Deletion of the C4-CYP21 Repeat Module Leading to the Formation of a Chimeric CYP21P/CYP21 Gene in a 9.3-kb Fragment as a Cause of Steroid 21-Hydroxylase Deficiency

Abstract

Gross gene deletions have been reported in 20% of alleles in patients with congenital adrenal hyperplasia (CAH) involving a 21-hydroxylase deficiency (1). This type of deletion occurs in the RCCX module, including the CYP21P, tenascin A (TNXA), RP2, C4B, CYP21, and tenascin B (TNXB) genes, as evidenced by a 30-kb deletion identified by pulse-field electrophoresis (2). Inactivation of the CYP21 gene may also occur through intergenic recombination with transfer of deleterious mutations from the neighboring CYP21P pseudogene. The frequency of gene deletions or conversions in CAH is controversial (3)(4)(5) and is dependent on the population studied. Evidence for gene deletions and/or conversions is traditionally obtained by Southern blot analysis. Multiple probes and separate restriction endonuclease digestions are used. TaqI generates 3.7-kb (functional) and 3.2-kb (pseudogene) fragments, and BglII produces 11-kb (functional) and 12-kb (pseudogene) fragments. These analyses have been used since 1984 (1)(3)(5)(6)(7)(8)(9). However, the method is indirect and time-consuming, and densitometry of fragments can be prone to error. To identify the interchange region and improve detection of gene deletions and conversions in the RCCX module (10)(11)(12)(13), we have developed a novel Southern blot analysis that uses two restriction endonucleases, AseI and NdeI, and requires only one probe. In addition, we use a PCR product amplified with locus-specific primers covering the TNXB gene to the 5′ end of CYP21P or CYP21 to directly analyze the 3.2/3.7-kb TaqI fragment and the status of the CYP21 gene. For the novel Southern blot analysis, 10 μg of genomic DNA was digested with the restriction endonucleases AseI and NdeI, resolved on a 0.65% agarose gel, blotted on a Hybond-N+ membrane (Amersham Pharmacia Biotech AB), hybridized with an [α-32P]dCTP-labeled probe, and autoradiographed. The blot was probed with a 2271-bp PCR product amplified with the paired primers Tena36F2/Tena43R. Tena36F2 (5′-AGGCGCTCGCTATGAGGTGAC-3′) is located in TNXB [nucleotides (nt) 81255–81257; GenBank accession no. AL049547], and Tena43R (5′-CTCCCTTCCTGACCCTCCGCT-3′) is located in TNXB and TNXA (nt 83511–83526; GenBank accession no. AL049547; Fig. 1A ). Strategy for identification of gene deletions in the RCCX module of the human histocompatibility (MHC) class III gene. (A), the wild-type gene is indicated by □; a nonfunctional gene is indicated by ▪. The probe is indicated by the black bar. The deleted region of RCCX is indicated by a solid line, which represents a 26- or 32-kb deletion. The chimeric CYP21P/CYP21 gene is indicated by ╡. Horizontal arrows indicate the direction and location of the primer for PCR amplification, and vertical arrows indicate the location of restriction endonuclease cleavage. (B), Southern blot analysis of AseI and NdeI digestion products in genomic DNA. Hybridization used the probe consisting of the 2271-bp PCR product derived by the paired primers Tena36F2/Tena43R. A wild-type individual (lane N) had two fragments of 11.3 and 21.6 kb, and 1 of 18 patients suspected of having a gene deletion (lane M) had an additional 9.3-kb fragment as well as the 11.3- and 21.6-kb fragments. (C), detection of an ACRS amplification product for the CYP21 gene from each locus, designated as (a) for P30L, (b1) for IVS-2 −12A/C→G, (c) for I172N, (d) for I236N, (g) for V281L, (j1) for Q318X, and (j2) for R356W. The structure of the CYP21 gene is indicated by □, and the CYP21P gene is indicated by ▪. Solid arrows show the mutation locus in the CYP21P gene. Dashed arrows indicate the chi-like sequence and minisatillite consensus as shown in the diagram. CH-1, CH-2, and CH-3 represent