Human Immunology 76 (2015) 30–35

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Seven novel HLA alleles reflect different mechanisms involved in the evolution of HLA diversity: Description of the new alleles and review of the literature Martina Adamek, Cornelia Klages, Manuela Bauer, Evelina Kudlek, Alina Drechsler, Birte Leuser, Sabine Scherer, Gerhard Opelz, Thuong Hien Tran ⇑ Department of Transplantation Immunology, Institute of Immunology, University of Heidelberg, Heidelberg, Germany

a r t i c l e

i n f o

Article history: Received 3 February 2014 Accepted 3 December 2014 Available online 11 December 2014 Keywords: Human leukocyte antigen Polymorphism Gene conversion Recombination New alleles

a b s t r a c t The human leukocyte antigen (HLA) loci are among the most polymorphic genes in the human genome. The diversity of these genes is thought to be generated by different mechanisms including point mutation, gene conversion and crossing-over. During routine HLA typing, we discovered seven novel HLA alleles which were probably generated by different evolutionary mechanisms. HLA-B*41:21, HLADQB1*02:10 and HLA-DQA1*01:12 likely emerged from the common alleles of their groups by point mutations, all of which caused non-synonymous amino acid substitutions. In contrast, a deletion of one nucleotide leading to a frame shift with subsequent generation of a stop codon is responsible for the appearance of a null allele, HLA-A*01:123N. Whereas HLA-B*35:231 and HLA-B*53:31 were probably products of intralocus gene conversion between HLA-B alleles, HLA-C*07:294 presumably evolved by interlocus gene conversion between an HLA-C and an HLA-B allele. Our analysis of these novel alleles illustrates the different mechanisms which may have contributed to the evolution of HLA polymorphism. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

1. Introduction The human leukocyte antigen (HLA) loci rank among the most polymorphic genes in the human genome [1]. According to the IMGT/HLA database release 3.15.0 (January 2014) (http://www.ebi.ac.uk/ipd/imgt/hla/stats.html), 10,533 HLA alleles have been recorded to date [2]. While the first catalogue of common and well-documented (CWD) alleles [3] included 721 alleles, the 2012 updated CWD catalogue [4] designated 1122 alleles of the HLA-A, -B, -C, DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as CWD alleles. Several molecular mechanisms are believed to play an important role in the generation of novel HLA alleles, such as point mutation, gene conversion and crossing-over [5–8]. Point mutation can occur as substitution or insertion/deletion of a single nucleotide. A nucleotide substitution can lead to synonymous (without amino acid altering) or non-synonymous (change of amino acid coding) nucleotide exchange. Insertion/deletion of one nucleotide or more ⇑ Corresponding author at: Department of Transplantation Immunology, Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany. Fax: +49 6221 564200. E-mail address: [email protected] (T.H. Tran).

(other than three or the multiple of three) nucleotides often causes a frame shift with subsequent generation of a premature stop. However, an insertion/deletion of three nucleotides, e.g. in HLADQA1 codon 56, can lead to a frameshift, but not a stop codon (http://www.ebi.ac.uk). Gene conversion and crossing-over both fall under the general heading of recombination [9]. While gene conversion is the donation of a DNA segment from one chromosome to its homolog, crossing-over is a bidirectional sequence exchange between homolog chromosomes. A gene conversion can be intragenic (also called intralocus or interallelic, occurring between alleles of the same gene locus) or, more rarely, intergenic (also called interlocus, occurring between different gene loci) [10,11]. We have recently identified seven novel HLA alleles which can be used as examples to illustrate how different mechanisms have shaped the genetic diversity of the HLA complex.

