Clonal Chromosomal Defects in the Molecular Pathogenesis of Refractory Hyperparathyroidism of Uremia
Yasuo Imanishi*,
Hideki Tahara*,
Nallasivam Palanisamy,
Sarah Spitalny*,
Isidro B. Salusky,
William Goodman,
Maria Luisa Brandi||,
Tilman B. Drüeke¶,
Emile Sarfati¶,
Pablo Ureña¶,
R. S. K. Chaganti and
Andrew Arnold*
*Center for Molecular Medicine, University of Connecticut Health Center, Farmington, Connecticut; Metabolism, Endocrinology, and Molecular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan; Memorial Sloan-Kettering Cancer Center, New York, New York; University of California, Los Angeles, Medical Center, Los Angeles, California; ||University of Florence, Florence, Italy; and ¶Hospitals Necker and St. Louis, Paris, France.
Correspondence to Dr. Andrew Arnold, Center for Molecular Medicine, University of Connecticut School of Medicine, 263 Farmington Ave., Farmington, CT 06030-3101. Phone: 860-679-7640; Fax: 860-679-7639; E-mail: aarnold{at}nso2.uchc.edu
ABSTRACT. Indirect X chromosome-inactivation analyses have demonstratedthat most parathyroid glands from patients with uremic refractorysecondary/tertiary hyperparathyroidism are monoclonal neoplasms.However, little is known regarding the specific acquired geneticabnormalities that must underlie such clonal expansion or themolecular pathogenetic features of this disorder, compared withprimary parathyroid adenomas. To address these issues in a uniquelypowerful manner, both comparative genomic hybridization (CGH)and genome-wide molecular allelotyping were performed with alarge group of uremia-associated parathyroid tumors. As indicatedby CGH, one or more chromosomal changes were present in 24%of the tumors, which is markedly different from the value forcommon sporadic adenomas (72%). Two recurrent abnormalitiesthat had not been previously described for sporadic parathyroidadenomas were noted with CGH, i.e., gains on chromosomes 7 (9%)and 12 (11%). Losses on chromosome 11 occurred in only one ofthe 46 uremia-associated tumors (2%); the tumor also containeda somatic mutation of the remaining MEN1 allele (221del18).A total of 13% of tumors demonstrated recurrent allelic losson 18q, with 18q21.1-q21.2 being defined as the putative tumorsuppressor-containing region. In conclusion, the powerful combinationof genome-wide molecular allelotyping and CGH has identifiedrecurrent clonal DNA abnormalities that suggest the existenceand locations of genes important in uremic hyperparathyroidism.In addition, genome-wide patterns of somatic DNA alterations,including disparate roles for MEN1 gene inactivation, indicatethat markedly different molecular pathogenetic processes existfor clonal outgrowth in severe uremic hyperparathyroidism versuscommon parathyroid adenomas.
Uremic refractory secondary/tertiary hyperparathyroidism ischaracterized by hyperfunctioning parathyroid tissue that nolonger responds appropriately to physiologic influences or conventionalmedical therapy. The resulting autonomous parathyroid hormone(PTH) secretion may cause clinical problems such as hypercalcemia,bone disease, or nephrocalcinosis (1). Parathyroid glands inthe earliest stages of chronic renal failure undergo multiglandulargeneralized hyperplasia (presumably true polyclonal expansion)in response to stimuli that may include chronic hypocalcemia,decreased serum 1,25-dihydroxyvitamin D3 levels, and hyperphosphatemia(2–4). However, in the late stage of this disease, usuallyafter many years of dialysis treatment, a subset of patientsdevelop refractory hyperparathyroidism, in which excessive PTHsecretion no longer responds to physiologic influences or standardmedical therapy; some patients, if they develop irreversiblehypercalcemia, are said to have tertiary hyperparathyroidism.Medically refractory secondary or tertiary hyperparathyroidismis therefore very different from the readily managed uremicsecondary hyperparathyroidism, is characterized by an abnormalPTH secretory "set point" (5–7), and is typically treatedwith surgical parathyroidectomy.
We and others previously reported that the majority of surgicallyremoved uremic parathyroid glands are monoclonal neoplasms,as assessed by X chromosome-inactivation analysis (8–10).Monoclonality suggests that somatic mutation of certain genescontrolling cell proliferation occurred in a single parathyroidcell and conferred a selective growth advantage upon it andits progeny. However, the major genes involved in the pathogenesisof these tumors are unknown. A few studies, which were limitedby either small sample size, lack of comprehensiveness in surveyingthe tumor genome, and/or reliance on a single analytical method[loss of heterozygosity (LOH) analysis], attempted to identifychromosomal regions harboring putative parathyroid tumor suppressorgenes. In one study, allelic loss of 11q13, the chromosomallocation of the multiple endocrine neoplasia type 1 (MEN1) gene,was detected in two of 12 parathyroid glands (16%) obtainedfrom dialysis patients (11). Other studies failed to confirmsignificant 11q13 losses or frequent MEN1 mutations in parathyroidtumors obtained from such patients (8,9,12–14). In addition,a limited (non-genome-wide) allelotyping study noted LOH on3q in a small subset of tumors obtained from uremic patients(12). Primary mutations in the calcium-sensing receptor geneon 3q and the vitamin D receptor gene on 12q have been directlysought and are not major contributors to clonal outgrowth inuremic hyperparathyroidism (15,16). Comparative genomic hybridization(CGH), a modern molecular cytogenetic method that examines theentire genome for regional losses or gains of chromosomal material,has not yet been applied in uremic hyperparathyroidism, eitheralone or in combination with the complementary approach of molecularallelotyping.
