Isolated Growth Hormone Deficiency Type Research Articles

A GHRHR founder mutation causes isolated growth hormone deficiency type IV in a consanguineous Pakistani family.

Isolated growth hormone deficiency (IGHD) is caused by a severe shortage or absence of growth hormone (GH), which results in aberrant growth and development. Patients with IGHD type IV (IGHD4) have a short stature, reduced serum GH levels, and delayed bone age. To identify the causative mutation of IGHD in a consanguineous family comprising four affected patients with IGHD4 (MIM#618157) and explore its functional impact in silico. Clinical and radiological studies were performed to determine the phenotypic spectrum and hormonal profile of the disease, while whole-exome sequencing (WES) and Sanger sequencing were performed to identify the disease-causing mutation. In-silico studies involved protein structural modeling and docking, and molecular dynamic simulation analyses using computational tools. Finally, data from the Qatar Genome Program (QGP) were screened for the presence of the founder variant in the Qatari population. All affected individuals presented with a short stature without gross skeletal anomalies and significantly reduced serum GH levels. Genetic mapping revealed a homozygous nonsense mutation [NM_000823:c.G214T:p.(Glu72*)] in the third exon of the growth-hormone-releasing hormone receptor gene GHRHR (MIM#139191) that was segregated in all patients. The substituted amber codon (UAG) seems to truncate the protein by deleting the C-terminus GPCR domain, thus markedly disturbing the GHRHR receptor and its interaction with the growth hormone-releasing hormone. These data support that a p.Glu72* founder mutation in GHRHR perturbs growth hormone signaling and causes IGHD type IV. In-silico and biochemical analyses support the pathogenic effect of this nonsense mutation, while our comprehensive phenotype and hormonal profiling has established the genotype-phenotype correlation. Based on the current study, early detection of GHRHR may help in better therapeutic intervention.

Abstract 80: A case of familial growth hormone deficiency

Here by presenting, a case of familial short stature where in 2 siblings presented with failure to gain height. Similar history was present in maternal uncle. They had no history of peri/ intra / post-natal complications. They had normal developmental milestones and had normal scholastic performance. On examination there were no other syndromic features. Height was - 6 SDS and weight was below 5 centiles. Upper to lower segment ratio was preserved. SMR was Prepubertal. Systemic examination was unremarkable. On evaluation, IGF 1 levels were low. Dynamic pituitary testing for growth hormone deficiency failed to stimulate growth hormone. Pituitary Imaging with contrast MRI showed hypoplastic pituitary. In view of both siblings having short stature and similar history in maternal uncle, a whole exome sequencing was done, which showed homozygous mutation for Growth hormone releasing hormone receptor, Exon 3, c.214G>T (p. Glu72Ter). Thus, the diagnosis was confirmed as isolated growth hormone deficiency type 4.

Isolated growth hormone deficiency type IA due to a novel GH1 variant: a case report

BackgroundA case of isolated growth hormone deficiency type IA (IGHD IA) caused by novel compound heterozygous mutation in the GH1 gene was reported in this study, which aimed to provide insights that will benefit future diagnosis and treatment.Case presentationWe analyzed and summarized the clinical data and genetic test results from a patient with IGHD admitted in March 2019 to the Department of Pediatrics Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. We described the results from a 1-year-9-months old female, whose chief complaint was “growth retardation for more than one year”. Her birth length was 49.0 cm, and her birth weight was 3.05 kg. Suboptimal intake (breastfeeding) jaundice lasted for approximately two months following birth. When evaluated at the age of 1-year-9-months old, the patient’s height was 61.0 cm (− 7.24 SD), and her weight was 6.4 kg (− 1.50 SD). The patient’s physical characteristics included yellowish hair, large and unclosed anterior fontanelles, raised forehead, and a low and flat nose. The major abnormalities observed from the auxiliary examinations included low GH (< 0.05 μg/l), low IGF-1 (16.99 μg/l), and elevated TSH (6.97 mIU/l). Genetic testing revealed two heterozygous variants: a splicing mutation (NG_011676.1(NM_022560.4): c.10 + 1G>T, inherited from her mother) in intron 1 of the GH1 gene and a deletion that encompassed the same gene (chr17: 61973811–61996255, inherited from her father). After hormone replacement therapy with L-thyroxine and recombinant human GH (rhGH), the patient’s thyroid function returned to normal, and her serum IGF-1 level significantly improved, which resulted in an accelerated increase in height.ConclusionThis study described a case of IGHD caused by novel compound heterozygous mutations in the GH1 gene. This study suggested that closer attention should be directed to genetic testing and diagnosis based on clinical characteristics to avoid misdiagnosis.

