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A Novel IVS2-1G>A Mutation Causes Aberrant Splicing of the HRPT2 Gene in a Family with Hyperparathyroidism-Jaw Tumor Syndrome

Departments of Internal Medicine (S.-D.M., J.-H.K., J.-H.H., S.-J.Y., K.-H.Y., M.-I.K., K.-W.L., H.-Y.S., S.-K.K., B.-Y.C.) and Clinical Pathology (K.-M.K.), and Research Institute of Molecular Genetics, Catholic Research Institutes of Medical Science, Department of Biomedical Sciences (E.-M.K., S.-J.K.Y.), The Catholic University of Korea College of Medicine, Seoul, 137-701 Korea; Korean Hereditary Tumor Registry (J.-H.P., J.-G.P., I.-J.K., H.C.K.), Laboratory of Cell Biology, Cancer Research Center and Cancer Research Institute, Seoul National University, Seoul, 151-742 Korea; Research Institute and Hospital (J.-G.P.), National Cancer Center, Goyang, Gyeonggi, 411-769 Korea; Departments of Pathology (S.-W.H.) and Internal Medicine (K.-R.K.), Yonsei University College of Medicine, Seoul, 120-749 Korea; and Departments of Internal Medicine (S.-D.M., J.-H.H.) and Surgery (S.-J.O.), Our Lady of Mercy Hospital, The Catholic University of Korea College of Medicine, Incheon, 403-720 Korea

Address all correspondence and requests for reprints to: Dr. Je-Ho Han, Division of Endocrinology and Metabolism, Department of Internal Medicine, Our Lady of Mercy Hospital, The Catholic University of Korea College of Medicine, 665 Pupyung-dong Pupyung-gu, Incheon 403-720, Korea. E-mail: hjh60103{at}dreamwiz.com.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
HRPT2, the gene associated with hyperparathyroidism-jaw tumor (HPT-JT) syndrome, was previously mapped to 1q24-q32. It was recently cloned, and several germline mutations were found to predispose to HPT-JT syndrome. We sequenced the complete HRPT2 coding sequence and splice-junctional regions in a Korean family with HPT-JT syndrome and identified a novel germline mutation, IVS2-1G>A in intron 2, that caused the autosomal dominant trait of HPT-JT syndrome in this family. RT-PCR and sequencing of the transcripts revealed that this splicing mutation generated alternative splicing errors leading to the formation of two different transcripts, one with exon 3 deleted, the other lacking the first 23 bp of exon 3 due to the use of an internal splice acceptor in exon 3. Translation of both transcripts results in premature termination. In addition, we detected two novel somatic mutations of HRPT2 in malignant parathyroid tumors from the affected individuals. One, 85delG, causes premature termination; the other, an 18 bp in-frame deletion of 13_30delCTTAGCGTCCTGCGACAG, suggests that this region may be important in the development of the parathyroid carcinomas in HPT-JT syndrome. These findings provide further evidence that mutation of HRPT2 is associated with the formation of parathyroid tumors in HPT-JT syndrome.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
HEREDITARY HYPERPARATHYROIDISM-JAW tumor (HPT-JT) syndrome (OMIM 145001) is an au-tosomal dominant disease with predisposition to the development of parathyroid tumors and ossifying fibromas of the jaw (1, 2, 3). The major features of HPT-JT syndrome are primary hyperparathyroidism in about 90% of cases and parathyroid carcinoma in 15% (3, 4, 5, 6). In addition, 30% of the affected individuals develop ossifying fibroma of the mandible or maxilla (2, 3, 7). Renal lesions may also occur in HPT-JT as bilateral cysts, renal hamartomas, or Wilms tumors (2, 7, 8, 9).

The gene responsible for HPT-JT syndrome, HRPT2, was identified (10) after being mapped to the chromosomal region 1q24-q32 (1, 7, 8, 11), and germline HRPT2 mutations have been shown to predispose to parathyroid malignancy (12). The HRPT2 gene consists of 17 exons and encodes a 531-amino acid protein known as parafibromin because of its involvement in the development of parathyroid tumors and ossifying jaw fibromas (10). HRPT2 is considered to be a tumor suppressor gene whose inactivation leads to HPT-JT syndrome (10).

