Identification of the new HLA-B*44:02:45, DQB1*02:85, DQB1*06:210, DRB1*01:01:30 alleles by monoallelic Sanger sequencing
Four new HLA alleles were identified using the monoallelic Sanger sequencing method: HLA-B*44:02:45, HLADQB1*02:85, HLA-DQB1*06:210, HLA-DRB1*01:01:30. A distinctive feature of the method is to implement the initial allele-specific PCR products for subsequent separate amplification of the target gene alleles. This, in turn, allows for sequencing of each allele separately and avoiding ambiguous HLA typing results observed when performing locus-specific sequencing. The isolated sequencing of specific gene alleles is a sufficient requirement for the registration of new HLA alleles, as prescribed by the World Health Organization Nomenclature Committee for Factors of the HLA System.
Major histocompatibility complex, novel HLA alleles, monoallelic Sanger sequencing.
The Major Histocompatibility Complex (MHC) is among the most polymorphic genetic systems in humans. Over last decade, extensive research in HLA (Human Leukocyte Antigens) has revealed hundreds of new HLA allelles by means of intensive application of immunogenetic sequencing methods, including monoallelic Sanger-sequencing method, or, more recently, next-generation sequencing (Fig. 1). In June 2017, the database of the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System (IPD-IMGT/HLA Database) contained information on the nucleotide sequences of 17331 different HLA alleles, of which 12631 were HLA class I, and 4700 were found for the HLA class II alleles [1-3].
The registration and name of new HLA alleles is carried out by the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System. The Committee developed a number of requirements ruling identification of the new HLA alleles (http://www.ebi.ac.uk/ipd/imgt/hla/subs/). In particular, the methods used to confirm new alleles should provide for separate sequencing of the alleles in the gene of interest. When performing HLA studies, appropriate primers should be used to determine the nucleotide sequence in forward and reverse directions. At the present time, sequencing of exons 2 and 3 is an obligatory requirement for identification of new HLA class I gene alleles. To verify any new alleles of HLA class II genes, one should perform the exon 2 sequencing.
The aim of this study was to evaluate the advantages of using monoallelic Sanger sequencing approach as a necessary step of identifying new HLA alleles.
Materials and methods
The test samples were obtained from potential donors of Bone Marrow Donor Registry of the I. P. Pavlov State Medical University in St. Petersburg and from a patient with acute myeloblastic leukemia who underwent HLA testing for subsequent allogeneic hematopoietic stem cell transplantation. Genomic DNA was isolated from peripheral blood leukocytes by a proteinase method using columns with a silica gel membrane and a kit PROTRANS DNA BOX reagents (Protrans, Germany). The target DNA concentration was 30 ng/ μL. Quantity and quality estimation of the isolated DNA was performed with Thermo Scientific NanoDrop 2000 Spectrophotometer. The quality of isolated DNA was estimated as an optical density ratio at of 260/280 nm wavelength, with reference range of 1.6-1.8.
Immunogenetic studies were done using the method of monoallelic Sanger sequencing. Initial and control studies were performed for each sample. To perform a control typing, the DNA was isolated from the newly collected biomaterial. The Protrans reagent kits (Germany) were used as follows: PROTRANS S4 HLA-A * Cyclerstrips, PROTRANS S4 HLA-B * Cyclerstrips, PROTRANS S4 HLA-C * Cyclerstrips, PROTRANS S4 HLA-DRB1 * Cyclerstrip, PROTRANS S3 HLADQB1 * Cyclerstrips. To identify HLA class I alleles (HLA-A, HLA-B, HLA-C), we sequenced exons 2-3; for class II HLA alleles (HLA-DRB1, HLA-DQB1) we sequenced exon 2.
Capillary electrophoresis was performed using Applied Biosystems 3500xl genetic analyzer (USA). To specify nucleotide sequences of the target alleles, the rough laboratory data were evaluated with Sequens Pilot software, version 4.1.2 (JSI Medical Systems, Germany).
The method of monoallelic Sanger sequencing is routinely applied at the tissue typing laboratory of St. Petersburg State Medical University I. P. Pavlov since 2010. A special feature of this method is carrying out allele-specific PCR at the initial stage, which usually leads to separate amplification of the analyzed gene alleles. After separate amplification, each allele may be subject to isolated sequencing. The allele-specific sequencing allows of avoiding the socalled cis-trans ambiguities which occur when interpreting HLA typing results. About 90% of people are known to be heterozygous for each HLA gene, thus causing cis-trans ambiguities precluding the exact HLA genotyping after locus-specific sequencing .
Using an allele-specific sequencing approach, the novel alleles of HLA-B, HLA-DRB1, HLA-DQB1 genes have been identified in three potential bone marrow donors from the Bone Marrow Registry at the St. Petersburg State I.P. Pavlov Medical University, and in one patient from the R. Gorbacheva Memorial Research Institute for Children Oncology, Hematology and Transplantation. Later on, this patient underwent unrelated bone marrow transplantation [5-7].