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.

SUN-245 Heterozygous GH1 p.R209H Variant within a Large Pedigree May Mimic a Growth Hormone Insensitivity Pattern

Isolated growth hormone deficiency (IGHD) is most frequently caused by mutations in the GH1 gene. Pathogenic mutations in the GHRHR and GHSR have also been reported to cause IGHD. Individuals with IGHD type II present with variable clinical phenotype. Autosomal dominant GH1 p.R209H variant impairs GH secretion despite normal GH synthesis. The aim of this work is to expand the clinical and biochemical phenotype of heterozygous GH1 p.R209H missense mutation (previously known as p.R183H) in a large Argentinean pedigree. We report a non-consanguineous 4 generation pedigree. Two affected members showed classical IGHD phenotype: short stature (height -4.13 and -2.6 SD), low GH peak after arginine/clonidine provocative tests (3.6 and 2.6 ng/ml, cut-off value 4.7 ng/ml, GH IS 98/574, Immulite), non-detectable IGF-1, and low IGFBP-3 (-2.83 and -2.28 SD). Brain MRI showed anterior pituitary gland of 3.3 mm and 2.5 mm, respectively. The use of a customized panel for congenital hypopituitarism using single molecule molecular inversion probes sequencing (smMIPS) revealed a c.626G>A transition in GH1 gene, predicted to result in p.R209H mutation. The variant was subsequently confirmed by Sanger sequencing. Parents affected with the same variant presented either short (-1.91 SDS) or normal stature (-0.72 SDS). The patients’ response to rhGH treatment was adequate (Δ heights were 2.49 and 1.37 SDS in 2.4 and 1.0 year of treatment, respectively). Additionally, 4/9 siblings within this pedigree had short stature (mean height -2.48 ± 0.62 SD) with mild or no clinical phenotype of GHD, normal GH response to provocative pharmacologic tests (maximum GH peaks 5.46 to 10.9 ng/ml), associated to low serum IGF-1 (mean -3.27 ± 0.89 SD) and IGFBP3 (mean -2.61 ± 0.42 SD), resembling a pattern of growth hormone insensitivity (GHI). IGF-1 generation tests (GH dose 33 µg/kg.day) showed a mean increment in IGF-1 of 3.25 times over basal, confirming adequate GH sensitivity. Despite the absence of complete GHD phenotype, data from the extended pedigree suggested GH1 as the initial candidate gene, identifying the same pathogenic GH1 variant in these four siblings. Brain MRI showed normal height of the anterior pituitary gland (range 3.9 to 4.6 mm). Their affected father had normal stature (-1.69 SDS) with low serum IGF-1 (64 ng/mL). We have shown that members of a large pedigree affected with the p.R209H GH1 variant showed either a classical clinical and biochemical pattern of GHD or an initial biochemical pattern resembling GHI. This variability could lead in certain cases to an elusive diagnosis of GHD when the classical phenotype is absent. Therefore, these results highlight the need to explore GH1 gene in children with short stature associated to low IGF-1 and IGFBP-3 serum levels even when GH response to pharmacological tests is normal.

Biological Significance of Anti-GH Antibodies in Children Treated with rhGH

Background: The occurrence of antidrug antibodies is common in children treated with recombinant human growth hormone (rhGH). However, their clinical significance is unclear. Objective: This study aimed to examine the clinical significance of anti-GH antibodies by analyzing the phenotype of patients who tested positive in relation to the quantity of anti-GH antibodies. Method: In this laboratory-based retrospective study encompassing a time span of 6 years, all positive samples were identified, and senders were contacted. Anti-GH antibodies were measured using a radioprecipitation assay; positive samples underwent a confirmatory assay. Results: Out of a total of 104 samples from 66 patients, positive test results were found in 28 samples from 13 patients. Clinical data were available from all but one. The group with positive test results comprised 6 patients with a normal response to GH provocative tests (group A) and 6 with an insufficient response or with isolated GH deficiency (IGHD) type 1A (group B). Diagnoses in group A were neurosecretory dysfunction, bioinactive GH syndrome and constitutional delay of growth and puberty. Diagnoses in group B were IGHD type 1A, septo-optic dysplasia, and cerebral midline defect with multiple pituitary hormone deficiency. Insufficient growth response to rhGH was absent except in one sibling pair with IGHD type 1A and a patient with cerebral midline defect. These patients had the highest concentrations of anti-GH antibodies. Conclusions: The biological significance of anti-GH antibodies seems to be limited to patients with high concentrations of anti-GH antibodies. For all other patients, we recommend a careful “wait and see” strategy and monitoring antibody titers.

