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Alerts NEW ALLELE C O N T R I B U T I O N S T O T H I S S E C T I O N WI L L NO T U N D E R G O P EE R RE V I EW , B U T W I L L B E R E V I E W E D B Y T H E ED I T O R * HLA-A*2313 is closest to A*2301 but is likely to stimulate T cells when mismatcheda P. A. Horn, M. Verboom & R. Blasczyk Institute for Transfusion Medicine, Hanover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany Key words: A*2313; HLA-A; sequencing-based typing We here describe the identification The novel allele of the novel allele HLA-A*2313 differs from A*2301 by the HLA-A*2313, amino acid which has been exchange detected in a potential German His70Gln. bone marrow donor. The complete typing results were A*2313, *3301; B*0702, *1402; DRB1*010201, *150101. Cycle sequencing of exons 2 and 3 was performed following haplotypespecific amplification essentially as previously described (1) using an Applied Biosystems 3730 sequencer, resulting in the detection of the new allele. The new allele is identical to A*2301 except for a non-synonymous nucleotide exchange at position 209 in exon 2 (T!C) changing codon 70 from CAC to CAG (Figure 1). Because this triplet is frequently found at codon 70 in a number of alleles (e.g., most A*03, A*11, A*29, A*34, A*66 and A*68 alleles), the new A*23 allele probably originated by gene conversion. The data have been submitted to the IMGT/HLA database (2), and the name A*2313 has been officially assigned by the WHO Nomenclature Committee in August 2005. The nucleotide sequence is available in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AM072344. The resulting amino acid exchange His!Gln at position 70 of the mature polypeptide (Figure 2) affects a position that is part of the alpha helix and that participates in A*2301 A*2302 A*2303 A*2304 A*2305 A*2306 A*2307N A*2308N A*2309 A*2310 A*2311N A*2312 A*2313 60 TGG ------------------------- GAC ------------------------- GAG --------------C---------- GAG ------------------------- ACA ------------------------- 65 GGG --------------C---------- AAA ----------------T --------- * the formation of pockets A, B and C of the peptide-binding groove. The rather high Risler amino acid substitution score (3) of 68 indicates that these two amino acids are relatively dissimilar. Accordingly, it can be expected that the peptide-binding properties are altered. If so, a possible mismatch with any other known A*23 variant may stimulate alloreactive T cells and thus impair the clinical outcome of stem cell transplantation. Correspondence Prof Dr Rainer Blasczyk Institute for Transfusion Medicine Hanover Medical School Carl-Neuberg-Str. 1 30625 Hannover Germany Tel.: þ49 511 532 6700 Fax: þ49 511 532 2079 e-mail: [email protected] GTG --------------A---------- AAG ------------------------- GCC ------------------------- 70 CAC ------------------------G TCA ------------------------- CAG ------------------------- ACT ------------------------- GAC --------------------C---- 75 CGA ------------------------- Figure 1 Nucleotide alignments of codon 60-75 of the new allele in comparison to the consensus of all other A*23 alleles. a The name A*2313 has been officially assigned by the WHO Nomenclature Committee in August 2005. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (4), 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. 166 A*2301 A*2302 A*2303 A*2304 A*2305 A*2306 A*2309 A*2310 A*2312 A*2313 60 Trp ------------------- Asp ------------------- Glu ----------Gln ------- Glu ------------------- Thr ------------------- 65 Gly ----------Arg ------- Lys ----------Asn ------- Val ----------Met ------- Lys ------------------- Ala ------------------- 70 His ----------------Gln Ser ------------------- Gln ------------------- Thr ------------------- Asp --------------His --- 75 Arg ------------------- Figure 2 Deduced amino acid sequence from the sequences as shown in A. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard NEW ALLELE Alerts Received 01 November 2005; accepted 09 November 2005 doi: 10.1111/j.1399-0039.2005.00541.x Acknowledgments The authors thank Kerstin Müller and Anja Hundt for excellent technical assistance. This study was supported in part by grant KF0483301KLF3 (BMWA). References 1. Kotsch K, Wehling J, Kohler S, Blasczyk R. Sequencing of HLA class I genes based on the conserved diversity of the noncoding regions: sequencing-based typing of the HLA-A gene. Tissue Antigens 1997: 50: 178–91. 2. Robinson J, Waller MJ, Parham P et al. IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic Acids Res 2003: 31: 311–4. 3. Risler JL, Delorme MO, Delacroix H, Henaut A. Amino acid substitutions in structurally related proteins. A pattern recognition approach. Determination of a new and efficient scoring matrix. J Mol Biol 1988: 204: 1019–29. 4. Marsh SG, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2004. Tissue Antigens 2005: 65: 301–69. Identification of a novel HLA-A*02 allele, A*0287, in the cord blood of a Korean baby H.-S. Yun*, J.-Y. Baek*, S.-S. Hur, O.-J. Kwon & K. Kwack Pochon CHA University, Seongnam 463-836, Korea Key words: HLA-A*0287; Korean; sequence-based typing (SBT) The HLA-A2 antigen is the most common HLA-A antigen among major ethnic groups, and HLA-A*02 is one of the most frequent and polymorphic alleles at this locus. *These authors contributed equally to this work. A novel HLAWe reported the A*02 allele, identification of a A*0287, was new HLA-A*02 detected in the allele, A*0287, cord blood of a with a codon Korean baby, change at position during HLA 77 in exon 2 in the genotyping of cord blood of a cord blood Korean baby. donated to the cord blood bank. The nucleotide sequence of HLA-A*0287 differs in exon 2 from that of the A*0281 allele by one nucleotide (a G to A substitution at codon 77) (Figure 1). Donor’s HLA types were HLA-A*020601, *0287; HLA-B*150101, *270502; HLACw*010201, *040101; HLADRB1*010101, *0406; HLADRB4*010301; HLA-DQA1*0101, *030101; HLA-DQB1*030201, *050101; HLA-DPA1*010301, *020202; HLA-DPB1*020102, *0501. The new HLA-A*02 allele, A*0287, was identified by the sequence-based typing method. Exons 2 and 3 of the HLA-A gene were sequenced with a BigDyeTM Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on an ABI PRISMÒ 3100 Genetic Analyzer or ABI PRISMÒ 3730XL DNA Analyzer (Applied Biosystems). Accession numbers for HLA*0287, DQ139310 (exon 2) and DQ139311 (exon 3) were assigned after deposition of the sequence in to the GenBank, and an official name, HLA-A0287, was assigned by the WHO nomenclature committee in August 2005. The novel HLA-A*0287 generated a single-nucleotide substitution in exon 2, resulting in a change in the amino acid sequence from serine (AGC) to asparagine (AAC) in the HLA-A*0281 allele. Most polymorphisms of HLA class I antigens are present in exons 2 and 3, which encode the a1 and a2 domains of HLA-A molecules, involved in both peptide-binding and HLA-restricted recognition by the T-cell receptor. Residue 77 is located on the longer curved a-helix in the helical region of a1 and may make contact with both peptide and TCR (1). Compared with the HLA-A*0281 allele, the introduction of a bulky amino acid, such as asparagine, instead of serine, into the peptidebinding groove may affect HLA functions. In conclusion, a novel HLAA*0287 allele, detected in the cord blood of a Korean baby, will help us understand the HLA function and the immune response. A*0281 A*0287 1 5 10 15 20 25 GC TCT CAC TCC ATG AGG TAT TTC TTC ACA TCC GTG TCC CGG CCC GGC CGC GGG GAG CCC CGC TTC ATC GCA GTG -- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- A*0281 A*0287 30 35 40 45 50 GGC TAC GTG GAC GAG ACG CAG TTC GTG CGG TTC GAC AGC GAC GCC GCG AGC CAG AGG ATG GAG CCG CGG GCG CCG --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- A*0281 A*0287 55 60 65 70 75 TGG ATA GAG CAG GAG GGT CCG GAG TAT TGG GAC GGG GAG ACA CGG AAA GTG AAG GCC CAC TCA CAG ACT GAC CGA --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- A*0281 A*0287 80 85 90 95 100 GAG AGC CTG CGG ATC GCG CTC CGC TAC TAC AAC CAG AGC GAG GCC G GT TCT CAC ACC GTC CAG AGG ATG TAT GGC --- -A- --- --- --- --- --- --- --- --- --- --- --- --- --- - -- --- --- --- --- --- --- --- --- --- A*0281 A*0287 105 110 115 120 125 TGC GAC GTG GGG TCG GAC TGG CGC TTC CTC CGC GGG TAC CAC CAG TAC GCC TAC GAC GGC AAG GAT TAC ATC GCC --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- A*0281 A*0287 130 135 140 145 150 CTG AAA GAG GAC CTG CGC TCT TGG ACC GCG GCG GAC ATG GCA GCT CAG ACC ACC AAG CAC AAG TGG GAG GCG GCC --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- A*0281 A*0287 155 160 165 170 175 CAT GTG GCG GAG CAG TTG AGA GCC TAC CTG GAG GGC ACG TGC GTG GAG TGG CTC CGC AGA TAC CTG GAG AAC GGG --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- A*0281 A*0287 180 AAG GAG ACG CTG CAG CGC ACG G --- --- --- --- --- --- --- - Figure 1 Alignment of nucleotide sequence of exons 2 and 3 of HLA-A*0281 and HLA-A*0287 allele. Dashes (-) indicate identity with the HLA-B*0281 sequence, and pipe (|) indicates the exon/ intron. Changed amino acid is indicated to a circle. