HLA-B新等位基因B*9536和B*4612的测序分析和确认

目的 研究人类白细胞抗原(human leukocyte antigen,HLA)新等位基因HLA_B*9536和B*4612的分子机制.方法 采用Invitrogen抽提试剂盒抽提标本DNA,利用单链特异性引物PCR方法扩增样本HLA-B基因第2～4外显子,对PCR产物直接进行HLA-B基因第2、3、4外显子双向测序分析.结果 先证者标本存在2个HLA-B等位基因,经HLA Blast验证均为新的等位基因,新的等位基因序列已递交GenBank(EU081878和EU081879),经世界卫生组织HLA命名委员会正式命名为HLA-B*9536和HLA-B*4612.HLA-B*9536第2～4外显子序列与最接近的B*1505相比,在第3外显子存在一个碱基的不同,即第544位G→A改变,导致第158位氨基酸Ala→Thr;HLA-B*4612第2～4外显子序列与最接近的B*4601相比,在第3外显子存在一个碱基的不同,即第363位G→C,导致第97位氨基酸Arg→Ser.结论 在同一标本中发现两个新的HLA-B等位基因,并被世界卫生组织HLA命名委员会正式命名.     

Identification of three novel HLA class I alleles: HLA-B*3928, HLA-B*400104 and HLA-B*4437

Three new human leukocyte antigen (HLA) class I alleles have been identified in the Tissue Typing Laboratory in Sydney, Australia. Sequence analysis of exon 2 and exon 3 of the HLA-B gene revealed the novel polymorphism. A silent substitution of C to T at nucleotide position 369 has been identified for the HLA-B*400104 allele when compared to the closest matched allele, HLA-B*400101. The HLA-B*3928 allele was identified with a nucleotide substitution of G to C at position 362 when compared to the closest matched allele, HLA-B*390101, resulting in an amino acid substitution of Arginine to Threonine. A nucleotide substitution of C to G at position 572 resulting in the amino acid change Serine to Tryptophan was identified in the new allele HLA-B*4437, when compared to the closest matched allele HLA-B*440301. Both amino acid substitutions implicate a different specificity and affinity of antigen binding for the alleles HLA-B*3928 and HLA-B*4437.     

New HLA-B alleles detected by polymerase chain reaction-sequence-specific oligonucleotide typing: HLA-B*0725, B*0728, and B*3808

Three novel alleles, human leukocyte antigen (HLA)-B*0725, B*0728, and B*3808, were discovered during routine genotyping of samples for the Australian Bone Marrow Donor Registry and Australian Cord Blood Bank. The new alleles contain amino acid changes in the antigen-binding site of the expressed HLA protein, which may alter the antigen-binding properties of the functional protein.     

Three newly identified HLA-B alleles: B*5124, B*5306, B*5307 and confirmation of B*0809 and B*5606

Five HLA-B sequences are described which have been detected as irregular patterns during routine molecular typing. Sequencing of HLA-B exon 2 and 3, both heterozygous and after group specific amplification, revealed three new HLA-B alleles: B*5124, B*5306 and B*5307, whereas the sequences of B*0809 and B*5606 were confirmed. Serological typing showed that B*0809 is expressed as a regular B8, B*5124 as a regular B51, B*5306 as a B51/B53-like variant and B*5606 as a B"blank"-Bw6.     

Identification of three novel HLA class I alleles: HLA-A*0261, HLA-B*1585 and HLA-B*1587

Three novel human leucocyte antigen (HLA) class I alleles have been characterized by means of direct DNA sequencing analysis. HLA-A* 0261 showed sequence variation at conserved codon. It differs from HLA-A* 020601 by a single-nucleotide substitution at codon 57 (CCG-->GCG) resulting in an amino acid change from Pro to Ala. The sequences of HLA-B*1585 are similar to those of HLA-B*15010101, but differed five nucleotides on exon 3 resulting in three amino acid changes at residues 94 (Thr-->Ile), 95 (Leu-->Ile) and 103 (Val-->Leu). Likewise, HLA-B*1587 is identical to HLA-B*15010101 except at codons 80-83 (Asn-Leu-Arg-Gly-->Ile-Ala-Leu-Arg) which has been replaced by HLA-Bw4 motif. These alleles seemed to be generated by either a point mutation or a gene conversion-like event from alleles existing in the population with high frequencies.     

