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Letters to the Editor
Analysis of CYP21A1P and the duplicated CYP21A2 genes
Li-Ping Tsai, a,b and Hsien-Hsiung Lee a,c*
a
Department of Pediatrics, Buddhist Tzu Chi General Hospital, Taipei Branch, 289
Jiangguo Road, Sindian, New Taipei City 231, Taiwan. b School of Medicine, Tzu Chi
University, 701 Chung Yang Road, Sec. 3, Hualien 970, Taiwan. c School of Chinese
Medicine, College of Chinese Medicine, China Medical University, 91 Hsueh-Shih
Road, Taichung 404, Taiwan.
Keywords: CYP21A1P pseudogene; Duplicated CYP21A2.
*Corresponding author: Hsien-Hsiung Lee
School of Chinese Medicine
College of Chinese Medicine
China Medical University
91 Hsueh-Shih Road
Taichung 404, Taiwan
Tel. & Fax: 886 3 9389073
E-mail: hhlee@mail.cmu.edu.tw and
leehsienhsiung@gmail.com
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Highlights:
Two homologous genes, a functional CYP21A2 and a non-functional CYP21A1P
pseudogene, share 98% nucleotide sequence homology. The gene located downstream
of the XA gene can possibly include the CYP21A2 as well as the CYP21A1P gene.
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Abstract
The RCCX module on chromosome 6p21.3 has 3 possible forms: monomodular,
bimodular, and trimodular. Chromosomes with 4 RCCX modules are very rare. In the
monomodule, most of the CYP21A1P gene does not exist. However, haplotypes of the
RCCX module with more than one CYP21A2 gene were observed. Obviously, the
gene located downstream of the XA gene can possibly include the CYP21A2 as well as
the CYP21A1P gene.
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Background:
The major histocompatibility complex (MHC) class III region on chromosome
6p21.3 contains C4 (C4A and C4B) (Shen et al., 1994), CYP21A2 (and CYP21A1P),
RP (RP1 and RP2) (Shen et al., 1994), and tenascin (TNXA and TNXB) (Gitelman et
al., 1992; Bristow et al., 1993). This comprise the most frequent bimodular RCCX (or
the C4-CYP21 repeat) of the RP1-C4-CYP21A1P-TNXA-RP2-C4-CYP21A2-TNXB
gene sequence in 69% of alleles in Caucasians (Blanchong et al., 2000). In this region,
two homologous genes, a functional CYP21A2 and a non-functional CYP21A1P
pseudogene, share 98% nucleotide sequence homology.
Method:
Recently we read with interest the article by Cantürk et al. (Cantürk et al. 2011)
published in Clinical Chemistry, in which the authors describe an analysis of the
CYP21A1P pseudogene and CYP21A2 gene in a German population using polymerase
chain reaction (PCR) amplification and a multiple ligation-dependent probe
amplification (MLPA) analysis (Concolino et al. 2009). The PCR described by
Cantürk et al. used 2 primers in the 5’ flanking sequence specific for CYP21A2 and
CYP21A1P genes, whereas the other 2 primers are positioned in the 3’ end were
specific for 2 genes. Although this PCR is indeed suitable for detecting CYP21A2,
CYP21A1P, and the copy number of these two genes, it did find out variants via
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transfer from the CYP21A2 to CYP21A1P gene (Cantürk et al. 2011), which is the
CYP21A2-like gene with the 5’end of the CYP21A2 sequence and 3’ end of the
CYP21A1P sequence (Tsai et al. 2011).
Cantürk et al. (Cantürk et al. 2011) successfully characterized 200 German
populations by this method and found there were 40 potentially variable positions in
CYP21A2 which were conserved in CYP21A1P, and 14 CYP21A1P variants were
detected. No variants occurring via transfer from CYP21A2 to CYP21A1P gene
indicates that it may be rare or non-existent in the German population. In a recent
study of ethnic Chinese (i.e., Taiwanese) in Tsai et al. (Tsai et al. 2011) by using a 6.1
kb PCR product, however, such a CYP21A2-like gene or duplicated CYP21A2 that
existed downstream of the TNXA gene showed as a 3.7-kb TaqI-produced fragment
and carried multiple mutations which present 4 haplotypes with frequencies of 2.5%.
