[Clinical and genetic analysis of an infant with aldosterone synthase deficiency].

OBJECTIVE: To analyze the clinical characteristics and genetic variants in a two-month-and-one-day male infant with aldosterone synthase deficiency. METHODS: Clinical data of the child was collected. Whole exome sequencing was carried out by next generation sequencing(NGS). Candidate variants were verified by Sanger sequencing. RESULTS: The infant had measured 54 cm (-2.1 SD) in length and 3.9 kg (-2.8 SD) in weight, and featured recurrent vomiting, poor feeding, apathetic appearance and failure to thrive. Blood electrolyte testing showed low sodium and increased potassium. Serum cortisol, adrenocorticotrophic hormone, 17-alpha-hydroxyl progesterone, androstenedione, and testosterone were all within the normal ranges. The plasma renin activity activity was increased, and plasma aldosterone level was low. NGS revealed that the infant has harbored compound heterozygous variants of the CYP11B2 gene, namely c.1334T>G(p.Phe445Cys) inherited from his father and c.1121G>A(p.Arg374Gln) inherited from his mother. Neither variant was reported previously, and both were predicted to be deleterious for the function of the protein product. CONCLUSION: The compound heterozygous variants of c.1334T>G (p.Phe445Cys) and c.1121G>A (p.Arg374Gln) of the CYP11B2 gene probably underlay the disease in this patient.

Aldosterone synthase deficiency type II: an unusual presentation of the first Greek case reported with confirmed genetic analysis.

OBJECTIVE: Aldosterone synthase deficiency (ASD) is a rare, autosomal recessive inherited disease with an overall clinical phenotype of failure to thrive, vomiting, severe dehydration, hyperkalemia, and hyponatremia. Mutations in the CYP11B2 gene encoding aldosterone synthase are responsible for the occurrence of ASD. Defects in CYP11B2 gene have only been reported in a limited number of cases worldwide. Due to this potential life-threatening risk, comprehensive hormonal investigation followed by genetic confirmation is essential for the clinical management of offsprings. CASE PRESENTATION: We herein describe an unusual case of ASD type II in a neonate with faltering growth as a single presenting symptom. To our knowledge, this is the first Greek case of ASD type II reported with confirmed genetic analysis. Next generation sequencing of her DNA revealed the homozygous mutation p.T185I (ACC-ATC) (c.554C>T) (g.7757C>T) in exon 3 of the CYP11B2 gene in the neonate, inherited from both parents who were heterozygotes for the mutation. CONCLUSIONS: Physicians handling neonates with faltering growth, particularly in the initial six weeks of life, should be suspicious of mineralocorticoid insufficiency either as isolated hypoaldosteronism or in the context of congenital adrenal hyperplasia. Essential investigations should be performed and appropriate treatment should be administered promptly without awaiting for the hormonal profile results. Interpretation of the clinical picture and the hormonal profile will guide the analysis of candidate genes. Primary selective hypoaldosteronism is a rare, life threatening disease, but still with an unknown overall population impact. Thus, reporting cases with confirmed gene mutations is of major importance.

Aldosterone synthase knockout mouse as a model for sodium-induced endothelial sodium channel up-regulation in vascular endothelium.

Recently, a novel feedforward activation of the endothelial epithelial sodium channel (ENaC) [endothelial sodium channel (EnNaC)] by sodium was reported that counteracts ENaC function in kidney. In the absence of aldosterone, a rise in extracellular sodium (>145 mM) increases EnNaC surface abundance, thereby stiffening the cortex of vascular endothelial cells (ECs) in vitro. The latter reduces the release of NO-the hallmark of endothelial dysfunction. Here, we test whether high extracellular sodium per se increases EnNaC expression and cortical stiffness in an aldosterone synthase (Cyp11b2)-deficient (AS(-/-)) mouse model. Therefore, we employed in situ ECs of ex vivo aorta preparations from wild-type (WT) and AS(-/-). EnNaC surface expression (-16%) and cortical stiffness (-22%) were reduced in AS(-/-), compared with WT, whereas NO secretion was exclusively detectable in AS(-/-). EnNaC inhibition with benzamil decreased stiffness in both, while mineralocorticoid receptor antagonism diminished stiffness only in the WT. In the absence of aldosterone, high sodium (150 mM) increased EnNaC surface expression ex vivo (plus 19%) and cortical stiffness ex vivo (plus 41%) and in vivo (plus 44%). Application of aldosterone adjusted the stiffness of AS(-/-) to the WT level. We conclude that high sodium per se determines EnNaC expression and consequently endothelial cortical nanomechanics, thus likely contributing to endothelial dysfunction.

