Unambiguous high resolution genotyping of human leukocyte antigens

Accepted Manuscript Unambiguous high resolution genotyping of human leukocyte antigens

Robert A. Bradshaw, Paul P.J. Dunn PII: DOI: Reference:

S0022-1759(16)30398-2 doi: 10.1016/j.jim.2017.02.011 JIM 12276

To appear in:

Journal of Immunological Methods

Received date:

28 December 2016

Please cite this article as: Robert A. Bradshaw, Paul P.J. Dunn , Unambiguous high resolution genotyping of human leukocyte antigens. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jim(2017), doi: 10.1016/j.jim.2017.02.011

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ACCEPTED MANUSCRIPT Unambiguous High Resolution Genotyping of Human Leukocyte Antigens

Robert A. Bradshaw, Paul P. J. Dunn

Transplant Laboratory

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University Hospitals Leicester Leicester General Hospital

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Gwendolen Road

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Leicester LE5 4PW

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United Kingdom

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Address for correspondence [email protected]

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Abstract We have developed a high resolution sequencing based typing method for genotyping Human Leukocyte Antigens (HLA) over a period of twenty years. The methods are based upon the separation of HLA alleles per locus at the initial amplification to simplify the analysis post-

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sequencing. The increasing discovery of polymorphism in HLA, manifested in new alleles,

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has necessitated the continuing development of this method. Here we present methods for the high resolution Sequence Based Typing of HLA-A, B, C (class I) and HLA-DQB1 and DRB1

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(class II). The purpose of this article is to provide a valuable resource of methods and primers

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for other laboratories engaged in HLA typing.

Keywords

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High resolution unambiguous HLA typing Amplification and sequencing primers

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HLA class I (HLA-A, B, C) genotyping

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HLA class II (DRB1, DQB1) genotyping

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1. Introduction The “Classical” Human Leukocyte Antigen system comprises six loci divided into class I (HLA-A, -B, and -C) and class II (HLA-DP, -DQ, and -DR). The principal function of these

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proteins is to present peptides which are derived from viral and intracellular proteins (class I) or those derived from processing of pathogens and extracellular proteins (class II)

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(McCluskey and Peh,1999). HLA are the most polymorphic protein and genetic systems

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found in humans and represent a significant challenge for successful allogeneic

and future applications (Taylor et al., 2011).

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transplantation of an organ or stem cells (Tait 2011; Tiercy 2002) and in stem cell banking

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The complexity and expanse of HLA polymorphism has attracted the adaption and development of new technologies in tissue typing laboratories (Dunn 2011, 2015). Various

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techniques have been developed and modified in the post-PCR era, including PCR sequence-

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specific primer (PCR-SSP) (Bunce et al 1995), PCR-sequence-specific oligonucleotide probe (PCR-SSOP) (Cao et al 1999), PCR-sequencing-based typing (PCR-SBT) (Dunn et al 2003),

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oligonucleotide arrays (Guo et al 1999), and, more recently, next generation sequencing (NGS) (Holcomb et al 2011). The un-abating march of HLA polymorphism, mainly

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discovered through DNA sequencing, has revealed the inadequacies of many of these technologies (Dunn 2011). DNA sequencing, using the Sanger method of di-deoxy chain termination (Sanger et al., 1977), for HLA typing became the preferred method for allelic HLA typing through the discovery of locus- and antigen-specific polymorphisms in the noncoding introns flanking the polymorphic exons (Cereb et al., 1995; Cereb & Yang, 1997; Kotsch et al., 1997, 1999; Dunn et al., 2005). The methods described here have evolved since 2000 in response to the

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ACCEPTED MANUSCRIPT discovery of increasing levels of HLA polymorphism. The initial methodology used amplification of exon 2 in class II genes (HLA-DPB1, DQB1 and DRB1) and exons 2 and 3 in class I genes (HLA-A, B, C), amplifying and sequencing both alleles together. As new alleles were reported with polymorphisms outside of these amplified regions we included these exons. Full amplification of HLA class I genes was commenced in 2000 and this was

