The majority of mutations causing 21OHD are the consequence of intergenic recombination within the RCCX module. The high recombination rate in the HLA III region along with the nucleotide identity shared across the RCCX locus promote unequal crossing- over and gene conversions between CYP21A2 and its pseudogene. Unequal crossing overs with break points outside the CYP21A locus cause deletions of the CYP21A2 gene. Chromosomes lacking the CYP21A2 gene frequently carry a TNXB/ TNXA chimeric gene. 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, which have in the past been regarded as large gene conversions. Deletions and chimeric genes account for approximately 20– 25% of mutant alleles in 21OHD. CYP21A2 gene duplications are relatively frequent in some populations. The majority of these alleles carrying a CYP21A2 gene duplication have a p.Gln318X mutation in the du plicated CYP21A2 gene next to the TNXB gene, and a wild- type CYP21A2 gene next to the TNXA pseudogene (Figure 1a). Such alleles are non- disease- causing.
Fig1. Organization of the RCCX module at chromosome 6p21. Representative Copy Number Variants at the RCCX locus (a) non- disease variants; (b) Configurations associated with 21- hydroxylase deficiency. C4A and C4B: complement component C4A and C4B genes; CYP21A1P: steroid 21- hydroxylase pseudogene; CYP21A2: steroid 21- hydroxylase gene; TNXB: tenascin- X gene. TNXA: tenascin- X pseudogene; RP1: serine/ threonine kinase 19 gene (other names: STK19); RP2: serine/ threonine kinase 19 pseudogene (other names: STK19P); intron splice, I2G, c.293- 13A/ C>G.
Approximately 75% of CYP21A2 disease- causing mutations, including seven point mutations, a deletion of 8 base pairs in exon 3 and a cluster of three point mutations in exon 6 originate from the transfer of genetic material from the CYP21A1P pseudogene to the CYP21A2 gene (Figure 2b). In addition, over 200 pseudogene- independent mutations are listed in the Human Gene Mutation Database (HGMD, http:// www.hgmd.cf.ac.uk; August 2018) and the Pharmacogene Variation Consortium (https:// www. pharmvar.org/ gene/ CYP21A2). Most of these rare mutations are sporadic. However, due to founder effects increased frequencies of some pseudogene- independent mutations are observed in some populations.
Fig2. CYP21A2 gene locus, most common CYP21A2 gene mutations and genotype- phenotype correlation. (a) Organization of the functional CYP21A2 gene and its non- functional CYP21A1P pseudogene in the RCCX module Grey arrows indicate for the approximate position of MLPA probes. (b) Nine out of 10 common mutations are transferred by microconversions from the CYP21A1P gene into CYP21A2. Grey arrows show the five MLPA probes specific for the CYP21A2 gene and the three probes specific for the CYP21A1P pseudogene. (c) 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 salt- wasting (SW) form of 21- hydroxylase deficiency (21OHD), group B with the simple virilizing (SV) form, and group C with the non- classic (NC) form. Positive predictive values are calculated from the cited publications. The variability in the degree of virilization 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. E6 cluster refers to the p.Ile236Asn, p.Val237Glu and p.Met239Leu mutation cluster at exon 6; intron splice refers to the c.293- 13A/ C>G mutation (other names: i2G, I2G, IVS2- 13A/ C>G): Δ8bp refers to the p.Gly110ValfsX21 mutation.
Pseudogene- derived mutations occur with similar frequencies in most populations (Table 1). Deletions and chimeric genes along with the splice site mutation in intron 2 (c.293- 13A/ C>G) and p.Ile172Asn are the most common mutations in most Caucasian populations. The p.Val281Leu is by far the most common mutation detected in NCCAH patients. Novel or rare mutations account for about 3– 5% of detected mutations in large cohorts. The vast majority of these rare mutations has been identified in single families or small populations. Approximately 1– 2% of CYP21A2- inactivating mutations arise de novo. If possible, carrier testing should be performed in the parents to con firm compound heterozygosity or homozygosity in the index case and estimate the reoccurrence risk.
Table1. Allele frequency of common 21OHD mutations in different populations
