Subjects

Genomic DNA samples were collected from 36 healthy, unrelated Hungarian subjects with European ancestry. The study protocol was approved by the Institutional Ethics Committee of Semmelweis University (local ethics committee) and was executed according to the Declaration of Helsinki principles. The subjects gave written informed consent.

Determination of RCCX CNV structures and CYP21A2 haplotypes

The two haplotypic RCCX CNV structures of a DNA sample were determined principally in the same way as described recently [13]. Briefly, a set of allele-specific long-range (ASLR) polymerase chain reactions (PCRs) [13], C4-type-specific quantitative PCRs (qPCR) [37], and HERV-K CNV allele-specific qPCR [38] were applied to the dissection of RCCX CNV. In addition to these published methods, a CYP21-type-specific qPCR was developed for the direct gene copy number determination of CYP21A1P and CYP21A2. The forward primers were CYP21-specific and the same as the primers applied to the nested-PCR (see below). The reverse primer was not allele-specific, and the Taqman minor groove binder probe (Applied Biosystems) was labeled with fluorescent dye 6-FAM (see primer and probe sequences in Table S1). RPPH1 labeled with dye VIC was used as an endogenous reference (RNase P reference assay, Applied Biosystems). Multiplex reactions were carried out in an Applied Biosystems 7500 Fast Real-Time PCR machine by TaqMan Fast Universal PCR master mix, and the reaction conditions were almost the same as that in the manufacturer's protocol, except the annealing temperature was 64 °C. To check the reliability of the Taqman based CYP21 gene copy number assay, the CYP21 qPCR primers were tested on some samples in a LightCycler 1.0 qPCR machine (Roche) by LightCycler FastStart DNA Master SYBR Green I mix using B2M and HMBS genes as references (Table S1).

To determine the CYP21A2 haplotypes, CYP21-specific nested PCRs were performed from particular ASLR-PCR products as described [13]. Using these nested PCR products, whole-gene haplotypes and genotypic alleles of full-length CYP21A2 on the CYP21A2 locus were sequenced mostly as described [13], but with some modified sequencing primers. The 5’-forward and the 3’-reverse sequencing primers (SEQ_11F and SEQ_21R) were moved to the ends of nested PCR products to extend the double-sequenced region to 3357 bp (GenBank NT_007592.15: 31946070-31949426). Two primers (SEQ_15F and SEQ_23F) which lie on some polymorphic sites of CYP21A1P were changed to ones avoiding the polymorphisms in CYP21A1P, and, finally, the SEQ_12F primer was changed to one which could cover the 5’-end of intron 2 in CYP21 genes (Table S1). The sequence calls of capillary sequencing were assembled with CLC DNA Workbench v6.5 (CLC bio) and were inspected manually by two different operators.

Because the haplotypic RCCX structures and CYP21A2 haplotypes could not be experimentally determined from all diploid combination of RCCX structures, bioinformatic haplotype reconstruction by PHASE software v2.1.1 [39,40] was applied to resolve the experimentally indeterminable haplotypic RCCX structures and CYP21A2 haplotypes from genotypic data [13]. CYP21A2 haplotype sequences encompassed the whole gene, 122 bp of 5’-flanking region (FR) and 7 bp of 3’-FR. To summarize, a human population dataset of RCCX structure-CYP21A2 haplotypes was generated from 36 unrelated European subjects by a combined molecular and inferred haplotyping approach.

Sequence and polymorphism data of CYP21 genes from great apes

The sequences of human CYP21A2 haplotype variants were used from a recent study [13], and full-length CYP21 sequences and intergenic sequences between C4 and CYP21 genes (100 bp from the 3’-end of C4 genes and 400 bp from the 5’-end of CYP21 gene to avoid the described and potential gene regulatory sites) from human leukocyte antigen (HLA) homozygous cell lines [41] (Table S2). Primate CYP21 sequences were collected from GenBank, and the status of gene (functional gene or pseudogene) was assessed (File S1). Human CYP21A1P polymorphism data was obtained from a previous study (The studied population was German [42], but Hungarians barely deviate from the German population, the European reference population (CEU) or the majority of European populations based on genome-wide polymorphisms [43,44].), but the segregating sites below 0.01 of minor allele frequency were excluded to preclude the possibility of bias, as suggested recently [45]. The polymorphism dataset from the most 3’-end of CYP21A1P (NT_007592.15: 31916635-31916691) is lacking, hence the CYP21A1P sequences from HLA-homozygous cell lines and a partial CYP21A1P sequence (GenBank: KC493621) were checked, but segregating sites were not found in this short segment. To summarize, a great ape CYP21 sequence dataset and a human CYP21A1P polymorphism dataset were compiled.

