Discussion

Classic CAH remains a rare disorder, with its prevalence ranging from 1 in 10,000 to 1 in 20,000 across most human populations. In contrast, non-classic CAH occurs far more frequently, with a prevalence ranging from 1 in 200 to 1 in 2,000. Non-classic (mild) CAH is caused by CYP21A2 variants that preserve 20%–50% of residual enzymatic activity. This subtype is often clinically asymptomatic and highly prevalent. Epidemiological data have revealed that the prevalence of non-classic CAH reaches approximately 1 in 200 in the United States, and its carrier frequency ranges from 4.0% to 7.5% in several European countries (Baumgartner-Parzer et al., 2005; Ezquieta et al., 2005; Phedonos et al., 2013; Hannah-Shmouni et al., 2017).

Neonatal screening for 21-OHD has been routinely performed in Fujian Province, China, for nearly 3 years. The widespread implementation of such newborn screening programs is critical for preventing neonatal adrenal crisis and reducing early mortality. Nevertheless, conventional biochemical screening fails to identify patients with non-classic CAH when their serum 17α-OHP levels stay within the normal reference range. Females affected by non-classic CAH tend to develop hyperandrogenism and infertility in adulthood, whereas most male patients with this subtype remain undiagnosed throughout their lives. Furthermore, given the relatively high false-positive rate of traditional biochemical assays, molecular genetic screening has emerged as an indispensable tool for accurate subtype classification and early definitive diagnosis of CAH.

Single-molecule long-read sequencing has become a mainstream sequencing technology. It enables real-time sequencing at the single-DNA-molecule level and generates substantially longer reads with higher sequencing efficiency (Hu et al., 2021). Owing to its robust detection capability, this technology has been widely applied in the diagnosis of cancers and human genetic disorder (Thibodeau et al., 2020; Xu et al., 2020; Chen et al., 2023; Mimosa et al., 2023). Compared with MLPA combined with Sanger sequencing, which presents obvious drawbacks in clinical performance and economic cost, LRS-based genotyping serves as a promising strategy for CAH newborn screening (Chen et al., 2012; Xu et al., 2013; Concolino, 2019). Firstly, it yields comprehensive genetic profiles, enabling direct analysis of copy number variations and cis-trans configurations of CYP21A2 and CYP21A1P, as well as precise identification of chimeric gene junction site (Liu et al., 2022; Li et al., 2023). Secondly, MLPA and Sanger sequencing suffer from low throughput and demand high-quality genomic DNA at concentrations above 10 ng/μL. By contrast, LRS-based CAH genotyping only requires DNA concentrations exceeding 1 ng/μL and is compatible with neonatal dried blood spot specimens, which better adapts to large-scale newborn genetic screening. Thirdly, LRS achieves faster genotyping turnaround than MLPA, shortening the interval from initial screening to final diagnosis and cutting overall costs in population-scale newborn screening (Liu et al., 2022; Adachi et al., 2024). Furthermore, integrating genetic testing into routine 17α-OHP biochemical screening and conducting integrated result analysis can markedly reduce unnecessary recall procedures and repeated dried blood spot sampling. Meanwhile, non-classic CAH and carrier status can be directly determined, facilitating timely clinical monitoring, early intervention and prenatal genetic counseling for subsequent pregnancies.