2. Materials and methods Genotyping was performed by polymerase chain reaction sequence-based typing (PCR-SBT). The sequences of exons 2 and 3 (exon 4 in addition for HLA-A*01:123N) were determined using

http://dx.doi.org/10.1016/j.humimm.2014.12.007 0198-8859/Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

M. Adamek et al. / Human Immunology 76 (2015) 30–35

the CTS-SEQUENCE HLA-A, HLA-B, HLA-C, HLA-DQB1, HLA-DQA1 Kits (CTS, Heidelberg, Germany) and confirmed by the Protrans S3 HLA-A, S4 HLA-B, S3 HLA-C, S3 HLA-DQB1 Kits (Protrans, Hockenheim, Germany) according to the manufacturers’ instructions. The alleles were sequenced separately in both directions. Sequences were analyzed using the Sequence Pilot software (JSI medical systems, Kippenheim, Germany). Nucleotide changes of the novel alleles were confirmed by PCR with sequence-specific primers (PCR-SSP) under previously described conditions [12]. Serological typing of HLA-A and HLA-B was carried out with the CTS-Trays HLA-AB1, HLA-AB2 (CTS, Heidelberg, Germany) and the Lymphotype HLA-AB 120 Special Trays (Bio-Rad Medical Diagnostics, Dreieich, Germany) according to the manufacturers’ instructions. 3. Results 3.1. Point mutations (single nucleotide substitutions) leading to nonsynonymous amino acid substitutions (HLA-B*41:21, HLADQB1*02:10 and HLA-DQA1*01:12) 3.1.1. Case 1 HLA-B*41:21 was identified in a male patient from the Middle East who was scheduled for hematopoietic stem cell transplantation at our hospital. The most similar allele is HLA-B*41:01, from which B*41:21 differs at nucleotide (nt) 412 in exon 3, where an exchange from A to G (codon 114, AAC to GAC) results in a coding change from asparagine, which is neutral-polar to the polar, negatively charged aspartic acid (114N to 114D). This residue forms part of the peptide binding pocket [13], hence the amino acid exchange could alter the binding properties and specificity of the HLA molecule. It is likely that HLA-B*41:21 emerged from HLAB*41:01 by point mutation. Serologically, HLA-B*41:21 was detected by commercially available antisera against the common HLA-B41 alleles. The name B*41:21 has been officially assigned by the WHO Nomenclature Committee in June 2012. The nucleotide sequence is available at GenBank (accession number HE817759). The complete typing of the patient was HLAA*01:01, *03:01; HLA-B*41:21, *44:02; HLA-C*16:04, *17:01; HLA-DRB1*04:04, *10:01; HLA-DQB1*03:02, *05:01; and HLADQA1*01:04, 03:01. 3.1.2. Case 2 HLA-DQB1*02:10 was identified in a Caucasian female living kidney donor. The most similar allele is HLA-DQB1*02:02:01, from which HLA-DQB1*02:10 differs by one nt at position 520 in exon 3, where an exchange from G to A (codon 142, GTT to ATT) results in a coding change: valine (142V) is substituted by isoleucine (142I). This site is part of the b-domain which interacts with the CD4 co-receptor [13]. Since both amino acids, valine and isoleucine, are non-polar, an influence on the stability of the complex with CD4+ T-lymphocytes is rather unlikely. HLA-DQB1*02:10 presumably emerged from HLA-DQB1*02:02:01 by point mutation. The name HLA-DQB1*02:10 has been officially assigned by the WHO Nomenclature Committee in November 2012. The nucleotide sequence is available at GenBank (accession number HF 545657). The complete typing of the donor was HLA-A*11:01, *30:04; HLA-B*41:01, *51:01; HLA-C*15:02, *17:01; HLA-DRB1*07:01, ; HLA-DQB1*02:02, *02:10, and DQA1*02:01, . 3.1.3. Case 3 HLA-DQA1*01:12 was identified in a deceased kidney donor of unknown ethnic origin. Compared with the most similar allele HLA-DQA1*01:01:01, HLA-DQA1*01:12 shows one nt change at position 574 in exon 3, where codon 169 exhibits a coding change