Sequence and population data analyses

All alignments were assembled by ClustalX2 v2.0.5 [46], and were edited by CLC DNA Workbench v6.5 and MEGA v5.05 [47]. The Hardy-Weinberg equilibrium and Slatkin's linearized fixation index were calculated by Arlequin v3.5 [48]. Pearson’s chi-square (χ²) and continuous Kolmogorov-Smirnov (KS) tests were calculated under the assumption of independent sites and events by STATISTICA 8 (Statsoft). Power for the χ² test was performed with G*Power v3.1.3 [49]. The MEGA v5.05 program [47] was used to construct neighbor-joining (NJ) trees with complete gap deletion and maximum likelihood (ML) trees with gap sites under the assumption that the substitutions followed the Jukes-Cantor (JC) model and uniform rates among sites. The bootstrap tests of NJ and ML trees were performed with 1000 bootstrap replications. Spatial heterogeneity in the phylogenetic signal was detected with the BootScan algorithm built in SimPlot v3.5.1 [50], using a 300 bp window size, 30 bp step size, NJ tree based on the JC model and 1000 bootstrap replications. The Robinson–Foulds tree distance metric [51] was calculated by TOPD/FMTS v3.3 software [52] in order to characterize the similarity among the tree topologies of great ape paralogues and orthologues (The metric skips branch length and bootstrap value, and it evaluates only topology.). Only one human paralogous sequence pair (DBB sequences) was taken into account, because the ambiguous relationships among human CYP21 sequences were confounding factors for this analysis (It should be noted that the genealogical network is suitable for the analysis of intraspecific sequences with high nucleotide identity [53].). 3Seq [54], BootScan [55], GENECONV [56], MaxChi [57] and RDP [58] algorithms implemented in RDP v3.44 software [59] were chosen to detect potential gene conversion events. The sequences of human CYP21 genes having high identities were removed following the instructions of RDP v3.44, and a human CYP21 sequence dataset (h08, h21, h30, h35, h41, h42, h44 (QBL), h47, h57, h58, CYP21A1P DBB and CYP21A1P PGF above a nucleotide difference threshold of 7) and a great ape CYP21 sequence dataset (h13 (MCF), h28 (COX), h37 (DBB), h44 (QBL), CYP21A1P DBB, CYP21A1P PGF and all of the chimpanzee, gorilla, orangutan and macaque CYP21 sequences above a nucleotide difference threshold of 8) were compiled. DnaSP v5.10.01 [60] was applied to perform sliding window analyses using a 300 bp window size and a 30 bp step size. This program was also utilized for assessing the expected haplotype heterozygosity (HHe), the ratio of the nonsynonymous and synonymous average number of pairwise nucleotide differences (π_A/π_S), linkage disequilibrium (LD) and neutrality tests; Tajima’s D test [61], Fu’s Fs test [62] and Fay and Wu’s H test [63] were used with the macaque CYP21A2 sequence as an outgroup if it was needed (H test relies on an outgroup.). Other neutrality tests, normalized Fay and Wu’s H (nH) test [64], Ewens-Watterson (EW) test [65] and the rejection probability of EW test were all carried out using the macaque outgroup by DH software (http://zeng-lab.group.shef.ac.uk). The rejection probabilities of D, Fs, H and nH tests under neutral model and European demography for a 5% significance level were calculated by mlcoalsim v1.42 software [66] based on a previous version of ms software [67]. The rejection probability criteria in DH and mscoalsim softwares were different [65], and the criterion of DH was used. The demography model of European population contained a bottleneck with an effective population size of 1861 from 51 thousand years to 21 thousand years before the present and an expansion from 21 thousand years (about from the retreat of the last glacial age) to the present [68-70]. The gene conversion events were also taken into account omitting the sites of minimum tract length [13] of statistically evident gene conversion events in a similar way to that was described previously [36]. The ratio of the number of nonsynonymous changes per site to the number of synonymous changes (Ka/Ks) and the McDonald-Kreitman test [71] were performed using the gorilla CYP21A2 sequences (Table S2) by DnaSP v5.10.01 (Hence there were more functional CYP21A2 coding sequences in gorillas [72] (File S1).).

