthermore, we asked whether mutations might be present in the 5′-untranslated region (5′-UTR) of HRPT2. We investigated a pool of tissue from 3 normal parathyroid glands, as well as 15 individual parathyroid tumor samples including 6 tumors with known HRPT2 mutations, for hypermethylation of the HRPT2 CpG island. Methylation was not identified in any specimens despite complete loss of parafibromin expression in two parathyroid carcinomas with a single detectable HRPT2 mutation and retention of the wild-type HRPT2 allele. Furthermore, no mutations of a likely pathogenic nature were identified in the 5′-UTR of HRPT2. These data strongly suggest that alternative mechanisms such as mutation in HRPT2 intronic regions, additional epigenetic regulation such as histone modifications, or other regulatory inactivation mechanisms such as targeting by microRNAs may play a role in the loss of parafibromin expression.

Abstract

The tumor suppressor HRPT2/CDC73 is mutated in constitutive DNA from patients with the familial disorder hyperparathyroidism–jaw tumor syndrome and in ∼70% of all parathyroid carcinomas. In a number of HRPT2 mutant tumors however, expression of the encoded protein parafibromin is lost in the absence of a clear second event such as HRPT2 allelic loss or the presence of a second mutation in this tumor suppressor gene. We sought to determine whether hypermethylation of a 713 bp CpG island extending 648 nucleotides upstream of the HRPT2 translational start site and 65 nucleotides into exon 1 might be a mechanism contributing to the loss of expression of parafibromin in parathyroid tumors. Furthermore, we asked whether mutations might be present in the 5′-untranslated region (5′-UTR) of HRPT2. We investigated a pool of tissue from 3 normal parathyroid glands, as well as 15 individual parathyroid tumor samples including 6 tumors with known HRPT2 mutations, for hypermethylation of the HRPT2 CpG island. Methylation was not identified in any specimens despite complete loss of parafibromin expression in two parathyroid carcinomas with a single detectable HRPT2 mutation and retention of the wild-type HRPT2 allele. Furthermore, no mutations of a likely pathogenic nature were identified in the 5′-UTR of HRPT2. These data strongly suggest that alternative mechanisms such as mutation in HRPT2 intronic regions, additional epigenetic regulation such as histone modifications, or other regulatory inactivation mechanisms such as targeting by microRNAs may play a role in the loss of parafibromin expression.

Introduction

The tumor suppressor HRPT2, also known as CDC73, maps to 1q31.2 (Ensembl release 55), and has 17 exons encoding the 531 amino acid protein parafibromin. Mutation of HRPT2 has been strongly associated with parathyroid tumorigenesis with mutations reported in patients with the familial disorder hyperparathyroidism–jaw tumor (HPT–JT) syndrome (OMIM #145001; Carpten et al. 2002, reviewed in Marsh et al. (2007)) and in a small subset of patients classified as familial isolated HPT (FIHP) (OMIM #145001; reviewed in Marsh et al. (2007)). Inactivating HRPT2 mutations are found in ∼70% of parathyroid carcinomas but only rarely in non-HPT–JT benign parathyroid tumors ( Howell et al. 2003, Shattuck et al. 2003, Cetani et al. 2004, Krebs et al. 2005, Gill et al. 2006). Mutations in HRPT2 have also been identified in sporadic kidney tumors ( Zhao et al. 2007). Approximately, 80% of mutations occur in exons 1, 2, or 7, with the majority predicted to inactivate parafibromin by prematurely truncating this protein (reviewed in Marsh et al. (2007)).

Loss of heterozygosity at the HRPT2 locus and ‘second-hit’ mutations in the coding region of HRPT2 have been reported and are consistent with Knudson's ‘two-hit’ hypothesis for inactivation of tumor suppressor genes ( Howell et al. 2003, Bradley et al. 2006, Kelly et al. 2006, Cetani et al. 2007). However, allelic loss at the HRPT2 locus is not always observed in parathyroid tumors with either a known or suspected HRPT2 mutation ( Carpten et al. 2002, Howell et al. 2003, Shattuck et al. 2003). Therefore, it would appear that despite the strong association with parathyroid tumorigenesis, mutation within the coding region of HRPT2 or the loss of heterozygosity at 1q31.2 does not account for all reported cases of HPT–JT or sporadic parathyroid tumors. Mutation in non-coding regions of HRPT2 or the involvement of other genes cannot be discounted. For example, mutation of the PTEN promoter has been reported in a number of patients with Cowden syndrome ( Zhou et al. 2003). Furthermore, hypermethylation of an HRPT2 CpG island may play a role in silencing the expression of parafibromin in some parathyroid tumors.

The majority of cancer-associated CpG islands are located in the 5′-untranslated region (5′-UTR) and the first exon of tumor suppressor genes ( Esteller 2007). In these islands, clusters of cytosines preceding guanines, CpGs, can undergo the addition of a methyl group to the 5 position of the cytosine ring. This process is mediated by DNA methyltransferases and leads to aberrant methylation resulting in down-regulation of gene expression. Well-known tumor suppressor genes associated with endocrine neoplasia syndromes where hypermethylation occurs in familial and sporadic counterpart tumors include VHL and PTEN ( Esteller 2007). Here, we report the results of investigations into the methylation status of a CpG island within the 5′-UTR and first exon of HRPT2 in normal parathyroid tissue, as well as benign and malignant parathyroid tumors. Furthermore, we report sequence analyses of an almost 2 kb segment of the 5′-UTR region of HRPT2. Data are presented in conjunction with HRPT2 mutation status, allelic status at the HRPT2 locus, and expression of parafibromin.

Materials and methods

CpG island identification

The web-based resource CpG plot (http://www.ebi.ac.uk/emboss/cpgplot) was used to analyse over 2500 nucleotides upstream of the HRPT2 translational start site, as well as the first exon of HRPT2, for the presence of regions rich in CpGs (clone RP11-239J11, human DNA sequence from chromosome 1 containing the 5′ end of the HRPT2 gene, NCBI Accession No. AL390863).

Tumor samples

A single pool of tissue from 3 normal parathyroid glands and 15 individual parathyroid tumor samples consisting of 6 adenomas, 3 primary hyperplasias, and 6 tumors harboring known HRPT2 mutations were studied ( Tables 1 and 2). Samples were obtained from Royal North Shore Hospital, Sydney, NSW, Australia and the Institute of Pathology, University of Halle, Halle, Germany, according to protocols in place for each centres' human ethics committee. DNA was extracted from tissue stored at −80 °C according to standard procedures. The HRPT2 mutation status and allelic loss in the HRPT2 region have been previously reported for the majority of these specimens ( Howell et al. 2003, Haven et al. 2004). For all samples, the entire coding and flanking intronic regions were sequenced.