HRPT2 mutations were found in the sporadic parathyroid carcinomas of 10 of 15 patients; unexpectedly, germline mutations were identified in three of 15 patients with sporadic parathyroid carcinomas (12). In another study, HRPT2 mutations were detected in each of four sporadic parathyroid carcinoma samples, and germline mutations were found in each of five parathyroid tumors in HPT-JT syndrome as well as in two parathyroid tumors from one case of familial isolated hyperparathyroidism (13). Therefore, HRPT2 mutation is thought to be an early event that may lead to parathyroid malignancy and also seems to play an important role in the development of parathyroid carcinomas in HPT-JT syndrome and in sporadic parathyroid carcinoma (12, 14).

However, the nature of the HRPT2 gene remains unclear, although it is considered a tumor suppressor gene (15, 16, 17). We describe here a family with HPT-JT syndrome manifesting with parathyroid carcinoma and jaw tumors and present a mutational analysis of HRPT2 in its members.

	Patients and Methods

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Patients and tissue specimens

Clinical and genetic details of the affected family members in this study are listed in Table 1. The proband was a 20-yr-old male presenting with hypercalcemia. Total serum calcium was 15.1 mg/dl (3.78 mmol/liter), 1.4 mg/dl phosphate (0.47 mmol/liter), and 1110.51 pg/ml parathyroid hormone (111.05 pmol/liter). Urinary calcium excretion was elevated to 940 mg/d (23.5 mmol/d). At the time of diagnosis, there was no sign of renal stones, but there were symptoms of polyuria or polydipsia. Computed tomography (CT) scan of the neck showed a hypodense mass in the right neck (Fig. 1A). Oral pantography revealed circumscribed radiolucency in the body of the mandible (Fig. 1B), and CT scan of the jaw showed effacement of the left mandible (Fig. 1C). Surgical exploration revealed a parathyroid carcinoma of the right upper parathyroid gland, which was removed. The parathyroid carcinoma was diagnosed according to detailed World Health Organization guidelines (Fig. 1, D and E). Histological examination of the ossifying fibroma revealed fibrous cellular tissue with a giant cell reparative granuloma (Fig. 1F). Postoperatively, the patient developed hungry bone syndrome. After receiving intravenous calcium gluconate and magnesium sulfate treatment, he took oral calcium and alfacalcidol for 2 months. Serum calcium and parathyroid hormone levels reverted to the normal range 3 months after the operation. Parathyroid carcinoma did not recur, although we did not use anticancer medicine up to now. The father of the proband was diagnosed with a parathyroid carcinoma at the age of 40, and he was also diagnosed with hypertension and type 2 diabetes mellitus. Four members of the family were investigated for HRPT2 germline mutations (Fig. 2A). Tumor and blood samples were obtained in accordance with protocols approved for human studies by our institutional review boards, and all the family members provided written informed consent as directed in these protocols.

View larger version (125K): [in this window] [in a new window]   FIG. 1. Radiographic and histological findings in the HPT-JT of the proband presented in the text. A, CT scan of the neck showing a parathyroid tumor (1.9 x 2.0 x 3.0 cm) on the right (arrow). B, Oral pantography showing circumscribed radiolucence in the body of the mandible (arrow). C, CT scan of the jaw tumor (arrow) showing effacement of the left mandible. D, Light microscopic picture showing hematoxylin-eosin staining of the parathyroid carcinoma with typical fibrous septae. Sheets of large tumor cells have infiltrated into the surrounding tissue. Islands of bland normal parathyroid gland are indicated by arrows (magnification, x200). E, Invasion of parathyroid carcinoma into adjacent vascular spaces. The tumor cells are highlighted by immunohistochemical staining for cytokeratin (x400). F, Histology of an ossifying fibroma with cellular fibrous tissue showing a giant cell reparative granuloma of the jaw. This representative area of the jaw tumor shows mononucleated and multinucleated tumor cells. The nuclei in both tumor components are similar in morphology, and there is no atypical mitosis (x200).

