Sequence-based HLA high-resolution typing of a bone marrow donor/recipient pair reveals the novel HLA allele HLA-C*07:208

Nucleotide sequence of HLA-C*07:208 allele was different from that of HLA-C*07:02:01:01 by a single-nucleotide substitution at position 475 G>C.     

Molecular cloning of two new HLA-C alleles: Cw*1801 and Cw*0706

Nucleotide sequence analysis of the HLA-C alleles of the GB92 cell line, heterozygous for B*8101 and B*4407, revealed the existence of two new allelic variants: Cw*1801 and Cw*0706. The former allele, initially detected as a PCR-SSP variant, displays a hybrid aspect, sharing sequence motifs with Cw*07 at exons 1 and 2, and with'Cw*04 at distal exons. In serological assays, Cw*1801 is only recognized by some cross-reactive sera. Cw*0706 shows a primary structure closely related to previously known Cw7 alleles, but carries new sequence motifs at its 3'-end. Preliminary data indicate that Cw*1801 is associated to B*8101 and that Cw*0706, B*4407 could account for a part of the Cw7, B44 haplotypes observed in African populations.     

Identification of a novel HLA-C allele, HLA-C*04:82, by sequence-based typing in a Chinese individual

A novel HLA allele, C*04:82, was identified in a Chinese individual, which has nine nucleotides insertion compared with the closest matching allele HLA-C*04:01:01 in exon 5, resulting in three amino acid insertions: 301 Ala, 302 Val and 303 Leu.     

HLA-C typing of eleven Papua New Guineans: identification of an HLA-Cw4/Cw2 hybrid allele

HLA-C polymorphism of 11 individuals from Papua New Guinea was studied by serology and DNA typing (SSP ARMS-PCR). To resolve certain discrepancies HLA-C alleles were cloned and sequenced. Five alleles were identified by sequencing, four of which; Cw*0304, Cw*0401, Cw*12022 and Cw*1502 have been identified previously in other populations. The fifth allele, which was found in four individuals is a novel HLA-C allele. The new allele, called HLA-Cw*0403 is most similar to HLA-Cw*0401, differing by 10 nucleotides, 9 of which are located in the region from nucleotide 98 to 218. This region of Cw*0403 is identical to both HLA-Cw*0201 and Cw*02022. The 9 nucleotide differences between Cw*0401 and Cw*0403 result in 6 amino acid differences in the α1 domain. These amino acids in Cw*0403 may contribute to the serological typing of some, but not all Cw*0403 expressing cells. The Final difference between Cw*0401 and Cw*0403 is a coding substitution at nucleotide 979 in exon 5. The guanine found in Cw*0403 is identical to all HLA-C alleles except HLA-Cw*0401, which has an adenine. The Cw*0403 allele was most likely formed by a gene conversion event between Cw*02 and Cw*04, involving a minimum of 121 to a maximum of 215 nucleotides.     

Identification of a novel HLA-C*07:02:25 allele by polymerase chain reaction sequence-based typing in a Chinese leukemia patient

Nucleotide sequence of HLA-C*07:02:25 allele was different from that of HLA-C*07:02:01:01 by a single nucleotide substitution at position 78C>G.     

Identification of two novel HLA-C alleles, Cw*070105 and Cw*1408, from East African women

HLA-C was shown to be a highly polymorphic gene that can be accurately typed by sequencing methodologies. We report two novel HLA-C alleles identified during sequence-based typing of East African populations; the novel alleles were confirmed by sequencing two separate polymerase chain reaction products and by molecular cloning and sequencing multiple clones. The first new allele is identical to Cw*0701 except for a single-nucleotide synonymous substitution at codon 182 (GCA-->GCG). The new allele has been named by the WHO nomenclature committee as Cw*070105. The second new allele is identical to Cw*1403 except for a nonsynonomous change at codon 21 (CAC-->CGC), changed from histidine to arginine. The new allele has been named by the WHO nomenclature committee as Cw*1408.     