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard 167 NEW ALLELE Alerts Acknowledgments This study was supported by grants from the Korea Science and Engineering Foundation (R01-2002-000-00267-0) and from the Ministry of Health and Welfare (0405-BC02-0604-0004). Correspondence KyuBum Kwack or Oh-Joong Kwon Pochon CHA University Seongnam 463-836 Korea Tel: 82 31 725 8376 Fax: 82 31 725 8350 e-mail: [email protected] Received 10 November 2005; accepted 15 November 2005 doi: 10.1111/j.1399-0039.2005.00544.x Reference 1. Bjorkman PI, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 1987: 329: 506–12. molecular typing. We further A new HLA-A allele, investigated the HLA-A*3308, has been sample by poly- identified in Chinese individual by merase chain sequence-based typreactionsequence-based ing. This allele shows five nucleotides typing (PCRdifference with HLASBT). A*330301 in exon 2 at The new codon 56(GGG!GGT), A*3308 allele codon 62(CGG!GGG), showed unexcodon 63(AAC!GAG) pected reaction and codon patterns in routine PCR-SSO 66(AAT!AAA), typing (Tepnel resulting in three amino acids change Lifecodes, Stamford, CT, from Arg to Gly(R62G), Asn to Glu(N63E) and USA). Asn to Lys(N66K). According to the data analysis, it can not assign to the final result. A*330301 A*3308 Identification of a new allele, HLA-A*3308, in the Chinese population† While we override the probe #1109(50 ACCGGAACACACGG.AATGT-30 ) from negative to positive, the assignment for A*02XX,33XX were obtained. The SSO testing results suggested that it might be something new in A*33 within probe #1109 region; therefore, we further investigate the sample by using SBT method after cloning. To confirm the presence of a novel allele in the sample, the entire coding region of HLA-A gene was amplified with PCR from genomic DNA. Amplified PCR product was directly ligated into pCR4-TOPO vector and cloned by using TOPO cloning sequencing kit (Invitrogen, Carlsbad, CA, USA). Many recombinant colonies of the sample on Luria-Bertani plates were selected at random, and plasmid DNA was extracted from 74 G C T C C C A C T C C A T G A G G T A T T T C A C C A C A T 103 ------------------------------ A*330301 104 C C G T G T C C C G G C C C G G C C G C G G G G A G C C C C 133 A*3308 ------------------------------ A*330301 134 G C T T C A T C G C C G T G G G C T A C G T G G A C G A C A 163 L.-X. Yan, F.-M. Zhu, Q.-F. Lv, W. Zhang & J. He HLA Typing Laboratory, Blood Center of Zhejiang Province, Hangzhou, Zhejiang Province 310006, China Key words: HLA-A*33; new allele; SBT A*3308 ------------------------------ A*330301 164 C G C A G T T C G T G C G G T T C G A C A G C G A C G C C G 193 A*3308 ------------------------------ A*330301 194 C G A G C C A G A G G A T G G A G C C G C G G G C G C C G T 223 More than 396 alleles of HLA-A have been officially recongnized to date (1, 2). In this report, we present a novel allele which has been detected by unusual probe reaction patterns during routine A*3308 ------------------------------ A*330301 224 G G A T A G A G C A G G A G G G G C C G G A G T A T T G G G 253 A*3308 ----------------T------------- A*330301 254 A C C G G A A C A C A C G G A A T G T G A A G G C C C A C T 284 †The name A*3308 has been officially assigned by the WHO Nomenclature Committee in June 2005. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (2), names will be assigned to new sequences as they are identified. List of such new names will be published in the following WHO Nomenclature Report. 168 A*3308 --G--G-G--------A------------- A*330301 284 C A C A G A T T G A C C G A G T G G A C C T G G G G A C C C 312 A*3308 ------------------------------ A*330301 313 T G C G C G G C T A C T A C A A C C A G A G C G A G G C C G 343 A*3308 ------------------------------ Figure 1 Alignment of the exon 2 of A*33 with the sequences of A*330301. Dashes indicated nucleotide identity with the A*330301 sequence. Numbers above the sequence correspond to nucleotide position. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard NEW ALLELE Alerts each colony and used as template for sequencing reaction. Both strands of exons 2, 3 and 4 of HLA-A were sequencing. The DNA sequence was determined with a cycle sequencing kit (BigDye terminator, Applied Biosystems, Foster City, CA, USA) and a genetic analyzer (ABI Prism 377, Applied Biosystems) according to the manufacturers’ instructions (3). The sequencing results showed HLA-A alleles of the sample as A*0201 and the variant allele. The variant allele has been officially named A*3308 by the WHO Nomenclature Committee. The new A*3308 allele shows a sequence identical to that of A*330301 in exons 3 and 4, but shows five nucleotides mismatches with A*330301 in exon 2 (Figure 1) at nucleotide positions 240G!T, 256C!G, 259A!G, 261C!G and 270T!A, which result in three amino acids change from Arg to Gly(R62G), Asn to Glu(N63E) and Asn to Lys(N66K). The sample was typed as A*0201,*3308; B*1511,5502; DRB1*1405,0803. phenotypes in Chinese persons, including a novel nonfunctional FUT1 allele. Transfusion 2005: 45: 725–30. A novel HLA-A*680104 varianta G. A. Heymann, A. Lassahn, C. Schönemann & A. Salama Institute of Transfusion Medicine, Campus Virchow-Klinikum, Charité – Universitätsmedizin Berlin, Berlin, Germany Key words: allele-specific amplification; DNA sequencing; SSO; SSP Correspondence Prof. Li-Xing Yan Director, Blood Center of Zhejiang Province Wulin Road 345 Hangzhou, Zhejiang Province 310006 China Tel: 86 571 85167801 Fax: 86 571 85065851 e-mail: [email protected] We describe a novel human leukocyte antigen A (HLA-A) allele, A*680104, which was initially identified by lowresolution HLA-typing techniques in an adult bone marrow transplant recipient of Caucasian origin. The variant A novel, silent human was recognized by low- leukocyte antigen A allele, A*680104, with resolution a variation of codon HLA-A typ181 (ACG!ACT) ing of the (conserved Thr) is DNA sample described. using the Dynal RELISSO HLA-A kit (batch no. PK622, DynalTM, Oslo, Norway). This testing resulted in positive reactions at positions 2, 3, 5, 9, 10, 11, 12, 14, 17, 19, 24, 26, 28, 30, 36, 38, 41, and 42 and did not match any allele Received 16 July 2005; revised 28 July 2005, 12 August 2005, 7 September 2005; accepted 20 September 2005 a doi: 10.1111/j.1399-0039.2005.00519.x References 1. Schreuder GM, Hurley CK, Marsh SG et al. The HLA Dictionary 2004: a summary of HLA-A, -B, -C, -DRB1/3/4/5 and -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR and -DQ antigens. Tissue Antigens 2005: 65: 1–55. 2. Marsh SG, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2004. Tissue Antigens 2005: 65: 301–69. 3. Yan L, Zhu F, Xu X, Hong X, Lv Q. Molecular basis for para-Bombay The nucleotide sequence data presented in this report have been submitted to the EMBL nucleotide sequence database and were assigned the accession number AM062639. The name HLA-A*680104 has been officially assigned by the WHO Nomenclature Committee in August 2005. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (1), 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. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard combination in the Dynal PATTERN MATCH program database. Without probe 41, a result (A*3201, 6808) was found, but probe 41 does not reach the codon 181. Finally, we cannot blame any oligonucleotide of this batch with a sequence in the user manual for a detection failure. Next, we performed HLA-A typing polymerase chain reaction sequence-specific primer SSP), including different SSP kits from GenovisionTM, Vienna, Austria [HLA-A low resolution (lot no. N28), HLA-A*32 (lot no. N06), HLA-A*68 (lot no. M67)]. When comparing the sequences of primers that resulted in amplification reactions in concordance with the assumed HLA-type A*3201,6801, the results seemed to originate from primers not affecting the nucleotide 618 of HLAA*6801. Serologic typing was performed using panels of antisera (OneLambda by BMT, Merbusch, Germany, Tray M144A, Lot 003). The M144A tray gave positive reactions with all antisera against A32 and A68. Finally, we performed a sequencebased analysis of the novel HLA-A variant in two different DNA probes of the same patient using the ProtransTM S4 HLA-A kit (lot no. 441E03, Protrans, Ketsch, Germany). Briefly, the Protrans S4 kit allele-specific amplification of a region containing exons 2 and 3 resulted in separate amplification products covering HLA-A*29/*32/*74 and HLAA*68/*69/*3401/*3405/*6602/*6603, respectively. The remaining primers and deoxyribonucleoside triphosphates were removed by ExoSAP-IT (Amersham Biosciences, Cardiff, UK) digestion. The amplification products were used for the cycle-sequencing reactions. For each sequencing reaction, 2 ml of the treated amplification products were mixed with 2 ml BigDye 1.1 (Applied Biosystems, Warrington, UK) and 6 ml sequencing primer and incubated [(96˚ C, 1 min), 25 (96˚ C, 10 s; 50˚ C, 5 s; 60˚ C, 4 min), (4˚ C)]. The following sequencing primers included in the Protrans S4 HLA-A kit were 169 NEW ALLELE Alerts Codon >A*680101 >A*680102 >A*680103 >A*680104 >A*6814 >A*2906 >A*010103 >A*240302 .CAGCGC ....... ....... ....... ....... ....... ....... ....... 181 ACG ... ..C ..T G.. G.. ..T ..T GACGCCCCC-.........-.---------.---------.---------.