Analysis of the CYP21A2 Gene with Intergenic Recombination
and Multiple Gene Deletions in the RCCX Module
Shwu-Fen Chang1and Hsien-Hsiung Lee2
The most frequent bimodular RCCX module of the RP1-C4A-CYP21A1P-TNXA-RP2-C4B-CYP21A2-TNXB gene sequence is located on chromosome 6p21.3. To determine RCCX alterations, we used the polymerase chain reaction (PCR) product containing the tenascin B (TNXB) and CYP21A2 genes with TaqI digestion and Southern blot analysis with AseI and NdeI endonuclease digestion of genomic DNA from congenital adrenal hyperplasia patients with common mutations resulting from an intergenic conversion of CYP21A1P, such as an I2 splice, I172N, V281L, F306-L307insT, Q318X, and R356W, and dual mutations of I236N/V237E in the CYP21A2 gene. The results showed that a 3.7-kb fragment of the CYP21A2 gene was detected in each case, and 21.6- and 11.3-kb DNA fragments were found in the RCCX region by a Southern blot analysis with these corresponding muta-tions. However, the IVS2-12A/C- > G (I2 splice) haplotype in combination with the 707–714delGAGACTAC (without the P30L mutation) mutation produced a 3.2-kb TaqI fragment in the PCR product analysis and a specific 9.3-kb fragment by the Southern blot method. Therefore, we concluded that the rearrangement in the RCCX region resulting from processing of either an intergenic recombination or multiple gene deletions can be identified by the PCR analysis and Southern blot method based on a fragment-distinguishing configuration without a family study.
Introduction
M
ore than 90% of congenital adrenal hyperplasia (CAH) cases are caused by mutations of the CYP21A2 gene. To date, 133 different CYP21A2 mutations have been reported (Human Gene Mutation Database, 2010). Among them, 15 mutations (Lee, 2001) identified so far are considered to be a result of the processing of an intergenic recombination of DNA sequences from the neighboring duplicate CYP21A1P gene. The other 92 mutations are believed to be spontaneous mutations. The CYP21A2 and CYP21A1P genes are located on chromosome 6p21.3 and are adjacent to and alternate with the C4A and C4B genes that encode the fourth component of se-rum complement (White et al., 1984). This region is composed of a portion of the RP gene (serine/threonine nuclear protein kinase) (Shen et al., 1994), a full-complement C4 gene (Shen et al., 1994), a full CYP21A2 (CYP21A1P) gene, and a portion of the TNX gene (Gitelman et al., 1992; Bristow et al., 1993). The C4 protein is coded by two genes, C4A and C4B. The occur-rence of a long (20.4 kb) or a short C4 gene (14.1 kb) is due to the presence of an endogenous retroviral sequence (of 6.7 kb), named HERV-K (C4), in intron 9 (Yu, 1991). The TNX gene contains XA (TNXA) and TNXB. TNXB, located downstream of the CYP21A2 gene, is partially duplicated in thedown-stream CYP21A1P gene, where a truncated gene exists, termed TNXA. Both TNXA and TNXB are transcribed on the opposite strand. There are two RP genes, RP1 and RP2. The RP2 gene is truncated and corresponds to RP1 adjacent to TNXA. These genes are arranged in the most frequent (*69%) bimodular RCCX module of the RP1-C4-CYP21A1P-XA-RP2-C4-CYP21A2-TNXB gene sequence in Caucasians (Blanchong et al., 2001) (Fig. 1A). Therefore, the bimodule may consist of a long module including part of RP1, C4 (long), CYP21A1P, and TNXA and a short one containing RP2, C4 (short), CYP21A2, and part of the TNXB gene (Koppens et al., 2002) (Fig. 1A). In ethnic Chinese (i.e., Taiwanese), 47% of C4 genes adjacent to the RP2 gene are the short gene and 53% are the long gene (Lee et al., 2006a). All of the C4 genes adjacent to the RP1 gene examined were the long gene (Lee et al., 2006a). In addition, 87% and 85% of the C4 genes adjacent to the CYP21A1P and CYP21A2 genes, respectively, carried the C4A and C4B genes (Lee et al., 2006d). The RCCX module has three possible forms: monomodular, bimodular, and trimodular. Chromosomes with four RCCX modules are very rare (Blanchong et al., 2000).
