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Endocrine Care |
Service de Médecine Interne et Endocrinologie (V.V.-G., A.F., C.B., M.D., J.-L.W.) and Laboratoire de Biochimie Endocrinologique (P.P.), Clinique Marc Linquette, USNA, and Service Central de Médecine Nucléaire, Hôpital Roger Salengro (M.D.’H.), CHU de Lille, 59037 Lille, France
Address all correspondence and requests for reprints to: Virginie Vlaeminck-Guillem, M.D., Ph.D., Service de Médecine Interne et Endocrinologie, Clinique Marc Linquette, USNA, 6 rue du Professeur Laguesse, 59037 Lille Cedex, France. E-mail: virginie.vlaeminck{at}wanadoo.fr
Abstract
Pseudohypoparathyroidism Ia (PHP Ia) is characterized by resistance to
PTH and many other stimuli because of deficiency of stimulatory G
protein 
-subunit. To determine the incidence, natural history, and
mechanism of C cell dysfunction in PHP, calcitonin assays were
performed in six patients with PHP Ia and four with
pseudopseudohypoparathyroidism from three unrelated families. Controls
included healthy subjects and patients with PHP Ib or
hypoparathyroidism. The mean basal level of calcitonin was higher in
PHP Ia patients than in controls (95.3 ± 112.7 vs.
3.7 ± 2.4 pg/mL; P = 0.005; n < 10). In
PHP Ia patients, calcitonin levels rose over the normal range (30
pg/mL) after pentagastrin infusion in five patients and remained normal
in one. Familial medullary thyroid carcinoma was clinically,
biologically, and ultrasonographically ruled out over a mean follow-up
exceeding 3 yr. Genomic screening for RET protooncogene
mutations failed to reveal any anomaly. The calcitonin infusion test,
which induced a significant increase in plasma cAMP in controls 30 and
60 min after infusion, failed to produce this response in PHP Ia
patients, suggesting that the action of calcitonin was specifically
impaired. PHP Ia may therefore be an independent etiology of
hypercalcitoninemia and hyperresponsiveness to pentagastrin infusion.
PSEUDOHYPOPARATHYROIDISM (PHP) is defined
as a form of end-organ resistance to PTH (1). Type Ia is
the most frequent type and includes Albright’s hereditary
osteodystrophy (AHO), characterized by short stature, brachymetacarpia,
brachymetatarsia, round face, obesity, sc calcifications and
developmental dental defects (1), and deficient expression
or function of the -subunit of the guanine nucleotide regulatory
protein (Gs) (2, 3). By contrast,
in some PHP Ia kindreds, some patients display normal endocrine
responsiveness despite Gs insufficiency, a
condition that has been called pseudopseudohypoparathyroidism (PPHP)
(4). The same genetic defect is responsible for both PHP
Ia and this variant phenotype, i.e. an inactivating mutation
of GNAS1, the gene encoding the Gs
protein (5, 6).
Calcitonin (CT) is a 32-amino acid hormone that is produced by thyroid parafollicular C cells. It reduces plasma calcium levels in acute situations by increasing renal calcium excretion and inhibiting osteoclast-mediated bone resorption. It is mainly known for being the most sensitive tumoral marker of medullary thyroid carcinoma (MTC), a C cell-derived cancer (7). The CT receptor belongs to the family of G protein-coupled heptahelical membrane-spanning receptors and, more specifically, to a subset including receptors for PTH/PTHrP, secretin, vasoactive intestinal peptide, GHRH, glucagon, glucagon-like peptide, pituitary adenylyl cyclase-activating peptide, CRH, and CT gene-related peptide (8).
Although hypercalcitoninemia in patients with PHP has been mentioned in a few case reports (9, 10, 11) and one clinical study (12), these features have not been emphasized in the literature, and the cause and significance of C cell dysfunction in PHP remain unknown. Other researchers found normal CT levels in patients with PHP (13, 14, 15). In all of these reports, clinical and biological data are often incomplete, and the mechanism of C cell dysfunction was not assessed. To determine the incidence, natural history, and mechanism of this C cell dysfunction in PHP, we therefore conducted a prospective study of C cell function in six patients with PHP type Ia from three genetically different families.
