HLA-B*35:233 , a novel B*35 allele found in a volunteer of the DONORMO – The Mexican Bone Marrow Registry of Unrelated Donors C Alaez, H Flores-A, A Mungu´ıa, ME Valencia & C Gorodezky Department of Immunology and Immunogenetics, Secretary of Health, Instituto de Diagn´ostico y Referencia Epidemiol´ogicas, Mexico City, Mexico Key words: B*35 antigenic group; HLA-B*35:233 ; Mexican Mestizo; novel allele
HLA-B*35:233 a novel human leukocyte antigen-B was identified in a Mexican Mestizo donor. Several new human leukocyte antigen (HLA) alleles have been published by us recently, corresponding to the very diverse antigen groups (1, 2). As of June 2013, 2934 alleles encoding 2211 proteins have been described for the HLA-B gene. The HLA-B*35 group is highly polymorphic, with 309 alleles, described nowadays (IMGT/HLA database) (3). A new B*35 allele, B*35:233 was found in a 22-year-old Mexican Mestizo male, born and living in the state of Puebla, Mexico. During a routine HLA-B typing for the Mexican Bone Marrow Registry of Unrelated Donors (DONORMO), the analysis software showed a perfect match with the combination B*39:48 , B*35:04/09/12 . The typing was performed using Luminex technology with the Lifecodes HLA-B typing kit (Gen-Probe Transplant Diagnostics Inc., Stamford, CT). DNA extracted from whole blood cells was used as template for the polymerase chain reaction (PCR) reaction. As B*39:48 is a rare allele, according to the Allele frequencies database (4), HLA-B sequence-based typing was performed on this sample to confirm the results. HLA-B exons 2, 3 and 4 were sequenced in both directions using the AlleleSEQR HLA-B kit (Celera, Alameda, CA) and an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Alameda, CA). The sequence analysis was performed with the assign 3.5+ software (ConexioGenomics, Applecross, Western Australia, Australia). No exact match was found for the consensus sequence of the sample. The closest typing was B*35:12:01 , B*39:05 with one mismatch at nucleotide 583, exon 3 corresponding to codon 171.1. At this position, the consensus of the sample showed Y (C and T) but only T was expected for a perfect match with the mentioned allele pair, suggesting the presence of a new allele. 436
B*35 and B*39 alleles were amplified and sequenced separately for exons 2–4, using two group-specific PCR primers: S4B7 (specific for B*35 , *48:02 , *51 , *52 , *53 , *56:06 , *58 , *78 ) and S4B8 (specific for B*14 , *38 , *39 , *67:01 , Protrans , Ketsch, Germany). An exact match for B*39:05:01 was found for the PCR product obtained with primer S4B8. However, for the S4B7 PCR product, the consensus sequence of the sample showed one mismatch with B*35:12:01 at nucleotide 583, exon 3 (Figure 1). This substitution affects aminoacid 171 that changes from the polar non charged Y, present in most of the B*35 alleles, to the positively charged H (codon 171 TAC→CAC). This substitution possibly does not affect the stability of the encoded B molecule, as histidine is found at this position, in most of the B*14 and B*18 alleles and in some B*15 and B*35 alleles. A point mutation occurring on B*35:12:01 , a very frequent B*35 allele in Mexican population (5), is the most probable mechanism that generated this new B*35 allele. B*35:233 was submitted to the GenBank; the accession number is KF032077. The name B*35:233 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee in May 2013. This follows the agreed policy that, subject to the condition stated in the most recent Nomenclature Report (6), 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. The complete HLA typing of this individual was A*02 , A*68 B*39:05:01 , B*35:233 , C*04 , C*07 , DRB1*04:07 , DRB1*04:07 , DQB1*03:02 , DQB1*03:02 . We were unable to set the gametic phase, however, as B*35-C*04 and B*39C*07 are typically in linkage disequilibrium. Therefore, we suggest that the new allele is part of the haplotype B*35:233C*04-DRB1*04:07-DQB1*03:02 . These demonstrate the importance of further confirming any HLA typing involving rare or less frequent alleles, using a different methodology, even when ‘a perfect match’ message seems to be clear from the software analysis.