Mechanisms of renal control of potassium homeostasis in complete aldosterone deficiency.

Aldosterone-independent mechanisms may contribute to K(+) homeostasis. We studied aldosterone synthase knockout (AS(-/-)) mice to define renal control mechanisms of K(+) homeostasis in complete aldosterone deficiency. AS(-/-) mice were normokalemic and tolerated a physiologic dietary K(+) load (2% K(+), 2 days) without signs of illness, except some degree of polyuria. With supraphysiologic K(+) intake (5% K(+)), AS(-/-) mice decompensated and became hyperkalemic. High-K(+) diets induced upregulation of the renal outer medullary K(+) channel in AS(-/-) mice, whereas upregulation of the epithelial sodium channel (ENaC) sufficient to increase the electrochemical driving force for K(+) excretion was detected only with a 2% K(+) diet. Phosphorylation of the thiazide-sensitive NaCl cotransporter was consistently lower in AS(-/-) mice than in AS(+/+) mice and was downregulated in mice of both genotypes in response to increased K(+) intake. Inhibition of the angiotensin II type 1 receptor reduced renal creatinine clearance and apical ENaC localization, and caused severe hyperkalemia in AS(-/-) mice. In contrast with the kidney, the distal colon of AS(-/-) mice did not respond to dietary K(+) loading, as indicated by Ussing-type chamber experiments. Thus, renal adaptation to a physiologic, but not supraphysiologic, K(+) load can be achieved in aldosterone deficiency by aldosterone-independent activation of the renal outer medullary K(+) channel and ENaC, to which angiotensin II may contribute. Enhanced urinary flow and reduced activity of the thiazide-sensitive NaCl cotransporter may support renal adaptation by activation of flow-dependent K(+) secretion and increased intratubular availability of Na(+) that can be reabsorbed in exchange for K(+) secreted.

Control of renin secretion from kidneys with renin cell hyperplasia.

In states of loss-of-function mutations of the renin-angiotensin-aldosterone system, kidneys develop a strong hyperplasia of renin-producing cells. Those additional renin cells are located outside the classic juxtaglomerular areas, mainly in the walls of preglomerular vessels and most prominently in multilayers surrounding afferent arterioles. Since the functional behavior of those ectopic renin cells is yet unknown, we aimed to characterize the control of renin secretion from kidneys with renin cell hyperplasia. As a model, we used kidneys from mice lacking aldosterone synthase (AS⁻/⁻ mice), which displayed 10-fold elevations of renin mRNA and plasma renin concentrations. On the absolute level, renin secretion from isolated AS⁻/⁻ kidneys was more than 10-fold increased over wild-type kidneys. On the relative level, the stimulation of renin secretion by the ß-adrenergic activator isoproterenol or by lowering of the concentration of extracellular Ca²âº was very similar between the two genotypes. In addition, the inhibitory effects of ANG II and of perfusion pressure were similar between the two genotypes. Deletion of connexin40 blunted the pressure dependency of renin secretion and the stimulatory effect of low extracellular Ca²âº on renin secretion in the same manner in kidneys of AS⁻/⁻ mice as in wild-type mice. Our findings suggest a high degree of functional similarity between renin cells originating during development and located at different positions in the adult kidney. They also suggest a high similarity in the expression of membrane proteins relevant for the control of renin secretion, such as ß1-adrenergic receptors, ANG II type 1 receptors, and connexin40.

Procollagen I-expressing renin cell precursors.

