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Original Studies |
Department of Pathology (C.J.H., H.M.), Department of Endocrinology (E.W.C.M.v.D.), Department of Clinical Genetics (C.v.A.), Department of Anthropogenetics (C.R.), Leiden University Medical Center, 2300 RC Leiden, The Netherlands; Department of Molecular Medicine (F.K.W., C.L.), Karolinska Hospital, Stockholm, S-17176 Sweden; Department of Medical Genetics (R.v.d.L., M.d.W.), Department of Internal Medicine (J.J., C.J.L.), Department of Internal Medicine and Pathology (J.R., J.H.), University Medical Center Utrecht, 3500 AB Utrecht, The Netherlands; and VanAndel Research Institute (B.T.T.), Grand Rapids, Michigan 49503
Address correspondence and requests for reprints to: Dr. Hans Morreau, Department of Pathology, Leiden University Medical Center, P.O. Box 9600, L1-Q, Building I, 2300 RC Leiden, The Netherlands. E-mail: jmorreau{at}pat.azl.nl
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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In conclusion, we report a family with hyperparathyroidism linked to chromosome 1q, and exhibiting several types of renal and endocrine tumors that have not been previously described.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A distinct disorder, hereditary hyperparathyroidism-jaw tumor syndrome (HPT-JT), with an autosomal dominant mode of inheritance, has been described in which primary hyperparathyroidism caused by parathyroid adenoma is associated with ossifying fibroma of the jaw (6). In a number of families, parathyroid carcinoma has been noted (7, 8, 9, 10, 11, 12). In addition, renal disease has also been described, including renal hamartomas, Wilms’ tumor, polycystic kidney disease, and degenerative cysts (8, 11, 12, 13, 14). The HPT-JT gene (HRPT2) has been mapped to the long arm of chr. 1q25-q31, later narrowed down to a 14.7-cm (centimorgan) region. Additionally, a 0.7-cm candidate region was recently suggested, based on shared haplotypes found in two Northern American families (15).
The nature of the HRPT2 gene is still unresolved, although [based on the presence or loss of the wild-type alleles in several renal hamartomas and some parathyroid tumors (13, 16)] the HRPT2 gene is considered a putative tumor suppressor gene. However, loss of heterozygosity (LOH) in HPT-JT is not always as evident as in MEN 1, in which the majority of familial tumors show loss at 11q13. For example, LOH has only been demonstrated in parathyroid tumors from a subset of families (11, 13, 14), possibly suggesting an alternative mechanism for tumorigenesis of parathyroid tumors in HPT-JT patients. To further understand the natural history and genetic involvement of this relatively new syndrome, we have studied the clinical, histopathological, and genotypic characteristics of a large Dutch kindred in which primary hyperparathyroidism is associated with other neoplasia.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. To date, 13 family members
have been documented with primary hyperparathyroidism caused by
parathyroid adenoma. Five individuals (III-2, III-5, III-8, III-10, and
III-11) had multiple renal cysts. Four of them (III-2, III-5, III-10,
and III-11) developed renal insufficiency requiring dialysis. The sex
distribution of affected individuals is roughly equal.
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), papillary renal cell carcinoma and
multiple cortical renal cell adenomas in the right kidney (III-5 at age
54, Fig. 2), parathyroid adenoma with atypia (III-11 at age 32) and
parathyroid carcinoma (III-11 at age 36, Fig. 2), and left testicular
mixed germcell tumor with a major seminoma component (IV-4 at age 32).
Clinically unaffected carriers with the disease haplotype that were
screened by renal ultrasonography (III-21, IV-7, IV-9, IV-12, and
IV-13) showed no apparent abnormalities. Also the renal ultrasonography
of carriers IV-11 and IV-14 was normal. Current biochemical testing of
Ca2+ and PTH of individuals III-4, III-21, IV-7,
IV-8, IV-9, IV-11, IV-12, IV-13, and IV-14 was normal. Of the four
individuals (IV-11, IV-12, IV-13, and IV-14) that were radiographically
screened, only two (IV-13 and IV-14) were suspect for carrying a
small jaw tumor; however, this has not been histologically
documented.
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Informed consent was obtained from all participating members of the family or their legal representatives.
High-molecular-weight DNA was isolated from leukocytes using standard methods. For two patients (III-2 and III-11) from whom a blood sample could not be obtained, constitutional DNA was extracted from nontumorous formalin-fixed paraffin-embedded tissue, as described (17). Eight microsatellite markers in and flanking the MEN1 gene in 11q13, cen-D11S4945-PYGM-D11S4946-D11S4940-D11S4947-D11S1783-D11S4937-D11S4936-tel, were used (4, 18). The 3–7th markers mentioned are intragenic, the other three are flanking the MEN1 locus. Eleven microsatellite markers from a 26-cm region in 1q25-q31, between D1S215 and D1S249, encompassing the HPRT2 locus (12), cen D1S215, D1S466, D1S191, D1S254, D1S422, D1S428, D1S222, D1S412, D1S413, D1S477, D1S510 (Genome Database: www. gdb.org) were used for linkage.
