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Link to original content: https://pubmed.ncbi.nlm.nih.gov/23241443
Intraspecific evolution of human RCCX copy number variation traced by haplotypes of the CYP21A2 gene - PubMed Skip to main page content
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. 2013;5(1):98-112.
doi: 10.1093/gbe/evs121.

Intraspecific evolution of human RCCX copy number variation traced by haplotypes of the CYP21A2 gene

Zsófia Bánlaki  1 , Julianna Anna Szabó, Ágnes Szilágyi, Attila Patócs, Zoltán Prohászka, George Füst, Márton Doleschall
Affiliations

Intraspecific evolution of human RCCX copy number variation traced by haplotypes of the CYP21A2 gene

Zsófia Bánlaki et al. Genome Biol Evol. 2013.

Abstract

The RCCX region is a complex, multiallelic, tandem copy number variation (CNV). Two complete genes, complement component 4 (C4) and steroid 21-hydroxylase (CYP21A2, formerly CYP21B), reside in its variable region. RCCX is prone to nonallelic homologous recombination (NAHR) such as unequal crossover, generating duplications and deletions of RCCX modules, and gene conversion. A series of allele-specific long-range polymerase chain reaction coupled to the whole-gene sequencing of CYP21A2 was developed for molecular haplotyping. By means of the developed techniques, 35 different kinds of CYP21A2 haplotype variant were experimentally determined from 112 unrelated European subjects. The number of the resolved CYP21A2 haplotype variants was increased to 61 by bioinformatic haplotype reconstruction. The CYP21A2 haplotype variants could be assigned to the haplotypic RCCX CNV structures (the copy number of RCCX modules) in most cases. The genealogy network constructed from the CYP21A2 haplotype variants delineated the origin of RCCX structures. The different RCCX structures were located in tight groups. The minority of groups with identical RCCX structure occurred once in the network, implying monophyletic origin, but the majority of groups occurred several times and in different locations, indicating polyphyletic origin. The monophyletic groups were often created by single unequal crossover, whereas recurrent unequal crossover events generated some of the polyphyletic groups. As a result of recurrent NAHR events, more CYP21A2 haplotype variants with different allele patterns belonged to the same RCCX structure. The intraspecific evolution of RCCX CNV described here has provided a reasonable expectation for that of complex, multiallelic, tandem CNVs in humans.

