In the present study, we recruited 70 infants and children with PAI and sequentially performed CYP21A2 gene Sanger sequencing, MLPA testing and biochemical plus clinical detailed examinations and found an overall 84.29% (59/70) diagnostic rate for CAH, with 91.53% (54/59) of CAH patients showing positive genetic findings. For uncharacteristic PAI, we found that 72.73% (8/11) of the cases had positive genetic findings by WES and Array-CGH. Thus, a total of 88.57% (62/70) of the children were determined to have a positive genetic test, which is higher than the rate in a previous review by Tulay Guran (80%)[12] but lower than the 94.2% positive rate detected by Rebecca Perry and colleagues[2].
Among our 21-OHD CAH patients, the most prevalent CYP21A2 variants were c.293-13C > G (31.36%), Del (18.64%), p.I173N (16.95%), E3 Δ8 (5.08%), p.R483PfsX58 (4.24%) and p.R357W (3.39%). The first three variants, c.293-13C > G, Del and p.I173N, were consistent with the most frequent variants in the Asian population[13–17]; however, our data showed higher E3 Δ8 and p.R483PfsX58 variant prevalence rates and a lower p.R357W variant prevalence. Ethnic differences were evident among different studies; the frequencies of p.P31L and p.V282L were 1.69% and 0.85% in our study, respectively, while the p.V282L variant had a dramatically high frequency in Argentina and Brazil[18–20] and the p.P31L variant was frequent in Serbia[21] (Table 1). Thus, the genetic background of 21-OHD differs by ethnicity.
Novel variants were found in the CYP21A2 gene in two children, c.651 + 2T > G and c.833dupT (p. 279GfsX17) (Table 2), which were predicted to be damaging. Variant c.651 + 2T > G located at the end of exon 5 was predicted to alter the wild-type donor site and mostly likely led to whole exon 5 skipping, which would result in the loss of 184–217 amino acid residues. With 9 highly conserved residues[22], these residues are essential to construct the important functional domains as steroid-binding sites (residues 203–207) and large hydrophobic areas ((residues 211–218). This splicing site variant should impair enzyme activity because the proband displayed the SW phenotype. The other novel variant, c.833dupT (p. 279GfsX17), most likely causes complete enzyme activity loss because another comparable frameshift variant c.923dupT (p.L308FfsX6), which retains more amino acid residues, causes complete enzyme activity loss[23]. However, more functional studies are required to confirm the exact impact of the novel variants on enzyme activity in the future.
In our research, SW females had higher PGS scores than SV females, as reported by previous studies[24, 25], and the reason may be the higher 17-OHP and TES levels discovered in SW patients (Fig. 1). Interestingly, compared with males, females had higher serum 17-OHP and cortisol levels in both the SW and SV groups, which is consistent with another study[15]. Regarding the genotype-phenotype correlation, the PPV for group 0 was 100%, which is higher than those in most similar studies, while the PPVs in group A (82.61%) and group B (84.62%) are in accordance with those in previous studies[10, 15, 18, 20, 21]. Surprisingly, we found that 8.47% (5/59) of the patients clinically presented with the SW (n = 4) and NC (n = 1) phenotypes without biallelic variants (Supplement Table 3). Studies have reported that the monoallelic variant and absent variants accounted for 2.2–24% of enrolled children[10, 16, 19–21, 26]. The aetiology is unknown. We deduced that an amplification allele dropout effect may occur due to the high similarity between the CYP21A1 and CYP21A2 genes. Additionally, the CYP21A2 gene promotor region and other intronic variants that have not been analysed in studies may also reduce transcriptional activity[4].
In our uncharacteristic PAI patients, we detected a total of 7 pathogenic variants, including one chromosome microdeletion and 5 novel variants in the NR0B1, AAAS, NNT and ABCD1 genes. The NR0B1 gene is the most frequent gene responsible for X-linked AHC in males (Table 4). The absence of NR0B1 causes progenitor cells to prematurely differentiate into steroidogenic cells without adequate maturity[27, 28]. We found two patients each with one novel variant (c.338–339 CG > GA, p. 108S > X; c.1231_1234delCTCA, p.L411Vfs*6) and one infant with an NR0B1 deletion that manifested as PAI, mineralocorticoid deficiency and diminished adrenal glands; however, no hypogonadotropic hypogonadism was noted due to the patient’s young age. We also found two novel homozygous pathologic variants in the AAAS gene (c.399 + 1G > A, p.V103Afs*8; c.250delT, p.W84Gfs*10) in 3 patients (Table 3, cases 6, 7, and 8) from 2 families, and both parents were consanguineous. Alacrima and AI were found in all three patients; however, achalasia was observed only in an 8-year-old boy, which manifested as swallowing difficulties. The other two sisters shared the same genotype with different phenotypes. These results suggest that careful estimation should be performed in every PAI child with alacrima or achalasia. The ABCD1 gene is another causative gene that is often observed in male patients and results in X-linked ALD[28]. The clinical presentation of X‐linked ALD is variable, and no phenotype-genotype correlation has been observed[29]. These phenotypes include cerebral, adrenal, spinal cord and peripheral nerve involvement[29]. Fifty percent of affected children will ultimately develop adrenomyeloneuropathy within 10 years[30]. Our patients showed clinical PAI, elevated serum long-chain fatty acid levels and normal neurological examination results but had white matter lesions detected by magnetic resonance imaging. We also found a novel pathogenic variant in the NNT gene (c.2274delT, p.I758Mfs*10). Researchers reported that 53% of patients with NNT gene variants presented with hyperpigmentation, while 17% had mineralocorticoid deficiency[31]. Cardiac and thyroid involvement may also exist[32]; therefore, a close long-term follow-up is still needed as our patient presented with only hyperpigmentation. Evident genetic defects were not detected in three PAI patients, and we deduced that our current test methods may fail to identify deep intronic variants and epigenetic changes[33–36].
Table 4
The gene spectrum of uncharacterized PAI in other ethnic groups
Country | Methods | STAR | NR0B1 | SMAD9 | AAAS | NNT | MC2R | CDKN1C | AIRE | CYP11A1 | MRAP | NR5A1 | ABCD1 | CYP11B1 | Unknown |
Japan [36] | targeted gene sequence | 19 | 18 | 7 | 2 | 2 | 1 | 1 | - | 0 | 0 | 0 | - | - | 9 |
Canada[35] | targeted gene sequence | 5 | 0 | - | 0 | 0 | 1 | - | 3 | 0 | - | - | 0 | - | 2 |
Turkey [34] | targeted gene panel + NGS | 11 | 12 | 0 | 1 | 7 | 25 | 0 | 0 | 9 | 9 | 1 | 2 | 0 | 18 |
UK [23] | WES | 0 | 4 | 0 | 2 | 2 | 1 | 0 | 2 | 6 | 0 | 0 | 0 | 1 | 26 |
China | WES+ Array-CGH | 0 | 2 | 0 | 3 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 3 |
WES; whole-exome sequencing; NGS: next generation sequence; CGH; array-based comparative genomic hybridization |