Glycoprotein hormone alpha-subunit production in somatotroph adenomas with and without Gs alpha mutations.

Activating mutations of the Gs alpha subunit have been identified in a subset of somatotroph adenomas. The mutant form of the Gs alpha subunit causes persistent activation of adenylyl cyclase and consequently results in high intracellular levels of cAMP. Because cAMP is known to stimulate the synthesis of the glycoprotein hormone (GPH) alpha-subunit as well as GH, we examined somatotroph tumors with and without Gs alpha mutations for GPH alpha-subunit production. GPH alpha-subunit production was assessed in vivo by measuring serum hormone levels and in vitro by analyzing hormone secretion by cultured pituitary tumor cells. DNA was extracted from the pituitary tumors of 26 acromegalic patients. The Gs alpha gene was amplified by the polymerase chain reaction and screened for mutations at codons 201 and 227 using oligonucleotide specific hybridization. Nine of the 26 tumors (35%) had point mutations at Arg 201. Seven of these tumors contained a CGT to TGT mutation (Arg to Cys) and 2 contained a CGT to CAT mutation (Arg to His). No mutations were detected at codon 227. There were no significant differences in age, sex distribution, tumor size, or serum levels of GH or insulin-like growth factor-1 between the groups of patients with or was Gs alpha mutations. The mean serum level of the free GPH alpha-subunit was 1.9-fold higher in the group with Gs alpha mutations (0.48 +/- 0.37 micrograms/L) than in patients without mutations (0.25 +/- 0.17) (P less than 0.05). In pituitary tumor cell culture, 75% of somatotroph tumors with Gs alpha mutations secreted free GPH alpha-subunit into the media compared with 45% of tumors without Gs alpha mutations. The amount of GPH alpha-subunit secretion was 12-fold greater in the group of tumors containing the Gs alpha mutation (P less than 0.05). Immunocytochemical detection of the free GPH alpha-subunit was similar in the two groups of patients with 75% positive for the GPH alpha-subunit in tumors with Gs alpha mutations and 67% positive in tumors without mutations (P = 0.69). We conclude that GPH alpha-subunit production occurs in somatotroph tumors with and without Gs alpha mutations. The increased levels of GPH alpha-subunit secretion in vivo and in vitro suggest that the Gs alpha mutation may increase the amount of preexisting GPH alpha-subunit biosynthesis in the tumors, perhaps via activation of the cAMP pathway.     

Identification of a mutation in the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase in McCune-Albright syndrome.

McCune-Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, café-au-lait lesions, and a variety of endocrine disorders, including precocious puberty, hyperthyroidism, hypercortisolism, growth hormone excess, and hyperprolactinemia. The diverse metabolic abnormalities seen in MAS share the involvement of cells that respond to extracellular signals through activation of the hormone-sensitive adenylyl cyclase system (EC 4.6.1.1). Mutations that lead to constitutive activation of Gs alpha, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase activity, have been identified in a subset of human growth hormone-secreting pituitary tumors and human thyroid tumors. We report here the identification of a mutation in the gene encoding Gs alpha in a patient with MAS. Denaturing gradient gel electrophoresis was used to analyze amplified DNA fragments including exon 8 or exon 9 of the Gs alpha gene. In one subject with MAS a G-to-A transition was found in exon 8 of one of the two alleles encoding Gs alpha. This single-base substitution results in the replacement of arginine by histidine at position 201 of the mature Gs alpha protein. Semiquantitative analysis of amplified DNA indicated that the mutant allele was less prevalent than the wild-type allele in peripheral leukocytes and was present in very low levels in skin. These findings support the previous contention that the segmental distribution and variable expression of the cutaneous, skeletal, and endocrine manifestations of MAS reflect an underlying somatic mosaicism. Further, these results suggest that the molecular basis of MAS is a postzygotic mutation in Gs alpha that causes constitutive activation of adenylyl cyclase.     

