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

Steroids 150 (2019) 108448

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

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Hui Miaoa,1, Zhongxun Yub,1, Lin Lua, , Huijuan Zhua, , Richard J. Auchusc, Jiayan Liuc, Jun Jiangd, Hui Pana, Fengying Gonga, Shi Chena, Zhaolin Lua a Key Laboratory of Endocrinology of National Health Commission, Department of Endocrinology, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100730, China b Department of Pediatrics, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100730, China c Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, Department of Pharmacology, and the Program for Disorders of Sexual Development, University of Michigan, Room 5560A, MSRBII, 1150 West Medical Center Drive, Ann Arbor, MI 48109, United States d The Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Aldosterone synthesis Hypoaldosteronism CYP11B2 mutations Next-generation sequencing

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 nextgeneration sequencing for patients with atypical clinical manifestations.

1. Introduction Aldosterone is produced in the zona glomerulosa of adrenal cortex and is the most important mineralocorticoid hormone in humans. Aldosterone is mainly responsible for electrolyte balance. Aldosterone not only regulates sodium absorption and intravascular volume but also potassium excretion via distal convoluted tubules and renal cortex collecting ducts [1]. Aldosterone synthase deficiency (ASD) is a rare autosomal recessive disorder that causes life-threatening electrolyte imbalance in infants. Affected children often display hyperkalemia, salt loss, vomiting, severe dehydration and failure to thrive in the first months of life [2,3]. This disease is mainly due to loss-of-functional

mutations in the gene CYP11B2, which is located on chromosome 8q24.3 and contains 9 exons and 503 amino acids [4]. CYP11B2 encodes the cytochrome P450 enzyme aldosterone synthase [1]. This enzyme catalyzes the final three steps in aldosterone biosynthesis as follows: the 11β-hydroxylation of 11-deoxycorticosterone (DOC) to form corticosterone, the 18-hydroxylation of corticosterone to form 18hydroxycorticosterone, and finally, the 18-oxidation of 18-hydroxycorticosterone to form aldosterone [5,6]. There are two types of ASD, formerly known as corticosterone methyl oxidase deficiency type 1 (CMO I) and type 2 (CMO II) based on the specific defects in aldosterone synthesis. These two types of ASD both present low aldosterone levels and elevated plasma renin activity

Abbreviations: ASD, aldosterone synthase deficiency; CMO, corticosterone methyl oxidase; DOC, 11-deoxycorticosterone; PRA, plasma renin activity; NGS, nextgeneration sequencing; HPLC, high-performance liquid chromatography ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Lu), [email protected] (H. Zhu). 1 These authors have contributed equally to this work. https://doi.org/10.1016/j.steroids.2019.108448 Received 21 March 2019; Received in revised form 1 July 2019; Accepted 3 July 2019 Available online 11 July 2019 0039-128X/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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2.2. Genetic analysis

but can be distinguished by the production of 18-hydroxycorticosterone. ASD type 1 or CMO I affects the hydroxylation of corticosterone and thus exhibits decreased levels of 18-hydroxycorticosterone, while ASD type 2 or CMO II patients show markedly elevated levels of 18-hydroxycorticosterone due to defects restricted to the oxidation of 18-hydroxycorticosterone to aldosterone [6,7]. Reduced mineralocorticoids can be found in other diseases, the most common cause of which is congenital adrenal hyperplasia due to 21hydroxylase deficiency (21OHD). Patients with salt-wasting 21OHD may show signs of severe renal salt loss, which can be life-threatening during the first weeks of life. Unlike ASD, 21OHD also features cortisol deficiency, genital virilization in affected females, and elevated serum 17-hydroxyprogesterone (17OHP). These patients often carry inactivating mutations in the 21-hydroxylase gene (CYP21A2). But Elevated 17OHP could also be found in newborns not only with pathological condition such as preterm birth, infection and stress but also with other types of congenital adrenal hyperplasia (CAH) such as 11hydroxylase deficiency (11OHD), some of 17-hydroxylase deficiency (17OHD), 3β-hydroxysteroid dehydrogenase deficiency (3βHSD) and occasionally cytochrome P450 oxidoreductase defects (PORD) [6,8,9]. In addition, when patients show hyponatremia with no signs of hyperandrogenism, defects in 3β-hydroxysteroid dehydrogenase/isomerase (caused by HSD3B2 mutations), cholesterol side-chain cleavage enzyme (P450scc, caused by CYP11A1 mutations) and the steroidogenic acute regulatory protein (StAR, caused by StAR mutations) should be considered [9]. Here, we report an infant boy with growth retardation, poor weight gain, failure to thrive, hyponatremia, and low plasma aldosterone. The diagnosis of ASD was finally confirmed by molecular analysis due to his atypical clinical features. This patient was found to have compound heterozygous mutations; one mutation was a reported three single nucleotide deletion, and the other was a novel frame shift mutation.

