A 4-month-old male infant was recruited to the Gastroenterology department of the Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine (Shanghai, China) due to frequent vomiting (6–8 times/day) without inducements, shortly following birth, and had not improved with age. The patient was a full-term baby and had an uncomplicated delivery with a birth weight of 2,960 g. His parents were physical healthy, with non-consanguineous marriage. Physical examination showed his weight was 3,300 g, height was 54 cm, heart rate was 168 bpm, respiratory rate was 42 bpm, blood pressure was 82/55 mmHg, and body temperature was 37.5°C. He had severe malnutrition and moderate dehydration. The patient had male-appearing genitalia, with marginally darkened color scrotal skin, and ~1 ml bilateral testicles. Neither heart murmur, nor hepatosplenomegaly were found. Congenital anomaly of the digestive tract and inborn metabolic diseases were excluded. The patient had initially been treated for gastroesophageal reflux and allergy to formula in another hospital, without a satisfactory improvement. The present study was approved by the ethics committee of the Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine.

The results of the laboratory examinations performed on the first visit to hospital are shown in Table I. Based on the examination results, the baby was treated intravenously for rehydration, supplementation with sodium salt, correction of acidosis and nutritional support. However, it was difficult to correct hyponatremia and hyperkalemia following shifting to oral therapy, even with sodium supplementation to 18 mEq/kg/day. Problems in adrenal hormone production were considered, although the laboratory data did not provide evidence to confirm a diagnosis, and hormone detection can be affected by several factors (9). In order to obtain a clinical diagnosis, mutant genes were screened using WES, and CYP11B2 gene mutations were identified, however, due to the high levels of aldosterone at the first visit, aldosterone levels were evaluated again. Unlike the first visit, a low aldosterone level (38.12 pg/ml) was found. Subsequently, the patient was simultaneously treated with 9α-fluorohydrocortisone, and electrolyte levels rapidly reached equilibrium (data not shown). The effectiveness of the treatment supported the genetic diagnosis and the use of molecular genetic assessments, the patient was finally diagnosed with ASD.

The steps of the WES experiment were based on the report by Wang J et al (10). The genomic DNA of the patient and parents were isolated from 2 ml peripheral blood samples collected from the cubital veins using a QIAamp Blood DNA Mini kit^® (Qiagen GmbH, Hilden, Germany). A total of 3 µg DNA from the patient was processed through shearing using a Covarias^® M220 Ultrasonicator system (Covaris, Inc. Woburn, MA, USA) to result in sizes of 150–200 bp. An adapter-ligated library was prepared, and enrichment of the coding exons and flanking intronic regions was performed. Clusters were then generated by isothermal bridge amplification using an Illumina cBot station, and sequencing was performed on an Illumina HiSeq 2000 system (Illumina, Inc., San Diego, CA, USA).

Base calling and sequence read quality assessment were performed using Illumina HCS 2.2.58 software (Illumina, Inc.) for the Illumina HiSeq 2000 system which included new versions of HiSeq control software and Real Time Analysis. Alignment of the sequence reads to a reference human genome (Human 37.3; SNP135) was performed using NextGENe^® (SoftGenetics LLC, State College, PA, USA). All single nucleotide variants (SNVs) and indels were saved in a VCF format file, and uploaded for Ingenuity^® Variant Analysis™ (Ingenuity Systems, Mountain View, CA, USA) for biological analysis and interpretation.

The primers for amplification of the CYP11B2 gene (GenBank accession no. NM_000498.3) were designed using UCSC ExonPrimer online software (http://genome.ucsc.edu/index.html) and synthesized by Map Biotechnology, Co., Ltd., Shanghai, China. The primers designed for exon 2 were as follows: Forward 5′-CAG AGA AAA CCC CAG CTC AC-3′ and reverse 5′-CAA CCC ACA GTG CAG ACG-3′; and the primers designed to amplify exon 6 were as follows: Forward 5′-CCC AAG TGG TCA TCA AGGTT-3′ and reverse 5′-GGT GTT GAA GAG GGA TTC CA-3. The exons and the exonintron boundaries were amplified using polymerase chain reaction (PCR; Takara Biotechnology, Co., Ltd., Dalian, China). The reaction mixture for each amplification contained 1X Premix Taq (Ex Taq Version 2.0; cat. no. RR003; Takara Biotechnology, Co., Ltd.), 100 ng genomic DNA, and 1 pmol forward and reverse primer in a final volume of 25 µl. The reaction was carried out with the following PCR conditions: Initial denaturation at 95°C for 5 min, then 19 cycles of 95°C for 30 sec, 65°C for 30 sec, 72°C for 45 sec, 14 cycles of 95°C for 30 sec, 55°C for 30 sec, 72°C for 45 sec, and a final elongation step at 72°C for 5 min using a C1000™ Thermal Cycler PCR instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR products (5 µl) were examined on a 1% agarose gel and purified using a QIAquick Gel Extraction kit (Qiagen GmbH). The resulting DNA was sequenced using the ABI3730XL sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with the forward and reverse primers. The sequence data were analyzed using Mutation Surveyor^® software version 4.0.4 (SoftGenetics, LLC).