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(GAG to CAG) from the polar, negatively charged glutamic acid (169E) to the neutral-polar glutamine (169Q). Since this polymorphic site is located in exon 3 which encodes the a2 domain, the amino acid exchange should have no influence on the peptide binding properties of the HLA molecule. It is likely that HLADQA1*01:12 emerged from HLA-DQA1*01:01:01 by point mutation. The name HLA-DQA1*01:12 has been officially assigned by the WHO Nomenclature Committee in June 2013. The nucleotide sequence is available at GenBank (accession number HG315526). The complete typing of the donor was HLA-A*11:01, *24:01; HLA-B*37:01, *55:01; HLA-C*03:03, *06:02; HLA-DRB1*10:01, *13:01; HLA-DQB1*05:01, *06:03; and HLA-DQA1*01:03, 01:12. 3.2. Point mutation (deletion) leading to a frame shift which results in a stop codon (HLA-A*01:123N) 3.2.1. Case 4 HLA-A*01:123N was identified in a Caucasian male patient awaiting kidney transplantation. The new allele attracted our attention because PCR-SSP and serology had shown discrepant results. While PCR-SSP typing indicated the allele combination HLA-A*01,*24, serological typing only revealed positive reaction with HLA-A24 antiserum. Sequencing confirmed the heterozygous result of the PCR-SSP, showing the combination of HLA-A*24:02 with a new HLA-A*01 null allele which was subsequently named HLA-A*01:123N. HLA-A*01:123N has one nt deletion (cytosine) at base position 610 in exon 3 of HLA-A*01:01:01:01. The deletion results in a coding change at codon 180, where CAG becomes – AG and the reading frame is changed to AGC. This frame shift leads downstream to a stop codon at codon 189 in exon 4 (TGA). The mutation is therefore associated with the generation of a truncated peptide, explaining the lack of expression (no detection of HLA-A1 in our serological testing) and confirming the new allele as a null allele (Fig. 1). The name HLA-A*01:123N has been officially assigned by the WHO Nomenclature Committee in October 2012. The nucleotide sequence is available at GenBank (accession number HF545655). The patient’s complete typing result was HLAA*01:123N, *24:02; HLA-B*27:05, *35:01; HLA-C*02:02, *06:02; HLA-DRB1*01:01, *11:01; HLA-DQB1*03:01, *05:01; and HLADQA1*01:01, *05:05. 3.3. Intralocus gene conversion (HLA-B*35:231 and HLA-B*53:31) 3.3.1. Case 5 HLA-B*35:231 was discovered during confirmatory typing of a female voluntary bone marrow donor of unknown ethnic origin. The most closely related allele is HLA-B*35:50. HLA-B*35:231 has one nucleotide change at position 419 in exon 3 of HLA-B*35:50, where an exchange of C to T (codon 116, TCC to TTC) results in a coding change from the neutral-polar amino acid serine (116S) to the neutral-nonpolar amino acid phenylalanine (116F). Since this residue is part of the peptide binding pocket [13], this amino acid exchange could alter the binding properties and specificity of the HLA-molecule. The novel allele was typed as HLA-B35 with serological reagents. HLA-B*35:231 may theoretically have evolved from HLAB*35:50 by point mutation at codon 116. However, this appears to be a rather unlikely event due to the low allele frequency of HLA-B*35:50 (allele frequency in European Caucasians < 0.00006). Compared to HLA-B*35:50, HLA-B*35:03:01 is much more common (allele frequency in European Caucasians = 0.01536) [14]. An analysis of the sequences of all known HLA class I alleles showed that all alleles of the HLA-B*35:03 group (HLA-B*35:03:01–HLAB*35:03:12) bear the sequence motif TTC at codon 116 (similar to the new allele HLA-B*35:231). HLA-B*35:03:01 differs from HLA-B*35:231 in three nucleotide positions: codon 9 (CAC), codon

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Fig. 1. Nucleotide alignment of the novel null allele HLA-A*01:123N with HLA-A*01:01:01:01. A single nucleotide deletion at codon 180 leads to a shift in the reading frame, which results in a stop-signal at codon 189. The alignment was generated using the IMGT/HLA Sequence Alignment Tool (http://www.ebi.ac.uk). Only part of exon 3 (codon 151 to codon 200) is shown.

