Predicting an HLA-DPB1 Expression Marker Based on Standard DPB1 Genotyping: Linkage Analysis of Over 32,000 Samples

2.1. Samples

Samples were provided by DKMS donor centers in Germany (69.3%), USA (13.2%), UK (11.9%), and Poland (5.2%) and by the BMST (Bangalore Medical Services Trust) donor center in India (0.4%) between March 2016 and May 2016. The samples approximately reflect the ethnic diversity encountered in these countries. (Detailed information about the diversity of provenance for each donor center is listed in Supplementary Material 1.) The majority of samples (98%) were collected with nylon FLOQSwabs™ hDNA free (Copan Italia Spa, Brescia, Italy) and 2% as 150 μl whole blood in Venosafe 4 ml EDTA tubes (Terumo, Tokyo, Japan). During registration, donors signed an informed consent approving HLA genotyping and other analyses to facilitate or improve donor search for stem cell transplantation. No ethics committee approval was obtained as the described genotyping is within the scope of this consent form and performed as a genotyping service.

2.2. Isolation and quantification of DNA

DNA was isolated from buccal swabs or whole blood using the magnetic-bead-based “Chemagic DNA Buccal Swab kit Special” or “Chemagic DNA Blood Kit Special” (Perkin Elmer, Baesweiler, Germany), respectively. The isolated DNA was eluted in 100 μl elution buffer (10 mM Tris-HCl pH8.0), and its concentration was measured by fluorescence (SYBR Green, Biozym, Hessisch Oldendorf, Germany) using the TECAN infinite 200Pro plate reader (Tecan, Männedorf, Switzerland). Samples with DNA concentrations of < 2 ng/μl were excluded from genotyping.

2.3. Amplicon-based sequencing of HLA-DPB1 exons 2 and 3 and rs9277534

2.4. Whole-gene sequencing of HLA-DPB1

Samples with unexpected linkage patterns or otherwise inconclusive results after primary analysis were selected for secondary analysis on a Pacific Biosciences RS II instrument (Pacific Biosciences, Menlo Park, USA) as previously described [15]. To this end, the entire DPB1 gene, inclusive of 5′ and 3′ UTRs, was amplified by long-range PCR (Fig. 1C). A barcoded sequencing library was prepared using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences, Menlo Park, USA) following standard protocols. Base calling and de-multiplexing was performed using Pacific Biosciences’ SMRT Portal. The whole-gene sequence data was analyzed with NGSengine (GenDx, Utrecht, The Netherlands).

2.5. Linkage verification of samples with phasing ambiguities

Due to the lack of physical phasing between exons 2 and 3 during primary analysis, some of the DPB1 genotyping results contained phasing ambiguities. These samples are characterized by having two distinct sequences in exons 2 and 3 each, of which each combination has been reported as allele in the IPD-IMGT/HLA database. Such genotyping results are described by NMDP codes leading to a loss of information. For example, the genotyping result DPB1*04:ADCGE (including DPB1*04:01 and DPB1*350:01)/DPB1*17:JECX (including DPB1*17:01 and DPB1*131:01) could represent a) DPB1*04:01/DPB1*17:01; b) DPB1*04:01/DPB1*131:01; c) DPB1*350:01/DPB1*17:01, or d) DPB1*350:01/DPB1*131:01. However, DPB1*04:01 and DPB1*17:01 and likewise DPB1*350:01 and DPB1*131:01 share the same exon 3 sequences. Therefore, neither a) nor d) would correctly reflect the genotype of the sample. On the other hand, samples actually harboring these allele combinations would not result in NMDP codes, but in straightforward two-field resolution genotyping (DPB1*04:01/DPB1*17:01 or DPB1*350:01/DPB1*131:01). Therefore, all samples with phasing ambiguities were resolved and only the valid allele combinations were used to assign the expected rs9277534 alleles for the linkage analysis. In the given example, all valid combinations (DPB1*04:01/DPB1*131:01 or DPB1*350:01/DPB1*17:01) led to an expected heterozygous rs9277534. In contrast, the invalid combinations (DPB1*04:01/DPB1*17:01 or DPB1*131:01/DPB1*350:01) would have led to an expected homozygous rs9277534 (AA or GG, respectively).

