105 research outputs found
Repair of Aberrant Splicing in Growth Hormone Receptor by Antisense Oligonucleotides Targeting the Splice Sites of a Pseudoexon
Context: The GH receptor (GHR) pseudoexon 6 Psi defect is a frequent cause of GH insensitivity (GHI) resulting from a non-functioning GH receptor (GHR). It results in a broad range of phenotypes and may also be present in patients diagnosed as idiopathic short stature.Objective: Our objective was to correct aberrant GHR splicing and inclusion of 6 Psi using exon-skipping antisense oligonucleotides (ASOs).Design and Setting: Three ASOs binding the 5' (ASO-5), 3' (ASO-3), and branch site (ASO-Br) of 6 Psi were tested in an in vitro splicing assay and a cell transfection system. The wild-type (wt) and mutant (mt) DNA minigenes (wt- and mtL1-GHR6 Psi-L2, respectively) were created by inserting the GHR 6 Psi in a well-characterized splice reporter (Adml-par). For the in vitro splicing assay, the wt- and mtL1-GHR6 Psi-L2 were transcribed into pre-mRNA in the presence of [alpha P-32]GTP and incubated with ASOs in HeLa nuclear extracts. For the cell transfection studies, wt-and mtL1-GHR6 Psi-L2 cloned into pcDNA 3.1 were transfected with ASOs into HEK293 cells. After 48 h, RNA was extracted and radiolabeled RT-PCR products quantified.Results: ASO-3 induced an almost complete pseudoexon skipping in vitro and in HEK293 cells. This effect was dose dependent and maximal at 125-250 nM. ASO-5 produced modest pseudoexon skipping, whereas ASO-Br had no effect. Targeting of two splice elements simultaneously was less effective than targeting one. ASO-Br was tested on the wtL1-GHR6 Psi-L2 and did not act as an enhancer of 6 Psi inclusion.Conclusions: The exon-skipping ASO approach was effective in correcting aberrant GHR splicing and may be a promising therapeutic tool. (J Clin Endocrinol Metab 95: 3542-3546, 2010
Three-Dimensional Model of Human Nicotinamide Nucleotide Transhydrogenase (NNT) and Sequence-Structure Analysis of its Disease-Causing Variations
Medical Research Council. Grant Numbers: MR/K020455/1, MR/K021613/
Severe loss-of-function mutations in the adrenocorticotropin receptor (ACTHR, MC2R) can be found in patients diagnosed with salt-losing adrenal hypoplasia
Objective: Familial glucocorticoid deficiency type I (FGD1) is a rare form of primary adrenal insufficiency resulting from recessive mutations in the ACTH receptor (MC2R, MC2R). Individuals with this condition typically present in infancy or childhood with signs and symptoms of cortisol insufficiency, but disturbances in the renin-angiotensin system, aldosterone synthesis or sodium homeostasis are not a well-documented association of FGD1. As ACTH stimulation has been shown to stimulate aldosterone release in normal controls, and other causes of hyponatraemia can occur in children with cortisol deficiency, we investigated whether MC2R changes might be identified in children with primary adrenal failure who were being treated for mineralocorticoid insufficiency.
Design: Mutational analysis of MC2R by direct sequencing.
Patients: Children (n = 22) who had been diagnosed with salt-losing forms of adrenal hypoplasia (19 isolated cases, 3 familial), and who were negative for mutations in DAX1 (NR0B1) and SF1 (NR5A1).
Results: MC2R mutations were found in three individuals or kindred (I: homozygous S74I; II: novel compound heterozygous R146H/560delT; III: novel homozygous 579-581delTGT). These changes represent severely disruptive loss-of-function mutations in this G-protein coupled receptor, including the first reported homozygous frameshift mutation. The apparent disturbances in sodium homeostasis were mild, manifest at times of stress (e.g. infection, salt-restriction, heat), and likely resolved with time.
Conclusions: MC2R mutations should be considered in children who have primary adrenal failure with apparent mild disturbances in renin-sodium homeostasis. These children may have been misdiagnosed as having salt-losing adrenal hypoplasia. Making this diagnosis has important implications for treatment, counselling and long-term prognosi
A Sphingosine-1-Phosphate Lyase Mutation Associated With Congenital Nephrotic Syndrome and Multiple Endocrinopathy.