11 (TCC) and codon 12 (GTG). These sequence motifs can be found in HLA-B alleles other than HLA-B*35, such as HLA-B*18:01, HLAB*27:02, HLA-B*37:01 or HLA-B*40:02. Therefore, we believe that HLA-B*35:231 may have arisen from HLA-B*35:03:01 (‘‘acceptor allele’’) by gene conversion with another HLA-B allele (‘‘donor allele’’). To determine whether a crossing-over may have created the new allele, we sequenced exon 1 of HLA-B*35:231. Whereas HLA-B*18:01, HLA-B*27:02, HLA-B*37:01 and HLA-B*40:02 all have a T at nt 72, HLA-B*35:231 has a C at nt 72 – similar to other HLA-B*35 alleles. Therefore, a crossing-over event can be excluded. Fig. 2a shows the putative gene conversion (‘‘sequence donation’’) from HLA-B*18:01:01:01 to HLA-B*35:03:01, giving rise to HLAB*35:231. The name HLA-B*35:231 has been officially assigned by the WHO Nomenclature Committee in April 2013. The nucleotide sequence is available at GenBank (accession number HF912276). The complete typing of the donor was HLA-A*03:01, *11:01; HLA-B*35:231, *51:01; HLA-C*03:03, *04:01; HLA-DRB1*14:54, *09:01; HLA-DQB1*03:03, *05:03; and HLA-DQA1*01:04, *03:02. 3.3.2. Case 6 HLA-B*53:31 was identified in a Caucasian male patient scheduled for hematopoietic stem cell transplantation at our hospital. The patient had previously been typed in another laboratory which reported HLA-B*51:78, *53:04. It is noteworthy that HLA-B*51 and

HLA-B*53 have similar sequences and many PCR-SBT-typing kits do not provide the appropriate primers for separating these alleles in the sequencing reaction, leading to erroneous allele assignment. By employing the CTS-SEQUENCE HLA-B kit, which includes sequencing primers for the separation of HLA-B*51 and HLA-B*53 alleles, we were able to identify the new HLA-B*53 allele, HLAB*53:31. In addition, we confirmed the sequence of the new allele by typing the patient’s brother, who was haploidentical and possessed HLA-B*53:31 in combination with a common HLA-B allele (HLA-B*18:01). Serological typing showed positive reactions of lymphocytes carrying the novel allele HLA-B*53:31 with commercial antisera for HLA-B35, HLA-B51 and HLA-B53. It is well known that these antisera often cross-react and therefore cannot reliably distinguish alleles which belong the HLA-B*35, HLA-B*53 or HLAB*51 allele groups. HLA-B*53:31 has one nt change from HLA-B*53:13 at position 419 in exon 3, where an exchange from C to T (codon 116, TCC to TTC) results in a coding change from the neutral-polar amino acid serine (116S) to the neutral-nonpolar amino acid phenylalanine (116F). As described for the new allele HLA-B*35:231 (case 5), this amino acid exchange could affect the binding properties. We hypothesize that the new allele has emerged either by point mutation from HLA-B*53:13 (rare in Caucasians, frequency < 0.00006) [14], or, more likely, by a recombination event such as gene conversion of alleles that are more commonly found

(a)

(b)

Fig. 2. (a) Schematic presentation of a putative intralocus gene conversion event, passing a gene region including codon 9–12 from HLA-B*18:01:01:01 to HLA-B*35:03:01 and creating the novel allele HLA-B*35:231. (b) Schematic presentation of a putative intralocus gene conversion event, passing a gene region including codon 77–83 from HLA*44:02:01 to HLA-B*35:03:01 and creating the novel allele HLA-B*53:31.