Examining gene deletions and conversions in the RCCX module has traditionally used the gold standard of the Southern blot method with a single restriction digestion of
1Graduate Institute of Cell and Molecular Biology, Taipei Medical University, Taipei, Taiwan.
2School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan.
ª Mary Ann Liebert, Inc. Pp. 1–8
DOI: 10.1089/gtmb.2010.0080
TaqI [Fig. 1B(a)] or double digestions of BglII/EcoRI [Fig. 1B(b)]. However, those analyses are based on the absence or presence of fragments seen with hybridization using a probe (or probes) (Fig. 1B), leading to two problems. First, a het-erozygously deleted XA-RP2-C4B allele [Fig. 1B(a-1)] cannot be detected in the presence of a normal allele (Morel and Miller, 1991). Second, it is possible that an allele with the trimodular RP1-C4-CYP21A1P-XA-RP2-C4-CYP21A1P-XA-RP2-C4-CYP21A2-TNXB sequence can interfere with the identification of a bimodule with deletion of XA-RP2-C4B (Morel and Miller, 1991). Therefore, a family study should be used in these two cases to deduce the lack of an unusual configuration of the RCCX module (Gitelman et al., 1992;
Bristow et al., 1993; Shen et al., 1994; Yang et al., 1999), because a healthy carrier cannot independently be distinguished from a normal individual. To overcome these disadvantages, we developed a Southern blot method based on a ‘‘fragment-distinguishing’’ configuration (Lee et al., 2003a) using AseI and NdeI restriction endonuclease digestions, which produced two fragments of 21.6 and 11.3 kb from the genome of normal individuals. This analysis can examine the interchange region between RP1-CYP21A1P-XA-RP2-C4-CYP21A2-TNXB gene loci and is free from the aforementioned interference.
In this study, we analyzed the CYP21A2 allele with a single mutation from CYP21A1P sequences (containing 11 common mutations), such as an I2 splice (nt 655 or IVS2-12A/C > G),
I172N, V281L, F306-L307insT, R316X (Lee et al., 1998), Q318X, and R356W, or dual mutations of I236N and V237E (Lee et al., 2006b) and others in Taiwanese CAH patients, using a 6.2-kb polymerase chain reaction (PCR) product containing the partial TNXB and entire CYP21A2 genes (Lee et al., 2003c, 2006c) and a fragment-distinguishing Southern blot analysis (Lee et al., 2003a) to examine whether these mutated alleles influenced the arrangement of the RCCX region.
Materials and Methods
We collected data on families with CAH patients who were discovered during a concerted search among hospitals throughout Taiwan (Lee et al., 2008). All families requested an extensive molecular diagnosis and provided informed con-sent. The study strictly followed the guidelines of the insti-tutional review boards of the hospitals involved.
