GH-1 Gene Mutation Research Articles

PMON52 Use of PDE5 Inhibitors as Potential Treatment for Isolated Growth Hormone Deficiency Caused by Alternate Splicing of GH1 Gene

Abstract Mutations in the GH1 gene cause isolated growth hormone deficiency (IGHD) by affecting production, secretion, and stability of growth hormone as well as its binding to GHR. Genetic mutations resulting from splicing errors are known to increase the production of a small 17.5 kD isoform of human growth hormone. This smaller isoform of GH1 is inactive and is linked to IGHD. While studying the impact of small GH isoform on GH production and secretion, we noticed that cells transfect with sequences carrying the mutated GH gene that resulted in splicing defects produce the short 17.5 kD version of GH, looked very different from the control cells with WT GH gene. We found that production of short GH isoform distorts the cell morphology, contributing to detrimental effects seen in the patients with mutations in GH1 gene causing alternate splicing. We hypothesized that cellular distortion caused by small GH isoform and overall GH secretion can be a target and a tool to experimental approaches for reversing the damaging effects of small GH protein, thereby providing potential treatments for IGHD caused by splicing errors. It is known for some time that increasing cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, and recently increase in cGMP levels has also been shown to regulate protein degradation. Inhibitors of phosphodiesterase 5 (PDE5; e.g., Viagra (sildenafil)), which raise cGMP by inhibiting its hydrolysis to GMP, are widely used for the treatment of erectile dysfunction and pulmonary hypertension, and stimulators of soluble guanylyl cyclases (riociguat), which stimulate cGMP synthesis, are used as treatments for pulmonary hypertension and heart failure. Using our model of cellular morphology changes, have investigated the impact of chemicals that enhance cGMP levels by inhibiting PDE5 or activating guanyl cyclases. The cells expressing small isoform of GH consistently show distorted rounded morphology. Treatment with BAY 41-2272, a cGMP inducer slightly improved the cell morphology in small isoform GH expressing cells. BAY 41-2272 did not show any adverse effects of normal and WT GH expressing cells. Treatment of sildenafil to cells expressing 17.5 kD isoform of GH markedly improved the cell morphology compared to untreated cells, especially after 48h when a larger percentage of cells that were distorted (rounder shapes) regained the shape of normal cells expressing WT GH and no damage to cells expressing WT-GH was observed. Another PDE5 inhibitor tadalafil also caused a marked improvement in cell morphology caused by expression of small GH isoform. Overall, it seems the strategy to regulate the cGMP levels in cells transfected in small GH isoform worked and needs further investigation. A detailed analysis of the impact of PDE5 inhibitors on GH expression and secretion will be presented. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Mutations in GH1 gene and isolated growth hormone deficiency (IGHD): A familial case of IGHD type I and systematic review

BackgroundIsolated growth hormone deficiency (IGHD) due to mutations in GH1 gene is a rare disease caused by deficient production of endogenous growth hormone (GH). MethodsWe reported the clinical manifestation and genetic diagnosis (whole exome sequencing [WES], nested PCR Sanger sequencing, and rtPCR) of a family with two children with IGHD type I. We conducted a systematic review of cases with IGHD and compared height, and treatment outcomes in subtypes of IGHD. ResultsThe patients were siblings born of nonconsanguineous parents from the Chinese Han population. The siblings both presented significantly short stature without other apparent abnormalities. The patients carry compound heterozygous mutations in GH1: a deletion and c.456 + 1G > A mutation that led to abnormal splicing. The systematic review identified 365 IGHD cases with GH1 mutations. Among these patients, their body height was most severely impaired in patients with IGHD type Ia, and the height standard deviation score decreased with the age of diagnosis in IGHD type Ia. Patients with IGHD type II had the longest duration of rhGH treatment, while patients with IGHD type Ib had the highest relative height improvement. ConclusionWe identified two patients with IGHD type I caused by compound heterozygotic GH1 deletion and splicing mutation. The analysis of previously published IGHD patients suggests differences in linear growth among subtypes of IGHD.