Table 1

HRPT2 mutation, putative HRPT2 promoter variant, and allelic loss in parathyroid tumor specimens

SampleTumor typeHRPT2 allelic loss−466C>AHRPT2 mutation
 1Sporadic adenomaLOHAnmd
 2Sporadic adenomaRCAnmd
 3Sporadic adenomaRAAnmd
 4Sporadic adenomaRCAnmd
 5Sporadic adenomaRCAnmd
 6Sporadic adenomaLOHAnmd
 7Primary hyperplasiaRAAnmd
 8Primary hyperplasiaRCAnmd
 9Primary hyperplasiaRAAnmd
 10HPT–JT adenomaLOHAc.191C>T (L64P)
 11HPT–JT carcinomaLOHac.76delA
 12HPT–JT carcinomaRac.76delA, c.686delGAGT
 13Sporadic carcinomaRac.76delA
 14Sporadic carcinomaRaIVS6-1delG, c.165C>A (Y55X)
 15Sporadic carcinomaRac.162C>G (Y54X)

All mutations have been previously reported (3, 12). Genotype is recorded for a variant identified in the putative HRPT2 promoter 466 nucleotides upstream of the HRPT2 translational start site. nmd, no mutation detected; LOH, loss of heterozygosity; R, retention of heterozygosity.

Reported in Table 2.

Table 2

Genotype of variants identified in the 5′-UTR of HRPT2 in parathyroid carcinoma

Parathyroid carcinoma−1850 T>C (rs10754042 a)−1849 G>T (rs72731058 a)−1504 T>G (rs10754043 a)−1302 T>C−466 C>A (rs10737621 a)
 11CTGTC
 12TCGTTGTTCC
 13TCGTTGTTCC
 14TCGGTGTCCA
 15TCGGGGTCCA

Reported in the NCBI human SNP database.

Immunohistochemical analyses for the expression of parafibromin were carried out as previously reported ( Gill et al. 2006).

Methylation analysis

Bisulfite treatment of extracted DNA was performed using the MethylEasy DNA Bisulfite Modification Kit (Human Genetic Signatures, Sydney, NSW, Australia) according to the manufacturer's instructions. The amount of starting DNA varied between 100 and 1000 ng per sample, and DNA was resuspended in 20 μl H2O after bisulfite treatment. PCR was performed using 1 μl bisulfite-treated DNA. CpGenome Universal Methylated and Unmethylated DNA (Chemicon Australia, Boronia, VIC, Australia) were used as controls for these assays.

Primers were designed to amplify fully converted (C→T) bisulfite-treated DNA. The primers did not contain any CpGs and therefore amplified both methylated and unmethylated DNA. A semi-nested approach was used in which a first round PCR was performed using the primers 5′-GTTATTATTTAAAGAATGAAGAGGGAGGATTT-3′ (primer 1, Fig. 1) and 5′-ACAATCTCCTTCTTCTAAATATTATACTATC-3′ (primer 3, Fig. 1). One microliter from the first round reaction was used as template in a second round PCR using the nested primer 5′-GTTGGGGATGGAAGTGTTGATTTATTGTTT-3′ (primer 2, Fig. 1) and the same reverse primer (primer 3) as previously. PCR was performed using the PCRx Enhancer System (Invitrogen Australia Pty Ltd) and the following cycling conditions: 95 °C for 3 min, followed by 30 cycles at 95 °C for 1 min, 50 °C for 2 min, and 72 °C for 2 min followed by a final extension at 72 °C for 10 min.

Figure 1

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Figure 1

HRPT2 5′-UTR. (A) Schematic representation of the conserved sequence alignment and conservation scoring of ∼2000 nucleotides of the HRPT2 5′-UTR across 17 different vertebrate species. Degree of sequence conservation is indicated by shading, with darker regions representing stronger conservation (UCSC Genome Browser with VISTA tracks, http://enhancer.lbl.gov/). The region represented by −284 to +1 contains the strongest areas of conservation within the 5′-UTR (area sequenced extends to −1953) and constitutes part of the CpG island. (B) HRPT2 contains a 713 nucleotide CpG island harboring 78 CpGs extending 648 nucleotides upstream of the translation start site and 65 nucleotides into exon 1. Individual CpGs are marked by solid circles. The positions of primers used for amplification of the CpG island are indicated.

Citation: Endocrine-Related Cancer 17, 1; 10.1677/ERC-09-0291

PCR products were purified using the Wizard PCR Preps Purification System (Promega Australia) and directly sequenced using primer 3 (SUPAMAC, The University of Sydney, NSW, Australia).

Analysis of the 5′-UTR of HRPT2

Primer pairs were designed to amplify two overlapping regions of the 5′-UTR and first exon of HRPT2; −2002 to −652 relative to the HRPT2 translational start site, 5′-TCTGTAGACACAGGGTCTAG-3′ and 5′-GACCTCTAGGACTTAGTGATCC-3′, and −1135 to +150, 5′-CACTGAGACTTCTGAGTACC-3′ and 5′-ACAGCCATGCCGGACTTA-3′. PCR amplification was performed using Platinum Taq (Invitrogen Australia Pty Ltd) and the following cycling conditions: 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min followed by a final extension at 72 °C for 10 min. Fragments were gel purified (UltraClean GelSpin DNA Purification Kit, MO BIO Laboratories, Inc., Carlsbad, CA, USA) and sequenced using respectively 5′-TCTGTAGACACAGGGTCTAG-3′ and 5′-GTACTGAGTGCTCTGACACAGG-3′, and 5′-ACAGCCATGCCGGACTTA-3′ and 5′-CGCACGTACGGGGAACTTTG-3′. A fifth internal primer was also used for sequencing 5′-TTAGATCGGACCCAGGCCTAGC-3′. These primer combinations facilitated the interrogation of 1953 nucleotides of the HRPT2 5′-UTR in tumor samples studied.