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used as the template for sequencing reactions. This has proved more difficult for class II

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genes because of their size but we have now included exons 2 and 3 for sequencing HLA-

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DQB1 and HLA-DRB1. As the numbers of HLA polymorphisms have increased so the number of ambiguities has increased. Allele ambiguities are due to polymorphisms outside of

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the region analysed and genotype ambiguities are combinations of alleles which have identical heterozygous sequences in the region analysed. Our strategy then changed to

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combine a group-specific primer with a locus-specific primer followed by cycle-sequencing the desired number of exons (on both strands). Ideally, each allele will be amplified and

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sequenced separately providing an unambiguous allelic HLA type. The number of exons sequenced depends on the locus: e.g. HLA-A, B exons 1-4; HLA-C exons 1-7; HLA-DQB1, DRB1 exons 2 and 3. We have not extended this strategy to include

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other loci such as HLA-DPA1, DPB1,DQA1 or DRB3/4/5 as typing resolution using

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Luminex is usually sufficiently high enough. Here we present a comprehensive HLA sequencing strategy for use in Sanger sequencing but could also be applied to current NGS technologies. The strategy includes all amplification and sequencing primers, in supplementary tables 2 and 3, to enable unambiguous high resolution typing of HLA-A, B, C, DRB1 and DQB1.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Samples received were whole blood or DNA. DNA was extracted from whole blood by various conventional methods and used at 20-50 ng/µl. Absorbance at A260/A280 was in the range of 1.5-2.0. 1st-field (‘low’) resolution HLA types were obtained by Luminex-based

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LifeCodes PCR-SSO (Immucor). 2.2 The class I PCR enzyme is a combination of proof reading and standard Taq DNA

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polymerases (‘Expand’, Roche Diagnostics) whilst class II uses AmpliTaq Gold DNA

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polymerase (Thermofisher). The PCR conditions and parameters are shown in Table 1. Many

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different thermal cyclers have been used for these protocols but must be optimised for each laboratory’s use.

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2.3 Primers were designed against IMGT alignment 3.23 and manufactured by Thermo Fisher custom DNA oligo service (ThermoFisher Scientific Inc, Waltham, MA USA).

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Amplification primers are recorded in Supplementary Table 1 with the list of allele groups

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predicted to be amplified by a given primer set according to sequence matches at the 3’ primer ends. Annealing locations are given according to the IMGT genomic DNA alignment

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for each gene. HLA class I sense-strand primers are group-specific and anneal within exon 1, intron 1 or the 5’UTR. The antisense primer is gene-specific (universal) annealing within

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the 3’UTR. HLA class II amplification primer pairs amplify exons 2 and 3 separately by using group-specific primer pairs in introns 1 and 2 (exon 2) and introns 2 and 3 (exon 3). The majority of HLA-DRB1 sense primers are mismatched to the target sequence at the 5th nucleotide from the 3’ end to increase stringency as according to Kotsch et al 1999. Cycle sequencing primers are described in Supplementary Table 2. HLA class I primers are universal for all allele groups. Class II primers may differ depending on the allele group of the fragment to be sequenced. 5

ACCEPTED MANUSCRIPT 2.4 Excess primers and dNTPs are removed from amplicons with exoSAP-IT (Affymetrix) using the manufacturer’s protocol with amplicons diluted 1:10 (class I) or 1:4 (class II). Cycle sequencing of treated amplicon (3µl) with dye-labelled di-deoxy chain terminating inhibitors was carried out according to manufacturer’s instructions (Thermofisher) with 1µl 3.2µM sequencing primer. The cycle sequencing protocol was the ‘standard’ one

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recommended by the manufacturer: 96oC for 1 minute followed by 25 cycles of 96oC for 10

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seconds, 50oC for 5 seconds and 60oC for 4 minutes. Unincorporated dyes were removed

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using CleanSeq™ beads (Beckman Coulter) according to manufacturer’s protocol automated with a Biomek 3000 robot (Beckman Coulter). Sequenced fragments were separated using an

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ABI3130 Genetic Analyser.

