GH-producing Pituitary Tumors Research Articles

Outcome of endoscopic transsphenoidal surgery for acromegaly: Comparison of using and not using the floor standing pneumatic powered endoscope-holder system

IntroductionEndoscopic transsphenoidal surgery can be performed by two surgeons, including an endoscopist (PE/2S), and by a single surgeon with an endoscope-holder system (PE/1S + H). We analyzed the surgical outcome, and outcome predictors in acromegaly patients in endoscopic transsphenoidal surgery using floor standing pneumatic endoscope-holder system. MethodsEndoscopic transsphenoidal surgery was performed with PE/1S+H (n = 51) and PE/2S (n = 20). Postoperative remission was evaluated by the 2010 consensus criteria for acromegaly. We compared the surgical results of PE/2S style and PE/1S+H style, and investigated the factors associated with favorable surgical outcomes. ResultsThere was no difference in clinical background between the PE/2S and the PE/1S + H groups. The remission rates for PE/2S and PE/1S+H were 65.0% and 82.4%, respectively, with no significant difference (p = 0.128). In consecutive 71 cases, statistically useful predictors of remission were low preoperative growth hormone (GH) level (<12 ng/mL), low Knosp grade (0–2), and low revised Knosp grade (0–3A). In the conventional Knosp grade 0–2 and 3/4, the sensitivity was 0.76 and the specificity was 0.81. In the revised Knosp grade 0–3A and 3B/4, the sensitivity was 0.96 and the specificity was 0.44. ConclusionThe outcome of GH-producing pituitary neuroendocrine tumors surgically removed by PE/1S+H could be almost equivalent to that by PE/2S. Preoperative low GH level and Knosp grades, including revised Knosp grades, are useful preoperative predictors for surgical remission of acromegaly.

THU044 The Subcellular Localization Of β-Catenin Is Associated With Tumor Size And The Surgical Remission Rate In Growth Hormone-producing Pituitary Adenomas

Abstract Disclosure: S. Kuwabara: None. H. Kameda: None. H. Nomoto: None. K. Cho: None. A. Nakamura: None. Y. Ishi: None. H. Motegi: None. F. Miki: None. H. Miyoshi: None. T. Atsumi: None. The Wnt/β-catenin pathway is the best understood Wnt signaling pathway and controls cell proliferation. In the presence of Wnt, the translocation of β-catenin from the cytoplasm to the nucleus leads to pathway activation. It has been reported that the nuclear accumulation of β-catenin is associated with Ki-67 expression in pituitary adenomas and that the invasiveness of growth hormone (GH)-producing pituitary adenomas is associated with β-catenin expression. We aimed to clarify the effects of the Wnt/β-catenin pathway in GH-producing pituitary adenomas. We performed immunofluorescence staining using antibodies against GH and β-catenin in pathological samples of pituitary adenoma from 62 patients with acromegaly who underwent pituitary surgery at our hospital between January 2008 and December 2021. We also obtained clinical data for the patients, including the results of endocrine testing, and evaluated the relationships of key indices with the intracellular localization of β-catenin. Samples from 26 cases (42%) showed strong punctate expression of β-catenin in the nuclei (dot pattern (DP) group), and samples from 36 cases (58%) showed β-catenin expression in the cytoplasm or cell membrane (non-DP group). There were no differences in the sex, age, GH concentration, or insulin-like growth factor I (IGF-1) SD scores of the two groups, but the DP group had larger tumors (diameters 18±8 vs. 13±7 mm) and a higher prevalence of macroadenomas (77% vs. 53%). In the DP group, a paradoxical preoperative GH response (an increase of ≥30% of the basal concentration) to 75-g oral glucose tolerance testing was more frequent (46 vs. 22%, p&amp;lt;0.05) and postoperative remission was less frequent (23% vs. 50%, p&amp;lt;0.05). Accordingly, the Wnt/β-catenin pathway appears to be more active in the DP group. The larger tumors and lower postoperative remission rate of the DP group suggest that the Wnt/β-catenin pathway may influence tumor growth. Wnt/β-catenin pathway could play a major role in GH-producing pituitary tumor growth, therefore this pathway would be a potential therapeutic target in the affected patients. Presentation: Thursday, June 15, 2023

Epithelial-Mesenchymal Transition in the Resistance to Somatostatin Receptor Ligands in Acromegaly.

