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Original Article |
Department of Surgical Sciences, Endocrine Unit, University Hospital, Uppsala S-751 85, Sweden
Address all correspondence and requests for reprints to: Eva Szabo, M.D., Department of Surgical Sciences, Endocrine Unit, University Hospital, S-751 85 Uppsala, Sweden. E-mail: . eva.szabo{at}kirurgi.uu.se
Abstract
Development of sporadic parathyroid tumors is accompanied by loss of heterozygosity (LOH) on several chromosomes like 1p, 1q, 6q, 11q, and 15q. Here, we investigate a unique variant of familial hypercalcemia, unrelated to multiple endocrine neoplasia and hyperparathyroidism-jaw tumor syndromes, with hypercalcemia due to a point mutation in the intracellular part of the calcium receptor (CaR) gene. The hypercalcemia and hypercalciuria of the family is accompanied by age-related growth of the parathyroid glands and transition from diffuse to nodular parathyroid hyperplasia. Genome-wide screening for allelic loss was performed on nine enlarged parathyroid glands (weighing 40–680 mg) from eight parathyroidectomized members of the family (aged 22–66 yr). Using 139 fluorescent- or 32P-labeled microsatellite markers, informative results were obtained on all examined chromosome arms and 1p, 1q, 6q, 11q, and 15q were investigated more closely. All parathyroid glands displayed allelic loss on at least one chromosomal arm (range 1–7). Most of the common loci for allelic loss corresponded to findings in sporadic parathyroid tumors, but the unique variant of familial hypercalcemia also exhibited frequent LOH on 12q (67%) and 7q (44%). LOH could not be detected at 3q, where the CaR gene is located, and additional somatic mutations in exons 2–7 of the CaR gene was not found by sequencing. The point mutation resulting in alteration of the intracellular portion of CaR seems to cause sensitivity to secondary genetic hits, with increased frequency of allelic loss (P < 0.01, r2 = 0.66) and weight of parathyroid tumors with age in this family.
SPORADIC PRIMARY HYPERPARATHYROIDISM (pHPT) is a common disorder characterized by hypercalcemia and hypercalciuria due to excessive PTH secretion. About 80% of the patients exhibit a single hypercellular parathyroid adenoma, whereas hyperplasia with two or more affected glands occurs in the remaining 20% (1). pHPT is less frequently caused by familial tumor-susceptibility disorders such as multiple endocrine neoplasia type 1 or 2a (MEN1/2a), familial hyperparathyroidism with jaw-tumor (HPT-JT) and, very rarely isolated familial pHPT (2, 3, 4). In nonfamilial cases, most adenomas and about 40% of the hyperplastic glands are monoclonal lesions, and the multiple nodules of a hyperplastic gland may represent multiple monoclonal lesions (5, 6, 7). The adenomas show loss of heterozygosity (LOH) particularly on chromosome 11q, which has been found in 25–40% of the glands in nonfamilial HPT and approximately 50% of these show somatic mutations of the MEN 1 tumor suppressor gene (8, 9, 10). Allelic loss is also frequently found on chromosomal arms 1p, 6q, 11p, and 15q (11, 12). About one tenth of adenomas exhibit allelic loss at the calcium receptor (CaR) gene locus on 3q, but so far no evidence support the existence of somatic mutations in the CaR gene (13, 14). This suggests that the CaR does not function as a classical tumor suppressor gene in parathyroid neoplasms. The parathyroid lesions of familial HPT also show frequent allelic loss, which differs only partially from that of sporadic cases (15).
Recently, a novel variant of familial hypercalcemia was detected in a large Swedish kindred. The disorder is characterized by hypercalcemia, relative hypercalciuria, inappropriately high serum PTH levels, and parathyroid hypercellularity ranging from diffuse to nodular hyperplasia (16). Moreover, normocalcemia can be attained after radical parathyroidectomy (16). The affected family members show a unique point mutation located in exon seven of the CaR gene, which results in amino acid substitution in the intracellular portion of the CaR. In this study, we analyze allelic loss by genome-wide screening in nine glands from eight members of this family.