Characterization and polymorphic analysis of 4.5 kb genomic full-length HLA-C in the Chinese Han population

This study used long-range polymerase chain reaction to sequence 4.5 or 4.3 kb of genomic DNA covering human leukocyte antigen C (HLA-C) and its flanks in 45 Chinese Han subjects to better characterize variation in the gene in a single population. Sequences of 35 HLA-C alleles were obtained from the population, including major alleles of 13 lineages of HLA-C. Four novel alleles, C*03:04:01:02, C*04:01:01:03, C*08:22, and C*17:01:01:02, were identified, and complete full-length sequences of 18 HLA-C alleles were obtained for the first time. All sequences herein reported also represent extensions through the promoter region and the 3'-untranslated region. Fourteen 5'-nucleotide sequences and 14 3'-nucleotide sequences were detected outside the coding region. In total, 316 single-nucleotide polymorphisms unequally distributed in HLA-C subregions were observed. In addition to exons 2 and 3, nucleotide variability was found to be particularly high in exon 5, which encodes the transmembrane region. The differentiation of the C*07 and C*17 lineages in this region accounts for the high variability. The congruence of phylogeny across most regions of the gene suggests that gene conversion or recombination has not markedly influenced divergence between lineages in the evolution of HLA-C.     

HLA-C locus allelic dropout in Sanger sequence-based typing due to intronic single nucleotide polymorphism

We report a novel HLA-C allele that was identified during routine HLA typing using sequence-based methods. The patient was initially typed as a C*06:02, 06:04 with two nucleotide mismatches in exon 3, (C to T and T to G changes) which would have resulted in a non-synonymous mutation of a leucine residue being replaced with tryptophan. Further resolution of the patient’s type by using sequence-specific primers (SSP) revealed that the companion allele to C*06:02 was a novel C*17:01. Confirmation of the existence of the new allele was performed across multiple platforms: Sanger sequencing, SSP, and Next Generation Sequencing (NGS) on the original sample and allele-specific clones for the entire HLA-C locus. The investigation revealed a single nucleotide mismatch within the Sanger sequencing primer binding site in intron 3. The mutation caused the initial C*17 dropout in exons 2 and 3. Further analysis of the Sanger and NGS data revealed that the C*17 had two additional unique positions in introns 2 and 7. The companion C*06:02 allele also possessed a novel position at intron 3. On August 31, 2013, the WHO nomenclature committee officially named the novel C*17:01 allele sequence as C*17:01:01:03 and the novel C*06:02 allele sequence as C*06:02:01:03.     

A new pair of HLA-C alleles, Cw* 12042 and Cw*1203, differing at the KIR-related dimorphism of codons 77–80

Abstract: A previously unknown HLA-C variant of the Cw*12 group was identified by PCR-SSP from genomic DNA of cell NDS-JD. Molecular cloning and nucleotide sequence analysis permitted the characterization of the complete coding region of this new allele, Cw*12042. The new variant differs from the recently reported Cw*12041 by two silent changes at exons 2 and 3, and from Cw*1203 by coding changes at codons 77 and 80. Cw*1203 (Ser-Asn) and Cw*12042 (Asn-Lys) constitute the second known example of HLA-C alleles only differing at the KIR-related dimorphism of residues 77–80. The new allele is associated in cell NDS-JD with the haplotype HLA-A*2403, Cw*12042, B*51, DRB1*1502, DRB5*0102, DQB1*0601, possibly related from the evolutionary aspect to the ancestral haplotype A*2402, Cw*1202, B*5201, DRB1*1502, DRB5*0102, DQBl*0601.     

