Minireview
Chimeric CYP21P/CYP21 and TNXA/TNXB genes in the RCCX module

https://doi.org/10.1016/j.ymgme.2004.09.009Get rights and content

Abstract

Two types of chimeric RCCX modules found in chromosome 6p21.3 are the chimeras CYP21P/CYP21 and TNXA/TNXB. The CYP21P-specific sequence of chimera CYP21P/CYP21 has the 5′-end in common, but differs in the 3′-end of CYP21-specific genes. The sequence organization of the gene array is C4A–CYP21P/CYP21–TNXB, whereas chimera TNXA/TNXB is caused by a CYP21 deletion, and a partial TNXB replaced by the TNXA gene shows the C4A–CYP21P–TNXA/TNXB sequence. Therefore, chimeras CYP21P/CYP21 and TNXA/TNXB are two distinct hybrid genes produced in the RCCX module in HLA class III. In addition, the haplotype of CYP21 with chimera CYP21P/CYP21 causes 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH), while chimera TNXA/TNXB is associated with Ehlers–Danols syndrome as well as CAH.

Introduction

Three categories of defective CYP21 genes in chromosome 6p21.3 in congenital adrenal hyperplasia (CAH) are: (a) small-scale conversions from CYP21P, (b) spontaneous mutations, and (c) the chimeric RCCX module including the chimeric CYP21P/CYP21 gene [1] and chimera TNXA/TNXB [2], [3], [4], [5]. To date, 77 mutations of categories a and b have been reported in the literature [6]. The RCCX module in chromosome 6p21.3 of the human MHC class III region is composed of a part of the RP gene (serine/threonine nuclear protein kinase) [7], a full-complement C4 gene, a full of CYP21 (P) gene, and a portion of the TNX gene [8], [9]. In normal individuals, the complement component C4 is coded by two genes, C4A and C4B. The size difference between these two genes is due to the presence of an endogenous retroviral sequence (6.7 kb), HERV-K, in intron 9 of the long C4 gene (long gene, 20.4 kb; short gene, 14.1 kb) [10]. The TNX gene contains tenascin-XA (TNXA) [8] and tenascin-XB (TNXB). TNXB, downstream of the CYP21 gene, is partially duplicated in the downstream of the CYP21P gene, where a truncated gene is 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. The XA, RP, CYP21 (P), C4, and TNXB genes are arranged in tandem in the order of RP1–C4A–CYP21P–XA–RP2–C4B–CYP21–TNXB and comprise the “RCCX module” on chromosome 6p21.3 (Fig. 1A). The sequence length of the gene cluster is about 120 kb (Fig. 1A). The RP1–C4A–CYP21P–XA–RP2–C4B–CYP21–TNXB gene cluster consists of a long RCCX module including parts of RP1, C4A (long), CYP21P, and XA; and a short RCCX module containing RP2, C4B (short), CYP21, and part of the TNXB gene, thus creating a bimodular form [4] (Fig. 1A). In Caucasians, the RCCX module has three possible forms: monomodular, bimodular, and trimodular; the bimodular form is the most frequently occurring form [11].

From previous studies [1], [2], [3], [4], [5], [12], [13], [14], [15], [16], [17], [18], the chimeric RCCX module with deletion of a 26 or 32 kb DNA sequence (depending on whether C4B is the long or short gene) contains eight kinds of CYP21 haplotypes with the TaqI-produced 3.2 kb fragment (Table 1) which shows a CYP21P like gene [19]. Among these, there are at least five different chimera CYP21P/CYP21s and three kinds of chimera TNXA/TNXBs produced (Table 1).

Chimera CYP21P/CYP21s (Fig. 1B), consisting of combinations of the CYP21P and CYP21 genes, have the 5′-end of the CYP21P-specific sequence in common, but differ in the 3′-end of CYP21-specific genes. This is caused by deletion of a 26 or 32 kb gene sequence (more commonly shown in the literature as being 30 kb) of 1/XCYP21P–XA–RP2–C4B–1/XCYP21 gene arrays (1/X indicates an uncertain fraction of the gene sequence) [1], [12], [13], [15], [17], [21] or large gene conversion [22] in the RCCX region to produce CYP21P/CYP21 haplotypes. However, formation of the gene sequence, C4A–CYP21P–TNXA/TNXB results from deletion of the RP2–C4B–CYP21–1/XTNXB gene array by a large, unequal gene crossover [2], [3], [4], [5], [16] in the RCCX module (1/X indicates an uncertain fraction of the gene sequence). Therefore, chimera CYP21P/CYP21 with deletion of the XA–RP2–C4B gene array (or large gene conversion) differs from chimera TNXA/TNXB containing a TNXB gene with the TNXA/TNXB hybrid and loss of the CYP21 gene. Both chimeras have a 26–32 kb deletion of the nucleotide sequence depending on whether C4B is the long (21 kb) or short (14 kb) gene in the RCCX region.