Renin-expressing cells in the kidney normally appear as mural cells of developing preglomerular vessels and finally impose as granulated juxtaglomerular cells in adult kidneys. The differentiation of renin-expressing cells from the metanephric mesenchyme in general and the potential role of special precursor stages in particular is not well understood. Therefore, it was the aim of this study to search for renin cell precursors in the kidney. As an experimental model, we used kidneys of aldosterone synthase-deficient mice, which display a prominent compensatory overproduction of renin cells that are arranged in multilayered perivascular cell clusters. We found that the perivascular cell clusters contained two apparently distinct cell types, one staining positive for renin and another one staining positive for type I procollagen (PC1). It appeared as if PC1 and renin expression were inversely related at the cellular level. The proportion of renin-positive to PC1-positive cells in the clusters was inversely linked to the rate of salt intake, as was overall renin expression. Our findings suggest that the cells in the perivascular cell clusters can reversibly switch between PC1 and renin expression and that PC1-expressing cells might be precursors of renin cells. A few of those PC1-positive cells were found also in adult wild-type kidneys in the juxtaglomerular lacis cell area, in which renin expression can be induced on demand.

Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice.

A dietary potassium load induces a rapid kaliuresis and natriuresis, which may occur even before plasma potassium and aldosterone (aldo) levels increase. Here we sought to gain insight into underlying molecular mechanisms contributing to this response. After gastric gavage of 2% potassium, the plasma potassium concentrations rose rapidly (0.25 h), followed by a significant rise of plasma aldo (0.5 h) in mice. Enhanced urinary potassium and sodium excretion was detectable as early as spot urines could be collected (about 0.5 h). The functional changes were accompanied by a rapid and sustained (0.25-6 h) dephosphorylation of the NaCl cotransporter (NCC) and a late (6 h) upregulation of proteolytically activated epithelial sodium channels. The rapid effects on NCC were independent from the coadministered anion. NCC dephosphorylation was also aldo-independent, as indicated by experiments in aldo-deficient mice. The observed urinary sodium loss relates to NCC, as it was markedly diminished in NCC-deficient mice. Thus, downregulation of NCC likely explains the natriuretic effect of an acute oral potassium load in mice. This may improve renal potassium excretion by increasing the amount of intraluminal sodium that can be exchanged against potassium in the aldo-sensitive distal nephron.

Two novel mutations of the CYP11B2 gene in a Japanese patient with aldosterone deficiency type 1.

Isolated hypoaldosteronism is a rare and occasionally life-threatening cause of salt wasting in infancy. A 2-month-old Japanese boy of unrelated parents was examined for failure to thrive and poor weight gain. Laboratory findings were hyponatremia, hyperkalemia, high plasma renin and low aldosterone levels. Spot urine analysis by gas chromatography-mass spectrometry (GC-MS) showed that urinary excretion of corticosterone metabolites was elevated. Whereas excretion of 18-hydroxycortricosterone metabolites was within the normal range, excretion of aldosterone metabolites was undetectable. The patient was therefore suspected to have aldosterone synthase deficiency type 1. Sequence analysis of CYP11B2, the gene encoding aldosterone synthase (CYP11B2), showed that the patient was a compound heterozygote for c.168G>A, p.W56X in exon 1 and c.1149C>T, p.R384X in exon 7. p.W56X was inherited from his mother and p.R384X was from his father. Since both alleles contain nonsense mutations, a lack of CYP11B2 activity was speculated to cause his condition. To our knowledge, this is the first Japanese patient in which the molecular basis of aldosterone synthase deficiency type 1 has been clarified. This case also indicates that spot urinary steroid analysis is useful for diagnosis.

Tissue kallikrein permits early renal adaptation to potassium load.

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K+ intake, we hypothesized that this enzyme might regulate plasma K+ concentration ([K+]). We showed in wild-type mice that renal K+ and TK excretion increase in parallel after a single meal, representing an acute K+ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK-/-) were used to assess the impact of the enzyme on plasma [K+]. A single large feeding did not lead to any significant change in plasma [K+] in TK+/+, whereas TK-/- mice became hyperkalemic. We next examined the impact of TK disruption on K+ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK-/- mice exhibit net transepithelial K+ absorption because of abnormal activation of the colonic H+,K+-ATPase in the intercalated cells. Finally, in CCDs isolated from TK-/- mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H+,K+-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K+ load.

Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron.