2.6. Estimation of DPB1 allele frequencies

From the observed genotypes, we formulated a Bayesian multinomial model and employed Gibbs sampling as described in [15] in order to estimate DPB1 allele frequencies. Since this algorithm assumes standard Hardy Weinberg equilibrium, we restricted the analysis to the subset of 19,301 samples of self-declared German ancestry.

3.1. Amplicon-based genotyping of 32,681 samples for DPB1 exons 2 and 3 and rs9277534

We examined 32,681 samples meeting all quality criteria as defined in Section 2.3.3. Evaluation of the reads obtained for the rs9277534 amplicon showed a high and even coverage of both alleles (Fig. 2). Read numbers averaged at about 1200 reads per sample in both heterozygous and homozygous samples (Fig. 2A). In heterozygous samples, both alleles were amplified in a balanced fashion with each allele covered by 40 to 60% of the reads (Fig. 2B). Therefore, there was no indication of an amplification bias for either allele.

3.2. Establishment of novel linkages

Due to the lack of physical phasing from the amplicon-based genotyping approach, identification of the linkage between rs9277534 and DPB1 exons 2 and 3 was performed indirectly (Fig. 3). In samples with a homozygous rs9277534, the SNP allele found was declared to be linked to both DPB1 alleles. Conversely, samples with a heterozygous rs9277534 were only used to define a novel linkage if one of the two alleles had an already established linkage. In these cases, the known linkage was assumed to be correct, and the remaining DPB1 allele and observed rs9277534 allele were defined as linked. Subsequently, newly established linkages were used in an iterative analysis until no further novel linkages could be defined. Using this procedure, we confirmed the previously reported linkages (originally based only on exon 2) and established novel linkages for 46 DPB1 alleles, which we confirmed in at least three samples, each. Of these, 22 alleles are linked to rs9277534-A, whereas 24 alleles are linked to rs9277534-G (Table 1).

3.3. Verification of known and novel linkages

Based on the linkages defined in Table 1, we re-evaluated all 32,681 samples for unexpected linkages. Due to the lack of physical phasing between exons 2 and 3 during our primary analysis, some of the DPB1 genotyping results contained phasing ambiguities which prevented two-field genotyping resolution. Since those ambiguities may include alleles of both linkage groups, they required a more sophisticated analysis (for more details, see Methods, section 2.5). Therefore, we separated our sample pool into three subsets: 1) samples predicted to be homozygous for rs9277534, 2) samples predicted to be heterozygous without phasing ambiguities, and 3) samples with phasing ambiguities (Table 2).

The first subset constituted the simplest case, in which a homozygous rs9277534 was expected based on the DPB1 genotyping. Some of those DPB1 genotyping results contained ambiguities due to the lack of phasing; however, those ambiguities were of no relevance to the rs9277534 linkage as all alleles belonged to the same linkage group. The homozygous subset contained 19,322 samples, of which 18 showed an unexpected linkage in the primary analysis. All but one of these unexpected linkages were disproved as artifacts during secondary analysis by full-length SMRT sequencing (artifact sources are explained below). The remaining unexpected linkage was a sample with DPB1*34:01 linked to rs9277534-A.

The second subset consisted of samples with an unambiguous DPB1 genotyping, for which a heterozygous rs9277534 was expected. Of the 7,481 samples in this group, four samples showed an unexpected linkage in the primary analysis. Two of these were disproved as artifacts during secondary analysis. Intriguingly, the remaining two samples both contained DPB1*34:01 linked to rs9277534-A, as already observed in the first subset.

The third subset consisted of samples with ambiguous DPB1 genotyping results due to the lack of phasing between exons 2 and 3. We resolved these codes into valid allele combinations and determined the expected rs9277534 alleles for all these combinations. We confirmed the expected heterozygous rs9277534 alleles in all of the 5,761 samples in this group.

