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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 9 4059-4069
Copyright © 2003 by The Endocrine Society

Special Feature

Growth Hormone Deficiency in Pseudohypoparathyroidism Type 1a: Another Manifestation of Multihormone Resistance

Emily L. Germain-Lee, Joshua Groman, Janet L. Crane, Suzanne M. Jan de Beur and Michael A. Levine

Department of Pediatrics, Division of Endocrinology (E.L.G.-L., J.L.C., M.A.L.); the Ilyssa Center for Molecular Endocrinology (E.L.G.-L., J.L.C., S.M.J.d.B., M.A.L.); the McKusick-Nathans Institute for Genetic Medicine (J.G., M.A.L.); and the Department of Medicine, Division of Endocrinology (S.M.J.d.B., M.A.L.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287

Address correspondence and request for reprints to: Emily L. Germain-Lee, M.D., Park 211, Division of Pediatric Endocrinology, Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, Maryland 21287. E-mail: egermain{at}jhmi.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Albright hereditary osteodystrophy (AHO) is a genetic disorder caused by heterozygous inactivating mutations in GNAS1, the gene encoding the {alpha}-chain of Gs, and is associated with short stature, obesity, brachydactyly, and sc ossifications. AHO patients with GNAS1 mutations on maternally inherited alleles also manifest resistance to multiple hormones (e.g. PTH, TSH, LH, FSH), a variant termed pseudohypoparathyroidism (PHP) type 1a, due to paternal imprinting of G{alpha}s transcripts in specific tissues. Recent evidence has shown that G{alpha}s transcripts are also imprinted in the pituitary somatotrophs that secrete GH. Because this imprinting could influence GHRH-dependent stimulation of somatotrophs, we hypothesized that maternally inherited GNAS1 mutations would impair GH secretion. We studied GH status in 13 subjects with PHP type 1a. GH responses to arginine/L-dopa and arginine/GHRH were deficient in nine subjects, all of whom were obese and had low serum concentrations of IGF-I. By contrast, none of the four GH-sufficient subjects were obese, and all had normal IGF-I levels. Our data indicate that GH deficiency is common (69%) in PHP type 1a and may contribute to the obesity and short stature typical of AHO. We propose that GH status be evaluated in all patients with PHP type 1a.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
ALBRIGHT HEREDITARY OSTEODYSTROPHY (AHO) is an autosomal dominant condition caused by heterozygous inactivating mutations in the GNAS1 gene. GNAS1 encodes the {alpha}-chain of the heterotrimeric G protein, Gs, that couples receptors for many hormones and neurotransmitters to activation of adenylyl cyclase (reviewed in Refs. 1 and 2). AHO is associated with characteristic developmental abnormalities that include short stature, obesity, round face, brachydactyly, sc ossifications, dental hypoplasia, and mental retardation (3). In some patients, many of these features may be subtle or absent. Within a given kindred, affected patients may have these defects associated with resistance to multiple hormones (e.g. PTH, TSH, gonadotropins, and glucagon) (1, 4, 5) whose receptors couple to Gs, a condition termed pseudohypoparathyroidism (PHP) type 1a, or may have the developmental defects alone, a variant termed pseudopseudohypoparathyroidism (pseudoPHP) (1, 6).

One explanation for the variable hormone responsiveness in AHO is genomic imprinting of GNAS1 at 20q13.3 (1, 2, 7). Analysis of published pedigrees demonstrates that maternal transmission of a GNAS1 mutation causing G{alpha}s deficiency leads to PHP type 1a, whereas paternal transmission of the same defect leads to pseudoPHP (6, 8, 9). Studies of transgenic mice in which one Gnas allele has been inactivated by homologous recombination (Ref. 10 , data not shown) have confirmed these clinical observations. In the Gnas knockout mouse, in which exon 2 was disrupted by insertion of the neomycin cassette (10), both the transcript for G{alpha}s and its paternally derived alternative transcript XL{alpha}s (which can functionally couple to receptors that act via G{alpha}s) were inactivated (2, 11, 12, 13). These mice demonstrate tissue-specific paternal imprinting of G{alpha}s transcripts in renal proximal tubule cells as well as in fat cells (10). Recent experimental evidence has shown that GNAS1 is also paternally imprinted in the human thyroid (14). This thyroid imprinting, however, is partial and contrasts markedly with the tightly regulated reciprocal imprinting of the alternative first exons, NESP55 and XL{alpha}s, located upstream of G{alpha}s exon 1 (11, 12, 15, 16, 17, 18, 19, 20). This partial imprinting provides a likely explanation for the mild TSH resistance and hypothyroidism in AHO patients (4, 14) and Gnas exon 1 knockout mice (Ref. 21 , data not shown) that inherit a defective maternal allele. In addition, it is possible that XL{alpha}s, which can functionally couple to TSH receptors (13), may partially compensate for the diminished G{alpha}s and also contribute to this mild TSH resistance. Development of pituitary somatotrophs and secretion of GH require GHRH, a hypothalamic hormone that binds to receptors on somatotrophs that are coupled via Gs to activate adenylyl cyclase. The critical role of G{alpha}s protein in controlling GH secretion is illustrated by the development of pituitary hyperplasia and GH excess in transgenic mice (22) and humans (23) with genetic activation of G{alpha}s in somatotropic cells.