2.5 A number of different software tools have been used to compile and analyse HLA

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nucleotide sequences derived from the strategies described here including MatchTools

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(Applied Biosystems), Assign (Conexio) and SBTengine (GenDx). Validation of the most

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3. Results

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recent update to this method has been performed with SBTengine v3.11.

In the early incarnations of this sequencing strategy, both HLA alleles were amplified together in a gene-specific manner, exons 2 and 3 for class I and exon 2 for class II. In this updated strategy, heterozygous alleles are amplified in isolation using group-specific primers. Exons 2-4 of HLA-A and B, exons 2-6 of HLA-C and exons 2+3 of HLA-DRB1 and DQB1 are sequenced in both directions. HLA-C exon 7 is sequenced twice on the sense strand, and

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ACCEPTED MANUSCRIPT HLA-A exon 4 is partially sequenced on the antisense strand as the antisense primer anneals within the 3’ end of the exon. The methods and strategies described here have been evaluated using Quality Assurance samples and the methods finally presented here have been evaluated using DNA provided by

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UK NEQAS for H&I Educational typing scheme (https://neqas.welsh-blood.org.uk ), templates that had previously been HLA typed to at least 2nd field and assessed by NEQAS

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according to the consensus of all participating laboratories. This method was also validated

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against 10 samples of unknown HLA type provided by the UCLA International DNA Exchange Scheme (http://pathology.ucla.edu/ ) August 2016 and November 2016 send-outs.

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results from the Quality Assurance schemes.

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These validations showed 100% concordance between our SBT results and the consensus

Despite best efforts, ambiguous allele combinations may still be found which require

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additional primer sets to resolve. Allele ambiguities arise when one or more more alleles

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differ outside of the region sequenced while genotype ambiguities are heterozygous combinations of alleles which have the same nucleotide sequence in the region sequenced.

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Figure 1 shows an example of genotype ambiguities and how to resolve these for a sample which is HLA-A*02, 24. This sample was sequenced, exons 1-4, in 2011 with both alleles

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present (Fig 1A) or the A*02 allele was amplified and then sequenced in isolation (Fig 1B). Sequencing both alleles gives 11 possible heterozygous combinations which is equivalent to a low resolution result (Fig 1A). Sequencing the amplified A*02 allele in isolation shows that A*02:01:01 is present and therefore the full result for this sample is A*02:01:01/02L, 24:02:01:02L. Admittedly, with this sequencing strategy we cannot exclude A*02:01:01:02L but this is a very rare allele.

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ACCEPTED MANUSCRIPT Discussion Over a period of almost 20 years variations of the SBT strategy described here have been used to unambiguously type HLA alleles and characterize new alleles. Incarnations of the methodology have been fully validated in laboratories in UK and New Zealand and passed

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EFI and ASHI Accreditations there, respectively In the initial development of this sequencing based typing strategy between 1998 and 2003

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many samples were received from UK HLA laboratories which gave equivocal PCR-SSP

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types. In this period such laboratories were experiencing DNA typing for the first time, many following the “Phototyping” SSP protocol developed in Oxford (Bunce et al, 1996) and

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comparing their DNA results with their established serological techniques. Such a ‘Reference

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Service’ helped these laboratories identify and characterise many new alleles which are listed in Table 2. The size of the class I gene is more amenable to full length sequencing which means we have been able to submit full length sequences in many cases, introns as well as

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exons. Samples from many ethnicities have been sequenced and almost all the main antigens and allele groups have been sequenced. Some new alleles discovered and characterized by sequencing are recombinants of two different alleles (e.g. A*02:65, B*15:42, B*15:68,

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B*40:16) which is not reflected in the final allele name. Another newly discovered allele,

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HLA-A*68:24 was discovered in 3 different samples, by 3 different UK laboratories in as many months (Davey et al 2004) showing that this allele, despite the name, is clearly common in the UK population at least. The methods described here represent many years of adapting a sequencing strategy in response to increasing HLA polymorphisms. This method is designed to enable the user to identify all previously unseen SNPs in critical exons, adding/removing exons as required by the addition/removal of cycle sequencing primer sets to the final plate. Resolving ambiguous combinations of alleles is also possible providing the alleles can be amplified in isolation. 8