Porvair plc, UK

Isolated growth hormone deficiency (IGHD) due to mutations in GH1 gene is a rare disease caused by deficient production of endogenous growth hormone (GH).We reported the clinical manifestation and genetic diagnosis (whole exome sequencing [WES], nested PCR Sanger sequencing, and rtPCR) of a family with two children with IGHD type I. We conducted a systematic review of cases with IGHD and compared height, and treatment outcomes in subtypes of IGHD.The patients were siblings born of nonconsanguineous parents from the Chinese Han population. The siblings both presented significantly short stature without other apparent abnormalities. The patients carry compound heterozygous mutations in GH1: a deletion and c.456 + 1G > A mutation that led to abnormal splicing. The systematic review identified 365 IGHD cases with GH1 mutations. Among these patients, their body height was most severely impaired in patients with IGHD type Ia, and the height standard deviation score decreased with the age of diagnosis in IGHD type Ia. Patients with IGHD type II had the longest duration of rhGH treatment, while patients with IGHD type Ib had the highest relative height improvement.We identified two patients with IGHD type I caused by compound heterozygotic GH1 deletion and splicing mutation. The analysis of previously published IGHD patients suggests differences in linear growth among subtypes of IGHD.

GENETIC FORMS OF GROWTH HORMONE INSUFFICIENCY, PECULIARITIES OF CLINIC AND DIAGNOSTIC

The article considers genetic forms of growth hormone insufficiency in children. Analysis of genes, defects of which can lead to severe growth retardation due to somatotropic insufficiency or syndromal disorders, is important not only to improve the diagnosis, but also for choosing of adequate therapy and prediction of final height. Choice of analyzed gene is determined by a combination of clinical data and features of hormone secretion.Are discussed primary IGF-1 deficiency (disturbance of synthesis and secretion of growth hormone (neurosecretory dysfunction), biologically inactive growth hormone (GH1 gene mutation), and partial insensitivity to growth hormone as a result of its receptor defects (GHR gene mutations).The article details the principles of diagnostic search in the genetic forms of growth hormone insufficiency, was analyzed molecular-genetic defects in genes of the growth axis system: growth hormone - insulin-like growth factor 1.

A novel gross indel in the growth hormone releasing hormone receptor gene of Indian IGHD patients

ContextCohort specific mutations in the growth hormone (GH1) and growth hormone-releasing hormone receptor (GHRHR) genes have been reported worldwide in isolated growth hormone deficiency (IGHD) patients. However, limited data is available on ethnically diverse Indian IGHD patients. ObjectiveThe aim of the study was to find GH1 and GHRHR gene mutations in Indian IGHD patients from two unrelated non-consanguineous families. DesignThe 5′ and 3′ untranslated regions (UTRs) and coding regions with splice sites of the GH1 and GHRHR genes were sequenced for all patients (n=6). Family members and 20 controls were evaluated for the sequence variants identified in the index patients. Online bioinformatics tools were used to confirm mutations and their pathogenicity. ResultsGHRHR gene mutations were observed in all patients. Interestingly, a novel indel g.30999250_31006943delinsAGAGATCCA was observed in both the unrelated families. Three patients were homozygous for the novel indel, two were homozygous for the previously reported p.E72X mutation and one was compound heterozygous with both the mutations (indel and p.E72X) in the GHRHR gene. The novel indel has resulted in the loss of 5′ regulatory region and exon 1 of the GHRHR gene impairing the GHRHR expression. All the normal family members were heterozygous either for the indel or p.E72X mutation. None of the patients had GH1 gene mutations. ConclusionsWe describe a novel gross indel in the GHRHR gene resulting in the loss of 5′ regulatory region and GHRHR exon 1 in four IGHD IB patients from two unrelated non-consanguineous Indian families.

Clinical challenges in the management of isolated GH deficiency type IA in adulthood

