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.

Elevated sphingosine-1-phosphate lyase leads to increased metabolism and reduced survival in adrenocortical carcinoma.

OBJECTIVE: Adrenocortical carcinomas (ACCs) are invasive tumours arising in the adrenal cortex, and steroidogenic tumours are associated with worse prognostic outcomes. Loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1) cause primary adrenal insufficiency and as a key degradative enzyme in the sphingolipid pathway, SGPL1 also influences the balance of pro-proliferative and pro-apoptotic sphingolipids. We, therefore, hypothesized increased SGPL1 may be linked to increased disease severity in ACC. DESIGN: Analyse SGPL1 expression impact on patient survival and adrenal cancer cell phenotype. We analysed two ACC cohorts with survival and corresponding transcriptomic data, focusing on SGPL1 and sphingolipid pathway genes. In vitro, we generated SGPL1-knockout and overexpressing H295R adrenocortical cells to investigate the role of SGPL1 in cell signalling in ACCs. RESULTS: We found increased expression of several sphingolipid pathway receptors and enzymes, most notably SGPL1 correlated with reduced patient survival in both cohorts. Overexpression of SGPL1 in the H295R cell line increased proliferation and migration while reducing apoptosis, while SGPL1 knockout had the opposite effect. RNA-seq revealed a global increase in the expression of genes in the electron transport chain in overexpressing cells, correlating with increased aerobic respiration and glycolysis. Furthermore, the opposite phenotype was seen in cells lacking SGPL1. We subsequently found the increased proliferation is linked to metabolic substrate availability and increased capacity to use different fuel sources, but particularly glucose, in overexpressing cells. CONCLUSIONS: We, therefore, propose that SGPL1-overexpressing ACC tumours reduce patient survival by increasing fuel usage for anabolism and energy production to facilitate growth and invasion.

Missplicing due to a synonymous, T96= exonic substitution in the T-box transcription factor TBX19 resulting in isolated ACTH deficiency.

SUMMARY: 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. LEARNING POINTS: Synonymous variants (a nucleotide change that does not alter protein sequence) usually thought to be benign may still have detrimental effects on RNA and protein function causing disease. Hence, they should not be ignored, especially if very rare in public databases. In vitro splicing assays can be employed to characterise the consequence of intronic and exonic nucleotide gene changes that may alter splicing. Establishing a diagnosis due to a TBX19 mutation is important as it defines a condition of isolated ACTH deficiency not associated with additional pituitary deficiencies.

Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant aldosterone-producing adenomas presenting in puberty, pregnancy or menopause.

Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1-mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ. Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR, the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1.

Mylk3 null C57BL/6N mice develop cardiomyopathy, whereas Nnt null C57BL/6J mice do not.

The C57BL/6J and C57BL/6N mice have well-documented phenotypic and genotypic differences, including the infamous nicotinamide nucleotide transhydrogenase (Nnt) null mutation in the C57BL/6J substrain, which has been linked to cardiovascular traits in mice and cardiomyopathy in humans. To assess whether Nnt loss alone causes a cardiovascular phenotype, we investigated the C57BL/6N, C57BL/6J mice and a C57BL/6J-BAC transgenic rescuing NNT expression, at 3, 12, and 18 mo. We identified a modest dilated cardiomyopathy in the C57BL/6N mice, absent in the two B6J substrains. Immunofluorescent staining of cardiomyocytes revealed eccentric hypertrophy in these mice, with defects in sarcomere organisation. RNAseq analysis identified differential expression of a number of cardiac remodelling genes commonly associated with cardiac disease segregating with the phenotype. Variant calling from RNAseq data identified a myosin light chain kinase 3 (Mylk3) mutation in C57BL/6N mice, which abolishes MYLK3 protein expression. These results indicate the C57BL/6J Nnt-null mice do not develop cardiomyopathy; however, we identified a null mutation in Mylk3 as a credible cause of the cardiomyopathy phenotype in the C57BL/6N.

Adrenocorticotropin resistance syndromes.

Familial glucocorticoid deficiency (FGD) and triple A syndrome belong to a rare group of autosomal recessive disorders characterized by adrenocorticotropin (ACTH) insensitivity. Unlike triple A syndrome which presents a range of clinical features, FGD is solely characterized by glucocorticoid deficiency. ACTH regulates steroid biosynthesis in the adrenal cortex by exerting its effects via the ACTH receptor (melanocortin- 2 receptor, MC2R). In FGD, mutations in the MC2R account for only approximately 25% of cases (FGD type 1). The inability to express a functional MC2R in non-adrenal cell lines had implied the presence of an adrenal specific accessory factor(s), essential for MC2R expression. More recently, this factor was identified as melanocortin receptor accessory protein (MRAP). Mutations in MRAP account for 20% of cases (FGD type 2). Like the receptor activity-modifying proteins (RAMPs) and receptor transporter proteins (RTPs), which are well-characterized accessory proteins for G-protein-coupled receptors (GPCRs), MRAP is a small single transmembrane domain protein. MRAP is essential for the functional expression of the MC2R. About 55% of FGD cases have no identifiable gene defect, implying the involvement of additional genes. This chapter briefly describes the clinical and biochemical features of ACTH resistance syndromes. However, we will focus on the recent progress made towards understanding the molecular defect underlying these conditions, in particular the interaction of MC2R and MRAP.