Epithelial-mesenchymal transition (EMT) is a dynamic process by which epithelial cells loss their phenotype and acquire mesenchymal traits, including increased migratory and invasive capacities. EMT is involved in physiological processes, such as embryogenesis and wound healing, and in pathological processes such as cancer, playing a pivotal role in tumor progression and metastasis. Pituitary tumors, although typically benign, can be locally invasive. Different studies have shown the association of EMT with increased tumor size and invasion in pituitary tumors, and in particular with a poor response to Somatostatin Receptor Ligands (SRLs) treatment in GH-producing pituitary tumors, the main cause of acromegaly. This review will summarize the current knowledge regarding EMT and SRLs resistance in acromegaly and, based on this relation, will suggest new biomarkers and possible therapies to SRLs resistant tumors.

Functional screen analysis reveals miR-26b and miR-128 as central regulators of pituitary somatomammotrophic tumor growth through activation of the PTEN–AKT pathway

MicroRNAs (miRNAs) have been involved in the pathogenesis of different types of cancer; however, their function in pituitary tumorigenesis remains poorly understood. Cyclic-AMP-dependent protein kinase-defective pituitaries occasionally form aggressive growth-hormone (GH)-producing pituitary tumors in the background of hyperplasia caused by haploinsufficiency of the protein kinase's main regulatory subunit, PRKAR1A. The molecular basis for this development remains unknown. We have identified a 17-miRNA signature of pituitary tumors formed in the background of hyperplasia (caused in half of the cases by PRKAR1A-mutations). We selected two miRNAs on the basis of their functional screen analysis: inhibition of miR-26b expression and upregulation of miR-128 suppressed the colony formation ability and invasiveness of pituitary tumor cells. Furthermore, we identified that miR-26b and miR-128 affected pituitary tumor cell behavior through regulation of their direct targets, PTEN and BMI1, respectively. In addition, we found that miR-128 through BMI1 direct binding on the PTEN promoter affected PTEN expression levels and AKT activity in the pituitary tumor cells. Our in vivo data revealed that inhibition of miR-26b and overexpression of miR-128 could suppress pituitary GH3 tumor growth in xenografts. Taken together, we have identified a miRNA signature for GH-producing pituitary tumors and found that miR-26b and miR-128 regulate the activity of the PTEN-AKT pathway in these tumors. This is the first suggestion of the possible involvement of miRNAs regulating the PTEN-AKT pathway in GH-producing pituitary tumor formation in the context of hyperplasia or due to germline PRKAR1A defects. MiR-26b suppression and miR-128 upregulation could have therapeutic potential in GH-producing pituitary tumor patients.

The Expression of Wnt4 Is Regulated by Estrogen via an Estrogen Receptor Alpha-dependent Pathway in Rat Pituitary Growth Hormone-producing Cells

Wnt signaling is important in many aspects of cell biology and development. In the mouse female reproductive tract, Wnt4, Wnt5a, and Wnt7a show differential expression during the estrus cycle, suggesting that they participate in female reproductive physiology. Although the pituitary is a major gland regulating reproduction, the molecular mechanism of Wnt signaling here is unclear. We elucidated the subcellular distribution of Wnt4 in the pituitary of estrogen-treated ovariectomized female rats. Expression of Wnt4 mRNA increased dramatically, particularly in proestrus compared with estrus and metestrus. Wnt4 protein was observed in the cytoplasm of almost all growth hormone (GH)-producing cells and in only a few thyroid-stimulating hormone β (TSHβ)-producing cells. In rat GH-producing pituitary tumor (MtT/S) cells, estrogen-induced expression of Wnt4 mRNA was completely inhibited by estrogen receptor antagonist ICI 182,780 in vitro. Thus, rat pituitary GH cells synthesize Wnt4 and this is induced by estrogen mediated via an estrogen receptor alpha-dependent pathway.

Improvement of Congestive Heart Failure after Octreotide and Transsphenoidal Surgery in a Patient with Acromegaly

A 59-year-old man was admitted because of congestive heart failure. He was suspected to have acromegaly, and magnetic resonance imaging revealed a pituitary macroadenoma. Endocrine examination revealed elevated plasma levels of growth hormone (GH) and insulin-like growth factor (IGF)-1, and an oral glucose tolerance test failed to suppress plasma GH levels, consistent with the diagnosis of GH-producing pituitary tumor. Treatment with octreotide, followed by transsphenoidal surgery resulted in normalization of plasma GH/IGF-1 levels, accompanied by the improvement of cardiac function. Thus, it is suggested that excess GH/IGF-1 axis is involved in the development of acromegaly-related cardiomyopathy in the present case.