Results

Identification of a CpG island in the 5′-UTR of HRPT2

Using CpG plot, a 713 bp CpG island containing 78 CpGs was identified beginning 648 nucleotides upstream of the HRPT2 translational start site and extending 65 nucleotides into exon 1 of HRPT2 ( Fig. 1). Although the promoter region of HRPT2 remains to be experimentally determined, this CpG island contains the most highly evolutionarily conserved regions of the HRPT2 5′-UTR predicted by sequence alignment of 17 vertebrate species (UCSC Genome Browser with VISTA tracks, http://enhancer.lbl.gov/; Fig. 1A). This computational prediction would suggest that this region, and in particular regions within the segment between −284 and +1, may play a significant role in regulating HRPT2 transcription.

Direct sequencing of bisulfite-converted DNA

We employed direct sequencing of PCR products amplified from bisulfite-converted DNA. A semi-nested PCR approach amplified a 563 bp fragment of the HRPT2 CpG island ( Fig. 1). Sequence data are presented using the reverse primer that allowed interrogation of 67 of the 78 CpGs within this island ( Fig. 2). An example of a sequence chromatogram obtained from bisulfite-treated DNA from a parathyroid carcinoma harboring a single HRPT2 mutation, Y54X, and displaying retention of heterozygosity (sample 15, Table 2) shows no methylation, i.e. complete conversion of all cytosines to thymines ( Fig. 2A).

Figure 2

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Figure 2

Sequence chromatograms, reversed and complemented, of bisulfite-treated DNA. (A) Complete sequence obtained from a PCR product amplified from bisulfite-converted DNA from a sporadic parathyroid carcinoma tissue sample (sample 15). CpGs within the island identified by CpG plot are numbered. The 5′-UTR variant −466C>A is indicated with *, and the translation start site is marked with an arrow. The region depicted in panel B is indicated by a black bar. (B) Representative sequence obtained from PCR products amplified from positive (methylated) and negative control (unmethylated) bisulfite-converted DNA (top two panels) and bisulfite-converted DNA obtained from a pooled normal, sporadic carcinoma, adenoma, and hyperplastic parathyroid tissue showing no methylation. CpGs 65–74 are shaded grey.

Citation: Endocrine-Related Cancer 17, 1; 10.1677/ERC-09-0291

Hypermethylation of the HRPT2 CpG island is not present in normal parathyroid tissue and parathyroid tumors

Sequence data obtained from PCR products of bisulfite-treated DNA from all 15 parathyroid tumors and pooled normal parathyroid tissue showed no methylation. This was consistent with analyses of unmethylated negative control DNA ( Fig. 2B). In contrast, sequencing of the methylated positive control DNA revealed methylation at each CpG, i.e. retention of the cytosine, and complete conversion of cytosines to thymines at all non-CpG sites. Representative sequence chromatograms spanning CpGs 65–74 for both the methylated positive and unmethylated negative control samples as well as an example of each type of parathyroid tumor examined are shown in Fig. 2B.

Two-hit mechanisms of HRPT2-associated parathyroid tumorigenesis

From our previous studies, six tumors were shown to harbor HRPT2 mutations ( Tables 1 and 2; Howell et al. 2003). Two tumors had both a single HRPT2 mutation and HRPT2 allelic loss (samples 10 and 11). An additional two tumors had two HRPT2 mutations and retention of heterozygosity at the HRPT2 locus (samples 12 and 14). Previously reported immunohistochemical analyses for these tumors showed weak or complete absence of nuclear parafibromin ( Gill et al. 2006) consistent with biallelic inactivation of HRPT2. These results suggest that either double mutation or mutation combined with allelic loss functions as two-hit mechanisms to silence parafibromin. Two additional carcinoma specimens (samples 13 and 15) harbored only single HRPT2 mutations and retention of heterozygosity; however, immunohistochemical analyses showed complete loss of expression of parafibromin in both cases ( Fig. 3D and F).

Figure 3

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Figure 3

Sequential H&E (A, C, and E) and parafibromin-stained (B, D, and F) sections of normal parathyroid tissue (A and B) and two parathyroid carcinomas (sample 13 – C and D, sample 15 – E and F) showing the loss of parafibromin in carcinoma specimens. Non-neoplastic cells in sample 15 adjacent to the tumor field shown (panel D) stained positive for parafibromin (not shown).

Citation: Endocrine-Related Cancer 17, 1; 10.1677/ERC-09-0291

Mutational analysis of the HRPT2 5′-UTR in five carcinoma samples

Mutation in the 5′-UTR of HRPT2 was investigated as a possible second-hit mechanism that may result in the loss of parafibromin staining. In carcinoma samples 11–15, 1953 nucleotides upstream of the HRPT2 translational start site were sequenced. Five single nucleotide alterations were identified, four of which are reported in the NCBI single nucleotide polymorphism (SNP) database (http://www.ncbi.nlm.nih.gov/snp): −1850T>C (rs10754042), −1849G>T (rs72731058), −1504T>G (rs10754043), and −466C>A (rs10737621). The last of these SNPs, −466C>A, was present in the fragment sequenced to assess HRPT2 methylation; therefore, data are available for all samples ( Tables 1 and 2; Fig. 2A). A fifth variant, −1302 T>C, was not present in the NCBI SNP database. None of the genotypes showed specific association with carcinoma samples harboring either one or two hits ( Tables 1 and 2), suggesting that these are likely polymorphisms not associated with disease and do not act as ‘second-hits’ to inactivate HRPT2. A stretch of 13 Ts, −810 to −798, was unable to be reliably assessed by these analyses; however, variants in this region have also been reported in the NCBI SNP database.

Discussion

We have identified a 713 bp CpG island in HRPT2 that extends 648 nucleotides upstream of the translation start site and 65 nucleotides into exon 1. Using direct sequencing of PCR products amplified from bisulfite-treated DNA, we have shown that the HRPT2 CpG island is not methylated in normal parathyroid tissue or benign or malignant parathyroid tumors. Two sporadic carcinomas harboring a single HRPT2 mutation showed complete absence of parafibromin. These tumors did not have a detectable second-hit event that in other HRPT2 mutant samples was either loss of the wild-type allele or biallelic inactivation due to a second HRPT2 mutation. While these tumors did harbor a number of variants in the 5′-UTR region, most have either been reported in the NCBI SNP database or were found in tumors where a two-hit mechanism had already been established. While this study does not exclude the possibility that these SNPs represent low-risk variants for the development of parathyroid carcinoma, we provide no evidence that they participate in a two-hit mechanism leading to the loss of parafibromin. Therefore, in the tumors studied, mutations in the 5′-UTR of HRPT2 or hypermethylation of the HRPT2 CpG island are not alternative second-hit mechanisms that might explain complete silencing of parafibromin.