Isolated Growth Hormone Deficiency Type II due to a novel GH1 mutation: A Case Report

Isolated Growth Hormone Deficiency Type II due to a novel GH1 mutation: A Case Report

Endoplasmic Reticulum (ER) Stress and Endocrine Disorders.

The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article.

Rescue of Isolated GH Deficiency Type II (IGHD II) via Pharmacologic Modulation of GH-1 Splicing.

Isolated GH deficiency (IGHD) type II, the autosomal dominant form of GHD, is mainly caused by mutations that affect splicing of GH-1. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH isoform that reduces the accumulation and secretion of wild-type-human GH (wt-hGH). Usually, isolated GHD type II patients 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 17.5-kDa GH isoform on the pituitary gland, which can eventually lead to other hormonal deficiencies. Here, we tested the possibility to restore the constitutive splicing pattern of GH-1 by using butyrate, a drug that mainly acts as histone deacetylase inhibitor. To this aim, wt-hGH and/or different hGH-splice site mutants (GH-IVS3+2, GH-IVS3+6, and GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GHRHR) (GC-GHRHR). Upon butyrate treatment, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed increased GH transcript level, intracellular GH content, and GH secretion when compared with the corresponding untreated condition. The effect of butyrate was most likely mediated by the alternative splicing factor/splicing factor 2. Overexpression of alternative ASF/SF2 in the same experimental setting, indeed, promoted the amount of full-length transcripts thus increasing synthesis and secretion of the 22-kDa GH isoform. In conclusion, our results support the hypothesis that modulation of GH-1 splicing pattern to increase the 22-kDa GH isoform levels can be clinically beneficial and hence a crucial challenge in GHD research.

Novel growth hormone-releasing hormone receptor gene mutations in Turkish children with isolated growth hormone deficiency.

Objective: Isolated growth hormone deficiency (IGHD) is defined as a medical condition associated with growth failure due to insufficient production of GH or lack of GH action. Mutations in the gene encoding for GH-releasing hormone receptor (GHRHR) have been detected in patients with IGHD type IB. However, genetic defects on GHRHR causing IGHD in the Turkish population have not yet been reported. To identify mutations on GHRHR gene in a population of Turkish children with IGHD.Methods: Ninety-six Turkish children with IGHD were included in this study. Exon1-13 and exon/intron boundaries of GHRHR were amplified by suitable primers. The polymerase chain reaction products for GHRHR gene were sequenced with primers.Results: We analyzed the GHRHR gene for mutations in ninety-six patients with IGHD based on sequence results. We identified novel p.K264E, p.S317T, p.S330L, p.G369V, p.T257A and C base insertion on position 380 (c.380inserC) mutations. In 5 of the patients, the mutation was homozygote and in 1-heterozygote (p.S317T).Conclusion: Six new missense mutations and one first case of insertion mutations for the GHRHR gene are reported.

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.

Growth Hormone (GH-1) Gene Deletions in Children with Isolated Growth Hormone Deficiency (IGHD)