Exploring the ancestry and admixture of Mexican Oaxaca Mestizos from Southeast Mexico using next-generation sequencing of 11 HLA loci

The Mestizos of Oaxaca resulted from the admixture of Zapotecan Natives with Spaniards and Africans. We selected 112 donors from Oaxaca and applied next-generation sequencing to characterize exon and intron variants in complete or extended HLA genes. Some alleles found, are unique to Mexican Natives and most likely will be absent in most major ethnicities, namely: Caucasians, Africans or Asians. Among these are HLA-A*68:03:01, HLA-A*68:05:01, HLA-C*03:04:01:02, HLA-C*15:09, HLA-C*3:05, HLA-C*03:06:01, HLA-B*39:05:01, HLA-B*35:14:01, HLA-B*35:12:01, HLA-B*35:43:01, HLA-B*40:05, HLA-B:40:08, HLA-B*51:02:01, HLA-B*35:24:01 and HLA-B*39:08. HLA-DQA1*05:05:01:05 and some HLA-DRB1 alleles were only present in Amerindians/Mestizos. Three haplotypes are unique to Mexican Natives, five to Middle-Eastern and Sephardi-Jews. We detected a novel HLA-DQA1*04:01:01 exon 4 variant. Any novel allele may have been positively selected to enlarge the peptide-binding repertoire, and some, like HLA-B*39:02:02 and HLA-B*39:05:01 were found with unique haplotype associations, suggesting convergent evolution events and/or allele lineage diversification. The allele frequencies were fairly evenly distributed in most HLA loci with the exception of HLA-DPB1. The application of NGS in Oaxaca is novel and will lead to better use in the clinical setting. It offers deep knowledge on the population structure, origins, migration, and discovery of new alleles and haplotypes that other techniques did not achieve.     

Identification of a novel HLA-C*01:61 allele by polymerase chain reaction sequence-based typing in a Chinese leukemia patient

HLA-C*01:61 allele was different from HLA-C*01:02:01 by a single nucleotide substitution at position 303 C>A.     

Characterization of a novel HLA-Cw*02 variant, Cw*0208, in a Caucasian individual

We describe an additional HLA-Cw*02 variant, HLA-Cw*0208, which has been identified in a renal transplant recipient of Caucasian origin (Italy). After performing preliminary serological typing, we analyzed exons 2 and 3 of the HLA-C locus polymorphism by cloning the amplified DNA and using a sequence-based typing method. The new allele differs from Cw*020202 by one nucleotide substitution at nucleotide 61 (G-->A) of exon 2, which translates to a difference of one amino acid at residue 21 (His-->Arg) of the HLA-C heavy chain. We propose that Cw*0208 was generated by a random point mutation in codon 21 from the Cw*020202 allele, or through gene conversion of Cw*020202 with another allele, probably the Cw*1205 and Cw*1602 alleles.     

Elongation of the cytoplasmic domain,due to a point deletion at exon 7, results in an HLA-C null allele,Cw*0409 N

The development of molecular techniques for HLA typing has allowed the identification of genes previously assigned as serologic blank alleles. Lack or poor cell surface expression has been found for molecules coded by HLA-A, -B, -DRB4, -DRB5, and -DPB1 genes. In this report we describe the first HLA-C gene encoding for a null cell surface molecule. HLA-Cw*0409 N shows a point deletion at position 1095 within exon 7. This mutation provokes a codon reading shift, generating a new translation stop codon 97 bp downstream to that described in alleles normally expressed. This new stop codon location implies the presence of 32 extra amino acid residues in the cytoplasmic domain. Transfection experiments suggest that elongation of the cytoplasmic domain in Cw*0409 N would be the cause of cell surface expression failure, although Cw*0409 N heavy chain is able to create stable complexes with beta2-microglobulin. HLA-C fragment length analysis in a small selected group of samples with B44-Cblk haplotypic associations allowed us to identify two additional subjects showing both a serologic silent Cw*04 allele and a point base deletion at the 3' end of the HLA-C gene. This finding indicates that the allele frequency of Cw*0409 N within serologic C blank alleles would be appreciable, although basically restricted to the (A23)-Cw*0409 N-B*4403-DR7-DQ2 haplotype.     

Characterization of a novel variant allele, HLA-C*07:01:19, identified in a Chinese Han individual

The novel HLA-C*07:01:19 allele differs from the closest allele C*07:01:01 by a single nucleotide change at coding sequence nucleotide 738 G>A (codon 222 GAG>GAA) in exon 4.     