In the present study, the estimated CAH prevalence was 1:20029, which is comparable to the national incidence of 1:23024 reported in China. The c.293-13C>G variant was the most prevalent mutation, accounting for 20% of all detected variants, in line with earlier findings (Hou et al., 2019). Within our study, CYP21A2/A1P duplication chimeras were categorized as benign variants in accordance with published LRS data for CAH (Liang et al., 2025), for the following reasons: These trimodular RCCX rearrangements harbor two intact wild-type copies of CYP21A2 without deleterious pseudogene-derived fusion breakpoints that abrogate enzymatic function; no loss-of-function sequences originating from CYP21A1P are incorporated into the duplicated functional gene, and in vitro enzymatic activity assays confirmed residual 21-hydroxylase activity exceeding 80%. Population genetic data revealed a high carrier frequency of pure duplication chimeras at 4.00%. Furthermore, long-read-based haplotype phasing verified that duplication chimeras only act as pathogenic alleles when they reside in cis with SNVs or indels. This retrospective analysis adopted LRS-based genotyping to establish a combined biochemical and genetic screening model, verifying its clinical value as a second-tier newborn screening approach. Of the 52 neonates who underwent LRS-based CAH genetic testing, no false-positive results were detected except for five individuals with confirmed CAH. Among the 175 newborns recalled due to elevated initial 17α-OHP screening values, only five were finally diagnosed with CAH, demonstrating that approximately 97.1% (170/175) of recall visits were clinically unwarranted. These observations indicate that the current 17α-OHP threshold generates abundant false-positive signals and triggers redundant clinical follow-up. Incorporating LRS-mediated CAH gene analysis as a second-tier newborn screening strategy could effectively minimize unnecessary recall procedures.

Globally, studies focusing on CYP21A2 mutation carrier screening remain limited. Here, we applied LRS-based CAH genotyping in primary newborn screening to investigate the carrier frequency of CYP21A2 mutations in Fujian region.

The primary objective of newborn screening is to avert neonatal adrenal crisis and mortality. This is especially critical for male infants, as 21-hydroxylase deficiency (21-OHD) rarely presents with abnormal external genitalia and thus tends to evade clinical detection (Nordenström et al., 2005; Gidlöf et al., 2014). Existing screening strategies generally achieve high sensitivity for identifying salt-wasting 21-OHD (Gidlöf et al., 2014). Serum 17α-OHP levels rise within the first weeks after birth and correlate closely with disease severity and CYP21A2 genotypes. Early diagnosis of milder subtypes including simple virilizing CAH helps curb premature androgen overexpression, supporting normal physical growth and development. Non-classic 21-OHD is commonly missed by conventional biochemical screening. Given the inevitable false-positive and false-negative results of biochemical assays, molecular genetic testing plays a vital role in CAH subtype classification and clinical management (Wang et al., 2021; Liu et al., 2022).

In this study, 2,100 newborns received simultaneous 17α-OHP detection and LRS-based CAH genotyping. Two neonates presenting normal 17α-OHP levels harbored biallelic pathogenic variants in the CYP21A2 gene, corresponding to a population prevalence of 1 in 1050. An additional 88 infants (4.2%) carried heterozygous alleles linked to CAH pathogenesis: 85 individuals harbored variants in CYP21A2, 2 bore variants in CYP17A1, and 1 carried a variant in StAR. LRS into first-tier CAH newborn screening enhances diagnostic sensitivity and specificity for NCCAH and yields robust molecular evidence to support genetic counselling. This advantage is particularly notable for families carrying alleles associated with classic CAH, who face elevated disease recurrence risks in future pregnancies. LRS broadens the spectrum of detectable disease-causing variants and enables identification of pathogenic alterations in CAH-related genes that cannot be captured by routine biochemical screening, including CYP11B1, CYP17A1, StAR, and HSD3B2. Earlier research focusing on Chinese CAH populations has indicated divergent phenotypic manifestations in individuals carrying biallelic CYP21A2 variants, even among subjects harboring identical pathogenic alleles. Such variability may stem from modifier gene effects, gestational hormonal milieu, and postnatal stress conditions. Nonetheless, allele-specific phenotypic stratification (SW/SV/NC), which is established on residual enzyme activity and large clinical population datasets, is the primary framework adopted to assess variant pathogenic potential in newborn genetic screening. Nevertheless, this stratification framework cannot be utilized to confirm the clinical CAH status of infants with heterozygous gene alterations.

