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CH-8 phenotype in steroid 21-hydroxylase deficiency: fact or fancy?

To the Editor:

The defective CYP21A2 (1) (cytochrome P450, family 21, subfamily A, polypeptide 2) genes downstream of the TNXB (tenascin XB) gene in congenital adrenal hyperplasia (CAH) (2) fall into 3 categories: (a) small-scale conversions from the CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene) gene, (b) spontaneous mutations, and (c) chimeric RCCX modules that include the chimeric CYP21A1P/CYP21A2 genes and chimeric genes of TNXA [tenascin XA (pseudogene)] and TNXB (1). Most of the CYP21A2 mutations of the 15 loci identified thus far are due to small-scale conversions from CYP21A1P during both meiosis and mitosis (2). These mutations account for approximately 70% to 80% of CAH cases. These 15 mutational loci include nucleotide (nt) -126 (OT), nt -113 (G>A), nt -110 (T>C), nt -103 (A>G), P30L, nt 655 (A/C>G), nt 707-714del, I172N, cluster E6 (1236N, V236E, and M239K), V281L, F306AL307insT, Q318X, and R356W. The chimeric CYP21A1P/CYP21A2 and TNXA/TNXB genes, which are caused by unequal crossing over (or deletions) during meiosis (2) and occur in approximately 20% of CAH alleles in most populations (1, 3) reflect, respectively, the deletion of the 1/XCYP21A1P-TNXA-RP2-C4B1/XCYP21A2 gene array [where 1/X indicates an uncertain fraction Of the gene sequence, RP2 is the retinitis pigmentosa 1 pseudogene (serine/threonine kinase 19) located on chromosome 6, and C4B is the complement component 4B (Chido blood group) gene] (1) and a deletion of the RP2-C4B-CYP21A21/XTNXB gene array (1).

I read with interest the recent report by Chen et al. (4), in which the authors described an analysis of the CYP21A1P/CYP21A2 chimeric gene with a 3.2-kb TaqI-produced fragment prepared from the amplicon amplified with the primer pair CYP779 and Tena32F (5). Chen et al. found 2 novel phenotypes of the chimeras, which they termed "CH-8" and "CH-9." According to the authors, CH-8 (Table 1) carried the 15 mutational loci in the defective CYP21A2 gene, but they obtained no data from an analysis of the 3' end, owing to a lack of variants to distinguish between CYP21A2 genes (4). A report of a prior study by Lee et al. (5) pointed out, however, that the defective CYP21A2 gene is a CYP21A1P gene that carries the 15 mutational loci. When the defective CYP21A2 gene was combined with the TNXA/ TNXB-1 chimeric form, which is due to a deletion of the RP2--C4B--CYP21A2--1/XTNXB gene array, the allele exhibited haplotypes of C4A-CYP21A1P-TNXA/TNXB (1, 5). Therefore, in my view CH-8 may be the CYP21A1P gene and should not be classified into a series of the chimeric CYP21A1P/CYP21A2 gene, because there was no further analysis of the 3' end sequence of CYP21A1P adjacent to and downstream of the TNXA gene. The TNXA/TNXB-1 chimeric formation (1) is a product that contains CYP21A1P with the 15 mutational loci and presents the polymorphic sites in the 3' end of CYP21A1P in nt 83 897 to approximately nt 83 475, which do not extend to the duplicated TNXA gene (5).

Chen et al. (4) successfully characterized 252 patients with CAH by a comprehensive molecular genetic analysis, in which the well-established CYP779/Tena32F amplicon (5) is an unequivocal strategy for detecting attenuated chimeric CYP21A1P/CYP21A2 genes and the junction site (4).I believe that a better understanding of the underlying genetic mechanisms will contribute to more-precise diagnoses. I suggest that preparing the PCR products with a full CYP21A2 gene containing the downstream sequence of the TNXB gene (5) can faultlessly and accurately detect the molecular defect in CYP21A2 gene of the RCCX module that causes the 21-hydroxylase deficiency in CAH.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

References

(1.) Lee HH. Chimeric CYP21P/CYP21 and TNXA/ TNXB genes in the RCCX module. Mol Genet Metab 2005;84:4-8.

(2.) Tusie-Luna MT, White PC. Gene conversions and unequal crossovers between CYP21 (steroid 21hydroxylase gene) and CYP21P involve different mechanisms. Proc Natl Acad Sci U S A 1995;92: 10796-800.

(3.) White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000;21:245-91.

(4.) Chen W, Xu Z, Sullivan A, Finkielstain GP, Van Ryzin C, Merke DP, McDonnell NB. Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency. Clin Chem 2012; 58:421-30.

(5.) Lee HH, Lee YJ, Lin CY. PCR-based detection of the CYP21 deletion and TNXA/TNXB hybrid in the RCCX module. Genomics 2004;83:944-50.