Pituitary-Specific Knockout of the Carney Complex Gene Prkar1a Leads to Pituitary Tumorigenesis

Carney complex (CNC) is an inherited neoplasia syndrome characterized by spotty skin pigmentation, myxomas, endocrine tumors, and schwannomas. Among the endocrine tumors that comprise the syndrome, GH-producing pituitary tumors are seen in approximately 10% of patients, although biochemical abnormalities of the GH axis are much more common. To explore the role of loss of the CNC gene PRKAR1A on pituitary tumorigenesis, we produced a tissue-specific knockout (KO) of this gene in the mouse. For these studies, we generated a mouse line expressing the cre recombinase in pituitary cells using the rat GHRH receptor promoter. These mice were then crossed with Prkar1a conditional null animals to produce tissue-specific KOs. Although prolactinomas were observed in KO and control mice, the KO mice exhibited a significantly increased frequency of pituitary tumors compared with wild-type or conventional Prkar1a(+/-) mice. Characterization of the tumors demonstrated they were composed of cells of the Pit1 lineage that stained for GH, prolactin, and TSH. At the biochemical level, levels of GH in the serum of KO animals were markedly elevated compared with controls, regardless of the presence of a frank tumor. These data indicate that complete loss of Prkar1a is sufficient to allow the formation of pituitary tumors and abnormalities of the GH axis, in close analogy to human patients with CNC.

A Growth Hormone Receptor Mutation Impairs Growth Hormone Autofeedback Signaling in Pituitary Tumors

Pituitary tumors are a diverse group of neoplasms that are classified based on clinical manifestations, hormone excess, and histomorphologic features. Those that cause growth hormone (GH) excess and acromegaly are subdivided into morphologic variants that have not yet been shown to have pathogenetic significance or predictive value for therapy and outcome. Here, we identify a selective somatic histidine-to-leucine substitution in codon 49 of the extracellular domain of the GH receptor (GHR) in a morphologic subtype of human GH-producing pituitary tumors that is characterized by the presence of cytoskeletal aggresomes. This GHR mutation significantly impairs glycosylation-mediated receptor processing, maturation, ligand binding, and signaling. Pharmacologic GH antagonism recapitulates the morphologic phenotype of pituitary tumors from which this mutation was identified, inducing the formation of cytoskeletal keratin aggresomes. This novel GHR mutation provides evidence for impaired hormone autofeedback in the pathogenesis of these pituitary tumors. It explains the lack of responsiveness to somatostatin analogue therapy of this tumor type, in contrast to the exquisite sensitivity of tumors that lack aggresomes, and has therapeutic implications for the safety of GH antagonism as a therapeutic modality in acromegaly.

The Role of Endogenous Growth Hormone-Releasing Hormone in Acromegaly

Some indirect evidence suggests hypothalamic control of GH secretion in acromegaly. The objective of the study is to examine whether GH secretion in acromegaly is dependent on endogenous GHRH. We studied eight patients with untreated acromegaly due to a GH-producing pituitary tumor. All patients received an iv infusion of normal saline for 24 h and GHRH-antagonist (GHRH-ant) at 50 microg/kg x h for 7 d. GH was measured every 10 min for 24 h during the normal saline infusion and on the last day of the GHRH-ant infusion. A group of nine different patients with untreated acromegaly served as the control group and underwent blood sampling for GH every 10 min for two 24-h periods to assess the day-to-day variability of GH secretion. The study was set in a university referral center. Twenty-four-hour mean GH was the main outcome measured. In six of eight subjects treated with GHRH-ant, 24-h mean GH decreased by 5.8-30.0% during iv GHRH-ant and, in three subjects, the change in the 24-h mean GH was greater than the upper limit of the 95% confidence interval of the spontaneous day-to-day variability of the mean GH in patients with acromegaly. Based on the binomial distribution, the probability of this magnitude of change to occur in three of eight subjects by chance alone is 0.0008. In some patients with acromegaly due to a pituitary adenoma, GH secretion is under partial control by endogenous GHRH.