M. Adamek et al. / Human Immunology 76 (2015) 30–35

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in Caucasians than HLA-B*53:13. We first asked whether the most common allele of the HLA-B*53 group, HLA-B*53:01:01 (frequency 0.04544) [14], may have served as ‘‘acceptor’’ for the gene conversion. HLA-B*53:01:01 and HLA-B*53:31 (the new allele) differ at two polymorphic sites in exon 2 and one polymorphic site in exon 3. As these polymorphic sites are distant from each other, it is difficult to believe that HLA-B*53:01:01 may have been the origin of the new allele. Further analysis of all HLA-B alleles let it appear more likely that HLA-B*53:31 may have emerged from HLAB*35:03:01 (frequency 0.01536) [14] from which HLA-B*53:01 differs in seven polymorphic sites in proximity with each other, all of which are located in the region between codon 77 and codon 83 in exon 2. This region (amino acid sequence motif SLRNLRG) encodes the Bw6 epitope in HLA-B*35:03:01, whereas the novel allele HLAB*53:31 exhibits a Bw4 epitope (amino acid sequence motif NLRTALR) at codons 77–83 (sequences of the Bw4 and Bw6 epitopes obtained from http://www.dorak.info/hla/bw4bw6.html). Subsequently, we identified the motif of the Bw4 epitope NLRTALR (found in HLA-B*53:31) in the sequence of exon 2 of two common alleles: HLA-B*44:02 and HLA-B*13:02:01. Therefore, we think that this region may have been transferred from either HLA-B*44:02 (frequency 0.09011) [14] or from HLA-B*13:02:01 (frequency 0.02644) [14] to HLA-B*35:03:01 by gene conversion (Fig. 2b), giving rise to the new allele HLA-B*53:31. The name HLA-B*53:31 has been officially assigned by the WHO Nomenclature Committee in May 2013. The nucleotide sequence is available at GenBank (accession number HF952171). The complete typing of the patient was HLA-A*01:01, *32:01; HLA-B*51:01, *53:31; HLA-C*04:01, *14:02; HLA-DRB1*08:01, ; HLA-DQB1*04:02, ; and DQA1*04:01, *04:02. The patient’s brother was typed as HLA-A*01:01, *25:01; HLA-B*18:01, *53:31; HLA-C*04:01, *12:01; HLA-DRB1*08:01, *15:01; HLA-DQB1*04:02, *06:02; and HLA-DQA1*01:02, *04:01.

exon 3) from the most similar allele HLA-C*07:27:02: first, nt position 363 (G to C); second, nt position 379 (C to G); third, nt position 387 (C to G); and fourth, nt position 409 (T to C). The consequences were non-synonymous and synonymous substitutions: first, a change of codon 97 AGG to AGC which results in a coding change from arginine (97R) to serine (97S); second, a change of codon 103 CTG to GTG which results in a coding change from leucine (103L) to valine (103V); third, a synonymous substitution at codon 105 CCC to CCG (proline, 105P); and finally, a change of codon 113 TAT to CAT which results in a coding change from tyrosine (113Y) to histidine (113H). Sequence comparison with all known HLA class I alleles showed that the combination of four polymorphic sites described above cannot be found in any other HLA-C allele, but is typical of some HLA-B alleles such as HLA-B*07, HLA-B*08, HLA-B*40:02, HLAB*42, HLA-B*48:01, HLA-B*48:04-30 and HLA-B*81. Fig. 3 shows, for example, that a 75 base pair (bp) region of exon 3 (codon 91 to codon 115) of HLA-B*07:02:01 is identical with the corresponding sequence of HLA-C*07:294, whereas exon 2 and the remaining 201 bp region of exon 3 (codon 116 to codon 182) of HLA-C*07:294 can be fully aligned with HLA-C*07:02:01:01. This suggests that the novel allele HLA-C*07:294 has probably evolved by interlocus gene conversion or crossing-over between HLA-C*07:02:01:01 and an HLA-B allele such as HLA-B*07:02:01 (Fig. 4). The name HLA-C*07:294 has been officially assigned by the WHO Nomenclature Committee in October 2012. The nucleotide sequence is available at GenBank (accession number HF545656). The complete typing of the donor was HLA-A*03:01, ; HLAB*07:02, *15:01; HLA-C*03:03, *07:294; HLA-DRB1*13:01, *15:01; HLA-DQB1*06:02, *06:03; and HLA-DQA1*01:02, *01:03.

3.4. Interlocus gene conversion or crossing-over (HLA-C*07:294)

HLA diversity presumably enhances immune surveillance by binding and presenting a variety of peptides, thereby inducing a wide array of immune responses to pathogens [7]. HLA polymorphism is thought to be maintained by balancing selection [15], but the impact of other mechanisms such as asymmetric overdom-

3.4.1. Case 7 HLA-C*07:294 was detected in a deceased kidney donor of unknown ethnic origin. HLA-C*07:294 has four nt changes (all in

4. Discussion

Fig. 3. Nucleotide alignment of the novel allele HLA-C*07:294 with HLA-C*07:27:02 (the most similar allele), HLA-C*07:02:01:01, and HLA-B*07:02:01. Whereas the entire exon 2 and part of exon 3 (codon 116–182) of HLA-C*07:294 and C*07:02:01:01 are identical, the remaining segment of exon 3 (codon 91–115) of HLA-C*07:294 can be completely aligned with the respective region of HLA-B*07:02:01. The alignment was generated using the IMGT/HLA Sequence Alignment Tool (http://www.ebi.ac.uk). Part of exon 2 (codon 76 to codon 90) and the entire exon 3 are shown.