Materials and Methods
Patients and tumor samples
The majority of affected family members were diagnosed by screening for hypercalcemia in 1995. Clinical characteristics of the disorder and identification of the underlying CaR gene mutation have been described previously (16). So far, 22 affected (hypercalcemic) and 56 unaffected (normocalcemic) members have been recognized, individuals under the age of 18 have not been screened for ethical reasons. The point mutation, a phenylalanine to leucine transition in codon 881 of exon seven, was found in all of the hypercalcemic individuals but not in any of the normocalcemic family members. The affected individuals displayed moderate hypercalcemia, inappropriate elevated serum PTH and mild hypercalciuria (Table 1
). As detailed elsewhere, 17 family members, 5 women and 12 men, have undergone parathyroidectomy (17). The parathyroid histopathology of the family varied from mild enlargement with diffuse hyperplasia to nodule formations and microscopic findings interpreted incorrectly as single adenoma. The present investigation is based on nine enlarged parathyroid glands (mean weight 174 ± 66 mg, range 45–680 mg) from eight of these parathyroidectomized family members. All subjects gave informed consent to participate in the study, which was approved by the local Ethical Committee.
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). All eight patients, in which allelic loss have been studied, have remained normocalcemic during the follow up of 1.2 ± 0.3 (range 0.4–2.9) years after operation. The parathyroid glands showed hyperplasia dominated by parathyroid chief cells, which formed noduli in two patients. One case contributed with two parathyroid glands with mild diffuse hyperplasia in one and a large dominating nodule in the other. The parathyroid tissue was immediately frozen in liquid nitrogen at time of operation and stored at –70 C. Genomic DNA was extracted from frozen tumor tissue and matched peripheral blood leukocytes using genomic DNA Purification kit (Wizard, Promega Corp., Madison, WI). Analysis of LOH
Genomic DNA from the nine parathyroid lesions and paired leukocyte DNA were subjected to PCR amplification with highly polymorphic microsatellite markers throughout the human genome. The PCRs were performed in a total volume of 5–10 µl containing 10–20 ng genomic DNA, 2–4 pmol of each forward and reverse primer, 0.2 mM deoxyribonucleoside triphosphate, 1x PCR buffer, 1.5 mM MgCl2 and 0.2 U DNA polymerase (Life Technologies, Inc., Gaithersburg, MD). Using an ABI 877 Integrated Thermal Cycler (Applied Biosystems, Foster City, CA), the PCR started with 2 min at 95 C, 30 cycles of denaturing at 95 C for 30 sec, annealing at 53–57 C for 30 sec and elongation at 72 C for 30 sec followed by a further elongation at 72C for 10 min. Fluorescent labeled microsatellite markers from Weber set 6 (Nordic Consortium Primer Resource Center, Department of Genetics and Pathology, Uppsala University Hospital) and version 9a of the Human Screening Set-ABI Dyes (Genetic Research, Huntsville, AL) were used to obtain informative results of all chromosomal arms, except the short arms of the acrocentric chromosomes (Table 2). The PCR products were quantified with GeneScan Software (Applied Biosystems) on an ABI 310 semiautomated sequencer with GeneScan TAMRA 350 as size marker. Chromosomal arms 1p, 1q, 6q, 11q, and 15q were investigated more closely using both fluorescent and 32P-labeled primers. When using 32P-labeled markers the PCR products were mixed with formamide gel loading solution, heat denatured, run on a denaturing 4.5% polyacrylamide sequencing gel and visualized on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Allelic loss was defined as reduction by 50% or more of the signal intensity of one allele in the tumor compared with the corresponding allele in leukocyte DNA.
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Exons 2–7 of the CaR gene were sequenced in four parathyroidectomized family members using primers 1F-1R, 2F-2R, 3AF-3AR, 3BF-3BR, 4F-4R, 5F-5R, 7GF-6AR, 6BF-7ER, 7FF-6BR, 6CF-6CR, and 6DF-6DR (KEBOLab, Stockholm, Sweden) (18, 19). Sequence of blood and tumor DNA was compared to identify any somatic mutations and/or smaller deletions second to the germline CaR mutation in the tumor DNA. PCR was performed using 200 ng genomic or tumor DNA and 25 pmol of each primer. Both strands of approximately 60 ng of the PCR product were sequenced using the ABI PRISM Dye terminator cycle sequence ready reaction kit (PE Applied Biosystems, Foster City, CA).