To examine the status of the CYP21A2 gene, a 6.2-kb ge-nomic fragment in which the CYP21A2 and TNXB genes were juxtaposed was amplified with the primers, CYP779f and Tena36F2 (Lee et al., 2003c, 2006c), in a PCR (Fig. 1A). Primer CYP779f (50-AGGTGGGCTGTTTTCCTTTCA-30) is located at the 50end of the CYP21A1P and CYP21A2 genes (nt 87,443–
87,463; GenBank accession no. AL049547), and the Tena36F2 (50-AGGCGCTCGCTATGAGGTGAC-30) primer is located in
exon 36 of the TNXB gene (nt 81,255–81,275; GenBank ac-cession no. AL049547), which contains a 121-bp deletion of the tenascin A gene (TNXA) (Gitelman et al., 1992). For mutational detection of the CYP21A2 gene, the 6.2-kb PCR product was used as a template for the secondary PCR amplification in the amplification-created restriction site (ACRS) primer analysis (Lee et al., 1996). The ACRS primer pairs and reaction condi-tions, including P30L, the I2 splice (IVS2-12A/C > G), I172N, the E6 cluster, V281L, F306-307insT, R316X (Lee et al. 1998), Q318X, and R356W, were described previously (Lee et al., 1996). The R483P mutation was determined by DNA se-quencing using the PCR product amplified with the S10 pri-mer pair as described previously (Lee et al., 1998). To examine the status of the CYP21A2 gene, the 6.2-kb PCR products were
digested with 10 units of TaqI enzyme in a 10 mL volume at 658C for 2 h (Lee et al., 2003c) and analyzed by electrophoresis on 0.65% agarose gels.
For the fragment-distinguishing Southern blot analysis, genomic DNA was digested with AseI and NdeI restriction endonucleases and then resolved on a 0.65% agarose gel. The protocols used for Southern blotting and the subsequent hy-bridization process were all as described in a previous study (Lee et al., 2003a). A 2301-bp fragment (nt 81,255–83,526; GenBank accession no. AL049547) was amplified with the paired primers, Tena36F2/Tena43R. Tena43R (50-CTCCCTTC
CTGACCCTCCGCT-30) is located in the TNXB and TNXA
genes (nt 83,506–83,526; GenBank accession no. AL049547) (Fig. 1A). The fragment covering the TNXB or TNXA gene was labeled with32P and used as a probe for the Southern hy-bridization analysis.
Results
To investigate intergenic recombinations and multiple gene deletions that occurred within the RCCX module in CAH patients, a 6.2-kb PCR fragment amplified with the allele-specific paired primers of CYP779f and Tena36F2 (Fig. 1A) was obtained from all CAH patients including normal indi-viduals (n > 200 chromosomes) (only one sample is shown here) (Fig. 2A, lane UC). After TaqI [T/CGA] digestion (2 TaqI sites at nt 87,404 and 83,662; GenBank accession no. AL049547) (Lee et al., 2006c), the 6.2-kb PCR product pro-duced three fragments of 3740, 2410, and 60 bp (the last one of which ran out of the gel) from one of the normal individuals (Fig. 2A, lane N). The 2410-bp fragment was derived from sequences containing the TNXB gene (nt 83,662–81,225; GenBank accession no. AL049547), whereas the 3740-bp fragment represented the CYP21A2 gene. In an analysis of a CAH patient with CYP21A2 mutations of an I2 splice in one chromosome (identified by the ACRS method; data not shown) and R483P (identified by sequencing; data not shown) in the other, the 6.2-kb PCR product produced three frag-ments of 3740, 2410, and 60 bp (the last one of which ran out of
FIG. 1. (A)Bimodular form (RP1-C4-CYP21A1P-XA-RP2-C4-CYP21A2-TNXB) of the RCCX region of chromosome 6p21.3. The white box represents the structure of the wild-type gene, whereas the black box represents a nonfunctional gene (CYP21A1P, XA, and RP2 genes) or a different functional gene (C4A). Sizes of the genes (in B) from the ATG start codon to the TGA stop codon, including RP1, C4A, CYP21A1P, XA, RP2, C4B, CYP21A2, and TNXB, in the RCCX module of the figure are based on the sequences in GenBank with accession nos. AL049547 and AF019413. The presence of C4A (the long gene of 20.4 kb) or C4B (the short gene of 14.1 kb) depends on the presence of the endogenous 6.7-kb retroviral sequence, HERV-K (C4), in intron 9 (indicated by