To date, there are scattered case reports that have examined GH status in small numbers of PHP type 1a patients. These reports describe a spectrum of GH reserve from GH deficiency to GH sufficiency (24, 25, 26, 27, 28, 29, 30), not unlike the variable responsiveness to other hormones in patients with PHP type 1a. Recent studies have demonstrated that G{alpha}s is paternally imprinted in the pituitary (31), thus raising the likelihood that subjects with a defective maternal GNAS1 allele could have marked G{alpha}s deficiency in somatotrophs and reduced GH responsiveness to GHRH. In the present study, we report that most subjects with PHP type 1a have GH deficiency, thus extending the abnormal endocrine phenotype of this disorder and providing a possible explanation for the obesity and short stature in these patients.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Experimental subjects

We studied 13 subjects with PHP type 1a while they were receiving treatment for TSH and PTH resistance (Table 1AGo). Informed consent was obtained from each subject, or a parent of each subject, before participation in these studies. All subjects were evaluated in the General Clinical Research Center at the Johns Hopkins Hospital (Baltimore, MD), and all studies were approved by the Joint Committee on Clinical Investigation of the Johns Hopkins University School of Medicine. The diagnosis of PHP type 1a was based on the following criteria: 1) evidence of multihormone resistance, including biochemical hypoparathyroidism with elevated serum levels of intact PTH, hypocalcemia and hyperphosphatemia, and hypothyroidism with elevated TSH and low or normal free T4; 2) clinical features of AHO, including short stature, obesity, round face, brachydactyly/brachymetacarpia, sc ossifications, and cognitive deficiency. All subjects who were taking vitamin D, calcium, or thyroid hormone continued these treatments throughout the course of the study.


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  		TABLE 1A. Clinical and biochemical characteristics of study subjects

 
GNAS1 gene analysis

Total genomic DNA was isolated from peripheral blood leukocytes by standard methods (32), and the 13 coding exons and intron-exon boundaries of GNAS1 were analyzed for genetic defects by a modification of previously described techniques (33, 34, 35, 36, 37). Primer sequences and PCR conditions are available on request.

Southern blot analysis

Ten micrograms of genomic DNA from each of the three subjects in whom no mutations were found (subjects 3, 10, and 13) were isolated as previously described (32). The DNA was digested with either PstI alone or PstI plus NgoMIV, and the total digest was run at 80 V for 6 h on a 1.5% agarose gel. Southern blotting was performed using standard procedures (32), and the blot was probed with a 32P-labeled 915-bp fragment of GNAS1 (position 29078-29993 of chromosome 20q; GenBank accession no. AL12197; generated by Asc/SacI digest).

Anthropometric evaluation

Standing height was determined using a Harpendon stadiometer (Holtain Ltd., Crymych, Dyfed, United Kingdom). Weight was determined using a Detecto scale (Detecto Scale Co., Webb City, MO). We calculated each subject’s height in terms of SD from the mean (adjusted for age and gender) and expressed results as the height SD score (SDS). Weight SDS was calculated for each subject as well as body mass index (BMI) in kilograms per square meter. The growth velocity was determined from height records over the previous year. Target heights in centimeters were calculated using the standard formula: boys = (height of mother + height of father + 13)/2; girls = (height of mother + height of father -13)/2. Predicted heights were determined from bone ages of the left hand and wrist according to the method of Bayley and Pinneau (38).

