Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive diseases in humans with an incidence, evaluated on neonatal screening in different populations during the last decade or so, of 1:6,084 to 1:26,727 live births (1). It comprises several steroid enzyme deficiencies, among which 21-hydroxylase deficiency (21OHD) (OMIM # 201910) is by far the most common, affecting about 95%–99% of all CAH patients (1–5). In 21OHD, the hormonal disbalances consist of variable low cortisol and aldosterone levels, and compensatory high levels of 17-hydroxyprogesterone (17OHP), which converts to androgens. CAH appears in two clinical forms, classic and non-classic (NC) phenotypes (6). The classic form is further classified as salt-wasting (SW) and simple-virilizing (SV) forms. Patients affected by the SW form have SW with severe dehydration, hypoglycemia, failure to thrive, and hyperandrogenism. This can be clinically recognized early in girls due to atypical genitalia, while boys may first be identified, if not neonatally screened, when presenting with a life-threatening SW crisis within 2 weeks postnatally (2). The SV form has enough aldosterone production to avoid SW crisis and, prior to the introduction of neonatal screening, was identified due to atypical genitalia in girls and in male toddlers due to signs and symptoms of excessive androgen production, even though cases of diagnosis in adulthood occasionally happened (2, 7). The NC form is mild and can appear in numerous variants of the clinical picture, ranging from no signs in the newborn period, to mild virilization later in childhood, up to polycystic ovary syndrome or isolated hyperandrogenism and decreased fertility in adulthood (8). NC 21OHD is sometimes identified in neonatal screening (9), but most cases are identified due to symptoms and signs in adolescence or young adulthood, even though the majority probably never gets diagnosed. NC CAH shows an incidence of 1:200 to 1:1,000 (10, 11).

However, despite the traditional and generally accepted classification of CAH in different forms, due to a large variety of CYP21A2 mutations, the phenotype can have many variants, and it is clear that CAH phenotype represents a continuum between non-classic and classic forms (12). This is important for tailoring appropriate therapy in individual patients. The 21-hydroxylase enzyme is encoded by CYP21A2 gene, and the clinical 21OHD presentation depends upon the combination of pathogenic variants affecting this locus (13). Both CYP21A2 gene and CYP21A1P pseudogene are located on the short arm of chromosome 6 (6p21.3), in the human leukocyte antigen (HLA) class III region of the major histocompatibility (MHC) locus. These genes contain ten exons spaced over 3.4 kb with a sequence homology reaching 98% (12, 14, 15). Intergenic recombination events represent more than 95% of pathogenic variants causing 21OHD. Approximately 75% of the deleterious variants are transferred by small conversions from the pseudogene during meiosis (16, 17). Of the cases of 21OHD, 20%–25% are due to gene deletions, gene duplications, and deletions involving CYP21A2 and other contiguous genes (18). Finally, CYP21A2 pathogenic variants that are not apparently gene conversions account for 5%–10% of CAH alleles in most populations (16, 17).

To date, more than 230 CYP21A2 pathogenic variants have been identified (18). Most patients are compound heterozygous; and in this case, the phenotype correlates with the variant that predicts the higher residual enzyme activity (19). Based on the residual activity of the mutant enzyme, CYP21A2 variants are classified into specific groups (null, A, B, and C) (20, 21). While variants of the null group show 0% enzyme activity during in vitro assay, group A variants preserve a minimal (<1%) residual activity. These two groups are both associated with the SW form of 21OHD. Differently, group B (1%–5% enzyme activity) and group C (20%–50% enzyme activity) variants are related to the SV and NC forms, respectively (12, 22–25). Although there is good agreement between clinical phenotype and patient genotype, it is well-known that some exceptions exist (18–29).

The prevalence of 21OHD is highly variable among populations; some countries show higher (China and India) (19, 20) or lower prevalence (Japan and New Zealand) (3, 21–27). However, in most of the analyzed studies, the In2G variant is found to be, with rare exceptions (30–50), among the most common CYP21A2 pathogenic variants. It is usually related to the SW 21OHD; however, patients with a non-correspondent phenotype have been widely reported. In this review, we collected the most relevant evidence showing the phenotypic variability of the In2G variant.