Subjects and Methods
Patients
Six consecutive patients with PHP Ia (five women and one man;
mean age, 24.3 ± 3.6 yr) from three unrelated families were
evaluated (Fig. 1
). All met criteria for
AHO (including short stature, brachymetacarpia, brachymetatarsia, round
face, obesity, sc calcifications, and developmental dental defects),
resistance to PTH (hypocalcemia, hyperphosphatemia, and high levels of
immunoreactive PTH, and failure of PTH infusion to trigger the expected
increase in urinary phosphate or cAMP), and decreased
Gs activity (16). At initial
testing, all were normocalcemic while taking calcium (0.5–1.5 g/day)
and vitamin D3 supplementation (0.5–1 µg/day
1,25-dihydroxyvitamin D3 for patients 1 and 4,
and 1–1.25 µg/day 1-hydroxyvitamin D3 for
patients 2, 3, 5, and 6). Clinical information, including data from
cervical examination, was obtained for a period of up to 69 months
after the first measure of calcitoninemia.
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activity and absence of
resistance to PTH (normal levels of calcemia, phosphatemia and
immunoreactive PTH) (4). They were studied for CT
levels. Controls comprised five healthy subjects, one patient with PHP Ib (isolated resistance to PTH, with no AHO or multiple hormone resistance), and three patients with spontaneously acquired hypoparathyroidism (one had isolated idiopathic hypoparathyroidism and two had type I autoimmune polyendocrinopathy).
Informed consent was obtained from all participating patients and controls.
Determination of erythrocyte Gs
activity
The biological activity of Gs was
determined using a complementation assay based on the ability of
solubilized erythrocyte membrane extracts to restore the responsiveness
of adenylyl cyclase in membranes prepared from turkey erythrocytes,
which lack functional Gs proteins. The result
of the assay is roughly proportional to the amount of extract
Gs protein added. Heparinized blood samples
were collected from patients and control subjects. Soluble extracts
were prepared as previously described (2, 17). cAMP was
determined by radioimmunological quantitative analysis. Results were
expressed as a percentage of the activity of a standard membrane
preparation consisting of pooled erythrocytes from normal subjects and
represent the means of triplicate analyses.
Hormone assays
Thyroid and gonadal functions were evaluated in all patients before any therapy. Free T3 and free T4 were measured by radioimmunometric assays, and TSH was measured by immunochemiluminometric assay. Basal plasma levels of reproductive hormones were determined by immunochemiluminometric assays for estradiol and progesterone, radioimmunometric assay for testosterone, and immunoenzymometric assays for FSH and LH.
Serum CT was measured by a two-site immunoradiometric assay, using the ELISA-hCT kit (CIS Bioindustries, Gif-Sur-Yvette, France). Antibodies recognized the 11–17 and 24–32 regions of the molecule. The minimal detectable value was 2 pg/mL. Normal basal plasma CT levels were below 10 ng/mL. Results were expressed as the means of at least four measures for all PHP Ia patients. The CT precursor pro-CT was also measured by an immunometric assay.
Screening for MTC and other etiologies of hypercalcitoninemia
MTC was diagnosed by clinical examination (cervical palpation), measurement of carcinoembryonic antigen (immunoenzymometric assay), and ultrasonography. These procedures were repeated several times during a mean follow-up of 37.8 months. Furthermore, the propositus of each family (patients 1, 5, and 6) was screened for germline mutation of the RET protooncogene. Briefly, genomic DNA was prepared from peripheral blood samples collected on ethylenediamine tetraacetate (EDTA) according to standard protocols. Sequencing analysis was performed on PCR-amplified RET exons. The sequences of primers and PCR protocols were obtained from previously reported sources (18). All of the exons known to harbor germline mutations in familial MTC were studied (exons 10, 11, 13, 14, and 15). Both strands of the PCR products were sequenced with an automated DNA sequencer (ABI310, Lille, France).
Blood samples were also taken to evaluate uremia and creatininemia, because renal failure has been shown to be an etiology of hypercalcitoninemia. For the same reason, plasma gastrin levels were measured by RIA.
Provocative tests
A standard TRH test (iv injection of 200 µg) was performed in PHP Ia patients, with measurement of TSH, free T3, free T4, and PRL up to 180 min after TRH injection. The patients also underwent GnRH stimulation tests (iv injection of 100 µg synthetic GnRH). Samples were drawn 0, 20, and 40 min after injection for measurement of FSH and LH levels. Results were expressed as basal values and values determined in pooled 20 and 40 min samples. A glucagon test was performed as previously described (19). After a bolus injection of 500 µg glucagon, blood samples were taken at 0, 15, 30, and 60 min to determine plasma cAMP levels by radioimmunometric assay.