---------...C.....-.---------- Figure 1 Nucleotide alignment of the alleles of human leukocyte antigen A (HLA-A). Codon 181 is labeled. A*680104 exhibits a sequence motif found in two other HLA-A alleles, but not A*68 group. Some bases are indicated by dots. For A*2906, A*010103, and A*240302, see explanation in the text. Six leading bases before codon 181 are given. used: A-E2F (forward, intron 1), AE2R (reverse, intron 2), A-E3F (forward, intron 2), and A-E3R (reverse, intron 3). By using this sequence-based approach, clear identification of the allele A*3201 and the new HLAA*680104 variant was possible. HLAA*680104, which is most closely related to A*680101, exhibits a substitution of codon 181 (ACG!ACT). This variation results in no amino acid substitution. The sequence motif at codon 181 is also found in A*010103 and A*240302 (Figure 1) (2). Besides those alleles, A*680103 shows a silent ACG!ACC exchange, and A*6814 and A*2906 (3) show an ACG!GCG exchange, resulting in an amino acid change Thr!Ala. In all other alleles of the HLA-A, the amino acid at codon 181 is found to be a chemically neutral threonine. Because A*680104 is structurally unaltered, finding a bone marrow donor may not be problematical. Correspondence Dr G. A. Heymann Institute of Transfusion Medicine Campus Virchow-Klinikum Charité – Universitätsmedizin Berlin Augustenburger Platz 1 13353 Berlin Germany Tel: 49 30 450 553089 Fax: 49 30 450 553988 e-mail: [email protected] Received 8 September 2005; revised 20 September 2005; accepted 30 September 2005 doi: 10.1111/j.1399-0039.2005.00525.x 170 References 1. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA System, 2004. Tissue Antigens 2005: 65: 301–69. 2. Ellis J, Steiner N, Kosman C et al. Seventeen more novel HLA-A locus alleles. Tissue Antigens 2000: 55: 369–73. 3. Steiner NK, Rizzo M, Bradshaw D et al. Seventeen novel HLA-A alleles. Tissue Antigens 2003: 62: 256ff. Identification of two new HLA-A variants, HLAA*2911 and HLA-A*6827† K. R. Alkharsah1, M. Dedicoat1, D. S. DeLuca2, T. F. Schulz1 & R. Blasczyk2 1 Institute of Virology, Hannover Medical School, Hannover, Germany 2 Institute of Transfusion Medicine, Hannover Medical School, Hannover, Germany Key words: HLA-A; new allele; South Africa; Zulu †The names A*2911 and A*6827 have been officially assigned by the WHO nomenclature committee in December 2003 and in June 2004, respectively. This follows the agreed policy that, subject to the conditions stated in the most recent nomenclature report (1), names will be assigned to new sequences as they are identified. List of such new names will be published in the following WHO Nomenclature Report. The HLA system comprises We report here the the most poly- identification of the morphic loci, new alleles HLAif not the most A*2911 and HLAA*6827 in two healthy, at all, in the human genome. unrelated Zulus Improvements (Hlabisa, South of new HLA- Africa). sequencing strategies contributed to the identification of new alleles and therefore to the expansion of the HLA diversity. About 340 HLA-A alleles have been reported until October 2004 according to the WHO Nomenclature Committee for Factors of the HLA System. Here we present the identification of two new variants, HLAA*2911 and A*6827 (1), in two unrelated female Zulus in Hlabisa, South Africa. The two new alleles were identified during HLA-A typing in a group of healthy mothers attending the vaccination health clinics in Hlabisa, South Africa. Genomic DNA was extracted from saliva samples of the two females by QIAamp DNA blood mini kit (QIAGEN, Hilden, Germany). Exon 2 and 3 of HLA-A were PCRamplified locus specifically and sequenced in forward and reverse directions (2). Sequencing analysis was performed with the 3730 Genetic Analyzer (Applied Biosystem, Foster city, CA, USA), and the data were analysed by the SEQPILOT program version 2.0 (Protrans, Ketsch, Germany). The sequence of HLA-A*2911 was confirmed by hemizygous sequencing. This new variant when compared to its closely related allele, HLA-A*290201, was found to be characterized by a non-synonymous nucleotide exchange (A!C) at nucleotide position 151 of exon 3. This resulted in the amino acid exchange Gln!Pro at position 141 of the mature polypeptide. This position, located in the H1 helix of the a2-domain, is probably not directly involved in peptide binding. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard NEW ALLELE Alerts (A) A*01010101 A*29010101 A*29010102N A*290201 A*290202 A*290203 A*2903 A*2904 A*2905 A*2906 A*2907 A*2908N A*2909 A*2910 A*2911 A*2912 A*2913 A*2914 135 GCG ----------------------------------- GCG ----------------------------------- GAC ----------------------------------- ATG ----------------------------------- 5 ATG --------------------------------------------------------------- AGG --------------------------------------------------------------- TAT --------------------------------------------------------------- TTC --------------------------------------------------------------- GCA --G --G --G --G --G --G --G --G --G --G --G --G --G --G --G --G --G 140 GCT ----------------------------------- CAG ---------------------------C------- ATC ----------------------------------- ACC ----------------------------------- TTC -A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A- 10 TTC --C --T --C --C --T --T --C --T --T --T --C --T --C --T --T --C --C --C --T --T --C --T --T --T --C --T --T --C --C --C --C TCC --------------------------------------------------------------- GTG ------A---------------------------A-----A-------------------A-- TCC --------------------------------------------------------------- AAG C-C-C-C-C-C-C-C-C-C-C---C-C-C-C-C-- 145 CGC ----------------------------------- AAG ----------------------------------- TGG ----------------------------------- GAG ----------------------------------- GCG ----------------------------------- 150 GTC -C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C- CGG --------------------------------------------------------------- 15 CCC --------------------------------------------------------------- GGC --------------------------------------------------------------- CGC --------------------------------------------------------------- GGG --------------------------------------------------------------- GAG --------------------------------------------------------------- 20 CCC --------------------------------------------------------------- (B) A*01010101 A*680101 A*680102 A*680103 A*6802 A*680301 A*680302 A*6804 A*6805 A*6806 A*6807 A*6808 A*6809 A*6810 A*6811N A*6812 A*6813 A*6814 A*6815 A*6816 A*6817 A*6818N A*6819 A*6820 A*6821 A*6822 A*6823 A*6824 A*6825 A*6826 A*6827 A*6828 Figure 1 Alignment of nucleotide sequence displayed in codons of the HLA-A*29 and A*68 showing the location of the new mutation. (A) Alignment of HLA-A*29 alleles. (B) Alignment of HLA-A*68 alleles. Yet, since proline is a helix breaker (3, 4), the structure of the neighbouring pocket F may be altered, which might result in a change of the peptide-binding characteristics of the new variant compared to the A*2902 molecule. The mutation occurred in a region which is highly conserved in all classical and non-classical HLA class I alleles (Figure 1A). As no donor allele could be identified, it is unlikely that this mutation was generated by gene conversion or other recombination events. Instead it is more likely that a point mutation has occurred. We found this allele in further seven individuals of the same group, which indicate that it is not uncommon in Zulu population. The sequence of the HLA-A*6827 variant is similar to A*6802 with a non-synonymous nucleotide exchange in exon 2 at position 33 (A!G), leading to amino acid replacement of methionine by valine (Met!Val) (Figure 1B) at position Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard 12 in the mature protein. This variation was further verified by the separation of the two haplotypes (A*6827 and A*680101) by sequencespecific sequencing primers after repeated locus-specific PCR amplification. These sequencing primer (A497S 50 -AGGTTCTCACACCAT CCAGAT-30 for A*680101 and A498S 50 -GGTTCTCACACCATCC AGAG-30 for the A*6827) were located between intron 2 position 240 and exon 3 position 19 because exon 2 was found not to have heterozygous positions. Amino acid position 12 is found on the beta sheet of the peptide-binding pocket of the HLAA class I molecule. Its side chain is facing away from the binding groove and in the direction of b2-microglobulin. Since methionine and valine are hydrophobic molecules, we do not expect this mutation to have an effect on the peptide binding of HLA-A*6827 compared to A*6802. Similar to A*2911, the A*6827 seems to have arisen by a point mutation. Correspondence Rainer Blasczyk Institute of Transfusion Medicine Hannover Medical School Carl-Neuberg-Str.1 30625 Hannover, Germany Tel: þ49 511 532 6700 Fax: þ49 511 532 2079 e-mail: [email protected] Received 13 August 2005; revised 18 August 2005; re-revised 26 August 2005; accepted 1 September 2005 doi: 10.1111/j.1399-0039.2005.00505.x References 1. Marsh SG, Albert ED, Bodmer WF et al. Nomenclature for factor of the HLA system, 2002. Tissue Antigens 2002: 60: 407–64. 