Primary GH insensitivity '(Laron syndrome) caused by a novel 4 kb deletion encompassing exon 5 of the GH receptor gene: effect of intermittent long-term treatment with recombinant human IGF-I

OBJECTIVE: GH insensitivity syndrome (GHIS; Laron syndrome) is clinically characterized by severe postnatal growth failure and very low serum levels of IGF-I despite increased secretion of GH. This mainly autosomal recessive syndrome is clinically indistinguishable from isolated GH deficiency (IGHD). Fifty-one different mutations in the GH receptor (GHR) gene have been discovered, whereas only three deletions causing the disorder have been reported so far. In this report, we describe a consanguineous family from Sri Lanka with a novel deletion of 4097 bp in length encompassing exon 5. SUBJECTS AND METHODS: Parents of normal phenotype presented their second child (boy) to our clinic at the age of 7 months with severe growth retardation and the clinical features of IGHD (58 cm, -6.1 standard deviation score (SDS); 5.7 kg, -3.4 SDS). Assessment, however, revealed GHIS with absent GH-binding protein. Thereafter, the patient received intermittent recombinant human IGF-I (rhIGF-I; 80 microg/kg twice daily) treatment prepubertally for 5.5 years. Genomic DNA was extracted for genetic analysis and each exon was PCR amplified individually. Further, in order to amplify the GHR gene from exon 4 to 6, Expand Long Template PCR (Roche) was carried out. In addition, RNA isolation and RT-PCR were performed. RESULTS: Separate PCRs of each of the exons of the GHR gene revealed that exon 5 in the patient was missing. Thereafter, "Long PCR" from exons 4 to 6 revealed a 4097 bp deletion encompassing exon 5, in a homozygous state in the patient and in a heterozygous state in both parents. RT-PCR analysis revealed an exact absence of exon 5 resulting in a frameshift, leading to a stop codon in exon 6, which predicts a truncated, non-functional GHR protein. CONCLUSION: Fifty-one different mutations within the GHR gene causing GHIS have been reported so far. In contrast, only three deletions within the GHR gene are known. We describe a patient suffering from GHIS caused by a novel 4 kb deletion of the GHR gene encompassing exon 5 and, additionally, we focus on the effect of intermittent rhIGF-I treatment during prepuberty.     

(AC)23 [Z-2] polymorphism of the aldose reductase gene and fast progression of retinopathy in Chilean type 2 diabetics

Mutations in the glucokinase (GCK) gene are considered to be a possible cause of maturity-onset diabetes of the young. The purpose of this study was to evaluate the contribution of this gene to the development of post-renal transplantation diabetes mellitus (PTDM). Identification of the GCK mutation was attempted in 58 selected renal allograft recipients with PTDM and 45 normal controls. The exons in the GCK gene were examined using polymerase chain reaction (PCR), followed by an analysis of single-stranded DNA conformational polymorphism (SSCP). The abnormal bands were then confirmed by DNA sequencing analysis. The family members of the patients affected with GCK mutation were also examined. Two of the 58 PTDM patients (3.4%) were found to have GCK mutations. One had the mutation on exon 5 and the other on intron 7. One control subject had the mutation on intron 9. The mutation on exon 5 was identified as a substitution of CCT (proline) for CTT (leucine) at codon 164, which has never been reported before. The family members of the PTDM patient with a mutation on exon 5 were analyzed by PCR, followed by SSCP, and two of them had the same mutation. The abnormal band seen on SSCP analysis of exon 7 was identified as the C→T substitution at the 39th nucleotide in intron 7. Two of the family members also displayed the same bands on the SSCP. One of the 45 normal controls had a known polymorphism located at the 8th nucleotide in intron 9. We found a GCK mutation on the exon in subjects with PTDM and we speculate that this mutation may be one of the possible contributing factors of PTDM, although variations of the GCK gene are not common causes of PTDM.     

Combined bromocriptine and growth hormone (GH) treatment in GH-deficient children with macroprolactinoma in situ.

Experience with PRL-secreting macroadenomas in the pediatric and adolescent population is limited. Although use of synthetic GH after treatment of central nervous system tumors in children without active disease is accepted practice, reports of GH use in patients with central nervous system tumors in situ are rare. Furthermore, the effect of GH on tumor growth is not known. We report GH treatment (10 and 11.5 months), concomitant with bromocriptine (BC; dopamine agonist) therapy in two children, a 15.5-yr-old male and a 15.5-yr-old female, with PRL-secreting macroadenomas in situ. Surgical resection was deemed undesirable because of the risk of major morbidity due to the large size of the tumors and the close proximity to major vessels. Both patients were GH deficient and had heights below the fifth percentile coupled with arrested pubertal progress. During BC therapy, a decrease in tumor size and a reduction in serum PRL levels occurred in both patients, which continued after the addition of GH treatment. Neither patient experienced changes in visual acuity during combined treatment, and both experienced marked improvement in growth velocity. We conclude that in children with PRL-secreting tumors and GH deficiency in whom surgery is not advised, combined treatment with BC and GH appears to be safe and efficacious. To our knowledge, these patients represent the first report of the combined therapeutic use of BC and GH as the primary mode of treatment in children with prolactinoma in situ with documented GH deficiency.     

