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Original Studies |
Endocrine Surgery Unit, Department of Surgery (P.C., J.R., G.W., E.L., G.Å., T.C.), and Department of Internal Medicine (A.K.), Uppsala University Hospital, S-751 85 Uppsala, Sweden; and the Department of Medicine (P.S.), Gentofte Hospital, University of Copenhagen, DK-2900 Hellerup, Denmark
Address all correspondence and requests for reprints to: Tobias Carling, Ph.D., Endocrine Surgery Unit, Department of Surgery, Uppsala University Hospital, S-751 85 Uppsala, Sweden. E-mail: tobias.carling{at}kirurgi.uu.se
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Abstract |
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Introduction |
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As active vitamin D [1,25-dihydroxyvitamin D3 (1,25-(OH)2D3], via its receptor (VDR), inhibits both parathyroid cell proliferation and PTH secretion (3, 4), it is appropriate to use VDR gene polymorphisms in genetic association studies of PHPT. Additionally, the pathogenesis of both primary and secondary HPT has been suggested to involve derangements in VDR function or expression (5, 6, 7). Consistent with the importance of VDR in parathyroid regulation, the VDR b, a, and T alleles were recently found to be overrepresented in patients with PHPT (8, 9, 10). These alleles were linked to enhanced dysregulation of PTH secretion (11) and reduced VDR messenger ribonucleic acid (mRNA) levels in parathyroid adenomas patients (12).
When the nucleotide sequence of the human VDR complementary DNA was reported, two potential translation initiation (ATG) sites were disclosed (13). A T/C polymorphism, detected by the FokI restriction enzyme, was discovered at the first potential start sites. An individual homozygous for ATG would have two putative start sites, with translation initiating from the first one (14), whereas translation would start at the second site in those homozygous for ACG. The resulting difference in VDR length by three amino acids may affect the function of the protein. This contrasts to the b, a, and T polymorphisms in intron 8 and exon 9 of the VDR gene, which do not alter the VDR amino acid sequence (15). Moreover, there seems to be no apparent linkage disequilibrium between these polymorphisms and the VDR-FokI polymorphism (14, 16, 17). By producing a reporter gene construct under the control of a vitamin D response element, the FF variant was suggested to cause increased VDR expression compared to the ff genotype (18). The current study investigates VDR-FokI genotype frequencies in patients with PHPT and control subjects, any associations with clinical signs of the disorder, as well as parathyroid VDR and PTH mRNA expression.
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Subjects and Methods |
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A total of 91 PHPT patients and 91 controls were used in the genetic association study of the VDR-FokI polymorphism. Sixty-four postmenopausal women with nonfamilial PHPT were recruited by a population-based screening for PHPT (2). Sixty-four postmenopausal controls were procured from the same screening by matching for age and quarter of year of diagnosis. An additional 27 postmenopausal women with sporadic PHPT, who were diagnosed and operated on in the clinical routine, were included. Matched controls for this PHPT subgroup were 27 normocalcemic postmenopausal females referred for neck exploration due to atoxic goiter or thyroid cysts. All 182 patients and controls have previously been genotyped for the VDR 3'-end polymorphisms using restriction enzymes BsmI, ApaI, and TaqI (8, 9).
To investigate relationships between the VDR-FokI polymorphism and VDR and PTH mRNA levels, an additional 42 consecutive patients with parathyroid adenoma of PHPT were genotyped. The clinical characteristics of these patients were previously described (12). Twenty-three consecutive Danish patients with PHPT were also recruited. They consisted of 20 females and 3 males (aged 40–86 yr) and have previously been studied for defects in the calcium-controlled PTH secretion, in vivo (19, 20). All PHPT patients demonstrated the absence of familial hypercalcemia, signs of multiple endocrine neoplasia syndromes, and substantially elevated serum creatinine levels.
Blood was collected after an overnight fast, and total serum calcium (reference range, 2.20–2.60 mmol/L) and serum creatinine (reference range, 64–106 µmol/L) were measured. Intact serum PTH (reference range, 12–55 ng/L) was analyzed in all but the goiter controls, as previously described (8, 9). The female cases and controls recruited by screening were subjected to measurement of bone mineral density (BMD) at the lumbar spine (L2–L4), femoral neck, and total body (8, 21).
Genotype analysis
Leukocyte DNA was prepared by standard methods or by using a genomic DNA isolation kit (Wizard, Promega Corp., Madison, WI). The primers VDR2a (5'-agctggccctggcactgactctgctct-3') and VDR2b (5'-atggaaacaccttgcttcttctccctc-3') and 2 µl of the leukocyte DNA were used in a PCR to amplify a 265-bp fragment containing the start codon polymorphism (14). The PCR products were digested with FokI at 37 C for 3 h, followed by electrophoresis in a 1.5% agarose-gel containing ethidium bromide. Homozygous cleavage by FokI generates two fragments, 69 and 196 bp, respectively, whereas the heterozygotes display all three bands. Thus, the genotypes FF, Ff, and ff could be identified.
Measurements of VDR and PTH mRNA levels
VDR and PTH mRNA levels were determined by the ribonuclease protection assay using total RNA from parathyroid adenomas of PHPT patients as part of a previous study (12). All 42 parathyroid adenomas were subjected to measurement of VDR mRNA levels, whereas PTH mRNA levels were determined in 34 of the adenomas due to lack of sufficient amounts of total RNA. Individual VDR and PTH mRNA values were corrected using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level as an internal standard.
