CYP21 and CYP21P variability in steroid 21-hydroxylase deficiency patients and in the general population in the Netherlands

Abstract

Steroid 21-hydroxylase deficiency is caused by defectiveness of the CYP21 gene. Such defects have presumably originated from interactions with the nearby CYP21P pseudogene during evolution. We studied these mechanisms by comparing the genetic variability of CYP21, CYP21P, and CYP21P/CYP21 hybrids (resulting from large-scale rearrangements) at eight mutation sites in a group of Dutch steroid 21-hydroxylase deficiency patients, their family members, and controls. The most common CYP21 defect in patients with salt-losing steroid 21-hydroxylase deficiency was a splice junction mutation in intron 2. The most common defect in the simple virilising form of the disease was ile72 → asn. CYP21P showed considerable sequence variation in its central and 3' sections; the 5' section was constant. A single nucleotide (T) insert in exon 7 was found in all CYP21P genes. During the course of evolution, this was probably the third defect introduced into CYP21P after the splice junction mutation in intron 2 and the 8 bp deletion in exon 3. Gene conversions introducing CYP21-like sequences contribute to CYP21P variability. Such an event has occurred de novo in one family. A comparison of CYP21 and CYP21P mutations on the same chromosome shows that at least some of the small-scale gene conversions that supposedly transfer defects to CYP21 involve interaction between homologous chromosomes. The majority of the putative CYP21P-CYP21 transitions in hybrid genes appears to occur in a distinct zone that lies 5' of nucleotide 2108, which is further downstream than previously hypothesised. The other transitions lie upstream of nucleotide 999. Apparent 'large-scale' CYP21-CYP21P gene conversions lead to hybrid genes that are very similar to those found in CYP21 deletions, so these haplotypes have probably resulted from a meiotic double unequal crossover.

The chimeric CYP21P/CYP21 gene and 21-hydroxylase deficiency

“…This finding further confirms that allele PCR dropout in previous PCR analyses was in fact caused by the presence of the 5' end region of the CYP21P sequence, which is consistent with our previous suggestion (Lee et al 2000). Since an antisense primer anchored at nt 707-714 of CYP21 has been used in most primary PCR amplifications (Tajima et al 1993;Day et al 1996;Koppens et al 2000) to eliminate CYP21P contamination, it may have led to PCR dropout in detecting CYP21 mutations of IVS2-12A/C>G combined with707-714delGAGAC-TAC and the chimeric CYP21P/CYP21.…”

“…There have been various studies (White et al 1988;Sinnott et al 1990;Levo and Partanen 1997;Koppens et al 2000) indicating hybrid genes with the 30-kb deletion between CYP21P and CYP21 genes in Caucasians. Several studies (Lee et al 2002;Lee et al 2003a) have shown that there are three distinct chimeric CYP21P/CYP21s in the CYP21 gene in the ethnic Chinese population in Taiwan.…”

“…Identification of the chimeric CYP21P/CYP21 gene Identification of gene deletions and conversions is still being studied by Southern blot analysis with oligonucleotides (Higashi et al 1988b;Donohoue et al 1989;Helmberg et al 1992;Tusie-Luna and White 1995) or DNA probes (Krone et al 1998;Koppens et al 2000;L'Allemand et al 2000) for hybridization. There are two established strategies of PCR amplification for identifying the CYP21 gene:…”

“…However, the population frequency is dependent on the population studied (White and Speiser 2000). Three reports (Levo and Partanen 1997;Koppens et al 2000;L'Allemand et al 2000) pointed out that such a gross 30-kb deletion consisted of a fused CYP21 gene, with its 5' and 3' ends corresponding to CYP21P and CYP21 respectively, and the product appearing as a 3.2-kb Taq I fragment in Southern blot analysis. In a recent study (Lee et al 2003a), it demonstrated that such a 30-kb gene deletion in fact is a chimeric CYP21P/CYP21 formation caused by multiple gene deletions, including XA, RP2, and C4B, and between unequal parts of the CYP21P and CYP21 genes in the C4-CYP21 repeat module (Fig.…”

“…Such a gene deletion or gene conversion is traditionally detected by Southern blotting with multiple isotopelabeled probes and RFLP analysis; Taq I generates the 3.7-kb (functional) and 3.2-kb (pseudogene) fragments, while Bgl II produces the 11-kb (functional) and 12-kb (pseudogene) fragments. These two approaches to fragment analysis have been used since 1984 (White et al 1984;Donohoue et al 1989;Koppens et al 2000;L'Allemand et al 2000). However, the method is laborious and indirect, and densitometric screening of fragments is error prone, although a nonisotopic Southern procedure was later described (Krone et al 1998).…”