ACCEPTED MANUSCRIPT Where no primer set is available to accomplish this, it is a relatively simple matter to design additional primers to ensure alleles can be separated by PCR (e.g. new reverse primer specific for HLA-C*07 which does not amplify C*18 (Supplementary Table 2)). The future of high resolution HLA typing lies in the world of Next Generation Sequencing

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which will eventually become the high throughput method of choice for all HLA typing. NGS has the potential to provide HLA genotypes in which the phase of the complex patterns

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of HLA polymorphisms is ensured. There is still a lot of development to be done in the

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commercial hardware and reagents available for HLA typing before this will become a routine tool. The amplification primers and strategies described here are suitable tools to help

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in the application of NGS and are thus a valuable resource for the future NGS method of high

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resolution HLA typing.

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References

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Bunce, M., O’Neill, C.M., Barnardo, M.C., Browning, M.J., et al 1995 Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by

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PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens 46,355–367.

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Cao, K., Chopek, M., Fernandez-Vina, M., 1999. High and intermediate resolution DNA typing systems for class I HLA-A, -B, -C genes by hybridization with sequence-specific oligonucleotide probes (SSOP). Rev Immunogenet 1,177–208. Cereb, N., Yang, S.Y., 1997. Dimorphic primers derived from intron 1 for use in the molecular typing of HLA-B alleles. Tissue Antigens, 50, 74 -76.

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ACCEPTED MANUSCRIPT Cereb, N., Maye, P., Lee, Y., Kong, S., Yang, S.Y., 1995. Locus-specific amplification of HLA class I genes from genomic DNA: locus-specific sequences in the first and third introns of HLA-A, -B, and –C alleles. Tissue Antigens, 45, 1-11. Davey S, Carter V, Goodman R, Day S, Brown C, Morris J, Key T, Bendukidze N & Dunn

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PPJ. 2005 A new HLA-A allele, HLA-A*6824, identified in three unrelated individuals. Tissue Antigens 65, 485-487.

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Dunn PPJ, Day S, Williams S, Bendukidze N (2003) HLA sequencing as a tissue typing tool.

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In: Goulding N, Seward C (eds) Pediatric hematology. Humana Press, Totowa, NJ. 233-

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236.

Dunn, P.P.J., Day, S., Bendukidze, N., 2005. A method for HLA-DQB1 sequencing based

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typing using antigen-specific nucleotide sequences in introns 1 and 2. Tissue Antigens

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66, 99-106.

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Dunn, P.P.J., 2011. HLA typing: techniques and technology, a critical appraisal. Int J Immunogenet 38,463–473.

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Dunn, P.P.J., 2015. Novel approaches and technologies in molecular HLA typing. Methods in Molecular Biology (Springer). 1310, 213-230.

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Guo, Z., Hood, L., Petersdorf, E.W., 1999. Oligonucleotide arrays for high resolution HLA typing. Rev Immunogenet 1, 220–230. Holcomb, C.L., Höglund, B., Anderson, M.W., Böhme, I.et al., 2011. A multi-site study using high resolution HLA genotyping by next generation sequencing. Tissue Antigens 77, 206–217.

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ACCEPTED MANUSCRIPT Kotsch, K., Wehling, J., Kohler, R., Blasczyk, R., 1997. Sequencing of HLA class I genes based on the conserved diversity of the non-coding regions: sequence-based typing of the HLA-A gene. Tissue Antigens. 50, 178-191. Kotsch, K., Wehling, J. Blasczyk, R, 1999. Sequencing of HLA class II genes based on the

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conserved diversity of the non-coding regions: sequencing based typing of HLA-DRB genes. Tissue Antigens, 53, 486-497.

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McCluskey, J., Peh, C.A., 1999. The human leukocyte antigens and clinical medicine: an overview. Rev Immunogenet 1, 3–20.