This study was approved by the ethics committee of the Peking Union Medical College Hospital. Informed consent and blood samples for DNA extraction were obtained from the parents on behalf of the infant. DNA was isolated using the EZNA Blood DNA Midi Kit (Omega, USA); a custom next-generation sequencing (NGS) panel, which was designed to target 23 hyponatremia genes with hot spot mutations for genetic testing, was used to analyze the isolated DNA. Three micrograms of DNA was used, and sequencing was processed on an Illumina HiSeq 2000 system. The primers of the CYP11B2 gene were designed by Oligo 7 Primer Analysis Software according to the NCBI reference sequence (NG_008374.1). The sequences of the primers to amplify fragments of exon 8 were as follows: forward: 5′-CCCCACACCCCTCGAGCTG-3′ and reverse: 5′-AAGGCCCCATCCACTGTTCCC-3′. Polymerase chain reaction (PCR) was conducted in 20 μL containing 2 μL genomic DNA, 1 μL (10 μmol/L) forward and reverse primers (each), 10 μL Taq SuperMix (10X reaction buffer, Taq DNA polymerase, deoxy-NTP), and 6 μL double distilled water. The cycling conditions were as follows: 94 °C for 4 min, then 36 cycles of 94 °C for 30 sec, 65 °C for 30 sec and 72 °C for 30 sec, and finally, 4 min at 72 °C. 2.3. CYP11B2 expression and enzymatic activity 2.3.1. Site-directed mutagenesis in pcDNA3-CYP11B2 pcDNA3 plasmid and full-length human CYP11B2 cDNA were obtained from OriGene (SC102224, Rockville, MD). QuikChange SiteDirected Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) was used to generate the CYP11B2 mutation constructs. Primers were designed using the Agilent QuikChange primer design tool software. Wildtype (WT) and mutated CYP11B2 plasmid DNA were generated in large quantities using a Qiagen (Valencia, CA) Maxi Prep Kit. Primer sequences are as follows: L464del (sense): 5′-GATGCTGCTGCTGCACCA CGTGCTGAAG-3′; L464del (antisense): 5′-CTTCAGCACGTGGTGCAGC AGCAGCATC-3′; R432Gfs*37 (sense): 5′-CTGGCTAGACATCGGGGCTC CGGCAG-3′; And R432Gfs*37 (antisense): 5′-CTGCCGGAGCCCCGATG TCTAGCCAG-3′.

2. Subject and methods 2.1. Clinical manifestation and initial tests A male infant was born apparently normal at full-term. The birth weight of this boy was 2.8 kg, and the length was 49 cm. His parents are Chinese and denied consanguinity. He was breastfed after birth. From 2 months of age, he was referred to various local hospitals due to recurrent vomiting, poor feeding and failure to thrive. Upon medical examination at 3 months old, his body weight was 4.25 kg (−3.14 SDS) and length was 58 cm (−1.75 SDS). The patient was initially diagnosed with an allergy to formula and was treated with different formulas, but he still had repeated vomiting and poor weight gain. At 11 months of age, he was referred to our hospital for further examination. On admission, the boy was mildly dehydrated with a weight of 6 kg (−3.45 SDS) and a length of 67 cm (−3.10 SDS). He had normal external genitalia, and no hyperpigmentation was found. Then, the endocrine laboratory test for this patient was performed. Serum DOC, corticosterone, 18OH-corticosterone and aldosterone were measured with an isotope dilution-LC–MS/MS method. Before analysis, serum samples, calibrators, and controls were thawed, mixed thoroughly, and equilibrated to room temperature. 200 μL of each sample was precisely withdrawn using a micropipette and transferred to a 5-mL glass tube. The LC–MS/MS analysis was performed using a Jasper HPLC system coupled with a triple quadruple mass spectrometer 4500MD system (Sciex Applied Biosystems, Foster City, CA, USA). Chromatographic separation was performed on an Ascentis Express F5 column (2.1 mm × 100 mm; 2.7 μm). MS/MS detection was carried out in the positive electrospray ionization mode with multiple reaction monitoring (MRM).