Growth hormone insensitivity and severe short stature in siblings: a novel mutation at the exon 13-intron 13 junction of the STAT5b gene.

BACKGROUND/AIMS: Growth hormone insensitivity (GHI) is characterized by severe short stature, high serum growth hormone (GH), low serum IGF-I and IGFBP-3 levels and is classically associated with genetic defects of the GH receptor (GHR). Recently, mutations of the STAT5b gene have been identified and shown to be associated with GHI and severe IGF deficiency. We investigated 2 sisters from a consanguineous family from Kuwait, with clinical and biochemical features of GHI, in whom no molecular defects in the GHR were identified. METHODS: Serum and DNA were analyzed. RESULTS: In addition to GHI, siblings 2 and 1 presented with, respectively, a diagnosis of juvenile idiopathic arthritis and recurrent pulmonary infections. Molecular analysis of the STAT5b gene revealed a novel homozygous deletion of a G at the junction of exon 13-intron 13. The parents, who are of normal height, were heterozygous for the mutation. CONCLUSIONS: This is the first STAT5b defect to be identified in siblings, further supporting the autosomal recessive mode of transmission of STAT5b deficiency. The results affirm that defective STAT5b is an etiology for IGF deficiency and the GHI phenotype, and emphasize the importance of considering this diagnosis in patients with IGF deficiency, especially when associated with diverse immunological problems.

In-frame seven amino-acid duplication in AIP arose over the last 3000 years, disrupts protein interaction and stability and is associated with gigantism.

OBJECTIVE: Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. DESIGN AND METHODS: Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP-region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to most recent common ancestor (tMRCA) of the derived allele; forward population simulations to estimate current number of allele carriers; proposal of mutation mechanism; protein structure predictions; co-immunoprecipitation and cycloheximide chase experiments. RESULTS: Nine European-origin, unrelated c.805_825dup-positive pedigrees (four familial, five sporadic from the UK, USA and France) included 16 affected (nine gigantism/four acromegaly/two non-functioning pituitary adenoma patients and one prospectively diagnosed acromegaly patient) and nine unaffected carriers. All pedigrees shared a 2.79 Mbp haploblock around AIP with additional haploblocks privately shared between subsets of the pedigrees, indicating the existence of an evolutionarily recent common ancestor, the 'English founder', with an estimated median tMRCA of 47 generations (corresponding to 1175 years) with a confidence interval (9-113 generations, equivalent to 225-2825 years). The mutation occurred in a small tandem repeat region predisposed to slipped strand mispairing. The resulting seven amino-acid duplication disrupts interaction with HSP90 and leads to a marked reduction in protein stability. CONCLUSIONS: The c.805_825dup allele, originating from a common ancestor, associates with a severe clinical phenotype and a high frequency of gigantism. The mutation is likely to be the result of slipped strand mispairing and affects protein-protein interactions and AIP protein stability.

Primary Adrenocortical Insufficiency Case Series: Genetic Etiologies More Common than Expected.

BACKGROUND/AIMS: Primary adrenal insufficiency (AI) is an important cause of morbidity in children. Our objectives were: (1) to describe the clinical presentation of children with new-onset primary AI, and (2) to identify monogenic causes of primary AI in children. METHODS: Chart review and mutation detection in candidate genes were conducted for 11 patients with primary AI. RESULTS: The likely cause of AI was determined in 9 patients. One had a homozygous MC2R mutation associated with familial glucocorticoid deficiency. Two had the same homozygous mutation in the AIRE gene which is associated with type 1 autoimmune polyglandular syndrome. One patient had a heterozygous change in this gene of undetermined significance. Five were homozygous for the previously reported p.R188C STAR mutation causing nonclassic lipoid congenital adrenal hyperplasia, representing the largest cohort of such patients from a single geographic area. In the remaining 2 patients, no clear etiology was identified. CONCLUSIONS: We recommend genetic testing for patients who have negative anti-adrenal antibodies or suggestive family history. Diagnosing a genetic etiology can provide information about prognosis and treatment, and is therefore beneficial for patients. Our high proportion of patients with nonclassic lipoid congenital adrenal hyperplasia likely represents a founder effect.

A novel homozygous mutation of the IGFALS gene in a female adolescent: indirect evidence for a contributing role of the circulating IGF-I pool in the pubertal growth spurt.