Reconciling the concerted evolution of CYP21 genes in great apes

The concerted evolution of orangutan CYP21 paralogues has been verified by a phylogenetic tree (paralogues resemble each other better than their orthologues) based on partial sequence data [20], hence this phenomenon was tested on the full-length sequences of the great ape CYP21 sequence dataset. Agreeing with this, the concerted evolution of orangutan paralogues was unequivocal in both the NJ tree and the ML tree, but the CYP21A2 sequences of human and chimpanzee were separated from CYP21A1P sequences with high bootstrap values (Figure 2A, Figure S1 and S2). Gorilla paralogues resided in their own clade with a moderate bootstrap value, also indicating concerted evolution, and the intercalating position of the gorilla clade could be explained by the incomplete lineage sorting of human, chimpanzee and gorilla [73]. The average nucleotide identities of CYP21 orthologues and paralogues reflected the relationships of phylogenetic trees (Table S3). For instance, the average nucleotide identity of human and chimpanzee CYP21A2 sequences was slightly higher than that in human paralogous CYP21A2 and CYP21A1P sequences (98.42% against 97.71%), and, vice versa, CYP21A1P orthologues in humans and chimpanzee were slightly closer to each other than chimpanzee CYP21 paralogues (97.85% against 97.09%).

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Figure 2. Rooted maximum likelihood phylogenetic trees constructed from selected full-length sequences, intron 2 sequences and CYP21 gene sequences without intron 2 of the great ape CYP21 sequence dataset.

The names of CYP21 sequences of HLA-homozygous cell lines available from public databases are represented at the human sequences. Bootstrap values are shown next to the corresponding nodes, but the values within human clades are not presented for clarity. The scale bar indicates genetic distance. (A) Rooted ML tree of great ape CYP21 full-length gene. (B) Rooted ML tree of great ape CYP21 intron 2. (C) Rooted ML tree of great ape CYP21 genes without intron 2.

https://doi.org/10.1371/journal.pone.0081977.g002

Because concerted evolution is based on homogenization by gene conversion, the unbalanced sequence transfers between the sections of CYP21 paralogues could lead to subregions with different evolutionary histories. The great ape alignment was scanned by a BootScan algorithm to dissect whether the spatial heterogeneity of the phylogenetic signal (phylogenetic signal is the likelihood of closely related sequences to resemble each other more than random sequences of the same phylogenetic tree) stayed in the background of straggly phylogenetic relationships between CYP21 sequences. Surprisingly, the most equivocal phylogenetic signal of human CYP21A2 was located in a narrow region around intron 2 and was related to the human and chimpanzee CYP21A2 orthologues indicating that there was no homogenization in this region, whereas the expected signs of homogenization between human paralogues were weaker, and the peaks of the signal were dispersed (Figure 3). The similarity of this phylogenetic signal pattern was demonstrated between the CYP21A1P orthologues of human and chimpanzee, and between chimpanzee paralogues; the CYP21A1P orthologous signal was very high around intron 2, but the chimpanzee paralogous signal ceased at this section. The signals of the paralogues showed fragmented lines with high peaks in each great ape species (Figure 3, Figure S3). These paralogous signals had different spatial patterns as compared to each other implying the different histories of transitions, but the loss of signal in paralogues around intron 2 proved to be a common feature, and therefore the homogenization was absent from all intron 2 of the great ape CYP21 genes.

