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Effectiveness and limitations of resolving HLA class I and class II by heterozygous ambiguity resolving primers (HARPs)—a modified technique of sequence-based typing (SBT)
Clinical Biochemistry 45 (2012) 1471–1478
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Effectiveness and limitations of resolving HLA class I and class II by heterozygous ambiguity resolving primers (HARPs)—a modified technique of sequence-based typing (SBT) Cherng-Lih Perng a, b, Lei-Fa Chang c, Wen-Chun Chien d, Tsung D. Lee d, Jin-Biou Chang a, e,⁎ a
Department of Pathology, National Defense Medical Center, Division of Clinical Pathology, Tri-Service General Hospital, Taipei, Taiwan, R.O.C Graduate Institute of Pathology, National Defense Medical Center, Taipei, Taiwan, R.O.C c Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu, Taiwan, R.O.C d Noustem Biotech. Inc., Taipei, Taiwan, R.O.C e School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taiwan, R.O.C b
a r t i c l e
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Article history: Received 1 February 2012 received in revised form 26 April 2012 accepted 15 May 2012 Available online 31 May 2012 Keywords: Ambiguity HARP HLA SBT
a b s t r a c t Objectives: The aim was to evaluate the use of combination of SBT (sequence based typing) and HARP (heterozygous ambiguity resolving primer) in HLA typing to acquire high resolution typing results. Design and methods: 167 DNA samples were analyzed by SBT. The web site HARPs Finder provided by Conexio Genomics, the developer of HARPs (http://www.harpsfinder.conexio-genomics.com/index.html) was then used to search for appropriate HARPs. Results: HARPs can resolve 95% of ambiguities for locus A; 86% for B and 60% for DRB1 locus. However, there are still limitations. Practically PCR products of un-separated alleles are used as templates for sequencing by HARP; sometimes, it is still impossible to get unambiguous typing. Conclusions: We outlined the advantages and disadvantages of SBT/HARP. A list of HARPs for choice to resolve ambiguity of SBT in Taiwanese population is concluded. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Human leukocyte antigen (HLA) plays an important role in modern medical and clinical science, involving in many fields such as immune system function, disease defense and reproduction. As of today, the numbers of HLA class I and class II alleles have reached 6403 as dated in April 2011 on IMGT. For class I, there are 4946 alleles, and 1457 alleles for class II [1]. In allogeneic organ or hematopoietic stem cell transplantation, the differences in HLA typing for donor and recipient could induce acute or chronic rejection, which might lead to transplantation failure or, in more serious cases, fatal consequence [2]. It is now widely accepted that high-resolution HLA typing is required for identification and selection of best-matched donor in order to minimize the probabilities of rejection. There are two main categories of HLA typing methodology: serological-based and molecular-based technique. Lots of studies indicate that serological technique may be unsatisfactory or unreliable in resolving HLA alleles in high resolution compared to molecular methods [3–5]. The most commonly used molecular HLA typing methods include sequence specific oligonucleotide probe (PCR-SSO), ⁎ Corresponding author at: Department of Pathology, National Defense Medical Center, Division of Clinical Pathology, Tri-Service General Hospital, No. 325, Sec. 2, Chenggung Rd., Taipei City, Taiwan R.O.C. Fax: + 886 2 87927226. E-mail address: [email protected] (J.-B. Chang).
sequence specific primer (SSP) and sequence-based typing (SBT). PCR-SSO can be a high-throughput and relatively inexpensive method, so it is usually used for large-scale, low-resolution HLA allele analysis involving large numbers of samples. SSP is typically used on samples that have failed to be analyzed by SSO, serological method or for situation that higher resolution is required [6]. Among all molecular typing techniques, SBT has the highest resolution and is the only way to directly sequence and identify new alleles. However, conventional SBT technique is much complicated and more expensive than PCR-SSO and SSP; thus its use as routine HLA typing in clinical lab is limited. However, recent developments and advances on automated genetic sequencer, labeled-dye chemistry and alignment analysis algorithms make it possible to regard SBT as part of the routine HLA-typing repertoire. Because of the advance in HLA typing methodologies, total number of HLA alleles increases rapidly over the past couple of years; hence the complexity of HLA allele ambiguities grows rapidly as well [7]. It is becoming more and more difficult to employ a single HLA typing technique to resolve different allele combinations. Commonly the SBT reagents amplify and sequence at least exon 2 and exon 3 of HLA class I as well as exon 2 of HLA class II. Even though SBT is the technique with highest resolution, it could encounter ambiguities in resolving heterozygous allele pairs. Ambiguous alleles occur when sequenced by common sequencing primers and the analyzing software cannot decide whether it is a cis or trans combination of
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2012.05.023
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polymorphic sites. For other times, ambiguities occur when there is only one DNA base different in two typing. Typically, a panel of specially designed SSP or an additional group-specific PCR amplification (GSA) followed by sequencing is employed to identify those ambiguities. The use of these supplemental methods could decrease HLA typing throughput and efficiency, could increase the probability of operation error and is also time-consuming. A modified SBT technique, heterozygous ambiguity resolving primer (HARP), is used to resolve cis/trans allele combinations in a one-step manner to avoid the drawbacks in group-specific PCR amplification or SSP. HARPs work differently from group-specific PCR amplification primers or cloning in resolving polymorphism in allele combinations. GSAs are primers which can only amplify certain alleles in the PCR phase of SBT; thus, only one allele shall be sequenced in the following sequencing phase. HARPs work in the sequencing phase and were specially designed in their annealing sequence to avoid sequencing of both alleles in the mixture of PCR products. Hence, allele separation is unnecessary, and extra processing time can be saved. Fig. 1 illustrates the principle of HARP. HLA class I is utilized here as an example. Yellow and green bars represent exon 2 and 3; purple vertical lines within the bar are the polymorphic sites. Two different HARPs (as labeled HARP 1 and 2) recognize specific polymorphic site, allowing for the subsequent haplotype sequencing to discriminate heterozygous allele combinations. In some ambiguous allele combinations, only one HARP might be required. For example, HARP 2 only anneals to one of the two alleles and reads out the sequence on exon 3 for that particular allele. The sequenced DNA alignment is compared to already existing DNA alignment on IMGT for identification. As a result, correct typing is obtained. Using HARP is a new way for resolving heterozygous ambiguity in sequence-based typing. It works in the same way as the sequencing primers, except one difference, that HARP only sequence one of the two alleles. Hence no allele separation before sequencing reaction is required. Human leukocyte antigen (HLA) typing is a requirement for matching of patient and donor in cord blood and bone marrow
transplantation. The match between patient and donor will influence the successfulness of transplantation. SBT/HARP is one of the ultimate solutions for high resolution HLA typing. Materials and methods Blood The sources of samples include cord blood units from our lab and specimens of international proficiency test/cell exchange program. DNA isolation Genomic DNA was extracted by NucleoSpin Blood kit (Macherey– Nagel GmbH & Co. KG, Düren, Germany). Low and intermediate-resolution HLA typing for cord blood units and DNA samples The low resolution HLA typing was performed by PCR-SSO (GenProbe Transplant Diagnostics, Stamford, CT, USA) and Luminex technology (Luminex Corporation, Austin, TX, USA). Samples displaying uncommon heterozygous allele combinations or those failing to generate SSO results were subsequently tested with PCR-SSP (Olerup SSP kits, Olerup SSP AB, Sweden) to obtain intermediate resolution for both HLA class I and class II. Genomic sequence-based typing for HLA class I and class II genes For cord blood units failed to be resolved either by SSO or SSP because of single nucleotide polymorphism in variable regions, SBT for HLA-A, B and DRB1 locus was performed to obtain highest resolution, unambiguous results. Complete PCR amplification and sequencing reaction for exon 2 and exon 3 of HLA class I as well as exon 2 of HLA class II was performed by reagents and primers provided by AlleleSEQR (Abbott, Germany, Fig. 2). The optimal PCR and purification conditions
Fig. 1. The difference on actions of GSA and HARP. HLA class I is utilized here as an example. Yellow and green bars represent exons on different chromosome; purple vertical lines within the bar are the polymorphic sites. Two different HARPs (as labeled HARP 1 and 2) recognize specific polymorphic site, allowing for the subsequent haplotype sequencing to discriminate heterozygous allele combinations.