2.3.2. Expression in HEK-293T cells for comparing to empty vector and WT HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Valley Biomedical). Cells were seeded in 12-well plates such that the confluency reached 60% on the day of transfection. WT and variant CYP11B2 constructs were transiently transfected into HEK-293T cells using TransIT-LT1 Transfection Reagent (Mirus Bio, Madison WI) with 1 μg plasmid DNA. 2.3.3. Substrate treatment and high-performance liquid chromatography (HPLC) analysis Twenty-four hours after transfection, the cells were incubated with 1 μM DOC for 24 h. Cell culture medium was collected extracted with 1 mL of dichloromethane and concentrated under nitrogen gas. HPLC analysis was performed using an Agilent 1260 HPLC system. Samples were reconstituted in 20 μL methanol, and 7 μL were injected into the HPLC. Steroids were separated using a 50 × 2.1 mm, 2.6 μm, C8 Kinetex column (Phenomenex, Torrance, CA) maintained at 30 °C, and samples were eluted using methanol/water gradients following methods described previously [10]. DOC, corticosterone, 18-hydroxycorticosterone and aldosterone peaks were identified by retention times of external standards chromatographed. DOC conversion was calculated using the total production of corticosterone, 18-hydroxycorticosterone and aldosterone divided by the sum of substrates and products. 2

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2.3 μg/dL) were also normal. Ultrasonography of the kidneys and adrenal glands were normal. There were no signs of salt-wasting nephropathy or enteropathy, urinary tract obstruction or infection. CAH with hyperandrogenism such as 21OHD and other adrenal disorders affecting adrenal glands such as 3β-HSD, CYP11A1 and StAR mutation were excluded due to normal plasma cortisol, testosterone and 17OHP. According to the clinical and laboratory results, treatment with oral sodium chloride and nutritional support was started immediately. However, serum sodium levels still ranged from 125 to 130 mmol/L. Considering the atypical clinical features of this patient, NGS was performed for molecular diagnosis. The patient was suspected to have ASD, and fludrocortisone (75 μg/day) was administered. The plasma sodium concentration increased to 133 mmol/L and potassium level dropped to 5.1 mmol/L after 4 days of treatment with fludrocortisone. After 5 months of therapy, the patient demonstrated weight gain of 3.2 kg, catch-up of growth (Table 2; Fig. 1), and normalization of serum sodium (137 mmol/L) with serum potassium of 4.8 mmol/L. He remained clinically well while receiving fludrocortisone replacement. On the first visit in our hospital at the age of 11 months old, the infant was in the life-threatening condition because of severe hyponatremia and dehydration. Intravenous fluid and sodium supplementation were given immediately in pediatric intensive care units. Steroid hormones and PRA were collected after 1 week of such treatment. The first test of PRA 0.01 ng/ml/h (Table 1) maybe impacted by volume and sodium supplementation. In the subsequent follow up after treatment of fludrocortisone, the second PRA was 0.6 ng/ml/h at the age of 20 months old (potassium 3.88 mmol/L and Na 138.7 mmol/L), and the third PRA was > 12 ng/ml/h at the age of 28 months old (potassium 5.09 mmol/L and Na 138.7 mmol/L). We measured the adrenal steroids of the stored sample before fludrocortisone treatment for this boy. The result showed DOC and corticosterone were obviously increased, while the 18OH-corticosterone was in normal range (Table 1). As a result, the boy could be identified as ASD 1.

