Abstract
CDC73 (HRPT2) germline mutations are responsible for more than half of cases of hyperparathyroidism-jaw tumor syndrome (HPT-JT) and for a subset of familial isolated HPT (FIHP). We performed a clinical, genetic, and histopathologic study in three unrelated Italian kindreds with HPT-JT and FIHP. We identified three germline inactivating mutations of the CDC73 gene in the probands and affected patients of the three kindreds, but also in some asymptomatic subjects. HPT-JT and FIHP patients had similar laboratory, clinical, and demographic features and shared primary HPT and other neoplasms, the most common of which was uterine polyposis. Genetic analysis of tumor samples demonstrated a second somatic CDC73 mutation only in a parathyroid adenoma and no cases with the loss of the wild-type allele or methylation of the CDC73 promoter, even though immunohistochemical analysis demonstrated the loss of nuclear parafibromin expression in all tumors, including a uterine polyp. In conclusion, our results indicate that FIHP and HPT-JT associated with CDC73 mutations do not have distinct clinical, genetic, and histopathologic features, but may represent variants of the same genetic disease. This study also confirms that uterine involvement represents a clinical manifestation of the syndrome.
Introduction
The hyperparathyroidism-jaw tumor syndrome (HPT-JT; online Mendelian inheritance in man (OMIM)#145001) is an autosomal dominant syndrome with incomplete penetrance, characterized by the occurrence of parathyroid tumors (with a relatively high prevalence of carcinomas and atypical adenomas), ossifying fibromas of mandible and/or maxilla (in 30% of patients), and, less frequently, uterine tumors, and a variety of renal lesions, such as Wilms tumors, hamartomas, and polycystic disease (Jackson et al. 1990, Carpten et al. 2002). HPT-JT is caused by mutations in the putative tumor suppressor gene CDC73 also known as (HRPT2) (Carpten et al. 2002), which encodes an ubiquitously expressed protein, named parafibromin (Porzionato et al. 2006). Parafibromin has been shown to inhibit cell proliferation by inducing cell cycle arrest (Woodard et al. 2005, Zhang et al. 2006) and to participate in transcription regulation by interacting with the polymerase II-associated factor 1 (PAF1) transcription elongation complex (Yart et al. 2005). Parafibromin has also been recognized as a component of the Wnt signaling pathway, with an unexpected activating role (Mosimann et al. 2006).
Germline mutations in CDC73 have been identified not only in about 60% of HPT-JT kindreds (Carpten et al. 2002) and in one-third of patients with apparently sporadic parathyroid carcinoma (Howell et al. 2003, Shattuck et al. 2003, Cetani et al. 2004), but also in 7% of kindreds with familial isolated HPT (FIHP; OMIM#145000; Simonds et al. 2004, Villablanca et al. 2004, Bradley et al. 2006, Mizusawa et al. 2006), a heterogeneous disease, which has also been related to MEN1 and CASR mutations (Hannan et al. 2008). Germline and somatic mutations identified so far are scattered throughout the coding region of the CDC73 gene and most are predicted to result in truncated or inactive forms of parafibromin (Carpten et al. 2002, Villablanca et al. 2004). Parafibromin inactivation has been confirmed by immunohistochemical and functional studies, which demonstrated that CDC73 mutations result in the loss of parafibromin expression (Tan et al. 2004, Gill et al. 2006, Juhlin et al. 2006) or abnormal subcellular localization (Bradley et al. 2007, Lin et al. 2007) and abolition of its anti-proliferative activity (Zhang et al. 2006), even though experiments with knockout mice indicate that expression of parafibromin is pivotal in mammalian development and survival in adults, whereas its loss leads to apoptosis in vitro (Wang et al. 2008).
This study reports the results of clinical, genetic, and histopathologic investigation of three unrelated Italian kindreds with HPT-JT and FIHP. The presence of germline and somatic CDC73 mutations in all kindreds, the occurrence of tumors other than parathyroid, and the loss of parafibromin expression in parathyroid and uterine tumors suggested that HPT-JT and FIHP could be variants of the same genetic disease.
Subjects and methods
Clinical investigation
The study population consists in a large HPT-JT kindred, including 6 clinically symptomatic and 9 asymptomatic subjects, and 2 unrelated FIHP kindreds, including a total of 10 symptomatic and 13 asymptomatic subjects (Fig. 1), which were evaluated at the Endocrine Surgery Department of the University of Padua and previously published in part (Iacobone et al. 2007). Informed consent for the collection of personal, genetic, and clinical data was obtained from all patients and the study was approved by the local ethics committee.
