Introduction
Sodium (Na) is the main determinant of serum osmolality concentration, and its concentration regulates the shifts between intracellular (ICF) and extracellular (ECF) fluid [1, 2]. Comparing with adults, infants have higher requirements for water and electrolyte. However, the ability to reabsorb sodium and water from urine are weaker in infants than in adults [3]. A good balance between water and sodium is essential to ensure infants’ healthy growth and development.
Severe hyponatraemia in infants is life-threatening and always represents the most typical symptoms of genetic forms of congenital primary adrenal failure [4]. Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (OMIM#201910) accounts for most cases, while other rare disorders including pseudohypoaldosteronism (PHA), aldosterone synthase deficiency (ASD), X-linked adrenal hypoplasia congenita (X-AHC), and familial glucocorticoid deficiency (FGD) can also cause severe hyponatraemia in infants [5, 6].
PHA type 1 (PHA 1) is a rare heterogeneous syndrome of functional abnormalities of the mineralocorticoid receptor (MR) (OMIM# 177735) or abnormalities in epithelial sodium channel (ENaC) (OMIM# 264350) of renal distal tubules leading to aldosterone (Ald) resistance [7]. PHA type 3 (PHA 3) comprises transient and secondary forms of salt wasting (SW) states caused by various pathologies of the renal, intestinal tract, or sweat glands, and the most common cause was nephropathies such as urinary tract infections and obstructive of the urinary tract [8–10]. Patients with PHA 1 or PHA 3 are usually presented with hyponatraemia, hyperkalaemia, metabolic acidosis, and elevated levels of plasma renin and aldosterone.
Aldosterone synthase (AS) is encoded by the CYP11B2 (OMIM#124080) gene and expressed in the adrenal cortex, which is a critical enzyme for aldosterone synthesis [11]. AS catalyses the 3 terminal sequential steps in aldosterone biosynthesis: the 11β-hydroxylation of 11-deoxycorticosterone (DOC), which forms corticosterone (B), the 18-hydroxylation of B to 18-hydroxycorticosterone (18-OHB), and the 18-oxidation of 18-OHB to form aldosterone [12, 13]. AS deficiency ASD- is an extremely rare autosomal recessive disorder mainly caused by inactivating mutations of the CYP11B2 gene [14]. The affected patients are typically associated with infant salt-wasting syndrome and mainly present with vomiting, poor feeding, and dehydration combined with failure to thrive in the first few weeks of life. Biochemical tests usually show hyponatraemia, hyperkalaemia, metabolic acidosis, and marked elevated plasma renin activity (PRA) with low or inappropriate normal aldosterone levels [15].
There were numerous differential aetiologies for infants with moderate or severe hyponatraemia. However, study on salt-wasting due to genetic aetiology is rare. We conducted a retrospective single-centre study, aiming to summarise the aetiologies and clinical features in infants (under one year old) with hyponatraemia due to genetic conditions. Meanwhile, we verified the pathogenicity of a novel compound heterozygous of CYP11B2 gene in in vitro experiments.
Material and methods
Patients and data collection
All children under the age of one year, admitted to the Paediatric unit of the First Affiliated Hospital of Guangxi Medical University from January 2012 to July 2022 were recruited. The inclusion criteria were as follows: (1) clinical symptoms relating hyponatraemia or suggesting inherited metabolic disorders, such as vomiting, poor weight gain, hyperpigmentation, and abnormal external genitalia. (2) hyponatraemia (serum sodium concentration < 130 mmol/L) during the first year of life; (3) a positive gene test result associated with hyponatraemia. Diagnosis was based on practical guidelines, and for rare cases, it mainly depended on the clinical manifestation, biochemical test, and genetic testing result. The exclusion criteria were as follows: (1) blood sample was contaminated with intravenous solution; (2) pseudohyponatremia due to hyperglycaemia or known hypertriglyceridaemia; and (3) patients with incomplete data.
Genetic analysis of CYP11B2 and NR3C2 gene
Written inform consent was obtained from each patient’s parents. Blood samples were collected from the patients and their parents. The genomic DNA was extracted using the RelaxGene Blood DNA system Kit (Tiangen Biotech Co. Ltd., Beijing China). Whole exome sequencing (WES) was performed on the MGISEQ-2000RS system (BGI Genomics Co., Ltd. Shenzhen, China). Sanger sequencing was performed to verify the mutations in the CYP11B2 and NR3C2 genes. The pathogenicity was predicted with SIFT, Mutation Taster, Provean, and Polyphen-2. DynaMut software was used to predict the stability of the mutant protein.