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were indicated by the manufacturer. The purified PCR products were then used as sequencing template and parameters for optimal sequencing reaction which is also provided by the manufacturer. Sequencing reaction products were subsequently purified by ethanol precipitation procedure and the purified product was analyzed by 3130 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Base calling and allele assignment were carried out with AssignSBT v3.5+ (Conexio Genomics, Perth, Australia).
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Table 1 Sample source and typing quantities of each method.
Resolution of heterozygous allele combination Though SBT is the typing technique of highest resolution, ambiguity still persists in certain heterozygous allele combinations. In order to resolve the ambiguities, we have selected a list of HARPs (Abbott, Germany) to clearly identify heterozygous alleles. PCR amplification products of SBT were used as template for sequencing reaction and common sequencing primers were replaced by selected HARP for haplotype sequencing. HARP can only amplify certain alleles so ambiguous allele combinations are distinguished. Haplotype sequencing results were also analyzed by AssignSBT v3.5+. Results and discussions Effectiveness of HARP We have sequenced 167 DNA samples for HLA class I and class II with the reagent AlleleSEQR, and have analyzed the sequencing results by AssignSBT v3.5+. Among the 167 specimens, 112 samples were cord blood units from our lab; 50 samples were specimens of international proficiency test/cell exchange program and 5 samples from other laboratories (Table 1). All of the sequencing results have been compared to the SSO and/or SSP results. HLA typings resolved by SBT are consistent with SSP and SSO, with two exceptions which we will discuss later, in either cord blood units of our lab or DNA samples of international proficiency test/cell exchange program. In the 167 sequenced samples, 110 samples (66%) show ambiguity in either A, B or DRB1 locus. In order to resolve these ambiguities and assign correct allele combinations for each sample, HARPs were chosen as the candidate. In order to find out what HARPs should we choose to resolve the heterozygous combinations in the 110 samples, we utilized the web site HARPs Finder provided by Conexio Genomics, the developer of HARPs (http://www.
harpsfinder.conexio-genomics.com/index.html) to search for appropriate HARPs. We have made a list of HARPs and calculate the frequencies of each HARP in our list (Fig. 3). A panel of HARPs was selected in an attempt to resolve most of the ambiguities. According to the allele frequencies observed in Taiwanese population, we have selected 4 HARPs for HLA-A, 3 for HLA-B and 4 for HLADRB1. The detailed information of the selected HARPs is shown in Table 2. By combining the selected HARPs with AlleleSEQR sequencing reagents, we can resolve most of the heterozygous ambiguities in either of the HLA-A, B and DRB1 locus of the 110 samples. Our data are in accordance with the conclusion that 4 to 6 HARPs are sufficient to identify most ambiguities for HLA-A, -B and -DRB1 (unpublished data, Conexio Genomics, Perth, Australia). Since the combinations of heterozygous alleles are highly correlated with ethnic groups, we can subsequently use the selected HARPs in Table 2 to resolve most of the heterozygous allele combinations in Taiwan in routine HLA typing process. With the utilization of HARPs, re-amplification with PCR primers such as GSA can be omitted. The used of HARP instead of GSA can save time and money. Typically redoing the PCR amplification step takes 3 hours. Following the PCR amplification step, sequencing reactions are done. Total time needed for PCR amplification and sequencing is about 5 hours. More than 3 hours can be saved if only HARP is used instead of GSA. As PCR amplification step can be omitted, the cost for PCR is saved. The use of HARP is very flexible. HARP which can resolve most frequent ambiguities can be used upfront to reduce the need for re-sequencing. With concerns of economy, HARPs are generally used retrospectively to resolve the ambiguities we encounter. Comparing the number of HARPs we use to reported GSSPs (group-specific sequencing primer) required in typical HLA typing
Fig. 2. The design of PCR and sequencing primers of AlleleSEQR.(a) PCR and sequencing primers of HLA class I. For HLA-A, PCR amplifies regions from mid exon 1 to intron 4, and then three sets of nested primers are used to sequence exon 2, 3 and 4. For HLA-B, PCR amplifies 2 fragments: one segment of intron 1 to intron 3 and the other from intron 3 to exon 7. Three sets of nested sequencing primers are also used to sequence exon 2, 3 and 4 of HLA-B.(b) PCR and sequencing primers of HLA-DRB1. PCR amplifies the most polymorphic region which is on exon 2, followed by a set of forward and reverse sequencing primers for resolving the sequence. Another sequencing primer, DRB86, is used to discriminate DRB alleles encoding amino acid residue glycine (GGT) versus valine (GTG) at codon 86 (Codon86 anneal to GTG sequence at position 86). DRB86 can be viewed as a HARP to identify a common DRB1 polymorphic site.
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Fig. 3. HARPs suggested by HARPs Finder for use in 110 samples with heterozygous ambiguity and numbers of ambiguous events each HARP can resolve. The height of each column indicates the frequencies of each HARP can be used. The pink colored bars indicate the HARPs we selected to resolve the ambiguities.
assignment, the number of GSSPs needed to resolve all these ambiguities is for HLA-A: 20, HLA-B: 36, and for HLA-C: 16 [8]. Lesser HARPs over GSSP give the advantages of easier operation and more economy in routine HLA typing laboratory.