). The bimodular form in the C4-CYP21 area, consisting of long and short RCCX modules, is also shown. Solid arrows indicate the orientation of transcription, and dashed arrows show the locations of the primers (Tena36F2/CYP779f) for the 6.2-kb PCR amplification. A 121-bp deletion in exon 36 of the XA gene is marked with an asterisk (*). Top: Scale in kb, with the TNXB gene starting at 0. (B) Strategies for identification of the CYP21A2 gene by the gold standard of the Southern blot method digested with TaqI (a), double digestions of BglII/EcoRI (b), and the fragment-distinguishing Southern blot method using AseI/NdeI double digestions (c) probed with different gene sequences. Re-presentations of the RCCX region with deletion of XA-RP2-C4B loci analyzed by these two Southern methods are shown in (a-1), (b-1), and (c-1), respectively. The dashed line (26/32 kb long) indicates the area of XA-RP2-C4B in the RCCX region. The Southern blot analysis based on a fragment-distinguishing configuration using AseI and NdeI digestion of genomic DNA indicates two fragments of 11.3 and 21.6 kb for normal individuals and a 9.3-kb fragment produced by a deletion of XA-RP2-C4B loci in the RCCX region. Hybridization used the probe consisting of the 2301-bp PCR product derived from the paired primers, Tena36F2/Tena43R. The probe is indicated by solid bars in A. (C) Representation of four sequence organizations of the RCCX module with multiple gene deletions: (a) C4(A/B)-CYP21A2-TNXB, (b) C4A-XCYP21A2-TNXB, (c) RP1-C4A-CYP21A1P/CYP21A2-TNXB; (d) RP1-C4A-YCYP21A2-TNXB. Mutation of the IVS-12A/C > G in combination with 707– 714delGAGACTAC (without the P30L mutation) in the CYP21A2 gene (XCYP21A2) is indicated by an asterisk (*). The chimeric CYP21A1P/CYP21A2 gene is indicated by a half-white, half-black box. Duplication of the 111-bp fragment (W21-P57dup) in exon 1 of the CYP21A2 gene is indicated by a black inverted triangle (!). PCR, polymerase chain reaction.the gel) (Fig. 2A, lane 1). Further, the 6.2-kb PCR fragment amplified from CAH patients with compound heterozygosity of I172N/R316X (identified by the ACRS method; data not shown) (Fig. 2A, lane 2), I172N/Q318X (identified by the ACRS method; data not shown) (Fig. 2A, lane 3), R356W/ F306-307insT (identified by the ACRS method; data not shown) (Fig. 2A, lane 4), and I172N/I236N and V237E mu-tations (identified by the ACRS method; data not shown) (Lee et al., 2006b) (Fig. 2A, lane 5) also generated three fragments of 3740, 2410, and 60 bp (the last one of which ran out of the gel) from two defective CYP21A2 alleles. In addition, the 6.2-kb PCR fragment amplified from CAH carriers with a mutation at V281L in one chromosome (identified by the ACRS method; data not shown) (Fig. 2A, lane 6) or the mutations of H392C-V397ins (Lee et al., 1998) (Fig. 2A, lane 7) and S329-R333Cdel (Lee et al. 1998) (Fig. 2A, lane 8) produced three fragments of 3740, 2410, and 60 bp (the last one of which ran out of the gel) from one mutated CYP21A2 allele. However, the CYP21A2 gene in a CAH carrier with dual mutations of IVS2-12A/ C- > G in combination with 707–711delGAGACTAC (without the P30L mutation) in one chromosome (Lee et al., 2003c) generated two extra fragments of 3207 and 533 bp (TaqI site at nt 86,870; GenBank accession no. AL049547) (Lee et al., 2006c), in addition to the 3740-, 2410-, and 60-bp DNA fragments (Fig. 2A, lanes 9) (both the 533- and 60-bp fragments ran out of the gel). Accordingly, the appearance of the 3740-bp fragments allele(s) either in the normal individual (Fig. 1A, lane N) or in CAH patients (Fig. 1A, lanes 1–8) indicates that the
juxta-posed sequence of the CYP21A2 and TNXB genes within the RCCX module is not impaired and maintains the order of RP1-C4A-CYP21A1P-XA-RP2-C4B-CYP21A2-TNXB (Fig. 1A) (Table 1), whereas a 3207-bp fragment of the CYP21A1P-like gene detected within the CYP21A2 region implies that the defective allele with dual mutations of IVS2-12A/C- > G in combination with 707–711delGAGACTAC (without the P30L mutation) exists in the XCYP21A2 structure, and genes within this module were organized in the order of RP1-C4A-XCY-P21A2-TNXB, Fig. 1B(a-1)], which may have resulted from multiple gene deletions of the XA-RP2-C4 sequence [a 26/32-kb deletion; Fig. 1B(a-1)] (Lee et al., 2003c). Therefore, the appearance of the 3740-bp fragment presenting the intact RCCX and the 3207-bp fragment presenting an impaired RCCX by the PCR method may be used as an indicator to examine the configuration of the rearrangement in the RCCX region. We also used the PCR amplification product to analyze Taiwanese CAH patients (n ¼ 396 chromosomes; Table 2).
