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.

Approach of Heterogeneous Spectrum Involving 3beta-Hydroxysteroid Dehydrogenase 2 Deficiency

“…Other authors consider that we should discuss of 3 distinct forms: SW, non-SW, and non-classical type as a different entity with pubertal onset especially in females [ 27 , 28 ]. Generally, 95% of cases diagnosed with paediatric adrenal insufficiency are caused by CAH, but 3βHSD2 deficiency has a modest epidemiological impact opposite to 21OH deficiency [ 29 ]. In teenagers and young adults, the most frequent cause of adrenal insufficiency is Addison’s disease followed by CAH, overall chronic adrenal failure having more than 30 aetiologies [ 30 ].…”

“…The advance of molecular and genetic diagnosis is mostly useful in different types of CAH, starting with the most frequent “mutation”, namely 21OH deficiency, since a level of genotype–phenotype correlation is expected. However, in 3βHSD2 deficiency the current level of knowledge makes this association less predictable due to the paucity of studies [ 29 ].…”

Congenital Adrenal Hyperplasia

“…CAH due to 21-hydroxylase deficiency (21OHD) represents approximately 90-95% of CAH cases encountered in clinical practice [3,4].…”

“…The gene that encodes CYP21A2 is located on the short arm of the 6 chromosomes within the HLA class III region (6p21.3) arranged in tandem with its inactive pseudogene (CYP21A1P) [1,3]. Both CYP21A2 and CYP21A1P genes share a high nucleotide homology of about 98% and 96% in exons and introns respectively, but only CYP21A2 gene encodes the active microsomal P450 enzyme, 21-hydroxylase (CYP21A2, P450c21) [1,6].…”

“…For this reason, the clinical phenotype is variable and depends on the severity of the enzyme deficiency. The most common enzyme deficiency that disrupt cortisol synthesis leading to CAH is that of 21 hydroxylase [1][2][3][4].…”