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2013, 82, 423–446
Figure 1 Nucleotide sequence of exons 2, 3 and 4 of HLA-B*07:02:01 (reference sequence), B*35:12:01 and B*35:233 . The nucleotide difference with B*35:12:01 is indicated by a square. Dashes indicate the identity with B*07:02:01.The figure is derived from IMGT/HLA sequence database (1).
Correspondence
References
Prof Dr Clara Gorodezky Head of The Department of Immunology and Immunogenetics Secretary of Health Instituto de Diagn´ostico y Referencia Epidemiol´ogicos, InDRE Carpio 470 1st floor M´exico D.F. 11340 Mexico Tel: (52 55) 5341 4569; (52 55) 5342 7555 Fax: (52 55) 5341 4418 e-mail: [email protected]
1. Alaez C, Mungu´ıa A, Flores HA, Gorodezky C. Identification of A*29:47, previously typed as A*29:19, in a Mexican bone marrow donor from the state of Hidalgo, Mexico. Tissue Antigens 2013: 81: 454–5. 2. Alaez C, Mungu´ıa A, Flores HA, Hilario PA, Gorodezky C. Identification of A*02:336 in a Mexican Mestizo acute lymphoblastic leukemia patient from the state of Veracruz. Tissue Antigens 2012: 80: 265–6. 3. Robinson J, Halliwell JA, McWilliam H, Lopez R, Parham P, Marsh SGE. The IMGT/HLA Database. Nucleic Acids Res 2013: 41: D1222–7. 4. Gonzalez-Galarza FF, Christmas S, Middleton D, Jones AR. Allele frequency net a database and online repository for immune gene frequencies in worldwide populations. Nucleic Acids Res 2011: 39: D913–9. 5. Gorodezky C, Alaez C, V´azquez-Garc´ıa MN et al. The genetic structure of Mexican Mestizos of different locations: tracking back
doi: 10.1111/tan.12219
Conflict of Interest
The authors have declared no conflicting interests. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2013, 82, 423–446
437
their origins through MHC genes, blood group systems, and microsatellites. Hum Immunol 2001: 62: 979–91.
438
6. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2010. Tissue Antigens 2010: 75: 291–455.
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2013, 82, 423–446
HLA-B*35:233 a novel human leukocyte antigen-B was identified in a Mexican Mestizo donor. Several new human leukocyte antigen (HLA) alleles have been published by us recently, corresponding to the very diverse antigen groups (1, 2). As of June 2013, 2934 alleles encoding 2211 proteins have been described for the HLA-B gene. The HLA-B*35 group is highly polymorphic, with 309 alleles, described nowadays (IMGT/HLA database) (3). A new B*35 allele, B*35:233 was found in a 22-year-old Mexican Mestizo male, born and living in the state of Puebla, Mexico. During a routine HLA-B typing for the Mexican Bone Marrow Registry of Unrelated Donors (DONORMO), the analysis software showed a perfect match with the combination B*39:48 , B*35:04/09/12 . The typing was performed using Luminex technology with the Lifecodes HLA-B typing kit (Gen-Probe Transplant Diagnostics Inc., Stamford, CT). DNA extracted from whole blood cells was used as template for the polymerase chain reaction (PCR) reaction. As B*39:48 is a rare allele, according to the Allele frequencies database (4), HLA-B sequence-based typing was performed on this sample to confirm the results. HLA-B exons 2, 3 and 4 were sequenced in both directions using the AlleleSEQR HLA-B kit (Celera, Alameda, CA) and an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Alameda, CA). The sequence analysis was performed with the assign 3.5+ software (ConexioGenomics, Applecross, Western Australia, Australia). No exact match was found for the consensus sequence of the sample. The closest typing was B*35:12:01 , B*39:05 with one mismatch at nucleotide 583, exon 3 corresponding to codon 171.1. At this position, the consensus of the sample showed Y (C and T) but only T was expected for a perfect match with the mentioned allele pair, suggesting the presence of a new allele. 436