2. Kotsch K, Wehling J, Kohler S, Blasczyk R. Sequencing of HLA class I genes based on the conserved diversity on the noncoding regions: sequencing based typing of the HLA-A gene. Tissue Antigens 1997: 50: 178–91. 3. Ballesteros JA, Weinstein H. Analysis and refinement of criteria for predicting 171 NEW ALLELE Alerts the structure and relative orientations of transmembranal helical domains. Biophys J 1992: 62: 107–9. 4. Barlow DJ, Thornton JM. Helix geometry in proteins. J Mol Biol 1988: 201: 601–19. A novel identification of HLA-B*07 allele, HLAB*0743, in the Korean population Y.-Y. Cha1*, J.-Y. Baek1*, Y.-S. Heo2, K. Kwack1 & O.-J. Kwon3 1 Pochon CHA University, Seongnam 463-836, Korea 2 CrystalGenomics Inc., Seoul, Korea 3 BioSenoom Institute of Bioscience & Biotechnology, Seoul, Korea Key words: HLA-B*07; HLA-B*0743; Korean; sequence-based typing To date, at least The novel HLA40 HLA-B*07 alleles have been B*0743 allele was identified from identified (1). cord blood of a Here, we report Korean baby. the new allele HLA-B*0743, which was detected during routine HLA genotyping of cord blood units donated to the Cord Blood Bank. During the routine HLA typing, the sequence-based typing method was employed. Thus, exons 2 and 3 of the HLA-B gene were sequenced directly using the BigDyeTM Terminator v3.1 Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISMÒ 3100 Genetic Analyzer or ABI PRISMÒ 3730XL Genetic Analyzer (Applied Biosystems). The sequence of the HLA-B gene was checked in the IMGT/HLA Sequence Database and was identified as a novel HLA-B *These authors contributed equally to this work. 172 allele. To confirm the presence of a new HLA-B allele, we sequenced the exon 3 again using the allele-specific sequence primers. The complete HLA genotype of the donor was HLA-A*020101, 240201; HLAB*0743, 150101; HLA-Cw*030301, 070201; HLA-DRB1*010101, 090102; HLA-DRB4*010301; HLADQA1*0101, 0302; HLADQB1*030302, 050101; HLADPA1*010301, Blank; HLADPB1*020102, 0402. We submitted the nucleotide sequence of the HLAB gene to GeneBank, and it has been assigned the accession numbers DQ088145 (exon 2) and DQ088146 (exon 3). The name HLA-B*0743 was officially assigned by the WHO nomenclature committee in June 2005. The nucleotide sequence of HLAB*0743 is identical to that of HLAB*0731 except for a single nucleotide change from G to T at position 539 in exon 3 (Figure 1). This change results in a non-synonymous amino acid change from arginine to leucine at residue 156. Residue 156 is located on the a2helix of the HLA class I molecule and is involved in peptide binding by constructing the D and E pockets of the peptide-binding groove (2). While a leucine instead of arginine at residue 156 of the B*0743 allele would not change the peptide preference of this allele from that of other B*07 alleles, it may elevate its peptidebinding affinity, because if the residues at P3, P5 and P7 of the bound peptide are bulky hydrophobic residues, Leu156 of B*0743 would be able to interact with these residues by van der Waals contact. These interactions would enhance the peptidebinding affinity of the B*0743 allele compared with that of other B*07 alleles bearing an arginine at residue 156. This tighter binding of peptides to B*0743 might also influence the recognition of the peptide–HLA complex by the T-cell receptor and thus may affect the immune response. In conclusion, we identified a novel HLA-B*07 allele from cord blood from a Korean baby. Further studies will be needed to determine whether this novel allele could affect the success of transplantation. B*070201 95 100 105 110 GG TCT CAC ACC CTC CAG AGC ATG TAC GGC TGC GAC GTG GGG CCG GAC GGG CGC CTC CTC B*0731 -- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- B*0743 -- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 115 120 125 130 B*070201 CGC GGG CAT GAC CAG TAC GCC TAC GAC GGC AAG GAT TAC ATC GCC CTG AAC GAG GAC CTG B*0731 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- B*0743 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 135 140 145 150 B*070201 CGC TCC TGG ACC GCC GCG GAC ACG GCG GCT CAG ATC ACC CAG CGC AAG TGG GAG GCG GCC B*0731 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- B*0743 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 155 160 165 170 B*070201 CGT GAG GCG GAG CAG CGG AGA GCC TAC CTG GAG GGC GAG TGC GTG GAG TGG CTC CGC AGA B*0731 --- --- --- --- --- --- --- --- --- --- --- --- AC- --- --- --- --- --- --- --- B*0743 --- --- --- --- --- -T- --- --- --- --- --- --- AC- --- --- --- --- --- --- --- 175 180 B*070201 TAC CTG GAG AAC GGG AAG GAC AAG CTG GAG CGC GCT G B*0731 --- --- --- --- --- --- --- --- --- --- --- --- - B*0743 --- --- --- --- --- --- --- --- --- --- --- --- - Figure 1 Comparison of the exon 3 nucleotide sequences of HLA-B*070201, HLA-B*0731 and HLA-B*0743. Dashes (–) indicate identity with the HLA-B*070201 sequence. The change in the nucleotide sequence is indicated in bold. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard NEW ALLELE Alerts Correspondence KyuBum Kwack Pochan CHA University, Seongnam 463-836, Korea Tel: 82 31 725 8376 Fax: 82 31 725 8350 e-mail: [email protected] Received 8 October 2005; revised 12 and 31 October 2005; accepted 8 November 2005 doi: 10.1111/j.1399-0039.2005.00535.x Acknowledgments This study was supported by grants from the Korea Science and Engineering Foundation (R01-2002-000-00267-0) and from the Ministry of Health and Welfare (0405-BC02-0604–0004). References 1. Marsh SG, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2004. Tissue Antigens 2005: 65: 301–69. 2. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 1987: 329: 506–12. Characterization of the novel allele HLA-B*3710y S. Frison, E. Longhi, A. Espadas de Arias, E. Andreini, F. Poli & M. Scalamogna Dipartimento Trasfusionale e di Riferimento per i Trapianti di Organi e Tessuti – Unità Operativa per il Prelievo e la Conservazione di Organi e Tessuti, †The allele B*3710 has EMBL nucleotide sequence Database Accession Numbers HWS10003117 and AJ969934. The name B*3710 has been officially assigned by the WHO Nomenclature Committee in June 2005. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (1), 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. Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena – Fondazione IRCCS di Natura Pubblica, Milan, Italy Key words: direct sequencing; HLA-B*3710; PCR-SSO HLA B*3710 differs from the In this report, we most homolo- describe the identification of the new gous allele B*3701 by four allele HLA-B*3710, nucleotide sub- which was found in a Caucasian individual stitutions in exon 2 at posi- on the waiting list for a kidney transplant in tions 300 the North Italy (G>A), 308 Transplant program (G>C), 312 (C>G) and 313 organization. (T>C). These mutations result in three codon changes: at codons 77 and 81 where an aspartic acid is substituted by an asparagine and an alanine by a leucine, respectively, and at codon 79 without an amino acid replacement. Allelic polymorphism of HLA-B locus is mainly located in exons 2 and 3 which encode for a1 and a2 domains within the antigenic peptidebinding site. We report herein the characterization of a new HLA-B*37, officially named HLA-B*3710 (1). This new variant was identified in a male patient (ID 108616) on the waiting list for a kidney transplant in the North Italy Transplant program organization. This patient was typed with serology for HLA-A,B as A1,32;B37,40 (GTI, Waukesha, WI, USA) and with polymerase chain reaction-reverse sequence-specific oligonucleotide (PCR-reverse SSO) typing using a commercially available kit (Lambda Array Beads Multi-Analyte SystemÒ – LABMAS, RSSO1B, One Lambda, Canoga Park, CA, USA) (2) for HLA-A,B,DRB1 as A*01,*32;DRB1*11,*13. With the interpretation software, no typing was deducible at B locus because of an anomalous hybridization Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard pattern. With PCR-sequence-specific primers (SSP) (Olerup SSP, Saltsjoebaden, Sweden) (3), B locus typing was B*40, X (w4, w6). HLA-B sequence-based typing was then performed with generic primers which amplify both the alleles from exon 1 to exon 5 (Atria Genetics, San Francisco, CA, USA) (4). Exons 2, 3 and 4 were sequenced in both directions, and the obtained heterozygous sequence revealed the presence of a new allele in combination with HLA-B*4001, as the nucleotide sequence did not match with any known allele combination. In order to separate the two alleles, exons 2 and 3 were amplified with group-specific primers provided by Atria Genetics and sequenced. The HLA-B new variant, officially named HLA-B*3710, differs from B*3701 by four nucleotide substitutions in exon 2 at positions 300 (G>A), 308 (G>C), 312 (C>G) and 313(T>C) (Figure 1). These mutations result in three codon changes: at codon 77 (GAC>AAC) where an aspartic acid is substituted by an asparagine, at codon 81 (CTG>GCG) where an alanine is substituted by a leucine and at codon 79 (CGG>CGC) with no amino acid substitution. The mutations are located in the a1 helix of the peptide-binding groove (5). These results explain the ambiguous hybridization pattern found while performing HLA-B low resolution with reverse SSO. In fact, the two probes (4, CTG CGC ACC GCG CTC CGC TAC TAC and 26, CAG ACT TAC CGA GAG AAC þ ACC GCG CTC CGC TAC TAC; Lot 006) that gave a positive result even though not complementary to B37 and the probe specific for B37 (83, CAG ACT TAC CGA GAG GAC CTG; Lot 006) that failed to hybridize are complementary to the DNA region spanning from codon 72 to codon 85, where the four mutations are located. 