Citrate and calcium clamp
Twenty-three PHPT patients underwent a CiCa clamp technique protocol to evaluate the PTH dynamics to sequential induction of hypo- and hypercalcemia (19, 20). Based on this method the calcium set-point (the B-Ca2+ concentration causing 50% inhibition of maximal PTH secretion) of each patient was determined (19, 20).
Statistical analysis
Statistical analyses were executed with 
2-test,
unpaired t test, or ANOVA, and P < 0.05 was
considered significant. All values are presented as the mean ±
SEM.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). As expected, PHPT
patients detected by screening displayed less extensive increases in
the biochemical signs of PHPT and lower parathyroid tumor weights. The
VDR-FokI genotype distribution in the screening-detected
PHPT patients, all PHPT patients, and their control groups are
presented in Table 2. Although
statistically not significant, there was a tendency to
underrepresentation of the ff genotype in the
screening-detected and all PHPT patients compared to the controls
(P = 0.07 and 0.09, respectively). When analyzing the
allele frequencies in all PHPT patients, the f and
F alleles were found in 38% and 62%, respectively,
vs. 45% and 54% in the controls (P =
0.05). The genotype distribution for the screening-detected controls
and all controls was similar to previous findings in Caucasian women
from California (14), Boston (17), and France (16). The
VDR-FokI genotypes were not significantly associated with
age of the individuals, serum calcium, serum PTH, BMD, or parathyroid
tumor weight in any of the groups of PHPT patients or controls. In the
PHPT patients, there was a nonsignificant trend toward higher serum PTH
and calcium levels and glandular weight in those with the FF
genotype. The VDR-FokI polymorphism was unrelated to those
detected by the BsmI, ApaI, and TaqI
restriction enzymes (not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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When analyzing the VDR-FokI genotype distribution, there was no significant difference between the PHPT patients and controls, which indicates no major contribution to the risk of developing PHPT. Upon comparison of allele frequencies, however, the F allele was overrepresented in the PHPT patients, which suggests a slightly increased risk of developing PHPT in individuals with this genetic variant. The FF genotype also demonstrated higher serum calcium and PTH levels and glandular weight, although these tendencies were not significant. Overall, the VDR-FokI polymorphism failed to show any significant association with the biochemical characteristics of PHPT. Additionally, BMD levels in both PHPT patients and controls were not significantly associated with the F/f alleles, which contrasts to the findings of some previous studies. In Mexican-American women, the ff genotype was associated with lower BMD at the lumbar spine (14) and an increased bone loss over a 2-yr period (14). Also in premenopausal Japanese and American women, the ff genotype related to lower BMD at the lumbar spine and the femoral neck, respectively (17, 18). In contrast, two European populations showed no association between the VDR-FokI polymorphism and BMD (16, 23). The discrepancy could be due to ethnic differences related to VDR-FokI allelic prevalences as well as to environmental and geographical variations related to calcium intake and exposure to sunshine, etc.
Earlier studies have implied a 1.7-fold increase in vitamin D-dependent transcriptional activation for a reporter gene construct for the F variant VDR compared to the f-type protein (18). This might relate to differences in the efficiency of the binding to other transcription factors, such as basal transcription factor IIB (24). As this could cause allele-specific differences in the expression of VDR in target tissues such as bone, intestine, and parathyroid, we investigated the VDR and PTH mRNA levels in parathyroid adenomas of PHPT patients. No association between mRNA levels and the F/f alleles were detected, which suggests that the VDR-FokI alleles are unlikely to interact strongly in the regulation of PTH and VDR gene transcription. This contrasts to the demonstration of a significant association between the VDR b, a, and T polymorphisms and VDR and PTH mRNA levels in parathyroid adenomas (12). It has been speculated that the latter polymorphisms are related to the stability of VDR mRNA, and this might be via linkage disequilibrium to the poly(A) repeat polymorphism further downstream on the VDR mRNA (25). Recently, it was demonstrated that both the VDR-FokI and the poly(A) repeat polymorphisms are coupled to VDR trans-activation ability, and that the former polymorphism demonstrated the strongest association (26). This might be the explanation for the rather strong association between PHPT and the VDR b, a, and T polymorphisms, whereas the VDR-FokI polymorphism seems to be of less importance to the risk of developing PHPT. The absence of relationships between the presently studied alleles and gene expressions do not exclude the possibility that the VDR-FokI polymorphism is related to VDR expression in other target cells, as the regulation of VDR expression seems highly tissue specific (27). Any relation to parathyroid VDR mRNA levels might also be concealed by confounders such as duration of the disease, tumor size, and serum PTH, calcium, and 1,25-(OH)2D3 levels. Furthermore, the calcium set-point was not associated with the VDR-FokI polymorphism, indicating no or only mild effects on the characteristic alteration of calcium homeostasis in PHPT.
The theoretically appealing mechanism of altered VDR function or expression due to polymorphisms in the start codon of the VDR gene seems to be at most weakly associated with PHPT and the clinical expression of the disease in postmenopausal females. Additionally, the VDR-FokI polymorphism may have less impact on BMD than originally suggested, at least in the Swedish population.
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Footnotes |
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Received October 21, 1998.
Revised December 10, 1998.
Revised February 12, 1999.
Accepted February 17, 1999.
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