Pedigrees of the kindreds with HPT-JT and FIHP. (A) HPT-JT kindred; (B) FIHP kindred 1; (C) FIHP kindred 2. Square symbols indicate males, and round symbols indicate females. A diagonal slash mark through the symbol means deceased. The generations are labeled in Roman numerals, and the individuals within each generation are designated with Arabic numerals. The black arrow indicates probands. Filled quadrants indicate a diagnosis or history of the trait indicated in the legend. The sign on the right of the symbol marks the subjects that have been tested for a germline CDC73 mutations: the plus sign (+) indicates the presence of a CDC73 mutation, and the asterisk (*) indicate the absence of the mutation. The interrupted line in B) indicates the lack of three siblings and of their descendance that refused their participation in the study.
Citation: Endocrine-Related Cancer 15, 4; 10.1677/ERC-08-0066
FIHP was diagnosed when 1) primary HPT (hypercalcemia, inappropriately normal or increased intact parathormone levels, normal or increased urinary calcium with normal renal function) was present in the proband and in at least one relative; 2) there was evidence of abnormal parathyroid gland at histology in at least one case; 3) no other clinical manifestations related to multiple endocrine neoplasm type 1 (MEN1) or MEN2 were present. HPT-JT was diagnosed when maxillary or mandibular ossifying tumors were also present in FIHP patients (Cetani et al. 2004).
In- and outpatient medical records were reviewed for clinical and biochemical details; complete follow-up data were obtained by extensive clinical and laboratory reevaluation or personal telephone interview designed to elicit all information regarding the patient's current state of health, the most recent laboratory determinations, and the presence of other eventually affected relatives.
Screening for MEN1- and MEN2-associated tumors included laboratory and imaging evaluation of the pancreas, pituitary, adrenals, and thyroid C cells component.
Complete bilateral neck exploration and selective parathyroidectomy of the macroscopically abnormal glands and biopsy of the normal appearing parathyroids were performed as reported (Iacobone et al. 2007).
Screening for jaw tumors used orthopantographic X-rays and/or CT of the mandible and maxilla. Evaluation of the kidneys used standard ultrasound, abdominal MRI or CT scan. Uterine abnormalities were assessed by standard ultrasound and/or hysteroscopic examination and eventually confirmed at biopsy.
Tissue samples and pathological investigation
Parathyroid specimens were carefully reviewed, and the diagnosis confirmed according to the World Health Organization guidelines (DeLellis et al. 2004). Frozen and paraffin-embedded specimens from four parathyroid adenomas (patients II-2 and II-3, HPT-JT kindred; patient IV-3, FIHP kindred 1; patient IV-1, FIHP kindred 2) and three normal parathyroid glands (patients II-2 and II-3, HPT-JT kindred; patient IV-1, FIHP kindred 2) were available for genetic and immunohistochemical studies. Other three paraffin-embedded parathyroid adenomas (patient III-2, HPT-JT kindred; patient IV-4, FIHP kindred 1; patient III-1, FIHP kindred 2), a parathyroid carcinoma (patient I-1, HPT-JT kindred), and tissues from multiple endometrial hyperplastic polyps from patient II-3 of the HPT-JT kindred were available for immunohistochemistry. Ten normal parathyroids, which were accidentally removed at the time of thyroid surgery for benign diseases from patients without clinical and biochemical evidence of HPT, and five sporadic endometrial hyperplastic polyps from age-matched patients without germline CDC73 mutations were taken as control groups.
Mutation analysis of the CDC73 gene
Genomic DNA was isolated from peripheral blood leukocytes of probands and available family members and from parathyroid frozen tissues using a QIA Amp DNA Mini Kit (Qiagen GmbH). The entire coding region and the intron–exon boundaries of the CDC73 gene were sequenced by PCR amplification using oligonucleotide primer sequences previously reported (Shattuck et al. 2003) and bidirectional sequencing of PCR products using an ABI PRISM BigDye terminators v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequences were run on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems) and compared with the reference sequence Gene ID 79577. All genetic alterations were confirmed by a second independent sequencing reaction performed on a second blood sample. Moreover, in order to better define heterozygous frameshift mutations, PCR amplicons carrying the mutations were subcloned into pGEM-T Easy vectors (Promega, Madison, WI, USA) to separate the two alleles and resequenced as above described.
Methylation analysis of the CDC73 promoter
The methylation status of the CDC73 promoter region including 65 CpG sites was assessed by bisulfite sequencing. To this aim, genomic DNA from leukocytes and tumor tissues was modified by bisulfite treatment and subsequently purified using an EpiTect Bisulfite Kit (Qiagen GmbH) according to the manufacturer's recommendations. Bisulfite-treated DNA was amplified with primers from Mizusawa et al. (2006) and PCR products were cloned into a pGEM-T Easy vector. Five clones were each sequenced as above described. Sequences from parathyroid tumors and corresponding leukocytes were compared.
Immunohistochemistry
Parafibromin expression was evaluated by immunohistochemical staining on a parathyroid carcinoma, seven parathyroid adenomas, and three normal parathyroids surgically removed from HPT-JT and FIHP patients, and on endometrial hyperplastic polyps from the HPT-JT patient. Anti-parafibromin immunohistochemistry was also performed on control normal parathyroids and sporadic endometrial hyperplastic polyps. Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissues as previously described (Porzionato et al. 2006), using a mouse monoclonal anti-parafibromin antibody (SC-33638, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) raised to target amino acids 87–100 of parafibromin. Slide sections were examined by scanning the entire tissue specimens under low power magnification (5–10×), later confirmed at higher power magnification (20–40×).