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.     

Study of GH sensitivity in chilean patients with idiopathic short stature

We hypothesized that some children with idiopathic short stature in Chile might bear heterozygous mutations of the GH receptor. We selected 26 patients (3 females, 23 males) from 112 patients who consulted for idiopathic short stature at the University of Chile. Their chronological age was 8.3 +/- 1.9, and bone age was 6.1 +/- 1.0 yr. Their height was -3.0 +/- 0.7 SDS; IGF-I, -1.2 +/- 1.1 SD; IGF binding protein 3, -0.7 +/- 2.0 SDS; and GH binding protein, 0.4 +/- 0.8 SDS. Patients were admitted, and blood samples were obtained every 20 min to determine GH concentrations overnight. Coding sequences and intron-exon boundaries of exons 2-10 of GH receptor gene were amplified by PCR and subsequently analyzed through single-strand conformational analysis. Mean serum GH concentration, over 12-h, was 0.20 +/- 0.08 nM; pulse amplitude, 0.40 +/- 0.15 nM; number of peaks, 5.8 +/-1.5 peaks/12 h; peak value of GH during the 12-h sampling, 1.03 +/- 0.53 nM; and area under the curve, 151.4 +/- 56.1 nM/12 h. There were positive correlations between mean GH vs. area under the curve (P < 0.001) and GH peak (P < 0.01). The single-strand conformational analysis of the GH receptor gene showed abnormal migration for exon 6 in 9 patients and for exon 10 in 9 patients, which (by sequence analysis) corresponded to 2 polymorphisms of the GH receptor gene: an A-to-G transition in third position of codon 168 in exon 6 and a C-to-A transversion in the first position of codon 526 in exon 10. We further sequenced all coding exons and intron-exon boundaries in the most affected patients (nos. 6, 9, 11, 14, 15, 16, and 23). This analysis revealed a C-to-T transition in codon 161 of exon 6 in patient 23, which results in an amino acid change (Arg to Cys) in an heterozygous form in the patient and his father. In conclusion, the results of our study suggest that, in Chilean patients with idiopathic short stature, GH receptor gene mutations are uncommon, although we cannot exclude mutations that were missed by single-strand conformational analysis or mutations within introns or in the promoter regions of the GH receptor gene.     

Prevalence of Gs alpha mutations in Korean patients with pituitary adenomas

The reported frequencies of Gs alpha mutations (gsp mutations) in growth hormone (GH)-secreting pituitary adenomas are variable (ranging from 4.4 to 43%), and the presence of these mutations in the other pituitary adenomas is still a matter of controversy. Previous clinical and biochemical analyses of patients with GH-secreting pituitary adenomas and gsp mutations produced conflicting results and did not demonstrate obvious characteristics. Therefore, we investigated the prevalence of gsp mutations in Korean patients with pituitary adenomas and elucidated the characteristics of these patients. Forty-four GH-secreting adenomas, 7 prolactin (PRL)-secreting adenomas and 32 clinically non-functioning adenomas were examined for the presence of point mutations in codon 201 and 227 of the Gs alpha gene using a nested PCR and direct sequencing of DNA extracted from fresh tissue or paraffin-embedded pituitary adenoma samples. Seven of the 44 GH-secreting pituitary adenomas had point mutations at codon 201 or 227; of these, five mutations were in codon 201 and two were in codon 227. In patients with gsp mutations, mean tumor size was significantly smaller than in patients without gsp mutations (15.9+/-8.7 mm vs. 24.9+/-14.9 mm, P<0.05). Age, sex, basal GH levels, GH response to oral glucose loading, GH response to octreotide and surgical outcome were not different in the two groups. One of the 32 clinically non-functioning pituitary adenomas had a point mutation at codon 201; none of the seven prolactinomas had these mutations. These results show that gsp mutations are not rare in Korean acromegalic patients and mean tumor size is significantly smaller in acromegalic patients with gsp mutations. Our results also confirm the low frequency of gsp mutations in clinically non-functioning pituitary adenomas and the absence of gsp mutations in prolactinoma.     