SummaryIsolated GH deficiency type IA (IGHDIA) is an infrequent cause of severe congenital GHD, often managed by pediatric endocrinologists, and hence few cases in adulthood have been reported. Herein, we describe the clinical status of a 56-year-old male with IGHDIA due to a 6.7 kb deletion in GH1 gene that encodes GH, located on chromosome 17. We also describe phenotypic and biochemical parameters, as well as characterization of anti-GH antibodies after a new attempt made to treat with GH. The height of the adult patient was 123 cm. He presented with type 2 diabetes mellitus, dyslipidemia, osteoporosis, and low physical and psychological performance, compatible with GHD symptomatology. Anti-GH antibodies in high titers and with binding activity (>101 IU/ml) were found 50 years after exposure to exogenous GH, and their levels increased significantly (>200 U/ml) after a 3-month course of 0.2 mg/day recombinant human GH (rhGH) treatment. Higher doses of rhGH (1 mg daily) did not overcome the blockade, and no change in undetectable IGF1 levels was observed (<25 ng/ml). IGHDIA patients need lifelong medical surveillance, focusing mainly on metabolic disturbances, bone status, cardiovascular disease, and psychological support. Multifactorial conventional therapy focusing on each issue is recommended, as anti-GH antibodies may inactivate specific treatment with exogenous GH. After consideration of potential adverse effects, rhIGF1 treatment, even theoretically indicated, has not been considered in our patient yet.Learning points Severe isolated GHD may be caused by mutations in GH1 gene, mainly a 6.7 kb deletion.Appearance of neutralizing anti-GH antibodies upon recombinant GH treatment is a characteristic feature of IGHDIA.Recombinant human IGF1 treatment has been tested in children with IGHDIA with variable results in height and secondary adverse effects, but any occurrence in adult patients has not been reported yet.Metabolic disturbances (diabetes and hyperlipidemia) and osteoporosis should be monitored and properly treated to minimize cardiovascular disease and fracture risk.Cerebral magnetic resonance imaging should be repeated in adulthood to detect morphological abnormalities that may have developed with time, as well as pituitary hormones periodically assessed.

Effect of Zinc Binding Residues in Growth Hormone (GH) and Altered Intracellular Zinc Content on Regulated GH Secretion

Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn(2+) binding to GH through amino acid residues His18, His21, and Glu174 are essential for GH dimerization and might mediate its aggregation and storage in secretory granules. To investigate whether GH-1 gene mutations at these positions interfere with this process, GH secretion and intracellular production were analyzed in GC cells (rat pituitary cell line) transiently expressing wt-GH and/or GH Zn mutant (GH-H18A-H21A-E174A) in forskolin-stimulated vs nonstimulated conditions. Reduced secretion of the mutant variant (alone or coexpressed with wt-GH) compared with wt-GH after forskolin stimulation was observed, whereas an increased intracellular accumulation of GH Zn mutant vs wt-GH correlates with its altered extracellular secretion. Depleting Zn(2+) from culture medium using N,N,N',N'-tetrakis(2-pyridylemethyl)ethylenediamine, a high-affinity Zn(2+) chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn(2+) to culture medium increased intracellular free Zn(2+) levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn(2+) levels after forskolin stimulation. Confocal microscopy analysis of the intracellular secretory pathway of wt-GH and GH Zn mutant indicated that both variants pass through the regulated secretory pathway in a similar manner. Taken together, our data support the hypothesis that loss of affinity of GH to Zn(2+) as well as altering intracellular free Zn(2+) content may interfere with normal GH dimerization (aggregation) and storage of the mutant variant (alone or with wt-GH), which could possibly explain impaired GH secretion.

Molecular characterization in a case of isolated growth hormone deficiency and further prenatal diagnosis of an unborn sibling

Familial isolated growth hormone deficiency (GHD) type 1 is characterized by an autosomal recessive pattern of inheritance with varying degrees of phenotypic severity. We report a proband, with isolated GHD (IGHD) with very early growth arrest and undetectable levels of GH. Homozygous complete deletion of the GH1 gene was identified by real-time/quantitative polymerase chain reaction (RT/q-PCR) and confirmed by an independent molecular genetic method; the multiplex ligation-dependent probe amplification (MLPA) technique. Prenatal diagnosis was offered for the subsequent pregnancy in the mother of our proband. Identical heterozygous deletion of the GH1 gene was detected in both parents. The fetus had a similar homozygous deletion of the GH1 gene. We thus report a unique case with a confirmed mutation in GH1 gene in the proband followed by prenatal detection of the same mutation in the amniotic fluid which to our knowledge hitherto has not been documented from India.