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Figure 3. Spatial distributions of phylogenetic signals derived from the different orthologous and paralogous pairs of the human and chimpanzee full-length CYP21 sequences.

Blue line indicates the phlyogenetic signal of human paralogues (hA2-hA1P), red dashed line indicates CYP21A2 orthologues (hA2-cA2), orange line indicates chimpanzee paralogues (cA2-cA1P) and green dashed line indicates CYP21A1P orthologues (hA1P-cA1P). The likelihood of closely related sequences to resemble each other more than random sequences of the same phylogenetic tree is expressed by ‘% of permutated trees’ in y axis. Schematic CYP21 genes are indicated below the plot, high white boxes symbolize the exons, low white boxes represent the untranslated regions, and black lines indicate the introns and flanking regions.

https://doi.org/10.1371/journal.pone.0081977.g003

The full-length CYP21 sequences were divided into two regions based on the spatial distribution of phylogenetic signals; one covered intron 2 and the other encompassed the remaining gene parts. The CYP21A2 and CYP21A1P clades were completely separated in the ML tree of intron 2 sequences, showing no signature of concerted evolution or homogenization between paralogous sequences, and the tree followed the model of divergent evolution (Figure 2B). The species in the CYP21A1P clade evolved according to the accepted lineage sorting of great apes [73], and only the positions of gorilla and orangutan sequences were vaguely resolved in the CYP21A2 clade. In the ML tree of the sequences without intron 2, the paralogues from each species were located in separated clades, confirming concerted evolution in each of them (Figure 2C). In addition, the tree topologies of CYP21 paralogues and orthologues were compared by the Robinson-Foulds metric in ML trees of the full-length gene, the intron 2 subregion and the gene without intron 2. The scale of the metric ranges from 0 to 1 (from complete concordance to complete dissimilarity). The calculated Robinson-Foulds metric was 0.57 for both whole genes vs intron 2 and the whole gene vs gene without intron 2 trees, indicating the medium similarity, whereas the metric was 1 between the trees of the intron 2 and the gene without intron 2, demonstrating the complete lack of similarity (Robinson–Foulds metric range was 0.94-0.95 with 95% confidence interval (CI) of 0.08-0.09 for 100 randomized trees).

To verify the validity of concerted evolution for the coding (cds) region and the non-coding region (non-cds) without intron 2, we further divided the sequences without intron 2 into cds and non-cds subregions, and phylogenetic analyses were performed on them. The clades of different great ape paralogues were separated from each other in both ML trees, however, some discrepancy appeared in the lineage sorting of the gorilla clade (Figure S4). The Robinson-Foulds metric was 0.86 between the cds subregion and the intron 2 subregion, demonstrating very low similarity, while the metric was 1 between the non-cds without intron 2 subregion and intron 2 subregion suggesting complete dissimilartity (for 100 random trees: 0.96 CI: 0.07-0.09). Furthermore, the ML tree of the intergenic region between C4 and CYP21 genes also showed the perfect sign of concerted evolution (Figure S4) supporting the idea that the homogenization occurred outside the genes of RCCX CNV as well as inside the CYP21 genes. The Robinson-Foulds metric was 0.29 for the cds vs. non-cds, the cds vs intergenic and non-cds vs ingergenic trees (for 100 random trees: 0.93-0.96 CI: 0.08-0.09) indicating high concordance. The lack of complete concordance presumably came from the different positions of the gorilla clade and the positions of gorilla paralogues relative to each other.

