A novel HLA-DRB1 allele, DRB1*01:54 , identified by sequence-based typing S. Barbuti 1 , M. Curcio 1 , L. Marchetti 2 , O. Vittorio 2 , M. L Mariotti 1 & F. Scatena 1 1 Laboratorio di Immunogenetica, U.O. Medicina Trasfusionale e Biologia dei Trapianti, Hospital of Pisa, AOUP – Pisa, Italy 2 NEST-Scuola Normale Superiore and Istituto Nanoscienze – CNR, Pisa, Italy Key words: DRB1*01:54; new allele; sequence-based typing The new HLA DRB1*01:54 differs from DRB1*01:02:01 by one nucleotide at exon 2. Human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation is a well-established therapy used to treat hematological malignancies and other hematology or immune disorders. HLA matching between donor and recipient is the major determinant of allogenic bone marrow transplantation outcome (1). If an HLA-matched sibling is not available, it becomes necessary to look for an unrelated donor of hematopoietic stem cells obtained from either bone marrow or peripheral blood. In this report wedescribe a new HLA-DRB1 allele, found in an Italian stem cell donor. Genomic DNA was extracted from whole blood, using a commercial kit, according to the manufacturer’s instructions (QIAamp  250 DNA Blood kit, QIAGEN GmbH, Hilden, Germany). The sample was sequenced at the HLA-A, B, C, DRB1 and DQB1 loci using AlleleSEQR HLA PCR/sequencing kit [sequence-based typing (SBT) SeCore  HLA Invitrogen’s kit of high resolution HLA typing products based on SBT, Invitrogen by Life Technologies, Carlsbad, CA]. The amplified products were purified with AMPure XP Agencourt (Beckman Coulter Genomics, Danvers, MA) and the purified products were forward and reverse sequenced using the sequencing mixes provided by the manufacturer. Allele assignment was performed with utype  6.0 (Invitrogen by Life Technologies, Carlsbad, CA), HLA sequencing software and the HLA alleles of the individual were genotyped as A*02:01:01 , A*33:01:01 ; B*14:02:01 , B*44:02:01 ; C*08:02:01 , C*16:04:01 ; DQB1*05:01:01 , and DRB1*01:02:01 with one heterozygous nucleotide mismatch at position 286 (C/A, IUPAC code M). This suggested the existence of a new variant allele combination. To confirm the presence of the new allele in the sample, the group-specific amplification of HLA-DRB1 locus was performed using the single specific sequencing set for HLA-DRB1 (S4 HLA-DRB1; Protrans GmbH, Ketsch, Germany). The sequence was then analyzed using sequence pilot software (JSI medical systems GmbH, Kippenheim, Germany) and the allele assignment confirmed a new variant allele of HLA-DRB1*01 . In order to isolate the new HLA-DRB1*01 allele, the sample was cloned using the TA cloning kit (Invitrogen, Carlsbad, CA). Multiple clones were subjected to restriction digestion in order to verify the presence of the insert; the positive clones were then sequenced. The sequence analysis showed the (A) (B) Figure 1 Alignment of the nucleotide sequences of exon 2 of human leukocyte antigen (HLA)-DRB1*01:02:01 and HLA-DRB1*01:54 alleles (A) and amino acid sequences (B). Numbers above the sequence correspond to the amino acids codon (Codon Pos.) in the mature protein. Dashes indicate identity between two alleles. The codon 67 highlighted in bold show the difference between the alleles DRB1*01:54 and DRB1*01:02:01. Sequences are numbered according to the IMGT/HLA sequence database (3). presence of two alleles: the first one was assigned to DRB1*01 allele which exhibit the single nucleotide substitution at nucleotide position 286 (C→A), codon 67; the second allele was assigned to DRB1*01:02:01 with no change at codon 67. Sequence alignment with HLA-DRB1*01:02:01 allele sequence downloaded from IMGT/HLA Sequence Database (2) display the novel DRB1*01 variant (Figure 1), resulting in a nonsynonymous conservative substitution at codon 67 (L→I) with the replacement of Leucine by Isoleucine, both apolar amino acids. The new variant sequence was submitted to the IMGT/HLA database (HWS10016932) and the name DRB1*01:54 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee in October 2012. This 80  2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2013, 82, 59–81