The lack of HRPT2 hypermethylation found in this study is consistent with two previous case reports. CpG hypermethylation was not identified in DNA extracted from five parathyroid tumors and four matched peripheral leucocyte specimens from members of a single FIHP kindred harboring the germline HRPT2 mutation c.518–521del ( Mizusawa et al. 2006). Two of these five tumors demonstrated biallelic inactivation with the identification of the somatic mutations c.70–73del and c.95–102del. All five specimens exhibited retention of heterozygosity; however, immunohistochemistry for parafibromin was not reported; therefore, it is unknown whether parafibromin was silenced in these tumors. In a second study of a HPT–JT kindred harboring the germline HRPT2 mutation c.433-442delinsAGA, two parathyroid tumors without additional somatic mutations as well as matched leucocyte DNA did not exhibit hypermethylation of the HRPT2 CpG island ( Masi et al. 2008). While allelic loss was not described in this study, the loss of parafibromin expression was shown by immunohistochemistry in these tumors.

When combining these reports with the present study, lack of HRPT2 CpG island hypermethylation has now been described in seven parathyroid tumors with an apparently single HRPT2 mutation as well as in non-HRPT2-associated benign parathyroid tumors. These findings differ from a single study reporting HRPT2 CpG island methylation in 2 of 11 parathyroid carcinomas and 1 of 6 adenomas from 2 patients with HPT–JT ( Hewitt et al. 2007). As that study did not investigate HRPT2 mutation status, loss of heterozygosity at the HRPT2 locus, or the expression of parafibromin, it is difficult to interpret these data in the context of hypermethylation being an important mechanism leading to the loss of parafibromin expression. Consistent with the present study that showed no evidence of HRPT2 methylation in benign non-HPT–JT tumors, hypermethylation of the HRPT2 CpG island was not reported in 37 sporadic parathyroid adenomas ( Hewitt et al. 2007).

Hypermethylation of growth suppressing genes has not been extensively studied in parathyroid tumors. The Rb-interacting zinc finger gene RIZ1 is however hypermethylated in 36% of non-familial, predominantly benign parathyroid tumors, occurring in conjunction with allelic loss ( Carling et al. 2003). Interestingly, RIZ1 is also hypermethylated in malignant pheochromocytoma ( Carling et al. 2003). Hypermethylation of the multidrug resistance 1 (MDR1) gene associated with silencing of the encoded ABC-transported protein has also been reported in benign parathyroid tumors ( Takeuchi et al. 2007).

HPT–JT and FIHP are part of the familial endocrine neoplasia syndromes with known germline mutations in tumor suppressor genes that include Cowden syndrome (OMIM #158350; PTEN), von Hippel–Lindau syndrome (OMIM #193300; VHL), familial pheochromocytoma and paraganglioma syndrome (OMIM #115310; SDHB, SDHC), Carney complex (OMIM #160980; PRKAR1A), and multiple endocrine neoplasia type 1 (OMIM #131100; MEN1; reviewed in Marsh & Zori (2002)). A sixth endocrine neoplasia syndrome, MEN2 (OMIM #171400), is caused by activating mutations of the RET proto-oncogene, excluding a role for gene silencing ( Mulligan et al. 1993). With the exception of PRKAR1A that is yet to be investigated for methylation, hypermethylation of these tumor suppressors has been studied in a number of familial and sporadic counterpart tumors of these syndromes ( Table 3). PTEN appears to be the most frequently hypermethylated gene with methylation reported in sporadic Cowden syndrome component tumors thyroid ( Alvarez-Nunez et al. 2006, Schagdarsurengin et al. 2006), endometrial ( Salvesen et al. 2001), and breast ( Garcia et al. 2004, Khan et al. 2004). Of interest, PTEN methylation appears to be correlated with more aggressive breast tumors ( Garcia et al. 2004, Khan et al. 2004) and with metastatic disease in endometrial carcinoma ( Salvesen et al. 2001). Hypermethylation of PTEN is also seen in sporadic tumors that are not observed in Cowden syndrome such as colorectal tumors ( Goel et al. 2004).

Table 3

Tumor suppressor genes implicated in endocrine neoplasia syndromes – frequency of methylation in counterpart tumors

GeneTumorMethylationMethylation detection methodStudy
HRPT2ParathyroidSequencing This study
 Adenoma (non-HPT–JT)0/6 (0%)
 Primary hyperplasia (non-HPT–JT)0/3 (0%)
 Adenoma (HPT–JT, HRPT2 mutant)0/1 (0%)
 Carcinoma (HPT–JT, HRPT2 mutant)0/2 (0%)
 Carcinoma (sporadic, HRPT2 mutant)0/3 (0%)
Parathyroid (FIHP-associated)0/5 (0%)Sequencing aMizusawa et al. (2006)
Parathyroid (HPT–JT-associated)0/2 (0%)Sequencing aMasi et al. (2008)
ParathyroidMSP and sequencingHewitt et al. (2007)
 Adenoma (non-HPT–JT)0/37 (0%)
 Adenoma (HPT–JT-associated)1/6 (17%)
 Carcinoma2/11 (18%)
VHLPheochromocytoma 0/53 (0%)Methylation-sensitive restriction endonuclease digestion Bender et al. (2000)
0/35 (0%)MSP and sequencingCascon et al. (2004)
0/25 (0%)MSPDammann et al. (2005)
Renal cell carcinoma5/26 (19%)Methylation-sensitive restriction endonuclease digestionHerman et al. (1994)
11/99 (11%)Methylation-sensitive restriction endonuclease digestionClifford et al. (1998)
13/77 (17%)Methylation-sensitive restriction endonuclease digestionBrauch et al. (2000)
11/202 (5%)MSPKondo et al. (2002)
19/93 (20%)MSPBanks et al. (2006)
16/185 (9%)MSPSmits et al. (2008)
17/205 (8%)SequencingNickerson et al. (2008)
Hemangioblastoma0/9 (0%)Methylation-sensitive restriction endonuclease digestionTse et al. (1997)
0/42 (0%)Methylation-sensitive restriction endonuclease digestionGlasker et al. (2001)
SDHBPheochromocytoma 9/28 (32%)Sequencing aAstuti et al. (2004)
0/35 (0%)MSP and sequencingCascon et al. (2004)
SDHDPheochromocytoma 0/35 (0%)MSP and sequencingCascon et al. (2004)
PTENbThyroidMSPAlvarez-Nunez et al. (2006)
 Papillary thyroid carcinoma21/46 (46%)
 Follicular thyroid carcinoma6/7 (86%)
 Follicular thyroid adenoma5/6 (83%)
ThyroidMSPSchagdarsurengin et al. (2006)
 Medullary thyroid carcinoma0/6 (0%)
 Follicular thyroid carcinoma0/10 (0%)
 Papillary thyroid carcinoma2/12 (17%)
 Undifferentiated thyroid cancer1/7 (14%)
 Follicular thyroid adenoma2/9 (22%)
Endometrial carcinoma 26/138 (19%)Methylation-sensitive restriction endonuclease digestion and MSPSalvesen et al. (2001)
Breast43/90 (48%)MSP and sequencingGarcia et al. (2004)
15/44 (34%)MSPKhan et al. (2004)
MEN1Pancreatic ductal adenocarcinoma5/24 (21%)MSPCavallari et al. (2009)