GSPalpha mutations in Mexican patients with acromegaly: potential impact on long term prognosis.

OBJECTIVE: The frequency of activating mutations of the GSPalpha gene as the etiology of GH-secreting pituitary adenomas has been the subject of important ethnogenetic variability. Whereas up to 40% of Caucasian patients with acromegaly have tumors which harbor these somatic mutations, their prevalence among Asian populations is much lower. The correlation between the presence of these mutations and the clinical and biological behavior of these tumors has also been a matter of controversy. In the present study, we investigated the prevalence of GSPalpha mutations in GH-secreting tumors obtained from a genetically homogenous population of Mexican patients with acromegaly. We also sought to establish whether or not the presence of these mutations correlates in any way with the clinical or biochemical characteristics of the disease. STUDY DESIGN AND METHODS: Fifty eight GH-secreting pituitary adenomas were examined for the presence of point mutations in either codon 201 or 227 of the GSPalpha gene, using PCR and direct sequencing of DNA extracted from either fresh or paraffin-embedded tissues. Patients were prospectively followed clinically and biochemically for up to nine years after pituitary surgery. RESULTS: Heterozygous point mutations in exon 8 (codon 201) were found in 11 patients (19%), and no molecular alterations were evident in exon 9. The frequency and severity of the different clinical features of acromegaly did not differ between patients with and without GSPalpha mutations. Patients with and without mutations had pre-operative GH and IGF-I elevations of similar magnitude, and although microadenomas appeared to be more frequent among patients with GSPalpha mutations, this did not reach statistical significance. Upon short-term follow-up, biochemical cure (normal age- and gender-adjusted IGF-I and post-glucose GH below 1 ng/mL) was similarly achieved in both groups. After 3-9 years of post-operative follow up however, a significantly greater proportion of patients with the mutation achieved a "safe" basal GH value (100% vs 33%, p=0.001) as well a lower nadir post-glucose GH (0.53+/-0.5 vs 2.9+/-6.2 ng/mL, p=0.04) although the rate of IGF-1 normalization did not differ between the 2 groups. CONCLUSIONS: Our results show that the prevalence of GSPalpha mutations in Mexican patients with acromegaly is intermediate between that found in Asian and Caucasian populations. In this well-defined genetic population the presence of codon 201 mutations appeared to be associated with a greater probability of achieving a "safe" GH value upon long-term follow-up.     

Morphological studies on mixed growth hormone (GH)- and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule

A morphological study was carried out on five mixed GH- and PRL-secreting pituitary adenomas, surgically removed from acromegalic patients with hyperprolactinemia, in order to verify whether the two hormones were contained in the same cell or in different cells. Double labeling with the protein A-gold immunotechnique was used to visualize the ultrastructural localization of the two hormones on ultrathin sections of the tumors. By means of this high resolution technique we found in all adenomas the presence of numerous (from 50-80% of the whole cell population) mammosomatotrophs, i.e. cells containing simultaneously PRL and GH. The occurrence of cells producing only GH (in four tumors) or only PRL (in one tumor) was also observed. In mixed cells GH and PRL were segregated in the same mixed granule. In one tumor granules positive only for GH together with mixed granules were found in the same cell. Immunofluorescence studies, at the light microscopic level, allowed us to clearly identify mammosomatotrophs only in two tumors. Double labeling using the gold immunotechnique appears therefore to be the most suitable experimental approach to detect the existence of mixed cells in plurihormonal adenomas. Our results support the idea that the frequency of mixed adenomas with mixed cells may be higher than that believed previously. The simultaneous presence of two hormones in the same secretory granule could explain why, in patients having mixed tumors, factors able to stimulate or inhibit the release of one hormone can also stimulate or inhibit the secretion of the other.     