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Figures

F<sc>ig</sc>. 1.—
Fig. 1.—
Scale representation of the alignment of the RCCX variable region sequences from the external database and the localizations of the developed ASLR-PCRs. The names of cell lines and the schematic abbreviation of RCCX structures are indicated on the left side. A module is abbreviated with two letters, the first represents the alleles of HERV-K CNV (L—the long allele or S—short allele), and the second symbolizes the type of C4 gene (A or B). The duplication of these two letters indicates the bimodular structure. The alignment of the RCCX variable region has been generated from six MHC haplotype sequences of HLA-homozygous cell lines (NG_005163.2, NT_007592.15, NT_167245.1, NT_167247.1, NT_167248.1, and NT_167249.1). The alignment spans from the telomeric end of exon 4 of STK19 to the centomeric end of exon 28 of TNXB. Dashed line indicates sequence absent from the MCF cell line. The RCCX structures of cell lines are monomodular and bimodular. The variable region of bimodular RCCX contains two pairs of complete genes, complement component 4 (C4A and C4B), steroid 21-hydroxylase (CYP21A1P and CYP21A2), and two pairs of partial genes, serine/threonine kinase 19 (STK19 and STK19P) and tenascin-X (TNXA and TNXB). The CNV of the HERV-K virus sequence is located in the C4 genes. The module breakpoint of bimodular structures and the direction of the ends of chromosome 6 are indicated under the scale bar. The positions and names of ASLR-PCR primers and the length of PCR products are shown at the bottom. The names of ASLR-PCRs are abbreviated by the first letter or the C4 gene type of forward and reverse primers.
F<sc>ig</sc>. 2.—
Fig. 2.—
Results of ASLR-PCRs, allele-specific nested PCRs and qPCRs demonstrated in four samples (samples 1–4). The names of ASLR-PCRs are abbreviated by the first letter or the C4 gene type of forward and reverse primers, in alphabetical order: C4A_F-STK19P_R (A-S), C4A_F-TNXB_R (A-T), C4B_F-STK19P_R (B-S), C4B_F-TNXB_R (B-T), HERV-K_F-TNXB_R (H-T), STK19_F-C4A_R (S-A), STK19_F-C4B_R (S-B), TNXA_F-C4A_R (T-A), TNXA _F-C4B_R (T-B), and TNXA_F-HERV-K_R (T-H). The names of CYP21A1P- and CYP21A2-specific nested PCRs are abbreviated by the specific tag of CYP21 genes (A1P and A2) and the corresponding half of the gene from where the products can be amplified (5′ and 3′). The copy numbers (CN) of C4 genes and the alleles of HERV-K CNV determined by qPCRs are abbreviated by the types of C4 (A or B) and the long and short CNV alleles of HERV-K (L or S). Haplotypic RCCX module is abbreviated with two letters, the first represents the alleles of HERV-K CNV (L or S) and the second symbolizes the types of C4 gene (A or B). The multiplication of the two letters in a structure indicates the module number. For CYP21A1P- and CYP21A2-specific nested PCRs, a portion of ASLR-PCR mix containing ∼4 ng ASLR-PCR product was used as template. Genomic control confirms that the nested PCR products could not be amplified from the same amount of genomic DNA of the particular sample as the amount being included in a mix with ASLR-PCR product. PCR-negative control (nc) signifies the traditional control of PCR (complete PCR mix without DNA). HindIII digested lambda DNA (New England Biolabs) and 1 kb DNA ladder (New England Biolabs) were used as markers.
F<sc>ig</sc>. 3.—
Fig. 3.—
Graphic representation of resolved 71 haplotypic RCCX structure-CYP21A2 haplotype variants. Haplotypic RCCX structures on the left side are abbreviated with the multiplication of the two letters of a module. In a module, the first represents the alleles of HERV-K CNV (L or S) and the second symbolizes the types of C4 gene (A or B). CYP21A2 haplotype variants are on the right side, and the names of the genes at the bottom. Eleven CYP21A2 haplotype variants are connected to more than one RCCX structure. The segregating sites of CYP21A2 haplotype variants with the related haplotypic RCCX structures can be also found in supplementary table S4, Supplementary Material online (In the figure, the CYP21A2 haplotype variants are grouped by the haplotypic RCCX structure, and in supplementary table S4, haplotypic RCCX structures are grouped by the CYP21A2 haplotype variant.).
F<sc>ig</sc>. 4.—
Fig. 4.—
Genealogical haplotype networks of CYP21A2 haplotype variants (the root is abridged.). (A) Haplotype network constructed from CYP21A2 haplotype variants. Red circles indicate the (sampled) CYP21A2 haplotype variants, light gray circles show the missing intermediates, and light gray arrows symbolize the allele-state changes (character-state changes) with the positions of segregating sites and the arisen allele. Two or more allele changes belonging to adjacent segregating sites have been considered as unambiguous gene conversion events: These allele-state changes are indicated together. (B) Haplotype network with projected RCCX structures constructed from CYP21A2 haplotype variants. Light gray circles indicate the CYP21A2 haplotype variants with their names. Monomodular CNV alleles (copy number on a chromosome) are indicated by yellow, bimodular by green, and trimodular by blue. Haplotypic RCCX module is abbreviated with two letters, the first represents the alleles of HERV-K CNV (L—long allele or S—short allele), and the second symbolizes the types of C4 gene (A or B). The multiplication of the two letters in a structure indicates the module number.
F<sc>ig</sc>. 5.—
Fig. 5.—
Unequal crossover events in RCCX CNV. (A) Scale and schematic representations of a hypothetical unequal crossover event in RCCX CNV (although both haplotypic RCCX structures with corresponding CYP21A2 haplotypes exist, it is improbable that they have been generated exactly by this unequal crossover event). The name of genes in the RCCX CNV and the insertion allele of HERV-K CNV are indicated at the top. Haplotypic RCCX module is abbreviated with two letters, the first represents the alleles of HERV-K CNV (L—long allele or S—short allele), and the second symbolizes the types of C4 gene (A or B). The multiplication of the two letters in a structure indicates the module number. (B) Unequal crossover of haplotypic RCCX structures on CYP21A2 haplotype networks. Red circles indicate the unequal crossover events, orange arrows indicate the unequal crossover or consecutive mutational events, and the numbers preceded by h represent the CYP21A2 haplotypes. Numbers are assigned to particular unequal crossover events, the detailed explanations of which can be seen at the bottom. The question marks indicate the unknown RCCX polymorphisms of the parental structures. If two possibilities are present at the generation of a particular structure, the two possible breakpoints of unequal crossover are on the border of the range where the breakpoint can be located.
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