Refers to sequencing of cloned PCR products from bisulfite-treated DNA. Sequencing otherwise refers to sequencing of a PCR product amplified directly from bisulfite-treated genomic DNA. MSP refers to ‘methylation-specific PCR’ where primers have been designed to amplify either methylated or unmethylated bisulfite-treated genomic DNA. Methylation-sensitive restriction endonuclease digestion involves digestion of genomic DNA with a methylation-sensitive enzyme followed by either PCR amplification where fragments will only amplify in the absence of digestion, or Southern blotting that recognizes a methylation-specific digest pattern.

Care must be taken when interpreting PTEN hypermethylation data due to 5′-UTR sequence similarities between PTEN and the PTEN pseudogene ( Zysman et al. 2002).

While VHL and SDHD do not appear to be methylated in pheochromocytoma ( Bender et al. 2000, Cascon et al. 2004, Dammann et al. 2005), one study does show approximately one-third of sporadic pheochromocytomas with methylation of the SDHB promoter ( Astuti et al. 2004). VHL is hypermethylated in between 5 and 20% of renal cell carcinomas ( Herman et al. 1994, Clifford et al. 1998, Brauch et al. 2000, Kondo et al. 2002, Banks et al. 2006, Nickerson et al. 2008, Smits et al. 2008), but not in hemangioblastoma ( Tse et al. 1997, Glasker et al. 2001). Limited studies have been conducted regarding a role for CpG island hypermethylation in the regulation of MEN1; however, a recent study in pancreatic ductal adenocarcinoma tumors, distinct from the endocrine pancreatic tumors present in MEN1, has shown methylation in 21% of these tumors ( Cavallari et al. 2009). In a number of studies of CpG island methylation of these tumor suppressor genes in familial and sporadic counterpart tumors, hypermethylation has been shown to function as a second-hit event important for biallelic inactivation ( Table 3).

If hypermethylation does play a role in silencing parafibromin in parathyroid tumors, it would appear to be infrequent and confined to a small number of parathyroid carcinomas or HPT–JT-associated adenomas that likely harbor a mutation in HRPT2 and have been previously described as being of increased ‘malignant potential’ ( Howell et al. 2003, Hewitt et al. 2007). Like VHL that is not hypermethylated in all tumor types seen in VHL, it is possible that hypermethylation of the HRPT2 CpG island may be seen at greater frequencies in kidney and jaw tumors that are also component tumors of HPT–JT.

Data presented in the current study suggest that parafibromin can be inactivated by as yet undescribed mechanisms. These may include mutation in intronic regions of HRPT2, alternative epigenetic mechanisms such as histone modifications (reviewed in Esteller (2007)), aberrant expression of genes that regulate parafibromin, or still other mechanisms of regulatory inactivation such as targeting by microRNAs ( Ross et al. 2007, Corbetta et al. 2009).

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the Australian National Health and Medical Research Council (NHMRC, 302161). D J Marsh is a Cancer Institute NSW Fellow (Australia) and V M Howell is a CJ Martin Fellow (NHMRC, Australia).

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  • CarptenJDRobbinsCMVillablancaAForsbergLPresciuttiniSBailey-WilsonJSimondsWFGillandersEMKennedyAMChenJD2002HRPT2, encoding parafibromin, is mutated in hyperparathyroidism–jaw tumor syndrome. Nature Genetics32676680.

  • CasconARuiz-LlorenteSFragaMFLetonRTelleriaDSastreJDiezJJMartinez Diaz-GuerraGDiaz PerezJABenitezJ2004Genetic and epigenetic profile of sporadic pheochromocytomas. Journal of Medical Genetics41e30.

  • CavallariISilic-BenussiMRendeFMartinesAFogarPBassoDVellaMDPedrazzoliSHermanJGChieco-BianchiL2009Decreased expression and promoter methylation of the menin tumor suppressor in pancreatic ductal adenocarcinoma. Genes Chromosomes & Cancer48383396.

  • CetaniFPardiEBorsariSViacavaPDipollinaGCianferottiLAmbroginiEGazzerroEColussiGBertiP2004Genetic analyses of the HRPT2 gene in primary hyperparathyroidism: germline and somatic mutations in familial and sporadic parathyroid tumors. Journal of Clinical Endocrinology and Metabolism8955835591.

  • CetaniFPardiEAmbroginiEViacavaPBorsariSLemmiMCianferottiLMiccoliPPincheraAArnoldA2007Different somatic alterations of the HRPT2 gene in a patient with recurrent sporadic primary hyperparathyroidism carrying an HRPT2 germline mutation. Endocrine-Related Cancer14493499.

  • CliffordSCProwseAHAffaraNABuysCHMaherER1998Inactivation of the von Hippel–Lindau (VHL) tumour suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: evidence for a VHL-independent pathway in clear cell renal tumourigenesis. Genes Chromosomes & Cancer22200209.

  • CorbettaSVairaVGuarnieriVScillitaniAEller-VainicherCFerreroSVicentiniLChiodiniIBiscegliaMBeck-PeccozP2009Differential expression of microRNAs in human parathyroid carcinomas compared with normal parathyroid tissue. Endocrine-Related Cancer17135146.