To further validate the observed genomic arrangement that occurred within the RCCX module in CAH patients as ana-lyzed by the PCR method, a fragment-distinguishing South-ern blot analysis was used to investigate the altSouth-ernative genomic organization. According to the genome sequences among CYP21A1P, TNXA, RP2, C4B, CYP21A2, and TNXB (Fig. 1A) from GenBank (accession nos. AF019413 and AL049547) (Fig. 1A), two DNA fragments of 21.6 and 11.3 kb [Fig. 1B(c)] were generated from the genome of normal indi-FIG. 2. (A)Analysis of the
6.2-kb PCR product by TaqI diges-tion on a 0.65% agarose gel. Lane UC is the 6.2-kb PCR product amplified with the paired primers, Tena36F2/ CYP779f, from a normal indi-vidual. Lane N is the 6.2-kb PCR product digested by TaqI from a normal individual, and lanes 1– 5 are from CAH patients with mutations of IVS2-12A/C > G/ R483P, I172N/R316X, I172N/ Q318X, R356W/F306-307insT, and I172N/I236N and V237E, respectively. Lanes 6–8 are from CAH carriers with V281L, H392C-V397ins, and S329-R333Cdel in one chromosome, respectively. Lane 9 is from a CAH carrier with dual muta-tions of IVS2-12A/C- > G in combination with 707–711del-GAGACTAC (without the P30L mutation). ‘‘mk’’ is a Lambda DNA-BstEII-digested marker (New England BioLabs). (B)
Southern blot analysis of AseI and NdeI digestion of genomic DNA. Hybridization used the probe consisting of the 2301-bp PCR product derived from the paired primers, Tena36F2/Tena43R. Lane 1 shows the genome of a normal individual who had two fragments of 11.3 and 21.6 kb, and lane 2 shows the genome of a normal individual who had one fragment of 21.6 kb. (C)Southern blot analysis of genomes of CAH patients with the mutations of IVS2-12A/C > G/R483P in lane 1, I172N/ R316X in lane 2, I172N/Q318 X in lane 3, R356W/F306-307insT in lane 4, and I172N/I236N and V237E in lane 5. Lanes 6–8 are genomes from CAH carriers with mutations of V281L, H392C-V397ins, and S330-R333Cdel in one chromosome, re-spectively. Lane 9 is genome from a CAH carrier with the dual mutations of IVS2-12A/C- > G in combination with 707– 711delGAGACTAC (without the P30L mutation). CAH, congenital adrenal hyperplasia.
