Subjects
A total of 68 Chinese patients with 17-OHD who were admitted to the Peking Union Medical College Hospital (Beijing) were recruited for this study between 2003 and 2021.
The inclusion criteria[6, 18, 19] for subjects with 17-OHD were based on the clinical manifestations and serum hormone levels as follows: 1. low cortisol level and high ACTH level, high gonadotropins, high progesterone(P) and low testosterone (T) or estradiol (E2). 2.Patients aged ≥18 years at the clinical assessment.
This study was approved by the Ethics Committee for Human Research of Peking Union Medical College Hospital (No.JS-2111), and informed consents were obtained from all subjects participating in the study.
Methods
Detailed medical data pertaining to age, sex, height, weight, the highest blood pressure ever measured, blood pressure at the assessment of function of heart, kidney and retina, medication, imaging results (adrenal computed tomography (CT), pelvic and inguinal ultrasonography (USG), non-invasive transthoracic echocardiogram and resting standard 12-lead electrocardiography) were collected and analyzed. Funduscopy tests were conducted and interpreted by experienced ophthalmologists. Serum potassium (K+) and sodium (Na+), adrenocorticotropic hormone (ACTH), plasma cortisol(F), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), progesterone(P), testosterone(T)and 17α-hydroxyprogesterone (17- OHP) at baseline and when assessing the function of heart, kidney and retina were measured.
Laboratory test
Plasma ACTH and serum cortisol at 8:00 AM were measured by chemiluminescence immunoassay (Advia Centaur XP, Bayer). Serum LH, FSH, estradiol(E2), progesterone(P) and testosterone(T) were measured with chemiluminescence (ACS:180; Automatic Chemiluminescence Systems, Bayer). 17-hydroxyprogesterone (17- OHP) concentrations was determined by radioimmunoassay (Active 17α-OHP Progesterone DSL-5000, DSL). All hormone indexes above were measured in the context of absent hormone replacement therapy or drug withdrawal. The intra and inter assay coefficients of variation were 2.3% and 2.8% for LH, 3.9% and 4.5% for FSH, 5.6% and 6.6% for T, 6.7% and 8.2% for ACTH,5.3% and 5.7% for cortisol, 3.9% and 5.6% for 17-OHP, respectively.
PCR and sequencing of CYP17A1 gene
Genomic deoxyribonucleic acid (DNA) from the peripheral blood leukocytes were obtained from all patients using a standard procedure (Omega Blood DNA Midi Kit, Omega Bio-Tek, USA). PCR combined with sequencing was employed to detect the mutations in all 8 exons and exon-intron boundaries of CYP17A1 gene, PCR primers were designed by the Oligo Primer Analysis Software (version 7.60, Molecular Biology Insights, Inc., USA) and amplification methods are described in Supplementary Information Table S1. PCR products were identified on 1.5% agarose gel electrophoresis and sent to the Beijing SinoGenoMas Company for purification and sequencing. The sequence was compared with the reference sequence NM_000102.4 of the CYP17A1 gene and NP_000093.1of P450c17 protein through the NCBI website to ascertain the mutations.
The enzymatic activity assessment of different CYP17A1 mutants
The enzymatic activity corresponding to different mutations of CYP17A1 was assessed according to the mutation characteristics and the functional data reported by previous literature. The enzymatic activity of mutants caused by the variants seriously affecting the gene transcription and translation (e.g., small deletions/insertions, large deletions, nonsense mutation, frameshift mutation and splicing mutation) was considered “Nil” (completely abrogating the P450c17 protein function). The enzymatic activity of mutations verified on the previous literature was recognized as “Nil” (enzymatic activity abolished completely) or partial activity (residual enzyme function remained).
Classification criteria for body-mass index, potassium grade and hypertension grade
Body-mass index (BMI) was calculated as weight in kg divided by the square of height in meters, and was classified into the following categories: underweight (<18.5 kg/m²), normal (18.5–23.9 kg/m²), overweight (24.0–27.9 kg/m²), and obese (≥28.0 kg/m²)[20].
The definitions of serum potassium grade were as follows: normal serum potassium: ≥3.5 mmol/L; mild hypokalemia: 3.0–3.5 mmol/L; moderate hypokalemia: 2.5–3.0 mmol/L; severe hypokalemia: <2.5 mmol/L[21-23].
The definitions of hypertension grades were as follows[10, 24]: Grade 1 (mild): SBP 140-159 and/or DBP 90-99; Grade 2 (moderate) : SBP 160-179 and/or DBP 100-109; Grade 3 (severe): SBP ≥180 and/or DBP ≥110.
Classification and assessment of HMOD
HMOD mainly involves the following organs[10]: heart: left ventricular hypertrophy, atrial fibrillation, and other arrhythmias, ischemic heart disease, and heart failure; kidney: proteinuria (decrease in albumin–creatinine ratio) and renal dysfunction (decreased estimated glomerular filtration rate); retina: retinal hemorrhages, microaneurysms, hard exudates, cotton wool spots, and papilledema detected by fundoscopy.
The results of routine urine, renal function, electrocardiography, echocardiography, fundoscopy were obtained to evaluate the damage to the heart, kidney, retina organs.
Statistical analysis
SPSS (version 26, SPSS, IBM) statistical software package was used to analyze the data. A normality test was performed to determine whether the continuous variables conform to normal distribution. The normally distributed variables were represented as mean ± standard deviation ( ±s), and the non-normally distributed variables were expressed as median (upper and lower quartiles) [M (Q1, Q3)]. Comparison between two groups was performed using independent t-test or Mann–Whitney U test. Enumeration variables were expressed as cases (n) and percentages and were compared by the Chi-square test or Fisher exact test. The risk factors for the occurrence of HMOD in patients with 17-OHD were analyzed by logistic univariate regression. Subsequently, logistic multivariate regression was employed to estimate the independent risk factors for the occurrence of HMOD and the specific damaged organs in the patients. P value less than 0.05 was considered statistically significant.