  • DammannRSchagdarsurenginUSeidelCTrumplerCHoang-VuCGimmODralleHPfeiferGPBrauckhoffM2005Frequent promoter methylation of tumor-related genes in sporadic and men2-associated pheochromocytomas. Experimental and Clinical Endocrinology & Diabetes11317.

  • EstellerM2007Cancer epigenomics: DNA methylomes and histone-modification maps. Nature Reviews. Genetics8286298.

  • GarciaJMSilvaJPenaCGarciaVRodriguezRCruzMACantosBProvencioMEspanaPBonillaF2004Promoter methylation of the PTEN gene is a common molecular change in breast cancer. Genes Chromosomes & Cancer41117124.

  • GillAJClarksonAGimmOKeilJDralleHHowellVMMarshDJ2006Loss of nuclear expression of parafibromin distinguishes parathyroid carcinomas and hyperparathyroidism–jaw tumor (HPT–JT) syndrome-related adenomas from sporadic parathyroid adenomas and hyperplasias. American Journal of Surgical Pathology3011401149.

  • GlaskerSBenderBUApelTWvan VelthovenVMulliganLMZentnerJNeumannHP2001Reconsideration of biallelic inactivation of the VHL tumour suppressor gene in hemangioblastomas of the central nervous system. Journal of Neurology Neurosurgery and Psychiatry70644648.

  • GoelAArnoldCNNiedzwieckiDCarethersJMDowellJMWassermanLComptonCMayerRJBertagnolliMMBolandCR2004Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers. Cancer Research6430143021.

  • HavenCJHowellVMEilersPHDunneRTakahashiMvan PuijenbroekMFurgeKKievitJTanMHFleurenGJ2004Gene expression of parathyroid tumors: molecular subclassification and identification of the potential malignant phenotype. Cancer Research6474057411.

  • HermanJGLatifFWengYLermanMIZbarBLiuSSamidDDuanDSGnarraJRLinehanWM1994Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. PNAS9197009704.

  • HewittKMSharmaPKSamowitzWHobbsM2007Aberrant methylation of the HRPT2 gene in parathyroid carcinoma. Annals of Otology Rhinology and Laryngology116928933.

  • HowellVMHavenCJKahnoskiKKhooSKPetilloDChenJFleurenGJRobinsonBGDelbridgeLWPhilipsJ2003HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours. Journal of Medical Genetics40657663.

  • KellyTGShattuckTMReyes-MugicaMStewartAFSimondsWFUdelsmanRArnoldACarpenterTO2006Surveillance for early detection of aggressive parathyroid disease: carcinoma and atypical adenoma in familial isolated hyperparathyroidism associated with a germline HRPT2 mutation. Journal of Bone and Mineral Research2116661671.

  • KhanSKumagaiTVoraJBoseNSehgalIKoefflerPHBoseS2004PTEN promoter is methylated in a proportion of invasive breast cancers. International Journal of Cancer112407410.

  • KondoKYaoMYoshidaMKishidaTShuinTMiuraTMoriyamaMKobayashiKSakaiNKanekoS2002Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: relationship to clinicopathological parameters. Genes Chromosomes & Cancer345868.

  • KrebsLJShattuckTMArnoldA2005HRPT2 mutational analysis of typical sporadic parathyroid adenomas. Journal of Clinical Endocrinology and Metabolism9050155017.

  • MarshDZoriR2002Genetic insights into familial cancers – update and recent discoveries. Cancer Letters181125164.

  • MarshDJHahnMAHowellVMGillAJ2007Molecular diagnosis of primary hyperparathyroidism in familial cancer syndromes. Expert Opinion on Medical Diagnostics1377392.

  • MasiGBarzonLIacoboneMVielGPorzionatoAMacchiVDe CaroRFaviaGPaluG2008Clinical, genetic, and histopathologic investigation of CDC73-related familial hyperparathyroidism. Endocrine-Related Cancer1511151126.

  • MizusawaNUchinoSIwataTTsuyuguchiMSuzukiYMizukoshiTYamashitaYSakuraiASuzukiSBenikoM2006Genetic analyses in patients with familial isolated hyperparathyroidism and hyperparathyroidism–jaw tumour syndrome. Clinical Endocrinology65916.

  • MulliganLMKwokJBHealeyCSElsdonMJEngCGardnerELoveDRMoleSEMooreJKPapiL1993Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature363458460.

  • NickersonMLJaegerEShiYDurocherJAMahurkarSZaridzeDMatveevVJanoutVKollarovaHBenckoV2008Improved identification of von Hippel–Lindau gene alterations in clear cell renal tumors. Clinical Cancer Research1447264734.

  • RossJSCarlsonJABrockG2007miRNA: the new gene silencer. American Journal of Clinical Pathology128830836.

  • SalvesenHBMacDonaldNRyanAJacobsIJLynchEDAkslenLADasS2001PTEN methylation is associated with advanced stage and microsatellite instability in endometrial carcinoma. International Journal of Cancer912226.

  • SchagdarsurenginUGimmODralleHHoang-VuCDammannR2006CpG island methylation of tumor-related promoters occurs preferentially in undifferentiated carcinoma. Thyroid16633642.

  • ShattuckTMValimakiSObaraTGazRDClarkOHShobackDWiermanMETojoKRobbinsCMCarptenJD2003Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma. New England Journal of Medicine34917221729.

  • SmitsKMSchoutenLJvan DijkBAHulsbergen-van de KaaCAWoutersKAOosterwijkEvan EngelandMvan den BrandtPA2008Genetic and epigenetic alterations in the von Hippel–Lindau gene: the influence on renal cancer prognosis. Clinical Cancer Research14782787.

  • TakeuchiHGreepNCHoonDSGiulianoAEHansenNMUmetaniNSingerFR2007Hypermethylation of adenosine triphosphate-binding cassette transporter genes in primary hyperparathyroidism and its effect on sestamibi imaging. Journal of Clinical Endocrinology and Metabolism9217851790.

  • TseJYWongJHLoKWPoonWSHuangDPNgHK1997Molecular genetic analysis of the von Hippel–Lindau disease tumor suppressor gene in familial and sporadic cerebellar hemangioblastomas. American Journal of Clinical Pathology107459466.

  • ZhaoJYartAFrigerioSPerrenASchramlPWeisstannerCStallmachTKrekWMochH2007Sporadic human renal tumors display frequent allelic imbalances and novel mutations of the HRPT2 gene. Oncogene2634403449.