Functional assays of novel CYP11B2 gene mutations
The negative control vector (Con 238), lentivirus vectors carrying the interference fragment (mutant types: R374Q/L464dup, R374Q, L464dup), and full-length sequence (wild type) to the CYP11B2 were constructed by GeneChem (Shanghai, China). HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 100 U/L penicillin-streptomycin antibiotics (Meilunbio, China) in a 5% carbon dioxide (CO2) incubator at 37˚C. Cell transfection was performed according to the manufacturer’s protocol.
The total protein was extracted from HEK-293T cells for western blotting analysis to demonstrate the CYP11B2 enzyme levels by using anti-human CYP11B2 rabbit antiserum (Abcam). After transfection, cells were incubated with 2μM DOC for 72 hours. The steroids were extracted from the cell culture supernatants. Ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was performed using the LCMS-8050CL (Shimadzu, Kyoto, Japan) system. DOC, B, and aldosterone were identified and quantified. DOC conversion was calculated using the total production of B and aldosterone divided by the sum of substrates and products.
Statistical analysis
Statistical analyses were performed with GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA, USA) software. Variables with normal distribution are expressed as mean ± standard deviation, while values with a large distribution are expressed as medians ± interquartile ranges. The independent samples t-test was used if data were normally distributed, and non-parametric test was used if data were not normally distributed. P-values < 0.05 were regarded as statistically significant.
Results
A total of 30 patients were finally enrolled in this study. Twenty-six infants (86.7%) were diagnosed with CAH due to 21-hydroxylase deficiency, and 4 infants (13.3%) were non-CAH (Tab. 1). We will go into detail regarding 2 boys who were affected by endocrine origin disease.
|
No. |
Sex |
Gene |
Mutation |
Diagnosis |
|
1 |
Female |
SLC26A3 |
c.1631T>A (p.Ile490Asn) |
Congenital chloride diarrhoea |
|
2 |
Female |
CLCNKB |
c.1469A>G (p.Glu490Gly) |
Barter’s syndrome |
|
3 |
Male |
CYP11B2 |
c.1121C>T (p.Arg374Gln)，c.1391_1393dupTGC (p.Leu464dup) |
Aldosterone synthase deficiency |
|
4 |
Male |
NR3C2 |
c.556_557del (p.Met186fs) |
Pseudo-hypoaldosteronism type 1 |
Patients with CAH
Twenty-six patients had an elevated level of serum 17-hydroxyprogesterone and were finally diagnosed with CAH. Nineteen patients were diagnosed with mutations in the CYP21A2 gene (Supplementary File — Table S1). Because the severity of the disease may be associated with the concentration of sodium, the patients were divided into 2 groups according to the concentration of sodium (group 1: serum sodium concentration ranged from 125 to 130 mmol/L, and group 2: serum sodium concentration < 125 mmol/L). Their clinical features are shown in Table 2. Median age at the onset of hyponatraemia was 26 days (interquartile range [IQR]: 16.5–56). The symptoms were mainly recurrent vomiting (61.5%), poor feeding (26.9%), poor weight gain (69.2%), diarrhoea (23.1%), dehydration (19.2%), and growth retardation (19.2%). It is worth mentioning that 2 infants (7.7%) had convulsions due to severe hyponatraemia (102.1 mmol/L and 104.1 mmol/L). Eight infants had positive family histories, and 5 patients’ (all in group 2) siblings who presented with similar symptoms died in the neonatal period.