Statistical analysis
Demographics, clinical, and laboratory features of HPT-JT and FIHP kindreds were compared by Fisher's exact test and Mann–Whitney test. A P<0.05 was considered statistically significant.
Results
Clinical characteristics of the kindreds
Clinical features of investigated subjects are summarized in Table 1. Laboratory and imaging evaluation of pancreas, pituitary, thyroid C cells, and adrenals did not show any abnormalities in all screened patients. In all patients, a single-gland parathyroid involvement was found at each surgical procedure.
Clinical characteristics of patients with hyperparathyroidism from hyperparathyroidism-jaw tumor syndrome and familial isolated hyperparathyroidism (FIHP) kindreds
| Family | Patient | Sex/age (years) | HPT, age at diagnosis (years) | Calcemiaa (mmol/l) at diagnosis | PTHb (pg/ml) at diagnosis | Parathyroid tumors (number and pathology) | Jaw tumor | Cystic and neoplastic renal lesions | Uterine involvement | Other diseases |
|---|---|---|---|---|---|---|---|---|---|---|
| HPT-JT | I-1 | M/59c | 56 | 4.12 | 313 | 1 Carcinoma | − | − | − | Papillary thyroid carcinoma |
| HPT-JT | II-1 | M/49 | 30 | 2.74 | 73 | 1 Atypical adenoma | − | − | − | Multinodular goiter |
| HPT-JT | II-2 | F/48 | 1846d | 2.802.65d | NA57d | 1 Adenoma1 Adenomad | + | − | + | Papillary thyroid carcinoma; Colon adenocarcinoma |
| HPT-JT | II-3 | F/45 | 42 | 2.64 | 78 | 1 Adenoma | − | − | + | − |
| HPT-JT | II-5 | F/40 | 21 | 3.30 | 116 | 1 Adenoma | − | − | + | − |
| 30d | 2.90d | 120d | 1 Adenomad | |||||||
| HPT-JT | III-2 | F/17 | 11 | 4.48 | 1128 | 1 Adenoma | − | − | − | − |
| FIHP 1 | III-4 | F/62 | 44 | 3.00 | 151 | 1 Adenoma | − | − | − | − |
| FIHP 1 | III-8 | F/50 | 32 | 3.43 | 255 | 1 Adenoma | − | − | − | − |
| FIHP 1 | IV-3 | F/43 | 38 | 2.90 | 155 | 1 Adenoma | − | − | + | − |
| FIHP 1 | IV-4 | F/42 | 25 | 3.15 | 211 | 1 Adenoma | − | − | + | Thyroid adenoma |
| 29d | 4.01d | 280d | 1 Adenomad | |||||||
| FIHP 1 | II-3e | F/75 | 52 | NA | NA | 1 Adenoma | − | − | + | − |
| FIHP 1 | IV-2e | M/45 | 43 | NA | NA | 1 Adenoma | − | − | − | − |
| FIHP 2 | III-1 | F/43 | 38 | 2.89 | 103 | 1 Adenoma | − | − | + | − |
| FIHP 2 | IV-1 | F/26 | 23 | 3.24 | 81 | 1 Adenoma | − | − | − | − |
| FIHP 2 | III-5e | F/45 | 37 | NA | NA | 1 Adenoma | − | − | + | − |
| FIHP 2 | II-4 | F/76 | 71 | 3.05 | 94 | Not operated | − | − | − | − |
F, female; M, male; HPT, hyperparathyroidism; NA: not available; +: present; −: absent.
Total serum calcium, reference range: 2.10–2.60 mmol/l.
PTH, reference range: 15–65 pg/ml.
Deceased.
At HPT recurrence.
Operated elsewhere.
HPT-JT kindred
The proband (II-2) had recurrent HPT due to single parathyroid adenomas, which were diagnosed at 18 and 46 years (Fig. 1A). A jaw tumor (maxillary ossifying fibroma) was diagnosed and excised at 26 years. Other clinical manifestations included papillary thyroid carcinoma (pT1N0M0), operated at 46 years, multiple benign uterine polyps (surgery performed at 23 and 41 years) and left colon adenocarcinoma (pT2N1M0; colectomy at 42 years). Multiple uterine polyps and single parathyroid adenomas were present also in subjects II-3 and II-5. Patient I-1, who had a parathyroid carcinoma and an incidentally detected multifocal papillary thyroid microcarcinoma (pT1N0M0), died at 59 years because of metabolic complications of HPT. Subject II-1 underwent surgery at 30 years because of parathyroid atypical adenoma and a multinodular goiter. Subject III-2 was operated for a parathyroid adenoma at 11 years, whereas other young subjects of the family (III-4, 20 years; III-6, 14 years; and III-9, 15 years) are carriers of germline CDC73 mutations without laboratory or clinical signs of HPT, nor jaw tumors or renal involvement. To date, no renal cystic or neoplastic lesions are evident in this kindred.