Laboratory evaluation

All subjects fasted overnight for at least 8 h before the start of GH testing. An indwelling catheter was placed in a forearm vein for collection of blood samples. Blood samples for GH were obtained at -15, 0, 30, 60, 90, 120, and 150 min relative to the initiation (time 0) of a 30-min infusion of arginine (0.5 g/kg of a 10% solution; maximum dose, 40 g; Pharmacia/Upjohn, Kalamazoo, MI). At 60 min, L-dopa (Roche, Nutley, NJ) was given orally (0–30 lb, 125 mg; 30–70 lb, 250 mg; >70 lb, 500 mg). Serum GH levels were measured by the Tosoh immunoenzymometric assay (Tosoh Medics, Inc., Foster City, CA) in our clinical laboratory. The sensitivity of this assay is 0.1 ng/ml, with a three-level intraassay precision [mean, 4.9 ng/ml, coefficient of variation (CV) = 1.7%; mean, 9.7 ng/ml, CV = 0.9%; mean, 16.9 ng/ml, CV = 1.0%], and a three-level interassay precision (mean, 3.60 ng/ml, CV = 3.5%; mean, 8.4 ng/ml, CV = 3.2%; mean, 15.2 ng/ml, CV = 2.9%). Using this test, the diagnosis of GH deficiency is established by a peak serum concentration of GH that is less than 10 ng/ml in children (39, 40) and less than or equal to 5 ng/ml in adults (39, 41).

Serum concentration of IGF-I was measured in the fasting state with a commercial two-site chemiluminescence immunoassay (Nichols Institute, San Juan Capistrano, CA) in our clinical chemistry lab. The sample was acidified to separate IGF-I from IGF binding proteins (IGFBP); excess IGF-II was added in the assay to block the IGFBP from recombining with IGF-I. This assay has a sensitivity of 6 ng/ml, a three-level intraassay precision (mean, 61.7 ng/ml, CV = 6.95%; mean, 269.6 ng/ml, CV = 3.4%; mean, 466.4 ng/ml, CV = 5.0%), and a three-level interassay precision (mean, 53.0 ng/ml, CV = 6.8%; mean, 220.4 ng/ml, CV = 6.3%; mean, 473.5 ng/ml, CV = 5.6%). Normal ranges for IGF-I for young children (age, 2 months to 5 yr) and adults (>18 yr) were based on those established by Quest Diagnostics/Nichols Institute. Normal values for children between 5 and 18 yr of age were normal ranges based on Tanner stages of pubertal development (as reported by Quest Diagnostics/Nichols Institute).

IGFBP-3 was measured by RIA (Nichols Institute); intact PTH was measured by a two-site immunoradiometric assay (Nichols Institute); free T4, TSH, and insulin were measured by enzyme immunoassay (Tosoh Medics); and glycosylated hemoglobin was measured by HPLC (Bio-Rad, Hercules, CA).

Subjects in whom GH deficiency was documented with the combined arginine/L-dopa stimulation test underwent a combined arginine/GHRH stimulation test at least 24 h later. Blood levels for GH were measured at -15, 0, 30, 60, 90, 120, and 150 min after an iv bolus of 1 µg/kg GHRH (maximum dose, 100 µg; Geref, Serono Laboratories, Randolph, MA) followed by an infusion of arginine (0.5 g/kg, 10% solution; maximum dose, 40 g) from 0–30 min. The cutoff used to establish the diagnosis of GH deficiency in children with the arginine/GHRH test, using a 95% confidence interval, was less than 20 ng/ml (range, 19.4–120.0 ng/ml; mean, 61.8 ± 2.8 ng/ml) (42, 43); for adults this cutoff was less than or equal to 9–10 ng/ml (41, 44).

Imaging studies

Bone age of the left hand and wrist (H/W) was determined according to the Gruelich and Pyle standards (45). Forearm or knee (A and K in Table 3Go) bone ages were also determined in subjects with GH deficiency because the H/W bone age is accelerated in many patients with PHP type 1a (46).


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  		TABLE 3. Anthropometric measurements and IGF-I/IGFBP-3 levels

 
A dual energy x-ray absorptiometry (DEXA) scan (QDR 4500, Hologic, Inc., Bedford, MA) was performed on each GH-deficient subject to measure adipose content (arms, trunk, legs, head, and total body). Fat content and lean body mass were calculated with proprietary Hologic QDR 4500 software.