SBT vs. SSP/SSO (typing assigned, confirmed by SBT) HLA typing analyzed by SBT/HARP has advantages over the conventional SSO or SSP. Because SBT sequenced the whole DNA sequence, it is possible in finding out new alleles and to have the correct HLA typing. Apart from SSO or SSP, which is limited in resolving distinct typing, they are also incapable of discovering new alleles. In all specimens done in our lab, there are two samples that have encountered the problem of discordance. In one of the samples, both SSO and SSP failed to give a valid result. Using SBT, a more accurate methodology, for HLA typing is needed (Fig. 4). Cord blood sample is from our lab. This sample failed to be analyzed by either SSO or SSP for its HLA-B locus. In the SSO, we got more than two possibilities for HLA-B typing. It was repeated twice, and same results were obtained. For SSP, missing bands caused the data interpretation to fail in assigning the correct typing. SBT was done for final typing. After confirmation by SBT, we observed that SSO failed because there were two false negative probes, where no Table 2 Detailed information of selected HARPs. Name
Alias
Sequence
Start
End
Limit
A2F98A A2F144A A3F363A A3F363G B2F106A B2F144C B3R603G DR2F124C DR2F124T DR2R286A DR2R256A C3R486G
98A 144A 363A 363G 106A 144C 603G 124C 124T 286A 256A 486G
GGTATTTCTA CTTCATCGCA TCCAGATA TCCAGAGG ACACCGCCA CTTCATCACC GACGCTGCAG CAGGTTAAAC TACTCTACGT ATCCTGGA AGCGCCGAGT GGCGGCTCAG
89 135 356 356 98 135 603 115 115 286 256 486
98 144 363 363 106 144 612 124 124 293 265 495
98 144 363 363 106 144 612 124 124 293 265 495
signal should be detected. SSP, on the other hand, failed to produce sufficient bands. Three missing bands caused the data analysis invalid. In the second sample, both SSO and SSP provide a possible typing, however, without accordance. SBT was used as confirmation test for correct typing assignment (Fig. 5). Blood sample was from CAP proficiency testing. SSO and SSP results are not concordant, where SSP resulted in a rare allele typing, A*23:14. To confirm for the correct typing, SBT was used. Again, SSP is incapable in producing correct typing due to two missing bands. Although SBT can solve the incorrect typing assignment of SSO and SSP, limitation and disadvantages still persist that we still keep the use of SSO and SSP in our daily HLA typing analysis. Limitations Despite the fact that HARPs, in combination with AlleleSEQR, can resolve 140 of the 185 ambiguities, some allele combinations still cannot be resolved by this modified SBT technique. All the ambiguities that cannot be resolved have common characteristics in their sequences alignments. As a pair, more than two allele combinations are undistinguishable in their sequencing region. For example, A*11:01:01/26:01:01 and A*11:19/26:13 (Fig. 6). Allele typing A*11:01:01 and A*26:01:01 is one allele combination, and A*11:19 and A*26:13 is another combination. As seen, these two allele typing cannot be differentiated by SBT/HARP when two alleles are sequenced together because they have same DNA bases alignments in every position for the sequenced region. Only if haplotype separation or other method (for example cloning) is done, can this type of problem be solved. Otherwise, in this specific configuration, the typing for the specimen fails to be analyzed by SBT/HARP. Other examples with this similar situation are outlined in Table 3. Other than the HLA-A*11/26 typing, there are three other combinations of allele pairing that cannot be distinguished by SBT/HARP. All combinations are similar in their combined sequences; they have the exact same DNA bases arrangement as the other combination(s). Although they are different in their individual DNA sequences; together, they can only be analyzed by either haplotype separation or by cloning.
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Fig. 4. SSO/SSP/SBT result comparison. This sample failed to be analyzed by either SSO or SSP for its HLA-B locus. In the SSO, we got more than two possibilities for HLA-B typing. It was repeated twice, and same results were obtained. For SSP, missing bands caused the data interpretation to fail in assigning the correct typing. SBT was done for final typing. After confirmation by SBT, we observed that SSO failed because there were two false negative probes, where no signal should be detected. SSP, on the other hand, failed to produce sufficient bands. Three missing bands caused the data analysis invalid.
Other deficiencies associated with AlleleSEQR are seen in primer sequence difference; this kind of limitation is as follows (also listed in Table 4). 1) Some alleles, including DRB1*01:07, 04:34, 04:49, 04:66, 07:01:02, 08:14, 08:21, 09:07, 11:30, 13:67, 14:39 and 14:46, have not been tested. 2) Non-amplification of HLA-A*68:02:01:02 has been observed. This may be because the allele has nucleotide difference in the primer region (Fig. 7). 3) Some allele pairs, including DRB1*12:01/12:06/12:10 and DRB1*14:01:01/14:54, cannot be distinguished by HARP because
the nucleotide difference is located in exon 1 and exon 3, outside the amplified exon 2 region (Fig. 8). The problems of the HARP and AlleleSEQR described above can be answered by designing new primer sequence that amplifies different exon 2 region to avoid nucleotide difference in the original primer, or amplifies exon 1 and exon 3 to identify differences located outside exon 2. The primary concern about the newly designed primers is that they might be incompatible with the existed AlleleSEQR and HARP system, so a new sequencing approach has to be established for the new primers and such movements might increase the complexity of daily operations and difficulty in accreditation process. Out of 185 SBT typing,
Fig. 5. SSO/SSP/SBT results comparison—second example. Blood sample was from CAP proficiency testing. SSO and SSP results are not concordant, where SSP resulted in a rare allele typing, A*23:14. To confirm for the correct typing, SBT was used. Again, SSP is incapable in producing correct typing due to two missing bands.
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Fig. 6. DNA sequence for A*11:01:01/26:01:01 and A*11:19/26:13. Adopted from IMGT/HLA Database [1].