Aldosterone is thought to be the main hormone to stimulate the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the entire collecting duct (CD). There is immunohistochemical evidence for an axial gradient of ENaC expression along the ASDN with highest expression in the DCT2 and CNT. However, most of our knowledge about renal ENaC function stems from studies in the cortical collecting duct (CCD). Here we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice maintained on different sodium diets to vary plasma aldosterone levels. Single-channel recordings demonstrated amiloride-sensitive Na(+) channels in DCT2/CNT with biophysical properties typical for ENaC previously described in CNT/CCD. In animals maintained on a standard salt diet, the average ENaC-mediated whole cell current (ΔI(ami)) was higher in DCT2/CNT than in CNT/CCD. A low salt diet increased ΔI(ami) in CNT/CCD but had little effect on ΔI(ami) in DCT2/CNT. To investigate whether aldosterone is necessary for ENaC activity in the DCT2/CNT, we used aldosterone synthase knockout (AS(-/-)) mice that lack aldosterone. In CNT/CCD of AS(-/-) mice, ΔI(ami) was lower than that in wild-type (WT) animals and was not stimulated by a low salt diet. In contrast, in DCT2/CNT of AS(-/-) mice, ΔI(ami) was similar to that in DCT2/CNT of WT animals both on a standard and on a low salt diet. We conclude that ENaC function in the DCT2/CNT is largely independent of aldosterone which is in contrast to its known aldosterone sensitivity in CNT/CCD.

Aldosterone deficiency and mineralocorticoid receptor antagonism prevent angiotensin II-induced cardiac, renal, and vascular injury.

Angiotensin II causes cardiovascular injury in part by aldosterone-induced mineralocorticoid receptor activation, and it can also activate the mineralocorticoid receptor in the absence of aldosterone in vitro. Here we tested whether endogenous aldosterone contributes to angiotensin II/salt-induced cardiac, vascular, and renal injury by the mineralocorticoid receptor. Aldosterone synthase knockout mice and wild-type littermates were treated with angiotensin II or vehicle plus the mineralocorticoid receptor antagonist spironolactone or regular diet while drinking 0.9% saline. Angiotensin II/salt caused hypertension in both the knockout and wild-type mice, an effect significantly blunted in the knockout mice. Either genetic aldosterone deficiency or mineralocorticoid receptor antagonism reduced cardiac hypertrophy, aortic remodeling, and albuminuria, as well as cardiac, aortic, and renal plasminogen activator inhibitor-1 mRNA expression during angiotensin II treatment. Mineralocorticoid receptor antagonism reduced angiotensin II/salt-induced glomerular hypertrophy, but aldosterone deficiency did not. Combined mineralocorticoid receptor antagonism and aldosterone deficiency reduced blood urea nitrogen and restored nephrin immunoreactivity. Angiotensin II/salt also promoted glomerular injury through the mineralocorticoid receptor in the absence of aldosterone. Thus, mineralocorticoid antagonism may have protective effects in the kidney beyond aldosterone synthase inhibition.

Novel CYP11B2 mutation causing aldosterone synthase (P450c11AS) deficiency.

UNLABELLED: Aldosterone synthase (P450c11AS) deficiency is a rare autosomal recessive disorder, presenting with severe salt-losing in early infancy. It is caused by inactivating mutations of the CYP11B2 gene. Here, we describe three unrelated Asian patients who have clinical and hormonal features compatible with aldosterone synthase deficiency and identify their CYP11B2 mutations. Patient 1 was a Thai female infant. Patient 2 was an Indian boy, and patient 3 was a Thai male infant. All subjects presented at the age of 1-2 months with diarrhea, failure to thrive, and severe dehydration. Their plasma electrolytes showed hyponatremia, hyperkalemia, and acidosis. All patients had normal cortisol response and had elevated plasma renin activity with low aldosterone levels. The entire coding regions of the CYP11B2 gene were amplified by polymerase chain reaction and sequenced. Patient 1 was homozygous for a previously described mutation, p.T318M. Patient 2 was homozygous for a novel c.666delC mutation inherited from both parents resulting in p.223F>Sfsx295. No CYP11B2 mutation was detected in patient 3. CONCLUSIONS: We report the first CYP11B2 defects in Southeast Asian families responsible for aldosterone synthase deficiency and identified a novel CYP11B2 mutation. However, the affected gene(s) responsible for primary hypoaldosteronism other than CYP11B2 remain to be determined.

Homozygosity for a mutation in the CYP11B2 gene in an infant with congenital corticosterone methyl oxidase deficiency type II.