Five different chimeric CYP21P/CYP21 genes have been found and characterized in recent studies (Table 1). Three of them found in ethnic Chinese (Taiwanese) are designated CH-1, CH-2, and CH-3 [1], [14], [20]. These molecules with a deteriorated mutation of deletion 707–714delGAGACTAC lead to a frameshift mutation which forms a TGA stop codon downstream at nt 830 and produces a truncated protein in translation. CH-4, found in Caucasians [12] is probably the same one reported earlier by Killeen et al. [23], which is identical in sequence to the CYP21P gene extending to exon 1 with a mutation at P30L. Chimera CH-4 retains minor 21-hydroxylase activities resulting in a non-classic form of CAH. Chimera CH-5, with the HLA-B 47, DR7 haplotype, is caused by 30 kb deletion leaving behind the C4A and a single CYP21A-like gene [21], which is common among CAH patients of Caucasian origin. Regarding chimera TNXA/TNXB, three different chimera TNXA/TNXBs (Table 1) have been reported. From recent studies [2], [3], [4], [5], [16], CH–TNXA/TNXB-1 was found in an ethnic Chinese person with TNXB replaced by TNXA of IVS 44 [5]. CH–TNXA/TNXB-2, found in about four cases [3], [4], is a deleted TNXB with replacement of the TNXA gene extending to exon 36 with a 121 bp deletion [8]. Although the data of the recombination region analyzed by Yang et al. [3] and Koppens et al. [4] somewhat differed, the formation of haplotypes of TNXA/TNXB may be identical. In CH–TNXA/TNXB-3 [2], [4], [16], chimera TNXA/TNXB identified by Koppens et al. [4] was a deleted TNXB with replacement of the TNXA gene forward to IVS 31, which retains a restriction site of PlfMI. Two other such cases with a 30 kb deletion reported by Schalkwijk et al. [16] may be as CH–TNXA/TNXB-3 chimeras according to the data analyzed. Such a TNXA/TNXB chimera is involved in the recessive genetic disorder, Ehlers–Danlos syndrome [2], [16], in addition to 21-hydroxylase deficiency. It is common in Caucasian people [2], [4], but has never been found in any ethnic Chinese to date.

Based on PCR product analysis [14] and the Southern blot method, five different structures of chimera CYP21P/CYP21 presented a 3.2 kb fragment with the CYP21P-like gene upon digestion with TaqI [12], [13], [14], [15], whereas three types of chimera TNXA/TNXBs also showed a 3.2 kb gene of CYP21P due to loss of the CYP21 gene as described by several studies [2], [3], [4], [5], [16]. Therefore, the haplotype of the 3.2 kb CYP21 allele of chimera RCCX shows diversity [19], but is not a unique feature of the CYP21P gene.

These five CYP21P-like genes of chimera CYP21P/CYP21 are formed by unequal crossovers of the duplicate CYP21P and CYP21 genes, which produces different CYP21 haplotypes and chimera TNXA/TNXB genes with the TNXB gene replaced by different pieces of the TNXA sequence to form three distinct hybrids of TNXB genes. Although, the cause of the formation of such fused genes is unknown, studies have pointed out that the presence of sequences such as Chi-like [1], [3], [24] and minisatellite consensus [1], [13], [25] may play a role in promoting recombination in the generation of small-scale gene conversions. However, the four genes of C4, RP, CYP21, and tenascin-X are arranged in tandem in the order of RP1–C4A–CYP21P–XA–RP2–C4B–CYP21–TNXB. The high degree of sequence homology of these genes in such arrangements as the long module and short module being adjacent to each other and alternating with each other in chromosome 6p21.3 seems the most possible way to increase the chance of misalignment at meiosis to generate genetic recombinations [26] in this area.