HARPs can only resolve 75% of the samples (Table 5). The main concern is DRB1, which has ambiguities outside the sequencing region. Limitation of HARPs still persists; however, by comparing the advantages and disadvantages, it is clearly to favor HARPs over other methodologies in resolving polymorphisms in HLA allele typing. Only when HARPs is insufficient and incapable, then other methods, which is more time-consuming and procedure-difficulties, might apply. According to our data analysis, 66% of the samples analyzed by SBT have encountered ambiguities. However, HARPs can resolve 95% of ambiguities for locus A and 86% for B, and for DRB1 locus 60% of the ambiguities can be solved. By using only 12 different HARPs, we can assign HLA typing for most of the Taiwanese population. Accurate HLA typing is required for selection of the best bone marrow and/or organ donor for transplantation. At present, as many as 6403 HLA class I and II alleles are defined; hence, it is essential for clinical lab to combine a variety of HLA typing methodology for accurate identification of HLA allele in a high throughput manner. Among all of the typing methods, sequence-based typing of HLA alleles can give the highest resolution compared to other typing methods. However, as both alleles at heterozygous loci are sequenced together, it can be difficult to assign an accurate type for some combinations of alleles [7,9]. Conventionally, this can be solved by groupspecific PCR amplification primer that specifically amplifies one of the heterozygous allele. The haplotype PCR product can then be sequenced and analyzed as normal samples. The disadvantage of the
Table 3 Ambiguous typing cannot be resolved by HARP. Locus Ambiguous typing cannot be resolved by HARP
HLA-A A*11:01/26:01:01 A*11:19/26:13
HLA-B B*40:01:01/48:01:01 B*40:01:02/48:01:01 B*40:80/48:03:01 B*54:01/56:01 B*45:03/55:02:01
HLA-DRB DRB1*03:01:01/03:14 DRB1*03:05:01/03:15
group-specific PCR amplification is that it is time-consuming, has the potential of operating error and is inefficient in a high throughput need. We have utilized a modified SBT method for resolving ambiguities, heterozygous ambiguity resolving primer (HARP). The HARP is designed to recognize cis/trans sequence motif on the haplotype and can be used together with conventional SBT technique in sequencing reaction process. The developer of HARP has designed a whole set of HARPs to resolve most of the ambiguities present in IMGT/HLA database. However, not all of the 6403 defined HLA alleles are present at equal frequencies in all populations, therefore it is wasteful to have HARPs to define HLA types which may never be encountered in specific ethnic groups. Different set of HARPs may need for different ethnic populations and based on the allele frequencies in current analyzed samples, we have selected a panel of HARPs (Four for HLA-A, three for HLA-B and four for HLA-DRB1) to resolve most of the heterozygous alleles in our lab. Though HARPs can resolve most of the heterozygous allele combinations in the IMGT/HLA database, there are problems remained for identifying certain HLA types. Those alleles all have common characteristics in their sequence alignments; in that, more than one combination of alleles
Table 4 Limitation of HARP and AlleleSEQR. The following alleles have not been tested but based on the primer sequences and the known allele sequences they are unlikely to be detected. The following alleles have been shown not to amplify with the DRB1 Multiplex kit because it has nucleotide difference in the primer region. Some alleles have heterozygous nucleotides in exon 1 and exon 3, thus cannot be distinguished by AlleleSEQR (only amplified exon 2).
DRB1*01:07, DRB1*07:01:02, DRB1*08:21, DRB1*14:39, DRB1*14:46
HLA-A*68:02:01:02
DRB1*12:01 and DRB1*12:06 and DRB1*12:10 (in exon 1 and exon 3), DRB1*14:01:01 and DRB1*14:54 (in exon 3)
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Fig. 7. The allele cannot be amplified by the AlleleSEQR because of the primer sequence difference. Adopted from IMGT/HLA Database [1].
Fig. 8. The alleles have heterozygous nucleotides in exon 1 and exon 3 (DRB1*12:01:01, 12:06 and 12:10) and exon 3 alone (DRB1*14:01:01 and 14:54). Adopted from IMGT/HLA Database [1].
can't be discriminate when allele separation is not applied. The ambiguous allele combinations include A*110101/260101 with A*11:19/26:13; B*40:01:01/48:01:01, B*40:01:02/48:01:01, and B*40:80/48:03:01; B*54:01/56:01 with B*54:03/55:02:01; DRB1*03:01:01/03:14 with DRB1*03:05:01/03:15. All these combinations have exactly the same DNA alignments although they are different individually as one allele. Other limitations belong to HLA typing of the DRB1 locus and the main cause of the problem is primer sequence design. The primer sequence of AlleleSEQR DRB1 kit locates in the segment of codon5 to codon 12, and this region is one of the polymorphic regions of DRB1 (major DRB1 polymorphic regions include codon 9 to 16; codon 25 to 34; codon 67 to 74 and codon 86 [10]), so the sequence difference between AlleleSEQR primers and certain HLA types may occur. Based on the primer sequence and the allele sequence in IMGT/HLA database, DRB1*01:07, DRB1*07:01:02, DRB1*08:21, DRB1*14:39, DRB1*14:46, DRB1*08:14, DRB1*04:49, DRB1*11:30, DRB1*13:67 are alleles that cannot be amplified by the HARP or AlleleSEQR kit. DRB1*12:01/ 12:06/12:10, as well as DRB1*14:01:01/14:54, are allele pairs that differ in the region outside exon 2 (DRB1*12:01, 12:06 and 12:10 differ in exon 1 and exon 3; DRB1*14:01:01 and 14:54 differ in exon 3). Since exon 2 is the most polymorphic regions in HLA class II DRB1 locus, many sequencing approaches, including the one we used, have developed sequencing methods only for exon 2. The lack of the data in other exons results in a considerable level of ambiguity and possible mistyping of DRB1 [11], so it is necessary to design new primers to resolve the nucleotide difference in exon 1 and exon 3. HARPs are sequencing primers extra to forward and reverse primers that sequence both alleles (Fig. 2). Using HARPs can save time for extra PCR as compared in Fig. 1. Not all the HARPs suggested from population frequency data are needed for resolving ambiguity.
Table 5 Statistics on HARPs' efficiency.
Total samples performed HARPs Ambiguities cannot be solved by HARPs HARP failing rate
A
B
DRB
Total
44 2 5%
50 7 14%
91 36 40%
185 45 24%
HARPs denoted as black bars in Fig. 3 were not chosen because the allele combinations some HARPs can solve are overlapping. SBT has indeed the highest resolution compared to SSP and SSO. In Fig. 4 we demonstrated an example of non-ideal reaction of SSOP probes and dropout for some SSP primers. The mal reacting probes make it impossible to generate a descent typing according to the probe hit chart. The missing PCR bands can cause mistyping (Fig. 5) or no match, i.e. no typing result is generated. Ambiguities are allele combinations sharing the same consensus sequence of the sequenced region (Fig. 6) or the site of polymorphism (resolving base) is outside of sequenced region (Fig. 8). Two criteria of the HARP to resolve an ambiguity are the following: the HARP being upstream of the resolving base, and the distance of HARP and the resolving base no longer than the sequence reading ability. With the knowledge of the ambiguous allele combinations, we can even design our own ambiguity resolving primers. Albeit HARPs have deficiencies in recognizing certain alleles, they still present as a powerful tool to resolve heterozygous allele combinations in high resolution in most cases, and those alleles that failed to be identified by HARP can be typed by other methods, such as a panel of specially designed SSP primers. In conclusion, HARP in combination with conventional SBT can be used as a golden standard to directly sequence and identify HLA alleles or discover new alleles from nucleotide base difference in sequencing results.
