The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 6 2726-2729
Copyright © 2003 by The Endocrine Society
Mutation of IVS2 –12A/C>G in Combination with 707–714delGAGACTAC in the CYP21 Gene Is Caused by Deletion of the C4-CYP21 Repeat Module with Steroid 21-Hydroxylase Deficiency
Hsien-Hsiung Lee,
Shwu-Fen Chang,
Fuu-Jen Tsai,
Li-Ping Tsai and
Ching-Yu Lin
King Car Food Industrial Co. (H.-H.L., C.-Y.L.), Yuan-Shan Research Institute, Ilan 264; Graduate Institute of Cell and Molecular Biology (S.-F.C.), Taipei Medical University, Taipei 110; Department of Medical Genetics (F.-J.T.), China Medical College Hospital, Taichung 404; and Department of Pediatrics (L.-P.T.), Taipei Municipal Women/Children’s Hospital, Taipei 100, Taiwan, Republic of China
Address all correspondence and requests for reprints to: Hsien-Hsiung Lee, M.D., King Car Food Industrial Co., Ltd., Yuan-Shan Research Institute, No. 326 Yuan-Shan Road, Section 2, Yuan-Shan, Ilan 264, Taiwan, Republic of China. E-mail: hhlee{at}ms2.kingcar.com.tw.
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Abstract
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More than 90% of the cases of congenital adrenal hyperplasia are caused by mutations of the CYP21 gene. Approximately 75% of the defective CYP21 genes are generated through intergenic recombination, termed apparent gene conversion, from the neighboring CYP21P pseudogene. Among them, mutation of the aberrant splicing donor site of IVS2 –12A/C>G at nucleotide (nt) 655 is believed to be a result derived from this mechanism and is the most prevalent case among all ethnic groups. However, mutation of 707–714delGAGACTAC rarely exists alone, although this locus is a distance of 53 nt away from IVS2 –12A/C>G. From the molecular characterization of the mutation of IVS2 –12A/C>G combined with 707–714delGAGACTAC in patients with congenital adrenal hyperplasia, we found that it appeared to be in a 3.2-rather than a 3.7-kb fragment generated by Taq I digestion in a PCR product of the CYP21 gene. Interestingly, the 5' end region of such a CYP21 haplotype had CYP21P-specific sequences. Our results indicate that the coexistence of these two mutations is caused by deletion of the CYP21P, XA, RP2, and C4B genes and intergenic recombination in the C4-CYP21 repeat module. Surprisingly, this kind of the haplotype of the mutated CYP21 gene has not been reported as a gene deletion.
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Introduction
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CONGENITAL ADRENAL HYPERPLASIA (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase gene (CYP21). More than 90% of cases of CAH are caused by mutations of the CYP21 gene (1, 2, 3). Most CYP21 mutations identified so far are attributable to conversion of DNA sequences from the neighboring duplicated nonfunctional CYP21P gene. To date, 65 different CYP21 mutations have been reported (4, 5). Among them, 15 such mutations are believed to occur in an intergenic recombination of DNA sequences between the CYP21 gene and the highly homologous CYP21P pseudogene. The other 50 mutations are spontaneous mutations. To detect mutations in the coding region or intron sequence of CYP21, many methods have been developed such as allele-specific oligonucleotide (ASO) and reverse dot-blot hybridization (6, 7) and PCR/single-strand conformational polymorphism or direct DNA sequencing (8, 9) and the amplification-created restriction site (ACRS) method (10). However, the primary PCR products of CYP21 need to be prepared by differential PCR amplification to distinguish between the CYP21 and CYP21P genes and avoid cross-contamination before beginning the mutational analysis. Most of the primary products of the CYP21 gene are derived by two/three-step PCR amplification (8, 11) in which the CYP21 gene is divided into two additional fragments of PCR products. Although this method may eliminate contamination by the CYP21P gene, a PCR product with a mutation of 707–714delGAGACTAC in the CYP21 gene may possibly be neglected because an antisense primer anchored at the nt 707–714 region is used. To resolve this, a single complete CYP21-specific amplification performed in a single mixture of TAQ/POW polymerases (Roche Diagnostics GmbH, Mannheim, Germany) in one reaction was developed (10). This PCR product contains the entire CYP21 gene, which can be cloned and characterized by an expression analysis (12).
Mutation of IVS2 –12A/C>G of the aberrant splicing donor at nucleotide (nt) 655 (13) is reported to be the most prevalent cases appearing in the CYP21 gene among all ethnic groups (3). However, mutation of 707–714delGAGACTAC rarely exists alone in CAH patients (3), although this locus is located at a distance of 53 nt (13) away from the IVS2 –12A/C>G locus. As discussed earlier (14), allele dropout occurring in detecting the IVS2 –12A/C>G mutation is due to preferential amplification of DNA segments and is an artifact caused by polymerase (15). Our previous study (16) pointed out that the allele dropout was associated with the presence of the chimeric CYP21P/CYP21, which lacks a specific primer for PCR amplification. In the present study, we used a PCR product amplified with locus-specific primers covering the tenascin B (TNXB) gene to the 5' end of CYP21 (17) to characterize the CYP21 gene containing the mutation IVS2 –12A/C>G combined with 707–714delGAGACTAC in CAH patients. We found that this kind of the haplotype of CYP21 has not previously been reported as a gene deletion in the C4-CYP21 repeat module.