Search for statistical evidence of the gene conversion in the CYP21 sequence

We attempted to reveal the potential gene conversion events using several algorithms implemented in the RDP v3.44 software. Two non-allelic conversions confirmed by more algorithms were detected below the 0.05 significance level in the human CYP21 sequence dataset for RDP v3.44, but their exact breakpoints were undetermined. One (from CYP21A1P DBB to h42, GENECONV p=0.0453, confirmed by 3Seq p=0.0271) was nearly identical with an event of the 3’-untranslated region (UTR) covering the alleles of six adjacent segregating sites on the converted sequence (site 3080, 3102-3186) described recently [13]. The other (from CYP21A1P to h41, BootScan, p=0.0182, confirmed by GENECONV p=0.0022, 3Seq p=0.0047) was also very similar to a described event of intron 2 spanning four adjacent sites (site 624-634). In the great ape CYP21 sequence dataset for RDP v3.44, there was no detected gene conversion from chimpanzee, gorilla, orangutan and macaque CYP21 sequences to human CYP21 sequences, and there was an nearly identical non-allelic conversion event of 3’-UTR (site 3080, 3102-3186) without clear breakpoints (from CYP21A1P PGF to h37, BootScan p=0.0206, confirmed by 3Seq p=0.0037). The parallel observations of these highly similar gene conversion events in the 3’-UTR originated in one gene conversion event, according to the CYP21A2 genealogical network [13].

Spatial distribution of CYP21 polymorphisms and related indexes

A total of 33 different CYP21A2 haplotype variants were observed in the human population dataset, including the determination of a new CYP21A2 haplotype variant (h62, GenBank: KC493622; Table S4). These 33 haplotype variants encoded 6 different protein variants, which consisted of amino acid changes caused by the alleles of 4 segregating sites. If the deletion mutation (rs61338903) affecting three adjacent nucleotides and causing the loss of one amino acid in exon 1 was considered (The deletion mutations in the human population dataset of CYP21A2 haplotypes were not examined, because they would violate the majority of population genetic models that form the background of the population genetic analyses.), the number of protein variants would not change (Table S5). The validity of experimental data on haplotypic RCCX CNV structures and CYP21A2 haplotypes was checked, and only the segregating sites of the human population dataset containing a total of 64 CYP21A2 haplotypes could be consider as segregating sites, because other sites and site frequencies, which have been disclosed in a recent study [13], did not derive from a homogeneous population (File S1, Table S6).

The association of phylogeny with the different regions might reflect the uneven distribution of polymorphic sites observed along the gene (Figure 4). Hence we further investigated the spatial distribution of the polymorphic sites and related indexes using sliding window analysis. The distribution curves of the number of polymorphic sites (S) and the average number of pairwise nucleotide differences (π) calculated from the human population dataset of CYP21A2 haplotypes ran parallel to each other (Figure 5A). The curves fluctuated, had a jutting peak around intron 2, and the polymorphic sites were not distributed uniformly along the CYP21A2 gene (KS: p<0.01, χ²: p<0.0001). For the assessment of duplicated genes, the classification of polymorphic sites based on their coexistence in paralogues is a routine procedure [74]. Therefore, the sites from the datasets of human CYP21A2 haplotypes and human CYP21A1P polymorphisms were classified into four types: CYP21A2-specific and CYP21A1P-specific polymorphic sites, at which polymorphisms were observed in either of the two genes; shared sites, at which polymorphisms were shared by the two paralogues; and fixed sites, at which each paralogue had different fixed alleles (Table S7) [75]. The curves of CYP21A2-specific and fixed sites had a peak around intron 2 that was much larger than the fluctuating baseline, while the curves of CYP21A1P-specific and shared sites did not have any sharp peaks (Figure 5B). Agreeing with this, the distribution of CYP21A2-specific and fixed sites deviated significantly from uniform distribution (both sites: KS: p<0.01, χ²: p<0.0001), whereas the distribution of CYP21A1P-specific and shared sites did not deviate significantly (KS: non-significant (ns), χ²: p=0.2730 and KS: ns, χ²: p=0.1009). Some neutrality tests detecting skewness from the neutral distribution of allele frequencies in segregating sites [76] were also calculated from the population dataset of human CYP21A2 haplotypes, and plotted (Figure 5C). The curve of Tajima’s D fluctuated around the null point along the full gene, but the curve of Fay and Wu’s H dropped sharply around intron 2, indicating that the excess of high-frequency polymorphisms, and not just the excess of CYP21A2-specific and fixed sites, characterized this subregion.

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Figure 4. Scale representation of the segregating sites of human CYP21 genes.