To date, 18 intronic splicing variants, representing 7.7% of all disease-causing variants, have been reported in CYP21A2 gene (18, 51). In silico analysis or functional studies showed that all these variants are associated with the severe form of 21OHD due to the changed reading frame of the gene producing a non-functional enzyme (21, 52, 53). Generally, an intronic splicing variant causes the disruption of the acceptor/donor site, inducing activation of an intronic cryptic acceptor/donor site, retention of a whole intron or part of it, and exon skipping (51). Regarding intron 2 of CYP21A2 gene, five pathogenic splicing variants have been reported (51). Two of these, c.292+1G>A and c.293-2A>G, cause the SW phenotype to disrupt the donor and acceptor splicing sites, respectively (54, 55). Differently, the c.292+5G>A and c.293-7C>G variants were described, in SW patients, as reducing the consensus value for the intron 2 splice donor and acceptor sites, respectively (52, 56).

c.293-13C/A>G (In2G) is the most common splicing variant in CYP21A2 gene. It is usually transferred by microconversion from CYP21A2 pseudogene. At the −13 position, before the end of intron 2, the wild-type nucleotide is A or C. Substitution to G creates an additional splice acceptor site, causing aberrant splicing of intron 2 with retention of 19 intronic nucleotides. This results in a shift in the translational reading frame (21) ( Figure 1 ). The In2G variant is typically related to the SW form of CAH. It is by far the most common CAH mutation in the majority of countries and ethnicities, predominantly Europeans followed by Middle Easterners and Hispanics (27), although there are exceptions ( Table 1 ). In different studies, the prevalence of the In2G variant was between 20.6% and 30.3% (25, 27, 61, 63). In the homozygous form, it is more common in those with European and Middle Eastern ancestries than in Hispanic Americans, Asians, or East Indians (27). Some authors even refer up to 60.4% prevalence of this variant in certain ethnicities (32) ( Table 1 ).

In some populations, the In2G variant could be considered a founder variant. It is a unique finding in some enclosed populations such as Alaskan and one of the Roma populations from the Balkan region, although the number of explored individuals was rather small (62). Finally, in other populations, such as the Spanish population, the In2G variant appears to be related to recent conversion events (64).

The In2G variant is frequent in patients with 21OHD. It normally causes severe disease; however, the clinical presentation can vary from the SW form, through the SV form and rarely the NC form. Thus, there is a difference in the severity of 21OHD within group A. The mechanism underlying the variation in the clinical phenotype of the In2G variant was widely discussed. The most accredited hypothesis is that a small number of transcripts avoid aberrant splicing, providing a small amount of the 21-hydroxylase enzyme, which is sufficient for a milder clinical presentation of the disease. For this reason, in some patients, the phenotype appears as SV or even NC. In vitro experiments showed that the CYP21A2 intron 2 c.292+1G>A variant produces two different transcripts: the type I fragment lacked the entire introns 1 and 2 and exon 2, whereas the type II, representing approximately one-third of the mRNAs produced and generated by the use of a cryptic splice acceptor site downstream from exon 3, had a deletion of intron 1, entire exon 2, and part of intron 2 (97). These results supported the evidence that splicing is not a homogenous mechanism and that a single splicing variant can generate alternative transcripts, which might explain some unusual phenotypes (97, 98). In addition, the potential influence of extra-adrenal 21-hydroxylation on the CAH patient’s phenotype might be an additional cause to consider. In fact, it was demonstrated that hepatic CYP2C19 and CYP3A4 have the ability to 21-hydroxylate progesterone and thus may modulate mineralocorticoid deficiency (99).

In conclusion, although a good genotype–phenotype correlation exists in 21OHD, a disparity in phenotypic appearance is present in a portion of patients carrying the In2G/In2G or In2G/null genotypes. This evidence represents the most challenging issue in prenatal diagnosis and familiar counselling since the predictive value for different phenotypes can be uncertain.

MK drafted the manuscript and participated in writing and editing. PC participated in writing and editing. HF participated in writing and editing. All authors contributed to the article and approved the submitted version.

This study was supported by Magnus Bergvall Foundation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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