All subjects who were diagnosed with GH deficiency were evaluated with a brain magnetic resonance image (MRI), both with and without contrast, to identify possible masses or structural abnormalities as the cause of GH deficiency.

Statistical analyses

Differences between GH-deficient and GH-sufficient subjects were analyzed by unpaired two-tailed Student t tests, with significance accepted as P < 0.05. Area under the curve (AUC) was calculated for each subject’s GH stimulation test (Sigmaplot 2001, for Windows, version 7.101; SPSS, Inc., Chicago, IL). For each test, the GH peak was then plotted vs. the AUC, and the Pearson correlation coefficient (r) was calculated using the CORR procedure in SAS version 8.2 (SAS Institute, Inc., Cary, NC). The Pearson correlation coefficient was also calculated for the PTH (before arginine/L-dopa stimulation testing) vs. the GH peak and GH AUC for each subject.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Subjects

We evaluated 13 treated subjects with PHP type 1a, who ranged in age from 3.33–51 yr at the time of the first GH stimulation test (Table 1AGo). We conducted a full laboratory evaluation at the time of GH testing. All subjects had features of AHO and evidence of PTH and TSH resistance at the time of original diagnosis. All three adult females had a normal age of menarche with subsequent development of secondary amenorrhea. Subjects 8 and 9 are siblings, and their mother has pseudoPHP. They had been previously diagnosed as GH-deficient at the approximate ages of 9.25 and 12.25 yr, respectively (24). Subject 8 had previously been treated with GH for 2.5 years (~ages 9.5–12 yr).

Serum biochemistry levels and creatinine clearance were normal in all subjects at the time of arginine/L-dopa testing, except in subject 7, who had a mildly depressed serum calcium concentration (8.0 mg/dl), and subjects 2 and 5, who had minimally elevated serum phosphorus concentrations (Table 1AGo). Serum calcium and phosphorus concentrations were normal in all subjects at the time of arginine/GHRH testing. No subjects were hypothyroid based on free T4 levels. Serum levels of intact PTH were elevated in all but two subjects (subjects 11 and 13). The markedly elevated PTH levels (>200 pg/ml) in subjects 1 and 7 were secondary to long-term noncompliance with medications. The need for compliance was stressed, and follow-up levels were greatly improved (Table 1AGo). Fasting lipid profiles revealed cholesterol, triglycerides, and/or LDL levels that were either elevated or at the upper end of the normal range in all subjects. Levels of glycosylated hemoglobin and fasting insulin were normal for all subjects except subject 7, who had acanthosis nigricans and a strong family history of type 2 diabetes mellitus.

GNAS1 mutation analysis

A heterozygous mutation that was predicted to inactivate G{alpha}s function was identified in 10 of 13 subjects (Table 1BGo and Fig. 1Go). Mutations resulted in premature termination signals (subjects 1, 2, 4–9, and 11) or replacement of an amino acid (subject 12) in the C-terminal region of G{alpha}s (E392K) that interacts with receptors (47). Subjects 2 and 5 were unrelated but had the same exon 1 mutation; subjects 4, 6, and 11 were unrelated but had the same exon 7, 4-bp deletion known to be a common deletion in AHO and progressive osseous heteroplasia (1, 2). Restriction endonuclease analysis of genomic DNA from the three subjects (subjects 3, 10, and 13) in whom no mutation was identified was normal, thus excluding a major gene rearrangement, deletion, or insertion. In addition, digestion of DNA from these subjects with PstI plus NgoMIV produced restriction fragment patterns that were consistent with normal maternal methylation of exon 1A (data not shown), thus excluding an imprinting defect (15, 48).


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  		TABLE 1B. GNAS1 mutation analysis

 


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  		FIG. 1. Structure of the GNAS1 gene and encoded protein, with subjects’ mutations and resulting protein changes. c.21insT in exon 1, insertion of T at codon (c.) 21 resulting in an immediate premature termination codon (PTC) at amino acid (aa) 8; c.85 C > T in exon 1, C to T transition resulting in a stop codon at amino acid 29; c.103 C > T in exon 1, C to T transition resulting in a stop codon at amino acid 35; c.565–568 delGACT in exon 7, 4-bp deletion (GACT) at codons 565–568 resulting in an alternative 13 amino acids and a PTC at amino acid 203; c.1083insC in exon 13, insertion of a C at codon 1083 causing a frameshift (fs) and 8 alternative amino acids ending in a PTC at amino acid 370; and c.1174 G > A, G to A transition resulting in a stop codon at amino acid 392.