Routine HLA-B genotyping with PCR-sequence-specific oligonucleotides (PCR-SSO) detects eight new alleles: B*0807, B*0809, B*1551, B*3529, B*3532, B*4025, B*5304 and B*5508

This paper describes eight new alleles (B*0807, B*0809, B*1551, B*3529, B*3532, B*4025, B*5304 and B*5508) that have been found by routine HLA-B genotyping with sequence-specific oligonucleotides (SSOs). All of the new alleles have variations which cause changes in residues that occur within antigen binding pockets and T-cell recognition sites of the antigen. The new polymorphisms within these new alleles may affect the nature and specificity of peptide binding and cause differential T-cell activation, which may have an affect in transplantation.     

Characterization of HLA-B*3921 and confirmation of HLA-B*4415, two variant HLA-B alleles identified in the Omani population

We describe a variant HLA-B*39 allele present in two individuals from Oman, which has been officially named HLA-B*3921. In addition we confirm the existence of HLA-B*4415, an allele closely related to HLA-B*4501 differing only at the Bw4/Bw6 epitope.     

Increased diversity within the HLA-B*07 group: identification of the two novel alleles B*0709 and B*0710

The identification of the two novel alleles, HLA-B*0709 and B*0710, is described. B*0709 differs from B*07021 by a nucleotide exchange at position 419 (A>C) which is located in exon 3. At the protein level the nucleotide exchange results in an amino acid residue difference (Tyr116Ser). The other newly detected allele, B*0710, differs from B*07021 by a nucleotide exchange at position 272 (A>G) which is located in exon 2. This nucleotide exchange also leads to an amino acid substitution (Tyr67Cys). The allogeneic potential in case of mismatch with other alleles of the B*07 group at bone marrow transplantation was assessed. The peptide motifs between B*0709 and B*0711 may be very similar and thus the alloreactive potential in case of mismatch may be low. In contrast, mismatches of B*0709 and B*0710 with other B*07 alleles are likely to stimulate alloreactive T cells.     

Identification of four novel HLA-B alleles, B*1590, B*1591, B*2726, and B*4705, from an East African population by high-resolution sequence-based typing

We report here four novel HLA-B alleles, B*1590, B*1591, B*2726, and B*4705, identified from an East African population during sequence-based HLA-B typing. The novel alleles were confirmed by sequencing two separate polymerase chain reaction products, and by molecular cloning and sequencing multiple clones. B*1590 is identical to B*1510 at exon 2 and exon 3, except for a difference (GCCGTC) at codon 158. Sequence differences at codon 152 (GAGGTG) and codon 167 (TGGTCG) differentiate B*1591 from B*1503 at exon 3. B*2726 is identical to B*2708 at exon 2 and exon 3, except for a difference (AAGCAG) at codon 70. B*4705 was identified in three Kenyan women. The allele is identical to B*47010101/02 at exon 2 and exon 3, except for differences at codon 97 (AGGAAT) and codon 99 (TTTTAT). These new alleles have been named by the WHO Nomenclature Committee. Identification of these novel HLA-B alleles reflects the genetic diversity of this East African population.     

Identification of two new HLA-B22 variants, HLA-B*5509 and B*5606

In our recent study using high-resolution HLA-B locus typing by sequence-based typing (SBT) we identified 9 new alleles in a total of 355 unrelated individuals (4). Three of them concerned an allele belonging to the B22 group. One of them, B*5607, showed the unusual presence of a Bw4 sequence motif, as described previously (5). In this report the other two B22 variants are described; one belonging to the B55 specificity and named B*5509; the other one being a B*56 allele and assigned B*5606, which brings the total number of alleles belonging to the B22 group to 18.     