UNLABELLED: Isolated aldosterone synthase deficiency can be the source of life-threatening salt wasting and failure to thrive in infancy. We studied an infant with failure to thrive and persistent hyponatremia despite oral sodium supplementation. Initial analyses revealed highly elevated plasma renin but normal values of plasma aldosterone. The biochemical diagnosis of corticosterone methyl oxidase deficiency type II was established by multisteroid analysis, revealing a pathognomonic pattern with a highly elevated ratio of 18-OH-corticosterone to aldosterone. This reflects an enzymatic defect in the aldosterone synthase that is responsible for the terminal steps in the aldosterone biosynthesis. Molecular genetic analysis supported the diagnosis revealing homozygosity for a pathogenic c.554C>T (p.T185I) variation in exon 3 of the CYP11B2 gene encoding aldosterone synthase. Homozygosity for two other polymorphic variations c.504C>T (p.F168F) and c.518A>G (p.K173R) were identified as well. Treatment with fludrocortisone resulted in catch-up growth. Discontinuation of treatment at the age of 9 years was later possible without any clinical or biochemical deterioration. CONCLUSIONS: Isolated deficiency in aldosterone biosynthesis should be considered in neonates and infants with failure to thrive and salt wasting. Normal levels of plasma aldosterone compared with highly elevated levels of plasma renin indicate an impaired aldosterone biosynthesis and suggest the disorder. Recognition of its existence is important as fludrocortisone replacement therapy effectively normalizes sodium balance and growth.

Catch-up Growth and Discontinuation of Fludrocortisone Treatment in Aldosterone Synthase Deficiency.

BACKGROUND: Aldosterone synthase deficiency (ASD) caused by mutations in the CYP11B2 gene is characterized by isolated mineralocorticoid deficiency. Data are scarce regarding clinical and biochemical outcomes of the disease in the follow-up. OBJECTIVE: Assessment of the growth and steroid profiles of patients with ASD at the time of diagnosis and after discontinuation of treatment. DESIGN AND METHOD: Children with clinical diagnosis of ASD were included in a multicenter study. Growth and treatment characteristics were recorded. Plasma adrenal steroids were measured using liquid chromatography-mass spectrometry. Genetic diagnosis was confirmed by CYP11B2 gene sequencing and in silico analyses. RESULTS: Sixteen patients from 12 families were included (8 females; median age at presentation: 3.1 months, range: 0.4 to 8.1). The most common symptom was poor weight gain (56.3%). Median age of onset of fludrocortisone treatment was 3.6 months (range: 0.9 to 8.3). Catch-up growth was achieved at median 2 months (range: 0.5 to 14.5) after treatment. Fludrocortisone could be stopped in 5 patients at a median age of 6.0 years (range: 2.2 to 7.6). Plasma steroid profiles revealed reduced aldosterone synthase activity both at diagnosis and after discontinuation of treatment compared to age-matched controls. We identified 6 novel (p.Y195H, c.1200 + 1G > A, p.F130L, p.E198del, c.1122-18G > A, p.I339_E343del) and 4 previously described CYP11B2 variants. The most common variant (40%) was p.T185I. CONCLUSIONS: Fludrocortisone treatment is associated with a rapid catch-up growth and control of electrolyte imbalances in ASD. Decreased mineralocorticoid requirement over time can be explained by the development of physiological adaptation mechanisms rather than improved aldosterone synthase activity. As complete biochemical remission cannot be achieved, a long-term surveillance of these patients is required.

Aldosterone decreases glucose-stimulated insulin secretion in vivo in mice and in murine islets.