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Effectiveness and limitations of resolving HLA class I and class II by heterozygous ambiguity resolving primers (HARPs)—a modified technique of sequence-based typing (SBT) Cherng-Lih Perng a, b, Lei-Fa Chang c, Wen-Chun Chien d, Tsung D. Lee d, Jin-Biou Chang a, e,⁎ a
Department of Pathology, National Defense Medical Center, Division of Clinical Pathology, Tri-Service General Hospital, Taipei, Taiwan, R.O.C Graduate Institute of Pathology, National Defense Medical Center, Taipei, Taiwan, R.O.C c Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu, Taiwan, R.O.C d Noustem Biotech. Inc., Taipei, Taiwan, R.O.C e School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taiwan, R.O.C b
a r t i c l e
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Article history: Received 1 February 2012 received in revised form 26 April 2012 accepted 15 May 2012 Available online 31 May 2012 Keywords: Ambiguity HARP HLA SBT
a b s t r a c t Objectives: The aim was to evaluate the use of combination of SBT (sequence based typing) and HARP (heterozygous ambiguity resolving primer) in HLA typing to acquire high resolution typing results. Design and methods: 167 DNA samples were analyzed by SBT. The web site HARPs Finder provided by Conexio Genomics, the developer of HARPs (http://www.harpsfinder.conexio-genomics.com/index.html) was then used to search for appropriate HARPs. Results: HARPs can resolve 95% of ambiguities for locus A; 86% for B and 60% for DRB1 locus. However, there are still limitations. Practically PCR products of un-separated alleles are used as templates for sequencing by HARP; sometimes, it is still impossible to get unambiguous typing. Conclusions: We outlined the advantages and disadvantages of SBT/HARP. A list of HARPs for choice to resolve ambiguity of SBT in Taiwanese population is concluded. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Human leukocyte antigen (HLA) plays an important role in modern medical and clinical science, involving in many fields such as immune system function, disease defense and reproduction. As of today, the numbers of HLA class I and class II alleles have reached 6403 as dated in April 2011 on IMGT. For class I, there are 4946 alleles, and 1457 alleles for class II [1]. In allogeneic organ or hematopoietic stem cell transplantation, the differences in HLA typing for donor and recipient could induce acute or chronic rejection, which might lead to transplantation failure or, in more serious cases, fatal consequence [2]. It is now widely accepted that high-resolution HLA typing is required for identification and selection of best-matched donor in order to minimize the probabilities of rejection. There are two main categories of HLA typing methodology: serological-based and molecular-based technique. Lots of studies indicate that serological technique may be unsatisfactory or unreliable in resolving HLA alleles in high resolution compared to molecular methods [3–5]. The most commonly used molecular HLA typing methods include sequence specific oligonucleotide probe (PCR-SSO), ⁎ Corresponding author at: Department of Pathology, National Defense Medical Center, Division of Clinical Pathology, Tri-Service General Hospital, No. 325, Sec. 2, Chenggung Rd., Taipei City, Taiwan R.O.C. Fax: + 886 2 87927226. E-mail address: [email protected] (J.-B. Chang).
sequence specific primer (SSP) and sequence-based typing (SBT). PCR-SSO can be a high-throughput and relatively inexpensive method, so it is usually used for large-scale, low-resolution HLA allele analysis involving large numbers of samples. SSP is typically used on samples that have failed to be analyzed by SSO, serological method or for situation that higher resolution is required [6]. Among all molecular typing techniques, SBT has the highest resolution and is the only way to directly sequence and identify new alleles. However, conventional SBT technique is much complicated and more expensive than PCR-SSO and SSP; thus its use as routine HLA typing in clinical lab is limited. However, recent developments and advances on automated genetic sequencer, labeled-dye chemistry and alignment analysis algorithms make it possible to regard SBT as part of the routine HLA-typing repertoire. Because of the advance in HLA typing methodologies, total number of HLA alleles increases rapidly over the past couple of years; hence the complexity of HLA allele ambiguities grows rapidly as well [7]. It is becoming more and more difficult to employ a single HLA typing technique to resolve different allele combinations. Commonly the SBT reagents amplify and sequence at least exon 2 and exon 3 of HLA class I as well as exon 2 of HLA class II. Even though SBT is the technique with highest resolution, it could encounter ambiguities in resolving heterozygous allele pairs. Ambiguous alleles occur when sequenced by common sequencing primers and the analyzing software cannot decide whether it is a cis or trans combination of
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2012.05.023
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polymorphic sites. For other times, ambiguities occur when there is only one DNA base different in two typing. Typically, a panel of specially designed SSP or an additional group-specific PCR amplification (GSA) followed by sequencing is employed to identify those ambiguities. The use of these supplemental methods could decrease HLA typing throughput and efficiency, could increase the probability of operation error and is also time-consuming. A modified SBT technique, heterozygous ambiguity resolving primer (HARP), is used to resolve cis/trans allele combinations in a one-step manner to avoid the drawbacks in group-specific PCR amplification or SSP. HARPs work differently from group-specific PCR amplification primers or cloning in resolving polymorphism in allele combinations. GSAs are primers which can only amplify certain alleles in the PCR phase of SBT; thus, only one allele shall be sequenced in the following sequencing phase. HARPs work in the sequencing phase and were specially designed in their annealing sequence to avoid sequencing of both alleles in the mixture of PCR products. Hence, allele separation is unnecessary, and extra processing time can be saved. Fig. 1 illustrates the principle of HARP. HLA class I is utilized here as an example. Yellow and green bars represent exon 2 and 3; purple vertical lines within the bar are the polymorphic sites. Two different HARPs (as labeled HARP 1 and 2) recognize specific polymorphic site, allowing for the subsequent haplotype sequencing to discriminate heterozygous allele combinations. In some ambiguous allele combinations, only one HARP might be required. For example, HARP 2 only anneals to one of the two alleles and reads out the sequence on exon 3 for that particular allele. The sequenced DNA alignment is compared to already existing DNA alignment on IMGT for identification. As a result, correct typing is obtained. Using HARP is a new way for resolving heterozygous ambiguity in sequence-based typing. It works in the same way as the sequencing primers, except one difference, that HARP only sequence one of the two alleles. Hence no allele separation before sequencing reaction is required. Human leukocyte antigen (HLA) typing is a requirement for matching of patient and donor in cord blood and bone marrow
transplantation. The match between patient and donor will influence the successfulness of transplantation. SBT/HARP is one of the ultimate solutions for high resolution HLA typing. Materials and methods Blood The sources of samples include cord blood units from our lab and specimens of international proficiency test/cell exchange program. DNA isolation Genomic DNA was extracted by NucleoSpin Blood kit (Macherey– Nagel GmbH & Co. KG, Düren, Germany). Low and intermediate-resolution HLA typing for cord blood units and DNA samples The low resolution HLA typing was performed by PCR-SSO (GenProbe Transplant Diagnostics, Stamford, CT, USA) and Luminex technology (Luminex Corporation, Austin, TX, USA). Samples displaying uncommon heterozygous allele combinations or those failing to generate SSO results were subsequently tested with PCR-SSP (Olerup SSP kits, Olerup SSP AB, Sweden) to obtain intermediate resolution for both HLA class I and class II. Genomic sequence-based typing for HLA class I and class II genes For cord blood units failed to be resolved either by SSO or SSP because of single nucleotide polymorphism in variable regions, SBT for HLA-A, B and DRB1 locus was performed to obtain highest resolution, unambiguous results. Complete PCR amplification and sequencing reaction for exon 2 and exon 3 of HLA class I as well as exon 2 of HLA class II was performed by reagents and primers provided by AlleleSEQR (Abbott, Germany, Fig. 2). The optimal PCR and purification conditions
Fig. 1. The difference on actions of GSA and HARP. HLA class I is utilized here as an example. Yellow and green bars represent exons on different chromosome; purple vertical lines within the bar are the polymorphic sites. Two different HARPs (as labeled HARP 1 and 2) recognize specific polymorphic site, allowing for the subsequent haplotype sequencing to discriminate heterozygous allele combinations.