View larger version (36K): [in this window] [in a new window]   FIG. 2. Mutation in a relative with HPT-JT syndrome. A, Pedigree of the family with HPT-JT syndrome. Shaded upper left quadrant, Hyperparathyroidism; upper right quadrant, ossifying fibroma of the jaw; lower right quadrant, parathyroid carcinoma. Completely open symbols, Individuals who are currently unaffected. Small superscript circles to the upper right of the symbols of the family members, those individuals for whom DNA was available for analysis. Small superscript circles with an asterisk in the middle, Individuals who have the confirmed mutation. The arrow indicates the proband. Family members are indicated by generation (Roman numbers) and individual (Arabic numbers). B, Genomic DNA sequence analysis (5') of the IVS2/exon 3 HRPT2 splice site. The heterozygous IVS2-1G>A mutation is in the 1st bp of the 3'-acceptor splice site of intron 2. The A G substitution is indicated by the bold A/G.

Parathyroid tumor tissue was frozen in liquid nitrogen immediately after surgical removal and stored at –70 C. Peripheral blood was collected into EDTA anticoagulant tubes and stored at –70 C. Total genomic DNA was extracted as previously described (18) from frozen or paraffin-embedded tumor tissue and peripheral blood lymphocytes of four members of the family (Fig. 2A). Total RNA was similarly extracted with TRI Reagent (Sigma-Aldrich Corporation, St. Louis, MO) according to the manufacturer’s protocol.

Mutational analysis of HRPT2

We searched for mutations of HRPT2 over 17 overlapping PCR amplicons covering its entire coding region and splice junctions. PCR amplification was performed in 25 µM reaction mixtures containing 100 ng genomic DNA, one of the 17 primer pairs, 0.2 mM dNTPs, and 1 U Taq polymerase (Qiagen, Hilden, Germany) in an automated thermal cycler (MWG Biotech, Ebersberg, Germany). The amplified products were analyzed for purity and size by electrophoresis on 2% agarose gels. To detect mutations, we performed denaturing HPLC (DHPLC) followed by cloning and sequencing. Running conditions were optimized with WAVEMAKER software, and DHPLC analyses were performed with the 17 amplicons described above in the WAVE system (Transgenomic, Omaha, NJ). Where DHPLC analysis revealed an abnormal pattern, we performed bidirectional sequencing and confirmed any change by cloning followed by resequencing of the cloned inserts, as previously described (18). In addition, we performed subcloning and sequencing of the RT-PCR products of total RNA extracted from proband’s tumor or those of the PCR products of tumor DNA extracted from proband’s father to search for somatic mutations using primers for exons 5'-untranslated region to 4.

Detection of splice site mutations by RT-PCR of HRPT2 mRNA

We investigated the effect of the mutation in the splice acceptor site of intron 2 (IVS2-1G>A) by RT-PCR with mRNA isolated from peripheral blood lymphocytes and parathyroid tumors. The cDNA was synthesized with the SuperScript First-Strand Synthesis System (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s instructions. We amplified a cDNA encompassing part of exons 2 to 4 with forward primer 5'-GGCGACAAGAGAAGAAGGA-3' and reverse primer 5'-TTTGACTTGAGTAGATCGCTGA-3'. The PCR products were cloned with a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA), and the cloned inserts were analyzed by PCR and Big Dye terminator cycle sequencing with an ABI3100 Prism automatic sequencer (Applied Biosystems, Foster City, CA).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
A splice site germline mutation of HRPT2

The entire HRPT2 gene from the four family members was sequenced. Germline DNA samples from the family members indicated in Fig. 2A were amplified by PCR and bidirectionally sequenced for mutations across the 17 exons of HRPT2 encompassing the entire coding region. The genomic DNA of the proband and his father contained a novel germline mutation in the splice acceptor site of intron 2 (IVS2-1G>A) that should lead to alternative splicing of the HRPT2 mRNA (Fig. 2B). This mutation was not present in the unaffected members of the family (Fig. 2A, I-2 and II-2), nor was it present in 100 control individuals as determined by allele-specific PCR.