AIMS/HYPOTHESIS: Aldosterone concentrations increase in obesity and predict the onset of diabetes. We investigated the effects of aldosterone on glucose homeostasis and insulin secretion in vivo and in vitro. METHODS: We assessed insulin sensitivity and insulin secretion in aldosterone synthase-deficient (As [also known as Cyp11b2](-/-)) and wild-type mice using euglycaemic-hyperinsulinaemic and hyperglycaemic clamps, respectively. We also conducted studies during high sodium intake to normalise renin activity and potassium concentration in As (-/-) mice. We subsequently assessed the effect of aldosterone on insulin secretion in vitro in the presence or absence of mineralocorticoid receptor antagonists in isolated C57BL/6J islets and in the MIN6 beta cell line. RESULTS: Fasting glucose concentrations were reduced in As (-/-) mice compared with wild-type. During hyperglycaemic clamps, insulin and C-peptide concentrations increased to a greater extent in As (-/-) than in wild-type mice. This was not attributable to differences in potassium or angiotensin II, as glucose-stimulated insulin secretion was enhanced in As (-/-) mice even during high sodium intake. There was no difference in insulin sensitivity between As (-/-) and wild-type mice in euglycaemic-hyperinsulinaemic clamp studies. In islet and MIN6 beta cell studies, aldosterone inhibited glucose- and isobutylmethylxanthine-stimulated insulin secretion, an effect that was not blocked by mineralocorticoid receptor antagonism, but was prevented by the superoxide dismutase mimetic tempol. CONCLUSIONS/INTERPRETATION: We demonstrated that aldosterone deficiency or excess modulates insulin secretion in vivo and in vitro via reactive oxygen species and in a manner that is independent of mineralocorticoid receptors. These findings provide insight into the mechanism of glucose intolerance in conditions of relative aldosterone excess.

Molecular Analysis of the CYP11B2 Gene in 62 Patients with Hypoaldosteronism Due to Aldosterone Synthase Deficiency.

CONTEXT: Isolated congenital hypoaldosteronism presents in early infancy with symptoms including vomiting, severe dehydration, salt wasting, and failure to thrive. The main causes of this rare autosomal recessive disorder is pathogenic variants of the CYP11B2 gene leading to aldosterone synthase deficiency. OBJECTIVE: To investigate the presence of CYP11B2 pathogenic variants in a cohort of patients with a clinical, biochemical, and hormonal profile suggestive of aldosterone synthase deficiency. DESIGN: Clinical and molecular study. SETTING: Tertiary academic Children's Hospital, Center for Rare Pediatric Endocrine Diseases. PATIENTS AND METHODS: Sixty-two patients (56 unrelated patients and 6 siblings), with hypoaldosteronism and their parents, underwent CYP11B2 gene sequencing after its selective amplification against the highly homologous CYP11B1 gene. In silico analysis of the identified novel variants was carried out to evaluate protein stability and potential pathogenicity. RESULTS: CYP11B2 gene sequencing revealed that 62 patients carried a total of 12 different pathogenic CYP11B2 gene variants, 6 of which are novel. Importantly, 96% of the 56 patients carried the previously reported p.T185I variant either in homozygosity or in compound heterozygosity with another variant. The 6 novel variants detected were: p.M1I, p.V129M, p.R141Q, p.A165T, p.R448C, and the donor splice site variant of intron 8, c.1398 + 1G > A. CONCLUSION: Molecular diagnosis was achieved in 62 patients with aldosterone synthase deficiency, the largest cohort thus far reported. Six novel genetic variants were identified as possibly pathogenic, extending the spectrum of reported molecular defects of the CYP11B2 gene.

Five novel mutations in CYP11B2 gene detected in patients with aldosterone synthase deficiency type I: Functional characterization and structural analyses.

CONTEXT: Aldosterone synthase deficiency (ASD) is an important differential diagnosis of diseases associated with salt wasting in early infancy. OBJECTIVE: The objective of this study was to investigate the molecular basis for the disorder by (1) molecular genetic analysis in the CYP11B2 from patients suffering from ASD type I. (2) Functional characterization of the missense mutant gene products. (3) Structural simulation of the missense mutations. RESULTS: Patient 1 was a homozygous carrier of a novel mutation located in exon 4 causing a premature stop codon (p.W260X). Patient 2 was analyzed to be compound heterozygous for two novel mutations: The first was an insertion mutation (p.G206WfsX51), and the second was a deletion mutation (p.L496SfsX169). Two siblings (patients 3 and 4) were compound heterozygous carriers of two novel missense mutations (p.S315R, p.R374W). The expression studies of the mutant proteins in COS-1 cells showed a complete absence of CYP11B2 activity of p.S315R and p.R374W mutants for the conversion of 11-deoxycorticosterone to aldosterone. A 3-D model of CYP11B2 p.S315R and p.R374W indicated a change of the hydrogen bond network which might explain the cause of the dysfunction. CONCLUSION: We have identified the first CYP11B2 gene defects in two Polish families associated with phenotypes of ASD type I. Analysis of the enzymatic function as a complementary procedure to genotyping revealed data for understanding the clinical phenotype of ASD. Molecular modeling of the mutated enzyme provided a rational basis for understanding the changed activities of the mutant proteins.