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were indicated by the manufacturer. The purified PCR products were then used as sequencing template and parameters for optimal sequencing reaction which is also provided by the manufacturer. Sequencing reaction products were subsequently purified by ethanol precipitation procedure and the purified product was analyzed by 3130 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Base calling and allele assignment were carried out with AssignSBT v3.5+ (Conexio Genomics, Perth, Australia).
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Table 1 Sample source and typing quantities of each method.
Resolution of heterozygous allele combination Though SBT is the typing technique of highest resolution, ambiguity still persists in certain heterozygous allele combinations. In order to resolve the ambiguities, we have selected a list of HARPs (Abbott, Germany) to clearly identify heterozygous alleles. PCR amplification products of SBT were used as template for sequencing reaction and common sequencing primers were replaced by selected HARP for haplotype sequencing. HARP can only amplify certain alleles so ambiguous allele combinations are distinguished. Haplotype sequencing results were also analyzed by AssignSBT v3.5+. Results and discussions Effectiveness of HARP We have sequenced 167 DNA samples for HLA class I and class II with the reagent AlleleSEQR, and have analyzed the sequencing results by AssignSBT v3.5+. Among the 167 specimens, 112 samples were cord blood units from our lab; 50 samples were specimens of international proficiency test/cell exchange program and 5 samples from other laboratories (Table 1). All of the sequencing results have been compared to the SSO and/or SSP results. HLA typings resolved by SBT are consistent with SSP and SSO, with two exceptions which we will discuss later, in either cord blood units of our lab or DNA samples of international proficiency test/cell exchange program. In the 167 sequenced samples, 110 samples (66%) show ambiguity in either A, B or DRB1 locus. In order to resolve these ambiguities and assign correct allele combinations for each sample, HARPs were chosen as the candidate. In order to find out what HARPs should we choose to resolve the heterozygous combinations in the 110 samples, we utilized the web site HARPs Finder provided by Conexio Genomics, the developer of HARPs (http://www.
harpsfinder.conexio-genomics.com/index.html) to search for appropriate HARPs. We have made a list of HARPs and calculate the frequencies of each HARP in our list (Fig. 3). A panel of HARPs was selected in an attempt to resolve most of the ambiguities. According to the allele frequencies observed in Taiwanese population, we have selected 4 HARPs for HLA-A, 3 for HLA-B and 4 for HLADRB1. The detailed information of the selected HARPs is shown in Table 2. By combining the selected HARPs with AlleleSEQR sequencing reagents, we can resolve most of the heterozygous ambiguities in either of the HLA-A, B and DRB1 locus of the 110 samples. Our data are in accordance with the conclusion that 4 to 6 HARPs are sufficient to identify most ambiguities for HLA-A, -B and -DRB1 (unpublished data, Conexio Genomics, Perth, Australia). Since the combinations of heterozygous alleles are highly correlated with ethnic groups, we can subsequently use the selected HARPs in Table 2 to resolve most of the heterozygous allele combinations in Taiwan in routine HLA typing process. With the utilization of HARPs, re-amplification with PCR primers such as GSA can be omitted. The used of HARP instead of GSA can save time and money. Typically redoing the PCR amplification step takes 3 hours. Following the PCR amplification step, sequencing reactions are done. Total time needed for PCR amplification and sequencing is about 5 hours. More than 3 hours can be saved if only HARP is used instead of GSA. As PCR amplification step can be omitted, the cost for PCR is saved. The use of HARP is very flexible. HARP which can resolve most frequent ambiguities can be used upfront to reduce the need for re-sequencing. With concerns of economy, HARPs are generally used retrospectively to resolve the ambiguities we encounter. Comparing the number of HARPs we use to reported GSSPs (group-specific sequencing primer) required in typical HLA typing
Fig. 2. The design of PCR and sequencing primers of AlleleSEQR.(a) PCR and sequencing primers of HLA class I. For HLA-A, PCR amplifies regions from mid exon 1 to intron 4, and then three sets of nested primers are used to sequence exon 2, 3 and 4. For HLA-B, PCR amplifies 2 fragments: one segment of intron 1 to intron 3 and the other from intron 3 to exon 7. Three sets of nested sequencing primers are also used to sequence exon 2, 3 and 4 of HLA-B.(b) PCR and sequencing primers of HLA-DRB1. PCR amplifies the most polymorphic region which is on exon 2, followed by a set of forward and reverse sequencing primers for resolving the sequence. Another sequencing primer, DRB86, is used to discriminate DRB alleles encoding amino acid residue glycine (GGT) versus valine (GTG) at codon 86 (Codon86 anneal to GTG sequence at position 86). DRB86 can be viewed as a HARP to identify a common DRB1 polymorphic site.
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Fig. 3. HARPs suggested by HARPs Finder for use in 110 samples with heterozygous ambiguity and numbers of ambiguous events each HARP can resolve. The height of each column indicates the frequencies of each HARP can be used. The pink colored bars indicate the HARPs we selected to resolve the ambiguities.
assignment, the number of GSSPs needed to resolve all these ambiguities is for HLA-A: 20, HLA-B: 36, and for HLA-C: 16 [8]. Lesser HARPs over GSSP give the advantages of easier operation and more economy in routine HLA typing laboratory.