C cell function was evaluated by specific tests, including pentagastrin and calcium stimulation tests and the EDTA inhibition test. For the pentagastrin test, 0.5 µg/kg pentagastrin, iv, was slowly injected over a period of 3 min, as previously recommended (7). Blood samples were collected 0, 2, and 5 min after the infusion started. In healthy subjects, a peak value of less than 30 pg/mL CT is expected, and values above 30 are considered abnormal (7). The calcium infusion test consisted of slow iv injection of 15 mg/kg calcium over 240 min. Blood samples were drawn 0 and 240 min after the infusion started. A moderate increase in CT levels is expected in healthy subjects. The sodium EDTA infusion test consisted of slow iv injection of 50 mg/kg sodium edetate for 120 min. Blood samples were collected 0 and 120 min after the infusion started. A moderate decrease in calcium and CT levels as well as an increase in PTH levels are expected in healthy subjects.
The sensitivity of peripheral tissue to CT was evaluated by a CT administration test, consisting of im injection of 0.5 mg (100 IU) synthetic human CT (Novartis, Rueil-Malmaison, France). Blood samples were drawn 0, 30, 60, 120, and 160 min after administration to measure plasma cAMP by RIA (kit from Immunotech, Marseille, France). We checked that plasma levels of CT did rise by monitoring them throughout the test.
Statistical analysis
Results are expressed as the mean ± SD. The
PHP Ia patients and the controls, comprising healthy subjects and PPHP
patients, were compared using the nonparametric unpaired Wilcoxon test,
ANOVA, or unpaired Student’s t test, depending on the
parameters. For the CT administration test, the mean levels of plasma
cAMP obtained 30, 60, 120, and 160 min after injection were compared
with the mean basal values using the nonparametric paired Wilcoxon
test. Plasma cAMP levels were normalized to the basal value, and the
variations in the PHP Ia and control groups were compared using the
nonparametric unpaired Wilcoxon test. Correlations between CT levels
and Gs activity were sought using linear
regression and Fisher’s exact test. Results are expressed as the
mean ± SD. Differences were considered
statistically significant when P < 0.05. We used the
Stata 6.0 package (Stata Corp., College Station, TX).
Results
Clinical profiles and Gs levels
Clinical examination of the six PHP Ia and four control PPHP patients only disclosed evidence of AHO. No patient exhibited clinical features of gross hypothyroidism. The menstrual histories of the five women with PHP Ia showed that none of them had been pregnant. Three had regular menses, and two had oligomenorrhea (patients 4 and 5). The male patient (patient 3) had bilateral cryptorchidism.
The six patients with PHP Ia had a reduction of approximately 50% in
erythrocyte Gs activity (54.08 ± 5.8%;
Fig. 1
). Reduced activity was also observed in the four PPHP patients
(67 ± 18%). In PHP Ia patients, there was no significant
correlation between the level of Gs activity
and CT levels.
Basal hormonal profiles
Basal hormone levels in the six PHP Ia patients (Fig. 1) showed
biochemical evidence of mild primary hypothyroidism (Table 1) with normal levels of both free
T3 and free T4 and slightly
elevated TSH levels. The two PHP Ia patients with oligomenorrhea
(patients 4 and 5) exhibited normal plasma estradiol and progesterone
levels and moderately elevated levels of FSH and/or LH. Basal serum PRL
levels were normal in the six patients with PHP Ia.
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),
whereas they remained within the normal range (<10 pg/mL) in the three
PPHP mothers studied, the patient with PHP Ib, the three patients with
spontaneous acquired hypoparathyroidism, and the five healthy subjects.
The mean CT level was significantly higher in the PHP Ia group than in
the control group, comprising the patient with PHP Ib, the five healthy
subjects, and the three hypoparathyroid patients (95.3 ± 112.7
vs. 3.7 ± 2.4; P = 0.005). The CT
precursor pro-CT was constantly normal in the PHP Ia patients.
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None of the six patients with PHP Ia had any symptom suggestive of
MTC. Cervical palpation was normal, and no nodules were revealed by
thyroid ultrasonography. Carcinoembryonic antigen remained stable in
the normal range (Fig. 1). Furthermore, we failed to identify any
germline mutation of the RET protooncogene by sequencing the
five exons involved in familial MTC (exons 10, 11, 13, 14, and 15).
Other conditions known to induce hypercalcitoninemia were also ruled out, such as renal failure (urea and creatinine levels were normal), hypergastrinemia (normal gastrin levels), and chronic lymphocytic thyroiditis (no antithyroid antibodies).
Provocative tests
The time course for TSH response to TRH injection was normal in all six PHP Ia patients, with normal peak TSH values 15–30 min after the injection. No clear increases in free T3 and free T4 levels were observed at 120 or 180 min. The peak PRL responses to TRH were as high, as expected, in patients 1, 4, 5, and 6, but were low in patients 2 and 3. Provocative GnRH tests produced the expected increases in FSH and LH levels in all patients.