173 NEW ALLELE Alerts 1 GC TCC CAC TCC ATG AGG TAT TTC CAC ACC TCC GTG TCC CGG B*3710 -- --- --- --- --- --- --- --- --- --- --- --- --- --- B*3710 2. Kellar KL & Iannone MA. Multiplex microsphere-based flow cytometric assays. Exp Hematol 2002: 30: 1227–37. 3. Olerup O & Zetterquist H. HLA-DR typing by PCR amplification with sequence specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor–recipient matching in cadaveric transplantation. Tissue Antigens 1992: 39: 225–35. 4. Pozzi S, Longo A, Ferrara GB. HLA-B locus sequencing-based typing. Tissue Antigens 1999: 53: 275–81. 5. Bjorkman PJ, Saper MA, Samraoui B et al. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 1987: 329: 506–12. 14 B*3701 -- Ser Tyr Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Exon 2 15 28 B*3701 CCC GGC CGC GGG GAG CCC CGC TTC ATC TCA GTG GGC TAC GTG B*3710 --- --- --- --- --- --- --- --- --- --- --- --- --- --- B*3710 Pro Gly Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Exon 2 29 42 B*3701 GAC GAC ACC CAG TTC GTG AGG TTC GAC AGC GAC GCC GCG AGT B*3710 --- --- --- --- --- --- --- --- --- --- --- --- --- --- B*3710 Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Exon 2 43 56 B*3701 CCG AGG ACG GAG CCC CGG GCG CCG TGG ATA GAG CAG GAG GGG B*3710 --- --- --- --- --- --- --- --- --- --- --- --- --- --- B*3710 Identification of HLAB*4905 in a Caucasian familya Pro Arg Thr Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly Exon 2 57 70 B*3701 CCG GAG TAT TGG GAC CGG GAG ACA CAG ATC TCC AAG ACC AAC B*3710 --- --- --- --- --- --- --- --- --- --- --- --- --- --- B*3710 E. Andreini1, E. Longhi1, S. Frison1, M. Bontempelli2, L. Castellani2 & F. Poli1 1 Dipartimento Trasfusionale e di Riferimento per i Trapianti di Organi e Tessuti – Unità Operativa Complessa Immunologia dei Trapianti di Organi e Tessuti, Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena – Fondazione IRCCS di Natura Pubblica, Milan, Italy 2 U.O. Servizio di Immunoematologia e Medicina Trasfusionale, Laboratorio di Immunologia e Immunogenetica, Azienda Ospedaliera OO.RR., Bergamo, Italy Pro Glu Tyr Trp Asp Arg Glu Thr Gln Ile Ser Lys Thr Asn Exon 2 71 84 B*3701 ACA CAG ACT TAC CGA GAG GAC CTG CGG ACC CTG CTC CGC TAC B*3710 --- --- --- --- --- --- A-- --- --C --- GC- --- --- --- B*3710 Thr Gln Thr Tyr Arg Glu Asn Leu Arg Thr Ala Leu Arg Tyr Exon 2 85 91 B*3701 TAC AAC CAG AGC GAG GCC G B*3710 --- --- --- --- --- --- - B*3710 Key words: DNA direct sequencing, group-specific amplification, HLA-B*49 Tyr Asn Gln Ser Glu Ala Exon 2 Figure 1 The exon 2 nucleotide sequence for HLA-B*3710 is compared to that of B*3701. Dashes indicate identity between the two alleles. In the protein sequence, aminoacidic substitutions are reported in bold and the silent mutation in italic. Correspondence Francesca Poli Dipartimento Trasfusionale e di Riferimento per i Trapianti di Organi e Tessuti – Unità Operativa per il Prelievo e la Conservazione di Organi e Tessuti Ospedale Maggiore Policlinico, Mangiagalli Regina Elena – Fondazione IRCCS di Natura Pubblica Via Francesco Sforza 35 20122 Milan, Italy Tel: 39 02 55034239 Fax: 39 02 55012573 e-mail: [email protected] 174 Received 30 August 2005; revised 20 September 2005; accepted 23 September 2005 This communication reports one previously undescribed HLA-B allele, officially named HLA-B*4905 (1), detected in a Caucasian family from a North Italian region, namely in a patient doi: 10.1111/j.1399-0039.2005.00520.x Acknowledgments a This study was supported by a grant from ‘Ricerca Corrente’ Ospedale Maggiore Policlinico di Milano IRCCS, project number 580/01, 2004. References 1. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2004. Tissue Antigens 2005: 65: 301–69. The name B*4905 has been officially assigned by the WHO Nomenclature Committee in September 2005. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (1), 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. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard NEW ALLELE Alerts (ID122142) and A novel allele, in his father HLA-B*4905, is (ID122139). described. The two individualswere HLA-B*4905 shows a Mendelian inheriserologically typed as HLA- tance in a Caucasian A23,24;B44,49; family. This variant has four nucleotide Cw7 (ID122142) and substitutions in exon 3. These HLAmutations result in A23,25;one silent substitution B49,51;Cw1,7 at codon 129 (ID122139) (CCC!CCG) and in (One Lambda two aminoacid Inc, Canoga changes at codons Park, CA, 138 (AAC!GAC) USA). Low and 140 (TTA!TCC), resolution Asn to Asp and from molecular typLeu to Ser, from ing was carried respectively. out with PCRSSO using a commercially available kit (Lipa HLAA,B, Innogenetics N.V., Murex Biotech Limited, Dartford, UK), but no typing was deducible because of an anomalous hybridization pattern. PCR-SSP did not resolve the ambiguity (Olerup SSPTM HLA-B low resolution/M26, Saltsjoebaden, Sweden). In fact, one couple of primers even not specific for B*4901 amplified DNA from both the subjects, while the other primer pair specific for B*4901 did not amplify. HLA-B sequence-based typing was then performed with locus-specific primers which amplify both the alleles from exon 1 to exon 5 (Atria Genetics, San Francisco, CA, USA) (2). Exons 2, 3, and 4 were sequenced in both directions and analyzed with Assign SBTTM Version 3.2.7 software (Conexio Genomics, Applecross, Western Australia); the obtained heterozygous sequence revealed the presence of a new allele in combination with HLA-B*40020101, as the nucleotide sequence did not match with any known allele combination. With the aim at separating the two alleles, the sample was amplified with group-specific primers (AlleleSEQR HLA-B GSA SBT Kit, Atria Genetics). nt 344 A*4901 A*4905 384 gg tct cac act tgg cag agg atg tat ggc tgc gac ctg ggg -- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3 385 A*4901 A*4905 426 ccc gac ggg cgc ctc ctc cgc ggg tat aac cag tta gcc tac --g --- --- --- --- --- --- --- --- g-- --- -cc --- --Exon 3 427 A*4901 A*4905 468 gac ggc aag gat tac atc gcc ctg aac gag gac ctg agc tcc --- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3 469 A*4901 A*4905 510 tgg acc gcg gcg gac acc gcg gct cag atc acc cag cgc aag --- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3 511 A*4901 A*4905 552 tgg gag gcg gcc cgt gag gcg gag cag ctg aga gcc tac ctg --- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3 553 A*4901 A*4905 594 gag ggc ctg tgc gtg gag tgg ctc cgc aga tac ctg gag aac --- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3 595 A*4901 A*4905 619 ggg aag gag acg ctg cag cgc gcg g --- --- --- --- --- --- --- --- Exon 3 Figure 1 The exon 3 nucleotide sequence for HLA-B*4905 is compared with nucleotide sequence for B*4901. Dashes indicate identity with B*4905. The allele B*4905 has EMBL Nucleotide Sequence Database Accession Numbers HWS10003268 – AM076839. The HLA-B new variant differs from B*4901 by four nucleotide substitutions in exon 3: at codon 129 (CCC!CCG), at codon 138 (AAC!GAC), and at codon 140 (TTA!TCC). These variations result in a silent mutation and two aminoacidic changes from Asn to Asp and from Leu to Ser, respectively. The nucleotide sequence of exon 3 of HLA-B*4905 is shown in Figure 1 aligned with that of HLA-B*4901. The identification of this new allele, HLA-B*4905, inherited in a Mendelian fashion, contributes to the heterogeneity of HLA-B*49 that, although quite frequent in the Caucasian population (3), was monomorphic till 2001 when the variant B*4902 was detected by our group (4). Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard Acknowledgment This study was supported by a grant from ‘Ricerca Corrente’ Ospedale Maggiore Policlinico di Milano IRCCS, project n. 580/01, 2005. Correspondence Francesca Poli Dipartimento Trasfusionale e di Riferimento per i Trapianti di Organi e Tessuti – Unità Operativa Complessa Immunologia dei Trapianti di Organi e Tessuti Ospedale Maggiore Policlinico Mangiagalli Regina Elena – Fondazione IRCCS di Natura Pubblica Via Francesco Sforza 35 20122 Milan Italy Tel.: þ39 02 55034239 Fax: þ39 02 55012573 e-mail: [email protected] 175 NEW ALLELE Alerts Received 08 November 2005; accepted 15 November 2005 doi: 10.1111/j.1399-0039.2005.00543.x References 1. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2004. Tissue Antigens 2005: 65: 301–69. 2. Pozzi S, Longo A, Ferrara GB. HLA-B locus sequencing-based typing. Tissue Antigens 1999: 53: 275–81. 3. Gjertson DW, Geer L, Lee S et al. Population studies. In: Gjertson DW, Terasaki PI, eds. HLA, 1998. Lenexa, Kansas: American Society for Histocompatibility and Immunogenetics, 1998: 103. 