P03-32 Height outcome in late diagnosed growth hormone (GH) deficiency type II with GH-1 gene mutation (GH-R178H) affecting secretion and action: Use of higher than standard dose GH and pubertal suppression to optimise final height

P03-32 Height outcome in late diagnosed growth hormone (GH) deficiency type II with GH-1 gene mutation (GH-R178H) affecting secretion and action: Use of higher than standard dose GH and pubertal suppression to optimise final height

A novel GH-1 gene mutation (GH-P59L) causes partial GH deficiency type II combined with bioinactive GH syndrome

Context and ObjectiveDespite the differences in the main characteristics between the autosomal dominant form of GH deficiency (IGHD II) and the bioinactive GH syndrome, a common feature of both is their impact on linear growth leading to short stature in all affected patients. DesignThe index patient, a boy, was referred for assessment of his short stature (−2.54 SD score) and a delayed bone age of 5.9yrs at the chronological age of 7.7yrs. The GHD was confirmed by standard GH provocation tests, which revealed modestly reduced GH and IGF-I concentrations. Further genetic analysis of GH-1 gene identified heterozygosity for GH-P59L mutation. The secretion of the GH-P59L following stimulation with forskolin was investigated and compared to that of the wt-GH after expression of both GH variants in AtT-20 cells. Based on the position of P59L mutation that lies within a patch of residues composing the GH binding site 1 for GHR, we performed the analysis of GH-P59L binding to GHR by in silico mutagenesis and molecular dynamics simulations, which suggested possible problems in correct binding of GH-P59L to the GHR. Therefore, the functional characterization of this GH mutant was assessed through studies of GHR binding and activation of Jak2/Stat5 signaling pathway. ResultsIn line with the clinical data of the patient GH deficiency is suggested, underlined by GH-secretion studies revealing a moderate difference in secretion between GH-P59L and wt-GH. In addition, further functional characterization of the GH-P59L by studies of GH-receptor binding and activation of Jak2/Stat5 pathway presented with a reduced binding affinity of GH-P59L for GHR and decreased bioactivity compared to the wt-GH. ConclusionsThe clinical data of the patient combined with the laboratory data support the diagnosis of partial IGHD type II. Since the GH deficiency was not total, additional binding and signaling studies were performed, which revealed that the GH-P59L variant displays some of the common features of bioinactive GH syndrome. Taken together, in this study we report a patient suffering from the combination of two growth disorders (alteration of secretion as well as bioactivity) caused by a GH-1 gene alteration highlighting the necessity of functional analysis of any GH variant, despite the presence of obvious clinical features of IGHD type II.

Isolated GH Deficiency Type II: Knockdown of the Harmful Δ3GH Recovers wt-GH Secretion in Rat Tumor Pituitary Cells

Isolated GH deficiency type II (IGHD II) is the autosomal dominant form of GHD. In the majority of the cases, this disorder is due to specific GH-1 gene mutations that lead to mRNA missplicing and subsequent loss of exon 3 sequences. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH (Delta3GH) isoform that reduces the accumulation and secretion of wild-type-GH. At present, patients suffering from this type of disease are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the Delta3GH mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. We developed a strategy involving Delta3GH isoform knockdown mediated by expression of a microRNA-30-adapted short hairpin RNA (shRNA) specifically targeting the Delta3GH mRNA of human (shRNAmir-Delta3). Rat pituitary tumor GC cells expressing Delta3GH upon doxycycline induction were transduced with shRNAmir-Delta3 lentiviral vectors, which significantly reduced Delta3GH protein levels and improved human wild-type-GH secretion in comparison with a shRNAmir targeting a scrambled sequence. No toxicity due to shRNAmir expression could be observed in cell proliferation assays. Confocal microscopy strongly suggested that shRNAmir-Delta3 enabled the recovery of GH granule storage and secretory capacity. These viral vectors have shown their ability to stably integrate, express shRNAmir, and rescue IGHD II phenotype in rat pituitary tumor GC cells, a methodology that opens new perspectives for the development of gene therapy to treat IGHD patients.