To comprehensively seek new locations of pathogenetically importantoncogenes or tumor suppressor genes, we performed both CGH andgenome-wide molecular allelotyping on a large series of uremia-associatedparathyroid tumors. We also hypothesized that comparison ofthe patterns of acquired chromosomal defects in uremia-associatedparathyroid tumors versus typical parathyroid adenomas mightfacilitate elucidation of their molecular pathogenetic relationships.
Patients and Tumor Specimens
We studied 51 resected parathyroid tumors from 30 uremic patientswith refractory secondary or tertiary hyperparathyroidism. Themean age of the patients was 45.9 yr (range, 19 to 71 yr). Allpatients were undergoing maintenance hemodialysis treatment.Parathyroidectomy was indicated because of severe secondaryhyperparathyroidism associated with pruritus, osteitis fibrosa,soft-tissue calcification, hyperphosphatemia, and/or other symptomsthat were resistant to medical treatment. In no case was chronicrenal failure a consequence of primary hyperparathyroidism.Fourteen of the 30 patients were hypercalcemic in the absenceof calcium or vitamin D therapy (tertiary hyperparathyroidism),and 16 were normocalcemic. Serum PTH levels were markedly elevatedfor all patients (an average of 15-fold greater than the upperlimit of the normal range). No patient had a family historyof hyperparathyroidism or multiple endocrine neoplasia or ahistory of head or neck irradiation. For all patients, multiplehypercellular parathyroid glands (i.e., tumors) were identifiedand resected. Many of these glands were categorized as nodularhyperplasia, generalized diffuse hyperplasia, or mixed-type(nodular and diffuse) hyperplasia, according to gross and histopathologiccriteria (17,18). For two of the 30 patients, pathologic parathyroidgraft tissue from the arm was available for study. One parathyroidgland was available for study for 15 patients, two glands wereavailable for 11 patients, three glands were available for twopatients, and four glands were available for two patients. Peripheralblood leukocytes, providing control non-tumor DNA, were availablefor all 30 patients. All tissue and blood specimens were obtainedin accordance with institutional human study procedures.
DNA Sample Preparation
All parathyroid specimens were frozen in liquid nitrogen aftersurgical removal and were stored at -80°C. High-molecularweight DNA was extracted from frozen tissue and leukocytes byusing standard procedures (19). For tumors, a large proportionof the total resected tissue was extracted, to avoid possiblesampling artifacts introduced by microdissection.
CGH
Forty-six parathyroid tumors from 28 uremic patients were analyzedby CGH. CGH was performed as described previously (20,21). Briefly,the probe tumor DNA and normal reference DNA were differentiallylabeled by standard nick translation using fluorescein-12-deoxyuridine-5-triphosphateand Texas Red-5-deoxyuridine-5-triphosphate (NEN-DuPont, Boston,MA), respectively. Equal amounts (500 ng) of tumor and normalreference DNA were coprecipitated with 25 µg of unlabeledhuman Cot-1 DNA (Life Technologies-BRL, Gaithersburg, MD). TheCot-1 DNA was included to suppress the binding of labeled DNAfrom both genomes to the centromeric and heterochromatic regionsof normal chromosomes. Probe DNA was resuspended in 15 µlof hybridization mixture (50% formamide, 2x SSC, 10% dextransulfate) and hybridized to normal human metaphase chromosomesprepared from phytohemagglutinin-stimulated peripheral bloodlymphocyte cultures. Hybridization was performed at 37°Cfor 48 h. The slides were washed three times (5 min each) at45°C in 50% formamide/2x SSC, followed by three washes (5min each) at 45°C in 2x SSC and one wash (10 min) at 45°Cin 0.1x SSC. Slides were counterstained with 4,6-diamino-2-phenylindolefor the identification of chromosomes.
Digital Image Analysis
The green and red fluorescence intensities of the hybridizationsignals and the 4,6-diamino-2-phenylindole staining patternswere recorded with a cooled charge-coupled device camera (Photometrics,Tucson, AZ) attached to a Nikon Microphot-SA microscope (Nikon,Natick, MA). Fluorescence ratio profiles for each chromosomewere calculated by using the Quantitative Image Processing System(Vysis Inc., Downers Grove, IL). For each hybridization, thedata from 10 to 14 representations of each chromosome were combinedto yield the mean and 95% confidence interval for that ratio,plotted next to the ideogram for that chromosome. Gains or lossesof chromosomes or chromosomal regions were detected on the basisof ratio profile deviations from the green/red balance valueof 1.0. The upper and lower threshold limits for defining chromosomalgains and losses were set at 1.20 and 0.80, respectively. Thesethreshold values were determined in CGH experiments using twodifferentially labeled, normal genomic DNA samples. In thesenegative control experiments, the mean green/red ratio was wellwithin the range of 1.20 to 0.80 for the entire length of allchromosomes, providing robust, highly stringent criteria forthe determination of gains and losses in tumor samples. Metaphasespreads with uniform high-intensity fluorescence in both greenand red colors, on both homologous chromosomes, and with nobackground spots were selected for evaluations. The centromericand heterochromatic regions and p arm of acrocentric chromosomesand telomeric regions were not included in the interpretationof gains and losses.