Boxes symbolize the exons, red indicates the coding region, pink shows the untranslated regions. Segregating sites are denoted by their position, numbered from the start of the CYP21A2 coding region in the sequence NT_007592.15: 31945792-31949720. The segregating site of the CYP21A2 gene can be seen above the depicted gene. The segregating site of the CYP21A1P gene derived from an external dataset can be found below the depicted gene.

https://doi.org/10.1371/journal.pone.0081977.g004

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Figure 5. Sliding window plots of different genetic features.

Schematic CYP21 genes are indicated below the plots, high white boxes symbolize the exons, low white boxes represent the untranslated regions, and black lines indicate the introns and flanking regions. (A) Spatial distributions of the number of polymorphic sites (S) and the average number of pairwise nucleotide differences (π) calculated from the population dataset of human CYP21A2 haplotypes. (B) Spatial distributions of polymorphic site classes (fixed, shared, CYP21A2-specific, CYP21A1P-specific) based on their coexistence in the human paralogues and calculated from the datasets of human CYP21A2 haplotypes and human CYP21A1P polymorphisms. (C) Spatial distribution of Tajima’s D and Fay and Wu’s H values calculated from the human population dataset of CYP21A2 haplotypes.

https://doi.org/10.1371/journal.pone.0081977.g005

Genetic features of the whole CYP21A2, intron 2, coding and non-coding without intron 2 subregions

Taking into account the results of the phylogenetic analyses and the spatial distributions of various genetic characteristics in CYP21A2, we further used the three subregions; an intron 2 subregion, a cds subregion and a non-cds without intron 2 subregion, and assessed the population genetic features of both the whole gene and the subregions using the population dataset of human CYP21A2 haplotypes. From all 44 polymorphic sites, 12 sites were observed in intron 2, 21 in the non-cds without intron 2 subregion and 11 in the cds subregion (Table 1). Haplotype heterozygosity was close to its maximum value in the whole gene (HHe=0.949, standard deviation (sd)=0.014), and its values were also high in the different subregions, but the value in intron 2 was only slightly larger than those in the cds and non-cds subregions. As expected from the spatial distribution of the average number of pairwise nucleotide differences, intron 2 and two other subregions showed different nucleotide diversity levels (π), and intron 2 was about 4 times more diverse than the remaining part of the gene. The occurrences of the fixed, shared and CYP21A2-specific sites in the subregions highly deviated from each other. This stunning difference in the numbers of sites of the three site classes were highly significant between intron 2 and the remaining part of the gene (χ²: p=0.0055, power=1.0000), and the differences between the subregions were also statistically significant (intron 2 vs. non-cds without intron 2, χ²: p=0.0106, power=1.0000, intron 2 vs. cds, χ²: p=0.0091, power=1.0000, cds and non-cds without intron 2, χ²: p=0.0423, power=0.9989).

In neutrality analyses, the selective neutralities of the whole gene and different subregions were first examined using Tajima’s D test. The D test gave no significant values, but there was deviation between the values of the different subregions (Table 1). Rejection probabilities under both neutral and European population demography models were investigated (Table S8). To consider the gene conversion, an additional dataset of human CYP21A2 haplotypes was generated by omitting the segregating sites affected by statistically evident gene conversion events, and rejection probabilities were also examined. In contrast to D test, Fu’s Fs test and the normalized H test, based on the rejection probabilities corrected with demography, produced significant evidence for the non-neutral distribution of the observed allele frequencies in the whole gene (Table 1). The EW test was also significant under neutral model of the DH software (the EW test was also significant under Slatkin’s implementation of neutrality [77]). For the subregions, the P-values of the Fs test under demography model could be accepted as significant in all subregions, while P-values of the H and nH tests were highly significant only in the intron 2 subregion. Rejection probability values of the EW test were significant only in the non-cds without intron 2 subregion. The significances of rejection probabilities in the dataset of omitted sites affected by gene conversion were identical to those of the human population dataset of CYP21A2 haplotypes with the demography model except for the H and nH tests of the intron 2 subregion and the H test of whole gene (Table S8). In addition, the synonymous and nonsynonymous polymorphic sites were also assessed in the cds subregion; π_A/π_S was 0.288, Ka/Ks was 0.389, and the McDonald-Kreitman test did not show any deviation.