Extended HLA-DPB1 polymorphism: An RNA approach for HLA-DPB1 typing

Transcription

1 Chapter 4 Extended HLA-DPB1 polymorphism: An RNA approach for HLA-DPB1 typing Judith Reinders, Erik Rozemuller, Rogier van Gent, Yvonne Arts-Hilkes, Jan van den Tweel and Marcel Tilanus Department of Pathology, University Medical Center Utrecht, The Netherlands Immunogenetics, 2005, 2005: 57: chapter 4.indd 73 1/5/ :19:51

2 Abstract Most of the 119 HLA-DPB1 alleles are defined by polymorphism in six hypervariable regions (HVRs) in exon 2 of the HLA-DPB1 gene. We questioned how DPB1 polymorphism was represented in the entire coding region. A RNA sequencing based typing (SBT) approach was developed for the identification of HLA-DPB1 polymorphism from the 5 untranslated region (UTR) through the 3 - UTR. B-cell lymphoblastoid cell lines, encoding 16 different DPB1 alleles, were studied. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5 and 3 -UTR. Four new HLA-DPB1 alleles were identified, DPB1*0502, DPB1*0602, DPB1*0802 and DPB1*0902, which have exon 2 sequences identical to other DPB1 alleles but differ in the extended region. The additional polymorphism represents two main polymorphic lineages in the DPB1 alleles. Among the HVRs in exon 2, only HVR F correlates with these two main lineages. Introduction The human leukocyte antigen (HLA) class II genes HLA-DR, -DQ, and -DP are located centromeric in the HLA region on the short arm of chromosome 6. HLA-DP is composed of the α-chain, encoded by the HLA-DPA1 gene and the β-chain, encoded by HLA-DPB1 [1]. DPB1 allelic polymorphism is concentrated in six hypervariable regions (HVRs) in exon 2, A through F [2, 3] contributing in the formation of the five pockets present in the peptide-binding groove. The DP molecule is capable of binding peptides and presenting those to T cells [4-7]. E.g. HLA-DPB1 amino acids 84 through 87 in HVR F are involved in the formation of pocket 1 and are crucial for peptide binding and T cell allorecognition [4]. The generation of DPB1 allelic polymorphism has mainly evolved from segmental exchange of sequence motifs between the six HVRs. Compared to other HLA class II genes, HLA-DPB1 evolution is thought to have evolved relatively rapidly. Within the human DPB1 phylogenetic tree three major branches of DPB1 alleles have been deduced based upon exon 2 polymorphism [8]. HLA-DPB1 has merely been investigated by the identification of polymorphism in exon 2, since exon 2 polymorphism determines the specificity for peptide binding in the groove. Polymorphism beyond exon 2, extended DPB1 polymorphism, has been addressed in this study. To facilitate DPB1 full length coding sequencing, an RNA based approach was developed. Material and methods EBV transformed human B-cell lymphoblastoid cell lines Table 1 provides an overview of the HLA-DPB1 alleles studied from fifteen EBV transformed human B-cell lymphoblastoid cell lines (International Histocompatibility Working Group, IHWG; and cell line JRPAY from our institute. Original reference cell lines were used if possible. B- 74 chapter 4.indd 74 1/5/ :20:05

3 Chapter 4 cell lymphoblastoid cell lines were cultured in RPMI1640 medium (Invitrogen Ltd., Paisley, United Kingdom), supplemented with 10% heat inactivated FCS (Invitrogen), 100 IE/µl penicillin (Yamanouchi, Leiderdorp, The Netherlands), 100 µg/ml streptomycine (Fisiopharma, Milan, Italy) and 2.7% NaHCO 3 (Invitrogen) in a 37 C, 5% CO 2 incubator. RNA isolation and cdna synthesis Total RNA was isolated from approximately 5 million cells of each cell line with Trizol reagent according to the manufacturer s protocol (Invitrogen). cdna synthesis was performed with the Superscript RNase H- MLV-Reverse Transcriptase kit (Invitrogen). The quality of the cdna was verified by the amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [9]. Table 1: HLA-DPB1 alleles identified in 15 International Histocompatibility Working Group (IHWG) cell lines Assigned HLA-DPB1 allele a Sample name Accession number DPB1* IHW09021 AY DPB1* IHW09049 AY DPB1* IHW09032, IHW09366, IHW09376 AY DPB1*0202 IHW09020 AY DPB1* IHW09005, IHW09014, IHW09051, IHW09095 AY DPB1*0402 IHW09291, IHW09373 AY DPB1*0501 JRPAY AY DPB1*0502 IHW09373 AY DPB1*0602 IHW09021 AY DPB1*0802 IHW09366 AY DPB1*0901 JRPAY AY DPB1*0902 IHW09076 AY DPB1*1301 IHW09095 AY DPB1*1401 IHW09376 AY DPB1*1501 IHW09047 AY DPB1*1901 IHW09061 AY a New alleles are shown in bold. The names listed for these sequences have been officially assigned by the WHO Nomenclature Committee. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report [11] names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report. 75 chapter 4.indd 75 1/5/ :20:05

