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ISSN: 2471-9552
Immunotherapy: Open Access
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PCR-SBT Characterization of A New HLA Allele: A*02:548

Valentina Cappuzzo*, Francesco Ingrassia, Rosalba Bavetta, Serena Mistretta, Maria Blando, Igea Vega, Emanuela Collura and Raimondo Marcenò

Laboratory of Tipizzazione Tessutale ed Immunogenetica, Medicina Trasfusionale – A.O, Ospedali Riuniti Villa Sofia-Cervello, via Trabucco, 180- 90146 Palermo, Italy

*Corresponding Author:
Valentina Cappuzzo
Laboratory of Tipizzazione Tessutale ed
Immunogenetica, Medicina Trasfusionale – A.O
Ospedali Riuniti Villa Sofia-
Cervello, via Trabucco
180- 90146 Palermo, Italy
Tel: 390916802980
E-mail: [email protected]

Received date: January 18, 2016; Accepted date: February 9, 2016; Published date: February 18, 2016

Citation: Cappuzzo V, Ingrassia F, Bavetta R, Mistretta S, Marceno R, et al. (2016) PCR-SBT Characterization of A New HLA Allele: A*02:548. Immunother Open Acc 2:112. doi:10.4172/2471-9552.1000112

Copyright: © 2016 Cappuzzo V, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The human Major Histocompatibility Complex (MHC) lies within the short arm of chromosome 6 and is responsible for the production of Human Leukocyte Antigens (HLA). The HLA genes are the most polymorphic genes in the human genome, encoding over 13000 allelic variants. Our laboratories are involved in HLA tissue typing of patients, their relatives and volunteer donors for the Italian Bone Marrow Register. During HLA-A high resolution typing of our Caucasian bone marrow volunteer donors, we found a sequence largely homologous to A*02:01 with a mutation at position 367 a C instead of a T. This mutation results in a coding change from Tyrosine to Histidine in exon 3 (codon 99 TAT→CAT). This allele was then submitted to the WHO Nomenclature Committee via the IMGT/HLA Database for naming and was assigned the official name A*02:548.

Introduction

The human Major Histocompatibility Complex (MHC) lies within the short arm of chromosome 6 and is responsible for the production of Human Leukocyte Antigens (HLA). The HLA genes are the most polymorphic genes in the human genome, encoding over 13,000 allelic variants. Characterization of HLA alleles is very important in the success of hematopoietic stem cell transplantation (HSCT) used to treat forms of blood and bone marrow cancers, for HLA-related diseases and for solid organ transplantation [1-3].

Genotyping of HLA-A, B, C, DRB1, DQB1, DQA1 and DPB1 at level of high resolution is a very important step for unrelated hematopoietic stem cell transplantation to minimize acute Graft versus Host Disease (aGvHD) [1,4,5].

Currently, there are several methods we can apply to determine HLA alleles in high resolution such as PCR-SSO, PCR-SSP and PCRSBT. The last one is the choice method to investigate HLA alleles giving the possibility to analyse base by base every allele.

Here, we report one of our cases in which a new HLA-allele has been characterized using PCR-SBT method.

Material and Method

DNA extraction

Genomic DNA, from our volunteer bone marrow donor peripheral blood, has been extracted using an automatic system Maxwell 16 (Promega).

Typing has been performed by PCR-SBT using Allele SEQR HLA-A (Celera) for exons 2, 3 and 4 according to the manufacturers’ instructions. Typing has been perfomed again using Protrans S4 HLA-A for allele separation according to the manufacturers’ instructions. The products were reconstituted with 25μl of Hi-Di Formamide and loaded on the ABI Prism 310 (PE) and analysed using SBTEngine.

Results

During HLA-A PCR-SBT typing for exons 2, 3 and 4, we found a sequence largely homologous to A*02:01 with a mutation at position 367 (Exon 3) a C instead of a T. The full HLA type was found to be: HLA-A*02: new, *33:03; B*15:03, *38:01; C*02:02, *12:03; DRB1*11:01, 13:02 (Allele SEQR HLA-A Celera Corporation). We repeated this sequence in isolation from the second allele using allele specific primers (Protrans S4 HLA-A CE).

Sequencing was performed in both directions and the data analyzed by SBTengine 3.5.2.2346 software. The novel allele was found to have, at exon 3, a Histidine instead of a Tyrosine codon 99 TAT→CAT (Figure 1).

immunotherapy-Alignment-new-allele

Figure 1: Alignment of the new allele against A*02:01:01:01 that was found to be most homologous to the new one. Dashes (-) represent identical sequence to A*02:548. The new A*02:548 has one nucleotide difference found at codon 99 (T>C), resulting in a coding change (Tyr>Hist).

This allele was then submitted to the WHO Nomenclature Committee via the IMGT/HLA Database for naming and was assigned the official name A*02:548. The name HLA-A*02:548 has been officially assigned by the World Health Organization (WHO) nomenclature committee in November 2014. This follows the agreed policy that subject to the conditions stated in the most recent nomenclature report [4].

Discussion

The described mutation is not unique but it has been observed in the alleles HLA-A*30:10 and A*30:88 but never in A*02 [2].

In our opinion PCR-SBT method is really useful both for genotyping HLA alleles and for the determination of new alleles, but using commercial kit we are able to amplify only exons 2, 3 and 4 for Class I and exon 2 for Class II so we surely lose all those mutations falling in introns and exons not amplified.

In conclusion, being HLA genotyping important in unrelated hematopoietic stem cell transplantation, we consider very helpful the introduction of the Next Generation Sequencing (NGS) in order to sequence the full length of every HLA locus improving HLA matching between donor and recipient and also not losing HLA new alleles.

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