Background: Loss of function mutations in SGPL1 are associated with Sphingosine-1-phosphate lyase insufficiency syndrome, comprising steroid resistant nephrotic syndrome, and primary adrenal insufficiency (PAI) in the majority of cases. SGPL1 encodes sphingosine-1-phosphate lyase (SGPL1) which is a major modulator of sphingolipid signaling. Case Presentation: A Pakistani male infant presented at 5 months of age with failure to thrive, nephrotic syndrome, primary adrenal insufficiency, hypothyroidism, and hypogonadism. Other systemic manifestations included persistent lymphopenia, ichthyosis, and motor developmental delay. Aged 9 months, he progressed rapidly into end stage oligo-anuric renal failure and subsequently died. Sanger sequencing of the entire coding region of SGPL1 revealed the novel association of a rare homozygous mutation (chr10:72619152, c.511A>G, p.N171D; MAF-1.701e-05) with the condition. Protein expression of the p.N171D mutant was markedly reduced compared to SGPL1 wild type when overexpressed in an SGPL1 knockout cell line, and associated with a severe clinical phenotype. Conclusions: The case further highlights the emerging phenotype of patients with loss-of-function SGPL1 mutations. Whilst nephrotic syndrome is a recognized feature of other disorders of sphingolipid metabolism, sphingosine-1-phosphate lyase insufficiency syndrome is unique amongst the sphingolipidoses in presenting with multiple endocrinopathies. Given the multi-systemic and progressive nature of this form of PAI/ nephrotic syndrome, a genetic diagnosis is crucial for optimal management and appropriate screening for comorbidities in these patients
Loss of Nnt Increases Expression of Oxidative Phosphorylation Complexes in C57BL/6J Hearts
Nicotinamide nucleotide transhydrogenase (NNT) is a proton pump in the inner mitochondrial membrane that generates reducing equivalents in the form of NAPDH, which can be
used for anabolic pathways or to remove reactive oxygen species (ROS). A number of studies have
linked NNT dysfunction to cardiomyopathies and increased risk of atherosclerosis; however, biallelic
mutations in humans commonly cause a phenotype of adrenal insufficiency, with rare occurrences
of cardiac dysfunction and testicular tumours. Here, we compare the transcriptomes of the hearts,
adrenals and testes from three mouse models: the C57BL/6N, which expresses NNT; the C57BL/6J,
which lacks NNT; and a third mouse, expressing the wild-type NNT sequence on the C57BL/6J
background. We saw enrichment of oxidative phosphorylation genes in the C57BL/B6J in the heart
and adrenal, possibly indicative of an evolved response in this substrain to loss of Nnt. However,
differential gene expression was mainly driven by mouse background with some changes seen
in all three tissues, perhaps reflecting underlying genetic differences between the C57BL/B6J and
-6N substrains
MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation.