4 RNA sequencing based typing of HLA-DPB1 Full-length HLA-DPB1 cdna, spanning 5 -UTR through 3 -UTR giving rise to a 927 bp PCR product, was amplified using amplification primers DPRNARG5.1 and DPRNARG3.1, located respectively in the 5 -UTR and the 3 -UTR. The PCR was performed in a volume of 50 µl, with final concentrations of 1.5 mm MgCl 2 (Applied Biosystems, Foster City, CA, USA), 1x PCR buffer (Applied Biosystems), 150 µm of each dntp (Invitrogen), 10 pmol of each amplification primer (Sigma-Genosys Ltd.), 0.05 units/µl of Taq Gold (Applied Biosystems) and 2 µl cdna. The mixture was denatured for 10 minutes at 95 C, followed by 32 cycles of 20 at 96 C, 30 at 55 C and 30 at 72 C, and ending with a hold at 4 C. The 927 bp HLA-DPB1 PCR product was purified with 1 unit of shrimp alkaline phosphatase (SAP) and 10 units of Exonuclease I (both from USB Corporation, Cleveland, USA), incubated at 37 C for 15 min., deactivated at 80 C for 15 min. and followed by hold at 4 C. Sequence reactions were performed with the Big Dye Terminator Ready Reaction Mix v1.1 (Applied Biosystems) and subsequently electrophoresed on an ABI 377 DNA sequencer. PCR products were sequenced in both directions with amplification and group specific sequencing primers (GSSP). The software package SBTengine (Genome Diagnostics, Utrecht, the Netherlands) was used for sequence analysis and subsequent allele assignment of DPB1 alleles. Cloning of HLA-DPB1 alleles Cloning of HLA-DPB1 alleles was performed with the TA Cloning Kit according to the manufacturer s protocol (Invitrogen). Results HLA-DPB1 alleles DPB1 allele names as assigned by the WHO Nomenclature Committee and GenBank accession numbers ( are indicated in table 1. Sequences submitted were confirmatory for exons 2 through 5 and in addition extended for exon 1, the 5 -UTR and 3 -UTR for the DPB1 alleles *010101, *020102, *030101, * and *0402. Confirmatory exon 2 sequences were obtained for all other alleles and extended with new sequences including additional polymorphism present in the other exons and the 5 -UTR and 3 -UTR. Extended HLA-DPB1 polymorphism Extended polymorphism in exons 1 through 5 and parts of the 5 -UTR and 3 -UTR of the DPB1 coding sequence was determined for 16 HLA-DPB1 alleles through PCR and sequence analysis. Table 2 summarizes the extended HLA-DPB1 nucleotide polymorphism identified in the alleles. Thirty polymorphic nucleotide 76 chapter 4.indd 76 1/5/ :20:05