The splice region (c.240-1G>A; intron 1) of the CYP11B2 gene was amplified from the patient's genomic DNA using the following specific primer pair: Forward 5′-GCgaattcAGA GTC TCA GGC AGG TCCA-3′ and reverse 5′-GCggatccGAT GTG CTT TTG GGT CCT AC-3′. This was performed using PrimeSTAR HS DNA Polymerase (Takara Biotechnology, Co., Ltd.). The forward and reverse primer sequences were located in the upstream of the promoter region and in intron 2, respectively. The length of the PCR product was 986 bp. The restriction enzyme sites EcoRI and BamHI were inserted into the primer sequences to enable directional cloning. DNA from the patient's father who did not carry the genetic variant was used as a control. Target fragments were ligated into the pcDNA3.1/Myc-His B vector (Invitrogen; Thermo Fisher Scientific, Inc.) using solution I ligase (Takara Biotechnology, Co., Ltd.), according to the manufacturer's protocol.

HEK 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO₂ incubator at 37°C. Cell transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

At 48 h post- plasmid transfection, the total RNA from the HEK 293 cells was isolated using an RNeasy Mini kit (Qiagen GmbH), and the RNA samples were treated with 5 U/µl DNase I (Takara Biotechnology, Co., Ltd.). cDNA chains were obtained from 1 µg RNA using C1000™ Thermal Cycler PCR instrument (Bio-Rad Laboratories, Inc.) with a Primer Script™ RT Reagent kit (cat. no. RR037A; Takara Biotechnology, Co., Ltd.). The following primers were used to amplify a 330 bp PCR product for the target cDNA fragment of the CYP11B2 gene: Forward 5′-GCA GAG GTG TGC GTG GCAG-3′ and reverse 5′-CCA GGG CTC CAG GAT CATC-3′. The forward and reverse primer sequences were located in exon 1 and exon 2, respectively. Subsequently, the PCR products were visualized by electrophoresis on a 2% agarose gel (Sangon Biotech, Co., Ltd., Shanghai, China) following staining with 0.5 µg/ml ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA). In addition, the PCR products were sequenced for further identification. GAPDH was used as a control for RNA quality, and the following primers were used to amplify a 245 bp PCR product for GAPDH: Forward 5′-GTC AGT GGT GGA CCT GAC CT-3′ and reverse 5′-TGC TGT AGC CAA ATT CGT TG-3′.

To obtain a rapid and accurate clinical diagnosis, the patient was screened for causal variants using WES. Exome sequencing yielded a total of 101,306,626 reads, and the mean target coverage was 136 reads with 95.48% having 20x coverage, and 99.71% having 1x coverage. The candidate variants were first screened with criterion of a minor allele frequency under 3% in the 1000 Genomes Project, the NHLBI exome variant server, or in 50 HapMap control exomes, the area of analysis area included each exon and ~20 bp of exonintron boundaries. Subsequently, clinical symptoms of frequent vomiting, hyponatremia, and hyperkalemia were chosen as the filtering indexes to analyze the candidate variants. Ultimately, the results demonstrated that the patient had compound heterozygous mutations in the CYP11B2 gene, which were considered to contribute to the patient's condition. Of the two mutations, one was a heterozygous point transition (c.1009C>T) in exon 6. Sequence analysis results showed that the c.1009C>T mutation at codon 337 of exon 6 was a nonsense mutation, which led to early termination of the protein translation process (p.Q337X). The other was a transversion of guanine to adenine (c.240-1G>A) in the first base of the 3′-splice site of intron 1 (Fig. 1). Further analysis revealed that c.240-1G>A was an intronic variation, which was involved in the agGT-aaGT splicing site, which occurs at a highly conserved 'ag-GT' site of the intron-exon junction region. The two mutations were identified as novel mutations, according to the Human Gene Mutation Database (HGMD; http://www.hgmd.cf.ac.uk). In addition, several genes associated with the clinical symptoms mentioned above are wild-type, including CYP11B1, CYP17A1, CYP21A2, 3βHSD, SCNN1A, SCNN1B, SCNN1 G, NR3C2, DAX1.

To assess the identified variations in the patient's parents, and further confirm the results of the WES, the proband and parents were examined using PCR Sanger sequencing. Direct sequencing results revealed that the patient's father was heterozygous for the c.1009C>T and p.Q337X mutation, and his mother was heterozygous for the c.240-1G>A mutation, which indicated that the gene mutations in the pediatric patient were inherited from her parents. These results led to the conclusion that these two novel compound heterozygous mutations in the CYP11B2 gene were the genetic cause for the patient's condition.

To evaluate the effect of the c.240-1G>A mutation on splicing, a minigene construct was generated, which was transiently transfected into cultured HEK 293 cells (Fig. 2A and B). The HEK 293 cells were transfected with either a mutant-type CYP11B2, wild-type CYP11B2 or empty pcDNA3.1 vector (negative control). As shown in Fig. 2C, a difference in the size of the cDNA fragment was observed between the wild-type and mutant-type CYP11B2, as expected. The wild-type CYP11B2 produced a PCR product of ~330 bp, whereas the mutant-type construct produced a longer band of 717 bp. GAPDH was used as a control for RNA quality and quantity. Clone sequencing verified that the mutant CYP11B2 construct contained the 387 nucleotides, which constituted intron 1. Therefore, it was concluded that the c.240-1G>A mutation of the 3′-splice site at the intronexon boundary resulted in the retention of intron 1 in the RNA.