BACKGROUND: Mutations of the IGFALS gene have been reported since 2004 in 24 patients, but only 5 of these are females. CASE REPORT: We describe a 14.7-year-old female of a consanguineous Moroccan family with growth retardation and normal-onset but slow progression of puberty without manifest pubertal height gain. RESULTS: At age 3.2 years, the patient's height was 85.5 cm (-2.9 SDS) and her weight 9.9 kg (-2.9 SDS) with a head circumference of 44.5 cm (-3.3 SDS). Serum IGF-I and IGFBP-3 concentrations were low with normal basal and stimulated growth hormone (GH) levels. An IGF-I generation test confirmed a lack of response to GH administration. While onset of puberty occurred at a normal age, no significant pubertal growth acceleration was observed despite progression of breast development. Sequencing of the IGFALS gene revealed a novel homozygous frameshift mutation (c.1291delT) with a stop codon (p.W431GfsX10) leading to undetectable serum levels of acid-labile subunit. CONCLUSION: We report the phenotype of an adolescent girl with primary IGF-I deficiency due to a novel homozygous mutation of the IGFALS gene, who presented with growth delay, normal pubertal onset with slow progression and no pubertal growth acceleration indirectly suggesting a contributing role of the circulating IGF-I pool in the pubertal growth spurt.

IGFALS gene dosage effects on serum IGF-I and glucose metabolism, body composition, bone growth in length and width, and the pharmacokinetics of recombinant human IGF-I administration.

CONTEXT: Acid labile subunit (ALS) deficiency, caused by IGFALS mutations, is a subtype of primary IGF-I deficiency (PIGFD) and has been associated with insulin resistance (IR) and osteopenia. Whether patients respond to recombinant human IGF-I (rhIGF-I) is unknown. OBJECTIVE AND DESIGN: This study determined the 14-hour pharmacokinetic response of free and total IGF-I and IGF binding protein 3 (IGFBP-3) to a single sc dose of rhIGF-I (120 µg/kg) in four ALS-deficient patients, compared with severe PIGFD, moderate PIGFD, and controls. Intravenous glucose tolerance tests, fasting blood levels, dual-energy X-ray absorptiometry, peripheral quantitative computed tomography, and metacarpal radiogrammetry were performed in the four patients and 12 heterozygous family members. RESULTS: IGF-I and IGFBP-3 increased above baseline (P < .05) for 2.5 hours, returning to baseline 7 hours after rhIGF-I injection. Mean (SD) IGF-I Z-score increased by 2.49 (0.90), whereas IGFBP-3 Z-score increased by 0.57 (0.10) only. IGF-I elimination rates in ALS deficiency were similar, but the IGF-I increment was lower than those for severe PIGFD. Significant gene dosage effects were found for all IGF-I peptides, height, forearm muscle size, and metacarpal width. Bone analysis showed that ALS deficiency creates a phenotype of slender bones with normal size-corrected density. Abnormal glucose handling and IR was found in three of four patients and 6 of 12 carriers. CONCLUSIONS: These gene dosage effects demonstrate that one functional IGFALS allele is insufficient to maintain normal ALS levels, endocrine IGF-I action, full growth potential, muscle size, and periosteal expansion. Similar gene dosage effects may exist for parameters of IR. Despite similar IGF-I elimination compared with severe PIGFD, ALS-deficient patients cannot mount a similar response. Alternative ways of rhIGF-I administration should be sought.

Reduced expression of the growth hormone and type 1 insulin-like growth factor receptors in human somatotroph tumours and an analysis of possible mutations of the growth hormone receptor.

OBJECTIVE: Clinical acromegaly is characterized by elevated GH secretion in the presence of high circulating IGF-I levels. We hypothesized that the physiological IGF-I/GH negative feedback loop may be reset in somatotroph adenomas, specifically in terms of the level of expression of these receptors or mutations of the GH receptor (GH-R) in such tumours. METHODS: We therefore investigated the full coding sequence of the GH-R in a series of somatotroph and other pituitary adenomas. We also investigated the mRNA expression of these putative feedback receptors in a series of pituitary adenomas and normal pituitary tissue, and their protein expression by immunostaining. Real-time RT-PCR assay was used for the quantification of the type 1 IGF receptor (IGF-R) and GH receptor (GH-R) mRNA, and sequence analysis was performed on the coding region of the GH-R gene. RESULTS: No somatic mutations of the GH-R mRNA were detected in 18 GH-secreting tumours or two nonfunctioning pituitary adenomas (NFPAs). However, the levels of GH-R mRNA were significantly lower in both somatotroph tumours and NFPAs compared to the normal pituitary (P < 0.05 for both). Immunostaining for GH-R also showed significantly less GH-R expression in somatotroph adenomas compared to normal pituitary tissue (P < 0.0001). IGF-R mRNA levels were significantly lower in somatotroph tumours compared to normal pituitary (P = 0.005), and trended lower in corticotroph tumours (P = 0.07), while the other tumour types showed no significant difference from normal pituitary. Immunostaining for IGF-R also showed less IGF-R protein in the somatotroph adenomas compared to the normal pituitary tissue (P < 0.01). CONCLUSIONS: Our findings suggest that decreased feedback inhibition of GH because of somatic mutations of the coding region of the GH-R are unlikely to be a common factor in the pathogenesis of these tumours. Nevertheless, decreased expression of the GH-R and of IGF-R in somatotroph tumours (both at the mRNA and protein level) may, at least in part, help explain the continuous secretion of GH from the tumour despite the high circulating levels of IGF-I and GH.