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Sanger, F., Nicklen, S. Coulson, A.R., 1977. DNA sequencing with chain-terminating

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inhibitors. Proceedings of the National Academy of Sciences of the USA. 74, 5463-5467. Tait, B.D., 2011 The ever expanding list of HLA alleles: changing HLA nomenclature its

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relevance to clinical transplantation. Transplant Rev 25, 1–8. Taylor, C.J., Bolton, E.M., Bradley, A.J., 2011 Immunological considerations for embryonic

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and induced pluripotent stem cell banking. Philos Trans Roy Soc B 366, 2312–2322.

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Tiercy, J-M., 2002. Molecular basis of HLA polymorphism: implications in clinical transplantation. Transpl Immunol 9,173–180.

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ACCEPTED MANUSCRIPT Table 1 PCR conditions and parameters for HLA class I and class II amplifications

HLA-A, B, C

HLA-DRB1, DQB1 Volume (µl)

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6.0 1.0 9.8 25

AmpliTaq 10x buffer dNTP 10mM each 25 mM MgCl2 Betaine 5M AmpliTaq Gold Polymerase DNA 20ng/µl Primers 5µM each H2O TOTAL

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DNA 20ng/µl Primers 5µM each H2O TOTAL

Volume (µl) 2.5 1.0 1.8 2.5 0.2 6.0 1.0 10.0 25

Class II PCR Program Initial hot start 96oC, 12 minutes 10 cycles of 96oC 30 seconds 65oC 30 seconds 72oC 1 minute 15 cycles of 96oC 30 seconds 61oC 30 seconds 72oC 1 minute 15 cycles of 96oC 30 seconds 57oC 30 seconds 72oC 1 minute Final extension 72oC, 5 minutes

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Class I PCR program Initial denaturation at 94oC, 2 minutes 5 cycles of 94oC, 20 seconds 70oC, 15 seconds 72oC, 3m, 30s 5 cycles of 94oC, 20 seconds 68oC, 15 seconds 72oC, 3m, 30s 25 cycles of 94oC, 20 seconds 66oC, 15 seconds 72oC, 3m, 30s Final extension 72oC, 10 minutes

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2.5 1.0 1.8 2.5 0.4

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Expand 10x buffer dNTP 10mM each 25 mM MgCl2 DMSO Expand Polymerase

Reagent

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Reagent

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T P

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Figure 1A

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Figure 1B

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Figure 1 sequencing based typing in the presence of both alleles (A) or sequencing a separated allele (B). Sequences were analysed using SBTengine (GenDx)

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ACCEPTED MANUSCRIPT Table 2. New and confirmed HLA alleles characterised by the methods described. Ethnicity is as listed with submission, blank is ‘unknown’ ethnicity. Confirmatory means sequence confirms previous submission. Allele names use the latest 2010 nomenclature. YEAR

CONTACT

Caucasoid Caucasoid

2011 2003 1997 1999 2003 2003 2003 2007 2003 2003 1997 2003 1999 1998 2004 2004 1997 1999 1998 2003 2007 2007 2003

Cardiff Dublin Cardiff Oxford Birmingham Newcastle Cambridge Auckland NZ Cardiff Cardiff Manchester Bristol Oxford Oxford Bristol Bristol London UCLA/Bristol Oxford Bristol Auckland NZ Auckland NZ Cambridge, Newcastle, London

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Caucasoid Hispanic African Afro-Caribbean Pacific Islander Pacific Islander Caucasoid

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Caucasoid Caucasoid Caucasoid Caucasoid Caucasoid African Asian Caucasoid Caucasoid Asian (Korea) Caucasoid Caucasoid Caucasoid Caucasoid Caucasoid Caucasoid

Asia (Vietnam) Pacific Islander Pacific Islander

Afro-Caribbean African

Confirmatory

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Caucasoid Caucasoid Caucasoid Caucasoid Asian (Philipino) Caucasoid Caucasoid