A total of 117 samples were excluded from the linkage analysis at this point, including 31 samples harboring novel DPB1 alleles and 23 samples with rare alleles observed only once or twice. Additionally, 63 samples, heterozygous for rs9277534, were excluded because they encoded one allele with an undefined exon 3 sequence and a second allele with remaining phasing ambiguities. Due to the lack of exon 3 sequence information, the second allele could only be resolved to a group of alleles which linked to both SNP alleles.

In total, our primary analysis identified 22 samples with an unexpected linkage, 19 of which were disproved as artifacts during secondary analysis. The majority of these artifacts resulted from contaminations acquired during the workflow or sample acquisition (17 samples). The contaminated samples were flagged as such during routine genotyping analysis of DPB1 based on the detection of more than two alleles in at least one HLA locus. Independent samples from the same individuals were subjected to SMRT analysis, which confirmed the predicted linkages in all 17 cases. In the remaining two artifact samples, secondary analysis by SMRT sequencing revealed a PCR drop out during primary analysis. In both samples, one of the rs9277534 alleles failed to amplify for unknown reasons.

In summary, the analysis of 32,564 samples revealed a concordance rate of 99.99% with the linkages proposed in Table 1. In only 117 samples (0.4% of all samples), a linkage could not be proposed and therefore neither confirmed nor disproved. These cases included very rare or novel alleles or particular phasing ambiguities. All samples with unexpected linkages confirmed by SMRT analysis carried a DPB1*34:01 allele linked to rs9277534-A instead of rs9277534-G.

3.4. Novel alleles stemming from evolutionary recombination events between exons 2 and 3

Most DPB1 alleles in the IPD-IMGT/HLA database lack sequence information for exon 3. One of those alleles with an unknown exon 3 sequence is DPB1*34:01. In our sample set, we identified 18 samples carrying a DPB1*34:01 allele. For nine of these, a linkage could not be proposed due to the phasing issue explained above (Section 2.5). Since in six of the remaining nine samples, DPB1*34:01 was linked to rs9277534-G, we defined the linkage accordingly in Table 1. In these six samples, the exon 3 sequences of the DPB1*34:01 alleles were identical to the DPB1*01:01 exon 3 sequence. In contrast, in the three samples with unexpected linkage, the exon 3 sequences of the DPB1*34:01-like alleles were identical to the DPB1*02:01 exon 3 sequence. Therefore, full-length sequencing revealed two distinct alleles sharing the same DPB1*34:01 exon 2 sequence but having distinct exon 3 sequences. Interestingly, the rs9277534 variant linked to each allele is in concordance with the linkage defined for other alleles with the same exon 3 sequence (Fig. 4).

The nine samples previously excluded because of the phasing issues confirmed this pattern (postulating the presence of the most frequent of the compatible alleles and the corresponding linkages; the odds of most frequent versus less frequent alleles ranged from 1:16 to 1:1200). Seven of these samples carried the DPB1*34:01-rs9277534-G allele with the DPB1*01:01 exon 3 sequence, while the two remaining samples harbored the DPB1*34:01-rs9277534-A allele with the exon 3 sequence of DPB1*02:01. Interestingly, according to self-declared ethnic origin, all 13 individuals carrying the DPB1*34:01-rs9277534-G allele were of German (11) or Turkish (2) descent, while the DPB1*34:01-rs9277534-A allele was identified in samples from a different range of ethnicities, i.e. three individuals of self-declared Iberian descent, one from Sub-Saharan Africa, and one from the UK/Ireland. This suggests that the frequency of the two DPB1*34:01 like alleles varies greatly between populations.

Additionally, ten of the 31 samples with novel alleles encountered during this study were characterized by hitherto unknown combinations of known exon 2 and 3 sequence features (Table 3). Interestingly, only for one sample, the detected rs9277534 allele matched the allele expected based on the exon 2 sequence. However, in all ten samples, the detected rs9277534 allele matched the expectation when only considering the observed exon 3 sequence. Therefore, we hypothesized that rs9277534 might be more accurately predicted from exon 3 than exon 2. The full sequences of all these novel alleles, including both variants of DPB1*34:01, are currently being submitted to the IPD-IMGT/HLA database. In addition, as part of a separate project, we are currently submitting full-length sequence data of many DPB1 alleles which are so far only partially defined.