SBT vs. SSP/SSO (typing assigned, confirmed by SBT) HLA typing analyzed by SBT/HARP has advantages over the conventional SSO or SSP. Because SBT sequenced the whole DNA sequence, it is possible in finding out new alleles and to have the correct HLA typing. Apart from SSO or SSP, which is limited in resolving distinct typing, they are also incapable of discovering new alleles. In all specimens done in our lab, there are two samples that have encountered the problem of discordance. In one of the samples, both SSO and SSP failed to give a valid result. Using SBT, a more accurate methodology, for HLA typing is needed (Fig. 4). Cord blood sample is from our lab. This sample failed to be analyzed by either SSO or SSP for its HLA-B locus. In the SSO, we got more than two possibilities for HLA-B typing. It was repeated twice, and same results were obtained. For SSP, missing bands caused the data interpretation to fail in assigning the correct typing. SBT was done for final typing. After confirmation by SBT, we observed that SSO failed because there were two false negative probes, where no Table 2 Detailed information of selected HARPs. Name
Alias
Sequence
Start
End
Limit
A2F98A A2F144A A3F363A A3F363G B2F106A B2F144C B3R603G DR2F124C DR2F124T DR2R286A DR2R256A C3R486G
98A 144A 363A 363G 106A 144C 603G 124C 124T 286A 256A 486G
GGTATTTCTA CTTCATCGCA TCCAGATA TCCAGAGG ACACCGCCA CTTCATCACC GACGCTGCAG CAGGTTAAAC TACTCTACGT ATCCTGGA AGCGCCGAGT GGCGGCTCAG
89 135 356 356 98 135 603 115 115 286 256 486
98 144 363 363 106 144 612 124 124 293 265 495
98 144 363 363 106 144 612 124 124 293 265 495
signal should be detected. SSP, on the other hand, failed to produce sufficient bands. Three missing bands caused the data analysis invalid. In the second sample, both SSO and SSP provide a possible typing, however, without accordance. SBT was used as confirmation test for correct typing assignment (Fig. 5). Blood sample was from CAP proficiency testing. SSO and SSP results are not concordant, where SSP resulted in a rare allele typing, A*23:14. To confirm for the correct typing, SBT was used. Again, SSP is incapable in producing correct typing due to two missing bands. Although SBT can solve the incorrect typing assignment of SSO and SSP, limitation and disadvantages still persist that we still keep the use of SSO and SSP in our daily HLA typing analysis. Limitations Despite the fact that HARPs, in combination with AlleleSEQR, can resolve 140 of the 185 ambiguities, some allele combinations still cannot be resolved by this modified SBT technique. All the ambiguities that cannot be resolved have common characteristics in their sequences alignments. As a pair, more than two allele combinations are undistinguishable in their sequencing region. For example, A*11:01:01/26:01:01 and A*11:19/26:13 (Fig. 6). Allele typing A*11:01:01 and A*26:01:01 is one allele combination, and A*11:19 and A*26:13 is another combination. As seen, these two allele typing cannot be differentiated by SBT/HARP when two alleles are sequenced together because they have same DNA bases alignments in every position for the sequenced region. Only if haplotype separation or other method (for example cloning) is done, can this type of problem be solved. Otherwise, in this specific configuration, the typing for the specimen fails to be analyzed by SBT/HARP. Other examples with this similar situation are outlined in Table 3. Other than the HLA-A*11/26 typing, there are three other combinations of allele pairing that cannot be distinguished by SBT/HARP. All combinations are similar in their combined sequences; they have the exact same DNA bases arrangement as the other combination(s). Although they are different in their individual DNA sequences; together, they can only be analyzed by either haplotype separation or by cloning.
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Fig. 4. SSO/SSP/SBT result comparison. This sample failed to be analyzed by either SSO or SSP for its HLA-B locus. In the SSO, we got more than two possibilities for HLA-B typing. It was repeated twice, and same results were obtained. For SSP, missing bands caused the data interpretation to fail in assigning the correct typing. SBT was done for final typing. After confirmation by SBT, we observed that SSO failed because there were two false negative probes, where no signal should be detected. SSP, on the other hand, failed to produce sufficient bands. Three missing bands caused the data analysis invalid.
Other deficiencies associated with AlleleSEQR are seen in primer sequence difference; this kind of limitation is as follows (also listed in Table 4). 1) Some alleles, including DRB1*01:07, 04:34, 04:49, 04:66, 07:01:02, 08:14, 08:21, 09:07, 11:30, 13:67, 14:39 and 14:46, have not been tested. 2) Non-amplification of HLA-A*68:02:01:02 has been observed. This may be because the allele has nucleotide difference in the primer region (Fig. 7). 3) Some allele pairs, including DRB1*12:01/12:06/12:10 and DRB1*14:01:01/14:54, cannot be distinguished by HARP because
the nucleotide difference is located in exon 1 and exon 3, outside the amplified exon 2 region (Fig. 8). The problems of the HARP and AlleleSEQR described above can be answered by designing new primer sequence that amplifies different exon 2 region to avoid nucleotide difference in the original primer, or amplifies exon 1 and exon 3 to identify differences located outside exon 2. The primary concern about the newly designed primers is that they might be incompatible with the existed AlleleSEQR and HARP system, so a new sequencing approach has to be established for the new primers and such movements might increase the complexity of daily operations and difficulty in accreditation process. Out of 185 SBT typing,
Fig. 5. SSO/SSP/SBT results comparison—second example. Blood sample was from CAP proficiency testing. SSO and SSP results are not concordant, where SSP resulted in a rare allele typing, A*23:14. To confirm for the correct typing, SBT was used. Again, SSP is incapable in producing correct typing due to two missing bands.
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Fig. 6. DNA sequence for A*11:01:01/26:01:01 and A*11:19/26:13. Adopted from IMGT/HLA Database [1].
