A novel HLA-C null allele, HLA-C*05:99N B. Cauwelier1 , F. Nollet1 , A. Gadisseur2 , W. Schroyens2 & Z. Berneman2 1 Laboratory Medicin, AZ St Jan Brugge-Oostende, Brugge, Belgium 2 Department of Hematology, Antwerp University Hospital, Antwerp, Belgium Key words: HLA-C*05:99N; new human leukocyte antigen-C; null allele; sequence-based typing
HLA-C*05:99N results from a single nucleotide loss compared with its closest allele HLA-C*05:01:01:01. Here we describe a new HLA-C null allele observed during routine HLA typing of a Caucasian hematopoietic stem cell recipient and her offspring. Blood samples were extracted using the QiaSymphony DSP DNA extraction kit on the Qiasymphony instrument (Qiagen, Hilden, Germany). Low resolution typing was performed for the human leukocyte antigen (HLA)-A, -B, -C, -DRB1 and -DQB1 loci using Lifecodes HLA SSO typing kits (Lifecodes, Immucor© , Heppignies, 420
Belgium) on a Luminex® platform (Luminex Corporation, Oosterhout, the Netherlands). The C-locus was typed as a HLA-C*05,07. Subsequent high resolution confirmatory typing was performed by sequence-based typing (SBT) of exons 1–7 using Gendx© SBT reagents (GenDx, Utrecht, the Netherlands) on a 3500xL Genetic analyzer (Thermo FisherScientific Inc., Erembodegem, Belgium). The following genotypic ambiguities were found for the HLA-C locus: C*05:05,07:01:01:01/C*05:01:01:01,07:18/C*05:01:03,07: 166. No gene-specific sequence primers (GSSPs) were available to resolve these ambiguities. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 405–437
Figure 1 Nucleotide (A) and protein sequence (B) of HLA-C*05:99N compared to its closest allele HLA-C*05:01:01:01.
Subsequent allele-specific sequencing of the HLA-C locus exons 1–4 was performed using the Protrans© HLA SBT kit (Protrans, Hockenheim, Germany). For the HLA-C*07 allele we identified an ambiguous result: HLA-C*07:01:01:01/07:18. Polymerase chain reaction-sequence specific primer (PCR-SSP, OleRup, Vienna, Austria) was used to determine the unique nucleotide position at exon 6, resulting in a typing of HLA-C*07:18. For the HLA-C*05 allele, homozygous sequencing identified a best but not complete match with HLA-C*05:01:01:01. Further analysis of exon 3 sequences of the obtained sequences in comparison with allele C*05:01:01:01 (Figure 1A) showed a single nucleotide loss of a G nucleotide at position 381 (codon 104) (IMGT/HLA database, version 3.11). This deletion causes a frameshift and premature TGA stop codon at codon 126 (Figure 1B), resulting in a truncated protein sequence with partial loss of the antigen-binding domain thus assigned as a HLA null allele. Sequences for exons 2 and 3 were submitted to GenBank (accession number KF806553). The name C*05:99N has been officially assigned by the World Health Organization (WHO) Nomenclature Committee in November 2013. 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. The complete typing of the individual possessing the new allele is: A*02:05, A*02:20, B*44:02, B*58:01, C*05:99N,
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 405–437
C*07:18, DRB1*04:07/04:92, DRB1*14:54, DQB1*03:01, DQB1*05:03, DPB1*03:01/124:01, and DPB1*04:01. The same C*05:99N null allele was observed in the HLA typing of the daughter of this patient. Correspondence
Barbara Cauwelier Laboratory Medicin AZ St Jan Brugge-Oostende Brugge Belgium Tel: 003250452610 Fax: 003250452619 e-mail: [email protected] doi: 10.1111/tan.12404
Conflict of Interests
The authors have declared no conflicting interests. Reference 1. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2010. Tissue Antigens 2010: 75: 291–455.
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HLA-C*05:99N results from a single nucleotide loss compared with its closest allele HLA-C*05:01:01:01. Here we describe a new HLA-C null allele observed during routine HLA typing of a Caucasian hematopoietic stem cell recipient and her offspring. Blood samples were extracted using the QiaSymphony DSP DNA extraction kit on the Qiasymphony instrument (Qiagen, Hilden, Germany). Low resolution typing was performed for the human leukocyte antigen (HLA)-A, -B, -C, -DRB1 and -DQB1 loci using Lifecodes HLA SSO typing kits (Lifecodes, Immucor© , Heppignies, 420
Belgium) on a Luminex® platform (Luminex Corporation, Oosterhout, the Netherlands). The C-locus was typed as a HLA-C*05,07. Subsequent high resolution confirmatory typing was performed by sequence-based typing (SBT) of exons 1–7 using Gendx© SBT reagents (GenDx, Utrecht, the Netherlands) on a 3500xL Genetic analyzer (Thermo FisherScientific Inc., Erembodegem, Belgium). The following genotypic ambiguities were found for the HLA-C locus: C*05:05,07:01:01:01/C*05:01:01:01,07:18/C*05:01:03,07: 166. No gene-specific sequence primers (GSSPs) were available to resolve these ambiguities. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 405–437
Figure 1 Nucleotide (A) and protein sequence (B) of HLA-C*05:99N compared to its closest allele HLA-C*05:01:01:01.
Subsequent allele-specific sequencing of the HLA-C locus exons 1–4 was performed using the Protrans© HLA SBT kit (Protrans, Hockenheim, Germany). For the HLA-C*07 allele we identified an ambiguous result: HLA-C*07:01:01:01/07:18. Polymerase chain reaction-sequence specific primer (PCR-SSP, OleRup, Vienna, Austria) was used to determine the unique nucleotide position at exon 6, resulting in a typing of HLA-C*07:18. For the HLA-C*05 allele, homozygous sequencing identified a best but not complete match with HLA-C*05:01:01:01. Further analysis of exon 3 sequences of the obtained sequences in comparison with allele C*05:01:01:01 (Figure 1A) showed a single nucleotide loss of a G nucleotide at position 381 (codon 104) (IMGT/HLA database, version 3.11). This deletion causes a frameshift and premature TGA stop codon at codon 126 (Figure 1B), resulting in a truncated protein sequence with partial loss of the antigen-binding domain thus assigned as a HLA null allele. Sequences for exons 2 and 3 were submitted to GenBank (accession number KF806553). The name C*05:99N has been officially assigned by the World Health Organization (WHO) Nomenclature Committee in November 2013. 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. The complete typing of the individual possessing the new allele is: A*02:05, A*02:20, B*44:02, B*58:01, C*05:99N,
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 405–437
C*07:18, DRB1*04:07/04:92, DRB1*14:54, DQB1*03:01, DQB1*05:03, DPB1*03:01/124:01, and DPB1*04:01. The same C*05:99N null allele was observed in the HLA typing of the daughter of this patient. Correspondence
Barbara Cauwelier Laboratory Medicin AZ St Jan Brugge-Oostende Brugge Belgium Tel: 003250452610 Fax: 003250452619 e-mail: [email protected] doi: 10.1111/tan.12404
Conflict of Interests
The authors have declared no conflicting interests. Reference 1. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature for factors of the HLA system, 2010. Tissue Antigens 2010: 75: 291–455.
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