Effect of gsp oncogene on somatostatin receptor subtype 1 and 2 mRNA levels in GHRH-responsive GH3 cells

Growth hormone releasing hormone (GHRH) signals via G protein-coupled receptors (GHRH-R) to enhance intracellular Galphas/adenylyl cyclase/cAMP signaling, which in turn has positive effects on GH synthesis and release, as well as proliferation of the GH-producing cells of the anterior pituitary gland. Some GH-producing pituitary tumors express a constitutively active mutant form of Galphas (gsp oncogene). It has been reported that these tumors are more responsive to octreotide therapy. In this study we used a rat GH-producing cell line (GH3) stably transfected with the human GHRH-R cDNA (GH3-GHRHR cells) as a model to study the effects of gsp oncogene on somatostatin (SRIH) receptor subtype 1 and 2 (sst1 and sst2) mRNA levels. Transient transfection of gsp oncogene in GH3-GHRHR cells for 48 h increased intracellular cAMP levels and GH release. Phosphodiesterase (PDE) 4, sst1 and sst2 mRNA levels were increased by G protein mutation as assessed by real-time RT-PCR. Increased PDE mRNA levels in gsp-transfected cells may be a compensatory mechanism to the constitutive activation of cAMP-dependent pathway by G protein mutation and is consistent with reports of higher PDE expression in human pituitary tumor that express gsp. Our data suggest that higher expression of sst1 and sst2 mRNA induced by the gsp oncogene may be a mechanism by which gsp-positive tumors show a greater response to SRIH. GH3 cells permanently transfected with GHRH-R can be used for in vitro studies of actions of GHRH.

Clinically Silent Somatotropinomas May Be Biochemically Active

The diagnosis of acromegaly is suspected based on the typical clinical presentation and is subsequently confirmed biochemically by elevated GH and IGF-I concentrations. We report three female patients with pituitary tumors who presented without any signs or symptoms of acromegaly but with elevated IGF-I levels. Plasma GH was measured every 10 min for 24 h, and an oral glucose tolerance test was performed. All patients had abnormally elevated mean and trough plasma GH levels as well as post-glucose nadir GH concentrations. All patients had magnetic resonance imaging scans revealing pituitary tumors and underwent transsphenoidal surgery. Histologically, they had GH-producing pituitary tumors. Plasma IGF-I levels returned to normal in two patients after surgery. Some pituitary adenomas are true GH-secreting tumors despite not being accompanied by obvious clinical stigmata of acromegaly. Natural history of this disease is unknown because of the small number of reported patients and inconsistent results of biochemical testing. Based on the results of this and previous reports, we propose that all patients with known pituitary tumors, especially younger women with normal or mildly elevated prolactin level, be evaluated for GH excess.

Absence of constitutively activating mutations in the GHRH receptor in GH-producing pituitary tumors.

The molecular events leading to the development of GH-producing pituitary tumors remain largely unknown. We hypothesized that activating mutations of the GHRH receptor might occur in a subset of GH-producing pituitary tumors. Genomic DNA samples from 54 GH-producing pituitary tumor tissues were screened for mutations of the GHRH receptor. Eleven homozygous or heterozygous nucleotide substitutions [169G > A (A57T), 338C > T (P113L), 363G > T (E121D), 409C > T (H137Y), 547G > A (D183N), 673G > A (V225I), 749G > A (W250X), 760G > A (V254M), 785G > A (S262N), 880G > A (G294R), 1268G > A (C423Y)] were found in 12 patients (22.2%). The 169G > A substitution (A57T) appears to be a polymorphism (4 patients, 7.4%). E121D and V225I were each found in 2 patients. In 1 patient with the V225I sequence, the substitution was not found in genomic DNA from peripheral leukocytes, suggesting a somatic mutation. A patient with a heterozygous W250X mutation was homozygous for the C423Y substitution. These variant GHRH receptors were studied in transfected TSA-201 cells to evaluate the functional consequences of the amino acid changes. None of the GHRH receptor variants was associated with basal elevation of intracellular cAMP. GHRH induced variable cAMP responses. With the W250X and G294R variants, there was no cAMP stimulation by GHRH, indicating that the mutations are inactivating. Expression of the W250X GHRH receptor on the cell membrane was severely decreased and GHRH binding to the G294R GHRH receptor was impaired. Although GHRH receptor variants are common in GH- producing pituitary adenomas, constitutively activating mutations, as a mechanism for GH-producing pituitary tumors appear to be rare.

