21-hydroxylase deficiency (21OHD)(OMIM: 201910) is an autosomal recessive disorder caused by pathogenic variants in the CYP21A2 gene, accounting for over 90% of congenital adrenal hyperplasia (CAH) cases (1). The clinical phenotype correlates with the residual 21-hydroxylase activity: salt-wasting (SW) (minimal/no activity), simple virilizing (SV) (moderate deficiency), and non-classic (NC) (mild impairment) (2–4). 21OHD disrupts adrenal steroidogenesis, leading to distinct hormone profiles across phenotypes and sexes.

Elevated 17-hydroxyprogesterone (17OHP) is a sensitive diagnostic biomarker for 21OHD. Newborn screening (NBS) for 17OHP has been progressively implemented across most cities since 2018 in Hebei Province, China. NBS has been shown to reduce hospitalization and prevent morbidity and mortality from adrenal crises (5, 6). However, province-specific outcome data remain limited.

The CYP21A2 gene resides at 6p21.3 within the RCCX module, a tandem arrangement of genes (RP1-C4A-CYP21A1P-TNXA-RP2-C4B-CYP21A2-TNXB) prone to recombination due to high homology between functional genes (CYP21A2, TNXB) and pseudogenes (CYP21A1P, TNXA) (5–7). These rearrangements generate pathogenic chimeric genes, complicating molecular diagnosis, which typically combines Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) (1, 8–10). Co-occurring CYP21A2 and TNXB variants cause CAH-X syndrome, presenting with combined 21OHD and Ehlers-Danlos syndrome (EDS) features (~10% of 21OHD cases) (8, 11, 12). CAH-X syndrome is classified into three subtypes based on breakpoints within the chimeric TNXA/TNXB gene: CH-1: 120-bp deletion in exon 35; CH-2: Pathogenic variants disrupting exon 40; CH-3: Multiple clustered variants in exons 41 and 43 (11–14).

In this study, we comprehensively analyzed the clinical phenotype, steroid hormone profiles, and genetic spectrum in 115 Chinese children with 21OHD and functionally validated a novel pathogenic variant.

This study advances beyond prior genetic-focused investigations of 21OHD (10, 21, 22) by integrating clinical, hormonal, and molecular profiling in 115 Chinese pediatric patients. We systematically compared phenotype-stratified clinical and steroid hormone characteristics, analyzed sex-specific differences, evaluated Prader score correlations with phenotypes/hormones in females, and quantified genotype-phenotype concordance, variant frequencies, and CAH-X CH-1 incidence.

Key clinical distinctions emerged at diagnosis: SW infants presented neonatally with classic electrolyte crises, enabling prompt diagnosis. SV children manifested early virilization but faced diagnostic delays (median diagnosis age: 4.32 years), reflecting low disease awareness for isolated masculinization. NC boys were typically identified during school-age growth assessments via advanced bone age—a consequence of increased developmental surveillance. Their historical underrepresentation in cohorts likely stems from underdiagnosis.NC girls presented peripubertally with menstrual irregularities ± virilization, aligning with existing reports (22). Simultaneously, the diagnosis of NC form also highlights the importance of genetic testing (23).

Steroid hormone analysis revealed significantly elevated levels of 17OHP and P in SW and SV patients compared to NC patients, consistent with enhanced ACTH stimulation. Sex-specific variations were observed: P levels were significantly higher in SW and SV females—as well as in SW males—compared to their NC counterparts. However, no significant sex-based differences in steroid levels were detected within the same phenotypic subgroups. In contrast, Chang et al. (24) reported sex-related hormonal differences within phenotypes—a discrepancy potentially attributable to our smaller sex-stratified subgroups or limited biomarker panel.

Notably, a male predominance (55.65%; 64/115) was observed, which was particularly pronounced in the SW subgroup (61.84%). This finding aligns with a Chinese cohort analysis by Xia et al. (22) and may be attributed to the fact that females with virilization often receive earlier diagnoses in primary care settings, while males with electrolyte crises require specialized referral to tertiary centers. Previous studies have reported that clinically diagnosed cohorts typically exhibit a female predominance (25, 26). Furthermore, a higher proportion of patients with severe genotypes were diagnosed after the implementation of NBS compared to the pre-screening era (27), highlighting significant pre-NBS mortality among severe phenotypes—especially males.