HLA-B allele (B*44:02:45). A new HLA-B allele was detected in a potential bone marrow female donor (Caucasoid, living in St. Petersburg). As based on immunogenetic studies, the following HLA phenotype was determined: HLA-A*02:01:01G, *30:01:01G, HLA-B*13:02, *44new, HLA-C*05:01:01G, *06:02:01G, HLA-DRB1*04:02:01, *10:01:01G, HLA-DQB1*03:02:01,*05:01:01G. According to the version 3.26.0 of the IPD-IMGT/HLA database, we have revealed that the nucleotide sequence of exon 2 in the new HLA-B *44 allele differs from the most close homologue HLA-B*44:02:01:01 at position 243 (guanine substituted for thymine). As shown in Fig. 2, the nucleotide sequence of codon 81 GCG is changed to GCT. The substitution is synonymous since it does not lead to an amino acid change (alanine). The nucleotide sequence of the new HLA-B*44 allele was submitted to the GenBank database being available under the accession number KY039061 (http://www.ncbi.nlm.nih.gov/Genbank/). The name of new HLA-B*44 allele has been officially assigned as HLA-B*44:02:45 by the WHO Nomenclature Committee for Factors of the HLA System .
New alleles of HLA class II genes
HLA-DQB1 allele (DQB1*02:85). A new HLA-DQB1 allele was identified in a potential bone marrow donor (female, Caucasoid, from Leningrad Region). Immunogenetic studies showed following results: HLA-A*01:01:01G, *24:02:01G, HLA-B*08:01:01G, *39:01:01G, HLA-C*07:01:01G, *07:02:01G,HLA-DRB1*03:01: 01G, *04:04:01, HLA-DQB1*02new, *03:02. According to the version 3.26.0 of the IPD-IMGT/HLA database, the exon 2 sequence of the new HLA-DQB1*02 allele differs from the most close homologue HLA-DQB1*02:01:01 at the position 141 (cytosine replaced by adenine). As seen from Fig. 3, codon 47 is changed from TTC to TTA, thus causing an amino acid coding change (phenylalanine to leucine). Nucleotide sequence of the new HLA-DQB1*02 allele is available under the accession number KY014073 in the GenBank database. The new HLA-DQB1*02 allele has been officially assigned as HLA-DQB1*02:85 by WHO Nomenclature Committee for Factors of the HLA System .
HLA-DQB1 allele (DQB1*06:210). Another new HLADQB1 allele was detected in a patient with acute myeloblastic leukemia (female, Caucasoid, from Sverdlovsk Region). High-resolution HLA typing was performed to select an HLA compatible donor for allo-HSCT.
The following HLA phenotype was determined: HLA-A*03:01,*23:01/17/69, HLA-B*44:03, *50:01, HLA-C*04:01/09N/30/82,*06:02/83, HLA-DRB1*07:01/34, *13:01/117/190, HLA-DQB1*02:02,*06new. According to the version 3.26.0 of the IPD-IMGT/HLA database, the nucleotide sequence of exon 2 of the new HLA-DQB1*06 allele differs from the nearest homologue HLA-DQB1*06:03:01 at position 112 (thymine instead of guanine). The sequence of nucleotides in the codon 38 GCG is changed to TCG, thus leading to the amino acid replacement at position 38 (alanine replaced by serine, see Fig. 4). The nucleotide sequence of the new HLA-DQB1*06 allele is available under the accession number KX988007 in the GenBank database. The new HLA-DQB1*06 allele has been officially assigned as HLA-DQB1*06:210 by WHO Nomenclature Committee for Factors of the HLA System .
HLA-DRB1 allele (DRB1*01:01:30). A new HLA-DRB1 allele was detected in a potential bone marrow donor (male, Caucasoid, from Lipetsk). The HLA phenotype showed following distribution: HLA-A*01:01, *24:02, HLAB* 07:02/61/161N,*57:01, HLA-C*06:02/83, *07:02/50/349, HLA-DRB1*01:01new, *17:01/34, HLA-DQB1*03:03, *05:01. According to the version 3.26.0 of the IPD-IMGT/HLA database, the nucleotide sequence of the exon 2 of the new HLA-DRB1*01 allele differs from the nearest homologue HLA-DRB1*01:01:01 in position 279 (thymine is determined instead of guanine), for details see Fig. 5.
The sequence of nucleotides in codon 93 CGG is changed to CGT. The substitution is synonymous since a change in the amino acid (arginine) does not occur. The nucleotide sequence of the new HLA-DRB1*01 allele is available under the accession number KY026176 in the GenBank database. The new HLA-DRB1*01 allele has been officially assigned as HLA-DRB1*01:01:30 by the WHO Nomenclature Committee for Factors of the HLA System .
The results of our work are in accordance with previously published data [4, 8], which demonstrate the advantage of monoallelic Sanger sequencing which provide opportunity of the separate sequencing for the initially studied gene alleles, that allowing to resolve the ambiguities when interpreting HLA typing results. Thus, we may fulfill an important requirement of the WHO Nomenclature Committee for Factors of the HLA System which regulates the new HLA allele identification procedure.
Conflict of interest
The authors have declared no conflicting interests.
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