5 Chapter 4 Table 2: Overview of the HLA-DPB1 extended nucleotide polymorphism 5 -UTR Exon 3 -UTR Nucleotide position Alleles * a,b t g C A c c a c T C c c G G T a c c a a c t g c t c g c g a * * * * *0902 e c a T Δ *1301 e Δ * * * * * * * * * * * * * * * * * * * * A a * A * * * * * * * * * * * * * A *0502 a A * * * *1901 d A * t - - C * * * * G t t g t C - t g t t g a c - t c t a * * * * G t t g t C - t g t t g a c - t c t a a t t * G t t g t C - t g t t g a c - t c t a a t t *0602 c G t t g t C - t g t t g a c - t c t a a t t *0402 c G t t g t C A t g t t g a c - t c * * * * * *0802 d G t t g t C - t g t t g a c - t c t a a t t Nucleotide positions, in vertical orientation, are based on the IMGT/HLA database ( - identity with the DPB1* allele, * sequence not determined, Δ nucleotide deletion. New alleles and new polymorphic positions are shown in bold. Synonymous substitutions or non-coding polymorphism is indicated in lower cases; non-synonymous substitutions in upper cases. a DPB1*0502 and DPB1* have identical exon 2 sequences b DPB1* sequence information was obtained from the IMGT/HLA database ( c DPB1*0602 and DPB1*0402 have identical exon 2 sequences d DPB1*0802 and DPB1*1901 have identical exon 2 sequences e DPB1*0902 and DPB1*1301 have identical exon 2 sequences 77 chapter 4.indd 77 1/5/ :20:05

6 positions, including 21 new polymorphic nucleotide positions, were observed in the regions beyond exon 2. Seven out of these 30 polymorphic nucleotide polymorphisms encode non-synonymous substitutions. The other 23 nucleotide polymorphisms are either synonymous substitutions or non-coding. In exon 5 a one nucleotide deletion was observed in the DPB1*0902 and DPB1*1301 alleles. DPB1 alleles have the nucleotide sequence TAAACA at nucleotide position 775 through 780. In DPB1*1301 and DPB1*0902, one adenine nucleotide is deleted: TAACA. This does not influence the stop codon TAA (codon 230). Table 3: Extended HLA-DPB1 amino acid polymorphism Exon Codon * a,b T DEAV K I L R V I * *0902 e M *1301 e * * Q - - * Q - - * M - *0502 a M - *1901 d M - * VGPM T * GGPM R T * GGPM R T * GGPM R T *0602 c - GGPM R T *0402 c - GGPM R T M *0802 d - - R T New alleles are shown in bold. Polymorphic codons in exons 1, 3 and 4 are shown. HVR F (amino acids 84-87) with the amino acid motifs DEAV, VGPM and GGPM is shown for exon 2. - identity with the DPB1* The characters indicate amino acids: T threonine, D aspartic acid, E glutamic acid, A alanine, V valine, K lysine, I isoleucine, L leucine, R arginine, M methionine, Q glutamine, G glycine, P=proline. a DPB1*0502 and DPB1* have identical exon 2 sequences b DPB1* sequence information was obtained from the IMGT/HLA database ( imgt/hla/) c DPB1*0602 and DPB1*0402 have identical exon 2 sequences d DPB1*0802 and DPB1*1901 have identical exon 2 sequences e DPB1*0902 and DPB1*1301 have identical exon 2 sequences 78 chapter 4.indd 78 1/5/ :20:05

7 Chapter 4 New HLA-DPB1 alleles Table 3 summarizes the extended amino acid polymorphism in the coding region for the 16 HLA-DPB1 alleles. Four new DPB1 alleles were identified with identical exon 2 sequences to other alleles, but have unique extended polymorphism. DPB1*0502 has an exon 2 sequence identical to DPB1*030101, but differs by one nucleotide in exon 4 resulting in a non-synonymous substitution. DPB1*0602 is identical to DPB1*0402 for its exon 2 sequence, but only differs in exon 3 resulting in a non-synonymous substitution. The DPB1*0802 allele, identical with DPB1*1901 for exon 2, differs at 21 nucleotide positions resulting in three non-synonymous substitutions. The DPB1*0902 allele, identical for exon 2 with DPB1*1301, has three unique nucleotides in the 5 -UTR and exon 1, resulting in one non-synonymous substitution (tables 2 and 3). HLA-DPB1 exon 2 and extended polymorphism DPB1 alleles can be grouped by characteristic amino acids KI and RT at codons 96 and 170 respectively (table 3). Lineage KI includes the DPB1*010101, *010102, *030101, *0501, *0502, *0901, *0902, *1301, *1401, *1501 and *1901 alleles. Lineage RT the DPB1*020102, *0202, *040101, *0402, *0602 and *0802 alleles. Within both lineages limited variation is observed in exon 1, 3 and 4 for several DPB1 alleles, present in codons -14, 178, 194, 205 and 215 (table 3). Subsequently, extended polymorphism and assigned lineages were correlated with exon 2 polymorphism. Among the 6 HVRs present in exon 2 only HVR F correlated with the KI and RT lineages defined by exons 3 through 6. HVR F is encoded by codons 84 through 87 where three different amino acid motifs are present: GGPM, VGPM and DEAV. All alleles with the GGPM motif have the RT motif in exons 3 through 6. DPB1*1501 has a VGPM motif in combination with the KI motif. All alleles with the DEAV motif, except the DPB1*0802 allele, have the KI motif in exons 3 through 6. The DPB1*0802 allele has the RT motif in exons 3 through 6 (table 3). Diversification of HLA-DPB1 alleles Based upon HVR F, KI and RT motifs and polymorphism in exon 4, diversification of the DPB1 alleles is illustrated in figure 1. The motifs DEAV-KI- RVI and GGPM-RT-RVI might reflect two ancient DPB lineages. From these two lineages, the exceptions DPB1*1501 and DPB1*0802 could have evolved by a recombination, occurring between HVR F and exon 3, and in case of DPB1*1501 an additional point mutation. 79 chapter 4.indd 79 1/5/ :20:05