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B*07:02:45 B*07:16 B*08:08N B*08:10 B*08:19N B*13:08 B*14:02:09 B*15:01:02 B*15:42 B*15:57 B*15:68 B*15:69 B*15:78 B*15:83 B*27:12 B*35:27 B*35:32 B*35:41 B*37:06 B*39:24 B*40:10 B*40:10:01 B*40:12 B*40:14:03 B*40:16 B*40:26

Asian (India) Caucasoid Caucasoid Caucasoid

NOTES Renamed from A*01:34N Confirmatory

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ETHNICITY

A*01:01:38L A*02:01:01:02L A*02:24 A*02:32N A*0262 A*02:65 A*02:67 A*02:113:01N A*03:01:01:02N A*03:11N A*11:05 A*1115 A*24:17 A*24:18 A*24:29 A*24:44 A*29:03 A*31:02 A*31:03 A*33:07 A*34:01:01 A*34:01:02 A*68:24

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HLA ALLELE

2015 1999 1999 1999 2003 2001 2013 1998 1998 1999 2001 2004 2003 2003 1997 1998 1999 2005 2004 1999 1997 2012 1997 2003 1997 1999

Auckland NZ Newcastle Newcastle Bristol Cardiff Newcastle Auckland NZ London Liverpool Oxford Dynal UK Bristol Bristol Newcastle Southampton Cardiff Oxford Bristol Cambridge Oxford London Auckland NZ UCLA/Bristol Newcastle London Newcastle

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Confirmatory Confirmed in3 UK labs in 3 months

Renamed from B*13:08Q

Confirmatory Confirmatory Confirmatory Confirmatory

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CONTACT

Pacific Islander

2014 2015 2014 2001 2004 1999 2008 2000 1998 2003 1997 1999 1997 1999

Auckland NZ Auckland NZ Auckland NZ Glasgow Bristol Oxford Auckland NZ Bristol Cardiff Bristol Cardiff Bristol Argentina Birmingham

1999 2001 2001 1999 2015 1999 1999 1999 2000 2004 1998 2003 2014

London Dynal Dynal Oxford Auckland NZ Bristol Oxford Oxford Bristol Bristol Bristol Newcastle Auckland NZ

Caucasoid Amerindian Caucasoid

African South Africa East African

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East African East African Caucasoid Caucasoid Asian (Pakistan)

Caucasoid Caucasoid

2002 1998 2001 2014 1998 2000 1997 1997 2001 2003 2002 2000

DQB1*03:05:03 DQB1*06:11:02 DQB1*06:15 DQB1*06:21

Caucasoid Caucasoid Caucasoid Caucasoid

2016 1998 1998 2014

Leicester London London Auckland NZ

DPB1*63:01 DPB1*96:01

Hispanic/Mexican

1999 2002

Manchester Munich Germ

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Caucasoid Asian (Korean) Pacific Islander Caucasoid Asian (Pakistan) Caucasoid Caucasoid

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DRB1*01:09 DRB1*07:04 DRB1*12:08 DRB1*14:08 DRB1*15:07 DRB1*15:11 DRB3*01:02 DRB3*01:01:03 DRB3*01:08 DRB4*01:03:04 DRB5*01:12 DRB5*02:05

Confirmatory Confirmatory

Confirmatory

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Afro-Caribbean Japanese/Black Caucasoid

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Caucasoid East African

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C*01:04 C*02:02:05 C*02:05 C*03:04:02 C*03:279 C*04:01:01:01 C*04:04 C*04:07 C*06:06 C*07:28 C*08:05 C*12:09 C*12:125

African

NOTES

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ETHNICITY

B*40:285 B*40:303 B*41:29 B*44:09 B*44:13 B*44:15 B*44:59 B*45:04 B*47:03 B*48:08 B*50:02 B*51:01:04 B*51:13 B*51:19

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ALLELE

London Cardiff Dynal Auckland NZ Cardiff Glasgow Cardiff Cardiff

Confirmatory

Confirmatory

Hamburg, Germ

Italy Birmingham Newcastle

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Confirmatory