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Fig. 4. Schematic presentation of a putative interlocus gene conversion event that transfers codon 91–115 of HLA-B*07:02:01 to the corresponding region in exon 3 of HLA-C*07:02:01:01, creating the novel allele HLA-C*07:294.

inant selection and ‘‘Associative Balancing Complex’’ (ABC) evolution has also been discussed [16]. In a neutral model of gene evolution, Klitz et al. estimated that new HLA alleles appear at a rate ranging from 0.3 (founding populations, rural Eurasia) to 600 (contemporary urban) per generation. According to their model, the number of new mutant HLA alleles in the current generation of humans is about 1.4 million [11]. In many cases, HLA polymorphisms appear to have arisen by point mutations (nucleotide substitution, insertion and deletion) [7]. As shown by Hughes & Nei for HLA class I [17] and class II [18], the rate of non-synonymous substitutions greatly exceeds the rate of synonymous substitutions within the antigen recognition site whereas the opposite is true for other regions of the HLA molecule. Point mutation as a motor driving HLA variation can be exemplified by four of our seven new alleles: HLA-B*41:21, HLA-DQB1*02:10, HLA-DQA1*01:12 and HLA-A*01:123N. Nucleotide substitutions can result in the generation of null alleles, e.g. by introducing an alternative splicing site which prevents translation into a correct and stable HLA molecule [19] or by affecting the interaction with b2-microglobulin [20]. Here, we describe a novel null allele, HLA-A*01:123N, in which the deletion of a single nucleotide abrogates the expression. Generally, the deletion or insertion of nucleotides can range from a single nucleotide up to regions of several kb including complete exons [21–23]. If the insertion/deletion affects a number of nucleotides that is not a multiple of three, the result will be a shift of the codon reading frame. In most cases, this shift will lead to an early stop codon and the generation of a null allele [24,25]. Similar to our new HLA-A null allele, HLA null alleles have been described that are characterized by a deletion or insertion causing premature stop, for example, HLA-A*01:15N [24], HLA-A*02:305N [26], HLA-B*08:08N [27], HLA-DRB1*01:33N [28], and HLA-DRB1*14:137N [29]. It has been reported that HLA null alleles occur at frequencies between 0.003% and 0.08% [21,30,31]. Beside point mutations, HLA diversity is further shaped by recombination between homolog chromosomes [32]. Parham et al. hypothesized that probably the majority of HLA-A, -B, and C alleles were generated by interallelic recombination, while a smaller number were likely to be products of point mutations [8,33]. A distinction is commonly made between crossing-over (bidirectional sequence exchange) and gene conversion (unidirectional sequence exchange), whereby both represent recombination events [10,34,35]. However, in the majority of cases it is not possible to delineate through which exact mechanism a recombinant allele evolved. Typical features of gene conversion are short nucleotide stretches which are transferred and shuffled between different alleles [36,37]. In contrast, crossing-over may play a greater role over longer stretches of DNA [38,39]. In a mouse model, crossing-over between two sister chromatids has been shown to occur at about 10-times lower frequency than gene conversion [40]. Based on sequence analysis, we think that three of our seven new alleles (HLA-B*35:231, HLA-B*53:31 and HLA-C*07:294) may have arisen from gene conversion or crossing-over. Since only