A novel splicing mutation in exon 4 (456G&gt;A) of the GH1 gene in a patient with congenital isolated growth hormone deficiency

Isolated Growth Hormone Deficiency (IGHD) due to GH1 gene defects has a variable inheritance pattern: autosomal recessive, autosomal dominant, and X-linked. the autosomal dominantly inherited form, IGHD II, is mainly caused by heterozygous mutations of splicing around the exon 3/IVs3 boundary region of the GH1 gene resulting in exon 3 skipping of transcripts. We have previously reported findings on GH1 gene mutations in 28 russian patients with severe congenital IGHD (-3.22+/-1.2 height sDs at the age of 1yr); five heterozygous dominant negative splice site mutations in intron 2, intron 3, and exon 4 of the GH1 gene were identified in 32.1% of the cohort. In the present report we describe a novel 456G>A heterozygous mutation of splicing of the last base of the 3'-acceptor splice site of exon 4 within the GH1 in a 4.2-year old, extremely short (-5.32 height sDs) girl with congenital IGHD. the mutation involves a highly conserved GGGgtg sequence of the exon 4/IVs4 boundary region of the GH1 gene. the predicted effect of the 456 G>A mutation is perturbed splicing with possible skipping of exon 4 of the GH1 gene. the novel heterozygous 456 G>A mutation in exon 4 expands the spectrum of dominant negative splicing defects within the GH1 gene, responsible for congenital IGHD.

Isolated Autosomal Dominant Growth Hormone Deficiency: Stimulating MutantGH-1Gene Expression DrivesGH-1Splice-Site Selection, Cell Proliferation, and Apoptosis

The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.

Clinical Characteristics, Etiologies and Pathophysiology of Patients with Severe Short Stature with Severe GH Deficiency: Questionnaire Study on the Data Registered with the Foundation for Growth Science, Japan

In this study, we sent questionnaires to doctors treating severe short stature with severe GH deficiency (GHD) (height SDS (HtSDS) below -4 and all peak GH to provocative stimuli below 2 micro/L) (abbreviated as Severe Case), and obtained effective replies of 51 cases. The clinical characteristics, etiologies, and pathophysiology of these patients were examined. Among the 51 Severe Cases no consanguinity was observed, 44 were IGHD (24 males and 20 females), 3 were GH-1 gene deletion, 2 were Pit-1 gene mutation, and 2 were achondroplasia. HtSDS in these Severe Cases was already remarkably low at 12 (-3.0) and 24 months old (-3.9), while their birth weight and birth length were within normal ranges. Among 44 patients with IGHD, 12 were isolated GHD, and the remaining 32 were combined pituitary hormone deficiency (CPHD). Pituitary MRI was undergone in 25 idiopathic GHD, and abnormal findings (pituitary atrophy, interruption of stalk, and ectopic posterior lobe) were observed in 21 patients with CPHD. More than half of these patients had the history of breech delivery. Three patients with GH-1 gene mutation showed normal pituitary MRI, whereas one of two patients with Pit-1 mutation showed pituitary atrophy and narrowing of pituitary stalk. In conclusion, Severe Cases tended to have CPHD, and the incidence of Severe Case was only 0.6% of total IGHD. Although GHD due to genetic disorders is considered to be extremely rare (0.06% of total IGHD), the incidence reaches high levels (9.8%) among Severe Cases. Growth disorders in these Severe Cases seem to occur soon after delivery. Much earlier diagnosis and hGH treatment are desirable to attain better final height in the Severe Cases. GH-1 and Pit-1 gene analyses are crucial, when genetic abnormalities other than achondroplasia are suspected.

The serum growth hormone (GH) response to provocative tests is dependent on type of assay in autosomal dominant isolated GH deficiency because of an ARG(183)HIS (R183H) GH-I gene mutation.

The clinical appearance and genetic basis of familial isolated growth hormone deficiency (IGHD) are heterogeneous and are associated with at least four types of Mendelian disorders: two forms with autosomal recessive inheritance (IGHD type 1A and 1B), one with autosomal dominant inheritance (IGHD type 2), and one with X-linked inheritance (IGHD type 3) (1)(2). Recently, Deladoey et al. (3) reported a new form of IGHD type 2 in four unrelated families. These patients carried an ARG183HIS (R183H) GH-1 mutation. On the basis of in vivo and in vitro experiments, the authors reported that the R183H mutant GH peptide severely impaired GH-regulated secretion. Furthermore, this GH mutant was found to have an effect on GH receptor/GH-binding protein (GHR/GHBP) transcription identical to that of the 22-kDa GH when tested in a human hepatoma cell line (4). Human GH represents a family of proteins rather than a single hormone. Indeed, GH is among the more heterogeneous polypeptide hormones, and the proportions of immunoreactive forms in plasma vary with time after a GH secretory pulse (5). The simultaneous use of an immunoassay that detects most serum GH isoforms (total GH) and one specific for the 22-kDa GH form makes it possible to uncover changes in the pattern of secretion of GH isoforms. Moreover, discrepancies between GH immunologic and biological activities have been reported (6). It has therefore been proposed that the ratio of non-22-kDa GH to 22-kDa GH isoforms may have important implications for normal and abnormal growth (7). We studied two nonrelated patients from Argentina with severe short stature and IGHD type 2 attributable to the R183H GH-1 gene mutation and found that the evaluation of GH response to provocative tests might be misleading in these patients, depending on the type of assay used to assess …