Allelic Loss Analysis Using Microsatellite Polymorphisms
Fifty-one uremia-associated parathyroid tumors from 30 patients,including the 46 glands on which CGH was performed, were extensivelyanalyzed for allelic losses with polymorphic microsatellitemarkers, as previously described (21–23). Primers forPCR amplification of microsatellite markers, which were chosento represent every chromosomal arm except the short arm of acrocentricchromosomes, were obtained from Research Genetics (Huntsville,AL) or synthesized with a DNA synthesizer (Applied Biosystems,Foster City, CA). The 43 primer pairs and genomic loci analyzedfor genome-wide allelotyping were identical to those examinedin our previous study (21), with the addition of D11S449 (11q13).Nine additional markers were used for detailed deletion mappingof chromosome 18 using 40 tumors from 23 patients, which werealso analyzed by genome-wide allelotyping. Primer labeling,PCR amplification of paired tumor and leukocyte genomic DNA,analysis of PCR products, and scoring of allelic losses wereperformed as described previously (23).
Sequence Analysis of the MEN1 Gene
The uremia-associated parathyroid tumor with 11q13 LOH was subjectedto full sequencing of the MEN1 gene coding region and splicejunctions. Primers flanking exons 2 to 10 of the MEN1 gene thatwere used for PCR amplifications and sequence reactions arelisted in Table 1. PCR products were directly analyzed via semiautomatedsequencing with an ABI 377 system, using the ABI PRISM BigDyePrimer Cycle Sequencing Ready Reaction kit with AmpliTaq DNApolymerase FS (PE Applied Biosystems, Foster City, CA). Theobserved internal deletion mutation was confirmed by analysisof the length of the PCR product as described above, using sequencingprimers.
Forty-six parathyroid tumor DNA samples from 28 uremic patientswith refractory secondary or tertiary hyperparathyroidism wereanalyzed by CGH. Representative CGH images, with fluorescenceratio profiles and corresponding ideograms for recurrent chromosomalgains and losses, are presented in Figure 1. Eleven tumors (24%)demonstrated clonal chromosomal imbalances that included gainsor losses of whole or partial chromosomes. Among the 11 tumors,six exhibited two or more chromosomal changes. Chromosomal gainswere more commonly observed than chromosomal losses (16 versusfive cases). Three abnormalities that had not previously beendescribed for uremia-associated parathyroid tumors were repeatedlyobserved, i.e., a gain on chromosome 7 (four of 46 samples,9%), a gain on chromosome 12 (five of 46 samples, 11%), anda loss on chromosome 21 (2 of 46 samples, 4%). A loss on chromosome11 (the location of the MEN1 gene) was observed in only oneof 46 uremia-associated tumors (2%), compared with 34% of adenomas(21). Gains on chromosomes 1q, 2, 6q14–24, 9p, 9q34, 18,22, and X and losses on chromosomes 11, 13q21–34, and22 were each observed in one of 46 tumors (2%). A comprehensivesummary of the CGH results for all tumors is presented in Figure 2.
Figure 1. Representative comparative genomic hybridization (CGH) results for parathyroid tumors from uremic patients. Individual examples of fluorescence ratio profiles (right) and digital images (left) of chromosomes with recurrent gains or losses are presented. The red vertical bar on the left side of the chromosome ideogram (middle) indicates regions of loss, and the green vertical bars on the right side of the ideograms indicate regions of gain. The human chromosome number is indicated below each displayed chromosome image. The number of representations of each chromosome that were used in digital image analysis to obtain the displayed data is indicated below each fluorescence ratio profile.
Figure 2. DNA copy number changes in 46 parathyroid tumors from 28 uremic patients. A summary of all gains and losses detected by CGH, with relevant human chromosome numbers, is presented for the ideograms. The vertical bars on the left side of the chromosome ideograms indicate losses and those on the right side indicate gains in the corresponding chromosomal region for each individual tumor (numbered above each vertical bar).
Fifty-one parathyroid tumors from 30 uremic patients, includingthe 46 tumors on which CGH was performed, were extensively analyzedfor allelic losses with polymorphic microsatellite markers.Among the loci that demonstrated LOH, the frequency ranged upto 12.5%. Apparent microsatellite instability was observed foronly two of 1997 alleles on which genome-wide allelotyping wasperformed. Fractional allelic loss (FAL) was calculated as theratio of the number of chromosomal arms that demonstrated lossto the total number of informative arms for each patient, asdescribed by Vogelstein et al. (24). FAL values varied from0 to 0.071, with a median of 0.000 (mean, 0.012), in 51 uremia-associatedtumors. In comparison, the median FAL value for a series ofbenign parathyroid adenomas was 0.087 (mean, 0.086) (23), andthat for colon cancers was 0.200 (24). Neither FAL values nordeletions in any particular chromosomal arm were significantlyassociated with any clinical parameter studied, including tumorsize, patient age, gender, serum PTH level, serum calcium level,and clinical symptoms (data not shown). Frequent allelic losswas observed on chromosomal arm 18q (four of 32 informativetumors, 13%), and lower-frequency losses were detected on chromosomalarms 7p (5%), 21q (9%), and 22q (5%).