FIHP kindred 1
The proband (IV-3) had parathyroid adenoma and uterine polyposis. Her sister (IV-4) had recurrent HPT due to parathyroid adenomas, uterine polyposis, and thyroid adenoma (Fig. 1B). The other effected family members (III-4, III-8) had parathyroid adenoma. Subjects II-3 and IV-2 had parathyroid adenomas and underwent surgery elsewhere; subject II-3 had also uterine involvement. Subjects III-5, IV-13, and V-2 (aged 60, 15, and 13 years) are unaffected carriers of germline CDC73 mutations. No renal or jaw tumors were detected in this kindred.
FIHP kindred 2
The proband (III-1) had parathyroid adenoma, which was operated on at 38 years, and multiple uterine polyposis (Fig. 1C). Her daughter (IV-1) underwent surgery for parathyroid adenoma at 23 years. Patient III-5 underwent excision of a single parathyroid adenoma elsewhere at the age of 38 years; she had also uterine involvement. Patient II-4 had a biochemical diagnosis of HPT, but refused surgery. Patient II-1 died at 67 years because of renal carcinoma. To date, no jaw tumors have been detected in this kindred.
No significant differences were found between HPT-JT and FIHP kindreds according to the occurrence of primary HPT (63 vs 67% respectively, in subjects carrying CDC73 mutations), age at diagnosis of HPT (median, 25.5 years; range, 11–56 years vs 38 years; range, 23–71 respectively), total serum calcium levels (mean, 3.2±0.7 mmol/l vs 3.2±0.4 mmol/l), and plasma parathyroid hormone (PTH) levels (mean, 269±388 pg/ml vs 166±75 pg/ml).
CDC73 mutation and methylation analysis
The proband of HPT-JT kindred (II-2) carried a previously unreported germline heterozygous frameshift mutation in exon 6 of the CDC73 gene (c.433_442delinsAGA), which predicts an alteration of the reading frame with a premature truncation at codon 201 (Fig. 2A). Genetic testing was extended to 13 members of the kindred, and the CDC73 mutation was detected in other 4 affected and 3 unaffected family members (Fig. 1A). Both normal and mutant alleles were retained in the two investigated parathyroid tumors and no additional somatic CDC73 mutations were identified.
Mutation analysis of the CDC73 gene (A–D). Electropherograms of PCR amplicons from (A) peripheral blood DNA of subject II-2 of the HPT-JT kindred (after subcloning into pGEM®-T Easy vectors) showing the wild type (upper panel) and mutant allele (lower panel) carrying the c.433_442delinsAGA mutation of the CDC73 gene; (B) peripheral blood DNA of subject IV-3 of FIHP kindred 1 showing the germline heterozygous mutation c.188T>C (Leu63Pro) of the CDC73 gene; (C) parathyroid adenoma of subject IV-3 of FIHP kindred 1 showing the somatic heterozygous frameshift mutation (c.375_376insAA) at exon 5 of the CDC73 gene, after subcloning into pGEM®-T Easy vectors; and (D) peripheral blood DNA of subject III-2 of FIHP kindred 2 showing the germline heterozygous mutation c.(136_144)del5 of the CDC73 gene, after subcloning into pGEM®-T Easy vectors.
Citation: Endocrine-Related Cancer 15, 4; 10.1677/ERC-08-0066
Germline CDC73 mutations were also identified in both FIHP kindreds. The proband of FIHP kindred 1 (IV-3) carried a novel germline heterozygous c.188T>C transition, resulting in the substitution Leu63Pro (Fig. 2B). The patient had also a somatic heterozygous frameshift mutation (c.375_376insAA) at exon 5 in the parathyroid adenoma, which is predicted to lead to the formation of a stop codon at residue 132 (Fig. 2C). Mutation analysis of the CDC73 gene was extended to 18 members of the kindred, which demonstrated the presence of the germline c.188T>C mutation in the three other affected patients, but also in three unaffected healthy individuals (Fig. 1B).
In FIHP kindred 2, a germline heterozygous five-nucleotides deletion c.(136_144)del5 in exon 2 was detected in the index case patient (III-1) and in her daughter (Fig. 2D), while it was not detected in the healthy subjects IV-2 and V-1. The mutation determines a frameshifting that leads to formation of a stop codon at residue 62. Heterozygosity of the mutation was retained in an investigated parathyroid adenoma (IV-1).
Methylation analysis of the CDC73 promoter in the DNA purified from two parathyroid adenomas without somatic CDC73 mutations, obtained from two patients (II-2; II-3) of HPT-JT kindred 1 and in the corresponding leukocytes did not demonstrate any methylated CpG site.
