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Letters to the Editor |
1 Department of Pediatrics, Erasmus MC/Sophia, PO Box 1738, 3000 DR Rotterdam, The Netherlands
aAddress correspondence to this author at: Erasmus MC-University Medical Center Rotterdam, Laboratory of Pediatrics, Room Ee1502b, PO Box 1738, 3000 DR Rotterdam, The Netherlands. Fax 31-10-408-9486; e-mail p.koppens{at}erasmusmc.nl.
To the Editor:
We read with interest the Technical Brief by Lee et al. (1), in which the authors describe a novel method to detect C4-CYP21 deletions in patients with steroid 21-hydroxylase deficiency. Such deletions result from an unequal crossover in the RCCX module (RP-C4-CYP21-TNX) on chromosome 6. In most cases, chromosome 6 carries two RCCX modules, one with a CYP21P (CYP21A1P) pseudogene and a truncated XA pseudogene, and one with a functional CYP21 (CYP21A2) gene (encoding steroid 21-hydroxylase) and a functional TNXB gene (encoding tenascin-X). Meiotic misalignment and recombination may occur at several locations and create a chromosome with a single chimeric RCCX module. The PCR described by Lee et al. uses one primer in the 5' flanking sequence of CYP21 and CYP21P (2), whereas the other primer is positioned in a 120-bp sequence of TNXB that is not present in the XA pseudogene (3). Although this PCR is indeed suitable for the detection of chimeric CYP21P/CYP21 genes, it would fail to detect any RCCX chimera in which the pseudogene-like region includes the 120-bp deletion of XA (4), as illustrated in Fig. 1
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Lee et al. (1) successfully characterized 18 patients by this method, finding three categories of CYP21P/CYP21 chimeras. Therefore, XA/TNXB chimeras may be rare in the Chinese population they studied. In The Netherlands, however, such hybrids are common (5)(6): in our patient group, the PCR would have yielded no product in four of nine chimeric RCCX modules on bimodular chromosomes (6) as well as in a recently described de novo deletion (7). Thus, this method fails to detect all CYP21 deletions.
To amend this problem, we recommend that the TNXB-specific primer be positioned beyond the RCCX duplication boundary, in the nonduplicated area of TNXB (see Fig. 1
). This will produce three additional TaqI fragments, but these are smaller than 1 kb and should not interfere with the agarose gel separation shown in Fig. 1D
of the Technical Brief by Lee et al. (1). In addition, CYP21P/CYP21 chimeras would then produce a 2.5-kb TaqI fragment and could be readily distinguished from XA/TNXB chimeras, which would produce a 2.4-kb TaqI fragment.
Southern blotting remains the established approach for comprehensive analysis of this highly complex and variable region of the human genome. Genomic TaqI digestion coupled with cohybridization with CYP21, TNX, and C4 probes provides direct information about these three genes and alerts the investigator to uncommon configurations that require further analysis by long-range restriction mapping (8)(9). Although we recognize the benefits of rapid nonradioactive detection methods, especially for diagnostic purposes, careful evaluation of such methods is necessary, notably if the method is recommended for general use and not limited to the population for which it was originally designed.
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References
R1 King Car Food Industrial Co., Yuan-Shan Research Institute, No. 326 Yuan Shan Rd., Sec. 2, Yuan Shan, Ilan 264, Taiwan, Republic of China, Fax 886-3-9228030, E-mail hhlee{at}ms2.kingcar.com.tw
To the Editor:
In their letter, Koppens and Degenhart present an interesting and feasible recommendation extending the procedure described previously by Lee et al. (1) using a primer (REC) located in the TNXB gene to amplify a PCR product, which could identify congenital adrenal hyperplasia patients with a CYP21 deletion (2) or a hybrid TNXA/TNXB gene (3). I agree with their method for use in future studies.
In fact, the primer REC in the nonduplicated area of TNXB was designed and used for amplification in our laboratory. The sequence of the REC primer, designated Tena32F (5'-CTGTGCCTGGCTATAGCAAGC-3'), is located in intron 32 of TNXB (nucleotides 78918–78938; GenBank accession no. AL049547). A 8515-bp PCR product was amplified with paired primers CYP749f/Tena32F (Fig. 1
, lane 2). The PCR conditions and reaction mixture were as described previously (1). This PCR product will be used further to identify the CYP21 mutation with amplification-created restriction site primers (4) or digested with TaqI to examine the status of the RCCX module on a 1.2% agarose gel (1).
I believe that this rapid, nonradioactive detection method will have benefits for diagnostic purposes that will not be limited to the population originally studied.
References
The following articles in journals at HighWire Press have cited this article:
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  S. Parajes, C. Quinterio, F. Dominguez, and L. Loidi A Simple and Robust Quantitative PCR Assay to Determine CYP21A2 Gene Dose in the Diagnosis of 21-Hydroxylase Deficiency Clin. Chem., September 1, 2007; 53(9): 1577 - 1584. [Abstract] [Full Text] [PDF]
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H.-H. Lee, Y.-J. Lee, P. Chan, and C.-Y. Lin Use of PCR-Based Amplification Analysis as a Substitute for the Southern Blot Method for CYP21 Deletion Detection in Congenital Adrenal Hyperplasia Clin. Chem., June 1, 2004; 50(6): 1074 - 1076. [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
120). CYP749f, CYP21/CYP21P primer (