Case 1, a 2-year-old girl with 46,XY DSD, attended our hospital. Physical examination of the female external genitalia was performed. B-ultrasound was carried out to detect male or female internal genital organs, including testis, uterus, and ovaries. In addition, hormone assay and karyotype analysis were performed, along with the sequencing of 219 DSD-related genes.
Case 2, case 1’s younger sibling, was suspected of having 46,XY DSD at 25 weeks’ gestation. Type B ultrasonic test, karyotype analysis, and SRD5A2 gene sequencing for 46,XY DSD were performed. Upon abortion of the fetus, physical examinations were performed for female external genitalia and HE staining for testis tissue and epididymis tissue.
Written informed consent for the genetic studies was obtained from the family, and all analyses were approved by the Medical Ethics Committee of the People’s Hospital of Xingtai City.
Serum levels of FSH, LH, estradiol (E2), progestin (PROG), T, and prolactin (PRL) of the proband were measured by radioimmunoassay.
Peripheral blood cells from the proband were cultured in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (FBS) for 72 h. Colchicine (20 mg/ml) was added for 2 h to arrest cells in metaphase and inhibit spindle body formation. Samples were incubated for 20 min with 75 mM potassium chloride to spread out the spindle body and fixed in Carnoy’s solution. The fixed cells were dropped onto glass slides and placed in an incubator at 75 °C for 3 h to air-dry. Giemsa solution was used to stain the G-bands of the chromosomes.
The uncultured amniotic fluid cells from the aborted sibling of the proband were obtained for FISH analysis. The probes were used to test chromosomes 13, 16, 18, 21, 22, X, and Y.
Peripheral blood samples of case 1, her aborted sibling, surviving sibling, father, mother, maternal uncle, grandfather, and grandmother were obtained for DNA sequencing. Genomic DNA was extracted using the TIANamp Blood DNA Kit (Tiangen), in accordance with the manufacturer’s instructions. Sequencing of 219 of the proband’s DSD-related genes was performed by BGI using Illumina Genome Analyzer IIx (Supplementary Table 1). The mutated genes in case 1 and her relatives as listed above were subjected to Sanger sequencing. Mutations were identified by comparing the sequencing results of the case to the UCSC reference genome using the BWA tool.
Human SRD5A2 site-directed mutagenesis
Human SRD5A2 cDNA was kindly provided by Professor Jiahuai Han (Xiamen University) and was subcloned into vector pcDNA3.1 (with 3Flag tag, purchased from Life Technologies). To generate 695A>G mutation in the SRD5A2 gene, the pcDNA3.1-SRD5A2 plasmid was used as a template and the QuikChange II Site-Directed Mutagenesis Kit (Catalog #200523; Agilent) was used to produce the mutation site, in accordance with the manufacturer’s instructions. The primers for cloning the 695A>G mutation in the SRD5A2 gene were as follows: SRD5A2 mF-695: gcgagcttttcaccaccGtaggttctacctcaagatgtttg, and SRD5A2 mR-695: catcttgaggtagaacctaCggtggtgaaaagctcgcag.
HEK293 cells (purchased from the Chinese Academy of Sciences Cell Bank in Shanghai) were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (Gibco) and 1% streptomycin/penicillin (Gibco). The cells were transiently transfected with 2.0 μg of wild-type or p.H232R mutant SRD5A2 plasmids in each well of a six-well plate using TurboFect™ Transfection Reagent (Thermo Scientific), in accordance with the manufacturer’s protocol. The transfected HEK293 cells were cultured in 5% CO2 at 37 °C for 48 h before the assays.
DNA sequencing of transfection plasmids
HEK293 cells were transfected with wild-type or p.H232R mutant SRD5A2 plasmids for 48 h and Sanger sequencing was used to assess the DNA sequence. Briefly, the DNA samples were isolated by TIANamp Genomic DNA kit (Cat# DP304-02; TIANGEN, China). The concentration and purity of the DNA were determined by NanoDrop (at 260/280 nm, ND2000C; Thermo). The DNA samples were amplified by polymerase chain reaction (PCR) and sequenced by Genewiz Corporation (Suzhou, China). Primer sequences for the PCR were as follows: SRD5A2-Fseq: AGCCCGTTAAGCAGTTGAGG, and SRD5A2-Rseq: CGGCTTCTTCCGCTTCTTGA.
Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was performed to assess the mRNA expression after transfection of the wild-type or p.H232R mutant SRD5A2 plasmid for 48 h. Total RNA was isolated by RNAiso Plus (Cat# 9109; TaKaRa) and reverse transcription was performed using the PrimeScript™ RT Reagent Kit (RR047A; Takara), in accordance with the manufacturer’s protocol. qPCR was performed using PrimeScript™ RT Reagent Kit (RR037A; Takara) with the ViiA7 Real-time PCR System (ABI). The PCR schedule was as follows: 95℃ for 30 s, followed by 40 cycles of 95℃ for 5 s and 60℃ for 34 s. For the relative quantification of SRD5A2 mRNA, the 2-ΔΔCt method was performed. The primer sequences for qPCR were as follows: SRD5A2F: GCCACTTTGGTCGCCCTT, SRD5A2R: CTCCGTGTGCTTCCCGTAG, β-actinF: AGAGCTACGAGCTGCCTGAC, and β-actinR: AGCACTGTGTTGGCGTACAG.
Western blot was performed as previously described . Briefly, after HEK293 cells has been transfected with wild-type or p.H232R mutant SRD5A2 plasmids for 48 h, they were collected from six-well plates and lysed with RIPA buffer (P0013; Beyotime Biotechnology). The whole-protein lysates were separated by 12% SDS-PAGE and then transferred to nitrocellulose membranes. After blocking with 5% milk, the membranes were incubated with the primary antibodies monoclonal anti-flag (F9291; Sigma) and β-actin (sc-69879; Santa) at 4℃ overnight. After rinsing with Tris-buffered saline containing 1% Tween-20 (TBST), the membrane was then incubated with appropriate HRP-conjugated secondary antibodies (sc516102; Santa Cruz) and detected with an ECL Plus kit (P1050; Applygen).
Kinetic assays were performed to assess the activity of steroid 5α-reductase 2 after transfection of the p.H232R mutant SRD5A2 plasmid into HEK293 cells. The cells were collected from six-well plates and resuspended in 200 μl of Tris-citrate buffer (pH 5.5). The resuspended cells were sonicated for 2 min (HD3100; Bandelin) and subsequently incubated at 37°C for 30 min with 500 μM NADPH (CAS: 100929-71-3, N302057; Aladdin) and various concentrations of T (0.25–8.0 μmol/L, CAS: 58-18-4, M163044; Aladdin). The steroids were extracted with chloroform, condensed by a freeze-drying apparatus, and re-dissolved in chromatographic methanol for liquid chromatography–mass spectrometry (LC-MS) analysis, using buffer A containing 1 mM formate (F112034; Aladdin) and buffer B containing 100% methanol. DHT was quantified according to the concentration and peak area of the standard DHT [CDCT-C10255010; ANPEL Laboratory Technologies (Shanghai) Inc.]. The protein concentration was determined using BCA assays (P0012S; Beyotime). The rate of enzyme production (nmol/mg protein/h) was calculated as an indicator of enzyme activity. The data were processed by Prism8 (GraphPad). The experiments on each group were repeated three times. The data are presented as mean ± SEM.
Sequence alignment of SRD5A2 in humans and other species
FASTA format SRD5A2 sequences of homologous species were downloaded from the NCBI database. Protein sequences of SRD5A2 were aligned between humans and the other homologous species using MEGA software.
Analysis of the pathogenicity of human SRD5A2 p.H232R mutation
The pathogenicity of the human SRD5A2 p.H232R mutation was analyzed using the following bioinformatic programs: Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org), and PROVEAN (http://provean.jcvi.org/seq_submit.php).