Mutations Of Gs Alpha Gene Research Articles

A new heterozygous mutation (D196N) in the Gs alpha gene as a cause for pseudohypoparathyroidism type IA in a boy who had gallstones

Pseudohypoparathyroidism (PHP) is characterized by hypocalcemia and hyperphosphatemia in association with an increased secretion of parathyroid hormone (PTH) due to decreased target tissue responsiveness to PTH. Patients with PHP type Ia are not only resistant to PTH, but also to other hormones that bind to receptors coupled to stimulatory G protein (Gsalpha). PHP Ia and Albright hereditary osteodystrophy (AHO) are caused by a reduced activity of the Gsalpha protein. Heterozygous inactivating Gs alpha (GNAS) gene mutations have been identified in these patients. We studied a boy with PHP Ia. During follow-up the patient developed elevated liver enzyme serum levels and abdominal discomfort. Gsalpha activity was measured in erythrocyte membranes from the patient and the GNAS coding region of Gsalpha sequenced. Gsalpha activity was reduced (62%) and molecular analysis revealed a new heterozygous GNAS gene mutation (D196N). Gallstones were diagnosed and cholecystectomy was performed. Biochemical analysis revealed cholesterol stones, a condition that was not reported before in PHP Ia. Cholesterol gallstones may rarely be associated with PHP Ia and should be taken into account.

Morphological, immunohistochemical and autoradiographic studies of thyroid autonomy.

The values and limits of morphological, immunohistochemical and autoradiographic methods in studies of thyroid autonomy are briefly discussed. For meaningful studies of molecular aspects of thyroid autonomy--such as for example TSH receptor and Gs-alpha gene mutations--it is absolutely crucial that the tissue analysed is well characterized and really is autonomous. This is particularly important in view of the well known heterogeneity of human goiter tissue in respect to many if not all functional and proliferative parameters. To prove functional and proliferative autonomy of thyroid tissue, autoradiography is a very helpful tool, while simple morphology and immunohistochemistry do not contribute substantially to this aim.

Pathogenesis of toxic thyroid adenomas and nodules: relevance of activating mutations in the TSH-receptor and Gs-alpha gene, the possible role of iodine deficiency and secondary and TSH-independent molecular mechanisms.

In iodine deficiency areas, activating mutations in the TSH receptor and Gs-alpha gene are found in the majority of toxic thyroid adenomas and in some toxic goiter nodules. Since TSH receptor and Gs-alpha gene mutations are very rare in areas with high iodine supply, iodine deficiency has been suspected to favor the occurrence of these mutations by yet unknown pathways. However, TSH receptor and Gs-alpha gene mutations alone are not sufficient to cause toxic adenomas and nodules. There is compelling evidence that other secondary and cAMP-independent mechanisms, including enhanced expression of various growth factors, their corresponding receptors and of signaling proteins, may affect the mutated cell and thus promote cell proliferation and in turn generation of the tumor.

TSH receptor and Gs(alpha) genetic analysis in children with Down's syndrome and subclinical hypothyroidism.

The prevalence of thyroid diseases in children with Down's syndrome (DS) is about 3%. The most frequently observed condition is autoimmune subclinical hypothyroidism (SH). Autoimmune SH must be distinguished from defects in the biological activity of the TSH molecule or from the rare inherited condition of thyroid resistance to TSH. To investigate this last aspect we studied 12 patients with DS that had moderately elevated TSH with normal free thyroid hormones without signs of autoimmunity. For the genetic analysis the genomic DNA was extracted from peripheral lymphocytes. All the exons of the TSH receptor (TSHr) and Gs(alpha) genes were sequenced. The genetic analysis of the TSHr gene revealed the presence of four polymorphic variants. In two patients there was an allelic variant in the exon 1 (Pro52Thr--in one patient in the heterozygous state and in the other as a homozygous substitution). In one patient there was an allelic variant in the exon 1 (Asp36His) in the heterozygous state. In 11 patients there was a silent polymorphism in the exon 7 at nucleotide 561. All patients were homozygous for a silent polymorphism in the exon 9 at nucleotide 855. No inactivating mutations of TSHr or Gs(alpha) genes were identified in the 12 patients. In conclusion, our results seem to exclude the role of TSHr or Gs(alpha) gene mutations in the pathogenesis of the non-autoimmune SH observed in some children with DS.