A PCR-based amplification method was used to detect multiple gene deletions in the RCCX region [1], [5], [14], [18]. One of the PCR products is 6.2 kb and covers the 5′end of the CYP21 (nt −779) and TNXB genes at exon 36 [1]. This 6.2 kb PCR product could only be used to identify chimera CYP21P/CYP21. However, an 8.5 kb fragment [5], [18], amplified to the TNXB fraction beyond the boundary of the duplicated TNXA gene is more useful for identifying both chimeras. The 8.5 kb PCR product was combined with RFLP analysis to identify a TNXA/TNXB hybrid gene [5]. These two PCR products were successfully used to examine the altered status of the RP2–XA–C4 locus with TaqI digestion. Since the trimodular CYP21P–XA–RP2–C4B–CYP21P–XA–RP2–C4B–CYP21–TNXB may interfere with identification of a bimodule with the XA–RP2–C4B deletion and because a heterozygous deleted XA–RP2–C4B allele cannot be detected in the presence of a normal allele by the conventional Southern blot method, the results may be hard to interpret without a family study [27]. Based on studies of gene organization [1], [2], [3], [4], [5], we conclude that chimeras CYP21P/CYP21 and TNXA/TNXB in the RCCX module in chromosome 6p21.3 have two distinct hybrid genes. From the aspects of gene formation and gene structure, the RCCX chimera in the chromosome 6p21.3 includes chimeras CYP21P/CYP21 and chimera TNXA/TNXB is diversity.

Section snippets

Acknowledgment

The author thanks Drs. H.T. Chao, Y.J. Lee, F.S. Lo, M.C. Chao, D.M. Nu, S.J. Lin, F.J. Tsai, and L.P. Tsai for donating blood samples of Taiwanese CAH patients from 1994 to the present.

References (28)

  • J. Bristow et al.

    Tenascin-X: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B

    J. Cell Biol.

    (1993)
  • C.Y. Yu

    The complete exon–intron structure of a human complement component C4A gene. DNA sequences, polymorphism, and linkage to the 21-hydroxylase genes

    J. Immunol.

    (1991)
  • C.A. Blanchong et al.

    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.

    (2000)
  • D. L’Allemand et al.

    How a patient homozygous for a 30-kb deletion of the C4-CYP21 genomic region can have a nonclassic form of 21-hydroxylase deficiency

    J. Clin. Endocrinol. Metab.

    (2000)
  • Cited by (35)

    • A de novo mutation in CYP21A2 gene in a case of in vitro fertilization

      2015, Molecular Genetics and Metabolism Reports
      Citation Excerpt :

      Those two mutations were identified in only 8–12% of cases of CYP21A2 deficiency in Brazil [23–25]. CYP21A1P/A2 chimeric genes with different sequence compositions have been described depending on the recombination breakpoint [22,26]. In this paper we describe a de novo large gene conversion identified in a patient conceived by in vitro fertilization (IVF).

    • Variants of the CYP21A2 and CYP21A1P genes in congenital adrenal hyperplasia

      2013, Clinica Chimica Acta
      Citation Excerpt :

      _CYP21A2 (Fig. 3A) [29] and RP2-C4B-CYP21A2-_? _TXNB gene arrays (Fig. 3B) [29], which leads to CAH and is associated with the recessive disorder of Ehler–Danlos syndrome [40,41]. Among the three most possible forms of RCCX modules, the CYP21A1P gene does not exist in the monomodule in 17% of Caucasians according to Blanchong et al. [17].

    • Impact of Genome Complexity of the CYP21A2 Gene on Adrenal Steroidogenesis

      2012, Journal of Experimental and Clinical Medicine
      Citation Excerpt :

      Thus, the high degree of sequence homology and multiple copies of these tandem-arranged genes seem to provide a hotspot area for genetic recombination or unequal crossover. Additional CYP21A2 mutations that occurred in the P450c21-hydroxylase deficiency were found to be the formation of CYP21A1P/CYP21A2 chimeric genes, resulting from unequal crossing-over.21–23 There are different chimeric CYP21A1P/CYP21A2, including the one containing the 5′-end of the CYP21A1P sequences and the 3′-end of CYP21A2 sequences or chimera containing a hybrid of TNXB with the loss of CYP21A2, reported in CAH patients,23 demonstrating the impact of the great diversity associated with gene copy number variation and high rate of unequal recombination on the genomic stability of the CYP21 gene, thereby affecting the activity of P450c21 hydroxylase.

    • Analysis of the CYP21A1P pseudogene: Indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes

      2011, Analytical Biochemistry
      Citation Excerpt :

      As a result, the most frequent steroid hormone abnormality occurs as a CYP21A2 deficiency that causes approximately 90–95% of all CAH cases. Genetic defects of the CYP21A2 gene in CAH may commonly lead to one of two categories of (i) small-scale conversions of the CYP21A1P sequence (commonly one of 15 mutations) and (ii) chimeras of the RCCX module, including the chimeric CYP21A1P/CYP21A2 and TNXA/TNXB genes [27–29]. The CYP21A1P pseudogene belongs to the nonprocessed type [23], for which CYP21A1P transcription itself was not detected under any culture conditions or with various regulatory factors [30] solely because of two deteriorated mutations of the 707–714del and I2 splice.

    View all citing articles on Scopus
    View full text