help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fischer, J. A.
Right arrow Articles by Born, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fischer, J. A.
Right arrow Articles by Born, W.
The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 3 935-938
Copyright © 1998 by The Endocrine Society

Original Studies

An Inherited Mutation Associated with Functional Deficiency of the {alpha}-Subunit of the Guanine Nucleotide-Binding Protein Gs in Pseudo- and Pseudopseudohypoparathyroidism1

J. A. Fischer, F. Egert, E. Werder and W. Born

Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery, Medicine, and Pediatrics, University of Zurich, Zurich, Switzerland

Address all correspondence and requests for reprints to: Prof. J. A. Fischer, Klinik Balgrist, Forchstrasse 340, 8008 Zurich, Switzerland.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Pseudohypoparathyroidism type Ia (PSP) is a disorder characterized by Albright’s osteodystrophy, secondary hyperparathyroidism, lowered Gs activity, and resistance of the urinary cAMP excretion to exogenous PTH. The patients had raised basal serum levels of TSH and/or excessive TSH response to TRH. Here we have described a 38-bp deletion at the exon 1/intron 1 boundary of one Gs{alpha} allele in two mothers with pseudo-PSP and in six offsprings with PSP of a kindred with Albright’s osteodystrophy. The deletion eliminates the splice donor site of exon 1. The pseudo-PSP patients presented decreased Gs activity, but normal urinary cAMP responses to PTH and normal TSH levels and responses to TRH. As monitored during 22 yr, they had normal serum levels of calcium and PTH. The findings demonstrate the same inherited functional defect of Gs{alpha} in two female patients with pseudo-PSP and in six of their offspring with PSP. The pathogenesis of clinical hypoparathyroidism remains to be clarified.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PSEUDOHYPOPARATHYROIDISM Ia (PSP) is a disorder with clinical hypoparathyroidism and the phenotype of Albright’s osteodystrophy, but raised plasma levels of bioactive and immunoreactive PTH (1; see Ref. 2 for additional references). Patients with PSP frequently present raised serum levels of TSH and excessive TSH response to TRH with sometimes overt clinical hypothyroidism (3 and references cited therein). The diagnosis of PSP-Ia was established on the basis of resistance of the urinary cAMP excretion to exogenous PTH, and decreased guanine nucleotide-binding protein Gs activity (4, 5, 6, 7, 8, 9, 10). Gs activity was decreased in the PSP patients reported here, but it was similarly decreased in their mothers with pseudo-PSP (8). The latter responded normally to exogenous PTH with raised urinary cAMP excretion; secondary hyperparathyroidism and excessive TSH response to TRH were not observed. A functional deficiency of Gs has been reported in PSP and pseudo-PSP (8, 9, 10).

A genetic deficiency of Gs{alpha} has been revealed in familial PSP and pseudo-PSP (11–15; for additional mutations, see Ref.16). As shown here, the patients with PSP-Ia as well as those with pseudo-PSP carry a novel deletion in one Gs{alpha} allele resulting in reduced Gs{alpha} activity. In healthy siblings with no signs of Albright’s osteodystrophy, both Gs{alpha} alleles were normal. It would seem, therefore, that the described genetic defect, resulting in reduced Gs{alpha} activity, is not an obvious cause of the resistance to exogenous PTH in the kindred reported here.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Figure 1Go shows the pedigree of a family with PSP, pseudo-PSP, and Albright’s osteodystrophy. Laboratory data, including serum calcium, serum PTH, urinary cAMP responses to parathyroid extract (Eli Lilly Co., Indianapolis, IN), serum TSH, TSH response to TRH, and Gs protein activity, for patients 7, 9, 14–17, 20, and 22 have been reported previously (3, 5, 8) (Table 1Go).