 
GH assessment (Table 2GoGo, A and B)

Six of eight children (subjects 1–6) had subnormal GH responses to arginine/L-dopa stimulation testing that ranged from 0.6–8.0 ng/ml. The AUC (GH vs. time) showed a statistically significant correlation to the GH peaks for each test (r = 0.993; P < 0.001) (Table 2BGo), thereby indicating that the GH peak values were representative of relative GH secretion over the entire 150 min of analysis.


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  		TABLE 2A. GH stimulation tests

 

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  		TABLE 2B. GH stimulation tests: Pearson correlation coefficients and P values

 
Five of the six children (subjects 1, 2, 4, 5, and 6) with deficient GH responses to arginine/L-dopa also had subnormal GH responses to arginine/GHRH (42). Subject 3 had a borderline response, with a peak (21.8 ng/ml) minimally above the normal lower limit (20 ng/ml) (43) but less than the 2.5th percentile for this test (mean = 61.8 ± 2.8 ng/ml) (42). Using time points in common with published values for 81 normal historical controls (42), we found that the AUC values after arginine/GHRH administration were markedly reduced (6.7–41.5% of normal) in all six PHP type 1a children with GH deficiency (subjects 1–6), including subject 3 (41.5% of normal) who had a borderline peak with arginine/GHRH. One of the two children (subject 11) who had normal GH responsiveness to arginine/L-dopa was also tested with arginine/GHRH and had a normal response.

Two of five adults (subjects 7 and 8) had subnormal GH responses with arginine/L-dopa testing (39, 41). One additional adult (subject 9) had a peak GH value of 6.3 ng/ml (borderline range), with other GH values that were very low. The AUC for this test was less than his sibling’s (subject 8), who had a clearly subnormal GH peak. This subject had a borderline low peak GH value after arginine/GHRH testing (9 ng/ml), consistent with partial GH deficiency (39, 44). By contrast, one adult with pseudoPHP (mother of subject 2) had a normal GH response (data not shown). The AUC in our adult subjects showed a statistically significant correlation to the GH peaks for each test (r = 0.926; P < 0.024) (Table 2BGo), thereby indicating that the GH peak values were representative of relative GH secretion over the entire 150 min of analysis.

AUC data for arginine/GHRH testing for all subjects (children and adults) also revealed a statistically significant correlation to the GH peaks (r = 0.988; P < 0.0001) and were therefore representative of GH secretion over the entire 150 min of testing for all ages (Table 2BGo).

There was no correlation of serum intact PTH levels to GH peaks or AUC values from arginine/L-dopa testing (r = -0.233, P = 0.44; and r = -0.207, P = 0.50, respectively). Arginine/GHRH testing was performed only on those subjects found to be GH deficient with arginine/L-dopa testing; therefore, these results could not be compared with serum PTH levels.

All nine subjects with GH deficiency underwent MRI of the brain and pituitary, which did not reveal any structural abnormalities or masses that could result in GH deficiency.

Anthropometric measures (Table 3Go) and statistical analyses (Table 4Go)

The height and growth velocities were more than 1.0 SD below the mean in nine of 13 (69%) PHP type 1a subjects and more than 2.0 SD below the mean in four of these nine subjects. There was no difference between the height SDS of the GH-deficient and GH-sufficient children, whether or not subject 10, who also had features of acrodysostosis, was included (Table 4Go). Moreover, there was no significant difference in the growth velocity over the previous year between children with GH deficiency and children with normal GH status. Finally, calculated target heights for GH-deficient subjects were not significantly different from those with normal GH secretion based on midparental heights, although subjects 2, 8, and 9 had mothers with pseudoPHP that lowered their midparental height determinations.


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  		TABLE 4. Statistical analyses of anthropometric measurements

 
Predicted height based on the Bayley and Pinneau algorithm varied depending on the skeletal site used for the bone age test. The H/W bone ages were advanced by more than 2.0 SD beyond chronological age for most PHP type 1a subjects. Adult height SD scores predicted by this method were significantly less for GH-deficient children (-2.21 ± 0.27) than for the GH-sufficient child (-0.94) who was old enough for determination of bone age (P = 0.041). Most importantly, GH-deficient children had significantly lower height predictions based on the method of Bayley and Pinneau than by calculated midparental target heights, regardless of the site (forearm, knee, or wrist) used to determine bone age.