  • ZhouXPWaiteKAPilarskiRHampelHFernandezMJBosCDasoukiMFeldmanGLGreenbergLAIvanovichJ2003Germline PTEN promoter mutations and deletions in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3-kinase/Akt pathway. American Journal of Human Genetics73404411.

  • ZysmanMAChapmanWBBapatB2002Considerations when analyzing the methylation status of PTEN tumor suppressor gene. American Journal of Pathology160795800.

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Figures

  • View in gallery

    HRPT2 5′-UTR. (A) Schematic representation of the conserved sequence alignment and conservation scoring of ∼2000 nucleotides of the HRPT2 5′-UTR across 17 different vertebrate species. Degree of sequence conservation is indicated by shading, with darker regions representing stronger conservation (UCSC Genome Browser with VISTA tracks, http://enhancer.lbl.gov/). The region represented by −284 to +1 contains the strongest areas of conservation within the 5′-UTR (area sequenced extends to −1953) and constitutes part of the CpG island. (B) HRPT2 contains a 713 nucleotide CpG island harboring 78 CpGs extending 648 nucleotides upstream of the translation start site and 65 nucleotides into exon 1. Individual CpGs are marked by solid circles. The positions of primers used for amplification of the CpG island are indicated.

  • View in gallery

    Sequence chromatograms, reversed and complemented, of bisulfite-treated DNA. (A) Complete sequence obtained from a PCR product amplified from bisulfite-converted DNA from a sporadic parathyroid carcinoma tissue sample (sample 15). CpGs within the island identified by CpG plot are numbered. The 5′-UTR variant −466C>A is indicated with *, and the translation start site is marked with an arrow. The region depicted in panel B is indicated by a black bar. (B) Representative sequence obtained from PCR products amplified from positive (methylated) and negative control (unmethylated) bisulfite-converted DNA (top two panels) and bisulfite-converted DNA obtained from a pooled normal, sporadic carcinoma, adenoma, and hyperplastic parathyroid tissue showing no methylation. CpGs 65–74 are shaded grey.

  • View in gallery

    Sequential H&E (A, C, and E) and parafibromin-stained (B, D, and F) sections of normal parathyroid tissue (A and B) and two parathyroid carcinomas (sample 13 – C and D, sample 15 – E and F) showing the loss of parafibromin in carcinoma specimens. Non-neoplastic cells in sample 15 adjacent to the tumor field shown (panel D) stained positive for parafibromin (not shown).

References

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AstutiDMorrisMKronaCAbelFGentleDMartinssonTKognerPNeumannHPVoutilainenREngC2004Investigation of the role of SDHB inactivation in sporadic phaeochromocytoma and neuroblastoma. British Journal of Cancer9118351841.

BanksRETirukondaPTaylorCHornigoldNAstutiDCohenDMaherERStanleyAJHarndenPJoyceA2006Genetic and epigenetic analysis of von Hippel–Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer. Cancer Research6620002011.

BenderBUGutscheMGlaskerSMullerBKirsteGEngCNeumannHP2000Differential genetic alterations in von Hippel–Lindau syndrome-associated and sporadic pheochromocytomas. Journal of Clinical Endocrinology and Metabolism8545684574.

BradleyKJCavacoBMBowlMRHardingBCranstonTFratterCBesserGMConceicao PereiraMDavieMWDudleyN2006Parafibromin mutations in hereditary hyperparathyroidism syndromes and parathyroid tumours. Clinical Endocrinology64299306.

BrauchHWeirichGBriegerJGlavacDRodlHEichingerMFeurerMWeidtEPuranakanitsthaCNeuhausC2000VHL alterations in human clear cell renal cell carcinoma: association with advanced tumor stage and a novel hot spot mutation. Cancer Research6019421948.

CarlingTDuYFangWCorreaPHuangS2003Intragenic allelic loss and promoter hypermethylation of the RIZ1 tumor suppressor gene in parathyroid tumors and pheochromocytomas. Surgery134932940.

CarptenJDRobbinsCMVillablancaAForsbergLPresciuttiniSBailey-WilsonJSimondsWFGillandersEMKennedyAMChenJD2002HRPT2, encoding parafibromin, is mutated in hyperparathyroidism–jaw tumor syndrome. Nature Genetics32676680.

CasconARuiz-LlorenteSFragaMFLetonRTelleriaDSastreJDiezJJMartinez Diaz-GuerraGDiaz PerezJABenitezJ2004Genetic and epigenetic profile of sporadic pheochromocytomas. Journal of Medical Genetics41e30.

CavallariISilic-BenussiMRendeFMartinesAFogarPBassoDVellaMDPedrazzoliSHermanJGChieco-BianchiL2009Decreased expression and promoter methylation of the menin tumor suppressor in pancreatic ductal adenocarcinoma. Genes Chromosomes & Cancer48383396.

CetaniFPardiEBorsariSViacavaPDipollinaGCianferottiLAmbroginiEGazzerroEColussiGBertiP2004Genetic analyses of the HRPT2 gene in primary hyperparathyroidism: germline and somatic mutations in familial and sporadic parathyroid tumors. Journal of Clinical Endocrinology and Metabolism8955835591.

CetaniFPardiEAmbroginiEViacavaPBorsariSLemmiMCianferottiLMiccoliPPincheraAArnoldA2007Different somatic alterations of the HRPT2 gene in a patient with recurrent sporadic primary hyperparathyroidism carrying an HRPT2 germline mutation. Endocrine-Related Cancer14493499.

CliffordSCProwseAHAffaraNABuysCHMaherER1998Inactivation of the von Hippel–Lindau (VHL) tumour suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: evidence for a VHL-independent pathway in clear cell renal tumourigenesis. Genes Chromosomes & Cancer22200209.

CorbettaSVairaVGuarnieriVScillitaniAEller-VainicherCFerreroSVicentiniLChiodiniIBiscegliaMBeck-PeccozP2009Differential expression of microRNAs in human parathyroid carcinomas compared with normal parathyroid tissue. Endocrine-Related Cancer17135146.

DammannRSchagdarsurenginUSeidelCTrumplerCHoang-VuCGimmODralleHPfeiferGPBrauckhoffM2005Frequent promoter methylation of tumor-related genes in sporadic and men2-associated pheochromocytomas. Experimental and Clinical Endocrinology & Diabetes11317.