Absence of Constitutively Activating Mutations in the GHRH Receptor in GH-Producing Pituitary Tumors

Absence of Constitutively Activating Mutations in the GHRH Receptor in GH-Producing Pituitary Tumors

Serum concentrations of 20K human growth hormone in normal adults and patients with various endocrine disorders. Study Group of 20K hGH.

The 20K hGH isoform is produced by alternative splicing of GH mRNA, and comprises approximately 10% of all GH in the pituitary. The physiological role of 20K hGH remains to be determined partly because of the lack of a simple and specific assay. We have established sensitive enzyme-linked immunoadsorbent assays (ELISAs) specific to 20K and 22K hGH. The serum levels of 20K hGH after overnight fasting was 118 +/- 178 pg/mL (N=282) in normal women, significantly higher than in normal men (64 +/- 170 pg/mL, N=226). However, there was no difference in the proportion of 20K hGH to 20K plus 22K hGH between men (6.3 +/- 2.6%, N=176) and women (6.3 +/- 2.1%, N = 263). No correlation was detected between the ratio of 20K hGH and age, body height, body weight or body fat mass in normal subjects. The proportion of 20K hGH was significantly (P < 0.001) higher in patients with active acromegaly (9.2 +/- 2.2%, N=33) and in patients with anorexia nervosa (9.0 +/- 1.9, N=8), both of which are characterized by chronic elevation of circulating GH levels. The proportion of the 20K hGH in successfully treated acromegalic patients did not differ from that in normal subjects, suggesting that GH-producing pituitary tumors secrete a higher proportion of 20K hGH, or chronic excess of 22K hGH altering the metabolic clearance rate of 20K hGH. The values in patients with adult growth hormone deficiency (GHD), hyperthyroidism, primary hypothyroidism, or GH-independent short stature did not differ from those in normal subjects. The 20K ratio did not change after acute GH provocative tests such as insulin tolerance test and GRH test. These results suggest that secretion of 20K hGH from the pituitary is under the same control as that of 22K hGH.

Serum concentration of 20K human growth hormone (20K hGH) measured by a specific enzyme-linked immunosorbent assay. Study Group of 20K hGH.

Several GH isoforms have been identified in pituitary and serum, the most abundant of which is the 22K human GH (hGH) isoform. The 20K hGH isoform is produced by alternative splicing of GH messenger ribonucleic acid and comprises approximately 10% of all GH in the pituitary. The physiological role of 20K hGH remains to be determined, partly because of the lack of a simple and specific assay. We have established sensitive enzyme-linked immunosorbent assays (ELISAs) specific to 20K and 22K hGH. To determine whether regulation of 20K hGH secretion is the same as that for 22K hGH, we measured serum concentrations of both species of hGH in normal subjects and patients with a variety of endocrine disorders. The serum levels of 20K hGH after overnight fasting was 118 +/- 178 pg/mL (n = 282) in normal women, significantly higher than that in normal men (64 +/- 170 pg/mL; n = 226). However, there was no difference in the proportion of 20K hGH to 20K plus 22K hGH between men (6.3 +/- 2.6%, mean +/- SD; n = 176) and women (6.3 +/- 2.1%; n = 263). No correlation was detected between the ratio of 20K hGH and age, body height, body weight, or body fat mass in normal subjects. The proportion of 20K hGH was significantly (P < 0.001) higher in patients with active acromegaly (9.2 +/- 2.2%; n = 33) and patients with anorexia nervosa (9.0 +/- 1.9; n = 8), both of which are characterized by chronic elevation of circulating GH levels. The proportion of 20K hGH in successfully treated acromegalic patients did not differ from that in normal subjects, suggesting that GH-producing pituitary tumors secrete a higher proportion of 20K hGH, or that a chronic excess of 22K hGH alters the MCR of 20K hGH. The values in patients with adult GH deficiency, hyperthyroidism, primary hypothyroidism, or GH-independent short stature did not differ from those in normal subjects. The 20K ratio did not change after acute GH provocative tests, such as the insulin tolerance test and the GHRH test. These results suggest that secretion of 20K hGH from the pituitary is under the same control as that of 22K hGH. This new assay may provide a tool for understanding the physiological or pathophysiological role of the 20K hGH isoform.