The results of molecular allelotyping were remarkably concordantwith CGH results for these tumors. Cumulatively, for all 1429informative markers examined in all 46 tumors, allelic losswas observed with 11 markers (0.8%) for which the correspondingchromosomal region was determined with CGH to be lost. For 1392total markers (97.4%), allelic loss was not observed and thecorresponding chromosomal region exhibited no loss with CGH.Molecular analysis demonstrated allelic loss in only six instances(0.4%) in which the corresponding region did not exhibit losswith CGH. In only 20 cases (1.4%) did the analyzed molecularmarkers fail to demonstrate allelic loss when loss of the correspondingchromosomal region was observed with CGH. Therefore, the overalllevel of concordance between molecular allelotyping and CGHwas 98.2% for all informative markers/chromosomal arms examined,and the discordance rate was only 1.8%. This concordance issimilar to our previous findings for parathyroid adenomas (21).Figure 3 summarizes our cumulative molecular allelotyping datafor the 51 uremia-associated parathyroid tumors.
Figure 3. Frequency of allelic loss, as assessed by molecular allelotyping, on each chromosomal arm in 51 parathyroid tumors from 30 uremic patients. Loss of heterozygosity (LOH) represents the percentage of tumors demonstrating allelic loss on each chromosomal arm. Forty-six of these 51 tumors were also analyzed with CGH.
Seventeen of 51 uremia-associated parathyroid tumors (33%),from 15 of 30 patients (50%), were proven to have monoclonalabnormalities detectable by either CGH or molecular allelotyping.The serum calcium levels among patients with at least one identifiablymonoclonal tumor versus those without an identifiably monoclonaltumor were 10.9 ± 0.9 mg/dl versus 10.4 ± 1.0mg/dl (mean ± SD), which was a nonsignificant difference(P = 0.24, Mann-Whitney U test). Serum PTH levels were 12.8± 5.9-fold versus 14.1 ± 5.7-fold greater thanthe upper limit of the normal range, respectively, which wasalso a nonsignificant difference (P = 0.75, Mann-Whitney U test).Four of 14 nodular hyperplasias (29%) contained monoclonal abnormalitiesdetectable by CGH or molecular allelotyping, and 10 of 29 diffusehyperplasias (34%) were identifiably monoclonal. Therefore,there was no correlation between detectable monoclonality andserum calcium or PTH concentrations or histopathologic categoriesof diffuse versus nodular hyperplasia. This lack of correlationmight well have been expected, because these patients (all ofwhom required surgical intervention) were already a highly selectedgroup. Patients with severe secondary or tertiary hyperparathyroidismare also known to exhibit a high prevalence of monoclonal parathyroidtumors (8,14), only some of which would be detectable by CGHor allelotyping.
Clonal losses were observed on chromosome 11 (the location ofthe MEN1 tumor suppressor gene) in only one of 46 uremia-associatedtumors (2%). In this tumor, DNA sequencing demonstrated somaticmutation of the remaining MEN1 allele (specifically, a smallinternal deletion, 221del18, within exon 2) (Figure 4a). Thismicrodeletion was confirmed by analysis of the length of thePCR product for MEN1 exon 2 (Figure 4b).
Figure 4. Mutation of MEN1 gene exon 2 in an uremia-associated parathyroid tumor. (a) Leukocyte DNA (upper) and tumor DNA (lower) were analyzed by direct sequencing. The somatic mutation 221del18 detected in the tumor DNA sequence created a frameshift, resulting in an early stop codon (168X) in the amino acid sequence. (b) This deletion mutation was confirmed by analysis of the length of the PCR product for exon 2. N, leukocyte DNA with a normal exon 2 fragment length of 220 bp. T, tumor DNA with a corresponding fragment length of 202 bp, representing an 18-bp deletion.
Thirteen percent of uremia-associated parathyroid tumors demonstratedLOH on chromosomal arm 18q in allelotyping analyses. To morespecifically define the smallest shared regions of losses, andthus help pinpoint the location at which a putative tumor suppressorgene (or genes) is expected to occur, we constructed a detaileddeletion map of chromosome 18 by using 40 tumors, from 23 patients,that were also analyzed by genome-wide allelotyping (Figure 5).All 40 tumors were informative at one or more loci, andfour tumors revealed LOH of at least one locus on chromosome18. The only marker that revealed allelic loss in all informativetumors among these tumors was DCC at 18q21.1. These resultssuggest that the smallest region of common deletion exists at18q21.1–q21.2.
Figure 5. Deletion map of chromosome 18. The four tumors demonstrating chromosome 18 loss are illustrated. Markers are listed at the left, in order according to the linkage-average genetic maps from the Genome data base (http://gdbwww.gdb.org). Tumor numbers are indicated at the top. At the given loci, closed rectangles indicate tumor-specific allelic loss, open rectangles indicate retention of both alleles, and thick bars indicate constitutional homozygosity.