Analysis of parafibromin expression
Anti-parafibromin nuclear immunostaining was absent in almost all tumor cells in the parathyroid carcinoma and in all parathyroid adenomas from HPT-JT and FIHP patients. At variance, intense nuclear immunostaining (>90% of parathyroid cells) was present in all normal parathyroids obtained at surgery from both HPT-JT and FIHP patients and controls (Fig. 3). In the HPT-JT-related uterine polyp, stromal cells did not show any anti-parafibromin nucleocytoplasmatic reactivity, whereas epithelial cells had no nuclear immunostaining but moderate cytoplasmic immunoreactivity. In the five sporadic uterine polyps from the control group, stromal cells showed positive nuclear anti-parafibromin immunostaining and epithelial cells showed intense nuclear immunostatining and moderate cytoplasmic positivity (Fig. 4).
Anti-parafibromin immunostaining of parathyroid tissues in HPT-JT (A–C): the loss of nuclear parafibromin immunostaining in parathyroid adenoma (B) and carcinoma (C) with respect to normal parathyroid (A). (Original magnification: 40×).
Citation: Endocrine-Related Cancer 15, 4; 10.1677/ERC-08-0066
Anti-parafibromin immunostaining of sporadic (A) and HPT-JT-related (B) uterine polyps, showing the absence of nuclear staining in stromal and epithelial cells of syndrome-related uterine polyp. (Original magnification: 40×).
Citation: Endocrine-Related Cancer 15, 4; 10.1677/ERC-08-0066
Discussion
HPT-JT and FIHP are usually considered different entities, since HPT-JT is classically identified according to clinical criteria (the presence of familial HPT and associated jaw tumors – constantly absent in FIHP – without other endocrine tumors related to MEN1 or MEN2; Cetani et al. 2004). Genetic analysis has shown that the most of HPT-JT cases, but only a minority of FIHP cases, are related to CDC73 mutations (Carpten et al. 2002, Simonds et al. 2004, Villablanca et al. 2004, Bradley et al. 2006, Mizusawa et al. 2006). FIHP is a heterogeneous disease, since it may represent a phenotypic variant with incomplete penetrance of other genetic syndromes, such as HPT-JT, MEN1, MEN2, and familial hypocalciuric hypercalcemia. Thus, the clinical diagnosis of FIHP should be considered only provisional, and it should be revised following mutational analysis of the CDC73, MEN1, RET, and CASR genes respectively.
The results of our study confirm the idea that HPT-JT and FIHP (when associated with CDC73 mutations) may be variants of the same disease, since our kindreds, besides sharing the same genetic background, characterized by germline inactivating mutations of the CDC73 gene, had also similar clinical features. In fact, the age of onset of HPT and laboratory data were not significantly different in FIHP and HPT-JT patients, even though parathyroid carcinoma and atypical adenoma were found exclusively in the HPT-JT kindred. On the other hand, a revision of the literature shows that parathyroid carcinoma occurs quite frequently also in FIHP kindreds (Carpten et al. 2002, Bradley et al. 2005b). Furthermore, other typical clinical features, such as uterine polyposis and other neoplasms, were present in both HPT-JT and FIHP kindreds, in agreement with the literature (Carpten et al. 2002, Bradley et al. 2005b). Besides primary HPT, which was diagnosed in 63% HPT-JT patients and in 67% FIHP patients carrying CDC73 mutations, uterine polyposis was the second most common clinical feature in our patients, being identified in 75% of affected HPT-JT female patients and 56% of FIHP female patients, as also reported in the literature (Cavaco et al. 2004, Bradley et al. 2005b, Guarnieri et al. 2006).
Other neoplastic lesions were less frequent. Only one out of six affected members of the HPT-JT kindred had a jaw tumor. Variable penetrance of this syndromic feature has been reported in the literature (Carpten et al. 2002, Cavaco et al. 2004, Bradley et al. 2005b). As previously reported (Haven et al. 2000, Carpten et al. 2002, Cavaco et al. 2004, Bradley et al. 2005b), we also identified two cases of papillary thyroid carcinoma in HPT-JT patients, even though the exclusive finding of microscopic foci at a preclinical stage might be considered an occasional finding during targeted neck investigation rather than a clinical feature of the syndromes. Cystic or neoplastic renal involvement has been reported in 0–70% of CDC73 mutation carriers (Carpten et al. 2002, Howell et al. 2003, Cavaco et al. 2004, Villablanca et al. 2004, Bradley et al. 2005b, Guarnieri et al. 2006); in this regard, we identified only one case of kidney cancer in a patient without HPT from a FIHP kindred. However, the CDC73 mutation status of this patient is unknown, so we cannot exclude the renal cancer may be unrelated to the syndrome. Finally, we found a locally advanced colon carcinoma in a relatively young HPT-JT patient, an unusual finding in HPT-JT (Simonds et al. 2002). Unfortunately, a tumor sample was not available for genetic testing and parafibromin immunostaining, so, even in this case, the association between occurrence of colon cancer and CDC73 mutation cannot be established.