|
|
Group 1 (%) |
Group 2 (%) |
Total (%) |
|
Number of subjects |
7 (26.9) |
19 (73.1) |
26 |
|
Sex (F/M) |
5/2 |
13/6 |
18/8 |
|
Full term |
5 (71.4) |
16 (84.2) |
21 (80.8) |
|
Age at onset (d) [p50 (p25, p75)] |
28 (11, 123) |
24 (17, 56) |
26 (16.5, 56) |
|
Positive family history |
3 (42.9) |
5 (26.3) |
8 (30.8) |
|
Clinical presentation and sign |
|||
|
Recurrent vomiting |
2 (28.6) |
14 (73.7) |
16 (61.5) |
|
Poor feeding |
1 (14.3) |
6 (31.6) |
7 (26.9) |
|
Poor weight gain |
2 (28.5) |
16 (84.2) |
18 (69.2) |
|
Growth retardation |
0 (0) |
5 (26.3) |
5 (19.2) |
|
Dehydration |
0 (0) |
5 (26.3) |
5 (19.2) |
|
Diarrhoea |
0 (0) |
6 (31.6) |
6 (23.1) |
|
Abdominal distension |
0 (0) |
3 (15.8) |
3 (11.5) |
|
Convulsions or seizures |
0 (0) |
2 (10.5) |
2 (7.7) |
|
Fever |
1 (14.3) |
4 (21.1) |
5 (19.2) |
|
Hyperpigmentation |
5 (71.4) |
12 (63.2) |
17 (65.4) |
|
Laboratory tests |
|
||
|
Hyperkalaemia |
6 (85.7) |
19 (100) |
25 (96.1) |
|
Sodium concentration (mmol/L) mean ± standard deviation |
126.77 ± 4.33 |
113.51 ± 7.85 |
117.08 ± 9.21 |
|
Metabolic acidosis |
5 (71.4) |
19(100) |
24 (92.3) |
|
1 (14.3) |
9 (47.4) |
10 (38.5) |
|
|
Adrenal imaging abnormal |
5 (71.4) |
19 (100) |
24 (92.3) |
Patient with pseudohypoaldosteronism type 1
The boy was born at term with a birth weight of 3.1 kg and length of 50 cm from non-consanguineous Chinese parents. He was admitted to local hospital due to recurrent fever at 27 days and diagnosed with pneumonia and suspected urinary infection due to the increase of white blood cells in urine. However, his urine sample presented with negative bacterial cultures. Ultrasound of the kidneys and adrenal glands was normal. The patient’s temperature dropped to normal with antibiotics treatment for 10 days, without other clinical symptoms. Remarkably, he was found to have hyponatraemia (Na 125.7 mmol/L, normal range (NR): 135–145 mmol/L), hyperkalaemia (potassium (K) 7.31 mmol/L, NR: 3.5–5.5 mmol/L), and metabolic acidosis (base excess: –7.1 mmol/L, bicarbonate (HCO3) 16.6 mmol/L) during these days. Despite receiving the intravenous sodium supplementation, correction of hyponatraemia and hyperkalaemia remained challenging. After stabilisation, he was registered in our department at 40 days old. At admission, his body weight was 4.22 kg [–0.5 standard deviation score (SDS)] with a length of 54 cm (–0.42 SDS). His blood pressure was 80/50 mmHg with normal external genitalia and no hyperpigmentation. The biochemical profile findings were as follows: plasma Na 131.7 mmol/L, and K 6.43 mmol/L. Meanwhile, marked elevated plasma levels of PRA (103.6 ng/mL/h, NR: 0.93–6.56 ng/mL/h) and Ald (2000 pg/mL, NR: 57.5–201.3 pg/mL) with normal levels of 17-hydroxyprogesterone (17-OHP), adrenocorticotropic hormone (ACTH), and cortisol were detected. Routine urinalysis was normal, but Enterococcus faecium was identified in his urine culture. Oral sodium supplementation (3–5 g/day) and antibiotics was administered for one week, and urine culture was negative with normalised electrolyte balance. Currently, he is 3 months old and has normal growth rate and normal serum electrolyte levels with oral sodium supplementation.