Absence of activating mutations in the hot spots of the LH receptor and Gs-alpha genes in Leydig cell tumors.

Leydig-cell tumors are functioning endocrine tumors that produce T autonomously leading to isosexual precocity in boys and virilization in female patients. Molecular abnormalities such as activating mutations of the luteinizing hormone receptor (LHR), a G protein-coupled receptor, and of the Gs-alpha subunit of G protein have recently been described in these tumors. Both mutations cause continuous activation of the cAMP signaling cascade, autonomous production of T and cell proliferation. We searched for activating mutations in exon 11 of the LHR gene and in exons 8 and 9 of the Gs-a gene, which contain all hot spots for those mutations, in 4 Leydig cell tumors obtained from 4 patients (one boy with LH-independent precocious puberty and 3 women with virilization). DNA was extracted from paraffin-embedded neoplastic and non-neoplastic tissues and from peripheral lymphocytes. Hot spot regions of exons 11 of LHR and exons 8 and 9 of Gs-alpha genes were amplified by PCR and the purified PCR products were directly sequenced. No LHR or Gs-alpha gene mutations were found in the 4 tumors studied. Considering the previously reported mutations found in Leydig cell tumors, the absence of activating mutations in the hot spot regions for activating mutations in these tumors indicate molecular heterogeneity among Leydig cell tumors.

Gsalpha gene mutations in monostotic fibrous dysplasia of bone and fibrous dysplasia-like low-grade central osteosarcoma.

A series of nine cases with monostotic fibrous dysplasia (FD) and five cases of fibrous dysplasia-like low-grade central osteosarcoma (fd-like lgcOSA) were applied in a mutational analysis. Restriction digestion analysis, single-stranded conformational polymorphism (SSCP) analysis, and repeated sequencing demonstrated a R201H mutation in six cases and a R201C mutation in three cases of patients with monostotic FD. These results demonstrate that the presence of Gsalpha gene mutations is a constant finding in monostotic FD. To our knowledge, this is the first report in the literature investigating Gsalpha gene mutations in lgcOSA, which is one of the most important differential diagnoses of FD because of its low-grade malignant behavior. In four of five cases of fd-like lgcOSA, no mutation has been detected. In one case of this tumor, a R201C mutation could be demonstrated. Because our results demonstrate a low prevalence of Gsalpha gene mutations in this tumor in contrast to monostotic FD, mutational analysis may be an additional helpful parameter in individual cases for the differential diagnosis of FD and fd-like lgcOSA.

Expression of cyclic adenosine 3',5'-monophosphate (cAMP)-responsive element binding protein and inducible-cAMP early repressor genes in growth hormone-secreting pituitary adenomas with or without mutations of the Gsalpha gene.

In about 30-40% of GH-secreting adenomas, gain-of-function mutations of the Gsalpha gene, which convert this gene into an oncogene termed gsp, occur. Gsalpha mutations have been related to pituitary tumorigenesis. We focused on 2 nuclear transcription factors that are final targets of the cAMP-dependent pathway and are positively regulated by cAMP signaling, i.e. the cAMP-responsive element binding protein (CREB) and the inducible cAMP early repressor (ICER), that derives from alternative splicing of cAMP-responsive element modulator gene. We examined 21 GH-secreting adenomas, 8 with (gsp(+)) and 13 without (gsp(-)) a mutated Gsalpha. Analysis of CREB and ICER I/II messenger RNA revealed that the levels of both transcripts were higher in gsp(+) than in gsp(-) tumors (CREB/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mean optical density +/- SE, 2.34 +/- 0.36 in gsp(+) vs. 0.99 +/- 0.22 in gsp(-), P = 0.003; ICER I/GAPDH, 0.53 +/- 0.15 in gsp(+) vs. 0.14 +/- 0.07 in gsp(-), P = 0.01; ICER II/GAPDH, 1.5 +/- 0.21 in gsp(+) vs. 0.83 +/- 0.13 in gsp(-), P = 0.01), although a few cases in both groups did not display this pattern of expression. Moreover, no positive correlation between the levels of CREB and ICER transcripts was observed, suggesting the possible presence of alterations in the mechanisms by which cAMP signaling directs the expression of CREB and/or ICER genes. Our results indicate a complex pattern of expression of nuclear transcription factors that mediate cAMP action in both gsp(+) and gsp(-) tumors, suggesting that, beside Gsalpha gene mutations, different and partially unknown molecular events may contribute to the pathogenesis of these tumors.