short DNA sequences are involved, gene conversion appears to be the more likely event in the evolution of these alleles than crossing-over. Intralocus gene conversion, as shown here for HLA-B*35:231 and HLA-B*53:31, has also been previously suggested as the underlying mechanism for the generation of other HLA alleles such as HLA-B*55:18 [41], HLA-B*27:102 [42], and HLA-DRB1*13:45 [43]. Furthermore, we suggest that there may be a difference between our two new HLA-B alleles regarding their evolution: while HLAB*35:231 presumably originated from a common allele of the same group (HLA-B*35:03:01) (Fig. 2a), the sequence of HLA-B*53:31 points out that this allele has arisen by a gene conversion event between two HLA-B alleles other than HLA-B*53 (Fig. 2b). This highlights the interesting feature that HLA-B*35:231 and HLAB*53:31 (which were discovered in two unrelated persons and belong to different HLA-B allele groups) could both have originated from one common allele (HLA-B*35:03:01). HLA-B35 and HLA-B53 peptides are structurally related. The amino acid sequences of HLAB*53:01:01 and HLA-B*35:01:01:01 differ only at positions 77 and 79–83, at which HLA-B*53:01:01 has a Bw4 motif in contrast to HLA-B*35:01:01:01 which has a Bw6 motif [44]. HLA-B35 and HLA-B53 peptides both belong to the 5C cross reactive group (CREG) [45]. Thus, it is not surprising that a gene conversion based on a common HLA-B*35 allele can give rise either to a new HLAB*35 allele or a new HLA-B*53 allele (allele assignment was performed under consideration of the DNA sequence of the most similar alleles). Rather than an intralocus gene conversion, an interlocus gene conversion could explain the structure of HLA-C*07:294 (Fig. 4). Similarly, HLA-B*07:13 [46], HLA-A*23:31 [47], have been postulated as products of interlocus gene conversions between HLA-B and HLA-C, and between HLA-A and HLA-B loci, respectively. No alleles resulting from a process of interlocus gene conversion between HLA-A and HLA-C have been reported so far. In summary, we found seven novel HLA class I and II alleles which appear to have developed via different mechanisms, such as point mutation, gene conversion or crossing-over. A brief review of the literature on the mutational events underlying the generation of new HLA alleles was given. References [1] Mungall AJ, Palmer SA, Sims SK. The DNA sequence and analysis of human chromosome 6. Nature 2003;425:805. [2] Robinson J, Halliwell JA, McWilliam H, Lopez R, Parham P, Marsh SG. The IMGT/ HLA database. Nucleic Acids Res 2013;41:D1222. [3] Cano P, Klitz W, Mack SJ, Maiers M, Marsh SG, Noreen H, et al. Common and welldocumented HLA alleles: report of the Ad-Hoc committee of the american society for histocompatiblity and immunogenetics. Hum Immunol 2007;68:392. [4] Mack SJ, Cano P, Hollenbach JA, He J, Hurley CK, Middleton D, et al. Common and well-documented HLA alleles: 2012 update to the CWD catalogue. Tissue Antigens 2013;81:194. [5] Carrington M. Recombination within the human MHC. Immunol Rev 1999;167:245. [6] Högstrand K, Böhme J. Gene conversion can create new MHC alleles. Immunol Rev 1999;167:305. [7] Yeager M, Hughes AL. Evolution of the mammalian MHC: natural selection, recombination, and convergent evolution. Immunol Rev 1999;167:45. [8] Parham P, Adams EJ, Arnett KL. The origins of HLA-A, B, C polymorphism. Immunol Rev 1995;143:141. [9] Arnheim N, Calabrese P, Tiemann-Boege I. Mammalian meiotic recombination hot spots. Annu Rev Genet 2007;41:369. [10] Bahr A, Wilson AB. The evolution of MHC diversity: evidence of intralocus gene conversion and recombination in a single-locus system. Genetics 2012;497:52. [11] Klitz W, Hedrick P, Louis EJ. New reservoirs of HLA alleles: pools of rare variants enhance immune defense. Trends Genet 2012;28:480. [12] Heinold A, Bauer M, Scherer S, Opelz G, Tran TH. Characterization of a new HLA-B allele, HLA-B⁄5312, and re-evaluation of the published sequences of the untranslated regions of HLA-B⁄35 and HLA-B⁄53. Tissue Antigens 2007;70:319. [13] Reche PA, Reinherz EL. Sequence variability analysis of human class I and class II MHC molecules: functional and structural correlates of amino acid polymorphisms. J Mol Biol 2003;331:623.

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Seven novel HLA alleles reflect different mechanisms involved in the evolution of HLA diversity: description of the new alleles and review of the literature.

The human leukocyte antigen (HLA) loci are among the most polymorphic genes in the human genome. The diversity of these genes is thought to be generat...
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