Analysis of previous growth curves of the three adults with GH deficiency indicated that they had normal stature as children (two were above the 50th percentile) until approximately age 12 yr, when they experienced premature growth cessation that led to the subsequent diagnosis of short stature (Fig. 2Go). The GH-deficient adults were all shorter than the GH-sufficient adults (Table 4Go), but this comparison did not reach statistical significance, possibly owing to the small sample size.



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  		FIG. 2. Growth curves of adult GH-deficient male subject (A) and adult GH-deficient female subjects (B) with PHP type 1a. B, Subject 8 (•) was treated with GH between the ages of approximately 9.5 and 12 yr. {Delta}, Bone age (there were no bone age results for subject 7).

 
Eight of nine subjects with PHP type 1a and deficient GH responses had weights that were +1.0 SD above the mean, whereas none of the PHP type 1a subjects with normal GH responses had weights greater than +1.0 SD (Tables 3Go and 4Go). Similarly, children and adults with GH deficiency had significantly greater BMI values compared with those with normal GH responses. BMI values of all subjects with GH deficiency were equal to or greater than the 95th percentile, and all subjects were therefore obese by definition (49). All of the PHP type 1a subjects with a normal GH response had BMI values less than the 95th percentile, and only one (subject 12) was considered overweight (BMI > 85 but < 95%) (Ref. 49). Total adipose content determined by DEXA compared with BMI was extremely significant (P = 5.78 x 10-3) indicating that the elevated BMI values were secondary to adipose and not muscle; truncal adiposity determined by DEXA compared with BMI was even more significant (P = 5.93 x 10-8).

Indices of GH action (Table 3Go)

All GH-deficient PHP type 1a subjects had serum levels of IGF-I that were below the normal range, whereas all subjects with normal GH responses had normal IGF-I levels. IGFBP-3 levels were normal in all subjects.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Secretion of GH requires binding of GHRH, a hypothalamic hormone, to specific heptahelical receptors on pituitary somatotrophs that are coupled via Gs to activation of adenylyl cyclase. On the basis of recent evidence that G{alpha}s transcripts are paternally imprinted in the pituitary (31), we reasoned that loss of G{alpha}s encoded by the maternal GNAS1 allele might impair the ability of GHRH to stimulate GH release. We investigated GH secretion in 13 subjects with PHP type 1a and found evidence of GH deficiency in nine subjects (69%). No structural defects were visualized by brain MRI that might account for GH deficiency or impaired responsiveness to GHRH. Although PHP type 1a is an uncommon disorder, the frequency of idiopathic GH deficiency in these subjects is far greater than the frequency of idiopathic GH deficiency in the United States (1:3,480) (Ref. 50) or in other countries (1:30,000 to 1:1,800) (Refs. 51, 52, 53, 54), thus implicating G{alpha}s deficiency rather than a second unrelated metabolic defect as the basis for GH deficiency in PHP type 1a.

GH deficiency in PHP type 1a is variable, and previously had been described only in isolated case reports (24, 25, 26, 27, 28, 29, 30). In our subjects, stimulated GH secretion was deficient in nine of 13 subjects, a frequency that is similar to the variable expression of other hormonal abnormalities associated with PHP type 1a (1, 2, 4). Moreover, similar to the partial resistance to TSH that occurs in subjects with PHP type 1a, all subjects with GH deficiency retained some degree of GH responsiveness to GHRH. This is consistent with relaxed paternal imprinting of G{alpha}s transcripts in the human pituitary (31) and thyroid (14). Although G{alpha}s expression appears to be biallelic in most tissues that have been studied (1, 16), specialized cells in some hormonally responsive tissues exhibit an unusual form of imprinting in which there is preferential expression of the maternal allele rather than complete suppression of allelic expression. The basis for the partial paternal imprinting of the paternal G{alpha}s allele is unknown but contrasts markedly with the tightly regulated, reciprocal imprinting of the alternative first exons that are located upstream of G{alpha}s exon 1 (11, 12, 15, 16, 17, 18, 19, 20). Expression of the paternal G{alpha}s transcription unit is variable and accounts for a maximum of 14% of the total G{alpha}s transcripts present in pituitary (31, 55), and an average of 30.9, 31.2, or 38.0% in the thyroid (14, 55), ovaries (55), and murine renal proximal tubular cells (10), respectively. In patients with PHP type 1a, who have defective maternal GNAS1 alleles, the partial expression of the paternal G{alpha}s transcripts in imprinted tissues permits production of some G{alpha}s and likely explains why hormone resistance in the pituitary somatotroph as well as the thyroid is incomplete.

