Molecular Diagnosis of Steroid 21-Hydroxylase Deficiency: A Practical Approach

Abstract

Adrenal insufficiency in paediatric patients is mostly due to congenital adrenal hyperplasia (CAH), a severe monogenic disease caused by steroid 21-hydroxylase deficiency (21-OHD, encoded by the CYP21A2 gene) in 95% of cases. CYP21A2 genotyping requires careful analyses that guaranty gene-specific PCR, accurate definition of pseudogene-gene chimeras, gene duplications and allele dropout avoidance. A small panel of well-established disease-causing alterations enables a high diagnostic yield in confirming/discarding the disorder not only in symptomatic patients but also in those asymptomatic with borderline/positive results of 17-hydroxyprogesterone. Unfortunately, the complexity of this locus makes it today reluctant to high throughput techniques of massive sequencing. The strong relationship existing between the molecular alterations and the degree of enzymatic deficiency has allowed genetic studies to demonstrate its usefulness in predicting/classifying the clinical form of the disease. Other aspects of interest regarding molecular studies include its independence of physiological variations and analytical interferences, its usefulness in the diagnosis of simple virilizing forms in males and its inherent contribution to the genetic counseling, an aspect of great importance taking into account the high carrier frequency of CAH in the general population. Genetic testing of CYP21A2 constitutes an irreplaceable tool to detect severe alleles not just in family members of classical forms but also in mild late-onset forms of the disease and couples. It is also helpful in areas such as assisted reproduction and preimplantation diagnosis. Molecular diagnosis of 21-OHD under expert knowledge definitely contributes to a better management of the disease in every step of the clinical course.

Keywords: 21-hydroxylase deficiency; CYP21A2 gene; ccongenital adrenal hyperplasia (CAH); classical forms of congenital adrenal hyperplasia; molecular diagnosis; non-classical forms of congenital adrenal hyperplasia.

Figure 1

[Adapted from Santomé et al., (21)]. Scheme of the RCCX module located on the short arm of chromosome 6 within the HLA class III region. Tandem duplication affects CYP21 and C4 genes. In humans only CYP21A2 gives rise to the functional protein, whereas CYP21P is a homologous pseudogene that includes several inactivating point variants that can be transferred to the active gene by small gene conversion events. Both C4A and C4B are functional. Tenascin, also duplicated, is encoded in the complementary chain. The bottom of the image shows the recurrent variants grouped according to how they affect the enzimatic functionality: severely (red), moderately severe (green) or mildly (blue). Recurrent variants in all populations are circled. The complete nomenclature of each variant including the cDNA position (NM_000500.9) would be: c.92C>T [p.Pro31Leu], c.292+5C>A, c.293-13C>G, c.332-339del, c.518T>A [p.Ile173Asn], c.(710T>A; 713T>A; 719T>A) p.[Ile237Asn; Val238Glu; Met240Lys], c.844G>T [p.Val282Leu], c.923dupT, c.955C>T [p.Gln319*], c.1069C>T [p.Arg357Trp], c.1280G>A [p.Arg427His] and c.1360C>T [p.Pro454Ser]). The arrows “Fragment A”, “Fragment B” and “Fragment C” represents specific amplicons for CYP21A2 amplification.

Figure 2

(2A) [Taken from Ezquieta et al., (56)]: Receiver operating characteristic (ROC) curves analyses in fully genotyped children affected with NCF of 21-OHD (mild/mild vs severe/mild genotype) for: (A) adrenocorticotropic hormone (ACTH)-stimulated 17-OHP, (B) basal 17-OHP, or (C, D) the combination of both parameters [(C): sum; (D): product]. Areas under the curves (SE): ACTH-stimulated 17-OHP, 0.908 (0.057); basal 17-OHP, 0.790 (0.081); sum 0.866 (0.068); product 0.884 (0.064). Cut-off values, nmol/L (A–C) and nmol²/L² (D). The cut-offs for maximum predictive values are represented by small, empty squares in the Figures. (2B) [From Ezquieta et al., (91)] Diagram of a hypothetical interaction between protective and sensitizing factors modulating the clinical expressivity of 21-OHD-related hyperandrogenism. CAPN10-UCSNP44C and TNFR2-R196 are proposed in this study to be sensitizing and protective factors, respectively.