The combined screening strategy integrating LRS and biochemical testing established in the present study yields an overall pipeline TAT of merely seven calendar days. By contrast, the conventional two-stage workflow involving initial biochemical screening, clinical recall, and subsequent MLPA or Sanger sequencing verification requires a total TAT of 26 calendar days (Wang et al., 2025). The 19-day reduction in diagnostic waiting window eliminates the critical risk window for neonatal adrenal decompensation in SW-CAH patients. In the national 21,239 newborn LRS first-tier screening cohort, only 0.08% of DBS samples required repeat sequencing due to poor DNA quality, confirming stable high-throughput laboratory throughput compatible with routine provincial newborn screening centers (Liang et al., 2025).

LRS genotyping covering the complete CAH gene panel (CYP21A2, CYP11B1, CYP17A1, HSD3B2, and StAR) costs roughly 20 USD per sample via batch sequencing on barcoded 384-plex PacBio Sequel II platforms. In comparison, single MLPA analysis costs around 16.7 USD per specimen. For samples yielding equivocal MLPA signals, mandatory confirmatory Sanger sequencing incurs an extra 50.1 USD per case; collectively, the combined MLPA and Sanger workflow costs nearly 67 USD for each newborn with an initially suspicious biochemical screening result (Wang et al., 2025). LRS can resolve genotype ambiguity without parental trio validation, removing the financial expenditure incurred by family-based genetic testing. Conventional biochemical screening yields abundant false-positive results, which necessitate repeat heel-prick DBS collection, secondary 17α-OHP testing, and stepwise MLPA plus Sanger molecular validation. Every false-positive infant brought back for clinical follow-up accumulates extra expenditures including nursing sampling manpower, cold-chain logistics, repeated biochemical reagents, and multiple rounds of molecular testing. As a first-tier screening modality, LRS substantially cuts expenditures from unneeded patient recalls and duplicate biochemical assays. Overall, adopting LRS as primary screening reduces auxiliary recall-associated costs by an estimated 62% among all infants with abnormal initial screening readouts (Wang et al., 2025).

This study identified 19 neonates harboring single heterozygous CAH alleles from the retrospective cohort of 52 screen-positive infants, and an additional 88 individuals with heterozygous CAH variants among the 2,100 prospective newborns who exhibited normal 17α-OHP levels. While data on heterozygous variant prevalence yields valuable population genetic evidence, universal NBS is principally designed to detect infants with biallelic pathogenic variants who require urgent early clinical intervention. This creates ethical concerns over routinely disclosing results indicating single heterozygous variants to parents. Nevertheless, such genetic findings carry meaningful value for families’ reproductive planning. If both parents carry a heterozygous pathogenic CYP21A2 allele, each subsequent pregnancy carries a 25% risk of offspring affected by classic CAH. Early detection of heterozygous variants in newborns allows timely genetic testing for parents and targeted prenatal diagnosis in future pregnancies, lowering the risk of recurrent affected infants. This advantage is particularly notable in populations with higher frequencies of pathogenic classic CAH alleles. Clinical screening centers should establish standardized genetic counseling workflows for carrier results. Dedicated genetic counselors provide separate, tailored interpretation of heterozygous variants to parents, clearly differentiating asymptomatic carrier status from clinically affected individuals with biallelic pathogenic variants, thereby mitigating undue parental anxiety stemming from ambiguous carrier reports.

This study has several key limitations. First, its single-center design restricts the external generalizability of our findings. Large multicenter prospective studies are thus needed to characterize the epidemiological profile of CAH in Fujian Province, verify the clinical performance of LRS-combined screening, and systematically evaluate the cost-effectiveness of integrated biochemical and genetic newborn screening algorithms. Second, the two genetically identified NCCAH neonates in the prospective cohort lacked confirmatory ACTH stimulation tests and adrenal androgen measurements in the neonatal period. Long-term outpatient follow-up through adolescence has been arranged, which will facilitate definitive phenotypic validation. The lack of neonatal functional assays creates mild diagnostic ambiguity for these two subjects. Third, we did not develop a comprehensive health economic model incorporating multi-dimensional expenditure indicators. Further multicenter prospective trials are warranted to calculate precise population-level cost-effectiveness ratios.