EstellerM2007Cancer epigenomics: DNA methylomes and histone-modification maps. Nature Reviews. Genetics8286298.

GarciaJMSilvaJPenaCGarciaVRodriguezRCruzMACantosBProvencioMEspanaPBonillaF2004Promoter methylation of the PTEN gene is a common molecular change in breast cancer. Genes Chromosomes & Cancer41117124.

GillAJClarksonAGimmOKeilJDralleHHowellVMMarshDJ2006Loss of nuclear expression of parafibromin distinguishes parathyroid carcinomas and hyperparathyroidism–jaw tumor (HPT–JT) syndrome-related adenomas from sporadic parathyroid adenomas and hyperplasias. American Journal of Surgical Pathology3011401149.

GlaskerSBenderBUApelTWvan VelthovenVMulliganLMZentnerJNeumannHP2001Reconsideration of biallelic inactivation of the VHL tumour suppressor gene in hemangioblastomas of the central nervous system. Journal of Neurology Neurosurgery and Psychiatry70644648.

GoelAArnoldCNNiedzwieckiDCarethersJMDowellJMWassermanLComptonCMayerRJBertagnolliMMBolandCR2004Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers. Cancer Research6430143021.

HavenCJHowellVMEilersPHDunneRTakahashiMvan PuijenbroekMFurgeKKievitJTanMHFleurenGJ2004Gene expression of parathyroid tumors: molecular subclassification and identification of the potential malignant phenotype. Cancer Research6474057411.

HermanJGLatifFWengYLermanMIZbarBLiuSSamidDDuanDSGnarraJRLinehanWM1994Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. PNAS9197009704.

HewittKMSharmaPKSamowitzWHobbsM2007Aberrant methylation of the HRPT2 gene in parathyroid carcinoma. Annals of Otology Rhinology and Laryngology116928933.

HowellVMHavenCJKahnoskiKKhooSKPetilloDChenJFleurenGJRobinsonBGDelbridgeLWPhilipsJ2003HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours. Journal of Medical Genetics40657663.

KellyTGShattuckTMReyes-MugicaMStewartAFSimondsWFUdelsmanRArnoldACarpenterTO2006Surveillance for early detection of aggressive parathyroid disease: carcinoma and atypical adenoma in familial isolated hyperparathyroidism associated with a germline HRPT2 mutation. Journal of Bone and Mineral Research2116661671.

KhanSKumagaiTVoraJBoseNSehgalIKoefflerPHBoseS2004PTEN promoter is methylated in a proportion of invasive breast cancers. International Journal of Cancer112407410.

KondoKYaoMYoshidaMKishidaTShuinTMiuraTMoriyamaMKobayashiKSakaiNKanekoS2002Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: relationship to clinicopathological parameters. Genes Chromosomes & Cancer345868.

KrebsLJShattuckTMArnoldA2005HRPT2 mutational analysis of typical sporadic parathyroid adenomas. Journal of Clinical Endocrinology and Metabolism9050155017.

MarshDZoriR2002Genetic insights into familial cancers – update and recent discoveries. Cancer Letters181125164.

MarshDJHahnMAHowellVMGillAJ2007Molecular diagnosis of primary hyperparathyroidism in familial cancer syndromes. Expert Opinion on Medical Diagnostics1377392.

MasiGBarzonLIacoboneMVielGPorzionatoAMacchiVDe CaroRFaviaGPaluG2008Clinical, genetic, and histopathologic investigation of CDC73-related familial hyperparathyroidism. Endocrine-Related Cancer1511151126.

MizusawaNUchinoSIwataTTsuyuguchiMSuzukiYMizukoshiTYamashitaYSakuraiASuzukiSBenikoM2006Genetic analyses in patients with familial isolated hyperparathyroidism and hyperparathyroidism–jaw tumour syndrome. Clinical Endocrinology65916.

MulliganLMKwokJBHealeyCSElsdonMJEngCGardnerELoveDRMoleSEMooreJKPapiL1993Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature363458460.

NickersonMLJaegerEShiYDurocherJAMahurkarSZaridzeDMatveevVJanoutVKollarovaHBenckoV2008Improved identification of von Hippel–Lindau gene alterations in clear cell renal tumors. Clinical Cancer Research1447264734.

RossJSCarlsonJABrockG2007miRNA: the new gene silencer. American Journal of Clinical Pathology128830836.

SalvesenHBMacDonaldNRyanAJacobsIJLynchEDAkslenLADasS2001PTEN methylation is associated with advanced stage and microsatellite instability in endometrial carcinoma. International Journal of Cancer912226.

SchagdarsurenginUGimmODralleHHoang-VuCDammannR2006CpG island methylation of tumor-related promoters occurs preferentially in undifferentiated carcinoma. Thyroid16633642.

ShattuckTMValimakiSObaraTGazRDClarkOHShobackDWiermanMETojoKRobbinsCMCarptenJD2003Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma. New England Journal of Medicine34917221729.

SmitsKMSchoutenLJvan DijkBAHulsbergen-van de KaaCAWoutersKAOosterwijkEvan EngelandMvan den BrandtPA2008Genetic and epigenetic alterations in the von Hippel–Lindau gene: the influence on renal cancer prognosis. Clinical Cancer Research14782787.

TakeuchiHGreepNCHoonDSGiulianoAEHansenNMUmetaniNSingerFR2007Hypermethylation of adenosine triphosphate-binding cassette transporter genes in primary hyperparathyroidism and its effect on sestamibi imaging. Journal of Clinical Endocrinology and Metabolism9217851790.

TseJYWongJHLoKWPoonWSHuangDPNgHK1997Molecular genetic analysis of the von Hippel–Lindau disease tumor suppressor gene in familial and sporadic cerebellar hemangioblastomas. American Journal of Clinical Pathology107459466.

ZhaoJYartAFrigerioSPerrenASchramlPWeisstannerCStallmachTKrekWMochH2007Sporadic human renal tumors display frequent allelic imbalances and novel mutations of the HRPT2 gene. Oncogene2634403449.

ZhouXPWaiteKAPilarskiRHampelHFernandezMJBosCDasoukiMFeldmanGLGreenbergLAIvanovichJ2003Germline PTEN promoter mutations and deletions in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3-kinase/Akt pathway. American Journal of Human Genetics73404411.

ZysmanMAChapmanWBBapatB2002Considerations when analyzing the methylation status of PTEN tumor suppressor gene. American Journal of Pathology160795800.

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