three distinct chimeric CYP21P/CYP21 genes as described in the text. (D), analysis of the TaqI-digested 6.1-kb PCR product on a 0.65% agarose gel. Lane 1, 6.1-kb PCR product from a wild-type individual amplified with paired primers Tena36F2/CYP749f; lane 2, TaqI-digested 6.1-kb PCR product from a wild-type individual; lanes 3–5, TaqI-digested 6.1-kb PCR products from patients with CH-1, CH-2, and CH-3, respectively; lane mk, BstEII-digested lambda DNA marker (New England BioLabs). For PCR amplification, genomic DNA, 0.4 U of Taq/Pow DNA polymerase (Expand Long Template PCR System; Roche Diagnostics GmbH), primers (7.5 pmol of each), deoxynucleotide triphosphates (200 μM each), and 10× PCR buffer (commercial supply) were used for PCR amplification in a 30-μL reaction. The primers Tena36F2 and CYP749f (Fig. 1A ) were used for 6.1-kb PCR product amplification encompassing the TNXB gene and the 5′ end of the CYP21 gene. The primer CYP749f (5′-ggtgctcctggtggctaaaag-3′) is located at the 5′ end of the CYP21P and CYP21 genes (nt 87412–87432; GenBank accession no. AL049547). The 6.1-kb PCR product was directly subjected to TaqI digestion and analyzed by electrophoresis on 0.65% agarose. For identification of CYP21 gene status, the PCR product was analyzed by the amplification-created restriction site (ACRS) method (14). The ACRS primer pairing and reaction conditions used for mutation detection, including P30L, IVS-2 −12A/C→G, I172N, I236N, V281L, Q318X, and R356W, have been described previously (14). The strategy for detecting a gene deletion in the RCCX module is shown in Fig. 1A . According to the sequence of the RCCX region among CYP21P, TNXA, RP2, C4B, CYP21, and TNXB obtained from GenBank (accession nos. AF019413 and AL049547) and a restriction map produced by LASERGEN Sequence Analysis Software (DNASTAR), there are two NdeI restriction sites. One is located in intron 31 of the TNXB gene [nt 77510; GenBank accession no. AL049547; Fig. 1A , (1st)] and the other is in exon 8 of the C4B gene [nt 101868; GenBank accession no. AL049547; Fig. 1A , (2nd)]. There are 11 AseI restriction sites in these regions. Nine (Fig. 1A ) are in intron 9 of the C4B long gene. However, these sites are not present in the C4A short gene (15). The other two sites are in exon 16 of the C4B gene [nt 99188; GenBank accession no. AL049547; Fig. 1A , (1st)] and in the 5′ end of the CYP21P gene [nt 86855; GenBank accession no. AL049547; Fig. 1A , (11th)]. Therefore, the distance between NdeI [TNXB; Fig. 1A , (1st)] and AseI [the 5′ end of the CYP21P gene; Fig. 1A , (11th)] is either 38.1 or 43.9 kb, depending on whether C4B is present as the long or short form (Fig. 1A ). From the map, two fragments of 21.6 and 11.3 kb were obtained by cleavage with AseI and NdeI in the region as calculated (Fig. 1A ). On probe analysis (with Tena36F2/Tena43R), two fragments of 21.6 and 11.3 kb were obtained in a wild-type individual, as expected (Fig. 1B , lane N). One patient with suspected gene deletions had an additional 9.3-kb fragment as well as the 21.6- and 11.3-kb fragments (Fig. 1B , lane M). Because the 9.3-kb fragment resulted from gene deletions from the TNXA, RP2, and C4B genes (Fig. 1A ), a 6.1-kb PCR product obtained by amplification using the paired primers Tena36F2/CYP749 (Fig. 1A ) was derived (Fig. 1D , lane 1) and used to characterize the CYP21 gene by ACRS analysis. Among 18 CAH families, three types of fused CYP21 genes were found, designated CH-1, CH-2, and CH-3 as shown in Fig. 1C . Both CH-1 and CH-2 had been characterized in a previous study (16) (Fig. 1C ). On ACRS analysis, CH-3 had mutations at P30L (Fig. 1C , a), IVS-2 −12A/C→G (Fig. 1C , b1), I172N (Fig. 1C , c), I236N (Fig. 1C , d), V281L (Fig. 1C , g), and Q318X (Fig. 1C , j1) as well as nucleotide substitutions at nt −100, −103, −110, and −123 within the promoter region, similar