Hsien-Hsiung Lee *

School of Chinese Medicine

College of Chinese Medicine

China Medical University

Taichung, Taiwan

* Address correspondence to the author at: School of Chinese Medicine College of Chinese Medicine China Medical University 91 Hsueh-Shih Rd. Taichung 404, Taiwan

Fax +886-3-9389073

E-mail hhlee@mail.cmu.edu.tw or leehsienhsiung@gmail.com

Previously published online at

DOI: 10.1373/clinchem.2012.190769

In Reply

In his letter, Lee questions our classification of a group of large CYP21A2 (1) (cytochrome P450, family 21, subfamily A, polypeptide 2) deletion alleles into a new CH-8 designation because of concern that the CH-8 group we described may be the CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene) gene itself, owing to a lack of further analysis of the 3' end sequence of the CYP21 genes and the region overlapping with the TNXB gene. We argue that CH-8 is a useful chimeric classification. Although the exact junction sites were not determined, those for these CH-8 alleles were downstream of R356W, which is the very last determined CYP21A1P-specific mutation at the 3' end of the CYP21 genes.

In our study (1), we found 22 CH-8 alleles that carried the full range of CYP21A1P-specific common mutations. As Lee suggests, CH-8 alleles may represent chimeric TNXA/TNXB genes [TNXA, tenascin XA (pseudogene)]. By carrying the 120-bp deletion of the TNXA gene, a subset of these 22 CH-8 alleles are indeed chimeric TNXA/TNXB genes, but that was not a focus of our study. This subject is currently being investigated in a separate study, however.

Uncertainties remain regarding the identity of the CYP21 genes in the CH-8 alleles when the large deletions do not cross the 120-bp deletion of the TNXA gene. Lee suggests that PCR products with the full CYP21A2 gene and containing the downstream sequence of the TNXB gene might accurately detect the junction sites. Lee et al. identified a chimeric TNXA/TNXB gene in a Chinese congential adrenal hyperplasia patient via analysis of a polymorphic region based on a comparison of TNX gene reference sequences (GenBank Accession Nos. AL049547 and S38953) (2, 3). This polymorphic region is located at the end of the 3' untranslated region of the CYP21 genes and is in the last intron of the TNX genes. Eight of the polymorphic sites described by Lee et al. correspond to 8 single-nucleotide polymorphisms (SNPs) in the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/; Table 1) (2).Toad dress the possibility that these 8 SNPs are informative to identify a junction site, we sequenced the 8515-bp PCR products in 4 CH-8 carriers and 14 CH-8-negative patients. The sequencing primers are 5'-TCTCAGCTTCATTTCCG TGA-3' and 5'-AAGAACTCCA GAGCTCTGGC-3', which bind both CYP21 genes. The sequencing results revealed 4 SNPs (rs1058152, rs415620, rs451652, and rs7774739) upstream in the CYP21A2 transcriptional direction. They were distributed irregularly in 6 CH-8 -negative patients (patients 5-10) and 1 CH-8 carrier (patient 17), whereas the remaining 4 SNPs were downstream and were linked owing to their physical proximity. These SNPs were present in all CH-8 alleles and in 17.8% (5 of 28) of non-CH-8 alleles (Table 1). Thus, our data indicate that these variants are not unambiguous sites for determining junction sites in this region.

In summary, we had several reasons for simplifying the classification of CH-8. First, the junction sites of these CH-8 alleles are downstream of R356W, the very last determined CYP21A1Pspecific mutation at the 3' end of the CYP21 genes. Second, the salt-wasting type of congenital adrenal hyperplasia is the phenotype commonly associated with these CH-8 alleles. Third, there are no clinically distinguishable variants between the CYP21A1P and CYP21A2 genes at the 3' end sequence; thus, there is no need for further classification. Finally, congenital adrenal hyperplasia was the clinical focus of our report. The chimeric TNXA/TNXB genes can be further subclassified or excluded in other contexts.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: National Institutes of Health, Intramural Program.

Expert Testimony: None declared.

Patents: None declared.

References

(1.) Chen W, Xu Z, Sullivan A, Finkielstain GP, Van Ryzin C, Merke DP, et al. Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21hydroxylase deficiency. Clin Chem 2012;58:421-30.

(2.) Lee HH, Lee YJ, Lin CY. PCR-based detection of the CYP21 deletion and TNXATNXB hybrid in the RCCX module. Genomics 2004;83:944-50.

(3.) Lee HH. Chimeric CYP21P/CYP21 and TNXA TNXB genes in the RCCX module. Mol Genet Metab 2005;84:4-8.

Wuyan Chen [2] ([dagger])

Zhi Xu [2] ([dagger])

Deborah P. Merke [3,4]

Nazli B. McDonnell [2,5] *

[1] Human genes: CYP21A2, cytochrome P450, family 21, subfamily A, polypeptide 2; CYP21A1P, cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene; TNXB, tenascin XB; TNXA, tenascin XA (pseudogene).