The splice site mutation disrupts normal splicing

To confirm that the splice site mutation alters splicing, we carried out RT-PCR on mRNA from the corresponding peripheral blood and tumor samples and identified an abnormal HRPT2 transcript resulting from the IVS2-1G>A mutation, indicated by arrowheads in Fig. 3A. Because the intron-1 G at the splice acceptor site is completely conserved (19), we expected the mutation to eliminate splicing at that site. TOPO-TA cloning of the RT-PCR products of total RNA extracted from peripheral blood of the proband and his father and proband’s tumor confirmed that aberrant splicing generates two aberrant transcripts. One eliminates exon 3, whereas the other lacks the first 23 bp of exon 3 due to use of an internal AG sequence in exon 3 as a splice acceptor (Fig. 3, B and C). However, subcloning and sequencing of the RT-PCR products of total RNA extracted from placenta revealed that no aberrant transcripts were generated (data not shown).

View larger version (33K): [in this window] [in a new window]   FIG. 3. Abnormal HRPT2 transcripts resulting from the mutation IVS2-1G>A. A, RT-PCR analysis of the transcripts. Total RNAs from the proband’s tumor (T1) and peripheral blood from the proband (P1) and from the proband’s father (P2) were reverse transcribed. Placental cDNA (C) and water (DW) were used as internal controls. Normal (n) and abnormal (M2) transcripts are indicated by arrow and arrowheads, respectively. Bold arrows represent that each thick band (n+M1) seen by one in lane P2 and T1 has normal and abnormal (23 bp deleted) transcripts at the same time. RT-PCR primers were chosen in exons 2 and 4. B, RT-PCR products of total RNA extracted from the peripheral blood of the proband and his father were cloned into a TOPO-vector (pCR2.1; Invitrogen). Three different types of clones were identified among the PCR products. M1, 23-bp deletion of exon 2; M2, 70-bp deletion of exon 2. C, Schematic diagram and sequence analysis of the splicing variants. The IVS2-1G>A substitution in the 1st bp of the 3' acceptor splice site is indicated by the underlined bold A in intron 2. The PCR products from the proband and his father carrying the IVS2-1G>A mutation [23-bp deletion (M1) and 70-bp deletion (M2)] from cDNA were subcloned using a TOPO-TA Cloning Kit and sequenced to obtain the sequences of both the mutated and normal alleles. The wild-type (WT) transcript and nonspecific band (nsb) are indicated.

Two novel HRPT2 mutations were detected in the tumors of the affected individuals. The mutation, 85delG, identified in the proband’s tumor, should lead to impaired protein function due to premature termination (Fig. 4A, left). An 18-bp in-frame deletion was identified in the tumor of the proband’s father. This deleted 18-bp sequence (13_30delCTTAGCGTCCTGCGACAG) may be important for suppressing the development of parathyroid carcinomas (Fig. 4A, right). Subcloning and sequencing of the RT-PCR products of total RNA extracted from proband’s tumor or those of the PCR products of tumor DNA extracted from proband’s father revealed that these somatic mutations were located in the opposite allele to the one harboring the germline mutation (Fig. 4B).

View larger version (45K): [in this window] [in a new window]   FIG. 4. HRPT2 mutations in parathyroid tumors. Chromatograms showing the normal allele and corresponding 85delG (A, left) and 13_30delCTTAGCGTCCTGCGACAG (A, right) mutant allele in exon 1. The somatic mutations involving exon 1 are located in the opposite allele to the germline mutation (B). Arrows indicate the allelic site opposite to the somatic mutation in exon 1. Allele 2a lacking the first 23 bp of exon 3 or allele 2b lacking the whole exon 3 from the cDNA of proband has no mutation at 85 in exon 1. Allele A, having splice acceptor site mutation (underlined AA) in intron 2 from the tumor DNA of proband’s father, has no in-frame deletion at 13_30 in exon 1. PCR primers were from the 5'-untranslated region and exon 4. RT-PCR products of total RNA extracted from proband’s tumor or PCR products of tumor DNA from proband’s father were subcloned and sequenced to obtain the sequences of mutated and normal alleles.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