Melanocortin 2 receptor accessory protein (MRAP) is a single transmembrane domain accessory protein and a critical component of the hypothamo-pituitary-adrenal axis. MRAP is highly expressed in the adrenal gland and is essential for adrenocorticotropin hormone (ACTH) receptor expression and function. Human loss-of-function mutations in MRAP cause familial glucocorticoid (GC) deficiency (FGD) type 2 (FGD2), whereby the adrenal gland fails to respond to ACTH and to produce cortisol. In this study, we generated Mrap-null mice to study the function of MRAP in vivo. We found that the vast majority of Mrap-/- mice died at birth but could be rescued by administration of corticosterone to pregnant dams. Surviving Mrap-/- mice developed isolated GC deficiency with normal mineralocorticoid and catecholamine production, recapitulating FGD2. The adrenal glands of adult Mrap-/- mice were small, with grossly impaired adrenal capsular morphology and cortex zonation. Progenitor cell differentiation was significantly impaired, with dysregulation of WNT4/β-catenin and sonic hedgehog pathways. These data demonstrate the roles of MRAP in both steroidogenesis and the regulation of adrenal cortex zonation. This is the first mouse model of isolated GC deficiency and reveals the role of MRAP in adrenal progenitor cell regulation and cortex zonation.-Novoselova, T. V., Hussain, M., King, P. J., Guasti, L., Metherell, L. A., Charalambous, M., Clark, A. J. L., Chan, L. F. MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation
Missplicing due to a synonymous, T96= exonic substitution in the T-box transcription factor TBX19 resulting in isolated ACTH deficiency
Congenital isolated ACTH deficiency (IAD) is a rare condition characterised by low plasma ACTH and serum cortisol with
normal production of other pituitary hormones. TBX19 (also known as TPIT) is a T-box pituitary restricted transcription
factor important for POMC gene transcription and terminal differentiation of POMC-expressing cells. TBX19 gene mutations
have been shown to cause neonatal-onset congenital IAD. We report a neonate of Romanian origin, who presented at 15
h of life with respiratory arrest and hypoglycaemia which recurred over the following 2 weeks. Biochemical investigations
revealed IAD, with undetectable serum cortisol (cortisol < 1 μg/dL; normal range (NR): 7.8–26.2) and plasma ACTH levels
within the normal range (22.1 pg/mL; NR: 4.7–48.8). He responded to hydrocortisone treatment. Patient DNA was
analysed by a HaloPlex next-generation sequencing array targeting genes for adrenal insufficiency. A novel homozygous
synonymous mutation p.Thr96= (Chr1:168260482; c.288G>A; rs376493164; allele frequency 1 × 10−5, no homozygous) was
found in exon 2 of the TBX19 gene. The effect of this was assessed by an in vitro splicing assay, which revealed aberrant
splicing of exon 2 giving rise to a mutant mRNA transcript whereas the WT vector spliced exon 2 normally. This was
identified as the likely cause of IAD in the patient. The predicted protein product would be non-functional in keeping with
the complete loss of cortisol production and early presentation in the patient
Long-term outcome of partial P450 side-chain cleavage enzyme deficiency in three brothers: the importance of early diagnosis.
Objective: CYP11A1 mutations cause P450 side-chain cleavage (scc) deficiency, a rare form of congenital adrenal hyperplasia with a wide clinical spectrum. We detail the phenotype and evolution in a male sibship identified by HaloPlex targeted capture array. Family study: The youngest of three brothers from a non-consanguineous Scottish family presented with hyperpigmentation at 3.7 years. Investigation showed grossly impaired glucocorticoid function with ACTH elevation, moderately impaired mineralocorticoid function, and normal external genitalia. The older brothers were found to be pigmented also, with glucocorticoid impairment but normal electrolytes. Linkage studies in 2002 showed that all three brothers had inherited the same critical regions of the maternal X chromosome suggesting an X-linked disorder, but analysis of NR0B1 (DAX-1, adrenal hypoplasia) and ABCD1 (adrenoleukodystrophy) were negative. In 2016, next-generation sequencing revealed compound heterozygosity for the rs6161 variant in CYP11A1 (c.940G>A, p.Glu314Lys), together with a severely disruptive frameshift mutation (c.790_802del, K264Lfs*5). The brothers were stable on hydrocortisone and fludrocortisone replacement, testicular volumes (15-20 mL), and serum testosterone levels (24.7, 33.3, and 27.2 nmol/L) were normal, but FSH (41.2 µ/L) was elevated in the proband. The latter had undergone left orchidectomy for suspected malignancy at the age of 25 years and was attending a fertility clinic for oligospermia. Initial histology was reported as showing nodular Leydig cell hyperplasia. However, histological review using CD56 staining confirmed testicular adrenal rest cell tumour (TART). Conclusion: This kinship with partial P450scc deficiency demonstrates the importance of precise diagnosis in primary adrenal insufficiency to ensure appropriate counselling and management, particularly of TART
Whole-exome sequencing gives additional benefits compared to candidate gene sequencing in the molecular diagnosis of children with growth hormone or IGF-1 insensitivity
Ipsen UK!(HLS).
Sandoz Pharmaceutical
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