Genotyping was carried out by two methods: one using radiolabeled markers and the other fluorescent markers. PCR reactions were performed in a total vol of 10 µl, containing 100 ng genomic tumor DNA, 50 mmol/L KCL, 10 mmol/L Tris-HCL (pH 8.3), 1,5 mmol/L MgCl2, 125 mmol/L of each deoxynucleotide triphosphate, 2 pmol of each oligodeoxynucleotide primer (one of which was end-labeled with 32Phosphate in the case of radiolabeled markers), and 0.2 U DNA C polymerase (Dynazyme/Taq polymerase). Samples were amplified for 30 cycles (denaturation at 96 C for 30 sec, annealing at 55 C for 30 sec, and elongation at 72 C for 30 sec; and the products were run on 1% polyacrylamide gels.
With the fluorescent markers, the PCR products were pooled into three panels, according to the emission spectra of fluorescent dyes and the expected sizes of the amplified products. Electrophoresis was performed on 6% polyacrylamide gels, running on an ABI 377 laser-fluorescent sequencer (Perkin-Elmer Corp., Foster City, CA), and electrophoresis data were analyzed with the Genescan 3.1 computer software (Perkin-Elmer Corp.).
Two point logarithm of odds (lod) scores were generated using the LINKAGE (version 5.1) program adopting a conservative approach. Only patients with primary HPT were scored as affected, whereas other members at risk were considered as unknown. An autosomal dominant mode of inheritance and a penetrance of 0.90 were assumed.
Tumor analysis
Formalin-fixed, paraffin-embedded tumor tissue was obtained from 4 patients (III-2, III-5, III-11, and IV-1) and fresh frozen tumor tissue samples from 2 other patients (IV-11 and IV-14). Whenever possible, different tumor foci, as selected by a pathologist (H. Morreau), were microdissected from 10 10-µm-thick hematoxylin-stained sections mounted on glass-slides. Genomic DNA was extracted from the paraffin-embedded and fresh frozen specimens using standard methods.
Matched pairs of constitutional and tumor DNA were genotyped and analyzed for allelic imbalance, using markers as described under linkage analysis. For the chr.1q region, additional markers D1S2125, D1S1653, D1S408, D1S1614, D1S533, and D1S1660 were used. Allele status was identified on autoradiographic films and confirmed by digital images, which permitted computerized calculations of relative allele intensities. LOH was considered present when the signal intensity of one allele was reduced by more than 50%, in comparison with the corresponding allele in normal DNA.
Comparative genomic hybridization (CGH)
DNA from tumor samples was labeled with Fluorescein-12-deoxyuridine 5-triphosphate and DNA from the normal control with Lissamine-5-deoxyuridine 5-triphosphate (both from NEN Life Science Products, Boston, MA) by standard nick-translation.
The CGH was then performed according to the protocol described by Kallioniemi et al.(19), with a few modifications. Briefly, 200 ng of each labeled tumor and control DNA and 10 µg of human Cot-1 DNA (Life Technologies/BRL, Gaithersburg, MD) were dissolved in 10 µl hybridization buffer (50% formamide/2 x SSC/10% dextran-sulphate) and hybridized to normal male metaphase spreads at 37 C for 4 days. Posthybridization washes were performed with 2 x SSC at 37 C (3 x 10 min) followed by 0.1 x SSC at 60 C (3 x 5 min). Chromosomes were counterstained with 4,6-diamidino-2-phenylindole (0.5 µg/mL) in Vectashield antifade solution (Vector Laboratories, Inc., Burlingame, CA). Images were captured with a DM microscope (Leica Corp., Heidelberg, Germany) equipped with three single excitation filters, a multi-bandpass dichroic mirror, a multiband pass emission filter (P-1 filter set; Chroma Technology, Brattleborough, VT), and a cooled CCD camera (Photometrics Inc., Tucson, AZ). The green, red, and blue images were collected sequentially by changing the excitation filter. Images were analyzed using the QUIPS XL software from Vysis (Downers Grove, IL).