We identified mutations in the GNAS1 allele in 10 of 13 of our subjects with PHP type 1a and in eight of nine subjects with GH deficiency (Table 1BGo). This is consistent with the frequency of identified mutations reported by others (56). Those subjects without an identified mutation did not have evidence of the methylation defect that is typical of PHP type 1b. Despite a wide range of mutations, we found no relationship between genotype and phenotype. Interestingly, GH deficiency was present in all subjects with exon 1 mutations despite the ability of the defective maternal allele to produce NESP55, consistent with reports that this protein is unlikely to play a signaling role (19, 57). Moreover, expression of XL{alpha}s from the normal paternal allele, which is highly expressed in the rodent pituitary (12) and is capable of functionally coupling to ß2-adrenergic, type 1 PTH, TSH, and corticotropin releasing factor receptors that act via G{alpha}s (13), was not able to fully compensate for loss of G{alpha}s production in the pituitary. However, we cannot distinguish between expression of XL{alpha}s and partial expression of paternal G{alpha}s transcripts as the basis for residual GH secretion in these subjects. Although we propose that pituitary deficiency of G{alpha}s is the basis for GH deficiency in patients with PHP type 1a, recent evidence has shown that adult patients with primary hyperparathyroidism have a reduction in both spontaneous and stimulated GH secretion (58), raising the possibility that the increase of serum levels of PTH in many of our subjects could contribute to GH deficiency. Over half (63.6%) of the patients with primary hyperparathyroidism were reported as GH deficient based on testing, but serum levels of IGF-I were variable. We found no correlation between the GH peak response and the PTH levels in our subjects, indicating that the GH response is independent of the PTH level.

Our diagnosis of GH deficiency was based on the GH response to provocative agents. Although obesity can blunt the GH response to a variety of pharmacological stimuli, all GH-deficient subjects also had a low serum concentration of IGF-I, consistent with a true deficiency in circulating GH levels, rather than obesity. The AUC for both the arginine/L-dopa and arginine/GHRH tests correlated significantly to the GH peaks, indicating that the GH peaks were representative of relative GH secretion over the entire testing period. Thus, our data document true biochemical deficiency of GH. On the other hand, there was no significant difference in height between PHP type 1a subjects with GH deficiency and subjects with normal GH secretory status. Thus, it is unlikely that GH deficiency is the primary cause of short stature in PHP type 1a. One pseudoPHP subject was studied and demonstrated a normal GH response (data not shown) consistent with normal responsiveness to other hormones in this variant. This normal response is consistent with the imprinting model of GH deficiency, rather than haploinsufficiency. However, some PHP type 1a subjects also had a normal GH response. Thus, a more comprehensive analysis of GH reserve in subjects with PHP type 1a and pseudoPHP patients will be necessary to distinguish between imprinting and haploinsufficiency as the basis for GHRH resistance.

It is widely known that patients with PHP type 1a are short as adults, with a reported incidence of short stature in 80% of adults with PHP type 1a (59). It is less well appreciated that PHP type 1a children are not short (60), with previous studies reporting only 6.7–57% of children as short (<2.0 SD) (27, 61). All GH-deficient adults in our study had normal to tall heights as children (Fig. 2Go). Although the difference in height between the GH-deficient and GH-sufficient adults in our study did not reach statistical significance, all of the GH-deficient adults had a lower height SDS than those who were GH sufficient. The H/W bone ages were significantly advanced beyond chronological age for all GH-deficient PHP type 1a subjects and was predictive of final height in our adult subjects. This pattern of normal childhood linear growth with abrupt premature cessation of growth during early puberty resulting in adult short stature is reminiscent of the premature fusion of epiphyses in tubular bones that accounts for brachydactyly.