Isolated GH deficiency (IGHD) type II: imaging of the pituitary gland by magnetic resonance reveals characteristic differences in comparison with severe IGHD of unknown origin.

To determine the specific morphology of the pituitary gland in children with severe isolated GH deficiency due to GH-1 gene mutations (IGHD type II). The pituitary gland morphology in magnetic resonance imaging (MRI) of children with IGHD type II was analyzed and compared with the findings in a group of children with comparably severe IGHD of unknown origin. In addition, the birth histories of both groups were studied. Thirteen children with IGHD type II were diagnosed in seven European children's hospitals and they carried a corresponding GH-1 gene mutation. For comparison, we selected from a group of 66 MRI-studied GH-insufficient subjects diagnosed in our clinic, all children with severe IGHD (all GH peaks <4 microg/l) who had no GH-1 gene mutation, no first-grade relative with IGHD and no septo-optic dysplasia. Sagittal and coronal images of the brain were analyzed for the presence of any malformation of the pituitary gland and the intracranium. The height of each adenohypophysis was measured in a strict midline sagittal image for quantification of the gland's size. In addition, patients' files were reviewed for birth trauma or breech delivery. Normal MRI morphology of the pituitary gland was observed in all patients of the familial IGHD type II group (P<0.003) in which, however, five of thirteen patients (38%) exhibited a mild hypoplasia of their gland (mean sagittal adenohypophysial height -1.0+/-0.03 SD score (SDS)). In contrast, the pituitary gland in the idiopathic group showed a definitive malformation with hypoplasia of pituitary stalk and adenohypophysis in all cases, while ectopia of the neurohypophysis was present in nine of the ten cases. The adenohypophysis was significantly smaller in the idiopathic group (mean sagittal adenohypophysial height -3.2+/-0.3 SDS) (P<0.0001). All thirteen birth histories in the familial group (IGHD type II) were unremarkable while, in the idiopathic group, three of eight available birth histories recorded a breech delivery or traumatic birth (37.5%) (P<0.05). This study shows for the first time that MRI pituitary morphology may correlate with the etiology of severe IGHD: normal morphology suggests the presence of GH-1 gene mutations, while severe hypoplasia with malformation have other causes which might include so far unknown genetic defects as well as traumatic insults.

Autosomal Dominant GH Deficiency Due to an Arg183His GH-1 Gene Mutation: Clinical and Molecular Evidence of Impaired Regulated GH Secretion

Autosomal Dominant GH Deficiency Due to an Arg183His GH-1 Gene Mutation: Clinical and Molecular Evidence of Impaired Regulated GH Secretion

Reduced human growth hormone (hGH) bioactivity without a defect of the GH-1 gene in three patients with rhGH responsive growth failure.