Mutation of the p53 tumor suppressor gene in transitional cell carcinoma of the urinary tract in Taiwan

Early detection of a mutated p53 gene is thought to provide useful information in a wide range of human tumors. The aim of this study was to identify the role of the p53 gene in transitional cell carcinoma of the urinary tract. From March 1992 to July 2003, 75 patients (54 men and 21 women) with a mean age of 66.85 years and pathologically diagnosed transitional cell carcinoma were enrolled in this study. Fifty-eight patients had bladder cancer, eight had ureteral cancer, and nine had renal-pelvic cancer. Rapid screening for mutation of the p53 gene was performed using polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and sequencing analysis. Primer sets were designed to amplify fragments within exons 4, 5, 6, 7, and 8 of the p53 gene. Pathology classified 37 tumors as low grade and 38 as high grade. Tumor stage was pT1 or less in 29 patients and at least pT2 in 46 patients. Of the 75 patients in this study, 47 (62.7%) had a p53 mutation. Of the patients with a p53 mutation, 33 (70.2%) had invasive tumors. Invasive tumors were associated with p53 mutation (p < 0.05). Noted in 20 patients (26.7%), exon 4 was the most common site of the mutation. Of the patients with exon 4 mutations, 15 (75%) had invasive tumors and nine (45%) had high-grade tumors. Additionally, among the 20 patients with a common polymorphism at codon 72, 16 (80%) had invasive tumors and 14 (70%) had high-grade tumors. In this study, 62.7% of patients with transitional cell carcinoma had a p53 mutation, suggesting that the p53 gene mutation may be used as a marker of transitional cell carcinoma. Invasive tumors are more likely to have a p53 gene mutation. A simple analysis of the p53 gene using PCR/SSCP is suitable for screening for p53 abnormalities in transitional cell carcinoma. The relationship between cancer risk and the codon 72 polymorphism of exon 4 needs further investigation.     

Mutational analysis of PRKAR1A and Gs(alpha) in sporadic adrenocortical tumors.

Little is known about the pathogenesis of adrenocortical tumors. The cAMP-dependent pathway is physiologically activated by ACTH in adrenocortical cells and different components of this cascade may be altered in some functioning adrenocortical tumors. Recently, mutations of the gene encoding the PKA type 1 A regulatory subunit (R1 A), PRKAR1A, associated with loss of heterozygosity (LOH) at PRKAR1A locus, have been demonstrated in primary pigmented nodular adrenocortical disease (PPNAD), either isolated or associated with Carney complex. Moreover, activating mutations of the Gs(alpha) gene (the gsp oncogene) have also been found in a small number of adrenocortical cortisol-secreting adenomas. Aim of this study was to investigate the presence of such genetic alterations on a series of 10 ACTH-independent Cushing syndrome due to non-PPNAD adrenocortical adenomas. The coding sequence of PRKAR1A, evaluated by PCR and direct sequencing analysis, revealed the absence of mutations while heterozygosity for at least 1 polymorphism excluded LOH in most tumors. In one single adenoma gsp mutation was detected. In conclusion, we provide additional evidence that the only mutational changes able to activate the cAMP pathway so far identified, i.e. PRKAR1A mutations and gsp oncogene, are a rare event in adrenocortical tumors.     

c-kit gene mutation at exon 17 or 13 is very rare in sporadic gastrointestinal stromal tumors

BACKGROUND AND AIM: Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the human gut. They frequently have gain-of-function mutations of the c-kit gene, which encodes a receptor, tyrosine kinase. The mutations were found at exon 11 in most cases, and either at exon 9 or at exon 13 in rare cases. Recently, we found a family with multiple GIST and a gain-of-function mutation at exon 17. The family was the first reported GIST case with c-kit gene mutation at exon 17 including sporadic GIST. Although we previously reported that the c-kit gene mutation at exon 17 was not detected in 124 sporadic GIST by single-strand conformation polymorphism (SSCP) analysis, the mutation at exon 17 observed in the familial GIST was detectable by the use of direct sequencing but not by our SSCP method. In the present study, we examined the mutations at exon 17 and exon 13 by using direct sequencing. METHODS: Genomic DNA was extracted from formalin-fixed, paraffin-embedded GIST tissues. We could obtain 143 sporadic GIST cases appropriate for DNA analysis at exon 17 and 141 at exon 13. Exons 17 and 13 were amplified by using polymerase chain reaction and direct sequencing was conducted. RESULTS: No mutation was found at exon 17, and only one case with the mutation at exon 13 was observed. The GIST with the mutation at exon 13 was large and showed frequent mitosis, and the patient died of the recurrent GIST 3 years after the first operation. CONCLUSION: The mutation at exons 17 or 13 was considered to be very rare in sporadic GIST.     