Parathyroid glands in the earliest stages of chronic renal failureundergo multiglandular generalized hyperplasia (presumably truepolyclonal expansion) in response to stimuli that may includechronic hypocalcemia, decreased serum 1,25-dihydroxyvitaminD3 levels, and hyperphosphatemia (4). However, the large majorityand perhaps all of the patients who ultimately develop refractorysecondary or tertiary hyperparathyroidism exhibit a true neoplasticdisorder of the parathyroid glands, as indicated by monoclonalitydocumented in X chromosome-inactivation analyses (8–10,14).The evolution to clonal parathyroid neoplasia is likely to playa key role in the refractoriness to standard medical therapythat is characteristic of this clinical entity. In addition,the abnormal PTH secretory set point observed in refractorydisease (5), as opposed to the normal set point that is generallynoted in the more common and tractable secondary hyperparathyroidism(25–27), may well be related to this clonal neoplasticstate. It may be relevant in this regard that, in a geneticallyengineered mouse model, one oncogenic stimulus that is knownto drive the clonal outgrowth of human parathyroid adenomas,namely cyclin D1 overexpression, can secondarily result in ashift of the PTH secretory set point in vivo (28). Whether therelative set point shift in uremia-associated (or other) monoclonalparathyroid tumors is an end result of a sufficiently disturbedstate of glandular growth or is more specifically related toeffects of particular oncogenes or tumor suppressors is opento speculation. Many apparent discrepancies in published invivo and in vitro functional studies of secondary hyperparathyroidismmay be attributable to the fact that parathyroid tissue forstudy can generally be obtained only from the most severelyaffected patients, i.e., those who require surgery, and suchcases are enriched in monoclonal parathyroid neoplasms. In contrast,most uremic patients studied in vivo do not have medically refractorysecondary or tertiary hyperparathyroidism, likely bear fewerclonal outgrowths, and thus retain hormonal dynamic patternsthat are closer to normal (25–27,29).
Although the X chromosome-inactivation approach can definitivelydetect monoclonal neoplasms, it does not directly detect thespecific genetic alterations that provide a selective advantageand underlie clonal expansion. However, evidence indicatingthe locations of such specific lesions was indeed obtained withthe methods used in this study, namely CGH and molecular allelotyping.Furthermore, the chromosomal gains and losses detected hereconstitute independent evidence for tumor monoclonality, becausethe DNA analyzed was extracted in toto from large portions ofthe glands. Therefore, our results provide independent directconfirmation that at least 50% of hemodialysis patients withuremic refractory hyperparathyroidism harbor at least one monoclonaltumor. Because many relevant types of oncogenic DNA damage wouldnot be expected to be identified with these methods, the truenumber of monoclonal tumors in this population is likely tobe much higher; these findings are quite consistent with ourprevious report and other reports (using X chromosome-inactivationanalysis) that at least 60 to 75% of these patients harbor monoclonaltumors (8–10,14). In this study, no correlation existedbetween the presence of microscopically evident nodules andthe clonal character of resected parathyroid tissue. The appearancesof several glands with histologic patterns indicating diffusehyperplasia also were unequivocally monoclonal in the absenceof detectable nodular formations, suggesting that the currentcriteria for pathologic diagnosis do not reflect the geneticdifferences between these two histopathologic types.
It might initially be hypothesized that the pathogenesis andfunction of monoclonal parathyroid tumors in severe refractorysecondary/tertiary hyperparathyroidism are equivalent to thosein common sporadic parathyroid adenomas, which are also benignclonal tumors with altered hormonal responses to extracellularcalcium. Although certain key functional aspects (as discussedabove) may indeed be shared, the major differences we observedbetween uremia-associated tumors (in this study) and parathyroidadenomas (in a previous study) (21), in their respective patternsof clonal chromosomal gains and losses, strongly suggest thatthese tumors arise and evolve through distinct oncogenic mechanisms.One explanation may be that, before monoclonal outgrowth inuremic patients, significant activation of proliferative pathwaysis already present in parathyroid tissue. In contrast, for parathyroidadenomas to evolve on a substrate without such underlying proliferativeactivity, different (and probably more) acquired genetic changesmay be needed to drive clonal growth.
Consistent with this hypothesis, our genome-wide allelotypingand CGH results revealed a much lower frequency of pathogeneticinvolvement of the MEN1 tumor suppressor gene in uremic hyperparathyroidism(biallelic MEN1 inactivation in one of 46 tumors, 2%), comparedwith that observed in sporadic parathyroid adenomas (biallelicinactivation in 12 to 17% of tumors) (30–32). Our resultsindicating a very minor role for clonal 11q13 loss and/or MEN1inactivation in uremic hyperparathyroidism contrast with thoseof an earlier smaller study (11) and are in agreement with thoseof a recent study limited to this specific question (13). Interestingly,the cyclin D1 oncogene, which is activated and overexpressedby clonal gene rearrangement or other mechanisms in 20 to 40%of parathyroid adenomas (33–35), has not been observedto be overexpressed in uremia-associated tumors (35).