Genetic investigation demonstrated that both HPT-JT and FIHP kindreds carried germline mutations of the CDC73 gene. Mutations were predicted to result in truncated or inactive parafibromin, in agreement with its tumor suppressor activity. In addition, somatic CDC73 mutations and/or loss of parafibromin expression were detected in parathyroid tumors from both HPT-JT and FIHP patients. Three of the CDC73 mutations we identified have never been reported before in the literature, i.e., c.433_442delinsAGA germline mutation (the first mutation reported to date at exon 6), c.375_376insAA, and the germline Leu63Pro missense mutation. The fourth mutation, i.e., the germline five-nucleotides deletion c.(136_144)del5 identified in FIHP kindred 2, has been previously described by Kelly et al. 2006 as c.140_144del5 in an Australian kindred with FIHP. While the three frameshifting mutations are predicted to result in truncated forms of parafibromin, the Leu63Pro germline missense mutation changes a conserved leucine at codon 63 with proline. A similar mutation in the adjacent codon, Leu64Pro, has been already reported in FIHP kindreds (Howell et al. 2003, Villablanca et al. 2004). This mutation, characterized by a substitution of the hydrophobic amino acid leucine with the helix breaker amino acid proline, might cause a significant alteration in the structure of parafibromin and impair its activity, as demonstrated in in vitro experiments (Woodard et al. 2005).
CDC73 mutations reported so far in the literature (Table 2) are scattered along the sequence of the gene, with a higher number of mutations in exons 1–2 and 7–8, but no mutations in exons 9–12 and 14–17. Germline frameshift and nonsense CDC73 mutations are the most frequent mutations, representing 88 and 57% of the mutations identified in HPT-JT and FIHP kindreds respectively, whereas germline missense mutations are rather infrequent and generally affect functionally important regions of parafibromin. No genotype–phenotype correlation has been identified so far.
Summary of all CDC73 germline and somatic mutations reported in the literature
| Exon | Mutation | Predicted effect | Clinical condition | Mutation status | Reference |
|---|---|---|---|---|---|
| 1 | 3G>A | Met1Ile | HPT-JT | Germline | Carpten et al. (2002) |
| 1 | 4C>T | Ala2Ser | Sporadic PTA | Somatic | Cetani et al. (2007b) |
| 1 | 13_30del18 | Frameshift | HPT-JT | Somatic | Moon et al. (2005) |
| 1 | 16delA | Frameshift | Sporadic PTC | ND | Shattuck et al. (2003) |
| 1 | 20A>G, 24delC | Val7Arg- frameshift | HPT-JT | Germline | Aldred et al. (2006) |
| 1 | 23TGCG>gtg | Frameshift | Sporadic PTC | Somatic | Shattuck et al. (2003) |
| 1 | 25C>T | Arg9X | HPT-JT | Germline | Carpten et al. (2002) |
| 1 | 25C>T | Arg9X | Sporadic PTC | Somatic | Cetani et al. (2004) |
| 1 | 41-bp delins | Frameshift | HPT-JT | Germline | Carpten et al. (2002) |
| 1 | 34delAACATCC | Frameshift | HPT-JT | Germline | Carpten et al. (2002) |
| 1 | 30delG | Frameshift | HPT-JT | Germline | Carpten et al. (2002) |
| 1 | 39delC | Frameshift | HPT-JT | Germline | Carpten et al. (2002) |
| 1 | 39delC | Frameshift | HPT-JT | Germline | Mizusawa et al. (2006) |
| 1 | 39delC | Frameshift | Sporadic PTC | Somatic | Shattuck et al. (2003) |
| 1 | 53delT | Frameshift | Sporadic PTA | Somatic | Carpten et al. (2002) |
| 1 | 53delT | Frameshift | Sporadic PTA | Somatic | Juhlin et al. (2006) |
| 1 | 60del10 | Frameshift | Sporadic PTC | ND | Shattuck et al. (2003) |
| 1 | 62_66del | Frameshift | FIHP | Germline | Mizusawa et al. (2006) |
| 1 | 70G>T | Glu24X | Sporadic PTC | Somatic | Shattuck et al. (2003) |
| 1 | 70G>T | Glu24X | Sporadic PTC | Somatic | Cetani et al. (2007a) |
| 1 | 70delG | Frameshift | Sporadic OF | Somatic | Pimenta et al. (2006) |
| 1 | 70_73del | Frameshift | FIHP | Somatic | Mizusawa et al. (2006) |
| 1 | 76delA | Frameshift | Sporadic PTC | Somatic | Howell et al. (2003) |
| 1 | 76delA | Frameshift | HPT-JT | Germline | Howell et al. (2003) |