Transcriptional effects in GH3 cells of Gs alpha mutants associated with human pituitary tumors: stimulation of adenosine 3',5'- monophosphate response element-binding protein-mediated transcription and of prolactin and growth hormone promoter activity via protein kinase A

Somatic mutations of the alpha-subunit of Gs (Gs alpha) have been detected previously at high frequency in human PRL- and/or GH-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection assays to analyze their genomic effects in GH3 rat pituitary cells. We first show that guanosine triphosphatase (GTPase)-deficient Gs alpha subunits (mutated at amino acid 201 or 227) stimulate transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of Gs alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant Gs alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these Gs alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient Gs alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.

Transcriptional effects in GH3 cells of Gs alpha mutants associated with human pituitary tumors: stimulation of adenosine 3',5'-monophosphate response element-binding protein-mediated transcription and of prolactin and growth hormone promoter activity via protein kinase A.

Somatic mutations of the alpha-subunit of Gs (Gs alpha) have been detected previously at high frequency in human PRL- and/or GH-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection assays to analyze their genomic effects in GH3 rat pituitary cells. We first show that guanosine triphosphatase (GTPase)-deficient Gs alpha subunits (mutated at amino acid 201 or 227) stimulate transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of Gs alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant Gs alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these Gs alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient Gs alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.

Screening for growth hormone (GH) gene splice-site mutations in sporadic cases with severe isolated GH deficiency using ectopic transcript analysis.

We screened 10 children with sporadic severe isolated GH deficiency (IGHD) for GH-1 gene splice site mutations using ectopic transcript analysis. None had a history of birth trauma, congenital defects, thyroid disorders, or PRL deficiency. The mean age of these patients at diagnosis was 3.5 yr; the mean height at diagnosis was -4.0 SD score. GH-1 gene deletion was excluded in all cases. Ribonucleic acid (RNA) from lymphocytes was reverse transcribed and amplified by nested polymerase chain reaction, using two primer pairs with annealing sites within exons 2 and 5 of the GH-1 gene. The main polymerase chain reaction fragment obtained was 460 basepairs and proved to be the amplification of the GH-1 transcript. We also found three shorter fragments which were alternatively spliced GH-1 transcripts, including a variant devoid of the first 45 basepairs of exon 3, a second lacking the whole exon 3, and a third one, not previously described, lacking both exon 3 and exon 4. We found the same pattern of alternative splicing in RNA from GH-producing pituitary tumor tissue, which served as a positive control. In 1 of 10 patients, a pathologically shortened main fragment lacking exon 3 was detected. As proved by sequencing genomic DNA, this was the result of a heterozygous splice site mutation, with transversion from G to C of the first base of the donor splice site of intron III generating a new DdeI recognition site. The other allele had no mutation. DdeI digestion enabled us to rule out the defect in the parents' DNA. Thus, the mutation was de novo. As the patient with the mutation displayed the most severe and earliest growth retardation in the study group and had virtually no GH in serum, it must be assumed that the heterozygous genetic defect resulted in a dominant negative effect. The reason for this is still unclear. Recently, within a family that exhibited the autosomal dominant phenotype of IGHD (IGHD-II), a heterozygous point mutation was located 5 bases down-stream from that we describe here. A similar effect on splicing was observed. In conclusion, analysis of ectopic GH-1 transcripts enabled us to detect 1) a new alternatively spliced GH-1 messenger RNA variant lacking exons 3 and 4, and 2) 1 of 10 sporadic cases of severe idiopathic IGHD due to a heterozygous de novo splice site mutation in the GH-1 gene that changes G to C in the first base of intron III.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum insulin-like growth factor-I (IGF-I) in normal Japanese adults.

To evaluate surgical and medical treatment of GH-producing pituitary tumors and GH supplement to GH-deficient patients, we determined the reference values for serum IGF-I in Japanese adults. The serum IGF-I concentration in 454 apparently healthy subjects (246 men aged 21-70 years and 208 women aged 21-72 years) was measured with a commercial RIA kit after acid-ethanol extraction. The concentration of serum IGF-I decreased with age in both sexes. The IGF-I in females was significantly higher than that in males in the group aged 21-30 years and lower in the group aged 61-72 years. We conclude that an age- or sex-related standard is necessary for comparing IGF-I values in patients.

Persistence of rapid growth hormone (GH) pulsatility after successful removal of GH-producing pituitary tumors

Persistence of rapid growth hormone (GH) pulsatility after successful removal of GH-producing pituitary tumors