Identification of three novel spectrin alpha I/74 mutations in hereditary elliptocytosis: further support for a triple-stranded folding unit model of the spectrin heterodimer contact site

Six individuals with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) from three unrelated families were evaluated. Defects in the ability of spectrin (Sp) to undergo self-association were present, and associated with increased recovery of the Sp alpha I 74-kD fragment after limited tryptic digestion (Sp alpha I/74 variant). Because mutations associated with the Sp alpha I/74 variant described to date have been localized to the 5' coding region of the alpha-Sp gene (exon 2) or at the 3' coding end of the beta-Sp gene (exon 30), the polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) method was used to detect mutations in these two regions. In one family with HE, an abnormal pattern of migration of PCR- amplified fragments containing exon 2 was observed, and led to the detection of a new mutation (Ile24Ser) in helix 3 of repeating segment alpha 1. In the two other families, an abnormal pattern of migration of PCR-amplified fragments containing exon 30 was observed in affected individuals, and sequencing led to the identification of two new mutations (Ala2023Val and Trp2024Arg) in helix 1 of repeating segment beta 17. The elliptogenic potential of these mutations emphasizes the importance of the conformational integrity of each of the three helices involved in the formation of the Sp heterodimer contact site, and will help identify critical amino acids involved in this interaction.     

GH-secreting pituitary adenomas infrequently contain inactivating mutations of PRKAR1A and LOH of 17q23-24

OBJECTIVE: The molecular events leading to the development of GH-secreting pituitary tumours remain largely unknown. Gsalpha (GNAS1) mutations are found in 27-43% of sporadic GH-secreting adenomas in the Caucasian population, but the frequency of GNAS1 mutations in Japanese and Korean acromegalic patients was reported to be lower, 4-9% and 16%, respectively. Other genes responsible for the tumourigenesis of GH-secreting pituitary adenomas have not been detected yet. PRKAR1A, which codes for the RIalpha regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) on 17q23-24, was recently reported to contain inactivating mutations in some Carney complex families, which involved GH-secreting adenomas in about 10%. We re-evaluated the frequency of GNAS1 mutations and investigated PRKAR1A on the hypothesis that it might play a role in the tumourigenesis of GH-secreting adenomas. DESIGN: We analysed exons 8 and 9 of GNAS1 and all exons and the exon-intron boundaries of PRKAR1A with the PCR and by direct sequencing using genomic DNA extracted from 32 GH-secreting pituitary adenomas (30 GH-secreting adenomas, two GH and PRL-secreting adenomas) and 28 corresponding peripheral blood samples, and performed loss of heterozygosity (LOH) analysis of 17q23-24 with four microsatellite markers and intragenic markers of PRKAR1A. RESULTS: Seventeen of 32 (53.1%) tumours showed somatic-activating mutations of GNAS1: 16 (53.3%) of 30 GH-secreting adenomas and one of two GH and PRL-secreting adenomas. Neither inactivating somatic mutations of PRKAR1A nor LOH of 17q23-24 were detected in any of the tumours examined. CONCLUSION: We reconfirm the important role of activating mutations of GNAS1 in GH-secreting adenomas, and conclude that PRKAR1A does not play a significant role in the tumourigenesis.     

Oncogenic mutations in the thyrotropin receptor of autonomously functioning thyroid nodules in the Japanese population