Table 1 Laboratory examinations of the patient. Levels

Normal range

Sodium Potassium

119 mmol/L 5.4 mmol/L

Cortisol ACTH 17-hydroxyprogesterone Dehydroepiandrosterone sulfate Testosterone Plasma renin activity

22.2 μg/dL 15.2 pg/mL 0.27 ng/mL 2.3 μg/dL < 0.1 ng/mL 0.01 ng/ml/h (11 mo) 0.6 ng/ml/h (20 mo) > 12 ng/ml/h (28 mo) 3.23 ng/mL > 20 ng/mL 1.25 ng/mL < 0.02 ng/mL

135–145 mmol/L 3.5–6.1 mmol/L (6 mo–1yr) 3.3–4.6 mmol/L (> 1 yr) 4.0–22.3 μg/dL 0–46 pg/mL 0.06–2.15 ng/mL 0.4–5.2 μg/dL 0.04–0.06 ng/mL 0.05–0.79 ng/ml/h

11-deoxycorticosterone* Corticosterone* 18-hydroxycorticosterone* Aldosterone*

0.07–0.57 ng/mL 0.6–12.93 ng/mL 0.039–2.5 ng/mL 0.03–0.16 ng/mL

mo:months; yr: years. * Assay method with LC/MS-MS.

2.4. Literature review The literature regarding ASD was reviewed by searching PubMed and MEDLINE with the following key words: ASD, hypoaldosteronism, CMO deficiency and CYP11B2 mutation. The selected literature was limited to case reports written in English. References of included articles were also searched. 2.5. Statistics analysis All statistics analysis was performed using SPSS software version 21.0. Pearson’s χ2 test/Fisher’s exact test was used to show the differences of patients with and without certain phenotypes. Non-parametric test was used if data were still not normally distributed.

3.2. Genetic analysis The patient was found to have compound heterozygous mutations with a known three single nucleotide deletion of c.1391_1393delTGC (p.Leu464del, rs776404064) and a novel frame shift mutation c.1294delA (p.Arg432Glyfs*37) in the CYP11B2 gene. Further confirmation of these mutations was performed by amplifying exon 8 of the CYP11B2 gene by PCR, followed by direct DNA sequencing (Fig. 2). Sanger sequencing of CYP11B2 in the parents revealed that the father was heterozygous for c.1391_1393delTGC (p.Leu464del), and the mother was heterozygous for c.1294delA (p.Arg432Glyfs*37).

3. Results 3.1. Clinical and biochemical data Initial laboratory examinations of the patient (Table 1) showed he had quite low sodium (119 mmol/L, normal range: 135–145 mmol/L), serum potassium of 5.4 mmol/L, metabolic acidosis (pH 7.337) and normal plasma creatinine. The 24 h urinary sodium excretion was 45 mmol/24 h (normal range, 18–60 mmol/24 h). Endocrinological evaluation showed low plasma aldosterone levels (< 0.02 ng/mL, normal range: 0.03–0.16 ng/mL) and low PRA (0.01 ng/ml/h, normal range, 0.05–0.79 ng/ml/h). The morning serum cortisol level was 22.2 μg/dL (normal range, 4.0–22.3 μg/dL), and the plasma adrenocorticotropic hormone (ACTH) level was 15.2 pg/mL (normal range, 0–46 ng/mL); both serum cortisol and ACTH levels were within normal ranges. Plasma 17-hydroxyprogesterone (0.27 ng/mL), testosterone (less than 0.1 ng/mL) and dehydroepiandrosterone sulfate (DHEAS,

3.3. Functional studies of novel CYP11B2 mutations Fig. 3 shows the results by HPLC analysis for the conversion of deoxycorticosterone into products by human CYP11B2. HEK-293T cell line lacks steroidogenic enzyme expression and is suitable for testing enzyme activities. After 24 h incubation with DOC, HEK-293T cells transfected with WT CYP11B2 converted nearly all substrate to 18hydroxycorticosterone and aldosterone. In contrast, products from cells

Table 2 Growth parameters progression. Growth parameters

2 months old

9 months old

11 months old*

14 months old

17 months old

24 months old

Weight (kg)

4.0 (−2.52SDS) 56 (−1.20SDS)

6.0 (−3.16SDS) 66 (−2.59SDS)

6.0 (−3.45SDS) 67 (−3.10SDS)

8.2 (−2.07SDS) 71 (−2.98SDS)

9.2 (−1.65SDS) 74 (−2.78SDS)

11.0 (−1.08SDS) 84 (−1.27SDS)

Length (cm)

* Fludrocortisone treatment started. 3

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Fig. 1. Growth chart with centile showing weight and length of the patient. Significant catch-up growth can be observed after fludrocortisone therapy. Time of initial treatment was marked by an arrow.