HARPs can only resolve 75% of the samples (Table 5). The main concern is DRB1, which has ambiguities outside the sequencing region. Limitation of HARPs still persists; however, by comparing the advantages and disadvantages, it is clearly to favor HARPs over other methodologies in resolving polymorphisms in HLA allele typing. Only when HARPs is insufficient and incapable, then other methods, which is more time-consuming and procedure-difficulties, might apply. According to our data analysis, 66% of the samples analyzed by SBT have encountered ambiguities. However, HARPs can resolve 95% of ambiguities for locus A and 86% for B, and for DRB1 locus 60% of the ambiguities can be solved. By using only 12 different HARPs, we can assign HLA typing for most of the Taiwanese population. Accurate HLA typing is required for selection of the best bone marrow and/or organ donor for transplantation. At present, as many as 6403 HLA class I and II alleles are defined; hence, it is essential for clinical lab to combine a variety of HLA typing methodology for accurate identification of HLA allele in a high throughput manner. Among all of the typing methods, sequence-based typing of HLA alleles can give the highest resolution compared to other typing methods. However, as both alleles at heterozygous loci are sequenced together, it can be difficult to assign an accurate type for some combinations of alleles [7,9]. Conventionally, this can be solved by groupspecific PCR amplification primer that specifically amplifies one of the heterozygous allele. The haplotype PCR product can then be sequenced and analyzed as normal samples. The disadvantage of the
Table 3 Ambiguous typing cannot be resolved by HARP. Locus Ambiguous typing cannot be resolved by HARP
HLA-A A*11:01/26:01:01 A*11:19/26:13
HLA-B B*40:01:01/48:01:01 B*40:01:02/48:01:01 B*40:80/48:03:01 B*54:01/56:01 B*45:03/55:02:01
HLA-DRB DRB1*03:01:01/03:14 DRB1*03:05:01/03:15
group-specific PCR amplification is that it is time-consuming, has the potential of operating error and is inefficient in a high throughput need. We have utilized a modified SBT method for resolving ambiguities, heterozygous ambiguity resolving primer (HARP). The HARP is designed to recognize cis/trans sequence motif on the haplotype and can be used together with conventional SBT technique in sequencing reaction process. The developer of HARP has designed a whole set of HARPs to resolve most of the ambiguities present in IMGT/HLA database. However, not all of the 6403 defined HLA alleles are present at equal frequencies in all populations, therefore it is wasteful to have HARPs to define HLA types which may never be encountered in specific ethnic groups. Different set of HARPs may need for different ethnic populations and based on the allele frequencies in current analyzed samples, we have selected a panel of HARPs (Four for HLA-A, three for HLA-B and four for HLA-DRB1) to resolve most of the heterozygous alleles in our lab. Though HARPs can resolve most of the heterozygous allele combinations in the IMGT/HLA database, there are problems remained for identifying certain HLA types. Those alleles all have common characteristics in their sequence alignments; in that, more than one combination of alleles
Table 4 Limitation of HARP and AlleleSEQR. The following alleles have not been tested but based on the primer sequences and the known allele sequences they are unlikely to be detected. The following alleles have been shown not to amplify with the DRB1 Multiplex kit because it has nucleotide difference in the primer region. Some alleles have heterozygous nucleotides in exon 1 and exon 3, thus cannot be distinguished by AlleleSEQR (only amplified exon 2).
DRB1*01:07, DRB1*07:01:02, DRB1*08:21, DRB1*14:39, DRB1*14:46
HLA-A*68:02:01:02
DRB1*12:01 and DRB1*12:06 and DRB1*12:10 (in exon 1 and exon 3), DRB1*14:01:01 and DRB1*14:54 (in exon 3)
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Fig. 7. The allele cannot be amplified by the AlleleSEQR because of the primer sequence difference. Adopted from IMGT/HLA Database [1].
Fig. 8. The alleles have heterozygous nucleotides in exon 1 and exon 3 (DRB1*12:01:01, 12:06 and 12:10) and exon 3 alone (DRB1*14:01:01 and 14:54). Adopted from IMGT/HLA Database [1].
can't be discriminate when allele separation is not applied. The ambiguous allele combinations include A*110101/260101 with A*11:19/26:13; B*40:01:01/48:01:01, B*40:01:02/48:01:01, and B*40:80/48:03:01; B*54:01/56:01 with B*54:03/55:02:01; DRB1*03:01:01/03:14 with DRB1*03:05:01/03:15. All these combinations have exactly the same DNA alignments although they are different individually as one allele. Other limitations belong to HLA typing of the DRB1 locus and the main cause of the problem is primer sequence design. The primer sequence of AlleleSEQR DRB1 kit locates in the segment of codon5 to codon 12, and this region is one of the polymorphic regions of DRB1 (major DRB1 polymorphic regions include codon 9 to 16; codon 25 to 34; codon 67 to 74 and codon 86 [10]), so the sequence difference between AlleleSEQR primers and certain HLA types may occur. Based on the primer sequence and the allele sequence in IMGT/HLA database, DRB1*01:07, DRB1*07:01:02, DRB1*08:21, DRB1*14:39, DRB1*14:46, DRB1*08:14, DRB1*04:49, DRB1*11:30, DRB1*13:67 are alleles that cannot be amplified by the HARP or AlleleSEQR kit. DRB1*12:01/ 12:06/12:10, as well as DRB1*14:01:01/14:54, are allele pairs that differ in the region outside exon 2 (DRB1*12:01, 12:06 and 12:10 differ in exon 1 and exon 3; DRB1*14:01:01 and 14:54 differ in exon 3). Since exon 2 is the most polymorphic regions in HLA class II DRB1 locus, many sequencing approaches, including the one we used, have developed sequencing methods only for exon 2. The lack of the data in other exons results in a considerable level of ambiguity and possible mistyping of DRB1 [11], so it is necessary to design new primers to resolve the nucleotide difference in exon 1 and exon 3. HARPs are sequencing primers extra to forward and reverse primers that sequence both alleles (Fig. 2). Using HARPs can save time for extra PCR as compared in Fig. 1. Not all the HARPs suggested from population frequency data are needed for resolving ambiguity.
Table 5 Statistics on HARPs' efficiency.
Total samples performed HARPs Ambiguities cannot be solved by HARPs HARP failing rate
A
B
DRB
Total
44 2 5%
50 7 14%
91 36 40%
185 45 24%
HARPs denoted as black bars in Fig. 3 were not chosen because the allele combinations some HARPs can solve are overlapping. SBT has indeed the highest resolution compared to SSP and SSO. In Fig. 4 we demonstrated an example of non-ideal reaction of SSOP probes and dropout for some SSP primers. The mal reacting probes make it impossible to generate a descent typing according to the probe hit chart. The missing PCR bands can cause mistyping (Fig. 5) or no match, i.e. no typing result is generated. Ambiguities are allele combinations sharing the same consensus sequence of the sequenced region (Fig. 6) or the site of polymorphism (resolving base) is outside of sequenced region (Fig. 8). Two criteria of the HARP to resolve an ambiguity are the following: the HARP being upstream of the resolving base, and the distance of HARP and the resolving base no longer than the sequence reading ability. With the knowledge of the ambiguous allele combinations, we can even design our own ambiguity resolving primers. Albeit HARPs have deficiencies in recognizing certain alleles, they still present as a powerful tool to resolve heterozygous allele combinations in high resolution in most cases, and those alleles that failed to be identified by HARP can be typed by other methods, such as a panel of specially designed SSP primers. In conclusion, HARP in combination with conventional SBT can be used as a golden standard to directly sequence and identify HLA alleles or discover new alleles from nucleotide base difference in sequencing results.
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