Ann Pediatr Endocrinol Metab.  2023 Jun;28(2):77-86. 10.6065/apem.2346108.054.

Molecular basis and genetic testing strategies for diagnosing 21-hydroxylase deficiency, including CAH-X syndrome

• Kim JH
• Kim GH
• Yoo HW
• Choi JH

• KMID: 2543288
• DOI: http://doi.org/10.6065/apem.2346108.054

Abstract

Congenital adrenal hyperplasia (CAH) is a group of autosomally recessive disorders that result from impaired synthesis of glucocorticoid and mineralocorticoid. Most cases (~95%) are caused by mutations in the CYP21A2 gene, which encodes steroid 21-hydroxylase. CAH patients manifest a wide phenotypic spectrum according to their degree of residual enzyme activity. CYP21A2 and its pseudogene (CYP21A1P) are located 30 kb apart in the 6q21.3 region and share approximately 98% of their sequences in the coding region. Both genes are aligned in tandem with the C4, SKT19, and TNX genes, forming 2 segments of the RCCX modules that are arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. The high sequence homology between the active gene and pseudogene leads to frequent microconversions and large rearrangements through intergenic recombination. The TNXB gene encodes an extracellular matrix glycoprotein, tenascin-X (TNX), and defects in TNXB cause Ehlers-Danlos syndrome. Deletions affecting both CYP21A2 and TNXB result in a contiguous gene deletion syndrome known as CAH-X syndrome. Because of the high homology between CYP21A2 and CYP21A1P, genetic testing for CAH should include an evaluation of copy number variations, as well as Sanger sequencing. Although it poses challenges for genetic testing, a large number of mutations and their associated phenotypes have been identified, which has helped to establish genotype-phenotype correlations. The genotype is helpful for guiding early treatment, predicting the clinical phenotype and prognosis, and providing genetic counseling. In particular, it can help ensure proper management of the potential complications of CAH-X syndrome, such as musculoskeletal and cardiac defects. This review focuses on the molecular pathophysiology and genetic diagnosis of 21-hydroxylase deficiency and highlights genetic testing strategies for CAH-X syndrome.

Keyword

21-hydroxylase deficiency; Congenital adrenal hyperplasia; Copy number variation

Figure

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  Fig. 1. Organization of the human RCCX module and copy number variations on chromosome 6p21. (A) Schematic representation of the RCCX module featuring 2 segments with the following gene arrangement: STK19 (formerly RP1)-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. The arrow indicates transcriptional orientation. (B) Three different RCCX module arrangements: monomodular, bimodular, and trimodular structures.

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  Fig. 2. Schematic representation of microconversion events and the location of common mutations within the CYP21A2 gene transferred by gene conversion. NC, nonclassical; SV, simple virilizing; SW, salt-wasting.

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  Fig. 3. Gene rearrangement due to unequal crossover during meiosis. (A) CYP21A2 gene rearrangement caused by a 30-kb deletion, leading to a CYP21A1P/CYP21A2 chimera. The figure illustrates the 9 types of chimera according to the different junction sites. Seven chimeras harboring the I2G mutation are associated with the classic salt-wasting phenotype, and 2 chimeras carrying a p.P31L mutation exhibit an attenuated phenotype. (B) Formation of a chimeric TNXA/TNXB gene that deletes the CYP21A2 gene. Three different chimeric genes are demonstrated, depending on the junction site.

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  Fig. 4. Classification of common mutations in CYP21A2 based on their residual enzyme activity.

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  Fig. 5. Schematic representation of a molecular genetic analysis of CYP21A2. (A) Southern blot analysis digested with TaqI restriction endonuclease. This method distinguishes the CYP21A2 (3.7 kb) and CYP21A1P (3.2 kb) genes by fragment size. (B) Sequence-specific PCR amplification of the CYP21A1P and CYP21A2 genes. Two set of primers create 4 possible pairwise reactions to obtain amplicons corresponding to CYP21A1P, CYP21A2, the fusion gene, and rearrangement products.31) (C) Sequencing for a CYP21A1P/CYP21A2 chimera using paired primers, CYP779f/Tena32F. These primers amplify the 8.5-kb fragment. The Tena32F primer is located in a nonduplicated area of TNXB in exon 32, and the CYP779f primer is complementary to the 5’-end of the CYP21A1P and CYP21A2 genes. (D) Sequencing for a TNXA/TNXB chimera using mixed paired primers. The forward primer (F1) contains complementary sequences for intron 43 of TNXA and TNXB, and the reverse primer (R2) is specific for intron 31 of TNXB.

Reference

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