The complete primary structure of Cw*1701 reveals a highly divergent HLA class I molecule

Genotyping of the HLA-C locus by PCR-SSP has previously shown 100% association of B41 and B42 with a new allelic variant. Partial sequencing studies (exons 2–4) demonstrated that this PCR-SSP variant corresponded to the new allele Cw*1701. In this study we have characterized the whole coding region of Cw*1701 from a Bubi individual of Equatorial Guinea. Our results partially confirm the previously reported sequence and reveal that Cw*1701 has many new polymorphisms at several exons, including a 18-bp insertion in exon 5. Cw*1701 is thus a most unusual HLA-C molecule denning a third allelic lineage of this locus.     

Characterization of the novel HLA-C*16:07:02 allele in a family of Benin origin

The novel allele HLA-C*16:07:02 differs from HLA-C* 16:07:01 by a silent nucleotide substitution at codon 220 (TAC → TAT).     

Discovery of the novel HLA‐DRB1*04:05:14 allele in a Taiwanese unrelated haematopoietic stem cell donor by a sequence‐based typing method

We report here a de novo HLA‐DRB1*04 allele, DRB1*04:05:14, discovered in a Taiwanese unrelated volunteer bone marrow stem cell donor by a sequence‐based typing method. In exon 2, the DNA sequence of DRB1*04:05:14 is identical to the sequence of DRB1*04:05:01 except the nucleotide at positions 321 where C is replaced by T (at codon 78; TAC→TAT). Due to the silent mutation, the nucleotide substitution produced no amino acid variation in comparison with DRB1*04:05:01. We assume DRB1*04:05:14 was derived from DRB1*04:05:01 via a point mutation. The probable HLA‐A, ‐B and ‐DRB1 haplotype in association with DRB1*04:05:14 may be deduced as A*11‐B*55‐DRB1*04:05:14. We here report the Taiwanese ethnicity of DRB1*04:05:14.     

Identification of a novel HLA‐B*27 allele,B*27:79 and the B*27 subtype polymorphism in the Hunan ethnic Han population of China

This article describes a novel HLA‐B*27 allele, HLA‐B*27:79, which was identified in a Hunan Han ethnic individual of China by a PCR sequence‐based typing method. The new sequence has one nucleotide mutation at position 437(A→T) compared with the allele B*27:04:01. This nucleotide change causes an amino acid substitution from Aspartate (Asp) to Valine (Val) at codon 122. This is the first report of mutation at this position in the HLA‐B locus. Then, we investigated the HLA‐B*27 subtype polymorphism of the Hunan Han population, and the results showed that B*27:04, B*27:05 and B*27:06 are the predominant subtypes with the allele frequencies 0.97%, 0.26% and 0.10% respectively.     

Identification of a novel HLA-C*01 variant allele, C*01:46

A novel HLA-C*01 variant allele differs from the closest allele C*01:02:01 by single nucleotide change at coding sequence nt 316 C>T (codon 82 CGC>TGC) in exon 2, which causes an amino acid change Arg82Cys.     

Discovery of a novel HLA‐DRB1*04:05 variant,DRB1*04:05:15, in a Taiwanese and the probable HLA haplotype in association with DRB1*04:05:15

Here, we report a novel HLA‐DRB1*04 allele, DRB1*04:05:15, found in a Taiwanese unrelated volunteer bone marrow hematopoietic stem cell donor by a sequence‐based typing (SBT) method. The DNA sequence of DRB1*04:05:15 is identical to the sequence of DRB1*04:05:01 in exon 2, except the nucleotide at the position 198 where C is substituted by T (TAC→TAT at codon 37). Due to the silent mutation, the nucleotide replacement generated no amino acid variation in comparison with DRB1*04:05:01. We postulate the allele DRB1*04:05:15 was probably derived from DRB1*04:05:01 via a nucleotide point mutation event. The probable HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1 haplotype in association with DRB1*04:05:15 may be deduced as A*02:01‐B*48:01‐C*08:03‐DRB1*04:05:15‐DQB1*04:01.