For the six PHP Ia patients, the pentagastrin infusion test showed a
clear increase in CT levels at 2 and 5 min (Fig. 2). Peak values were almost 30 pg/mL for
patient 5, higher than 50 pg/mL for patient 4 and higher than 100 pg/mL
for patients 1, 2, 3, and 6. The calcium infusion test showed that
regulation of CT secretion was conserved, with a mean 2.84-fold
increase at 240 min (2.84 ± 1.18). A foreseeable result was also
obtained during the EDTA infusion test, with a mean 3.35-fold decrease
at 120 min (3.35 ± 1.86).
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].
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Long-term information on clinical symptoms and CT levels was obtained for the six patients with PHP Ia. The mean follow-up period was 37.8 ± 28.2 months (range, 4–69 months). CT levels remained high in all patients. All remained symptom free. In particular, cervical examination was unremarkable. Iterative thyroid ultrasonography failed to reveal any thyroid nodules. Levothyroxine therapy was given to all patients, with satisfactory control according to both clinical symptoms and TSH levels.
Discussion
Our study demonstrates that high levels of plasma CT may be found in patients with PHP Ia. The thyroid C cells appeared to respond adequately to both stimulatory and inhibitory factors. In particular, a marked rise in CT was induced by the pentagastrin infusion test.
The type Ia diagnosis was precisely defined and clearly established in
all of our patients. Firstly, the following characteristics were
present: AHO (1), resistance to PTH as demonstrated by the
PTH infusion test (16), associated resistance to TSH
(normal free thyroid hormone levels, high TSH levels, and weak thyroid
hormone response to TRH infusion), and associated resistance to
gonadotropins (oligomenorrhea and high LH and/or FSH levels)
(19). Secondly, biochemical assays showed significantly
reduced Gs activity that is specifically
related to PHP Ia and PPHP (2, 3, 5, 6). Thirdly, the
inheritance pattern of the disorder among our families was consistent
with the usual autosomal dominant trait (16) and paternal
imprinting (20) described in both PHP Ia and its related
disorder, PPHP. Lastly, artifactual events altering CT measures were
unlikely in the present study, because we performed at least five
measurements for each patient using immunoradiometric assays, which are
currently the methods of choice for measuring CT (7, 21).
C cell function in PHP patients has been poorly studied. A few case reports alluded to normal (13, 15) or increased CT levels (9, 12) in PHP patients, frequently without providing clear data about the type of the disease. Only one study reported experiments in PHP Ia patients in 1973 (14). Those researchers found normal CT levels in nine patients but used a 100 pg/mL detectability threshold. Consistent with our study, they described an increase in CT levels after calcium infusion in the nine patients and an abnormally marked increase after pentagastrin infusion in the one subject tested.
We showed that PHP Ia probably constitutes an independent etiology of hypercalcitoninemia, because the usual etiologies could be ruled out. In particular, although the hypercalcitoninemia and hyperresponsiveness triggered by pentagastrin are highly suggestive of MTC (7, 21), this etiology is unlikely, because clinical, biological, and ultrasonographic examinations were always normal. In addition, CT levels remained high despite repeated careful assessment over long follow-up periods of up to 5 yr, and genomic screening failed to reveal any of the RET protooncogene germline mutations found in familial MTC (18). Pathological examination has constantly failed to show any evidence of tumors in surgical specimens from PHP patients undergoing thyroidectomy (9, 22, 23). In our patients, other pathological conditions known to induce hypercalcitoninemia (7, 21) were also ruled out, such as chronic renal failure, chronic lymphocytic thyroiditis, nonmedullary malignant or benign thyroid tumors, neuroendocrine tumors originating from neural crest cells, and hypergastrinemia.
The mechanism by which PHP Ia is responsible for hypercalcitoninemia
has never been precisely assessed. It may be an associated resistance
to CT, as suggested by the usual impairment of
Gs-mediated hormone signal transduction in PHP
Ia (5, 6). The ubiquitous coupling of G proteins to the
heptahelical membrane-spanning receptors sheds light on the well known
associated resistance to multiple hormones, including resistance to
TSH, gonadotropins, glucagon (19), ACTH (24),
GH (25), and sensorineural, olfactory, and gustatory
stimuli (26). Resistance to CT may fit into the same
pattern, as CT acts on its target tissues via a specific receptor that
belongs to the family of G protein-coupled heptahelical
membrane-spanning receptors and more specifically to a subset of
receptors including the PTH/PTHrP receptor (8).