To detect growth hormone GH-1 gene deletions (6.7 kb, 7.6 kb, 7 kb) in familial/nonfamilial isolated growth hormone deficiency (IGHD) and note their clinical and investigative profile. Thirty (M16,F14) prepubertal IGHD patients aged 0.25 to 14 y, from 25 families were screened. Duration of growth failure, relevant history, clinical phenotype, and height SDS were recorded. Peak GH response to Clonidine (0.15 mg/m(2)), IGF-1, IGFBP-3 and pituitary/target gland hormones were studied. Genomic DNA of patients and family was analysed by PCR and DNA fragments were visualized on agarose gel electrophoresis. This series was divided into deletion +ve, Group I (n=12,40%) inclusive of six familial/six nonfamilial patients, and deletion -ve Group II (n=18,60%), 5 familial/13 nonfamilial cases; in total 11/30 were familial. Onset of growth failure was earlier in Group I (p<0.001) mean 1.1 vs 4.7 y. Mean height SDS was -7 vs. -4.5 in Groups I/II (p<0.01), age at presentation 5.1 vs 8.6 y. Overhanging forehead, prominent eyes, hypoplastic facies characterized Group I with FBS <50 mg/dl in 50% and very low peak GH <0.04 vs 2.04 ng/ml (p<0.001) in Group II. In both groups IGF-1 and IGFBP3 were low, other hormones were normal and MRI showed hypoplastic adenohypophysis. 40% had GH-1 gene deletion (6.7 kb deletion in 83%, 7.6 kb and a compound heterozygote in 8% each). In this series of 30 IGHD patients, frequency of GH-1 gene deletions (12/30) was 40%, and 54% among familial patients, and 31% with height SDS>-4. 83% had 6.7 kb deletion. Height SDS>-4, clinical phenotype, peak GH<1 ng/ml and hypoglycemia characterised IGHD Type IA.

Pharmacologic Correction of Dominant‐Negative GH1 Deficiency Causing Mutations

Dominant-negative growth hormone gene (GH1) mutations cause familial isolated growth hormone deficiency type II (IGHD II), which is characterized by GH deficiency, occasional multiple anterior pituitary hormone deficiencies, and anterior pituitary hypoplasia. We have previously shown that 17.5-/22-kDa GH1 transcript ratios correlate with the severity of the IGHD II phenotype. We hypothesized that different pharmaceutical agents could affect the GH1 transcript ratio by modulating alternative splicing. We exposed peripheral blood mononuclear cells from IGHD II patients and unaffected family members to different pharmacologic agents and then determined the 17.5-/22-kDa transcript ratios by real-time PCR. Dexamethasone and digoxin significantly increased the 17.5-/22-kDa transcript ratio, while sodium butyrate and 5-iodotubericidin significantly decreased the ratio. Since we have previously shown that the ratio of the 17.5-/22-kDa GH1 transcripts correlates with severity of the IGHD II phenotype, our findings here suggest that selected previously unconsidered agents could possibly reduce the severity of IGHD II, while other agents could possibly exacerbate the disease phenotype.

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 Molecular Basis for Variation in Clinical Severity of Isolated Growth Hormone Deficiency Type II

Dominant-negative GH1 mutations cause familial isolated growth hormone deficiency type II (IGHD II), which is characterized by GH deficiency, occasional multiple anterior pituitary hormone deficiencies, and anterior pituitary hypoplasia. The basis of the variable expression and progression of IGHD II among relatives who share the same GH1 mutation is poorly understood. We hypothesized that the cellular ratios of mutant/normal GH1 transcripts would correlate with the severity of the IGHD II phenotype. We determined the relative amounts of mutant and normal GH1 transcripts in cell lines and correlated transcript ratios with severity. Members of the same IGHD II kindred were genotyped for the GH1 E3+1 G/A mutation by DNA sequencing. Ratios of their 17.5-kDa (mutant)/22-kDa (normal) GH1 transcripts were determined in cultured lymphocytes (CLs), and these ratios were correlated with height sd scores obtained before GH replacement therapy. Ratios of 17.5-/22-kDa GH1 transcripts in CLs from family members with the same IGHD II mutation correlated with differences in their height SD scores. Our findings suggest that expression levels of both the mutant and normal GH1 allele are important in the pathogenesis of IGHD II, that the ratio of mutant/normal transcripts may be a predictive marker of the penetrance and severity of IGHD II, and that CLs may be useful as surrogates to study GH1 transcript expression of subjects whose anterior pituitary cells are not available.

Mutation Analysis of the Muscarinic Cholinergic Receptor Genes in Isolated Growth Hormone Deficiency Type IB