Moreover, in Caucasians such as Tunisian (Kharrat et al. 2011), Spanish (Parajes et al.
2008), Swedish (Wedell et al. 1994), Austria (Kleinle et al., 2009) and Dutch
populations (Koppens et al. 2000), respective frequencies were 12.5%, 7%, <2%,
13.2% and 1%, respectively (Table 1). Therefore, we question that the result implies
that German population might be “independent” in race or such a study needs to
assess by more specific method. In addition, analysis of alleles 1 and 2 of CYP21A1P
(Cantürk et al. supplemental data) of the 200 German samples in Cantürk et al.
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(Cantürk et al. 2011) indicated that frequencies of the CYP21A1P gene containing the
CYP21A2 sequence including P30, nt 707-714 (GAGACTAC), I172, V281, Q318,
and R356 loci were 4%, 0.3%, 3.6%, 31%, 8.5%, and 46%, respectively. There are
some differences with the population of ethnic Chinese (i.e., Taiwanese) in Tsai et al.
(Tsai et al. 2011) who showed frequencies of P30, V281, Q318, and R356 loci of 24%,
21%, 11%, and 34%, respectively. From these two datasets, no loci of nt 707-714
(GAGACTAC) and I172 in 2 CYP21A1P alleles were found to occur in Taiwanese.
Interestingly, the frequency of R356 was highest among these loci in both populations.
In addition, an analysis of the P30 locus by Tsai et al. (Tsai et al. 2011) point out that
the presence of a normal locus P30 (CCG) in the CYP21A1P gene was attributed to
production of L62 (CTC), and the L30 (CTG) mutation goes with H62 (CAC) in the
gene sequence (3). However, Cantürk et al. (Cantürk et al. 2011) presents a normal
locus P30 (CCG) (Allele-1, c.89, Cantürk et al. supplemental data) in the CYP21A1P
gene that go with H62 (CAC) (Allele-1, c.185, Cantürk et al. supplemental data)
rather than L62 (CTC). This shows an opposite result compared to the study by Tsai
et al. (Tsai et al. 2011). We consider that this might have been caused by data
misinterpretation between cis/trans allele of the PCR product.
Conclusion:
Tsai et al. (Tsai et al. 2011) study indicated that CYP21A2-like genes and
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duplicated CYP21A2 genes downstream of the XA gene showed a chimeric structure
with the 5’-end of the CYP21A2 structure and the CYP21A1P sequence in the 3’-end.
Therefore, identification of CYP21A2-like genes and duplicated CYP21A2 genes
using either a two-step CYP21A2 amplification (Kharrat et al. 2011; Parajes et al.
2008; Wedell et al. 1994; Kleinle et al., 2009; Koppens et al. 2000) or a allele-specific
PCR amplification of the CYP21A2 gene (Cantürk et al. 2011) may produce PCR
drop-off, because it might not be able to distinguish whether CYP21A2 exists
downstream of the XB or XA gene. Predictably, the MLPA assay might simultaneously
“catch” both the CYP21A2-like (or duplicated CYP21A2) downstream of the TNXA
and the CYP21A2 genes downstream of the TNXB. A Southern blot analysis by TaqI
digestion might also show fragments of the 3.7 kb which present the CYP21A2 gene
downstream of the TNXB gene, and 2.3 kb may be present as from the CYP21A1P
fraction in the RCCX region. Therefore, these two methods might lead to a
misinterpretation of CYP21A2-genotypeing. We believe that a better understanding of
the underlying genetic mechanisms will contribute to more-precise diagnoses. We
suggest that preparing two PCR products with a full CYP21A2 gene containing the
downstream sequence of the XB gene (Lee et al. 2011) and a full CYP21A1P gene
containing the downstream sequence of the XA gene (Tsai et al. 2011) can faultlessly
and accurately detect the molecular CYP21A2 gene of the RCCX module in CAH due
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to 21-hydroxylase deficiency.