OBJECTIVE: Constitutively activating mutations of the thyrotropin receptor (TSHR) have been found in the majority of autonomously functioning thyroid nodules (AFTNs) in European patients. The reported frequency of these mutations varies among reports but amounts to 50-80%. To date, only one such mutation responsible for AFTNs has been identified in the Japanese population and the pathogenic role of such mutations in Japanese AFTNs has been questioned. In the present study, we evaluated the frequency of activating mutations in the TSHR and G(alpha)s in 10 Japanese AFTNs. DESIGN: Genomic DNA was extracted from fresh frozen tissue. The TSHR and the almost entire sequence of the gene coding for the alpha subunit of Gs have been amplified and sequenced. RESULTS: In sequence analysis, four mutations in the TSHR (T632A, I486M, M453T and L512R) were found. To complete our analysis, we searched mutations in the gene coding for the alpha subunit of Gs, in the samples negative for TSHR mutations. In one case a mutation (R201H) affecting GTPase activity was found. CONCLUSIONS: If we focus on the solitary nodules, we obtain the same mutation proportion as in European patients (70%). The absence of TSHR and G(alpha)s mutations in a significant proportion of autonomous adenomas in multinodular goiters suggests that other causes may also play a role in the genesis of these lesions.     

Identification of two novel deletion mutations within the Gs alpha gene (GNAS1) in Albright hereditary osteodystrophy.

Albright hereditary osteodystrophy (AHO) is a genetic disorder characterized by short stature, skeletal defects, and obesity. Within AHO kindreds, some affected family members have only the somatic features of AHO [pseudopseudohypoparathyroidism (PPHP)], whereas others have these features in association with resistance to multiple hormones that stimulate adenylyl cyclase within their target tissues [pseudohypoparathyroidism type Ia (PHP Ia)]. Affected members of most AHO kindreds (both those with PPHP and those with PHP Ia) have a partial deficiency of Gs alpha, the alpha-subunit of the G protein that couples receptors to adenylyl cyclase stimulation, and in a number of cases heterozygous loss of function mutations within the Gs alpha gene (GNAS1) have been identified. Using PCR with the attachment of a high melting domain (GC-clamp) and temperature gradient gel electrophoresis, two novel heterozygous frameshift mutations within GNAS1 were found in two AHO kindreds. In one kindred all affected members (both PHP Ia and PPHP) had a heterozygous 2-bp deletion in exon 8, whereas in the second kindred a heterozygous 2-bp deletion in exon 4 was identified in all affected members examined. In both cases the frameshift encoded a premature termination codon several codons downstream of the deletion. In the latter kindred affected members were previously shown to have decreased levels of GNAS1 messenger ribonucleic acid expression. These results further underscore the genetic heterogeneity of AHO and provides further evidence that PHP Ia and PPHP are two clinical presentations of a common genetic defect. Serial measurements of thyroid function in members of kindred 1 indicate that TSH resistance progresses with age and becomes more evident after the first year of life.     

Analysis of <Emphasis Type="Italic">GNAS</Emphasis> mutations in 60 growth hormone secreting pituitary tumors: correlation with clinical and pathological characteristics and surgical outcome based on highly sensitive GH and IGF-I criteria for remission

Although the molecular mechanisms underlying GH secreting pituitary tumor formation are not well understood, mutations in the α-subunit of the stimulatory G gene, GNAS, have been identified in up to 40%. As these mutations could play a role in tumor growth, we screened 60 GH secreting tumors for GNAS mutations and assessed whether mutation status correlated with their clinical and pathological characteristics. Tumor specimens obtained at surgery were snap frozen. Tumor DNA was extracted, and PCR was used to amplify regions containing 2 sites of recurrent activating somatic mutations in codons 201 and 227 in GNAS. Amplicons were bi-directionally sequenced and analyzed. GNAS mutations were present in 24/60 (40%) of tumors; these were arg201cys(n = 15), arg201ser(n = 2), arg201his(n = 2), gln227leu(n = 4), gln227arg(n = 1). Preoperative IGF-I levels (age-adjusted) were higher (p = 0.01), but GH levels were slightly higher (p = 0.18) in mutation positive vs. negative groups. Mutation positive tumors were somewhat smaller than negative tumors (p = 0.07). The proportion of tumors >2 cm was somewhat less among positive (8.3%) vs. negative tumors (25%) (p = 0.10). Neither mib proliferation index, the proportion of invasive tumors nor surgical remission rates differed in the groups. IGF-I normalization rate with somatostatin analog therapy was similar in positive (3 of 6) vs. negative (3 of 7) patients. GH secreting tumors harboring GNAS mutations had higher preoperative IGF-I levels, somewhat higher preoperative GH levels and tended to be smaller than tumors without mutations. Presence of a GNAS mutation did not predict a difference in a proliferation marker, surgical remission or response to somatostatin analog therapy.