Critically, NBS significantly advanced the age at diagnosis for SW infants, although no significant differences were observed in electrolyte and biochemical parameters at the time of diagnosis, early detection mitigated long-term homeostatic imbalances and associated physiological damage, particularly for SW males. Family histories revealed neonatal deaths in undiagnosed cases, emphasizing NBS’s role in preventing mortality.

Female patients exhibited distinct virilization patterns across phenotypes: Prader 3 (44.83%) predominated in SW, while Prader 2 (56.25%) was most frequent in SV, contrasting with NC’s minimal virilization (Normal external genitalia: 83.33%). This phenotypic gradient aligned with elevated ACTH and P levels in SW/SV versus NC girls, though 17OHP and T showed non-significant trends. The higher prevalence of severe virilization (Prader ≥3) in SW versus SV girls may reflect comparatively elevated testosterone in this subgroup, consistent with prior reports (24, 28).

Critically, virilized girls (Prader ≥1; 90.20%) demonstrated accelerated diagnosis timelines and significantly higher ACTH, 17OHP, and progesterone. Surgical intervention occurred earlier and more frequently with increasing severity–92.59% of Prader ≥3 girls underwent surgery at median age 2.10 years versus 57.89% at 3.80 years in Prader 1~2. The overall surgical rate (78.26%) was slightly lower than previously reported (85%) (29). Studies demonstrated that patients undergoing surgery after 2 years of age required significantly fewer additional major surgeries and had higher long-term satisfaction rates than those operated on before 2 years of age (30). No significant association was observed between Prader scores and postnatal steroid hormone levels in this study. This may be attributed to several factors: postnatal steroid concentrations are susceptible to confounding variables such as stress and may not accurately reflect peak androgen exposure during the critical window of urogenital sinus differentiation. Additionally, individual tissue responsiveness to androgens is influenced by factors such as 5α-reductase activity, androgen receptor sensitivity, and coregulator expression (31, 32). Finally, the scope of androgen assessment was limited; key biomarkers including free testosterone, dihydrotestosterone, and 11-oxygenated androgens were not analyzed (33).

Our MLPA assay, employing six TNXB-specific probes, identified exon 35 deletions in 30/106 patients (28.30%). Of these, 12 patients (11.3% overall; 40% of CYP21A2-deleted cases) harbored contiguous deletions extending from CYP21A2 to TNXB exon 35, generating TNXA/TNXB chimeras (exons 1–34 of TNXB + exons 35–44 of TNXA) diagnostic of CAH-X CH-1. This prevalence exceeds prior reports (7–8.2%) (8, 11), suggesting population-specific variability, and chimeric mutations demonstrate incomplete penetrance for EDS features (8, 11), mild EDS manifestations may occur even without chimeras (14). Patients with missense-driven CH-2/3 exhibit greater joint hypermobility than CH-1 cases (8), likely reflecting distinct pathomechanisms: CH-1 causes an extracellular matrix protein TNX haploinsufficiency, whereas CH-2/3 exert dominant-negative effects (12, 13). A limitation of this study is the absence of systematic EDS assessments and lack of TNXB exons 40, 41, and 43 sequencing.

Genotype-phenotype correlations demonstrated high PPV for severe phenotypes (Group 0: 100%; Group A: 87.50%), while moderate PPV was observed for Groups B (76.92%) and C (75.00%). This largely aligns with established literature, although the PPV of Group B varies across studies involving different populations (10, 34–36), suggesting greater phenotypic heterogeneity in moderate forms. Such variability likely stems from modifier genes influencing 21-hydroxylase expression and steroidogenesis (4, 22), as well as divergent genetic backgrounds among different ethnic groups, further supporting the clinical continuum observed among the three phenotypes.