View larger version (67K):
[in this window]
[in a new window]
  		Figure 1. Pedigree of a family with ({blacksquare} and •) and without ({square} and {circ}) Albright’s osteodystrophy. {square} and {circ}, Not tested. Diagonal lines denote deceased subjects (top). Results of agarose gel electrophoresis analysis of DNA products obtained by PCR amplification of DNA isolated from blood samples of selected family members (bottom). The numbers below individual lanes of the agarose gel correspond to the numbers of individual family members, as indicated in the pedigree. M, DNA size markers ({Phi}X/HaeIII).

 

View this table:
[in this window]
[in a new window]
  		Table 1. Summary of laboratory data

 
Briefly, serum levels of calcium were measured by ethylene glycol-bis (ß-aminoethyl ether)-N,N'-tetraacetate titration, using calcein as an indicator, and by flame spectrophotometry (6, 9). Immunoreactive serum PTH was estimated with antibodies to bovine PTH recognizing predominantly intact human PTH-(1–84) in the serum of patients with PSP on gel permeation chromatography. [131I]Bovine PTH-(1–84) was used as radioligand, and human PTH-(1–84) as standard (8, 17). The urinary cAMP response to parathyroid extract was evaluated according to a slightly modified protocol of Chase et al. (4, 5). Immunoreactive TSH and the TSH response to TRH (Hoffmann-La Roche, Basel, Switzerland) were estimated as previously reported (3). Gs protein activity was assessed in erythrocyte ghosts as previously described (8).

Genomic DNA isolated from peripheral leukocytes of individual subjects was analyzed for Gs{alpha} gene insertions or deletions by PCR amplification of gene subdomains. The primer pairs indicated in Fig. 2Go were used to localize and analyze in detail the deletion described here in exon 1 and flanking regions of the Gs{alpha} gene. PCR products were separated by electrophoresis in 2% agarose (Seakem HGT, Flowgen Instruments, Sittingbourne, UK) and visualized by ethidium bromide staining. Nucleotide sequence analysis of PCR-amplified Gs{alpha} gene fragments was performed by cycle sequencing. The primers used were the same as those used for amplification of gene fragments.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 2. Schematic illustration of the Gs{alpha} gene region analyzed by PCR with indicated forward (DV-157 and MET1F) and reverse (MAL-2 and MET1R) primers. The expected sizes (base pairs) of products amplified from a normal allele (top) and the location and sequence of the identified 38 nucleotide deletion (underlined; bottom) are shown.

 

   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Figure 1Go shows the pedigree of the affected family with Albright’s osteodystrophy. At first presentation, patients 14–17, 20, and 22 suffered from PSP, with hypocalcemia in four and normocalcemia in two siblings (Table 1Go). Serum PTH levels were raised, and the patients were classified as PSP-I on the basis of absent or low urinary cAMP responses to exogenous PTH. Five of the six siblings presented raised levels of TSH, and all had excessive TSH response to TRH. Gs activity was decreased in all of them, but Gs was also decreased in patients 7 and 9 with pseudo-PSP exhibiting normal urinary cAMP excretion in response to the administration of parathyroid extract and normal serum levels of TSH and TSH response to TRH at first presentation. They had normal serum levels of calcium and PTH as observed over 22 yr.