Seventeen different HRPT2 germline mutations including this novel splice acceptor site mutation have now been reported in HPT-JT syndrome (10, 13, 17). We were able to demonstrate that the IVS2-1G>A mutation involved caused aberrant splicing of the HRPT2 gene. The idea that the IVS2-1G>A mutation is pathogenic is supported by the following evidence: the splice acceptor site of exon 3 is 100% conserved in normal controls and the abnormal splicing event generates aberrant transcripts that are predicted to produce truncated parafibromin, this hypothesis provides a consistent explanation for the clinical and pathological findings in the affected family, and the IVS2-1G>A mutation was not present in 100 control individuals.

RT-PCR analyses of the HRPT2 transcripts disclosed that loss of a 23-bp sequence and deletion of exon 3 of the HRPT2 gene occurred in parallel during mRNA splicing in the IVS2-1G>A mutant (Fig. 3). The first of these splicing variants appeared to be generated using a false splice acceptor site in exon 3 (AG at +22 bp indicated in bold), whereas the second resulted from use of the legitimate acceptor site of exon 4. Translation of both transcripts presumably results in premature termination of translation. Splicing mutations have been identified in a number of genes and shown to have a variety of consequences including exon deletion and intron retention (20, 21). The most frequent splicing defects are caused by sequence changes in invariant splice donor and acceptor sites at positions +1 and +2 and –1 and –2, respectively (20), and generally lead to exon skipping (22). Alternatively, cryptic splice sites can be activated in an adjacent exon or intron sequence (23). Deletion of an entire exon will have a substantial effect on protein function if an essential domain is affected, even if the reading frame is not changed. The effects of the splicing defects in the HRPT2 gene in HPT-JT syndrome remain to be evaluated.

The two somatic mutations shown in Fig. 4 were both in exon 1, as is the case for all known inactivating somatic mutations of the HRPT2 gene (10). 85delG should lead to impaired protein function due to premature termination, whereas the other, generating the 18-bp in-frame deletion of 13_30delCTTAGCGTCCTGCGACAG, may delete a region of HRPT2 important for preventing the development of parathyroid carcinomas in HPT-JT syndrome. The observation that the somatic mutations involving exon 1 occurred in the opposite allele to the site of the germline mutation in this family is consistent with the two-hit model of hereditary cancer proposed by Knudson (24). The genetic and clinical findings in this case support the view that the mutation in the HRPT2 splice acceptor site is involved in the development of parathyroid malignancies in HPT-JT syndrome. At the same time, it suggests the need for early risk assessment in individuals with HRPT2 mutations in families with HPT-JT syndrome. So, given the insensitivity of genetic screening and the poor prognosis associated with parathyroid carcinoma, we recommend that genetically affected individuals be tested periodically for hypercalcemia. If the proband gives birth to children, his descendants should be offered genetic analysis of the HRPT2 gene to determine which of them needs periodic serum calcium testing. Whether selective, subtotal, or total parathyroidectomy should be performed in affected offspring to prevent the future occurrence of parathyroid carcinoma is still open to debate.

In conclusion, we suggest that a novel IVS2-1G>A splicing germline mutation in the HRPT2 gene predisposes to parathyroid malignancy in a new case of HPT-JT syndrome. In addition, our results provide further evidence that HRPT2 mutations are associated with parathyroid carcinomas in this syndrome.

	Acknowledgments

 
We thank the family members in this study for selfless participation.

	Footnotes

 
First Published Online December 21, 2004

Abbreviations: CT, Computed tomography; DHPLC, denaturing HPLC; HPT-JT, hyperparathyroidism-jaw tumor.

This work was supported by the Catholic Medical Center Research Foundation in the program year 2004.

Received May 27, 2004.

Accepted November 18, 2004.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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