4. Poli F, Longhi E, Frison S et al. Identification of a novel HLA-B allele – HLA-B*4902. Tissue Antigens 2001: 57: 173–4. HLA-B*1586 is a novel, hybrid HLA-B15/B22 allele with unique serology and haplotypic association Y. Yang1, M. T. Aubrey2, G. Zhang1, Y. Ji1 & B. M. Freed2 1 Immunogenetics Laboratory, Shanghai Blood Center, Shanghai, 200051, China 2 ClinImmune Laboratories, University of Colorado Health Sciences Center, Aurora, CO 80010, USA Key words: gene conversion; HLA-B; SBT HLA-B*15 is HLA-B*1586 is a novel the most allele with hybrid polymorphic sequence and of all HLA serology. groups having over 100 members (1, 2). Numerous HLA-B alleles are presumptive hybrids of more common alleles (3, 4). By nature, hybrid alleles present challenges to our classification schemes, and this is reflected in their intermediate serology. In this communication, we report on a new hybrid HLA-B*15 allele first characterized by our group in a Chinese family. While 176 officially designated HLA-B*1586 (the name B*1586 has been officially assigned by the WHO Nomenclature Committee in May 2004), this novel allele displays serological reactivity overlapping the HLA-B15 and HLA-B22,B59 groups and an HLA-C linkage more consistent with HLA-B59 than seen with HLA-B62. Unusual serological reactivity (Figure 1), along with anomalous SSO patterns, suggested the possible presence of a new allele in a Chinese leukemia patient. Additional samples were available on the sibling and mother, which allowed SSP characterization. SSP results were also anomalous and did not match any known allele. Heterozygous sequencing (Applied Biosystems, Foster City, CA, USA) identified a novel polymorphism in the 5-prime area of exon 3. The novel allele was then separated by HaploPrep technology (Genovision, West Chester, PA, USA) using probes B317T and B355C (targeting the T and C at positions 317 and 355, respectively). Haploprep separations followed by hemizygous sequencing in the mother and sibling of the B22/59 Sera Score B17/15/35 Sera Score 9E B54, 55 6D 9F B54 6F B55 B7 9D B22 B67, ±B7 B8 B59 8F B54,B55 B16,67 B59 B59 8 1 8 1 1 1 B63 8 8C 10C 10B 10A 11A 12D 7C B17 B48,±44, 60 B51,53,18,13,46,78, 5102 B18,51,53,78 B53 B46 B57 B35 B35,±75 B35,1508,1511,62,75 B1508, 1511 B15,1508 8 B35,1508, 1511 6 8 1 1 1 2 7E 7F 12C 9B B55,56 B7,27,42,67, 73,81,2708 7D 9A ±B56 B21 12B Bw4 12A Bw6 8 8 B53,38,48,57, B22/15/35 Sera Score patient confirmed the B*1586 sequence. B*1586 is a classic hybrid allele; positions 74–362 of the nucleotide sequence match B*15010101 while the remaining nucleotides from 363 to the end of exon 4 match B*5401, B*5502, B*5901 and others. As a consequence, B*1586 has a unique CACCCTCCAG AGGATGTATG motif beginning at position 351. The hybrid nature is reflected by the unique serology of the patient’s sibling depicted in Figure 1. While there is clear reactivity with B22, B62, and B59, only a fraction of the sera representing those specificities was reactive. Identical hybrid serological patterns were observed in the patient and mother. The availability of the patient’s sibling and mother allowed us to confirm haplotypes. B*1586 was inherited in this family as A*1101-B*1586-Cw*0102DRB1*0901-DQB1*0303. Reference to the IMGT/HLA database (2) revealed that at least three separate groups have observed HLA-B*1586 (5). In all cases, the allele was observed in the B63 62, ±72,76 1 1 B35,B62,1508,1511, 70,75,77 8 1 B13 B62, 75,76 1 9C B22 B17 B70,75 1 Figure 1 Serological pattern of an Asian serological tray (One Lambda of Canoga Park, CA) observed in the sibling of the Chinese leukemia patient. The sibling has a molecular B-locus type of B*1586, B*5801. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard NEW ALLELE Alerts Chinese population and in combination with A11 and DR9. Cw*0102 was reported as present from the Australian Red Cross Blood Service as well as our group. Identical apparent linkages from three separate groups suggest that HLAB*1586 has a conserved haplotype association as observed in other rare alleles (4). Correspondence Michael T. Aubrey ClinImmune Laboratoratories University of Colorado Health Sciences Center Aurora CO 80010 USA Received 12 September 2005; revised 06 October 2005 and 12 October 2005; accepted 13 October 2005 doi: 10.1111/j.1399-0039.2004.00531.x References 1. Marsh SG, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2004. Tissue Antigens 2005: 65: 301–69. 2. Robinson J, Waller MJ, Parham P et al. IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic Acids Res 2003: 31: 311–4. 3. Marcos CY, Fernández-Vina MA, Lázaro AM, Nulf CJ, Raimondi EH, Stastny P. Novel HLA-B35 subtypes: putative gene conversion events with donor sequences from alleles common in native Americans (HLA-B*4002 or B*4801). Hum Immunol 1997: 53: 148–55. 4. Lebedeva TV, Huang A, Ohashi M et al. The recombinant HLA-B*5518 allele supports the evidence of conserved haplotype association of rare alleles. Tissue Antigens 2005: 66: 156–9. 5. Yan L-X, Zhu FM, Lv Q-F, Zhang W. Identification of a new HLA-DRB1 allele, HLA-DRB1*1212, and confirmation of HLA-B*1586. Tissue Antigens 2005: 65: 582–3. Identification of two novel HLA-C alleles, HLA-Cw*1217 and HLA-Cw*030404, in Singapore Chinese† S. W. Pang1, S. H. Chan1,2 & E. C. Ren2,3 1 WHO Collaborating Center for Research and Training in Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 2 Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 3 Genome Institute of Singapore, Singapore Key words: HLA polymorphisms; Singapore Chinese; Cw*030404 allele; Cw*1217 allele The major histocompatibility complex (MHC) is the most polymorphic region of our genome. Human leukocyte antigen-C (HLA-C) genes are located in the class I region of MHC. According to the latest version of the IMGT/HLA Sequence Database (release 2.11.0, October 2005) (2), there are 2184 HLA alleles identified, of which 210 are HLA-C alleles. Accurate DNA sequencing for typing and individual profiling is now commonplace and with high-throughput typing methods, sequence-based typing (SBT) is easily performed in most laboratories, resulting in numerous new alleles †The nucleotide sequences reported in this paper have been deposited in GenBank, Accession Numbers DQ206990 and DQ206991 for Cw*1217 and DQ200948 and DQ200949 for Cw*030404. The names Cw*1217 and Cw*030404 were officially assigned by the WHO Nomenclature Committee in September 2005. This follows the agreed policy that subject to the conditions stated in the most recent Nomenclature Report (1), 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. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard being discovIn this article, we ered each report two novel year. In this human leukocyte article, the antigen-C (HLA-C) sequences of alleles, HLAtwo novel HLA-C alleles Cw*030404 and HLACw*1217. Both are are reported. In perform- characterized by a single nucleotide ing population studies of change in exon 2 when compared to HLA polytheir most homolomorphisms, gous Cw alleles, but we identified only the nucleotide two novel change in Cw*1217 HLA-C resulted in an amino alleles, a acid change. Cw*12 varCompared to iant and a Cw*0304 var- Cw*120201, Cw*1217 had a single nucleoiant in two different indi- tide substitution C>G at nucleotide 295 of viduals of exon 2, resulting in a Singapore coding change, as Chinese desArg75 is changed to cent. Gly75. Cw*030404 had Cw*1217 was a synonymous nucleodiscovered in tide substitution of a Singapore Chinese male, A>G at nucleotide 264 compared to designated Cw*030401 and the CM924, and Cw*030404 in amino acid remains as Thr64. a Singapore Chinese male, designated CM795. Their HLA profiles are as follows: CM924 – HLAA*0207, 1101; HLA-B*1502, 2704; HLA-Cw*0801, 1217; HLADRB1*1101, 1202; CM795 – HLAA* 1101, 1102; HLA-B*1301, 1527; HLA-Cw*030404, 0401; HLADRB1*0406, 1602. Novel nucleotide polymorphisms were discovered during HLA-C sequence-based typing (SBT) of the samples, implying possible new alleles. To verify the new alleles, a nested polymerase chain reaction (PCR) was performed. Specific primers were then designed to amplify the novel alleles in isolation from their counterpart HLA-C alleles. The full-length 177 NEW ALLELE Alerts Cw*010201 Cw*030401 Cw*030404 Cw*120201 Cw*1217 80 GCTCCCA ------------------------- 90 CTCCATGAAG --------G--------G--------G--------G- 100 TATTTCTTCA -------A--------A--------A--------A-- 110 CATCCGTGTC -CG-T-----CG-T-----CG-------CG------- 120 CCGGCCTGGC ------C--------C--------C--------C--- 130 CGCGGAGAGC -----G--------G---------------------- 140 CCCGCTTCAT ---A--------A------------------------ Cw*010201 Cw*030401 Cw*030404 Cw*120201 Cw*1217 150 CTCAGTGGGC -G--------G--------G--------G-------- 160 TACGTGGACG ------------------------------------- 170 ACACGCAGTT ------------------------------------- 180 CGTGCGGTTC ------------------------------------- 190 GACAGCGACG ------------------------------------- 200 CCGCGAGTCC ------------------------------------- 210 GAGAGGGGAG ------------------A--------A--------- Cw*010201 Cw*030401 Cw*030404 Cw*120201 Cw*1217 220 CCGCGGGCGC ------------------------------------- 230 CGTGGGTGGA ------------------------------------- 240 GCAGGAGGGG ------------------------------------- 250 CCGGAGTATT ------------------------------------- 260 GGGACCGGGA ------------------------------------- 