Analysis by PCR amplification of exon 1 and flanking regions of the Gs{alpha} gene with primers DV-157 and MET1R (Fig. 2Go) revealed a normal-sized 535-bp product in all family members investigated (Fig. 1Go). In the 6 patients with PSP and the 2 patients with pseudo-PSP, an additional smaller PCR product, indicating a deletion in 1 Gs{alpha} allele, was also observed. Subsequent separate PCR and nucleotide sequence analysis of the 5'- and 3'-ends of exon 1 and corresponding flanking regions with primer pairs DV-157/MAL-2 and MET1F/MET1R, respectively, revealed a 38-bp deletion comprising 21 nucleotides of the 3'-end of exon 1 and 17 nucleotides of intron 1 in the mutated allele. This eliminates the donor splice site of exon 1, giving rise to a transcript that includes intron 1. As a result, termination of translation is predicted to occur within intron 1 leading to the incorporation of at least 116 alternative amino acids into a protein product of the mutated Gs{alpha} gene.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Albright et al. (1) made the discovery that PSP is not caused by a lack of PTH, but by an inability to respond to it. This has led to the concept of end-organ resistance to exogenous PTH caused by a defect of the PTH receptor and/or its signaling pathways. Mutations of the PTH/PTHrP receptor have not been detected to date in PSP type Ib (18, 19, 20) and have not been reported in PSP type Ia. The findings that urinary cAMP excretion in response to exogenous PTH is reduced or absent and of an excessive TSH response to TRH are consistent with inadequate activation of the PTH- and TRH-responsive adenylyl cyclase and different isotypes thereof, and activation of phosphodiesterase (4, 16). To this end, Gs activity was shown to be reduced (7, 8, 9, 10, 11). Subsequently, several mutations of the Gs{alpha}-encoding gene have been discovered (12, 13, 14, 15, 16). The mutations are equally present in patients with PSP and pseudo-PSP.

In the present report, a deletion in one Gs{alpha} allele has been identified in a large kindred with PSP, pseudo-PSP, and Albright’s osteodystrophy, but not in unaffected siblings. Formation of an inactive protein with an altered carboxyl-terminus is predicted. Maintenance of approximately 50% Gs activity in PSP and pseudo-PSP is provided by the normal allele. The findings resemble those obtained in mouse embryonic stem cells, in which one Gs{alpha} allele was disrupted by homologous recombination (21). The fact that two mothers with pseudo-PSP presented equally deficient Gs activity compared to their six offsprings with PSP suggests that Gs deficiency may be necessary, but not sufficient, for the development of clinically overt hypoparathyroidism and hypothyroidism. Serum levels of PTH and calcium have been normal in the two mothers between their ages of 36–58 yr and 34–56 yr, respectively. It seems improbable that secondary hyperparathyroidism would develop at a later age.

Circulating bioactive and immunoreactive PTH levels are normal or raised in PSP, but not in pseudo-PSP (2). Plasma from patients with PSP revealed higher PTH inhibitory activity, assessed in a renal cytochemical bioassay, than that in their mothers with pseudo-PSP presented here (22). Intact PTH was separated from a putative inhibitor on gel permeation chromatography of plasma samples. A postulated PTH antagonist whose structure remains to be elucidated may be responsible for the renal resistance to PTH observed in PSP. Yet, some patients with PSP and secondary hyperparathyroidism have osteitis fibrosa (23).

In conclusion, the hypothesis of target organ resistance to PTH being caused by an inactivating mutation of one Gs{alpha} allele is questionable in the kindred reported here.


   Acknowledgments
 
We thank M. Oude-Luttikhuis and R. Trembath for technical help with the DNA analysis.


   Footnotes
 
1 This work was supported by the Swiss National Science Foundation (Grant 31-43094.95) and the Kanton of Zurich. Back

Received October 6, 1997.

Revised December 1, 1997.

