Congenital Adrenal Hyperplasia (CAH) is the largest classified group in Disorders of Sexual Developments (DSDs). CAH has two forms; the severe classical /salt wasting (SW-CAH) and the milder non-classical/ simple virilizing (SV-CAH). Both forms are caused by deficient or decreased cortisol biosynthesis [1-4]. Inefficient cortisol biosynthesis leads to increased production of corticotrophin-releasing hormone, adrenocorticotropic hormone and hyperplasia of the adrenal glands. Thus this leads the adrenals to produce excessive amounts of androgens as early as 6–7 weeks of gestation [5-7]. Therefore, most of the CAH patients are unable to synthesize sufficient amounts of aldosterone and are prone to the life-threatening SW crises. So in SV-CAH, the aldosterone is normal and is usually ignored in boys because excess of androgen during childhood results in overgrowth and early signs of puberty [8-10].

Individuals with CAH have a deficiency in 21-hydroxylase enzyme (21-OHD) [11]. This enzyme, encoded by the CYP21A2 gene [12-20] is mutated in 95% of CAH cases [21-23, 15] and is located within the HLA region of the short arm of chromosome 6 (6p21.3), next to its inactive pseudogene (CYP21A1P) [11, 24, 25]. The CYP21A2 and CYP21A1P have 98% homology in exons and 96% in introns [21, 25, 26]. Both gene and pseudogene contain 10 exons with 3.1 kb in length [27].

The molecular genetics of CAH has identified different mutations which have been classified based on 21-hydroxylase activity [28]. Over 100 mutations in CYP21A2 have been discovered to cause 21-hydroxylase deficiency. Approximately, 65–70% of these occur due to micro-conversions derived from its pseudogene [25] such as the most common c.293-13A/C>G in intron 2 or p.I172L gene changes [29]. The rest are caused by copy number variants (CNV) (25-30%) or point mutations [25]. Compound heterozygous mutations have been also identified in the SW form, even though a single mutation could have a mild effect [30]. Moreover, previous studies have shown that p.Q318Y and p.R356W had 0% enzyme activity, while c. 293-13A/C>G and p.I172L presented minimal residual activity and were associated with the classic form of the syndrome [31].

Here we present a 12 years old boy, with 46, XY karyotype who developed early signs of puberty at age 6, including pubic hair, Tanner stage II without axillary hair, acne, and dark facial skin. His voice also changed to be more like an adult male voice. Penile length and testicular volume were 7.5 cm and 2 ml, respectively. His testes were not diagnosed with pathological condition. Testosterone level was 2.8 ng/ml and normal level of electrolytes. Accordingly; the boy was diagnosed with SV-CAH and administered hydrocortisone 17 mg (10 mg in the morning and 7 in the evening). At age 8 years old, his bone age was equal to that of 12-13 years old male. Testicular volume was 4 ml, FSH was 7.67mU/ml and LH 1.21 mU/ml. His weight was 37 Kg and was 137 cm tall. Furthermore, there wasn’t a family history of this particular condition; however, the patient had a sister who presented with ambiguous genitalia at an early age but died right after that. Their biological parents were not relatives and there was no information of a similar conditions in their extended family.

Genomic DNA was isolated from peripheral blood leukocytes using the automated MagPurix 12S system from Zinexts (Zinexts Life Science Corp., Taiwan) and the MagPurix Blood DNA Extraction Kit 200 (Zinexts Life Science Corp). Genetic alteration causing Disorders of Sexual Development (DSD) was analyzed using a customized gene panel designed with the Ion AmpliSeq Designer software (https://www.ampliseq.com) that included all exons and exon-intron boundaries of the selected 48 genes (Table 1). Libraries were prepared according to the manufacturer’s instructions and samples were sequenced using the Ion Torrent PGM platform (Thermo Fisher Scientific). Amplicons (9.3%) not appropriately covered (<20x fold) were assessed by Sanger sequencing (Sequences of the primers are available under request).

The sequencing data were analyzed using the Ion Reporter software (Thermo Fisher Scientific). Variants were filtered to include those with a p-value <0.001 and a Minor Allele Frequency (MAF) ≤0.001 in the annotation settings of the Ion Reporter software (dbSNP, ClinVar and 5000 exomes databases). Moreover, the allele frequency was further checked for each ethnic group in 1000 genomes browser (http://browser.1000genomes.org/) and others. Variants were discounted if they were common. Accordingly, there were 12 variants in different genes (Table 2). The impact of protein functionality of missense alterations was evaluated using in silico prediction programs (Table 2), including SIFT (http://sift.jcvi.org/), PolyPhen2 (http://genetics.bwh.harvard .edu/pph2/), PROVEAN (http://provean.jcvi.org/index.php), Mutation Taster (http://www.mutationtaster.org/), SNPs & GO (https://snps-and-go.biocomp.unibo.it/snps-and-go/), and Panther (http://www.pantherdb.org). We identified the pathogenic homozygous missense mutation c.878G>A (p.G293D) in exon 7 of the CYP21A2 gene (NM_000500.7) (Table 2). Sanger sequencing was then used for confirmation of the mutation.

Diagnosis of congenital adrenal hyperplasia (CAH) is challenging especially within clinical settings of the developing countries. Here, the patient was diagnosed with SV-CAH, based on his clinical presentation and available laboratory investigations. Genotyping analysis showed that the patient had the G293D variant in homozygosis in CYP21A2. The patient’s sister might have been another carrier of this point mutation as she was born with ambiguous genitalia. To the best of our knowledge, a single study has detected the G293D protein change in a female with CAH. Tardy et al. described a newborn girl with Prader stage 4 and severe virilization of genitalia [32]. Although biochemical values were not determined, the patient was diagnosed with SW-CAH form.

It is well-known that the severity of the disease correlates well with the level of enzymatic deficiency and the location of the residue [33]. For example, point mutations that disrupt the binding of heme cofactor cause an SW phenotype, while alterations in hydrophobic residues lead to SV-CAH [34]. The highly conserved G293 residue, covering the proximal substrate-binding site is responsible for the flexibility of the I-helix. Therefore, the G293D mutation makes the bending and swiveling of the pocket difficult [30, 33]. Certainly, in vitro studies revealed that the mutation decreases the residual activity to <1% [32].

The main difference between the SW and SV-CAH forms is the insufficient aldosterone secretion that leads to a fatal drop of electrolytes in the first [35-39]. The normal electrolyte levels observed in SV-CAH might be explained by the role of testosterone as a down-regulator of aldosterone secretion [40]. Unfortunately, we were not able to measure the aldosterone level because the test is not available in Sudan. Functional studies by Toot et al in 2008, concluded that testosterone influences the excretion of Na (sodium) and K (potassium) through an androgen receptor dependent mechanism [41]. Similar findings were later published [42, 43]. However, CAH, either SW or SV, displays variable degrees of sodium depletion which is not always significant [44-49]. This might depend on the function of the testes. Cabrera et al found that patients with SW-CAH had a higher frequency of developing testicular nodules compared to SV-CAH [44]. Our patient was referred to early puberty signs but no nodules or testicular abnormalities affecting his function were observed. Thus, we can speculate that although the G293D mutation was found first in SW-CAH, his elevated testosterone levels could have played a role in electrolyte balance since gonads were normal.

In this study, the c.878G>A (p.G293D) variant in CYP21A2, previously related to CAH-SW, was identified in homozygous state in a patient diagnosed with SV-CAH. We believe that testosterone had balanced electrolytes through one of the androgen biological pathways. Thus, the level of electrolytes is not only affected by the severity of the enzymatic activity, but also by androgen levels in individuals diagnosed with CAH.