Patient with aldosterone synthase deficiency
A baby boy was born at 40 weeks of gestation with a birth weight of 3.95 kg and length of 50 cm. He was the third child of non-consanguineous Chinese parents. From one month since birth, he suffered from recurrent vomiting, as well as poor breastfeeding and reduced weight gain. The symptoms gradually worsened, and at the age of 3 months he was brought to our hospital. Upon physical examination, the boy was found to be mildly dehydrated and had a severely decreased amount of subcutaneous adipose tissue. His body weight was 3.9 kg (–4.34 SDS) with a length of 58.5 cm (–2.06 SDS). His blood pressure was 84/50 mmHg, and the external genital appearance was normal without hyperpigmentation. Laboratory investigations revealed that he had a low serum sodium level (124.7 mmol/L), elevated serum potassium level (6.6 mmol/L), metabolic acidosis (base excess: –10.6 mmol/L), normal levels of adrenocorticotropic hormone, cortisol, 17-hydroxyprogesterone (17-OHP), with marked elevated plasma levels of DOC (4.047 ng/mL, NR: 0.07–0.57 ng/mL), B (28.218 ng/mL, NR: 0.78–17.50 ng/mL), PRA (19.5 ng/mL/h, NR: 0.93–6.56 ng/mL/h), and an inappropriate normal level of Ald (0.119 ng/mL, NR: 0.02–0.7 ng/mL). Ultrasound investigation of the kidneys and adrenal glands showed no abnormalities. Oral fludrocortisone replacement (FC; 0.1 mg per day, in 2 equal doses) and salt supplementation was administered. The patient’s response to the treatment was excellent, with the balance of electrolyte and acid-alkali restored. He is currently at 25 months old and has a satisfactory weight of 10.5 kg (–1.42 SDS) and normalised electrolyte balance with FC therapy treatment.
Genetic analysis
The first boy was suspected of PHA, and DNA sequencing revealed a novel pathogenic heterozygous mutation of c.556_557del (p.M186fs) in the NR3C2 gene, which was inherited from his father (Fig. 1). The father was asymptomatic without salt-wasting history. His biochemical testing showed normal plasma sodium and potassium concentration (Na: 136.1 mmol/L, K: 3.77mmol/L) with elevated levels of plasma renin and aldosterone (renin: 83.27 pg/mL, NR: 1.8–24.5 pg/mL; aldosterone: 640.15 pg/mL, NR: 30–250 pg/ml).
The second infant was suspected to have ASD. Genetic test results revealed that the boy had compound heterozygous mutations in the CYP11B2 gene (Fig. 2). One variant (c.1121C>T, p.R374Q) was inherited from his mother. The other variation (c.1391_1393dupTGC, p.Leu464dup) was inherited from his father. The variant of c.1121C>T was found to be a possible pathologic mutation by using multiple bioinformatic tools including SIFT, Mutation Taster, Provean, and Polyphen-2 software and previously reported in 2 young boys with ASD [5]. However, the predictions of the novel mutation (c.1391_1393dupTGC) with Mutation Taster and Provean tools were controversial, which were polymorphism and deleterious, respectively. So, we performed in vitro experiments to explore the pathogenicity of the novel mutation in CYP11B2 gene.
Functional studies of novel CYP11B2 gene mutations
Western blots were performed to analyse the levels of protein expression in wild-type and the mutant-types (R374Q/Leu464dup, R374Q, Leu464dup). A single, non-specific band of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at approximately 41 kDa revealed that the levels of protein expression loaded were approximately equivalent for all lanes. A band at approximately 50 kDa was detected in both wild type and the mutants (Fig. 3A). A comparison of the expression levels of the wild type and the mutants (Fig. 3B) showed that the latter were significantly lower than the former (p < 0.05). This showed that the mutants of R374Q/Leu464dup, R374Q, and Leu464dup caused decreases in AS protein expression in vitro and that this could eventually lead to ASD in the human body.
Transfected cells were lysed after 72 hours incubation with medium containing a DOC substrate, and the steroid metabolites were measured by UPLC-MS/MS. The results (Fig. 3C) showed that the HEK 293T cells transfected with wild type CYP11B2 converted nearly all the DOC to its products (B and Ald). However, the R374Q/Leu464dup, R374Q, and Leu464dup mutants could barely catalyse DOC to B and Ald. This was also observed in the negative control and HEK-293T groups, suggesting that the R374Q and Leu464dup were functionally deleterious, and this was consistent with the diagnosis of ASD.