Accepted December 5, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Albright F, Burnett CH, Smith PH, Parson W. 1942 Pseudohypopara-thyroidism–an example of "Seabright-Bantam syndrome." Endocrinology. 30:922–932.
  2. Nagant de Deuxchaisnes C, Fischer JA, Dambacher MA, et al. 1981 Dissociation of parathyroid hormone bioactivity and immunoreactivity in pseudohypoparathyroidism type I. J Clin Endocrinol Metab. 53:1105–1109.[Abstract]
  3. Werder E, Illig R, Bernasconi S, et al. 1975 Excessive thyrotropin response to thyrotropin-releasing hormone in pseudohypoparathyroidism. Pediatr Res. 9:12–16.
  4. Chase LR, Melson GL, Aurbach GD. 1969 Pseudohypoparyathyroidism: defective excretion of 3',5'-AMP in response to parathyroid hormone. J Clin Invest. 48:1832–1844.
  5. Werder EA, Fischer JA, Illig R, et al. 1978 Pseudohypoparathyroidism and idiopathic hypoparathyroidism: relationship between serum calcium and parathyroid hormone levels and urinary cyclic adenosine-3',5'-monophosphate response to parathyroid extract. J Clin Endocrinol Metab. 46:872–879.[Abstract]
  6. Farfel Z, Brickman AS, Kaslow HR, Brothers VM, Bourne HR. 1980 Defect of receptor-cyclase coupling protein in pseudohypoparathyroidism. N Engl J Med. 393:237–242.
  7. Levine MA, Downs Jr RW, Singer M, Marx SJ, Aurbach GD, Spiegel AM. 1980 Deficient activity of guanine nucleotide regulatory protein in erythrocytes from patients with pseudohypoparathyroidism. Biochem Biophys Res Commun. 94:1319–1324.[CrossRef][Medline]
  8. Fischer FA, Bourne HR, Dambacher MA, et al. 1983 Pseudohypoparathyroidism: inheritance and expression of deficient receptor-cyclase coupling protein activity. Clin Endocrinol (Oxf). 19:747–754.[Medline]
  9. Levine MA, Jap T-S, Mauseth RS, Downs RW, Spiegel AM. 1986 Activity of the stimulatory guanine nucleotide-binding protein is reduced in erythrocytes from patients with pseudohypoparathyroidism and pseudopseudohypoparathyroidism: biochemical, endocrine, and genetic analysis of Albright’s hereditary osteodystrophy in six kindreds. J Clin Endocrinol Metab. 62:497–502.[Abstract]
  10. Saito T, Akita Y, Fujita H, et al. 1986 Stimulatory guanine nucleotide binding protein acitivity in the erythrocyte membrane of patients with pseudohypoparathyroidism type I and related disorders. Acta Endocrinol (Copenh). 111:507–515.[Medline]
  11. Levine MA, Ahn TG, Klupt SF, et al. 1988 Genetic deficiency of the {alpha} subunit of the guanine nucleotide-binding protein Gs as the molecular basis for Albright hereditary osteodystrophy. Proc Natl Acad Sci USA. 85:617–621.[Abstract/Free Full Text]
  12. Patten JL, Johns DR, Valle D, et al. 1990 Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright’s hereditary osteodystrophy. N Engl J Med. 322:1412–1419.[Abstract]
  13. Weinstein LS, Gejman PV, Friedman E, et al. 1990 Mutations of the Gs {alpha}-subunit gene in Albright heredidatry osteodystrophy detected by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA. 87:8287–8290.[Abstract/Free Full Text]
  14. Farfel Z, Iiri T, Shapira H, Roitman A, Mouallem M, Bourne HR. 1996 Pseudohypoparathyroidism, a novel mutation in the ß{gamma}-contact region of Gs{alpha} impairs receptor stimulation. J Biol Chem. 271:19653–19655.[Abstract/Free Full Text]
  15. Iiri T, Farfel Z, Bourne HR. 1997 Conditional activation defect of a human Gs{alpha} mutant. Proc Natl Acad Sci USA. 94:5656–5661.[Abstract/Free Full Text]
  16. Levine MA. 1996 Pseudohypoparathyroidism. In: Bilezikian JP, Raisz LG, Rodan GA, eds. Principles of bone biology. San Diego: Academic Press; 853–876.
  17. Loveridge N, Tschopp F, Born W, Devogelaer J-P, Nagant de Deuxchaisnes C, Fischer JA. 1986 Separation of inhibitory activity from biologically active parathyroid hormone in patients with pseudohypoparathyroidism type I. Biochim Biophys Acta. 889:117–122.[Medline]
  18. Suarez F, Lebrun JJ, Lecossier D, Escoubet B, Coureau C, Silve C. 1995 Expression and modulation of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in skin fibroblasts from patients with type Ib pseudohypoparathyroidism. J Clin Endocrinol Metab. 80:965–970.[Abstract]
  19. Schipani E, Weinstein LS, Bergwitz C, et al. 1995 Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene. J Clin Endocrinol Metab. 80:1611–1621.[Abstract/Free Full Text]
  20. Fukumoto S, Suzawa M, Takeuchi Y, et al. 1996 Absence of mutations in parathyroid hormone (PTH)/PTH-related protein receptor complementary deoxyribonucleic acid in patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab. 81:2554–2558.[Abstract]
  21. Schwindinger WF, Reese KJ, Lawler AM, Gearhart JD, Levine MA. 1997 Targeted disruption of G-nas in embryonic stem cells. Endocrinology. 138:4058–4063.[Abstract/Free Full Text]
  22. Loveridge N, Fischer JA, Nagant de Deuxchaisnes C, et al. 1982 Inhibition of cytochemical bioactivity of parathyroid hormone by plasma in pseudohypoparathyroidism type I. J Clin Endocrinol Metab. 54:1274–1275.[Abstract]
  23. Kolb FO, Steinbach HL. 1962 Pseudohypoparathyroidism with secondary hyperparathyroidism and osteitis fibrosa. J Clin Endocrinol Metab. 22:59–64.