Furthermore, hypercalcitoninemia is specific to type Ia PHP, as
suggested by previous reports (9, 10, 11) and the results of
the present study; it was not detected in our PHP Ib patient, whose
resistance was typically restricted to PTH only (16). The
normocalcitoninemia found in our PPHP patients does not argue against
resistance to CT, because multiple hormone resistance is rarely
observed in this condition (16). Most important, the
resistance to CT is supported by the fact that whether CT
administration is iv (27), endonasal (28), or
im (our study), it raises plasma cAMP levels in healthy subjects, but
fails to induce a rise in PHP Ia patients, consistent with the
unresponsiveness of target tissues to CT.
The great variability in the basal and stimulated CT values in our
patients with PHP Ia may be explained by the fact that
hypercalcitoninemia results from the imbalance of a complex regulatory
system. Further studies are required to identify the strong stimulating
factor involved in PHP Ia, but we postulate that the
hypercalcitoninemia found in these patients is favored by low levels of
1,25-dihydroxyvitamin D3
[1,25-(OH)2D3]. The
production of 1,25-(OH)2D3
by renal 1-hydroxylase is indeed defective in PHP. This is due to
resistance to PTH, which normally stimulates 1-hydroxylase
production (29), but such resistance may be strengthened
by resistance to CT, because CT also promotes renal production of
1,25-(OH)2D3
(30) by interacting with the promoter of the
1-hydroxylase gene (31). As
1,25-(OH)2D3 down-regulates
C cell production of CT (32, 33), probably by interacting
with its nuclear receptor (34), low levels of
1,25-(OH)2D3 may constitute
a major stimulus of the hypercalcitoninemia found in PHP Ia
patients.
Besides its involvement in vitamin D metabolism, resistance to CT may also contribute to other phenotypic features of PHP Ia. Thus, the CT receptor is expressed in osteoclasts (35), and resistance to CT may worsen the bone abnormalities due to resistance to PTH and PTHrP. In addition, CT inhibits tubular absorption of phosphorus, and the related defect in kidney CT function may be a cause of the hyperphosphatemia usually observed in PHP Ia. Lastly, as CT is known to be a neuromediator (36), resistance to CT may contribute to the mild to moderate mental retardation frequently observed in PHP Ia.
From this study we conclude that PHP Ia is an independent etiology of hypercalcitoninemia. Hyperresponsiveness to CT may even be observed after pentagastrin injection. To deal with hypercalcitoninemia in patients with PHP Ia, physicians must first systematically rule out MTC by repeating careful clinical, biological and ultrasonographic examinations. Germline mutations of the RET protooncogene should also be sought by appropriate genomic screening. When a thyroid nodule is identified or genetic study is positive, surgical thyroidectomy is appropriate. By contrast, when all of these explorations are negative, our results suggest that cervicotomy and follow-up may be avoided.
Received September 12, 2000.
Revised February 7, 2001.
Accepted March 15, 2001.
References
-subunit gene in Albright
hereditary osteodystrophy detected by denaturing gradient gel
electrophoresis. Proc Natl Acad Sci USA. 87:8287–8290.-hydroxylase gene by parathyroid hormone,
calcitonin, and
1,25(OH)2D3 in intact
animals. Endocrinology. 140:2224–2231.This article has been cited by other articles:
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  A. Plagge, G. Kelsey, and E. L Germain-Lee Physiological functions of the imprinted Gnas locus and its protein variants G{alpha}s and XL{alpha}s in human and mouse J. Endocrinol., February 1, 2008; 196(2): 193 - 214. [Abstract] [Full Text] [PDF]
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 M. d'Herbomez, P. Caron, C. Bauters, C. Do Cao, J.-L. Schlienger, R. Sapin, L. Baldet, B. Carnaille, J.-L. Wemeau, and the French Group GTE (Groupe des Tumeurs Endocrine Reference range of serum calcitonin levels in humans: influence of calcitonin assays, sex, age, and cigarette smoking Eur. J. Endocrinol., December 1, 2007; 157(6): 749 - 755. [Abstract] [Full Text] [PDF]
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 M. Bastepe and H. Juppner Pseudohypoparathyroidism and Mechanisms of Resistance toward Multiple Hormones: Molecular Evidence to Clinical Presentation J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4055 - 4058. [Full Text] [PDF]
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 L. Hegedus, S. J. Bonnema, and F. N. Bennedbaek Management of Simple Nodular Goiter: Current Status and Future Perspectives Endocr. Rev., February 1, 2003; 24(1): 102 - 132. [Abstract] [Full Text] [PDF]
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) than in the PHP Ia group (
;
P = 0.0042 and P = 0.0164,
respectively).