Discussion
Multiple aetiologies can cause hyponatraemia in infants such as lung disease, liver disorders, renal injury, and central nervous system pathology. For infants, poor weight gain, failure to thrive, hyponatraemia, hyperkalaemia, and metabolic acidosis are usually related to primary adrenal insufficiency (PAI). PAI in children is usually associated with genetic aberrations [16]. CAH due to 21-hydroxylase deficiency was the most common genetic form [17]. Other extremely rare forms of CAH included deficiencies of 3β-hydroxysteroid dehydrogenase, steroidogenic acute regulatory protein, or cholesterol side-chain cleavage enzyme [18, 19]. In this study, CAH accounted for 86.7% of all the cases, and all of them were 21-hydroxylase deficient. Untreated SW type 21-hydroxylase deficiency may be deadly in infancy. Two infants (7.7%) with severe hyponatraemia presented with convulsions, and 5 patients’ siblings (19.2%) with severe hyponatraemia and similar symptoms died in the neonatal period.
Despite CAH, several other factors were found in this study. We found one boy with a renal form of PHA1 complicated with urinary tract infection and one patient with ASD. PHA 1 is marked by Ald resistance due to abnormalities in MR or ENaC. PHA 3 is transient and usually caused by urinary tract infections or obstruction of the urinary tract. Electrolyte disturbances can be normalised with the recovery of the primary disorder. The boy found in this study was initially suspected to have PHA 3 due to urinary tract infection caused by Enterococcus faecium. Correct employment of antibiotics was supplemented, but the infant was still presented with hyponatraemia and hyperkalaemia. Finally, the patient was found to have renal PHA 1 caused by a novel mutation in the NR3C2 gene that encodes MR, which was passed down by his father. Renal PHA 1 is a heterogeneous disorder, and the clinical feature is variable, ranging from severe salt-wasting to asymptomatic. As in our study, the boy showed SW presentation in infancy, but his father was asymptomatic and had no history of SW. The biochemical profile testing of the father still showed elevated levels of plasma renin and aldosterone with normal serum electrolyte. Elevated aldosterone levels can be the only biochemical marker of renal PHA 1 in adulthood [7]. The mechanism of phenotype and genotype is still unclear. Infection, intercurrent events, or living circumstances may cause the disease to worsen [20]. Sodium supplementation is an essential treatment for patients who present with SW in early infancy due to PHA 1, whereas the salt loss may ameliorate during childhood in patients with renal PHA1 [7].
We also identified a baby boy with ASD in this study. ASD is a very rare autosomal recessively inherited disease, and inactivating mutations in the CYP11B2 gene are the main reasons for this disorder. The estimated incidence of ASD is probably < 1/ 1,000,000 per year [21, 22]. Two different types of ASD, types 1 and 2, have been described based on the specific defects in aldosterone biosynthesis. ASD type 1 affects the hydroxylation of B, and thus the patient presents with decreased levels of 18-OHB, while ASD type 2 retains some residual enzymatic catalytic activity that can catalyse DOC to B, but there is a failure to oxidise the 18-OHB to form aldosterone. Hence, the patient presents with elevated levels of 18-OHB. However, the clinical manifestations, hormonal levels, and genotypic traits may show overlapping features in the 2 types. Therefore, to consider 2 distinct types of ASD may be of limited clinical value, and it would be better to recognise these as a continuous spectrum of the same disorder [15].
To our knowledge, approximately 65 pathogenic mutations in the CYP11B2 gene associated with ASD were identified. The correlations between genotype and phenotype in ASD cases are complex. Merakou et al. [23] compared the clinical data of ASD patients having homozygous and heterozygous mutations at p.T185I with any other variants identified in their cohort, and the results showed no significant differences among any of the genotypes identified and the corresponding biochemical examinations. Some mutations (such as R181W, D335G, and V386A) were observed in both types of ASD [24]. In addition, patients with homozygous mutations at E255X typically showed clinical features of ASD type 1 [25]. However, heterozygous mutations of E255X and Q272X revealed clinical features consistent with both ASD types 1 and 2 [26]. All in all, more detailed data regarding ASD are still needed to accurately and convincingly describe the relationships between the genotype and phenotype of this disease.