This article has been cited by other articles:


Home page
 Eur J EndocrinolHome page
A.-S. Balavoine, M. Ladsous, F.-L. Velayoudom, V. Vlaeminck, C. Cardot-Bauters, M. d'Herbomez, and J.-L. Wemeau
Hypothyroidism in patients with pseudohypoparathyroidism type Ia: clinical evidence of resistance to TSH and TRH
Eur. J. Endocrinol., October 1, 2008; 159(4): 431 - 437.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
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]

Home page
 J. Clin. Endocrinol. Metab.Home page
L. de Sanctis, S. Vai, M. R. Andreo, D. Romagnolo, L. Silvestro, and C. d. Sanctis
Brachydactyly in 14 Genetically Characterized Pseudohypoparathyroidism Type Ia Patients
J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1650 - 1655.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
J. Liu, B. Erlichman, and L. S. Weinstein
The Stimulatory G Protein {alpha}-Subunit Gs{alpha} Is Imprinted in Human Thyroid Glands: Implications for Thyroid Function in Pseudohypoparathyroidism Types 1A and 1B
J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4336 - 4341.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
K. Freson, J. Jaeken, M. Van Helvoirt, F. de Zegher, C. Wittevrongel, C. Thys, M. F. Hoylaerts, J. Vermylen, and C. Van Geet
Functional polymorphisms in the paternally expressed XL{alpha}s and its cofactor ALEX decrease their mutual interaction and enhance receptor-mediated cAMP formation
Hum. Mol. Genet., May 15, 2003; 12(10): 1121 - 1130.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
K. Freson, C. Thys, C. Wittevrongel, W. Proesmans, M. F. Hoylaerts, J. Vermylen, and C. Van Geet
Pseudohypoparathyroidism type Ib with disturbed imprinting in the GNAS1 cluster and Gs{alpha} deficiency in platelets
Hum. Mol. Genet., October 15, 2002; 11(22): 2741 - 2750.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
L. S. Weinstein, S. Yu, D. R. Warner, and J. Liu
Endocrine Manifestations of Stimulatory G Protein {alpha}-Subunit Mutations and the Role of Genomic Imprinting
Endocr. Rev., October 1, 2001; 22(5): 675 - 705.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
W. Ahrens, O. Hiort, P. Staedt, T. Kirschner, C. Marschke, and K. Kruse
Analysis of the GNAS1 Gene in Albright's Hereditary Osteodystrophy
J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4630 - 4634.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fischer, J. A.
Right arrow Articles by Born, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fischer, J. A.
Right arrow Articles by Born, W.

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