patients with ASD type 1 and 2 (p > 0.05). Laboratory tests indicated that elevated PRA levels were more likely to be found in the ASD type 1 group. For patients with ASD type 2, serum 18-hydroxycorticosterone values are much higher than those in patients with ASD type 1 (median, 11.4-fold versus 1.2-fold above the upper limit of normal range). For mutations, genetic analyses of CYP11B2 have found missense, nonsense mutations, splicing mutations, small insertions/deletions, gross deletions and complex rearrangements; the most common mutations were missense and nonsense mutations (Fig. 4, Supplementary Table). Some variants, such as p.Q170X, p.E198D, c.1398+2T > A, p.F223Sfsx295, p.L462R, p.Q337X and p.Q272X, were identified in patients without an ASD classification subtype. Treatment with fludrocortisone was effective for patients with ASD, often at a dosage of 0.1 mg/day, and symptoms improved in all patients who underwent fludrocortisone therapy (Table 3).

expressing the variant p.Leu464del and p.Arg432Glyfs*37 were not detected (Fig. 3A, B), consistent with ASD type 1.

3.4. Literature review of clinical features and mutations of CYP11B2 gene Since the ASD was a rare disease, we try to find some different clinical features in 2 subtypes in previous reported cases of ASD. Their clinical features and mutations of CYP11B2 were shown in Table 3 and Fig. 4, respectively. 44 patients presented symptoms in the first year of life. Failure to thrive, recurrent vomiting and dehydration were most frequently encountered in ASD patients. Clinical characteristics including the clinical manifestations, biochemical results and hormonal assays showed no significant difference in ASD type 1 and 2 (p > 0.05), other than 18-hydroxycorticosterone (see below). Thus, it is not possible to reliably distinguish ASD types 1 and 2 solely based on the clinical features. In total, 44 out of 45 individuals had hyponatremia, including 95% of patients having ASD type 1 and all ASD type 2 or undefined subtypes. Hyperkalemia was observed in 89% of patients, and the sodium and potassium levels showed no difference between

4. Discussion ASD is an extremely rare autosomal recessive disease in infants with 4

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Fig. 2. Heterozygous mutation detected in exon8 of CYP11B2. The patient had compound heterozygous: c.1294delA (pArg432Glyfs*37) inherited from the mother and c.1391_1393delTGC (pLeu464del) from the father. The mutations are marked by arrows and lines. Note that the sequence shown in panel B is the reverse strand.

between ASD subtypes, but serum 18-hydroxycorticosterone values in patients with ASD type 2 are much higher than those in ASD type 1 patients (median, 11.4-fold versus 1.2-fold above the upper limit of normal range). In our patient, the boy showed remarkably increasing DOC and corticosterone values, while the 18OH-corticosterone was in normal range (Table 1). In vitro tests also demonstrated undetectable intermediates or final product (Fig. 3), which is consistent with ASD 1. Taken together, this patient could be identified as ASD 1. To our knowledge, approximately 40 mutations in the CYP11B2 gene have been reported in cases of ASD, and missense and nonsense mutations are most common among them (Fig. 4). A majority of mutations have been found to completely destroy enzymatic activity, while in some mutations, double homozygosity is required for clinical phenotype (for instance, V386A and R181W, which are common mutations in Iranian Jewish patients with ASD). In vitro experiments indicated

typical manifestations of frequent vomiting, salt loss, dehydration and failure to thrive. Biochemical examinations often reveal hyponatremia, hyperkalemia, elevated PRA and low levels of aldosterone. We described a Chinese boy who presented with vomiting, poor weight gain, growth retardation, and hyponatremia; however, our patient did not have an elevated PRA or markedly elevated serum potassium typical of ASD in the beginning. Similar features have been reported in other cases of ASD [11,12], which illustrates the variability of serum potassium in ASD. One mechanism to conserve potassium involves renal outer medullary potassium and epithelial sodium channels, which can be activated independent of aldosterone [13]. For rare hyponatremia, steroids measurement, such as DOC, corticosterone and 18OH-corticosterone is of great significance in the diagnosis. In reported literature, approximately 50% patients underwent test (Table 3). It is found that DOC showed no remarkable distinction 5