A GH deficiency-like phenotype with normal or high hGH secretion, pathologically low IGF-I serum levels, and catch-up growth under treatment with recombinant hGH is suggestive of the presence of biologically inactive hGH syndrome, whose presumably heterogenous molecular basis is substantially unknown. Serum samples from patients who fulfilled the above criteria and from controls with idiopathic short stature were measured by polyclonal hGH-RIA, Nb2 rat lymphoma proliferation assay, and hGH immunofunctional assay (IFA). If assays were suggestive of the presence of bioinactive GH, mutational analysis of the hGH-1 gene was performed. Three patients were selected because of clinical and biochemical evidence. At the time of diagnosis mean age was 3.4 (2.2, 3.5, 4.5) years, mean height -3.5 (-2.8, -3.6, -4.2) SD score (SDS) and mean growth rate -1.5 (-1.4, -1.5, -1.6) SDS. Mean IGF-I serum levels were -1.9 (-0.7, -2.4, -2.5) SDS and mean IGFBP-3 serum levels -1.2 (-1.1, -1.2, -1.2) SDS. Stimulated and spontaneous GH peaks measured by a polyclonal radioimmunoassay were all above 14 microg/l. GHBP serum levels were normal, and antihGH antibodies were not detected. Therapy with rhGH was effective in causing catch-up growth of the three children with an initial mean growth rate of + 2.9 (+ 1.7, + 2.1, + 5.0) SDS, and normalization of IGF-I (mean: -0.66 SDS: -1.8, - 1.2, + 1.1 SDS) and IGFBP-3 serum levels (mean: + 0.81 SDS: -0.2, + 0.8, + 1.8 SDS). In comparison to controls, the patients' serum hGH levels were much lower when measured by Nb2 rat lymphoma cell proliferation bioassay (mean: -2.3 SDS, range: -1.7- -4.1) and by the immunofunctional assay (IFA) (-1.5 SDS, range: -0.2- -3.1) than by RIA. Retesting of two of the three patients including an one year break of therapy confirmed the rhGH dependence of growth in spite of normal endogenous GH secretion. Radioactive direct sequencing of both strands of PCR-amplified genomic DNA and cDNA excluded a GH-1 gene mutation in all three children. Mutations of the GH-1 gene are probably not the main genetic defect in children with biologically inactive hGH syndrome. Posttranslational processing of hGH might reduce the biological activity of the normal translation product.

Hereditary isolated growth hormone deficiency caused by GH1 gene mutations in Japanese patients

Most patients with hereditary isolated growth hormone deficiency (IGHD) are either heterozygous or homozygous for a growth hormone (GH) gene abnormality. GH1 gene deletions (6.7 and 7.6 kb) from eight Japanese families with IGHD type IA has been detected by Southern blot analysis or polymerase chain reaction and Smal digestion. Heterozygous point mutations at the donor splice site of intron 3 in the GH1 gene have been identified among autosomal dominant IGHD type II patients. Recently, we have identified two kinds of splicing mutations in intron 3 in four Japanese families with IGHD type II. We believe a newly diagnosed G to A mutation at the fifth base of intron 3 in a Japanese family is responsible for the IGHD type II phenotype.

Prevalence of human GH-1 gene alterations in patients with isolated growth hormone deficiency.

Human GH is encoded by the GH-1 gene which belongs to the GH gene cluster encompassing a distance of about 65 kb on the long arm of chromosome 17. Familial isolated growth hormone deficiency (IGHD) is associated with at least four Mendelian disorders. These include two forms that have autosomal recessive inheritance (IGHD types IA and IB) as well as autosomal dominant (IGHD type II) and X-linked (IGHD III) forms. The aim of our study was to evaluate the prevalence of all GH-1 gene alterations by sequencing the whole GH-1 gene after PCR amplification among 151 affected subjects from 83 families with severe IGHD (height: <-4.5 SD score). A high frequency of GH-1 gene alterations was found in families with IGHD type IA (8/12, 66.7%), whereas only a low frequency of GH-1 gene defects was present in all the other GH-deficient families (7/71, 9.9%). The absolute frequency of GH-1 gene deletions was 8.7% (6/69), 11.8% (4/34), and 18.7% (9/48) in Northern Europeans, Mediterraneans, and Asians, respectively, giving an overall frequency of 12.5% (19/151). The sizes of the deletions were heterogeneous with the most frequent (78%) being 6.7 kb. In addition, 6% (9/151) of the patients presented GH-1 gene mutations such as frameshift, stop codon and splicing error. Furthermore, total GH-1 gene abnormalities varied among different populations from 11.6% in Northern Europe, 14.7% in Mediterranean countries and 31.2% in Asia. Most striking, however, was the low frequency rate of 1.7% (2/119) of GH-1 gene mutations responsible for the most common phenotype of IGHD, namely type IB, among the subjects characterized by the production of deficient but detectable amounts of GH after provocative stimuli. This finding underlines the necessity to focus rather on the promoter region of the GH-1 gene (cis-acting elements and trans-acting factors), and on other candidate genes specific for the GH axis than the GH-1 gene itself to define genetically the IGHD type IB phenotype in more detail.