8 Figure 1: Evolutionary relationship of extended HLA-DPB1 polymorphism 80 chapter 4.indd 80 1/5/ :20:05

9 Chapter 4 Discussion HLA-DPB1 polymorphism was initially identified by six DP specificities with the primed lymphocyte typing test (PLT) [1, 10]. With the introduction of molecular techniques six HVRs were identified in exon 2 of the DPB1 molecule [2, 3], leading to the highly polymorphic HLA-DPB1 gene [11]. Additional polymorphism in other exons of DPB1 was revealed by SBT of cdna, using RNA as template, resulting in two main lineages, which correlate with HVR F in exon 2. Additional variation within each lineage defines the individual alleles. The RVI motif in exon 4 is shared by alleles in both lineages, indicating that this might be an ancient motif in DPB1 polymorphism. Several DPB1 alleles share the RMI motif in exon 4. Four new HLA- DPB1 alleles were identified based upon differences beyond exon 2. As a result of the new nomenclature strategy of the nomenclature committee, allele names in DPB1 do not reflect a relation between the alleles, which is the case for the first two digits of the allele names of the classical HLA loci. No direct relationship between the alleles DPB1*0501 and DPB1*0502 exists as the allele names might suggest, whereas DPB1*0502 and DPB1* have identical exon 2 sequence, and are expected to present the same peptide repertoire and that is not reflected in the allele names. References 1. Marsh SG, Parham P, and Barber LD, The HLA Factsbook. 2000, San Diego, San Francisco, New York, Boston, London, Sydney, Tokyo: Academic Press. 2. Laux G, et al., A new epitope-based HLA-DPB matching approach for cadaver kidney retransplants. Transplantation, (9): p Naruse TK, et al., Cloned primed lymphocyte test cells recognize the fourth, fifth, and sixth hypervariable regions at amino acid positions of the DPB1 molecule. Hum Immunol, (2): p Diaz G, et al., Functional analysis of HLA-DP polymorphism: a crucial role for DPbeta residues 9, 11, 35, 55, 56, 69 and in T cell allorecognition and peptide binding. Int Immunol, (5): p Berretta F, et al., Detailed analysis of the effects of Glu/Lys beta69 human leukocyte antigen-dp polymorphism on peptide-binding specificity. Tissue Antigens, (6): p Diaz G, et al., HLA-DPbeta residue 69 plays a crucial role in allorecognition. Tissue Antigens, (1): p Castelli FA, et al., HLA-DP4, the most frequent HLA II molecule, defines a new supertype of peptide-binding specificity. J Immunol, (12): p Gyllensten U, et al., Rapid allelic diversification and intensified selection at antigen recognition sites of the Mhc class II DPB1 locus during hominoid evolution. Tissue Antigens, (3): p chapter 4.indd 81 1/5/ :20:05

10 9. Ercolani L, et al., Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. J Biol Chem, (30): p Shaw S, Johnson AH, and Shearer GM, Evidence for a new segregant series of B cell antigens that are encoded in the HLA-D region and that stimulate secondary allogenic proliferative and cytotoxic responses. J Exp Med, (3): p Marsh SG, et al., Nomenclature for Factors of the HLA System, Hum Immunol, (5): p chapter 4.indd 82 1/5/ :20:05