Statistical analysis
Wilcoxon nonparametric rank sum test and Pearson’s correlation coefficients were used for statistical evaluation. Values are presented as the mean ± SEM, and a P value less than 0.05 was considered significant.
Results
One hundred thirty-nine microsatellites gave informative results from all 39 examined chromosomal arms in all tumors (Table 3). Each parathyroid gland had allelic loss at one or more chromosomal arms; the frequency ranged from 1 to 7 and was highest in the gland with a very large, dominating nodulus. The other parathyroid gland with noduli formations, although not as pronounced as in the first one, had the second highest LOH frequency with allelic loss on six chromosomal arms. Allelic loss on chromosomal arm 12q was most common and occurred in 67% of glands. Furthermore, allelic loss was found on 6q and 7q in 44%, on 15q and 17q in 33% each, and on 1p, 3p, 7p, and 19q in 22% each. Allelic loss for all microsatellites analyzed on chromosome arms 12q and 7q is shown in Table 4. The pattern of allelic loss is not uniform but two groups can be discerned on 12q; one potential minimally deleted region (MDR) may be located between markers D12S824 and D12S81 as exemplified by tumor no. 1 and no. 7, and one telomeric of D12S821 as exemplified by tumor no. 2. No tumor showed allelic loss close to the centromere on chromosome 12 q. LOH analyzes of chromosome 7q failed to detect any putative MDR on this limited tumor material. Allelic loss on 11q could only be detected with one marker (INT2) at 11q13 in one tumor. The LOH pattern differed between the glands and no uniform pattern could be detected. However, the pattern for two glands from one patient was similar and the gland with mild diffuse hyperplasia shared all LOH sites with the larger, nodular gland (LOH at 6q, 12q, 15q, and 17q). In addition the nodular one displayed LOH at sites as 7q, 11p and 19q. The frequency of allelic loss correlated to age of the patient at operation (P < 0.01, r2 = 0.66), but not to the preoperative serum calcium level, intact serum PTH level, daily urine calcium excretion, or the total weight of the excised parathyroid glands.
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). Because no deletions could be detected in this area, exons 2–7 of the CaR gene were sequenced to investigate the presence of smaller deletions or mutations. The results revealed no somatic mutations in the exons of the gene.
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Previous studies on tumorigenesis of parathyroid glands have not provided any evidence for a characteristic sequence of events comparable to, for example, colon cancer. Nevertheless, studies using molecular allelotyping and comparative genomic hybridization have shown some patterns of genetic abnormalities related to both histopathology and pathogenesis. The genetic aberrations of sporadic parathyroid adenomas have been studied in detail, and two genes of importance to the tumorigenesis have been identified. Rearrangement and/or overexpression of the PRAD1/cyclin D1 oncogene has been substantiated in 18–40% of parathyroid adenomas, but is less common in primary hyperplasias (20, 21). The MEN 1 tumor suppressor gene seems to be homozygously inactivated in up to 40% of the parathyroid adenomas (8, 22, 23). Additional abnormalities include variably frequent allelic loss on chromosomal arms 1p, 1q, 6q, 11p, and 15q (11, 12). Allelic loss of the tumor suppressor gene Rb1 on chromosome 13 has been detected in about 20% of the parathyroid adenomas and are even more frequently found in parathyroid carcinomas (24, 25). Whether benign parathyroid tumors develop into malignant forms is still a subject of debate and, in any event the exquisite rarity of parathyroid cancer indicates that this must be exceptional (26). Comparative genomic hybridization indicates losses on several chromosomal arms, but the highest frequencies are found on 1p, 6q, 11p, 11q, 15q, 17p, and 22q, whereas chromosomal gains have been described on chromosome 7 and on chromosomal arms 16p and 19p (25, 27, 28).