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References
Blanchong, C.A., et al, 2000. Deficiencies of human complement component C4A
and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX (RCCX)
modules in Caucasians: the load of RCCX genetic diversity on major
histocompatibility complex-associated disease. J Exp Med 191, 2183-2196.
Bristow, J., et al, 1993. Tenascin-X: a novel extracellular matrix protein encoded by
the human XB gene overlapping P450c21B. J Cell Biol 122, 265-278.
Cantürk C, et al., 2011. Sequence analysis of CYP21A1P in a German population to
aid in the molecular biological diagnosis of congenital adrenal hyperplasia. Clin
chem 57, 511-517.
Concolino P, et al., 2009. Multiple ligation-dependent amplification (MLPA) assay for
the detection of CYP21A2 gene deletions/duplications in congenital adrenal
hyperplasia: first technical report. Clinca Chimica Acta 402, 164-170.
Gitelman, S.E., Bristow, J., Miller, W.L., 1992. Mechanism and consequences of the
duplication of the human C4/P450c21/gene X locus. Mol Cell Biol 12, 2124-2134.
Kharrat, M., et al., 2011. Detection of a frequent duplicated CYP21A2 gene carrying
a Q318X mutation in a general population with quantitative PCR method. Dign
Mol Pathol 20, 123-127.
Kleinle, S., et al., 2009. Duplications of the Functional CYP21A2 Gene Are Primarily
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Restricted to Q318X Alleles: Evidence for a Founder Effect, J. Clin. Endocrinol.
Metab. 94, 3954-3958.
Koppens, P.F.J., Hoogenboezem, T., Degenhart, H., 2000. CYP21 and CYP21P
variability in steroid 21-hydroxylase deficiency patients and in the general
population in Netherlands. Eur J Hum Genet 8, 827-836.
Lee, H.H., Lee, Y.J., Lin, C.Y., 2004. PCR-based detection of the CYP21 deletion and
TNXA/TNXB hybrid in the RCCX module. Genomics 83, 944-950.
Parajes, S., et al., 2008. High frequency of copy number variations and sequencing
variants at CYP21A2 locus: implication for the genetic diagnosis of
21-hydroxylase deficiency. Plos ONE 3, 5.e2138.
Shen, L., et al., 1994. Structure and genetics of the partially duplicated gene RP
located immediately upstream of the complement C4A and the C4B genes in the
HLA class III region. Molecular cloning, exon-intron structure, composition
retroposon, and breakpoint of gene duplication. J Biol Chem 269, 8466-8476.
Tsai, L.P., et al., 2011. Analysis of the CYP21A1P pseudogene: indication of
mutational diversity and CYP21A2-like and duplicated CYP21A2 genes. Anal
Biochem 413, 133-141.
Wedell, A., Stengler, B., Luthman, H., 1994. Characterization of mutations on the rare
duplicated C4/CYP21 haplotype in steroid 21-hydroxylase deficiency. Hum
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Genet 94, 50-54.
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Table 1. Frequency of the duplicated CYP21A2 gene in different nationalities
Detection
Duplicated CYP21A2
Frequency (%)
method
(or CYP21A2-like)
(chromosome)
Nationality
Ref.
Aa
No
0 (n = 400)
German
Cantürk et al.
Bb
Yes
2.5 (n = 200)
Taiwanese
Tsai et al.
Cc
Yes
12.5 (n =272)
Tunisian
Kharrat et al.
Dd
Yes
7 (n =288)
Spanish
Parajes et al.
Ee
Yes
<2 (n = 186)
Swedish
Wedell et al.
Ff
Yes
13.2 (n = 38)
Austria
Kleinle et al.
Gg
Yes
1 (n = 286)
Dutch
Koppens et al.
a
PCR, MLPA, and sequencing;
b
PCR, amplification-created restriction site
(ACRS), and sequencing; c PCR, real-time PCR, MLPA, and sequencing; d PCR,
real-time PCR, Southern blotting, and sequencing; e PCR, and sequencing; fPCR,
HLA typing, MLPA, and Southern blotting;
sequencing.
g
PCR, Southern blotting, and