Our CGH and genome-wide allelotyping survey of uremia-associatedparathyroid tumors was performed with the goal of identifyingthe chromosomal locations of oncogenes and tumor suppressorgenes that are potentially involved in the acquisition of aclonal selective advantage. In this context, areas of recurrentlyobserved alterations are most likely to harbor such pathogeneticallyrelevant genes. Recurrent gains on chromosome 7 (9%) and chromosome12 (11%) were observed, suggesting the presence of key oncogenesat those sites. The highest frequency of recurrent losses wasobserved for chromosome 18. Allelic loss on chromosome 18q hasbeen reported for other types of tumors, including colorectalcarcinoma (36), lung cancer (37), pancreatic adenocarcinoma(38), prostate cancer (39), and esophageal cancer (40). We constructeddeletion maps for secondary uremic hyperplasias and identifiedone region of overlapping common deletion among the tumors.On the basis of the available physical mapping data for themarkers, this area of minimal common deletion corresponds to18q21.1. Therefore, several tumor suppressor genes could beconsidered candidates for involvement in uremia-associated hyperparathyroidism,including DCC (41), Smad4 (also known as DPC4) (38), and Smad2(also known as JV18-1, MADR2, or hMAD-2) genes (42–44).Inactivating mutations in the Smad genes are thought to disruptthe signal transduction pathway between cell membrane receptorsfor the tumor growth factor- family and the nucleus, and Smadgenes are key transducers of information transmitted by tumorgrowth factor- that, when inactivated, enhance tumor growth(42–44). Further studies are needed to determine whetherthese genes or others on chromosome 18q21 are commonly alteredand inactivated in parathyroid neoplasms.
In conclusion, the powerful combination of genome-wide molecularallelotyping and CGH has identified recurrent clonal DNA abnormalitiesthat suggest the existence and locations of genes importantin uremic hyperparathyroidism. Oncogenes on chromosomes 7 and12, as well as tumor suppressor genes on 18q and perhaps otherloci, may potentially be important in the pathogenesis of clonalhyperparathyroidism of uremia. In addition, genome-wide patternsof somatic DNA alterations, including disparate roles for MEN1gene inactivation, indicate that markedly different molecularpathogenetic processes exist for clonal outgrowth in severeuremic hyperparathyroidism versus common parathyroid adenomas.These observations provide new insights into the molecular pathogenesisof uremic hyperparathyroidism, as well as guideposts for futureefforts at positional cloning of genes that might eventuallyserve as novel therapeutic targets in this challenging clinicaldisorder.
Acknowledgments
We thank Pamela Vachon for invaluable assistance with the preparationof the manuscript.
Indridason OS, Quarles LD: Tertiary hyperparathyroidism and refractory secondary hyperparathyroidism.In: Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, 4th Ed., edited by Favus MJ, Philadelphia, Lippincott Williams & Wilkins, 1999,pp 198–202
Slatopolsky E, Brown A, Dusso A: Role of phosphorus in the pathogenesis of secondary hyperparathyroidism. Am J Kidney Dis 37 [Suppl 2]: S54–S57, 2001[Medline]
Silver J: Molecular mechanisms of secondary hyperparathyroidism. Nephrol Dial Transplant 15: 2–7, 2000[Free Full Text]
Drueke TB: Parathyroid gland hyperplasia in uremia. Kidney Int 59: 1182–1183, 2001[CrossRef][Medline]
Brown EM, Wilson RE, Eastman RC, Pallotta J, Marynick SP: Abnormal regulation of parathyroid hormone release by calcium in secondary hyperparathyroidism due to chronic renal failure. J Clin Endocrinol Metab 54: 172–179, 1982[Abstract]
Goodman WG, Veldhuis JD, Belin TR, Van Herle AJ, Juppner H, Salusky IB: Calcium-sensing by parathyroid glands in secondary hyperparathyroidism. J Clin Endocrinol Metab 83: 2765–2772, 1998[Abstract/Free Full Text]
Cardinal H, Brossard JH, Roy L, Lepage R, Rousseau L, D’Amour P: The set point of parathyroid hormone stimulation by calcium is normal in progressive renal failure. J Clin Endocrinol Metab 83: 3839–3844, 1998[Abstract/Free Full Text]
Arnold A, Brown MF, Urena P, Gaz RD, Sarfati E, Drueke TB: Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia. J Clin Invest 95: 2047–2053, 1995
Chudek J, Ritz E, Kovacs G: Genetic abnormalities in parathyroid nodules of uremic patients. Clin Cancer Res 4: 211–214, 1998[Abstract]
Tominaga Y, Kohara S, Namii Y, Nagasaka T, Haba T, Uchida K, Numano M, Tanaka Y, Takagi H: Clonal analysis of nodular parathyroid hyperplasia in renal hyperparathyroidism. World J Surg 20: 744–750, 1996[CrossRef][Medline]