| 1 | 82del4 | Frameshift | Sporadic PTC | Somatic | Shattuck et al. (2003) |
| 1 | 85delG | Frameshift | HPT-JT | Somatic | Moon et al. (2005) |
| 1 | 95_102del8 | Frameshift | FIHP | Somatic | Mizusawa et al. (2006) |
| 1 | 96G>A | Trp32X | HPT-JT | Germline | Sarquis et al. (2008) |
| 1 | 100A>C | Lys34Gln | Renal tumor | Somatic | Zhao et al. (2007) |
| 1 | 126del24 | Frameshift/splice mutation | Sporadic PTA | Somatic | Carpten et al. (2002) |
| 1 | 127insC | Frameshift | Sporadic PTC | Germline | Juhlin et al. (2007) |
| 1 | 128G>A | Trp43X | FIHP | Somatic | Carpten et al. (2002) |
| 2 | (136_144)del5 | Frameshift | FIHP | Germline | Present study |
| 2 | (136_144)del5 | Frameshift | FIHP | Germline | Kelly et al. (2006) |
| 2 | 162C>G | Tyr54X | Sporadic PTC | Somatic | Howell et al. (2003) |
| 2 | 162C>G | Tyr54X | Sporadic PTC | Somatic | Shattuck et al. (2003) |
| 2 | 162C>G | Tyr54X | Sporadic PTA | Somatic | Cetani et al. (2007b) |
| 2 | 165C>G | Tyr55X | HPT-JT | Germline | Carpten et al. (2002) |
| 2 | 165C>A | Tyr55X | Sporadic PTC | Somatic | Howell et al. (2003) |
| 2 | 176C>T | Ser59Phe | Sporadic PTC | Germline | Haven et al. (2007) |
| 2 | 182T>A | Leu61X | Sporadic PTC | Somatic | Cetani et al. (2007a) |
| 2 | 188T>C | Leu63Pro | FIHP | Germline | Present study |
| 2 | 191T>C | Leu64Pro | FIHP | Germline | Howell et al. (2003) |
| 2 | 191T>C | Leu64Pro | FIHP | Germline | Villablanca et al. (2004) |
| 2 | 195insT | Frameshift | Sporadic PTC | Somatic | Cetani et al. (2004) |
| 2 | 195insA | Frameshift | Sporadic PTC | Somatic | Cetani et al. (2004) |
| 2 | 226C>T | Arg76X | Sporadic PTC | Somatic | Shattuck et al. (2003) |
| 3 | 272G>C | Arg91Pro | Sporadic PTA | Germline | Cetani et al. (2007b) |
| 3 | 284T>C | Leu95Pro | FIHP | Somatic | Bradley et al. (2006) |
| 3 | 306delGTgtgagtacttttt | Frameshift/splice site mutation | HPT-JT | Germline | Carpten et al. (2002) |
| 4 | 356delA | Frameshift | HPT-JT | Germline | Carpten et al. (2002) |
| 4 | 356delA | Frameshift | FIHP | Germline | Bradley et al. (2006) |
| 5 | 373insA | Frameshift | Sporadic PTC | Germline | Shattuck et al. (2003) |
| 5 | c.375_376insAA | Frameshift | FIHP | Somatic | Present study |
| 5 | 406A>T | Lys136X | HPT-JT | Germline | Carpten et al. (2002) |
| 5 | 415C>T | Arg139X | Sporadic PTC | Germline | Cetani et al. (2007a) |
| 6 | c.433_442delinsAGA | Frameshift | HPT-JT | Germline | Present study |
| 7 | 518_521 del4 | Frameshift | FIHP | Germline | Mizusawa et al. (2006) |
| 7 | 636delT | Frameshift | HPT-JT | Germline | Carpten et al. (2002) |
| 7 | 664C>T | Arg222X | Sporadic PTC | Germline | Shattuck et al. (2003) |
| 7 | 669delAT/insG | Frameshift | HPT-JT | Germline | Bradley et al. (2005b) |
| 7 | 679 insAG | Frameshift | HPT-JT | Germline | Bradley et al. (2005b) |
| 7 | 679insAG | Frameshift | HPT-JT | Germline | Carpten et al. (2002) |
| 7 | 679insAG | Frameshift | HPT-JT | Germline | Carpten et al. (2002) |
| 7 | 679insAG | Frameshift | Sporadic PTC | Germline | Shattuck et al. (2003) |
| 7 | 679insAG | Frameshift | FIHP | Germline | Simonds et al. (2004) |
| 7 | 679del AG | Frameshift | HPT-JT | Germline | Howell et al. (2003) |
| 7 | 679del AG | Frameshift | HPT-JT | Germline | Sarquis et al. (2008) |
| 7 | 685delAGAG | Frameshift | FIHP | Germline | Guarnieri et al. (2006) |
| 7 | 686delGAGT | Frameshift | HPT-JT | Somatic | Howell et al. (2003) |
| 7 | 692_693insT | Frameshift | Sporadic PTC | Germline | Haven et al. (2007) |
| 7 | 693_694insG | Frameshift | Sporadic PTC | ND | Haven et al. (2007) |
| 7 | 700C>T | Arg234X | Sporadic PTC | Germline | Cetani et al. (2004) |
| 7 | 700C>T | Arg234X | Sporadic PTC | Germline | Cetani et al. (2004) |
| 7 | 700C>T | Arg234X | HPT-JT | Germline | Bradley et al. (2006) |
| 7 | 700C>T | Arg234X | Sporadic PTC | ND | Shattuck et al. (2003) |
| 8 | 732delT | Frameshift | Sporadic PTC | Somatic | Shattuck et al. (2003) |
| 8 | 745dup 1 | Frameshift | FIHP | Germline | Bradley et al. (2006) |