Somatostatin receptor subtype 2 and 5 in human GH-secreting pituitary adenomas: analysis of gene sequence and mRNA expression.

The role of somatostatin receptor subtypes 2 and 5 (SSTR2 and SSTR5) in determining the secretory and proliferative phenotype as well as the sensitivity to somatostatin analogue treatment is not clearly established. We quantified the expression of SSTR2 and SSTR5 mRNA using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 19 human growth hormone (GH) -secreting adenomas. Tumour characteristics and in vivo sensitivity to somatostatin analogues were assessed; tumours were screened for Gsalpha gene mutations. PCR products of SSTR2 and SSTR5 DNA from tumours resistant to somatostatin analogues were directly sequenced. All tumours expressed both SSTR2 and SSTR5 mRNA at variable levels. No significant correlation between SSTR2 and SSTR5 expression and the presence of Gsalpha mutation, GH levels, or tumour size and invasiveness was observed. A negative correlation between SSTR2 and SSTR5 mRNA levels was observed (r = 0.5; P < 0.05). No significant correlation between the levels of SSTR2 and SSTR5 expression and the in vivo responsiveness to somatostatin analogues was observed, although a tendency to a low SSTR2 expression in resistant tumours was found. No mutations in the coding or bordering regions of either SSTR2 or SSTR5 adenomatous DNA from patients totally or partially resistant to somatostatin analogues were found. The study shows that the different expression of SSTR2 and SSTR5 in GH-secreting adenomas is not significantly correlated with the secretory and proliferative phenotype, although the large, hypersecretory tumours and those with a poor sensitivity to somatostatin analogues seem to express low levels of SSTR2 mRNA. Moreover, both SSTR2 and SSTR5 DNA from tumours resistant to somatostatin analogues were found to possess intact coding sequences.

Constitutively active Gs alpha is associated with an increased phosphodiesterase activity in human growth hormone-secreting adenomas.

Because phosphodiesterase (PDE) expression and activity are controlled by cAMP, we investigated whether activating mutations of Gs alpha gene that occur in human GH-secreting adenomas are associated with increased PDE activity. We studied 10 adenomas with wild-type Gs alpha (gsp-) and 8 with mutant Gs alpha (gsp+). Although, in the absence of PDE inhibitors, intracellular cAMP levels were similar in gsp+ e gsp- adenomas, the PDE blockade with 3-isobutyl-1-methylxanthine induced a marked increase in cAMP in all but one gsp+ adenoma (% increase: from 77 to 2900) and a slight rise in only 2 gsp-. Similar results were obtained with the PDE4 selective inhibitor 4-[3-(cyclopentyloxy)-4-methoxyphenyl)]-2-pyrrolidinone. In vitro GH release was significantly higher in gsp+ than in gsp- adenomas (315 +/- 158 vs. 82 +/- 53 micrograms/well; P < 0.01), and PDE blockade caused a further increase in 3 of 5 gsp+ adenomas but not in gsp- tumors. By direct measurement, PDE activity was about 7-fold higher in gsp+ than in gsp- adenomas (320 +/- 213 vs. 48 +/- 23 pmol/min.mg protein; P < 0.05) and was largely 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone sensitive. This study first demonstrates that activating mutations of the Gs alpha gene that naturally occur in pituitary adenomas is associated with an increased PDE activity that might, at least partially, counteract the constitutive activation of the cAMP-dependent pathway.