One possible explanation of this premature epiphyseal fusion in PHP type 1a is ineffective Gs-mediated signal transduction in the growth plate. Previous evidence has shown that PTHrP and the PTH/PTHrP receptor play essential roles in skeletal development (62, 63, 64, 65, 66). Studies in mice have shown that Indian hedgehog (IHH) stimulates expression of PTHrP, which inhibits terminal chondrocyte differentiation (67). Humans with inactivating mutations of the genes encoding the PTH/PTHrP receptor (i.e. Blomstrand chondrodysplasia; Refs. 68 and 69) and transgenic mice with targeted ablation of the genes encoding PTHrP or the PTH/PTHrP receptor exhibit multiple skeletal defects and advanced endochondral bone formation (62, 66, 67, 70). Therefore, ineffective PTHrP signal transduction in subjects with G{alpha}s deficiency could lead to accelerated chondrocyte differentiation and premature fusion of the growth plate. In fact, a novel mutation in human IHH has been recently reported as the cause of brachydactyly type A1 (71). The more moderate skeletal phenotype in subjects with PHP type 1a is likely due to haploinsufficiency rather than partial imprinting of the paternal GNAS1 allele, because similar bone defects occur in patients with pseudoPHP who inherit a defective paternal GNAS1 allele (1, 2).

Although GH deficiency did not correlate with short stature in our limited cohort, all subjects with GH deficiency in our study were obese, defined as a BMI value of at least 95% (49), whereas all GH-sufficient subjects were not obese. Obesity can blunt the GH response to stimulation testing (72). However, serum levels of IGF-I and IGFBP-3 are normal or elevated in obesity (72). Thus, the low IGF-I levels in the nine obese subjects who had abnormal GH responses provide compelling evidence that GH deficiency and not obesity was the cause of the abnormal GH stimulation tests.

The etiology of the obesity in PHP type 1a is as yet undefined. Initial studies of Gnas +/- knockout mice, in which exon 2 was disrupted by insertion of the neomycin cassette with resulting inactivation of both G{alpha}s and XL{alpha}s, had proposed tissue-specific imprinting of G{alpha}s in adipose tissue as the basis for obesity (10). However, later studies demonstrated that hormone-responsive adenylyl cyclase was normal in fat cells from these animals (73). Thus, it is unlikely that G{alpha}s deficiency in fat cells accounts for obesity. On the other hand, GH deficiency is associated with increased accumulation of body fat, and it is possible that GH deficiency is a cause of obesity in PHP type 1a. This is similar to the GH deficiency documented in subjects with Prader-Willi syndrome, in whom short stature and obesity are in part due to GH deficiency (74, 75) and consequent low serum levels of IGF-I (76). Indeed, patients with Prader-Willi syndrome show significant weight loss and growth acceleration in response to exogenous GH treatment, with marked decreases in their BMI (77). These results suggest that PHP type 1a patients with GH deficiency might also benefit from GH treatment.

Additional studies will be required to establish an unequivocal basis for GH deficiency in PHP type 1a. The availability of knockout mice in which one Gnas allele has been disrupted now makes it possible to investigate the biochemical basis and physiological consequences of GH deficiency. The high frequency of GH deficiency in PHP type 1a provides justification to include GH testing as part of the routine management of these patients. Finally, taken in the context of similar studies in children with Prader-Willi syndrome, our results provide a rationale for evaluating these metabolic and growth responses of GH-deficient PHP type 1a patients to treatment with exogenous GH.


   Acknowledgments
 
We thank Kathryn A. Carson, Sc.M., Biostatistician (Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland), for her help with statistical analyses.


   Footnotes
 
This work was supported by grants from the Genentech Center for Clinical Research and Education (to E.L.G.-L.), United States Public Health Service Grant R01 DK56178 (to M.A.L.), Supplement PA-99106 to RO1 DK56178 (to E.L.G.-L.), Human Growth Foundation (to E.L.G.-L.), and National Institutes of Health/National Center for Research Resources Grant M01 RR00052 (to the Johns Hopkins University School of Medicine General Clinical Research Center).

Abbreviations: AHO, Albright hereditary osteodystrophy; AUC, area under the curve; BMI, body mass index; CV, coefficient of variation; DEXA, dual energy x-ray absorptiometry; H/W, hand and wrist; IFGBP, IGF binding protein; MRI, magnetic resonance image; PHP, pseudohypoparathyroidism; pseudoPHP, pseudopseudohypoparathyroidism; r, Pearson correlation coefficient; SDS, SD score.

Received January 6, 2003.

Accepted April 14, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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