viduals after cleavage with the AseI [AT/TAAT] and NdeI [CA/TATG] restriction endonucleases detected by the Southern blot analysis when probed with a fragment of the TNXA and TNNB sequences. The 21.6-kb fragment contained sequences spanning exon 16 of the C4B gene (AseI, nt 99,188; GenBank accession no. AL049547), the intergenic sequences between the C4B and CYP21A2 genes, the entire CYP21A2 gene, and the end of the TNXB gene at intron 31 (NdeI, nt 77,510; GenBank accession no. AL049547). The 11.3-kb frag-ment was composed of sequences upstream of the CYP21A1P gene (AseI, nt 86,855; GenBank accession no. AL049547) to exon 8 of the C4B genes (NdeI, nt 101,868; GenBank accession no. AL049547), which includes a truncated TNXA gene (4.5 kb) and the RP2 gene (913 bp) [Fig. 1B(c)]. From the Southern blot analysis, these two fragments were detected in most samples from healthy individuals (Fig. 2B, lane 1) (n > 50 chromosomes; data not shown) (only one sample is shown here). However, the genome from another healthy individual showed only the 21.6-kb fragment without the 11.3-kb one in two alleles (Fig. 2B, lane 2). Genomes of CAH patients with mutations of an I2 splice/R483P (Fig. 2C, lane 1), I172N/ R316X (Fig. 2C, lane 2), I172N/Q318X (Fig. 2C, lane 3), R356W/F306-307insT (Fig. 2C, lane 4), I172N/I236N, and V237E (Fig. 2C, lane 5) were detected as having both 21.6- and 11.3-kb fragments. In addition, genomes of CAH carriers with a mutation in one chromosome of V281L (Fig. 2C, lane 6), H392C-V397ins (Fig. 2C, lane 7), and S329-R333Cdel (Fig. 2C, lane 8) also displayed 21.6- and 11.3-kb fragments, whereas an additional 9.3-kb fragment (Fig. 2C, lanes 9) was detected
Table1. Status of the Proposed RCCX Module in CYP21A2 Deficiency Caused by Intergenic Recombination and Multiple Gene Deletions
Mutation locus
TaqI fragment (kb)
AseI/NdeI
fragmen t (kb) Sequence of the RCCX module
Normal individual A 3.7 21.6 þ 11.3 RP1-C4A-CYP21A1P-XA-RP2-C4B-CYP21A2-TNXB Normal individual B 3.7 21.6 RP1-C4A/B-CYP21-TNXB
Intergenic recombination 3.7 21.6 þ 11.3 RP1-C4A-CYP21A1P-XA-RP2-C4B-CYP21A2-TNXB I2 splice I172N I236N,V237Ea V281L Q318X R356W F306-L307insT
Spontaneous mutation 3.7 21.6 þ 11.3 RP1-C4A-CYP21A1P-XA-RP2-C4B-CYP21A2-TNXB H62L
IVS2 þ 1G > Ab R316Rb
S329-R333delCb
H392C-V397insb
Multiple gene deletions
Chimera CYP21A1P/CYP21A2c 3.2 9.3 RP1-C4A-CYP21A1P/CYP21A2-TNXB
IVS2–12A/C- > G combined with 707-714delGAGACTAC (without the P30Lmutation)d
3.2 9.3 RP1-C4A-XCYP21A2-TNXB
W21-P57 duplicatione 3.3 9.4 RP1-C4A-YCYP21A2-TNXB
aBased on Lee et al. (2006b). b
Based on Lee et al. (1998). cBased on Lee (2004). d
Based on Lee et al. (2003c). eBased on Lee et al. (2003b).
Table2. CYP21A2 Analysis of Taiwanese Congenital Adrenal Hyperplasia Patients Using the6.2-kb
Polymerase Chain Reaction Product with TaqI Digestion
Mutation locus Chromosome (a) Intergenic recombination
I2 splice 136
I172N 94
707-714del 1
I236N and V237E 3 I236N,V237E and M239k 1 F306-L307insT 8 Q318X 25 R356W 47 (b) Spontaneous mutation H62L 7 IVS2 þ 1G > A 5 W21-P57dup 5 C201W 1 V304M 1 R316R 3 S329-R333delC 1 H392C-V397ins 1
(c) Multiple gene deletions (or unequal cross-over recombination)
IVS2-12A/C > G in combination with 707-714del (without the P30L mutation)
26 Chimeric CYP21A1P/CY21A2 (CH-1)a 1 Chimeric CYP21A1P/CY21A2 (CH-2)a 2 Chimeric CYP21A1P/CY21A2 (CH-3)a 1 Total 369 aBased on Lee (2005).