Molecular profiling revealed three variant classes: micro-conversions predominated (70.89%), followed by large deletions/conversions (19.25%) and bona fide point mutations (9.86%), consistent with Asian populations (22, 37). The first three common variants were: I2G (31.46%); large deletions/conversions (19.25%) and p.I173N (13.62%). The first three common variants were consistent with the most common variants in Asian populations (9, 22, 24, 37). It should be noted that the MLPA approach used here, while effective for confirming deletions, may miss complex rearrangements and does not provide breakpoint resolution. Further analysis with advanced sequencing methods is warranted to fully elucidate the structure of these variants in the future.

Notably, p.V282L frequency (5.16%) exceeded previous similar reports yet remained below Argentinian/high-admixture cohorts (23.9–26.2%) (36, 38), indicating ethnogeographic variation. The p.V282L variant dominated our NC subgroup (5/9 cases), including two homozygotes, establishing it maybe a regional hotspot. While p.V282L and p.P31L share comparable residual activity in vitro, p.P31L carriers exhibit poorer genotype-phenotype concordance and higher symptom penetrance (4, 29)—consistent with its rarity (1/9 NC patients) in our cohort.

Two male patients (1.89%) in Group D exhibited monoallelic variants: one NC phenotype with I2G and one SW with p.Q319X, despite comprehensive screening revealing no second pathogenic allele. This aligns with reported monoallelic detection rates (2.2–24%) (4, 10, 34, 36, 39), potentially relating to pseudogenes affecting amplification or some unidentified intron region variants affecting transcriptional activity (24, 40). Notably, three male patients (2.83%) harbored paternally inherited cis double mutations: two brothers with discordant SW/SV phenotypes carried the first-reported discontinuous mutations (E3del8bp & p.V282L), while one SW patient had adjacent mutations (p.L308Ffs*6 & p.Q319X) previously hypothesized to be minor conversions (4, 24, 39, 41). The phenotypic divergence between siblings sharing identical paternal mutations implies the contribution of factors beyond the primary genetic defect, such as modifier genes, epigenetic influences, or stochastic developmental processes, which alter enzyme activity, hormone levels, or tissue responsiveness. Future studies employing genome-wide approaches and family-based designs are warranted to elucidate the complex interplay between primary mutations, modifier genes, and epigenetics in shaping the phenotypic landscape of 21OHD (38).

Additionally, three patients (2.83%) possessed de novo mutations absent in maternal blood, suggesting germline mosaicism (4). This incidence exceeds the 1% literature baseline (4), underscoring de novo events as non-trivial contributors. Finally, we validated a novel splice variant (c.203-1G>A) in an SW female. Splice-AI predicted exon skipping (probability=0.99), confirmed in vitro to cause exon 2 exclusion (NM_000500.9: c.203_292del) and protein truncation, explaining the severe phenotype when co-occurring with a deletion allele.

In conclusion, this study comprehensively analyzed clinical phenotypes, steroid hormone profiles, and molecular genetics in 115 Chinese patients with 21OHD. We identified and characterized a novel pathogenic splicing mutation (c.203-1G>A), demonstrating it induces exon 2 skipping during mRNA processing. We report the first documented case of paternally inherited, discontinuous cis double mutations (E3del8bp and p.V282L) in siblings exhibiting an intriguing discordant SW/SV phenotype. Significantly elevated levels of ACTH, AD, and P were demonstrated in the SW and SV groups compared to the NC group. Girls with vulvar masculinization exhibited higher ACTH, 17OHP, and P levels than non-virilized girls, and severe virilization was more prevalent in the SW than SV group. NBS substantially reduced time-to-diagnosis, particularly in male SW infants, underscoring the value of implementing nationwide NBS in China. We established genotype-phenotype correlations and defined the variant frequency, confirming I2G, large deletions/conversions, and p.I173N as predominant CYP21A2 mutations in this Chinese cohort. Notably higher frequencies of p.V282L and CAH-X CH-1 compared to previous reports suggest enhanced clinical recognition, particularly of NC form.