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Keywords:

  • allele-specific amplification;
  • new allele;
  • sequence-based typing

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgment
  4. Conflict of Interests
  5. References

The newly detected HLA-B*08:111 allele shows two nucleotide differences from B*08:01:01 in codons 113 and 114.

Human leukocyte antigens (HLA) genes, encoded within the human major histocompatibility complex, are the most polymorphic loci in the genome; HLA-B genes, especially, to date are represented by 2934 HLA-B alleles, 129 for HLA-B*08, according to the IMGT/HLA database (release 3.12.0, April 2013). During routine HLA genotyping of potential bone marrow donor, from Italian Bone Marrow Donor Registry (IBMDR) [1], we found a new HLA-B*08 allele in a male donor. HLA typing was performed by polymerase chain-reaction-sequence specific oligonucleotide (PCR-SSO) using LABType® (OneLambda Inc., Canoga Park, CA) following the manufacturer's protocol for classes I and II and produced the following results: B*08:28/35/69 and B*39:01/01L/15/19/25N/26/27/38Q/40N/44/45/46/51/5354/59/60/61.

To confirm this issue, high-resolution typing of HLA-B was executed using PCR-sequence specific primers (SSP; Olerup SSP AB, Stockholm, Sweden). The pattern of amplification did not confirm the previous analysis for the allele B*08 who was, at this time, B*08:74 in association with B*39:15 (Helmberg-SCORE™ 5.0).

Because of the sequence-based typing (PCR-SBT) represent the best high-resolution approach to discover a nucleotide polymorphism, we performed an allele specific amplification followed by a sequencing of exons 2, 3 and 4 using TBG HLAssure™ SE SBT locus B (Texas BioGene, Inc., Richardson, TX) on an AB3500DX Genetic Analyzer (Life Technologies Corporation, CA).

Sequencing data was analyzed using ASSIGN™ SBT 3.5.1.45 software (Conexio Genomics, Fremantle, Australia).

Sequencing of both alleles separately confirmed the presence of B*39:01 and showed a new B*08 allele that presents all the polymorphisms in exon 3. The allele with greatest similarity is B*08:01:01; however, the new allele differs from alleles B*08:01:01 and B*08:07 by one nucleotide at position 409 (C[RIGHTWARDS ARROW]T) resulting in an amino acid substitution (codon 113.1: CAT[RIGHTWARDS ARROW]TAT) from His to Tyr; from B*08:01:01 and B*08:74 and at position 412 (A[RIGHTWARDS ARROW]G) resulting in an amino acid substitution (codon 114.1: AAC[RIGHTWARDS ARROW]GAC) from Asn to Asp; from B*08:87 at position 418 (T[RIGHTWARDS ARROW]G) showing an amino acid switch (codon 116.1: TAC[RIGHTWARDS ARROW]GAC) from Tyr to Asp and shows a nucleotide variation at position 419, from B*08:28, which altered codon 116.2 (TAC[RIGHTWARDS ARROW]TCC), Tyr[RIGHTWARDS ARROW]Ser (Figure 1).

image

Figure 1. Alignment of exons 2, 3 and 4 of HLA-B*08:01:01, B*08:07, B*08:28, B*08:74, B*08:87 and B*08:111.

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The complete donor HLA typing was: A*03:01,*24:02, B*08:111,*39:01, C*07:01,*12:03 and DRB1*03:01,*16:01.

The new allele was submitted to GenBank and IMGT/HLA [2] and assigned the accession number: KF229774.

The name B*08:111 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee in June 2013. This follows the agreed policy that, subject to the conditions started in the most recent Nomenclature Report [3], names will be assigned to the new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature report.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Acknowledgment
  4. Conflict of Interests
  5. References

We want to thank Dr Angeloni Silvia of Medical Genetics Unit, S. Camillo-Forlanini Hospital, for her assistance in this work.

Conflict of Interests

  1. Top of page
  2. Abstract
  3. Acknowledgment
  4. Conflict of Interests
  5. References

The authors have declared no conflicting interest.

References

  1. Top of page
  2. Abstract
  3. Acknowledgment
  4. Conflict of Interests
  5. References