from a CAH carrier in one of the chromosomes with dual mutations of IVS2-12A/C- > G in combination with 707– 711delGAGACTAC (without the P30L mutation). The above data indicate that gene arrangements in the RCCX module may be diverse in normal individuals in either the RP1-C4A-CYP21A1P-XA-RP2-C4B-CYP21A2-TNXB sequence of the bimodule (Fig. 1A) or the RP1-C4A/B-CYP21A2-TNXB se-quence of the monomodule [Fig. 1C(a)]. The fragment of 21.6 kb carrying the CYP21A2 gene is an essential genetic in-dicator of healthy individuals. In addition, the defective CYP21A2 allele(s) with mutations including I2 splice, I172N, V281L, Q318X, R356W, F306-307insT, and E6 cluster (I236N and V237E) or the spontaneous mutations of R316X, H392C-V397ins, and S329-R333Cdel showing the 21.6-kb fragments in the Southern blot analysis indicate a lack of disruption of the RP1-C4A-CYP21A1P-XA-RP2-C4B-CYP21A2-TNXB se-quence as in most of the healthy individual alleles. The ap-pearance of the 9.3-kb fragment, a defective CYP21A2 allele with a 26/32-kb sequence deletion (a short C4 gene or a long C4 gene) [Fig. 1B(c-1)], resulted from disruption of the RCCX region by multiple gene deletions to become the proposed RP1-C4A-XCYP21A2-TNXB genomic arrangement [Fig. 1C(b)] as in the case of IVS2-12A/C- > G in combination with the 707–711delGAGACTAC mutation (without the P30L mutation).
Discussion
The most frequent mutations of CYP21A2 deficiency in ethnic Chinese (i.e., Taiwanese) are the I2 splice (nt 655, IV2-12A/C > G), I172N, and R356W (Table 2), which show high similar incidences worldwide in different races (White and Speiser, 2000; Dain et al., 2002; Stikkelbroeck et al., 2003). The spontaneous mutations of IVS2 þ 1G > A, R316X, S329-R333delC, and H392C-V397ins (Table 1) were unique in our CAH population (Human Gene Mutation Database). The H62L mutation producing a nonclassical form (Speiser, 2008) was also present in Brazilian and Scandinavian patients (Soardi et al., 2008), and the V281L mutation, the most com-mon nonclassical disease appearing in high frequencies in patients in France, Austria, Italy, Spain, Turkey, Argentina, and Portugal (Dain et al., 2002; Stikkelbroeck et al., 2003; Friaes et al., 2006), was not found in Japanese (Koyama et al., 2002), Taiwanese (Tsai et al., 2009), or Tunisian patients (Kharrat et al., 2004). At present, six types of chimeric CYP21A1P/ CYP21A2 genes exist in different populations (Concolino et al., 2009). Interestingly, the haplotype of IVS2-12A/C > G in combination with the 707–714del mutation (without the P30L mutation) is most prevalent in ethnic Chinese CAH patients (Lee et al., 2004).
Several possibilities for the mechanism of microconver-sions were proposed, such as the existence of a chi-like se-quence (GCTGGTGG) (Smith et al., 1981) and a minisatellite consensus ( Jeffreys et al., 1985). From our examination (Table 3), three chi-like sequences upstream of intron 2 might explain how the highest frequency of the I2 splice mutation occurred in CAH patients with a 21-hydroxylase deficiency (White and Speiser, 2000). In addition, two chi-like sequences upstream of intron 4 might also account for the formation of the Q318X and R356W mutations for the greatest population next to the I2 splice mutation (White and Speiser, 2000). However, a high degree of sequence homology and the tandem repeating
order of these genes seem the most likely causes for mis-alignment to occur at meiosis, which would generate illegiti-mate genetic recombinations or unequal crossovers (Koppens et al., 2003).