3.5. Relevance of exon 2 or exon 3 for the linkage to rs9277534

In the previous section, we described several cases of alleles sharing the same exon 2 sequences but linked to different rs9277534 alleles. However, in all these cases, the alleles with alternative rs9277534 alleles also featured alternative exon 3 sequences. Furthermore, the DPB1 gene structure is characterized by a large intron between exons 2 and 3 (4 kb, Fig. 1A). Therefore, the distance between rs9277534 and exon 2 is 6 kb, whereas the distance between rs9277534 and exon 3 is only 1.8 kb. Taken together, this suggests that the exon 3-rs9277534 linkage might be more reliable than the exon 2-rs9277534 linkage.

On the basis of the linkages defined for 64 alleles (Table 1), we performed a theoretical sequence analysis to establish the linkage between exon 2 or exon 3 sequences and rs9277534 (Supplementary Material 2). This analysis revealed that several alleles sharing the same exon 2 sequences are linked to different rs9277534 alleles. Based on allele frequencies (as determined for the subset of samples with German origin), however, we estimated an error probability of 0.9% for predicting the rs9277534 variant based on exon 2 sequence information alone. In contrast, our sequence analysis revealed no alleles which share the exon 3 sequences but are linked to alternative rs9277534 alleles. However, this linkage analysis disregarded all alleles with undefined exon 3 sequences, which account for a large portion of known alleles (541 of 630 DPB1 alleles in the IPD-IMGT/HLA database), albeit with low frequencies.

We therefore re-analyzed the complete dataset of 32,681 samples for the linkage of exon 3 sequences to rs9277534. This analysis was performed purely on observed sequence features (ignoring allele naming and associated limitations) and included alleles with undefined exon 3, the 23 rare alleles with undefined linkage, and the 31 novel alleles with SNPs or uncommon exon 2/exon 3 combinations. This analysis revealed 100% concordance of rs9277534 linkage for all alleles sharing the same exon 3 sequence features in all 32,681 samples (Table 4, Supplementary Table 4). These results demonstrate that exon 3 alone provides sufficient information for inferring the association of rs9277534 alleles.

Furthermore, this analysis underscored the limited sequence variability in exon 3 in the analyzed sample set. The three most common sequence features (DPB1*02:01, DPB1*01:01 and DPB1*04:01) accounted for 99.15% of the observed alleles in our study. Inclusion of DPB1*15:01 increases coverage to 99.89% of the observed alleles. For each exon 3 sequence feature, Table 4 lists the alleles observed in this study and those that were not observed but share the same exon 3 sequence. Based on the perfect linkage between exon 3 and rs9277534 observed in our sample set, we assume that the linkage will extend to these unobserved alleles as well. In summary, we found 100% linkage between the exon 3 sequence and the rs9277534 allele, whereas a prediction of rs9277534 based on exon 2 sequences alone yields an error rate of about 0.9%.

3.6. Exon 3 sequence analysis

Four described exon 3 sequence features were not observed in this study (Table 4). However, sequence similarity analyses may suggest a linkage for these based on the observed correlations. Therefore, we aligned all known exon 3 sequence features of DPB1 to reveal common motifs (Fig. 4). The sequences define two separate groups that are consistent with regard to their linkage to rs9277534. We conclude that they probably represent phylogenetically separate clades for rs9277534-A and rs9277534-G. Interestingly, the two primary sequences, distinguished by seven SNPs, are also the two most common sequence features. All other known exon 3 sequences deviate only by one SNP from one of those two root sequences, including the four not observed alleles with unique exon 3 sequences. Based on this clear segregation, we postulate that DPB1*168:01 and DPB1*534:01 are linked to rs9277534-A while DPB1*498:01 and DPB1*533:01 are linked to rs9277534-G. We hypothesize that novel exon 3 sequence features will align clearly with one or the other rs9277534 allele. Indeed, the 16 samples with novel exon 3 sequences observed in this study support this hypothesis, confirming the perfect linkage between rs9277534 and exon 3.