Isolated GH deficiency (IGHD) is familial in 5-30% of patients. The most frequent form (IGHD-IB) has autosomal recessive inheritance, and it is known that it can be caused by mutations in the GHRH receptor (GHRHR) gene or in the GH gene. However, most forms of IGHD-IB have an unknown genetic cause. In normal subjects, muscarinic cholinergic stimulation causes an increase in pituitary GH release, whereas its blockade has the opposite effect, suggesting that a muscarinic acetylcholine receptor (mAchR) is involved in stimulating GH secretion. Five types of mAchR (M(1)-M(5)) exist. A transgenic mouse in which the function of the M(3) receptor was selectively ablated in the central nervous system has isolated GH deficiency similar to animals with defective GHRH or GHRHR gene. We hypothesized that mAchR mutations may cause a subset of familial IGHD. After confirming the expression of M(1)-M(5) receptor mRNA in human hypothalamus, we analyzed the index cases of 39 families with IGHD-IB for mutations in the genes encoding for the five receptors. Coding sequences for each of the five mAchRs were subjected to direct sequencing. In one family, an affected member was homozygous for a M(3) change in codon 65 that replaces valine with isoleucine (V65I). The V65I receptor was expressed in CHO cells where it had normal ability to transmit methacholine signaling. mAchR mutations are absent or rare (less than 2.6%) in familial IGHD type IB.

Isolated growth hormone deficiency type II caused by a point mutation that alters both splice site strength and splicing enhancer function

A heterozygous single base mutation in the human growth hormone (GH) gene (GH-1) was identified in a family presenting with isolated GH deficiency type II (IGHD II). Affected individuals have a guanine to adenine transition at the first nucleotide of exon 3 (E3+1 G-->A) that results in exon skipping and production of a dominant-negative 17.5-kDa isoform. We show that the mechanistic basis for exon skipping is due to the unique position of this mutation because it weakens the 3' splice site and simultaneously disrupts a splicing enhancer located within the first seven bases of exon 3. A G-->T mutation at this same position not only affects splicing but also results in a premature stop codon for those transcripts that include exon 3. Thus, mutations that alter the first nucleotide of exon 3 illustrate the various mechanisms by which changes in sequence can cause disease: splice site selection, splicing enhancer function, messenger RNA decay, missense mutations, and nonsense mutations. For IGHD II, only exon skipping leads to production of the dominant-negative isoform, with increasing skipping correlating with increasing disease severity.

Growth Hormone Deficiency and Splicing Fidelity: TWO SERINE/ARGININE-RICH PROTEINS, ASF/SF2 AND SC35, ACT ANTAGONISTICALLY

The majority of mutations that cause isolated growth hormone deficiency type II are the result of aberrant splicing of transcripts encoding human growth hormone. Such mutations increase skipping of exon 3 and encode a 17.5-kDa protein that acts as a dominant negative to block secretion of full-length protein produced from unaffected alleles. Previously, we identified a splicing regulatory element in exon 3 (exonic splicing enhancer 2 (ESE2)), but we had not determined the molecular mechanism by which this element prevents exon skipping. Here, we show that two members of the serine/arginine-rich (SR) protein superfamily (ASF/SF2 and SC35) act antagonistically to regulate exon 3 splicing. ASF/SF2 activates exon 3 inclusion, but SC35, acting through a region just downstream of ESE2, can block such activation. These findings explain the disease-causing mechanism of a patient mutation in ESE2 that creates a functional SC35-binding site that then acts synergistically with the downstream SC35 site to produce pathological levels of exon 3 skipping. Although the precedent for SR proteins acting as repressors is established, this is the first example of a patient mutation that creates a site through which an SR protein represses splicing.

Rescue of Pituitary Function in a Mouse Model of Isolated Growth Hormone Deficiency Type II by RNA Interference

Splicing mutations in the human GH (hGH) gene (GH-1) that cause skipping of exon 3 result in a form of GH deficiency termed isolated GH deficiency type II (IGHD II). The GH-1 gene contains five exons; constitutive splicing produces the wild-type 22-kDa hormone, whereas skipping of exon 3 results in transcripts encoding a 17.5-kDa isoform that acts as a dominant-negative to block secretion of the wild-type hormone. Common characteristics of IGHD II include short stature due to impaired bone elongation, growth, and, in severe cases, anterior pituitary hypoplasia. Typically, IGHD II is treated by sc delivery of hGH, which can rescue stature but, unfortunately, does not inhibit pituitary hypoplasia. Direct destruction of transcripts encoding the dominant-negative 17.5-kDa isoform should both rescue stature and prevent hypoplasia. Here, we have used delivery of short hairpin RNAs to rescue a murine model of IGHD II by specifically targeting transcripts encoding the 17.5-kDa isoform using RNA interference. To our knowledge, this is the first example where a short hairpin RNA has been expressed to specifically degrade an incorrectly spliced transcript and rescue a dominant-negative disease phenotype in vivo.