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.
HRPT2 mutation, putative HRPT2 promoter variant, and allelic loss in parathyroid tumor specimens
| Sample | Tumor type | HRPT2 allelic loss | −466C>A | HRPT2 mutation |
|---|---|---|---|---|
| 1 | Sporadic adenoma | LOH | A | nmd |
| 2 | Sporadic adenoma | R | CA | nmd |
| 3 | Sporadic adenoma | R | AA | nmd |
| 4 | Sporadic adenoma | R | CA | nmd |
| 5 | Sporadic adenoma | R | CA | nmd |
| 6 | Sporadic adenoma | LOH | A | nmd |
| 7 | Primary hyperplasia | R | AA | nmd |
| 8 | Primary hyperplasia | R | CA | nmd |
| 9 | Primary hyperplasia | R | AA | nmd |
| 10 | HPT–JT adenoma | LOH | A | c.191C>T (L64P) |
| 11 | HPT–JT carcinoma | LOH | – a | c.76delA |
| 12 | HPT–JT carcinoma | R | – a | c.76delA, c.686delGAGT |
| 13 | Sporadic carcinoma | R | – a | c.76delA |
| 14 | Sporadic carcinoma | R | – a | IVS6-1delG, c.165C>A (Y55X) |
| 15 | Sporadic carcinoma | R | – a | c.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.
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) |
|---|---|---|---|---|---|
| 11 | C | T | G | T | C |
| 12 | TC | GT | TG | TT | CC |
| 13 | TC | GT | TG | TT | CC |
| 14 | TC | GG | TG | TC | CA |
| 15 | TC | GG | GG | TC | CA |
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.
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).
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).
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).
Tumor suppressor genes implicated in endocrine neoplasia syndromes – frequency of methylation in counterpart tumors
| Gene | Tumor | Methylation | Methylation detection method | Study |
|---|---|---|---|---|
| HRPT2 | Parathyroid | Sequencing | 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 a | Mizusawa et al. (2006) | |
| Parathyroid (HPT–JT-associated) | 0/2 (0%) | Sequencing a | Masi et al. (2008) | |
| Parathyroid | MSP and sequencing | Hewitt et al. (2007) | ||
| Adenoma (non-HPT–JT) | 0/37 (0%) | |||
| Adenoma (HPT–JT-associated) | 1/6 (17%) | |||
| Carcinoma | 2/11 (18%) | |||
| VHL | Pheochromocytoma | 0/53 (0%) | Methylation-sensitive restriction endonuclease digestion | Bender et al. (2000) |
| 0/35 (0%) | MSP and sequencing | Cascon et al. (2004) | ||
| 0/25 (0%) | MSP | Dammann et al. (2005) | ||
| Renal cell carcinoma | 5/26 (19%) | Methylation-sensitive restriction endonuclease digestion | Herman et al. (1994) | |
| 11/99 (11%) | Methylation-sensitive restriction endonuclease digestion | Clifford et al. (1998) | ||
| 13/77 (17%) | Methylation-sensitive restriction endonuclease digestion | Brauch et al. (2000) | ||
| 11/202 (5%) | MSP | Kondo et al. (2002) | ||
| 19/93 (20%) | MSP | Banks et al. (2006) | ||
| 16/185 (9%) | MSP | Smits et al. (2008) | ||
| 17/205 (8%) | Sequencing | Nickerson et al. (2008) | ||
| Hemangioblastoma | 0/9 (0%) | Methylation-sensitive restriction endonuclease digestion | Tse et al. (1997) | |
| 0/42 (0%) | Methylation-sensitive restriction endonuclease digestion | Glasker et al. (2001) | ||
| SDHB | Pheochromocytoma | 9/28 (32%) | Sequencing a | Astuti et al. (2004) |
| 0/35 (0%) | MSP and sequencing | Cascon et al. (2004) | ||
| SDHD | Pheochromocytoma | 0/35 (0%) | MSP and sequencing | Cascon et al. (2004) |
| PTEN b | Thyroid | MSP | Alvarez-Nunez et al. (2006) | |
| Papillary thyroid carcinoma | 21/46 (46%) | |||
| Follicular thyroid carcinoma | 6/7 (86%) | |||
| Follicular thyroid adenoma | 5/6 (83%) | |||
| Thyroid | MSP | Schagdarsurengin et al. (2006) | ||
| Medullary thyroid carcinoma | 0/6 (0%) | |||
| Follicular thyroid carcinoma | 0/10 (0%) | |||
| Papillary thyroid carcinoma | 2/12 (17%) | |||
| Undifferentiated thyroid cancer | 1/7 (14%) | |||
| Follicular thyroid adenoma | 2/9 (22%) | |||
| Endometrial carcinoma | 26/138 (19%) | Methylation-sensitive restriction endonuclease digestion and MSP | Salvesen et al. (2001) | |
| Breast | 43/90 (48%) | MSP and sequencing | Garcia et al. (2004) | |
| 15/44 (34%) | MSP | Khan et al. (2004) | ||
| MEN1 | Pancreatic ductal adenocarcinoma | 5/24 (21%) | MSP | Cavallari 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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