Congenital hyperreninemic hypoaldosteronism due to aldosterone synthase deficiency type I in a Portuguese patient - Case report and review of literature.

Hyperreninemic hypoaldosteronism due to aldosterone synthase (AS) deficiency is a rare condition typically presenting as salt-wasting syndrome in the neonatal period. A one-month-old Portuguese boy born to non-consanguineous parents was examined for feeding difficulties and poor weight gain. A laboratory workup revealed severe hyponatremia, hyperkaliaemia and high plasma renin with unappropriated normal plasma aldosterone levels, raising the suspicion of AS deficiency. Genetic analysis showed double homozygous of two different mutations in the CYP11B2 gene: p.Glu198Asp in exon 3 and p.Val386Ala in exon 7. The patient maintains regular follow-up visits in endocrinology clinics and has demonstrated a favourable clinical and laboratory response to mineralocorticoid therapy. To our knowledge, this is the first Portuguese case of AS deficiency reported with confirmed genetic analysis.

Analysis of novel heterozygous mutations in the CYP11B2 gene causing congenital aldosterone synthase deficiency and literature review.

Aldosterone synthase deficiency (ASD) is a rare autosomal recessive disorder characterized by severe hyperkalemia, salt loss, vomiting, severe dehydration and failure to thrive. ASD is a life-threatening electrolyte imbalance in infants resulting from mutations in CYP11B2. We described ASD in a Chinese male infant with vomiting, poor feeding and failure to thrive. He was mildly dehydrated, with a weight of 6 kg (-3.45 SDS) and length of 67 cm (-3.10 SDS). Laboratory tests showed hyponatremia (119 mmol/L), serum potassium 5.4 mmol/L, low plasma aldosterone and plasma renin activity (PRA) levels. Next-generation sequencing of his DNA revealed compound heterozygous mutations in CYP11B2, a known variant c.1391_1393delTGC (p.Leu464del, rs776404064) and a novel variant c.1294delA (p.Arg432Glyfs*37). The HEK-293T expression system was used to investigate the variants, demonstrating negligible aldosterone synthesis compared with WT CYP11B2. The patient started fludrocortisone and subsequently gained 3.2 kg of weight and normalized serum sodium (137 mmol/L). We further reviewed reported cases of ASD, summarizing clinical features and CYP11B2 mutations; missense and nonsense mutations are most frequent. Fludrocortisone treatment is essential for ASD, and the need for mineralocorticoid replacement wanes with age; eventually, therapy can be discontinued for many affected children. Our study broadens the ASD phenotypic spectrum and shows the efficiency of next-generation sequencing for patients with atypical clinical manifestations.

Aldosterone synthase deficiency caused by a homozygous L451F mutation in the CYP11B2 gene.

Isolated hypoaldosteronism is a rare cause of salt wasting in infancy and may be life-threatening, especially in the newborn infant. In a 3wk-old-boy with hyponatremia and hyperkalemia a GC-MS steroid profile on a spot urinary sample showed no 18-oxygenated steroid metabolites indicative for aldosterone synthase deficiency type I. Sequence analysis of the CYP11B2 gene revealed that the patient was homozygous for a novel missense mutation (L451F) caused by a T to C transition at position c.1351 in exon 8, whereas each non-symptomatic parent possessed only one mutated allele. The mutant cDNA was transiently expressed in a human cell line, HCT116 p53(-/-), and activity of the expressed protein optimized by co-expression of different adrenodoxin species, showing complete aldosterone deficiency with 11-deoxycorticosterone or corticosterone as substrates. The L451F mutation is the first mutation found located immediately adjacent to the highly conserved heme-binding C450 of the cytochrome P450. Computer modeling shows that replacement of leucine by phenylalanine leads to a steric effect in the immediate vicinity of the heme thereby preventing the activity of CYP11B2. Thus, by combining highly sensitive hormone detection in a spot urine sample with expression of the mutated cDNA in cell culture the phenotype of the patient can be correlated with a particular molecular defect.