to those of the CYP21P gene (data not shown). There was no mutation at R356W in CH-3 (Fig. 1C , and data not shown). Therefore, these three partial CYP21P-like genes were distinct chimeric CYP21P/CYP21 genes. To examine the reaction of these three distinct chimeric molecules to TaqI digestion, the 6.1-kb PCR product was directly subjected to TaqI cleavage analysis. In a wild-type control, 3.7- and 2.4-kb fragments were obtained (Fig. 1D , lane 2). The 2.4-kb fragment is a product of exon 45 (nt 83661; GenBank accession no. AL049547) to intron 36 (nt 81171; GenBank accession no. AL049547) of the TNXA and TNXB genes. The three chimeric CYP21P/CYP21 genes, CH-1 (Fig. 1D , lane 3), CH-2 (Fig. 1D , lane 4), and CH-3 (Fig. 1D , lane 5), each contained a 3.2-kb fragment as well as a 3.7-kb fragment. We have characterized 18 CAH families (17) with a suspected deletion of the CYP21 gene by one Southern blot using AseI and NdeI cleavage (data not shown). Each had a 9.3-kb fragment generated by AseI and NdeI cleavage. The novel Southern analysis with one probe demonstrated that the 9.3-kb fragment is a product of deletion in the RCCX module, including the XA, RP2, C4B, and CYP21P/CYP21 genes (Fig. 1A ). With this method, it is clearly unnecessary to perform a Southern blot twice with both TaqI and BglII digestions. We therefore suggest that the 9.3-kb fragment is a marker indicating the existence of the chimeric CYP21P/CYP21 gene. Given the potential for variations and the lack of information about the C4B gene (18) in Chinese populations, the size of the RCCX modular deletion may be either 26 or 32 kb (Fig. 1A ). The three distinct chimeric CYP21P/CYP21 genes may result from unequal recombination between the CYP21P and CYP21 genes. We previously suggested (16) that both the chi-like sequence GCTGGGG (19) and the tandem-repetitive minisatellite consensus GGGCAGGAXG (20) might be responsible for gene recombination in the formation of CH-1 and CH-2. In the case of CH-3, a high congruity of the sequence GGGCAGGACT with GGGCAGGAXG located in intron 7 (nt 1950–1959) probably contributes to the formation of the chimera. We suggest that the base change from C to T (nt 1994) in Q318X is evidence for a breakpoint of unequal crossover in this case. In addition, we found no CYP21 gene deletions. We therefore suggest that what has been assumed to be a deletion of the CYP21 gene may in fact be a chimera. Southern blot analysis using the TaqI restriction endonuclease-generated 3.7- and 3.2-kb fragments for detection of the CYP21 deletion is time-consuming and involves the use of radioisotopes. Our method is simpler and more convenient in that the 6.1-kb fragment derived by the locus-specific primer is directly subjected to TaqI digestion and is analyzed on an agarose gel without radioisotopic hybridization. It also appears to be more accurate because all three distinct chimeric CYP21P/CYP21 genes had 3.2-kb fragments (Fig. 1C , lanes 3 and 4). Simply assuming that the 3.2-kb fragment from conventional Southern analysis (6) is a deletion of the CYP21 gene has been shown to be incorrect. The authors of one report have stated that the 3.2-kb fragment analyzed by Southern blotting identified a fused CYP21 gene, but in this study, Southern blotting was performed with radioisotope hybridization and a different endonuclease (8). In conclusion, the coexistence of 9.3- and 3.2-kb fragments is consistent with a chimeric CYP21P/CYP21 gene. The method of analysis we have described is a novel tool for identification of such a molecule. These chimeric genes were found to occur frequently in CAH caused by steroid 21-hydroxylase deficiency in a Taiwanese (ethnic Chinese) population. We thank Dr. Bun-chu Chung for helpful discussions. This work was supported by the King Car Research Foundation from King Car Food Industrial Cooperation, Taiwan, Republic of China.