Both the PCR product and fragment-distinguishing Southern blot method (Fig. 2A, B) reveal that the defective CYP21A2 allele, when processing a micro-interchange of se-quences from the CYP21A1P gene, or dual mutations of I236N/V237E, and either a 10-base deletion (S329-R333delC) or a 16-base duplication (H392C-V397ins), showed a 3.7-kb TaqI fragment as the functional CYP21A2 gene and a 21.6-kb DNA fragment of the gene array (Table 1) in the RCCX region. This indicates that the intact gene arrangement remains in the order of C4A-CYP21A1P-XA-RP2-C4B-CYP21A2-TNXB (Fig. 1A). However, the case of 111-bp duplication of W21-P57 (Table 1) presenting a 3.3-kb TaqI-PCR fragment (data not shown) and a 9.4-kb genomic fragment [Fig. 1C(d)] in our previous study (Lee et al., 2003b) imply that the genomic RCCX organization was altered to the RP1-C4A-YCYP21A2-TNXB sequence (YCYP21A2 indicating the mutated CYP21A2 haplotype with a W21-P57 duplication) [Fig. 1C(d)], which was caused by multiple gene deletions [Fig. 1C(d)]. The CYP21A2 haplotype with dual mutations of IVS2-12A/C- > G in combination with 707–711del GAGACTAC (without the P30L mutation) (Fig. 2A, lanes 9) and the chimeric CYP21A1P/ CYP21A2 genes (data not shown) (see Lee, 2004) appearing with the 3.2-kb TaqI-fragment and 9.3-kb DNA fragment re-sulted from multiple gene deletions [Fig. 1B(a-1)]. This led us to conclude that a 26/32-kb deletion of XA-RP2-C4B (Fig. 1A) generated the particular gene sequences in the order of RP1-C4A-XCYP21-TNXB [Fig. 1C(b)], RP1-C4A-YCYP21A2-TNXB [Fig. 1C(d)], and RP1-C4A-CYP21A1P/CYP21A2-TNXB [Fig. 1C(c)]. It is worth noting that genomes from healthy indi-viduals without the 11.3-kb fragment (Fig. 2B, lane 2) carrying the CYP21A1P pseudogene do not influence the physiological activity of 21-hydroxylase, because the CYP21A2 gene exists and is functional.
As described above, we can use the PCR product and the Southern blot method based on a fragment-distinguishing configuration to propose the gene order in the RCCX module
Table3. Distribution of the Chi-Like Sequence inCYP21A2 Genes CYP21A1P and CYP21A2
CYP21A1P and CYP21A2 Initiator for the chimera Location Sequence (nt)a Chi sequence: GCTGGTGG Chi-like sequences GCTGGGGC 50end 54 to 48 GCTGGGGG IVS2 393 to 400 GCTGGTCT IVS2 461 to 468 GCTGGAGG IVS2 649 to 642 GCTGGGCT IVS3 828 to 835 GCTGGGGG IVS4 1062 to 1055 GCTGGGGT IVS5 1264 to 1271 GCTGTGGG IVS6 1449 to 1441 GCTGGAGG IVS7 1921 to 1928 GCTGCTGG IVS7 1943 to 1950 GCTGGGTG 30end 2830 to 2838
after intergenic recombination and multiple gene deletions without a family study.
Acknowledgment
This work was supported by grants (NSC-93-2320-B-038-046, NSC-94-2320-B-038-052, and NSC-95-2320-B-038-044) from the National Science Council, Taiwan.
Disclosure Statements
No competing financial interests exist.
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Address correspondence to: Hsien-Hsiung Lee, B.S. School of Chinese Medicine College of Chinese Medicine China Medical University 91 Hsueh-Shih Road Taichung 404 Taiwan E-mail: [email protected]; [email protected]