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Subjects and Methods
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Subjects
As in our earlier study (18), we collected CAH families from hospitals in Taiwan as described previously (9). There were 10 CAH families with the mutations IVS2 –2A/C>G and 707–714delGAGACTAC in the CYP21 gene.
Amplification of 6.2-kb fragments
To amplify 6.2-kb PCR products encompassing the TNXB to CYP21 genes (Fig. 1
), the PCR conditions and reaction mixture were as described previously (17). The paired primers Tena36F2/CYP779f (Table 1) were used. Primer CYP779f is located at the 5' end of the CYP21P and CYP21 genes (nt 87,443–87,463, GenBank accession no. AL049547) and primer Tena36F2 was described previously (17). Primer Tena36F2 is located in TNXB in exon 36 (nt. 81,255–81,275, GenBank accession no. AL049547), a 121-bp deletion of tenascin A.
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Figure 1. A schematic diagram of the strategy for the PCR amplification of a 6.2-kb fragment encompassing the TNXB gene to the CYP21 gene. The structure of the CYP21 gene is shown by a white box; the black box represents exons of the TNXB gene. Horizontal arrows represent the direction and location of the primers (CYP779f and Tena36F2) for PCR amplification. There is a 121-bp deletion in exon 36 in the tenascin A gene marked by an asterisk.
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Cloning of the 5' end of the CYP21 gene
To examine the 5'end of the CYP21 gene, the primary 6.2-kb PCR product was used as a template for secondary PCR amplification with the paired primers CYP749f/In3R (Table 1) in a 50-µl reaction. The PCR product of 1602 bp covering the sequences of CYP21 from nt -768 to 827 (13) was generated and subcloned into the pGEM-T vector system (Promega Corp., Madison, WI). The identified clone was directly used as a template for DNA sequencing.
ACRS method
ACRS primer pairs for detection of mutations of the CYP21 gene and reaction conditions were described previously (10). ACRS primer pairs C3B/C4 used for mutational detection at IVS2 –12A/C>G and 707–714delGAGACTAC were described previously (16).
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Results
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CYP21 carrying IVS2 –12A/C>G combined with the 707–714delGAGACTAC mutations is not a chimeric CYP21P/CYP21 gene as evidenced by the absence of a mutation at P30L
A 6.2-kb PCR product (Fig. 2A, lane 1) amplified using the paired primers CYP779f/Tena36F2 was derived and analyzed by the ACRS method (10). To detect the IVS2 –12A/C>G and 707–714delGAGACTAC mutations, a 132-bp fragment from a normal individual (Fig. 2B, lane C) was generated by ACRS primers C3B/C4 (16) amplified from the 6.2-kb PCR product. However, two fragments of 123 and 103 bp were produced after SacI digestion of a 132-bp fragment on a carrier with the IVS2 –12A/C>G mutation (Fig. 2B, lane 1), whereas 123- and 96-bp fragments (Fig. 2B, lane 2) were obtained in 1 of the 10 families that carried mutations of IVS2 –12A/C>G and 707–714delGAGACTAC on one allele only (data from only one family). There was no P30L mutation found (Table 2) in any of these 10 families, indicating that CYP21 with dual mutations might not be a chimeric CYP21P/CYP21 gene (17, 19) with its 5' and 3' ends corresponding to CYP21P and CYP21.
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Figure 2. Amplification of a 6.2-kb PCR product and analysis of Taq I digestion. A, Analysis on a 0.65% agarose gel. Lane 1, A 6.2-kb PCR product amplified with pair primers CYP779f/Tena36F2 from a normal individual; lane 2, a 6.2-kb PCR product digested by Taq I from an individual with the mutation IVS2 –12A/C>G in one chromosome and a CAH patient carrying the mutations IVS2 –12A/C>G and 707–714delGAGACTAC in one chromosome, respectively. mk, A Lamda DNA-BstE II-digested marker. B, ACRS analysis on 2.5% metaphor (FMC Bioproducts, Rockland, ME). Lane 1, A 132-bp PCR product generated by ACRS primers of C3B/C4; lane 2, two bands of 132- and 103-bp fragments digested with SacI from an individual with the mutation IVS2 –12A/C>G in one chromosome; lane 3, two bands of 132- and 93-bp fragments digested with SacI from a CAH patient carrying the mutations IVS2 –12A/C>G and 707–714delGAGACTAC in one chromosome.