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Fig. 3. HPLC chromatogram of CYP11B2 wild type and mutations. A. Steroid standards resolved by HPLC and detected by absorbance at 254 nm. Y-axis indicates absorbance units × 10−3 (mAU). Each steroid is presented as a single peak except 18-hydroxycorticosterone due to spontaneous formation of cyclic tautomer during storage (later eluting peak). B. Stacked chromatograms of HPLC analysis of metabolites by CYP11B2 wild type (blue line) and mutants pLeu464del (red line) or pArg432Glyfs*37 (green line). Wild type CYP11B2 consumed 97% of the substrate DOC and produced mainly 18-hydroxycorticosterone and aldosterone. Mutant CYP11B2 enzymes yielded no detectable intermediates or final product. C. Total activity comparison between wild type and mutant CYP11B2. The conversion rate was calculated as products of corticosterone, 18-hydroxycorticosterone and aldosterone in total divided the substrate DOC.

In contrast to the common dose of 100 μg/d in other cases, our patient underwent fludrocortisone therapy initiated at 75 μg/d, and gradually reduced to 50 μg/d. Fludrocortisone treatment is essential for patients with ASD, but symptoms of salt wasting improve with age; eventually, the therapy can be discontinued for most children when they grow older. There are two main reasons why the mineralocorticoid requirements of children with ASD decrease with age. Firstly, mineralocorticoid receptors are poorly expressed in the renal epithelium of newborns and this increases with age, thus increasing sensitivity to mineralocorticoids [30,31]. Secondly, newborn diets (especially mothers' milk) had low content of sodium, and dietary sodium increased with age with transition to 'table foods'. Other reasons mentioned before including increased sodium reabsorption due to mature renal tubules and alternative pathway of mineralocorticoid biosynthesis [32,33]. Recent studies recommend an initial dose of 100–200 μg/day, and the dose should be adjusted regularly according to clinical and laboratory data, such as blood pressure, serum sodium and potassium, and plasma renin activity [30,34]. Hypoaldosteronism and hyponatremia can be seen in many diseases. ASD is readily suspected based on typical clinical manifestations, but accurate diagnosis can be difficult when patients present atypical features, such as for our child. Given our limited ability to measure diagnostic steroids, we employed NGS technologies with a custom panel to arrive at a molecular diagnosis, while fludrocortisone therapy was instituted.

that p.V386A or p.R181W alone was insufficient for the disease phenotype, and only double homozygosity could cause aldosterone deficiency, with 0.2% WT activity [14–16]. ASD is a rare disease, and as far as we know, only two cases have been reported in China. One patient was compound heterozygous for c.977C > A and c.523_525delAAG, and the other patient was a compound heterozygote for mutations c.1009C > T and c.240‑1G > A [17,18]. In this study, our patient was identified as compound heterozygous for two frame shift mutations in exon 8. One mutation was the novel c.1294delA (p.Arg432Glyfs*37) and inherited maternally, and the other mutation was c.1391_1393delTGC (p.Leu464del) and inherited paternally. The latter mutation is known as rs776404064 and has a minor allele frequency (MAF) of 0.00003 in Exome Aggregation Consortium (ExAC), while c.1294delA (p.Arg432Glyfs*37) is a novel mutation leading to a premature stop codon and thus yields a truncated protein. Aldosterone synthesis of the encoded enzymes is impaired, as shown in Fig. 3. The truncated enzyme lacks the heme-binding motif containing Cys449, which precludes enzymatic activity [19,20]. A prompt diagnosis of ASD is necessary for successful treatment of the disorder. In our case, sodium chloride supplementation alone was able to maintain a serum sodium above 130 mmol/l; however, after treatment with fludrocortisone, the plasma sodium level normalized (137 mmol/L). In addition, the patient showed significant growth catch-up; after the treatment, the height and weight of the patient were −1.08 SDS and −1.27 SDS at the age of 24 months, compared with −3.45 SDS and −3.10 SDS before the treatment (Table 2). Other reported patients have also shown normal growth and development after treatment with fludrocortisone [21–25]. Consistent with previous studies, this patient illustrates a relationship between hyponatremia and failure to thrive. Salt wasting could be a factor leading to impaired growth [26,27], possibly due to insulin-like growth factor-1 (IGF-1) suppression or reduced extracellular fluid volume [28,29], but the exact mechanism of this association is not clear.