Up to now, mutations of R374Q, G435S, F445C, Q337X, T326K, L496fs, Leu464del, Lys175del, c.1200+1G > A, c.240-1G > A, and Arg432Glyfs*37 of CYP11B2 have been identified in the Chinese population [5, 15, 27, 28]. In our study, we found compound heterozygous mutations in the CYP11B2 gene, a previously reported missense mutation of R374Q in exon 6 and a novel variant, Leu464dup, in exon 8. When the missense mutation of R374Q was previously reported, there were no in vitro experiments to verify its pathogenicity. The novel variant had not been previously reported by dbSNP, genomAD, ExAC, or the Human Gene Mutation Database. The R374 residue was found to be the motif of Glu-X-X-Arg in the K-helix, which may contribute to the stability of the core structure of CYP11B2 [29]. The mutation Leu464dup caused only an amino duplication in the peptide chains, which produced some puzzling outcomes according to the in silico prediction tools. Herein, it was necessary to conduct in vitro cell experiments to verify the pathogenicity of these mutations and to further explore their pathogenesis. The studies showed that these compound heterozygous mutations could not only lower the expression levels of AS protein but could also decrease the activity of AS thereby leading to ASD.
It should be noted that not all infants with ASD reveal elevated PRA levels or low to normal aldosterone levels. Previously, a low level of PRA combined with a high plasma aldosterone level was observed in a 4-month-old boy with ASD during his initial hospital visit [29]. More interestingly, Martín-Rivada et al. [30] described an infant with salt-wasting syndrome showing high levels of PRA and plasma aldosterone, and then a presumptive diagnosis of PHA 1 was established. However, the PHA 1-associated genes, NR3C2, SCNN1A, B, and G, were studied by Sanger sequencing, which revealed no pathogenic variants. The patient was finally confirmed with ASD at 3 years of age due to his younger brother presenting with typically abnormal biochemical and hormonal assay results, which was consistent with ASD. It was difficult to explain the circulating hormone levels, particularly the adrenal steroids during early infancy, because these were important in making the clinical diagnosis and the eventual treatment. Partial and transient aldosterone resistance is sometimes observed in newborns and infants due to the immaturity of nephron. These findings should act as a reminder to the physician that hormonal assays should be repeated several times when the results are not consistent with the clinical symptoms, especially in infants, and even high aldosterone levels cannot always rule out the possibility of ASD. Gene analysis may be used to confirm the disorder, but the methods used for genetic testing should be chosen carefully.
FC and oral salt supplementation are essential treatments for most patients with ASD during infancy and childhood, although severely symptomatic infants may also need intravenous fluids [26]. Electrolyte disturbances could be corrected quickly, but PRA and levels of steroid precursors may not return to normal for a very long time. Catch-up growth can be achieved in infants whose ASDs have resulted in failure to thrive or poor weight gain after receiving the treatment [31]. Similarly, our patient displayed normal electrolytes in biochemical tests and showed a marked weight gain after receiving the oral salt supplementation and FC replacement. Oral salt supplementation could have been terminated when the PRA or renin levels had decreased to normal. However, no biochemical indicators were found to guide the precise termination time of FC replacement [32]. The decision for withdrawing the FC treatment should be individualised. Intensive monitoring and long-term surveillance is necessary for these patients due to the incompletely biochemical remission, and if necessary, resuming the treatment should always be under consideration.
Conclusions
Infants with SW were mostly caused by 21-hydroxylase deficiency. However, the rare disorders of pseudohypoaldosteronism and aldosterone synthase deficiency should also be considered possibilities in infants who present with salt-wasting syndrome. Normal or high plasma aldosterone level cannot be the factor to rule out the possibility of ASD in infancy. Genetic analysis can be used to confirm the disorder.
Data availability statement
The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.
Ethics statement
This study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (2022-KY-E-(175)). Written informed consent was obtained from the patients’ parents.
Author contributions
Y.X., X.L. and D.L. participated in study design, data collection, and data analysis. Y.X. and X.L. drafted the manuscript. Y.X., X.L., and J.T. completed the experimental part.
Funding
We thank the Scientific Research Foundation of the First Affiliated Hospital of Guangxi Medical University (grant no. 2010219) and the Guangxi Clinical Research Centre of Paediatric Diseases (grant no: GUI KE AD22035219) for funding this project.
Acknowledgments
We are very grateful to the patients and their families without whom this research could not have been performed.
Conflict of interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Supplementary material
Supplementary File — Table S1 Characteristics of infants and young children with CAH, PAI, and Xp21 contiguous gene deletion syndrome
