Five novel HLA-A alleles, HLA-A*030108, A*2491, A*2498, A*330303, A*3317 were identified by polymerase chain reaction sequence based typing

Five HLA-A variant alleles HLA-A*030108, A*2491, A*2498, A*330303, A*3317 were identified in Chinese individuals.     

A new HLA-A*02 null allele, HLA-A*9213N

A novel HLA-A*02 null allele, differing from HLA-A*02010101 at codon 60 (TGG tryptophan-->TAG stop), is described.     

Identification and nucleotide sequence of HLA-A*02015

A novel HLA-A*02 allele, A*02015, is described that possess a unique non-coding substitution of G to A at position 113 in exon 3.     

Identification and nucleotide sequence of HLA-A*03013

We have identified a further HLA-A*03 allele, A*03013, in three unrelated Caucasoid individuals. The allele was initially detected by the polymerase chain reaction using sequence-specific primers (PCR-SSP) and was shown to differ from HLA-A*03011 by a single non-coding substitution of G to T at position 167 in exon 2 by DNA sequencing. The A*03013 bearing haplotype in one of the three individuals, was HLA-A*03013, B*07, Cw*0702, DRB1*1101, DRB3*0202, DQA1*05, DQB1*0301. The estimated phenotype and gene frequencies for A*03013 was <0.02% and <0.00007, respectively.     

Identification of a novel HLA-A allele, HLA-A*9216

The new human leukocyte antigen (HLA)-A*9216 differs from A*02010101 by a nonsynonymous nucleotide change at condon 76 (GTG>CTG).     

Polarity of the P1 anchor residue determines peptide binding specificity between HLA-A*3101 and HLA-A*3303

A previous pool sequence analysis showed that HLA-A*3101 and HLA-A*3303 binding peptides have the same anchor residues at P2 and the C-terminus, the only difference being that HLA-A*3303 binding peptides have two additional P2 anchor residues. Using a stabilization assay with RMA-S transfectants expressing HLA-A*3101 and human beta2-microglobulin, we tested the binding of 232 8- to 11-mer peptides carrying HLA-A*3303 anchor residues to HLA-A*3101. One hundred of these peptides (43.1%) bound to HLA-A*3101, confirming that these residues are also anchors for HLA-A*3101. Although aromatic hydrophobic P2 residues were previously shown to be stronger anchors than aliphatic hydrophobic P2 residues in HLA-A*3303 binding peptides, we detected no significant difference in HLA-A*3101 binding affinity between peptides carrying aromatic or aliphatic hydrophobic P2 residues. Statistical analysis previously showed a positive effect of negatively charged P1 residues and a negative effect of positively charged P1 residues for peptide binding to HLA-A*3303. In contrast such analysis demonstrated a positive effect of positively charged P1 residues and a negative effect of negatively charged P1 residues for peptide binding to HLA-A*3101. Analysis using mutated peptides confirmed these results. The present study therefore demonstrates that peptide binding specificity between HLA-A*3101 and HLA-A*3303 is determined by the polarity of the P1 anchor residue.     

Detection of a novel HLA-A allele by polymerase chain reaction-sequence-specific oligonucleotide typing: HLA-A*0252

Anew human leukocyte antigen (HLA) class I allele, HLA-A*0252, has been found during routine typing of samples for the Australian Bone Marrow Donor Registry. A*0252 differs from A*020101 at four codon positions, with all the new polymorphisms resulting in an amino acid change. The amino acids involved are located in the antigen-binding region of the HLA protein.     

Novel HLA-A*11 allele, A*1120, identified by sequence-based typing

In this report, we describe the identification of a human leucocyte antigen-A*11 (HLA-A*11) nucleotide sequence variant, a new HLA-A*1120 by using sequence-based typing (SBT). The new allele was detected during routine HLA typing by high-resolution SBT. Allele A*1120 showed one nucleotide difference with A*110101 at codon 152 (GCG-->GAG) resulting in an amino acid change from alanine to glutamate. Residue 152 is located on alpha(2)-helix of HLA class I molecule and involved in peptide binding by constructing E pocket of peptide-binding groove, implying that the change of the residue 152 would affect the binding affinity of peptides to A*1120 allele.     

Identification of a novel HLA-A*24 allele, HLA-A*2467

In this report, we describe the identification of a novel human leukocyte antigen-A*24 (HLA-A*24) allele, designated HLA-A*2467. The new allele differs from the most closely related allele HLA-A*2408 at five nucleotide positions all located in exon 2. Four of the five nucleotide changes result in amino acid substitution.     