TSH receptor and Gs-alpha gene mutations in the pathogenesis of toxic thyroid adenomas--a note of caution

TSH receptor and Gs-alpha gene mutations in the pathogenesis of toxic thyroid adenomas--a note of caution

Distribution of Gs-alpha activating mutations in human thyroid tumors measured by subcloning.

In the search for the prevalence and distribution pattern of Gs-alpha gene mutations in differentiated thyroid tumors we examined 66 tumor tissue samples for the presence of mutations at "hot-spot" codons 201 and 227 using methods based on the polymerase chain reaction, subcloning and sequencing. The prevailing type of single-base substitution at codon 201 (71.4%) corresponded to the replacement of the wild-type sequence CGT (Arg) with TGT (Cys). The fragments of the Gs-alpha gene, including codon 201 or 227 from five follicular carcinomas and one follicular adenoma, were subcloned in Escherichia coli and it was found that the proportion of alleles with mutated codon 201 varied from 3.2% to 43%. Sequencing of the corresponding region has confirmed preliminary data indicating that the single-base changes CGT (Arg) to TGT (Cys) or CGT to CAT (His) occurred. There was only a weak correlation between the prevalence of cells bearing a mutation in the Gs-alpha gene and the level of Gs-alpha protein expression in the corresponding thyroid tumors.

Increased expression of the c-fos proto-oncogene in bone from patients with fibrous dysplasia.

Fibrous dysplasia is characterized by intense marrow fibrosis and increased rates of bone turnover. The lesions of fibrous dysplasia resemble those described in the long bones of transgenic mice overexpressing the c-fos proto-oncogene. Activating mutations in the alpha subunit of the stimulatory guanine-nucleotide-binding protein (GS alpha) linked to adenylate cyclase have recently been described in bone cells from patients with the McCune-Albright syndrome and fibrous dysplasia. We used in situ hybridization to determine the level of expression of c-fos in bone-biopsy specimens from two normal subjects, eight patients with fibrous dysplasia, and six patients with other bone disorders characterized by high rates of bone turnover. The probe used corresponded to the fourth exon of the c-fos gene. High levels of c-fos expression were detected in the bone lesions from all eight patients with fibrous dysplasia. No expression of c-fos was detected in bone specimens from the normal subjects or from specimens of normal bone obtained from patients with fibrous dysplasia. The cells that expressed c-fos in the dysplastic lesions were fibroblastic and populated the marrow space. A very low level of c-fos expression was detected in the biopsy specimens from the patients with other bone diseases. One patient with polyostotic fibrous dysplasia and one patient with the McCune-Albright syndrome were tested for the previously described GS alpha gene mutations and were found to express these mutations in bone. Increased expression of the c-fos proto-oncogene, presumably a consequence of increased adenylate cyclase activity, may be important in the pathogenesis of the bone lesions in patients with fibrous dysplasia.

Parental origin of Gs alpha gene mutations in Albright's hereditary osteodystrophy.

Heterozygous mutations of the Gs alpha gene leading to reduced Gs alpha activity have been identified in patients with Albright's hereditary osteodystrophy (AHO). However, AHO may be associated with hormone resistance (pseudohypoparathyroidism type Ia, PHPIa) or a normal response (pseudo-pseudohypoparathyroidism, PPHP). As both disorders may occur within the same family, the relationship between Gs alpha genotype and phenotype remains unresolved. The AHO phenotype may be dependent upon the sex of the parent transmitting the Gs alpha mutation, perhaps through a gene imprinting mechanism. We have used an intragenic Gs alpha FokI polymorphism to determine the parental origin of Gs alpha gene mutations in sporadic and familial AHO. We now show that a de novo G-->A substitution at the exon 5 donor splice junction in a child with PPHP was paternally derived. Furthermore, in a female with PPHP, the Gs alpha abnormality was shown to be of paternal origin, while subsequent maternal processing and transmission resulted in PHPIa in two offspring. As transmission of PPHP has rarely been reported, determining parental origin of the disease allele in sporadic cases may provide insight into the mechanism of hormone resistance in AHO.