Results

LRS of CAH genes in second-tier newborn screening for CAH

A total of 100,145 newborns were recruited from the Fujian Provincial Newborn Screening Center between January 1 and December 31, 2019. All newborns underwent CAH NBS using a 17α-OHP screening assay. A total of 190 cases were identified as screen-positive, with a positive rate of 0.19% (190/100,145; 95% confidence interval [CI], 0.17%–0.21%). Among the screen-positive neonates, 175 cases were successfully recalled, and an additional dried blood spot specimen was collected for re-evaluation. Of these 175 neonates, 20 cases remained positive in the second screening and underwent genetic diagnosis. Ultimately, 5 neonates were definitively diagnosed with CAH, corresponding to an estimated prevalence of 1:20,029.

A total of 52 full-term neonates (gestational age at delivery ≥ 37 weeks and birth weight ≥ 2,500 g) with positive screening results were selected and subjected to LRS-based CAH genotyping. Among the 52 samples analyzed, 5 cases of pathogenic CYP21A2 mutations were detected, including 3 samples with SNVs on both alleles, 1 sample with a 30-kb deletion on both alleles, and 1 sample with an SNV on one allele and complex SNVs on the other allele; these findings were consistent with the results of genetic diagnosis (Table 3). We additionally identified 10 neonates harboring monoallelic CYP21A2 variants. Collectively, these 15 individuals (5 patients with biallelic CYP21A2 variants plus 10 subjects carrying single CYP21A2 alleles) exhibited 13 distinct CYP21A2 genotypes, generating a total of 20 CYP21A2 alleles for frequency estimation (10 alleles derived from subjects with monoallelic variants and 10 alleles from patients with biallelic variants). Furthermore, two neonates carried variants in CYP11B1, three carried variants in StAR, and four carried variants in HSD3B2. Detailed variant information is summarized in Table 1.

Patient ID | Sex | Initial 17α-OHP concentration | Allele 1 (LRS) | Allele 2 (LRS) | Final Genotype (MLPA + Sanger)
1 | M | 349.50 | c.293-13C>G | c.293–13C>G | c.293-13C>G/c.293-13C>G
2 | M | 532.50 | CYP21A1P/A2-CH-3 | CYP21A1P/A2-CH-1 | CYP21A1P/A2-CH-3/CYP21A1P/A2-CH-1
3 | F | 60.95 | c.293-39_293-38delinsGG | c.371C>T | c.293-39_293-38delinsGG/c.371C>T
4 | F | 101 | c.293-13C>G | c.1280G>A | c.293-13C>G/c.1280G>A
5 | M | 17.7 | c.293-13C>G | c. [710T>A,713T>A,719T>A] | c.293-13C>G/c.[710T>A, 713T>A,719T>A]

LRS of CAH genes in first-tier newborn screening for CAH

Based on the excellent performance of LRS in CAH genetic detection for second-tier NBS, we further explored its clinical feasibility as a first-tier screening strategy. A total of 2,100 newborns (1,123 males and 977 females) underwent parallel detection of serum 17α-OHP and LRS-based CAH genotyping. Two newborns with normal 17α-OHP biochemical screening results were found to carry biallelic pathogenic variants in the CYP21A2 gene, yielding a prevalence of 1 in 1050 (Table 4). Furthermore, heterozygous variants were detected in 88 neonates (4.2%) exhibiting unremarkable 17α-OHP levels: 85 individuals harbored variants in CYP21A2, 2 carried variants in CYP17A1, and 1 bore a variant in StAR. Thirty-two distinct genotypes were observed among the 85 subjects with CYP21A2 heterozygous alterations (Table 2). The overall carrier rates were 1:78 (27/2,100) for classic CAH (CCAH) and 1:40 (53/2,100) for non-classic CAH (NCCAH). The allelic variant c.371C>T was the most prevalent among NCCAH carriers, with an allelic frequency of 0.524% (22/4200). Among the 27 carriers with classic phenotype-associated genotypes, c.293-13C>G was the dominant allele, with an allelic frequency of 0.190% (8/4200).

Case ID | Sex | Initial 17α-OHP concentration | Allele 1 genotype | Allele 2 genotype
1 | F | 1.8 | c.[-126C>T,-113G>A] | c.844G>T
2 | M | 3.1 | c.844G>T | c.913G>A