Falchetti A, Bale AE, Amorosi A, Bordi C, Cicchi P, Bandini S, Marx SJ, Brandi ML: Progression of uremic hyperparathyroidism involves allelic loss on chromosome 11. J Clin Endocrinol Metab 76: 139–144, 1993[Abstract]
Farnebo F, Teh BT, Dotzenrath C, Wassif WS, Svensson A, White I, Betz R, Goretzki P, Sandelin K, Farnebo LO, Larsson C: Differential loss of heterozygosity in familial, sporadic, and uremic hyperparathyroidism. Hum Genet 99: 342–349, 1997[CrossRef][Medline]
Tahara H, Imanishi Y, Yamada T, Tsujimoto Y, Tabata T, Inoue T, Inaba M, Morii H, Nishizawa Y: Rare somatic inactivation of the multiple endocrine neoplasia type 1 gene in secondary hyperparathyroidism of uremia. J Clin Endocrinol Metab 85: 4113–4117, 2000[Abstract/Free Full Text]
Shan L, Nakamura Y, Murakami M, Nakamura M, Naito A, Kawahara K, Utsunomiya H, Mori I, Kakudo K: Clonal emergence in uremic parathyroid hyperplasia is not related to MEN1 gene abnormality. Jpn J Cancer Res 90: 965–969, 1999[CrossRef][Medline]
Hosokawa Y, Pollak MR, Brown EM, Arnold A: Mutational analysis of the extracellular Ca2+-sensing receptor gene in human parathyroid tumors. J Clin Endocrinol Metab 80: 3107–3110, 1995[Abstract]
Brown SB, Brierley TT, Palanisamy N, Salusky IB, Goodman W, Brandi ML, Drueke TB, Sarfati E, Urena P, Chaganti RS, Pike JW, Arnold A: Vitamin D receptor as a candidate tumor-suppressor gene in severe hyperparathyroidism of uremia. J Clin Endocrinol Metab 85: 868–872, 2000[Abstract/Free Full Text]
Harach HR, Jasani B: Parathyroid hyperplasia in tertiary hyperparathyroidism: A pathological and immunohistochemical reappraisal. Histopathology 21: 513–519, 1992[Medline]
DeLellis RA: Tumors of the parathyroid glands.In: Atlas of Tumor Pathology, 3rd Series, Fascicle 6,edited by Rosai J, Washington, DC, Armed Forces Institute of Pathology, 1993,pp. 85–92
Cryns VL, Thor A, Xu HJ, Hu SX, Wierman ME, Vickery AL Jr, Benedict WF, Arnold A: Loss of the retinoblastoma tumor-suppressor gene in parathyroid carcinoma. N Engl J Med 330: 757–761, 1994[Abstract/Free Full Text]
Houldsworth J, Chaganti RS: Comparative genomic hybridization: An overview. Am J Pathol 145: 1253–1260, 1994[Abstract]
Palanisamy N, Imanishi Y, Rao PH, Tahara H, Chaganti RS, Arnold A: Novel chromosomal abnormalities identified by comparative genomic hybridization in parathyroid adenomas. J Clin Endocrinol Metab 83: 1766–1770, 1998[Abstract/Free Full Text]
Cryns VL, Yi SM, Tahara H, Gaz RD, Arnold A: Frequent loss of chromosome arm 1p DNA in parathyroid adenomas. Genes Chromosomes Cancer 13: 9–17, 1995[Medline]
Tahara H, Smith AP, Gas RD, Cryns VL, Arnold A: Genomic localization of novel candidate tumor suppressor gene loci in human parathyroid adenomas. Cancer Res 56: 599–605, 1996[Abstract/Free Full Text]
Vogelstein B, Fearon ER, Kern SE, Hamilton SR, Preisinger AC, Nakamura Y, White R: Allelotype of colorectal carcinomas. Science (Washington DC) 244: 207–211, 1989[Abstract/Free Full Text]
Ramirez JA, Goodman WG, Gornbein J, Menezes C, Moulton L, Segre GV, Salusky IB: Direct in vivo comparison of calcium-regulated parathyroid hormone secretion in normal volunteers and patients with secondary hyperparathyroidism. J Clin Endocrinol Metab 76: 1489–1494, 1993[Abstract]
Goodman WG, Belin T, Gales B, Juppner H, Segre GV, Salusky IB: Calcium-regulated parathyroid hormone release in patients with mild or advanced secondary hyperparathyroidism. Kidney Int 48: 1553–1558, 1995[Medline]
Messa P, Vallone C, Mioni G, Geatti O, Turrin D, Passoni N, Cruciatti A: Direct in vivo assessment of parathyroid hormone-calcium relationship curve in renal patients. Kidney Int 46: 1713–1720, 1994[Medline]
Imanishi Y, Hosokawa Y, Yoshimoto K, Schipani E, Mallya S, Papanikolaou A, Kifor O, Tokura T, Sablosky M, Ledgard F, Gronowicz G, Wang TC, Schmidt EV, Hall C, Brown EM, Bronson R, Arnold A: Primary hyperparathyroidism caused by parathyroid-targeted overexpression of cyclin D1 in transgenic mice. J Clin Invest 107: 1093–1102, 2001[Medline]
Ramirez JA, Goodman WG, Belin TR, Gales B, Segre GV, Salusky IB: Calcitriol therapy and calcium-regulated PTH secretion in patients with secondary hyperparathyroidism. Am J Physiol 267: E961–E967, 1994[Abstract/Free Full Text]
Heppner C, Kester MB, Agarwal SK, Debelenko LV, Emmert-Buck MR, Guru SC, Manickam P, Olufemi SE, Skarulis MC, Doppman JL, Alexander RH, Kim YS, Saggar SK, Lubensky IA, Zhuang Z, Liotta LA, Chandrasekharappa SC, Collins FS, Spiegel AM, Burns AL, Marx SJ: Somatic mutation of the MEN1 gene in parathyroid tumours. Nat Genet 16: 375–378, 1997[CrossRef][Medline]