[2] Laboratory of Clinical Investigation National Instituteon Aging Baltimore, MD

[3] National Institutes of Health Clinical Center Bethesda, MD

[4] National Institutes of Health Program in Developmental Endocrinology and Genetics (PDEGEN) Eunice Kennedy Shriver National Institute of Child Health and Human Development Bethesda, MD

[5] Clinical Research Branch National Institute on Aging National Institutes of Health Baltimore, MD

([dagger]) W. Chen and Z. Xu contributed equally to this work.

* Address correspondence to this author at: Laboratory of Clinical Investigation Biomedical Research Center National Institute on Aging 251 Bayview Blvd. Baltimore, MD 21224

Previously published online at

DOI: 10.1373/clinchem.2012.194118

[1] Human genes: CYP21A2, cytochrome P450, family 21, subfamily A, polypeptide 2; TNXB, tenascin XB; CYP21A1P, cytochrome P450, family 21, sub family A, polypeptide 1 pseudogene; TNXA, tenascin XA (pseudogene); RP2, pseudogene of retinitis pigmentosa 1 pseudogene; C4B, complement component 4B (Chido blood group).

[2] Nonstandard abbreviations: CAH, congenital adrenal hyperplasia; nt, nucleotide(s); 1/X, uncertain fraction of the gene sequence.
Table 1. Genotypes of 8 SNPs at the 3' untranslated region
of the CYP21A2 gene in 18 CAH patients.

                                      SNP (a)

                      rs1058152     rs415620 (c)   rs451652
CAH patient          (83897) (b)      (83581)       (83576)

Non-CH-8
1                       -- (d)           --           --
2                         --             --           --
3                         --             --           --
4                         --             --           --
5                         A              --           --
6                         A              --           --
7                       R (e)            --           --
8                         --             C            Ye
9                         --             --           --
10                        R              Y            --
11                        A              C            --
12                        R              Y            --
13                        R              Y            --
14                        R              Y            --

CH-8 on 1 allele
15                        R              Y            --
16                        R              Y            --
17                        R              C             Y

CH-8 on 2 alleles
18                        --             C            --

                                     SNP (a)

                     rs7774739    rs6457475 (c)   rs6457476 (c)
CAH patient           (83559)        (83509)         (83506)

Non-CH-8
1                        --            --              --
2                        --            --              --
3                        --            --              --
4                        --            --              --
5                        --            --              --
6                        --            --              --
7                        --            --              --
8                        --            --              --
9                        R             --              --
10                       R             --              --
11                       G              T               C
12                       R              Y               Y
13                       R              Y               Y
14                       R              Y               Y

CH-8 on 1 allele
15                       R              Y               Y
16                       R              Y               Y
17                       G              T               C

CH-8 on 2 alleles
18                       G              T               C

                              SNP (a)

                     rs7756934 (c)   rs7383707 (c)
CAH patient             (83485)         (83475)

Non-CH-8
1                         --              --
2                         --              --
3                         --              --
4                         --              --
5                         --              --
6                         --              --
7                         --              --
8                         --              --
9                         --              --
10                        --              --
11                         C               T
12                         Y               Y
13                         Y               Y
14                         Y               Y

CH-8 on 1 allele
15                         Y               Y
16                         Y               Y
17                         C               T

CH-8 on 2 alleles
18                         C               T

(a) SNPs are listed from left to right in the direction of
CYP21A2 transcription.

(b) Numbers in parentheses denote corresponding polymorphic sites
based on reference sequences (GenBank Accession nos. AL049547 and
S38953) [Lee et al. (2)].

(c) These SNPs are flagged as "suspected" in the dbSNP database.

(d) "--" denotes the reference sequence of the CYP21A2 gene
(ENSG00000206338).

(e) International Union of Biochemistry code: R = A/G; Y = C/T.

Table 1. Analysis of the mutational locus in CH-8 and TNXA TNXB-1.

Study                Mutations (a)   3' End sequence    Designation

                                     From nt 83 897
                                     to approximately
                                     nt 83 475 (b)

White and            15 Loci         TNXA sequence      CYP21A1P
  Speiser (3) (b)
Chen et al. (4)      15 Loci         Not done           CH-8 (?)
Lee et al. (5)       15 Loci         TNXA sequence      CYP21A1P and
                                                          TNXA/TNXB-1
                                                          formation

(a) The 15 loci include nt -126 (C>T), nt -113 (G>A), nt -110 (T>C),
nt -103 (A>G), P30L, nt 655 (A/C>G), nt 707-714del, I172N,
cluster E6 (1236N, V236E, and M239K), V281L, F306AL307insT,
Q318X, and R356W.

(b) Based on GenBank accession no. S38953.
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Title Annotation:Letters to the Editor
Author:Lee, Hsien-Hsiung
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Nov 1, 2012
Words:2216
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