5. Conclusion Here, we described a rare case of ASD in a Chinese male infant with molecular analysis, and functional studies in vitro further confirmed the impaired aldosterone synthesis by the encoded enzymes. ASD should be considered in infants presenting with poor feeding, salt wasting and failure to thrive but not virilism. Our study indicates that typical 6

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Table 3 Characteristics of cases with ASD from published literatures. ASD1 (%)

ASD2 (%)

ASD (undefined subtype) (%)

ASD total (%)

20 9/11

12 7/5

13 6/4

45 22/20

10 (50) 9 (45) 1 (5) 1d-90d, median, 21d (n = 19) 21d-47y, median, 525d (n = 12)

8 (67) 2 (17) 2 (17) 14d-1.83 yr, median, 38.5d (n = 12) 30d-14.5 yr, median, 315d (n = 10)

3 (23) 10 (77) – 3d-270d, median, 49d (n = 13) NA

21 (47) 21 (47) 3 (7)

Clinical features Vomiting Poor feeding Failure to thrive Dehydration Diarrhea Hypovolemic shock Growth retardation Poor weight gain

9 (45) 4 (20) 12 (60) 5 (25) 2 (10) – 3 (15) 11 (55)

4 (33) 3 (25) 10 (83) 3 (25) 2 (17) 1 (8) 4 (33) 7 (58)

8 1 8 4 4 1 3 6

21 (47) 8 (18) 30 (67) 12 (27) 8 (18) 2 (4) 10 (22) 24 (53)

Laboratory tests Hyponatremia Decreased sodium level (mmol/l) Median (25, 75 percentiles) Hyperkalemia Increased potassium level Median (25, 75 percentiles) Elevated PRA Low aldosterone 11-deoxycorticosterone Elevated folds* Median (25, 75 percentiles) Corticosterone Elevated folds* Median (25, 75 percentiles) 18-hydroxycorticosterone Elevated folds* Median (25, 75 percentiles)

19 (95) 125.4 (121.8, 128.5) 17 (85) 6.7 (6.1, 7.6) 19 (95) 13 (65) 8 (40) 2.5 (0.9, 6.0) 7 (35) 2.6 (1.4, 9.3) 11 (55) 1.2 (0.8, 2.1)

12 (100) 126.0 (114.0, 131.0) 10 (83) 6.4 (6.3, 6.8) 11 (92) 8 (67) 11 (92) 2.3 (1.1, 6.2) 9 (75) 5.3 (2.1, 6.1) 12 (100) 11.4 (4.6, 35.3)

13 (100) 124.0 (118.5, 129.0) 13 (100) 6.5 (6.3, 7.1) 11 (85) 9 (69) – – 1 (8) 4.6 1 (8) 1.2

Treatment Fludrocortisone Daily maximum dose of fludrocortisone

12 (60) 0.1

11 (92) 0.2

10 (77) 0.2

Number of subjects Gender (F/M) Geographical features Europe Asia North America Age at onset Age at diagnosis

(62) (8) (62) (31) (31) (8) (23) (46)

44 (98) 40 (89) 41 (91) 30 (67) 19 (42) 17 (38) 24 (53)

33 (73)

PRA: plasma renin activity; d, days; m: months; w, weeks; NA: limited data [11,12,16–19,21–25,35–52]. * Above the upper limit of normal range.

features of ASD are not always observed, and fludrocortisone treatment is required in infants to improve clinical features and to enable catchup growth. The in vitro tests of enzyme function were consistent with ASD type 1.

Acknowledgements This work was supported by “The National Key Research and Development Program of China” (No. 2016YFC0901501) and CAMS Innovation Fund for Medical Science (CAMS-2016-I2M-1-002). It’s our pleasure to give deep thanks to Ling Qiu, Songlin Yu and Yicong Yin (Clinical Laboratory, Peking Union Medical College Hospital) for giving us key support for further measurement of steroids with LC/MS-MS assay.

Declaration of Competing Interest All authors declare no conflict of interest.

Fig. 4. Mutations of CYP11B2 from literature review. ASD: Aldosterone synthase deficiency.[11,12,14–20,23–25,35–37,39–44,46–50,52–54] 7

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Appendix A. Supplementary data

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