Carling T, Correa P, Hessman O, Hedberg J, Skogseid B, Lindberg D, Rastad J, Westin G, Akerstrom G: Parathyroid MEN1 gene mutations in relation to clinical characteristics of nonfamilial primary hyperparathyroidism. J Clin Endocrinol Metab 83: 2960–2963, 1998[Abstract/Free Full Text]
Farnebo F, Teh BT, Kytola S, Svensson A, Phelan C, Sandelin K, Thompson NW, Hoog A, Weber G, Farnebo LO, Larsson C: Alterations of the MEN1 gene in sporadic parathyroid tumors. J Clin Endocrinol Metab 83: 2627–2630, 1998[Abstract/Free Full Text]
Hsi ED, Zukerberg LR, Yang WI, Arnold A: Cyclin D1/PRAD1 expression in parathyroid adenomas: An immunohistochemical study. J Clin Endocrinol Metab 81: 1736–1739, 1996[Abstract]
Vasef MA, Brynes RK, Sturm M, Bromley C, Robinson RA: Expression of cyclin D1 in parathyroid carcinomas, adenomas, and hyperplasias: A paraffin immunohistochemical study. Mod Pathol 12: 412–416, 1999[Medline]
Tominaga Y, Tsuzuki T, Uchida K, Haba T, Otsuka S, Ichimori T, Yamada K, Numano M, Tanaka Y, Takagi H: Expression of PRAD1/cyclin D1, retinoblastoma gene products, and Ki67 in parathyroid hyperplasia caused by chronic renal failure versus primary adenoma. Kidney Int 55: 1375–1383, 1999[CrossRef][Medline]
Thiagalingam S, Lengauer C, Leach FS, Schutte M, Hahn SA, Overhauser J, Willson JK, Markowitz S, Hamilton SR, Kern SE, Kinzler KW, Vogelstein B: Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nat Genet 13: 343–346, 1996[CrossRef][Medline]
Shiseki M, Kohno T, Nishikawa R, Sameshima Y, Mizoguchi H, Yokota J: Frequent allelic losses on chromosomes 2q, 18q, and 22q in advanced non-small cell lung carcinoma. Cancer Res 54: 5643–5648, 1994[Abstract/Free Full Text]
Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE: DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science (Washington DC) 271: 350–353, 1996[Abstract]
Latil A, Baron JC, Cussenot O, Fournier G, Soussi T, Boccon-Gibod L, Le Duc A, Rouesse J, Lidereau R: Genetic alterations in localized prostate cancer: Identification of a common region of deletion on chromosome arm 18q. Genes Chromosomes Cancer 11: 119–125, 1994[Medline]
Aoki T, Mori T, Du X, Nisihira T, Matsubara T, Nakamura Y: Allelotype study of esophageal carcinoma. Genes Chromosomes Cancer 10: 177–182, 1994[Medline]
Fearon ER, Cho KR, Nigro JM, Kern SE, Simons JW, Ruppert JM, Hamilton SR, Preisinger AC, Thomas G, Kinzler KW, Vogelstein B: Identification of a chromosome 18q gene that is altered in colorectal cancers. Science (Washington DC) 247: 49–56, 1990[Abstract/Free Full Text]
Eppert K, Scherer SW, Ozcelik H, Pirone R, Hoodless P, Kim H, Tsui LC, Bapat B, Gallinger S, Andrulis IL, Thomsen GH, Wrana JL, Attisano L: MADR2 maps to 18q21 and encodes a TGF--regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell 86: 543–552, 1996[CrossRef][Medline]
Riggins GJ, Thiagalingam S, Rozenblum E, Weinstein CL, Kern SE, Hamilton SR, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B: Mad-related genes in the human. Nat Genet 13: 347–349, 1996[CrossRef][Medline]
Riggins GJ, Kinzler KW, Vogelstein B, Thiagalingam S: Frequency of Smad gene mutations in human cancers. Cancer Res 57: 2578–2580, 1997[Abstract/Free Full Text]
Received for publication December 26, 2001.
Accepted for publication February 23, 2002.
This article has been cited by other articles:
T. Drueke, D. Martin, and M. Rodriguez Can calcimimetics inhibit parathyroid hyperplasia? Evidence from preclinical studies
Nephrol. Dial. Transplant.,
July 1, 2007;
22(7):
1828 - 1839.
[Full Text][PDF]
Y. Hibi, F. Kambe, Y. Tominaga, Y. Mizuno, H. Kobayashi, K. Iwase, T. Imai, and H. Seo Up-Regulation of the Gene Encoding Protein Kinase A Type I{alpha} Regulatory Subunit in Nodular Hyperplasia of Parathyroid Glands in Patients with Chronic Renal Failure
J. Clin. Endocrinol. Metab.,
February 1, 2006;
91(2):
563 - 568.
[Abstract][Full Text][PDF]
C. Morrison, W. Farrar, J. Kneile, N. Williams, Y. Liu-Stratton, A. Bakaletz, M. A. Aldred, and C. Eng Molecular Classification of Parathyroid Neoplasia by Gene Expression Profiling
Am. J. Pathol.,
August 1, 2004;
165(2):
565 - 576.
[Abstract][Full Text][PDF]
H. Sowa, H. Kaji, R. Kitazawa, S. Kitazawa, T. Tsukamoto, S. Yano, T. Tsukada, L. Canaff, G. N. Hendy, T. Sugimoto, et al. Menin Inactivation Leads to Loss of Transforming Growth Factor {beta} Inhibition of Parathyroid Cell Proliferation and Parathyroid Hormone Secretion
Cancer Res.,
March 15, 2004;
64(6):
2222 - 2228.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
family and the nucleus, and Smad genes are key transducers of information transmitted by tumor growth factor-