| 8 | 746delT | Frameshift | Sporadic PTC | Somatic | Shattuck et al. (2003) |
| 8 | 765delTG | Frameshift | HPT-JT | Germline | Cavaco et al. (2004) |
| 8 | 815A>G | Asn272Ser | Sporadic PTA | Germline | Juhlin et al. (2006) |
| 9 | 875G>A | Arg292Lys | renal tumor | Somatic | Zhao et al. (2007) |
| 13 | 1126insTT | Frameshift | Sporadic OF | Germline | Pimenta et al. (2006) |
| 13 | 1135G>A | Asp279Asn | HPT-JT | Germline | Bradley et al. (2006) |
| 14 | 1230delC | Frameshift | Sporadic PTC | ND | Shattuck et al. (2003) |
| 14 | 1238delA | Frameshift | HPT-JT | Germline | Carpten et al. (2002) |
| 14 | 1245del18 | Frameshift | Sporadic OF | Somatic | Pimenta et al. (2006) |
| Intronic | IVS1+1 G>A | Splice site mutation | FIHP | Germline | Bradley et al. (2005a) |
| Intronic | IVS1+1 G>A | Splice site mutation | Sporadic PTA | Somatic | Cetani et al. (2004) |
| Intronic | IVS1+1 G>A | Splice site mutation | FIHP | Germline | Cetani et al. (2004) |
| Intronic | IVS2 -1 G>A | Splice site mutation | HPT-JT | Germline | Moon et al. (2005) |
| Intronic | IVS2+1 G>C | Splice site mutation | FIHP | Germline | Villablanca et al. (2004) |
| Intronic | IVS6 -1delG | Splice site mutation | Sporadic PTC | Somatic | Howell et al. (2003) |
PTA, parathyroid adenoma; PTC, parathyroid carcinoma; OF, ossifying fibroma; ND, not determined.
Immunohistochemical analysis showed that parafibromin expression was uniformly lost in all investigated parathyroid tumors from both HPT-JT and FIHP patients, but not in normal parathyroids from the same subjects. This result is in agreement with reports in the literature which consistently demonstrated the loss of parafibromin immunoreactivity in parathyroid adenomas and carcinomas from HPT-JT and FIHP patients, despite a second somatic inactivating mutation or loss of heterozygosity at the CDC73 locus have been recognized only in a subset of tumors (Tan et al. 2004, Gill et al. 2006, Juhlin et al. 2006, Cetani et al. 2007a,b, Juhlin et al. 2007, present study). Our immunohistochemical study showed the absence of parafibromin immunoreactivity also in the patient with missense mutation (Leu63Pro) although the mutation involved an amino acid outside the sequence (aa 87–100) recognized by the antibody used in the present study. This result is in agreement with other studies reporting the mutations Ser59Phe (Haven et al. 2007) and Arg91Pro (Cetani et al. 2007b) and may be ascribed to modification of parafibromin conformation and/or stability. Mutations in regulatory regions of the CDC73 gene or epigenetic events, such as promoter methylation, could be involved, but their occurrence has not yet been demonstrated.
In the literature, association of uterine tumors with the HPT-JT syndrome has been reported (Fujikawa et al. 1998, Bradley et al. 2005b). Our study supports this association and, to the best of our knowledge, is the first one which compares parafibromin expression in HPT-JT-related polyps with sporadic ones. The loss of parafibromin nuclear immunostaining in both stromal and epithelial components of HPT-JT polyps, with respect to sporadic ones, supports the pathogenetic role for CDC73 mutations in uterine polyposis associated with this syndrome.
In conclusion, our results indicate that FIHP and HPT-JT associated with CDC73 mutations do not have distinct genetic, clinical, and pathological features, but may represent variants of the same genetic disease, which could be defined CDC73-related familial HPT. Penetrance of mutations is high, but disease expression may be incomplete. The most common clinical presentation is primary HPT, that may occur very early, but other tumors are also frequently diagnosed, such as uterine polyposis.
Declaration of interest
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
Funding
This work was supported by grant n. 526D/40 from Istituto Superiore di Sanità to Giorgio Palù.
The authors thank the patients for their participation in this study.
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