A PCR-SSP method to specifically select HLA-A*0201 individuals for immunotherapeutic studies

HLA-A*0201 is an important restriction element for peptide presentation to T cells in disease and cancer. Mutation studies and analyses using cytotoxic T lymphocytes have shown the functional relevance of subtype-specific differences in HLA-A2 molecules for peptide binding and T-cell receptor recognition. Therefore, many immunotherapeutic studies need to accurately select HLA-A*0201-positive individuals. We designed an easy, robust approach based on the polymerase chain reaction using sequence-specific primers (PCR-SSP) to specifically distinguish A*0201-positive individuals from other HLA-A2 subtypes described to date. The first step includes reactions that give information whether the sample donor is HLA-A2 and, if so, whether the individual is homozygous or heterozygous for HLA-A2. Further, it is determined whether the sample has an HLA-A*0209 or an HLA-A*0201 sequence at the corresponding position in exon 4. Samples that may contain an HLA-A*0201 allele according to the results of this first step are subtyped in a second step nested PCR. Here the strategy is focussed on the discrimination of HLA-A*0201 from the other subtypes by considering divergent nucleotide positions in two ways. One SSP combination amplifies the HLA-A*0201 sequence while a corresponding SSP combination specifically amplifies the subtype or group of subtypes differing from HLA-A*0201 at this position. Thus, at relevant polymorphic nucleotide positions the HLA-A*0201 sequence is both directly and indirectly confirmed. This strategy strongly enhances the reliability of the subtyping and allows better verification of HLA-A*0201-positive patient selection for clinical studies.     

Identification and characterization of three novel HLA alleles, HLA-A*240214, HLA-A*3215 and HLA-DQB1*060302

We describe three novel human leukocyte antigen (HLA) alleles found in three different Caucasians, HLA-A*240214, HLA-A*3215 and HLA-DQB1*060302. As compared with HLA-A*24020101, HLA-A*240214 has a synonymous nucleotide (nt) exchange in codon 132. HLA-A*240214 may have arisen from intergenic recombination between HLA-A*24020101 and an HLA-B or HLA-C allele. The second novel allele, HLA-A*3215, has three nucleotide exchanges as compared with HLA-A*320101. These variations result in amino acid exchanges in codons 62 and 63, generating the public epitope of the serological HLA-A10 group. The third novel allele, HLA-DQB1*060302, has one synonymous nucleotide exchange within codon 38 as compared with HLA-DQB1*060301. In a family segregation study, we found that HLA-DQB1*060302, similar to the known HLA-DQB1*060301 allele, cosegregates with HLA-DRB1*1301.     

HLA-A*0206-BSP和-A*0207-BSP融合基因的构建与表达

采用RT-PCR技术从HLA-A*0206和-A*0207阳性个体的PBMC中分别克隆出HLA-A*0206和-A*0207基因的全长cDNA序列,构建HLA-A*0206和-A*0207克隆载体。再利用PCR技术从构建的克隆载体中扩增HLA-A*0206和-A*0207的α链(重链)胞外段序列,分别经双酶切置换本室保存的HLA-A*0201-BSP重组体中的HLA-A*0201胞外段序列,使HLA-A*0206和-A*0207与BirA酶底物肽(BirA substrate peptide,BSP)序列融合,构建HLA-A*0206-BSP和-A*0207-BSP融合基因的表达载体,经限制性酶切和DNA测序证实。然后将该表达载体转化E.coliBL21(DE3)后获得表达产物,通过体外稀释复性,初步纯化的表达产物通过ELISA和Western blot检测证明能够与β2微球蛋白(HLA I类分子轻链)及HLA-A2限制性抗原肽(HBV core 18-27)折叠形成具有HLA I类分子天然构象的抗原肽/HLA-A2复合物单体。为进一步构建HLA-A*0206和-A*0207四聚体,探讨相应HLA-A2亚型的功能特点提供了物质基础。     

A novel HLA-A*33 (A*3309) allele in a Caucasian family that differs at protein position 66 from HLA-A*3308

A novel human leucocyte antigen (HLA)-A33 (HLA-A*3309) allele has been identified in a Caucasian family from Middle Europe using single allele-specific sequencing strategy. This allele is identical to the HLA-B*3308 allele except for one point mutation in exon 2 at codon 66 (AAA-->AAT), resulting in an amino acid change from lysine (K) to asparagine (N).     

Detection of HLA-A*24 null alleles by DNA typing methods

Abstract: A number of cases have been identified (seven unrelated individuals from the Northern Ireland bone marrow donor registry and two family groups) where an HLA-A*24 allele fails to express the normal HLA-A24 antigen. Family information has revealed common haplotypes with respect to each non-expressed allele indicating that the occurrence of these mutations has been a recent event. Two methods for the clinical typing of these alleles have been evaluated - PCR-SSOP and PCR-SSCP analysis.