270 GACACAGAAG ------------G------------------------ 280 TACAAGCGCC ------------------------------------- Cw*010201 Cw*030401 Cw*030404 Cw*120201 Cw*1217 290 AGGCACAGAC --------------------------G--------G- 300 TGACCGAGTG -------------------------------G----- 310 AGCCTGCGGA ------------------------------------- 320 ACCTGCGCGG ------------------------------------- 330 CTACTACAAC ------------------------------------- 340 CAGAGCGAGG ------------------------------------- 350 CCG|GGTCTCA ---|---------|---------|---------|------- Cw*010201 Cw*030401 Cw*030404 Cw*120201 Cw*1217 360 CACCCTCCAG --T-A------T-A----------------------- 370 TGGATGTGTG A------A-A------A-A------ACA------AC- 380 GCTGCGACCT --------G--------G------------------- 390 GGGGCCCGAC ------------------------------------- 400 GGGCGCCTCC ------------------------------------- 410 TCCGCGGGTA ------------------------------------- 420 TGACCAGTAC --------------------------C--------C- Cw*010201 Cw*030401 Cw*030404 Cw*120201 Cw*1217 430 GCCTACGACG ------------------------------------- 440 GCAAGGATTA ------------------------------------- 450 CATCGCCCTG ------------------------------------- 460 AACGAGGACC --------T--------T------------------- 470 TGCGCTCCTG ------------------------------------- 480 GACCGCCGCG ------------------------T--------T--- 490 GACACCGCGG -----G--------G--------G--------G---- Cw*010201 Cw*030401 Cw*030404 Cw*120201 Cw*1217 500 CTCAGATCAC ------------------------------------- 510 CCAGCGCAAG ------------------------------------- 520 TGGGAGGCGG ------------------------------------- 530 CCCGTGAGGC ------------------------------------- 540 GGAGCAGCGG --------T--------T-------T--------T-- 550 AGAGCCTACC ------------------------------------- 560 TGGAGGGCAC --------CT --------CT ------------------- Cw*010201 Cw*030401 Cw*030404 Cw*120201 Cw*1217 570 GTGCGTGGAG ------------------------------------- 580 TGGCTCCGCA ------------------------------------- 590 GATACCTGGA --------A--------A------------------- 600 GAACGGGAAG ---T--------T------------------------ 610 GAGACGCTGC ------------------------------------- 620 AGCGCGCGG --------------------------------- Figure 1 Nucleotide sequences of exons 2 and 3 of HLA-Cw*030404 and HLA-Cw*1217 compared with the sequences of similar HLA alleles. Dashes (–) indicate the homology with the sequence of Cw*010201. Numbering is from the beginning of the coding region. 178 Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard NEW ALLELE Alerts HLA-C gene was amplified using primers described by Cox et al. (3), followed by allele-specific amplification of the novel alleles. Exons 2 and 3 of the PCR products were then sequenced in both forward and reverse directions using primers described by Cereb et al. (4) and Dormoy et al. (5) to confirm the novel polymorphisms. In CM924, SBT revealed a novel allele that is similar to Cw*120201 within exons 2 and 3, except for a single nucleotide substitution in nucleotide 295 of exon 2 (Fig. 1). The C>G substitution resulted in an amino acid change from Arg75 to Gly75 and the allele has been officially named Cw*1217. Such a C>G substitution remains unique amongst all the known HLA-C alleles to date, with the exception of Cw*0412. In CM795, SBT revealed a novel allele, Cw*030404, which is similar to Cw*030401within exons 2 and 3, except for a single nucleotide substitution (A>G) in nucleotide 264 of exon 2 (Fig. 1), but there is no resulting amino acid change and the amino acid remains as Thr64. This is a unique polymorphism amongst all the known HLA-C alleles. databases for the study of the major histocompatibility complex. Nucleic Acids Res 2003: 31: 311–4. 3. Cox ST, McWhinnie AJ, Robinson J et al. Cloning and sequencing full-length HLA-B and -C genes. Tissue Antigens 2003: 61: 20–48. 4. Cereb N, Kong Y, Lee S, Maye P, Yang SY. Nucleotide sequences of MHC class I introns 1, 2 and 3 in humans and intron 2 in nonhuman primates. Tissue Antigens 1996: 47: 498–511. 5. Dormoy A, Froelich N, Leisenbach R, Weschler B, Cazenave J-P, Tongio M-M. Mono-allelic amplification of exons 2–4 using allele group-specific primers for sequence-based typing (SBT) of the HLA-A, B and -C genes: Preparation and validation of ready-to-use pre-SBT mini-kits. Tissue Antigens 2003: 62: 201–16. Correspondence Ee Chee Ren Genome Institute of Singapore Genome #02-01 60 Biopolis Street Singapore 138672 Tel: þ65 68743316 Fax: þ65 67775720 e-mail: [email protected] To date, at least The novel HLAfive HLADRB1*09 alleles DRB1*0904 allele was identified have been identified (1). In this from cord blood of two unrelated report, we Korean babies. describe a novel allele, DRB1* 0904, identified from two unrelated Korean babies whose cord blood was donated to the cord blood bank and detected during routine HLA typing by highresolution sequence-based typing (SBT). Received 11 October 2005; accepted 24 October 2005 doi: 10.1111/j.1399-0039.2004.00533.x References 1. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA System, 2004. Tissue Antigens 2005: 65: 301–69 [Int J Immunogenet 2005: 32: 107–159; Hum Immunol 2005: 66: 571–636]. 2. Robinson J, Waller MJ, Parham P et al. IMGT/HLA and IMGT/MHC: sequence Identification of a novel HLA-DRB1*09 allele, DRB1*0904, in the Korean population, by sequencebased typing C.-H. Song*, J.-Y. Baek*, Y.-S. Heo, K. Kwack & O.-J. Kwon Pochon CHA University, Seongnam 463-836, Korea Key words: HLA-DRB1*09; HLA-DRB1*0904; sequencebased typing (SBT) *These authors contributed equally to this work. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard For SBT, exon 2 of DRB1 from Korean cord blood units was amplified by polymerase chain reaction for sequence-based HLA-DRB1 typing. Sequencing was performed with a BigDyeTM Terminator v3.1 Cycle Sequencing Ready Reaction Kit on an ABI PRISMÒ 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). We identified a novel DRB1 allele in two unrelated individuals. SBT of these individuals was performed with other class I and II loci. Notably, the individuals had common alleles in the HLA class II group, with the genotypes DRB4*010301, DQA1*0302, DQB1*030302, DPA1*020201, and DPB1*1901, and the novel HLADRB1*09 allele, DRB1*0904. The nucleotide sequence of DRB1*0904 was submitted to GenBank and assigned the accession number AY772533 (exon 2). The name DRB1*0904 was officially assigned by the WHO Nomenclature Committee. As shown in Figure 1, allele DRB1*0904 differed from DRB1*090102 at one nucleotide position in codon 67 (TTC!CTC), resulting in an amino acid change from phenylalanine to leucine at residue 67 in exon 2 of the mature protein, as deduced from the nucleotide sequence. Residue 67 is located in the b1helix of the HLA class II molecule and forms the E pocket of the peptide-binding groove involved in peptide binding (2). This implies that the change at residue 67 affects the peptide preference of the DRB1*0904 allele (3). Leu67 of DRB1*0904 can interact with the labeled leucine of the bound peptide via van der Waals contact. However, Phe67 of other DRB1*09 alleles cannot accommodate a peptide with leucine at this position due to an inevitable collision between the leucine in the bound peptide and Phe67. Therefore, other 179 NEW ALLELE Alerts A DRB1*090102 DRB1*090103 DRB1*0902 DRB1*0903 DRB1*0904 DRB1*0905 5 CA ------ CGT ----------- TTC ----------- TTG ----------- AAG ----------- 10 CAG ----------- GAT ----------- AAG ----------- TTT ----------- CAG ----------- 15 TGT ----------- CAT ----------- TTC ----------- TTC ----------- AAC ----------- 20 GGG ----------- ACG --C --------- GAG ----------- CGG ----------- GTG ----------- 25 CGG ----------- TAT ----------- CTG ----------- CAC ----------- AGA ----------- DRB1*090102 DRB1*090103 DRB1*0902 DRB1*0903 DRB1*0904 DRB1*0905 30 GGC ----------- ATC ----------- TAT ----------- AAC ----------- CAA ----------- 35 GAG ----------- GAG ----------- AAC ----------- GTG ----------- CGC ----------- 40 TTC ----------- GAC ----------- ACC ----------- GAC ----------- GTG ----------- 45 GGG ----------- GAG ----------- TAC ----------- CGG ----------- GCG ----------- 50 GTG ----------- ACG ----------- GAG ----------- CTG ----------- GGG ----------- DRB1*090102 DRB1*090103 DRB1*0902 DRB1*0903 DRB1*0904 DRB1*0905 55 CGG ----------- CCT ----------- GTC ---A------- GCC ----T ------- GAG ----------- 60 TCC ---A------- TGG ----------- AAC ----------- AGC ----------- CAG ----------- 65 AAG ----------- GAC ----------- TTC ------C---- CTG ----------- GAG ------A ----- 70 CGG ----GAC ----- AGG ----------- CGG ------C ----- GCC ----------- GAG ----------- 75 GTG ----------- GAC ----------- ACC ----------- GTG --------TAC TGC ----------- DRB1*090102 DRB1*090103 DRB1*0902 DRB1*0903 DRB1*0904 DRB1*0905 80 AGA ----------- CAC ----------- AAC ----------- TAC ----------- GGG ----------- 85 GTT ----------- GGT ----------- GAG ----------- AGC ----------- TTC ----------- 90 ACA ----------- GTG ----------- CAG ----------- AGG ----------- CGA ----------- 95 G - B DRB1*090102 DRB1*090103 DRB1*0902 DRB1*0903 DRB1*0904 DRB1*0905 10 GDTQPRFLKQ *****----*****----*****----*****----*****----- 20 DKFECHFFNC ---------------------------------------------- 30 TERVRYLHRG ---------------------------------------------- 40 IYNQEENURF ---------------------------------------------- 50 DSDVGEYRAV ---------------------------------------------- 60 TELGRPVAES ---------------D--Y ---------------------------- 70 WNSQKDFLER ---------------------------D ------L------------ 80 RRAEVDTVCR -------------------------------------------Y-- 90 HNYGVGESFT ---------------------------------------------- 100 VQRRVHPEVT ----****** ----****** ----****** ----****** ----****** Figure 1 The exon 2 nucleotide and amino acid sequences of HLA-DRB1*0904, compared with the sequences of all HLA-DRB1*09 alleles. Dashes (-) indicate identity with the HLA-DRB1*090102 sequence and asterisks (*) indicate unsequenced regions. The numbers above the sequence refer to codon position. DRB1*09 alleles would prefer peptides with smaller residues at this position. In conclusion, we identified a novel HLA-DRB1*09 allele, DRB1*0904, that was found in two unrelated Korean babies. The new allele contains an aminoacid change in the antigenbinding site of the HLA protein, which may alter its antigen-binding properties. Correspondence KyuBum Kwack Pochon CHA University Seongnam 463-836 Korea Tel: 82 31 725 8376 Fax: 82 31 725 8350 e-mail: [email protected] Received 21st October 2005; Revised 7th November 2005; Accepted 8th November 2005 doi: 10.1111/j.1399-0039.2005.00537.x Acknowledgments This study was supported by grants from the Korea Science and Engineering Foundation (R01-2002-000-00267-0) and 180 from the Ministry of Health and Welfare (0405-BC02-0604-0004). References 1. Marsh SG, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2004. Tissue Antigens 2005: 65: 301–69. 2. Brwon JH, Jardetzky TS, Gorga JC et al. Three-dimensional structure of the human class II histocompatibility antigen HLADR1. Nature 1993: 364: 33–9. 3. Stern LJ, Brown JH, Jardetzky TS et al. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 1994: 368: 215–21. Identification of a novel HLA-DRB1*11 allele: DRB1*1152 G. Ozzella1, P. I. Monaco1, A. Tessitore2, A. Piazza1, D. Piancatelli3 & D. Adorno1 1 Organ Transplant and Immunocitology, C.N.R. Institute, Rome, Italy 2 Department of Experimental Medicine, University of L’Aquila, Italy 3 Organ Transplant and Immunocitology, C.N.R. Institute, Section of L’Aquila, Italy Key words: HLA-DRB1; new allele; polymorphism transplantation; polymorphism The novel New HLA-DRB1*1152 HLAallele differs from DRB1*11 DRB1*1117 by a single allele was nucleotide substitudetected in tion at codon 60 three mem(T!C). bers of a Moroccan Berber family. It was suspected from DRB1 genotyping discrepancies. Low-resolution PCR-SSP typing of the father’s DRB1 locus (DRB1, Olerup SSP, Genovision, Vienna, Austria) (1) revealed an ambiguity because it was not possible to discriminate between rare DRB1*11 homozygosity (*1113/1117/1122, *1123/1125) and DRB1*11, DRB1*14 heterozygosity (*1101/ 1104/1106/1112/1115/1124/1127– 1129/1132/1133/1135/1137–1139/ 1143, *1408/1434). DRB1 typing of the patient and his matched brother were specific for DRB1*14 (inherited from the mother) and DRB1*1113/1117/1122 (inherited from the father). This seemed to exclude the father from being heterozygous for DRB1*11. DRB1 typing of the sister was specific for DRB1*14 inherited from the mother and DRB1*11 inherited from the father (*1101/1104/1106/ 1112/1115/1124/1127–1129/1132/ 1133/1135/1137–1139/1143 alleles). This result excluded the possibility that the father was homozygous for DRB1*11. The family was then studied by high-resolution PCR-SSP typing. The father’s DRB1*11 typing permitted to assign DRB1*1104 allele. It was not possible to define the second allele because of an ambiguity between DRB1*1117 and DRB1*14: both showed one false positive reaction. However, it proved possible to assign the DRB1*1117 allele to the patient and his matched brother without any mismatch. In fact, the false positive reaction seen in the Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard NEW ALLELE Alerts DRB1*1117 DRB1*1152 DRB1*1117 DRB1*1152 DRB1*1117 DRB1*1152 DRB1*1117 DRB1*1152 DRB1*1117 DRB1*1152 DRB1*1117 DRB1*1152 DRB1*1117 DRB1*1152 DRB1*1117 DRB1*1152 5 10 15 20 25 CA CGT TTC TTG GAG TAC TCT ACG TCT GAG TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA -- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --P * R - F - L - E - Y - S - T - S - E - C - H - F - F - N - G - T - E - R - V - R - F - L - D - R - 30 35 40 45 50 TAC TTC CAT AAC CAG GAG GAG TTC GTG CGC TTC GAC AGC GAC GTG GGG GAG TAC CGG GCG GTG ACG GAG CTG GGG --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --Y - F - H - N - Q - E - E - F - V - R - F - D - S - D - V - G - E - Y - R - A - V - T - E - L - G - 55 65 70 75 60 CGG CCT GAT GAG GAG TAC TGG AAC AGC CAG AAG GAC CTC CTG GAG CGG AGG CGG GCC GAG GTG GAC ACC TAT TGC --- --- --- --- --- C-- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --R - P - D - E - E - Y H W - N - S - Q - K - D - L - L - E - R - R - R - A - E - V - D - T - Y - C - 80 85 90 95 AGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA G --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- R - H - N - Y - G - V - V - E - S - F - T - V - Q - R - R - V * Figure 1 Exon 2 nucleotide and predicted amino acid sequences alignment of DRB1*1117 and the new DRB1*1152. Dashes indicate identity with DRB1*1117; stars denote undefined amino acid. father could now be used for the assignment of the DRB1*1401 allele inherited from the mother. The typing of the sister’s DRB1 alleles permitted to assign unambiguously DRB1*1401 inherited from mother, with DRB1*1104 inherited from father. All these results supported the hypothesis of a novel DRB1*11 allele in the father, the patient and his matched brother. To confirm this, DRB1 sequence-based typing was carried out after a preliminary PCRSSP screening of the major DRB1 allele groups using DRBAMP-52 and DRAMP-B primers (twelfth IHW) (2). PCR products were sequenced in both directions by Big Dye Terminator chemistry. The complete sequence of exon 2 of the DRB1 gene was amplified using primers within intron 1 and intron 2 (3), and the PCR product was cloned using the pGEM-T Easy Vector System II Cloning Kit (Promega, Madison, WI, USA) to distinguish the alleles. DNA inserts from the cloned allele were amplified and sequenced. The new sequence obtained differs from DRB1*1117 by single nucleotide substitution at codon 60 (TAC!CAC), changing the amino acid from tyrosine (Y) to histidine (H). The nucleotide sequence was submitted to GenBank in March 2004 and given the accession number AY574194. The name DRB1*1152 was officially assigned by the WHO Nomenclature Committee for Factors of the HLA System in May 2004 (4) (Fig. 1). This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report, names are to be assigned to new sequences as they are identified. Comparison of DRB1*1152 with all DRB1*14 sequences on the IMGT/HLA database showed that the triplet CAC at codon 60 is shared with some DRB1*14 alleles such as DRB1*1408 and DRB1*1434, as suggested by low-resolution DR genotyping. The novel DRB1*1152 allele segregated together with DQB1*0503; this DR11-DQ5 phenotype was in contrast to already known HLA linkages. The alteration in the amino acid sequence encoded by DRB1*1152 changes the amino acid produced by codon 60 from a neutral tyrosine to a positively charged histidine. This mutation is located on the a-helix portion of the b1 chain of DRB1 that contributes to form the side of the Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard peptide-binding groove (5). Histidine often contributes to the hydrogen bond network formed between the peptide and a1/b1 a helical domains (6). This tyrosine-to-histidine substitution might interfere with binding energy, contributing to stabilize or destabilize peptide–HLA II interactions and thus might affect antigen presentation. Correspondence Giuseppina Ozzella Istituto C.N.R. peri Trapianti D’Organo e l’lmmunocitologia, P. le dell’Umanesimo 10, 00144 Rome, Italy e-mail: [email protected] Received 3 October 2005; revised 17 October 2005; accepted 2 November 2005 doi: 10.1111/j.1399-0039.2005.00534.x References 1. Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor recipient matching in cadaveric transplantation. Tissue Antigens 1992: 39: 225–35. 2. Fernandez-Vina M, Bignon JD. Primers and oligonucleotide probes (SSOP) used foe DNA typing of HLA class II 181 NEW ALLELE Alerts alleles. In: Charron D, ed. Genetic Diversity of HLA-Functional and Medical Implications. Paris: EDK, 1997, 596–632. 3. Kotsch K, Wehling J, Blasczyk R. Sequencing of HLA Class II genes based on the conserved diversity of the non- 182 coding regions: sequencing based typing of HLA-DRB genes. Tissue Antigens 1999: 53: 486–97. 4. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA System, 2002. Tissue Antigens 2002: 60: 407–64. 5. Brown JH, Jardetzky TS, Gorga JC et al. Three-dimensional structure of the human class II histocompatibility antigen HLADR1. Nature 1993: 364: 33–9. 6. Abderson MW, Gorski J. Cooperativity during the formation of peptide/MHC class II complexes. Biochemistry 2005: 44: 5617–24. Ó 2006 The Authors Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard