Molecular Genetics of 21‐Hydroxylase Deficiency

Abstract

Steroid 21‐hydroxylase deficiency (21OHD) accounts for approximately 95% of cases of congenital adrenal hyperplasia, one of the most common inherited metabolic disorders. It can be clinically classified into classic and nonclassic 21OHD. Classic 21OHD is associated with glucocorticoid deficiency and, in 46,XX patients, with disorder of sex development. Mineralocorticoid synthesis may also be significantly impaired in two‐thirds of patients, leading to life‐threatening salt‐wasting (SW) crises. Nonclassic 21OHD is milder and presents mainly with hyperandrogenism. 21OHD is caused by mutations in the CYP21A2 gene. The vast majority of CYP21A2 mutations are a result of unequal crossing overs and gene conversions with its highly homologous pseudogene, CYP21A1P. Molecular genetic analysis is challenging and requires the combination of different methods to detect gene deletions, chimeric genes, gene duplications and point mutations. An overall good genotype–phenotype correlation exists for the SW phenotype. Therefore, molecular diagnosis of 21OHD complements clinical diagnosis and allows for clinical and genetic counselling.

Key Concepts:

  • Steroid 21‐hydroxylase deficiency (21OHD) is an autosomal recessive disorder caused by mutations in the CYP21A2 gene and represents the most common form of congenital adrenal hyperplasia.

  • A good genotype–phenotype correlation exists for the mineralocorticoid deficiency phenotype.

  • Although a trend exists, the severity of 46,XX disorder of sex development as well as of other hyperandrogenic signs and symptoms correlates less strongly with the CYP21A2 genotype.

  • Common pseudogene‐derived mutations account for the majority of 21OHD disease‐causing alleles; these include gene deletions, chimeric genes, seven single point mutations, an 8 base‐pair deletion and a cluster of three point mutations.

  • Molecular genetic analysis of the CYP21A2 gene is challenging due to the high‐sequence homology with its pseudogene (CYP21A1P), the high rate of large rearrangements with different break points, and the presence of complex alleles carrying various mutations.

  • Some less common mutations are highly prevalent in specific populations due to founder effects. These should be considered in the genetic diagnosis of 21OHD when only screening for otherwise common CYP21A2 mutations.

  • CYP21A2 gene duplications should be considered in carriers of the p.Gln318X mutation to provide the correct molecular genetic diagnosis.

  • Molecular genetic diagnosis is essential to provide the correct diagnosis and allows for appropriate clinical and genetic counselling.

Keywords: CYP21A2; 21OHD; 21‐hydroxylase; CAH; congenital adrenal hyperplasia; adrenal insufficiency; disorder of sex development; RCCX

Figure 1.

Organisation and copy number variation at the RCCX locus at chromosome 6p21.3. Representative RCCX haplotypes non‐disease causative or associated with 21‐hydroxylase deficiency are shown.

  • C4A and C4B: complement component C4A and C4B genes/

  • 21A1P: steroid 21‐hydroxylase pseudogene (CYP21A1P)

  • 21A2: steroid 21‐hydroxylase gene (CYP21A2)

  • CYP21A1P/21A2: chimeric gene containing pseudogene‐like and CYP21A2‐like sequences at the 5′ and 3′ ends, respectively.

  • CYP21A2/21A1P: chimeric gene containing CYP21A2‐like and pseudogene‐like sequences at the 5′ and 3′ ends, respectively.

  • TNXB: tenascin‐X gene

  • TNXA: tenascin‐X pseudogene

  • TNXB/TNXA: chimeric gene containing TNXB‐like and TNXA‐like sequences at the 5′ and 3′ ends, respectively.

  • RP1: serine/threonine kinase 19 gene (other names: STK19)

  • RP2: serine/threonine kinase 19 pseudogene (other names: STK19P)

  • Δ120 bp refers to a deletion of 120 bp at intron 36 of the TNXB/TNXA chimeric gene, rendering the encoded tenascin protein non‐funtional.

  • i2G: c.293‐13A/C>G (other names: I2G, IVS2‐13A/C>G)

  • E6 cluster refers to the p.Ile236Asn, p.Val237Glu and p.Met239Leu mutation cluster at exon 6.

  • Arrangement of C4A and C4B are based on the most common haplotypes reported by Blanchong et al. ().

  • *Haplotype associated with 21OHD and type II Ehlers–Danlos syndrome.

Figure 2.

CYP21A1P/CYP21A2 chimeric genes reported in patients with 21‐hydroxylase deficiency. (a) Misalignment of chromosomes carrying different RCCX copy numbers promote unequal crossing overs within the CYP21A locus leading to the formation of CYP21A1P/CYP21A2 chimeric genes. (b) Break points in CYP21A1P/CYP21A2 chimeric genes. Black arrows denote the most common chimeric genes found in 21OHD patients. i2G refers to the c.293‐13A/C>G mutation (other names:, I2G, IVS2‐13A/C>G), Δ8 bp refers to the p.Gly110ValfsX21 mutation, E6 cluster to the p.Ile236Asn, p.Val237Glu and p.Met239Leu mutation cluster at exon 6; and insT refers to the p.Leu307PhefsX6 mutation.

Figure 3.

Localisation of the CYP21A2 gene at the RCCX module, Southern blot analysis, most common CYP21A2 gene mutations and genotype–phenotype correlation. (a) Organisation of the functional CYP21A2 gene and its non‐functional CYP21A1P pseudogene in the RCCX module. Red arrows indicate the approximate annealing site of MLPA probes. Grey arrows show direction of transcription. (b) Restriction fragment lengths after TaqI or BglII digestion of genomic DNA for Southern blot analyses. Digested DNA is used for hybridisation with probes for CYP21 genes and C4 genes. (c) Nine out of ten common mutations are transferred by gene conversions from the CYP21A1P gene into CYP21A2. Red arrows show the five MLPA probes specific for the CYP21A2 gene and the three probes specific for the CYP21A1P pseudogene. (d) Genotype–phenotype correlations in CAH due to 21‐hydroxylase deficiency based on in vitro CYP21A2 activity. Mutation groups Null and A are associated with the SW form of 21OHD, group B with the SV form, and group C with the NC form. Positive predictive values are calculated from the cited publications. The variability in the degree of virilisation of the female external genitalia in the different mutation groups (grading according to Prader genital stages) is shown in the lower panel. Modal values are provided in brackets where possible. i2G refers to the c.293‐13A/C>G mutation (other names:, I2G, IVS2‐13A/C>G), Δ8 bp refers to the p.Gly110ValfsX21 mutation, E6 cluster to the p.Ile236Asn, p.Val237Glu and p.Met239Leu mutation cluster at exon 6; and insT refers to the p.Leu307PhefsX6 mutation.

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Further Reading

Arlt W and Krone N (2007) Adult consequences of congenital adrenal hyperplasia. Hormone Research 68(suppl. 5): 158–164.

Gomes LG, Huang N, Agrawal V et al. (2008) The common P450 oxidoreductase variant A503V is not a modifier gene for 21‐hydroxylase deficiency. Journal of Clinical Endocrinology and Metabolism 93: 2913–2916.

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Parajes, Silvia, and Krone, Nils(Sep 2014) Molecular Genetics of 21‐Hydroxylase Deficiency. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0023845]