Losses of DNA sequences were defined as chromosomal regions where the average green-to-red ratio and its 95% confidence interval were below 0.8, whereas gains were above 1.2. These threshold values were based on measurements from a series of normal controls
Mutation analysis of the MEN1 gene
Mutation analysis was performed using single-stranded conformational polymorphism analysis and direct sequencing of all 10 exons and flanking intronic sequences after amplification of genomic DNA, as described (20).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Analysis of 11 markers indicated that primary HPT in this kindred was
linked to the 1q25–31 HPT-JT region. The maximum 2-point
lod score of 2.41 was obtained with markers D1S466, D1S191, D1S254,
D1S422, D1S412, and D1S413 (Table 1).
Considering that the most conservative approach was used in our
calculation, i.e. all individuals at risk were labeled as
unknown, the lod score obtained is the maximum possible in this family.
All affected individuals carried the disease haplotype (Fig. 1
).
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). Her
clinically unaffected son (IV-12) exhibited a recombination between
D1S222 and D1S428. The unaffected male III-21 (at age 50) exhibited a
recombination between D1S428 and D1S422.
LOH studies (Table 2) with markers from
the HPT-JT genomic region identified no LOH at chr. 1q21–32 in any of
the parathyroid adenomas or in a renal cell cortical adenoma studied.
Only different foci of the pancreatic adenocarcinoma (III-2), the
papillary renal cell carcinoma (III-5), and the parathyroid carcinoma
(III-11) exhibited LOH in the chr.1q region, involving either the
defective or wild-type chromosome (Table 2). CGH of fresh-frozen tissue
from two parathyroid adenomas of the family (IV-11 and IV-14) revealed
amplification of chr.16 for the tumor from IV-11 and deletion of
chr.13q, as well as amplification of chr.1q and chr.17p for the tumor
from IV-14.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The HRPT2 region has been narrowed to 14.7 cm by the
identification of recombinations in affected cases (15). A
0.7-cm candidate region was proposed, based on shared haplotypes
in two Northern American families, but this data warrants confirmation.
In the present studies, a number of recombinants have been identified.
Three clinically affected cases (III-8, IV-11, and IV-14) carry a
telomeric recombination between D1S413 and D1S477 which is the closest
telomeric border found in affected cases. Therefore, when combining
previously published data, the HPT-JT locus can be delineated to 14 cm
bordered by marker CHLC.12F10 (15) and D1S1632 (present study) (Table 3). Interestingly, two critical
re-combinants were identified in two members who remain
disease-free to date. The first one is in a 50-yr-old man (III-21) who
carries a telomeric recombination between markers D1S428 and D1S422,
and the second is in a 34-yr-old man (IV-12) with recombination between
D1S222 and D1S428. However, reduced penetrance is not uncommon in
HPT-JT (9, 11), as evidenced by six other diseased haplotype carriers:
III-4 (aged 62), IV-4 (aged 30), IV-7 (aged 24), IV-8 (aged 32), IV-9
(aged 30), and IV-13 (aged 33). As such, we are treating the two
disease-free recombinants with great caution, although they may
potentially further narrow the region. The HRPT2 gene has
been proposed as a putative tumor suppressor gene based on LOH
involving the wild-type alleles in a subset of 1q-linked tumors (11, 13, 16). However, in the present study, we were unable to demonstrate
consistent LOH of 1q21–32 in the tumors analyzed. This is in keeping
with other published data that indicates that an imbalance at 1q is not
always found in HPT-JT-related tumors (13, 14). It may be that the LOH
detected in our study on chr.1 is a late somatic event during
tumorigenesis, because only the malignant tumors exhibited this
pattern. In addition, our CGH results, which showed amplification of
chr.1q in one of the two parathyroid adenomas, are consistent with the
CGH results of two other 1q-linked parathyroid tumors (16). Taken
together, these complex data may suggest a different scenario: the
involvement of an oncogene in which loss of the wild-type copy was a
secondary event and followed by duplication of the mutated copy. A
similar mechanism has been found in hereditary papillary renal cell
carcinomas involving the MET locus on chr. 7 (21, 22, 27).
Finally, the gain at chr. 16q and the loss at chr.13q by CGH analysis
in the parathyroid adenomas in our family have also been described by
others (16), suggesting a role for these regions in parathyroid
tumorigenesis and progression. Future identification of the
HRPT2 gene will lead to a better understanding of the
mechanisms causing this interesting disease to have a wide spectrum of
clinical features.
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Acknowledgments |
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Footnotes |
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Received July 30, 1999.
Revised November 8, 1999.
Accepted December 20, 1999.
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References |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
,
Hyperparathyroidism; |KR,
, renal cysts, |Kr,
, jaw tumor; 
,
, unaffected; |Kl,
, additional
neoplasm; S, mixed germcell tumor with major seminoma component; RCC,
renal cell carcinoma; PC, parathyroid carcinoma; HC, Hürthle cell
adenoma of the thyroid. The affected haplotype is indicated by
black bars.