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The CYP21 allele with IVS2 –12A/C>G combined with 707–714delGAGACTAC mutations appears in a 3.2-kb fragment analyzed by Taq I digestion
The 6.2-kb PCR product generated by the paired primers CYP779f/Tena36F2 was directly subjected to Taq I digestion and analyzed by electrophoresis on a 0.65% agarose gel (Fig. 2A
). From the analysis of an individual with mutation of IVS2 –12A/C>G, 3.7- and 2.4-kb fragments of genuine CYP21 and the product of exon 45 (nt 83,664, GenBank accession no. AL049547) to intron 36 (nt 81,171, GenBank accession no. AL049547) of the TNXB gene were obtained, respectively (Fig. 2A, lane 2). However, a CAH patient carrying both IVS2 –12A/C>G and 707–714delGAGACTAC in one chromosome showed 3.2-, 3.7-, and 2.4-kb fragments (Fig. 2A, lane 3). This finding indicated that such a CYP21 gene with dual mutations is present on the 3.2-kb fragment analyzed by Taq I digestion, but the CYP21 gene having IVS2 –12A/C>G mutation shows up as a 3.7-kb fragment.
The 3.2-kb fragment results from deletion of the CYP21P, XA, RP2, and C4B genes in the C4-CYP repeat module, instead of a single CYP21 gene deletion
To analyze the 5' end of the mutant CYP21 allele for those 10 CAH families, a 1.6-kb PCR product amplified with the paired primers CYP749f/In3R was generated and then subcloned into a pGEM-T vector. The colony containing these two mutations was subjected to DNA sequencing analysis. The results (Table 2) showed that the 5'end of the CYP21 gene with IVS2 –12A/C>G and 707–714delGAGACTAC mutations had a CYP21P-specific base sequence from nt -103 to -306 (Table 2). Interestingly, the base at nt -4 of C appeared to be retained in the CYP21 sequence (Ref. 13 ; Table 2). Furthermore, base T at nt -209 changed to C, producing a Taq I restriction site on this defective CYP21 gene and subsequently led to a 3.2-kb fragment by Taq I analysis. All these results indicated that this CYP21 haplotype gene most probably results from deletion of the CYP21P, XA, RP2, and C4B genes in the C4-CYP21 repeat module.
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Discussion
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Nucleotide change of IVS2 –12A/C>G at nt 655 of CYP21 is the most frequent mutation in CAH among all ethnic groups (3). Our data here suggest that the CYP21 gene with the mutation IVS2 –12A/C>G is truly a case caused by intergenic recombination (containing a 3.7-kb fragment, Fig. 2A), but dual mutations of IVS2 –12A/C>G combined with 707–714delGAGACTAC (containing a 3.2-kb fragment, Fig. 2A) in the CYP21 are a case of deletion including the CYP21P, XA, RP2, and C4B genes in the C4-CYP21 repeat module. This kind of the CYP21 allele has not been reported as a gene deletion in the C4-CYP21 repeat module. This finding further emphasizes that the allele PCR dropout in a previous study (16) was in fact caused by the presence of the CYP21P sequence of the 5' end and the coexistence of the mutations IVS2 –12A/C>G and 707–714delGAGACTAC in the CYP21 gene, which are consistent with our previous suggestion (16). In addition, a base change at nt -198 from C to T (Table 2) generates an Ase I site, which produces a 9.3-kb fragment (data not shown) after digestion with AseI and NdeI as the chimeric CYP21P/CYP21 gene caused by deletion of the CYP21P, XA, RP2, and C4B genes in the C4-CYP21 repeat module (17). Because the fact that CYP21P-specific sequences present in the 5'end and the sequence at nt -4 showed C as the CYP21 gene, we suggest that recombination of the 5' end part of this haplotype of CYP21 may be initiated by a 
-like sequence of GCTGGGG (20) located at the region of nt -48 to -54. The breakpoint could be located between nt -4 and -103.
The coding frame at P10L (CTGins), L39 (TTG>CTG), and P45 (CCA>CCC) (Table 2) is a variation of polymorphic sequences appearing in the CYP21 gene (21). Because there was no change from P30 to L30, it indicated that such a haplotype of the CYP21 gene was not chimeric CYP21P/CYP21 (17, 19). Interestingly, the location between nt 395 (T>C) and S113 (TCC>TCT) (Table 2), considered to be the most polymorphic region and a hotspot of recombinations and microconversion (22), appeared to be the CYP21P gene. We suggest that this part of the CYP21 gene with the dual mutations IVS2 –12A/C>G and 707–714delGAGACTAC may be caused by intergenic recombination.
Taken together, the CYP21 allele with mutation IVS2 –12A/C>G in combination with 707–714delGAGACTAC is not a chimeric CYP21P/CYP21 gene but is similar to a chimeric one. Because this kind of CYP21 allele shows a 3.2-kb fragment by Taq I analysis; therefore, we believe that there may be various haplotypes of the CYP21 genes with the 3.2-kb fragment found in 21-hydroxylase deficiency. In addition, our PCR-based method covering the TNXB gene to the CYP21 gene is a useful tool for routine analysis and provides the status of the C4-CYP21 repeat modules in every CAH patient without having to use Southern blotting (23, 24).
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Footnotes
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This work was supported by the King Car Research Foundation from King Car Food Industrial Co. (Taiwan, Republic of China).
Abbreviations: ACRS, Amplification-created restriction site; ASO, allele-specific oligonucleotide; CAH, congenital adrenal hyperplasia; TNXB, tenascin B.
Received January 9, 2003.
Accepted March 10, 2003.
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Abstract
Introduction