Immunogenetics of two new HLA-B alleles: B*4414 and B*5708

Two new alleles, HLA-B*4414 and B*5708, were identified in north-western European Caucasoid blood donors. B*4414 differed from B*440201 by two nucleotide substitutions in exon 3 [positions 66 (T to C) and 69 (G to A)] producing two amino acid differences between the B*440201 and B*4414 specificities (tyrosine to histidine at codon 113 and aspartic acid to asparagine at codon 114). B*5708 differed from B*570101 by a single substitution (G to C) at position 247 in exon 2 causing an amino acid difference between B*570101 and B*5708 products of arginine to proline at codon 83. The likely haplotypes bearing these alleles were identified. Both alleles occurred once in approximately 25,000 random blood donors so both have a frequency of approximately 0.00002 (carriage frequency 0.004%). B*4414 and B*5708 specificities both gave 'short' serological reactivity for their expected specificities and Bw4. The likely reasons for this are discussed in relation to the epitopes of B44 and B57.     

HLA-A and HLA-B in Kenya,Africa: allele frequencies and identification of HLA-B*1567 and HLA-B*4426

HLA-A and HLA-B alleles of a population from Kenya, Africa were examined by sequencing exon 2 and exon 3 DNA and typing using a Taxonomy-based Sequence-analysis (TBSA) method. Extensive diversities were observed at both HLA-A and HLA-B loci in this population. Forty-one HLA-A alleles were identified from 159 unrelated individuals. The most frequently observed alleles were A*6802 (11.64%), A*02011/09 (9.75%), A*7401/02 (9.43%), A*3001 (7.86%), A*3002 (7.23%) and A*3601 (6.6%). Forty-nine HLA-B alleles were identified in 161 unrelated individuals, including two novel alleles, B*1567 and B*4426. The most frequently observed HLA-B alleles were B*5301 (9.01%), B*5801 (8.38%), B*4201 (7.76%), B*1503 (7.14%), B*1801 (6.21%), and B*5802 (5.90%). The most frequently observed HLA-A-B haplotypes were A*3601-B*5301 (3.55%) and A*3001-B*4201 (3.19%), followed by A*7401/02-B*5801 (2.84%), A*7401/02-B*5802 (2.84%) and A*02011/09-B*1503 (2.13%). Linkage disequilibrium and chi2 analysis showed the association of these HLA-A-B haplotypes at the antigen level to be significant. The frequencies of HLA-A and HLA-B alleles from the Kenyan population were compared with that of a population from Cameroon. The difference in allele and haplotype frequency distributions partly reflected the different ethnic composition of these two African populations.     

Two novel alleles HLA-B*9536 and B*4612 were identified in a healthy Chinese individual

The novel HLA-B*9536 and B*4612 alleles were identified by sequence-based typing in China.     

Two new HLA DPB1 alleles identified by sequence-based typing: DPB1*8201 and DPB1*8301

Two new HLA DPB1 alleles were identified by sequence-based typing and are reported. Both alleles differ from DPB1*0402 by a single nucleotide: DPB1*8201 has a difference at position 359 (codon 91) leading to an amino acid change from arg to his, making this position a new polymorphic site; DPB1*8301 has a difference at position 280 (codon 65) changing the amino acid from ile to phe.     

Further diversification of the HLA-B locus in Central American Amerindians: new B*39 and B*51 alleles in the Kuna of Panama

Several new HLA-B locus alleles have been discovered in South American Amerindians. By contrast, analysis of the MHC class I alleles of North American native populations has revealed few new HLA-B alleles. This suggests that the HLA-B locus is evolving rapidly in South American populations. Here we describe the HLA-B locus alleles present in individuals from a Central American tribe, the Kuna of Panama. Using a sequencebased typing technique that separates alleles by denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we determined the HLA-B alleles from eight Kunas. Two of the HLA-B alleles present in the Kuna have been previously described in other South American Amerindian populations; one allele has been characterized in a Mexican-American. We characterized two new HLA-B alleles in the Kuna, HLA-B*3911 and HLA-B*5110 . HLA-B*3911 differed from HLA-B*3905 by only a single nucleotide substitution in exon 3. This substitution resulted in an amino acid replacement of leucine by arginine at residue 156 in the alpha 2 domain. Such a change may affect the repertoire of peptides that are bound by this molecule. HLA-B*5110 differed significantly from other HLA-B*51 alleles in that it is the result of an unusually large intra-locus recombination event of minimally 216 nucleotides. This recombination results in an allele that is part HLA-B*51 and part HLA-B*40. Thus, more dramatic recombination events may also play a role in the rapid evolution of the HLA-B locus in Amerindians.