In this genome-wide screening for LOH, we conclude that the parathyroid lesions of our familial variant of hypercalcemia are monoclonal tumors as they display frequent allelic loss, particularly at chromosomal arms 12q, 7q, and 6q. The parathyroid glands of patients with familial hypocalciuric hypercalcemia (FHH), a disorder due to heterozygous inactivating mutations in the CaR gene, have not been investigated for chromosomal abnormalities. However, these parathyroid glands are generally normal or only very mildly hyperplastic, and normocalcemia is rarely achieved by surgery, suggesting that transformation to a monoclonal tumor would be rare (29). Interestingly, chromosomal arms 12q and 7q were frequently deleted in contrast to findings in other types of parathyroid neoplasms, suggesting that other, yet unidentified parathyroid tumor suppressor genes may be involved in the development of the parathyroid lesions in the present family. We attempted to identify any putative MDRs, and even though such findings should not be overemphasized in a small tumor populations, there may exist two loci on chromosome 12q harboring putative tumor suppressor genes of importance in monoclonal differentiation of the presently studied neoplasias. One may be located closely and centromerically of 12q21, and another telomerically of 12q23. Albeit no classic tumor suppressor gene is localized to these loci, it is interesting to note that LOH close to these regions have been detected in a number of other neoplasias such as those arising in the gall bladder, ovary, and pancreas carcinomas (30, 31, 32).
As there is a relationship between age at operation and the number of loci showing allelic loss in the present family, is it tempting to speculate that the germline CaR mutation cause an initial polyclonal proliferation. Such polyclonal hyperplasias would then be more susceptible to other genetic hits, involving e.g. allelic loss at various tumor suppressor gene loci. This would coincide with the possible sequence of events leading to monoclonality in both the parathyroid tumors of MEN 1 and HPT secondary to renal deficiency. It is unknown whether the CaR mutation of the present family is coupled to such susceptibility in contrast to those occurring in FHH families. However, CaR mutations identified so far in FHH do generally not involve coding domains for the intracellular part of the receptor. One specific FHH family has though exhibited an inserted Alu repetitive sequence with predicted truncation of the intracellular part of the CaR, and 3 of 36 heterozygous gene carriers developed operatively verified parathyroid gland enlargement (33, 34).
The CaR gene does not seem to act as a classical tumor suppressor gene in this particular family because there was no evidence of allelic loss at markers in the vicinity of the CaR gene on chromosome 3q. This coincides with the absence of CaR gene mutations in parathyroid adenomas of pHPT, although one study reported LOH on chromosome 3q in 2 of 35 adenomas (13, 14). Allelic loss on chromosomes 1p, 1q, 6q, 11q, and 15q has been found in 20–30% of parathyroid adenomas of pHPT (11, 12). This differs from the deletion pattern of the present variant of familial hypercalcemia with clinical signs of both FHH and pHPT, where 12q was the most frequently lost locus. Nevertheless, the frequency of LOH on 1p, 6q, and 15q were approximately the same as in nonfamilial pHPT, whereas LOH on 12q, 7q, and 17q were more frequent. Moreover, only one gland displayed LOH at 11q13 and for one marker only, indicating that the MEN1 gene is of minor importance for the development of parathyroid tumors in the family.
The present family exhibits autosomal dominant hypercalcemia with hypercalciuria that has been functionally coupled to a heterozygous inactivating mutation of the intracellular domain of CaR that is exclusive even to FHH. The monoclonal parathyroid lesions of this family may develop secondary to deletion of novel parathyroid tumor suppressor genes on chromosome arms 7q and 12q. Hypothetically, the germline mutation in the CaR gene of the affected family members may promote parathyroid cell proliferation and susceptibility to additional potential specific genetic hits.
Acknowledgments
The extensive cooperation of the family members is gratefully acknowledged.
Footnotes
Present address for J.R.: AstraZeneca R & D, 151 85 Södertälje, Sweden.
This work was supported by the Swedish Medical Research Council and the Fredrik and Ingrid Thuring Foundation.
Abbreviations: CaR, Calcium receptor; FHH, familial hypocalciuric hypercalcemia; HPT-JT, hyperparathyroidism with jaw; LOH, loss of heterozygosity; MDR, minimally deleted region; pHPT, primary hyperparathyroidism.
Received November 7, 2001.
Accepted May 6, 2002.
References
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  S. J. Marx and W. F. Simonds Hereditary Hormone Excess: Genes, Molecular Pathways, and Syndromes Endocr. Rev., August 1, 2005; 26(5): 615 - 661. [Abstract] [Full Text] [PDF]
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