B*35 and B*39 alleles were amplified and sequenced separately for exons 2–4, using two group-specific PCR primers: S4B7 (specific for B*35 , *48:02 , *51 , *52 , *53 , *56:06 , *58 , *78 ) and S4B8 (specific for B*14 , *38 , *39 , *67:01 , Protrans , Ketsch, Germany). An exact match for B*39:05:01 was found for the PCR product obtained with primer S4B8. However, for the S4B7 PCR product, the consensus sequence of the sample showed one mismatch with B*35:12:01 at nucleotide 583, exon 3 (Figure 1). This substitution affects aminoacid 171 that changes from the polar non charged Y, present in most of the B*35 alleles, to the positively charged H (codon 171 TAC→CAC). This substitution possibly does not affect the stability of the encoded B molecule, as histidine is found at this position, in most of the B*14 and B*18 alleles and in some B*15 and B*35 alleles. A point mutation occurring on B*35:12:01 , a very frequent B*35 allele in Mexican population (5), is the most probable mechanism that generated this new B*35 allele. B*35:233 was submitted to the GenBank; the accession number is KF032077. The name B*35:233 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee in May 2013. This follows the agreed policy that, subject to the condition stated in the most recent Nomenclature Report (6), 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. The complete HLA typing of this individual was A*02 , A*68 B*39:05:01 , B*35:233 , C*04 , C*07 , DRB1*04:07 , DRB1*04:07 , DQB1*03:02 , DQB1*03:02 . We were unable to set the gametic phase, however, as B*35-C*04 and B*39C*07 are typically in linkage disequilibrium. Therefore, we suggest that the new allele is part of the haplotype B*35:233C*04-DRB1*04:07-DQB1*03:02 . These demonstrate the importance of further confirming any HLA typing involving rare or less frequent alleles, using a different methodology, even when ‘a perfect match’ message seems to be clear from the software analysis.
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2013, 82, 423–446
Figure 1 Nucleotide sequence of exons 2, 3 and 4 of HLA-B*07:02:01 (reference sequence), B*35:12:01 and B*35:233 . The nucleotide difference with B*35:12:01 is indicated by a square. Dashes indicate the identity with B*07:02:01.The figure is derived from IMGT/HLA sequence database (1).
Correspondence
References
Prof Dr Clara Gorodezky Head of The Department of Immunology and Immunogenetics Secretary of Health Instituto de Diagn´ostico y Referencia Epidemiol´ogicos, InDRE Carpio 470 1st floor M´exico D.F. 11340 Mexico Tel: (52 55) 5341 4569; (52 55) 5342 7555 Fax: (52 55) 5341 4418 e-mail: [email protected]
1. Alaez C, Mungu´ıa A, Flores HA, Gorodezky C. Identification of A*29:47, previously typed as A*29:19, in a Mexican bone marrow donor from the state of Hidalgo, Mexico. Tissue Antigens 2013: 81: 454–5. 2. Alaez C, Mungu´ıa A, Flores HA, Hilario PA, Gorodezky C. Identification of A*02:336 in a Mexican Mestizo acute lymphoblastic leukemia patient from the state of Veracruz. Tissue Antigens 2012: 80: 265–6. 3. Robinson J, Halliwell JA, McWilliam H, Lopez R, Parham P, Marsh SGE. The IMGT/HLA Database. Nucleic Acids Res 2013: 41: D1222–7. 4. Gonzalez-Galarza FF, Christmas S, Middleton D, Jones AR. Allele frequency net a database and online repository for immune gene frequencies in worldwide populations. Nucleic Acids Res 2011: 39: D913–9. 5. Gorodezky C, Alaez C, V´azquez-Garc´ıa MN et al. The genetic structure of Mexican Mestizos of different locations: tracking back
doi: 10.1111/tan.12219
Conflict of Interest
The authors have declared no conflicting interests. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2013, 82, 423–446
437
their origins through MHC genes, blood group systems, and microsatellites. Hum Immunol 2001: 62: 979–91.
438
6. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2010. Tissue Antigens 2010: 75: 291–455.
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2013, 82, 423–446
