Identification of a novel HLA-B*44 variant (B*4441) in three unrelated Caucasian individuals

Here, we report the identification of a new human leukocyte antigen (HLA)-B*44 allele found almost simultaneous in three DNA samples which were part of routine bone marrow donor typing by order of the German registry 'Aktion Knochenmarkspende Bayern'. The samples appeared noticeable in different polymerase chain reactions using sequence-specific primers (PCR-SSP) or sequence-specific oligonucleotides (PCR-SSO). Sequence-based typing revealed a novel allele officially designated as B*4441*. This sequence differs from HLA-B*44020101/4427 by two nucleotide positions at the beginning of exon 3: by position 353 (T to C) and by position 355 (A to C). These differences in sequence result in deviant amino acids at codon 94 (Ile94Thr) and codon 95 (Ile95Leu).     

B*4440: a novel HLA-B allele identified by sequence-specific oligonucleotide hybridization and sequence-specific amplification

A novel human leukocyte antigen B (HLA-B) allele, B*4440, is described. The allele was identified in an adult stem cell donor of Caucasian origin. HLA-B*4440 most closely matches to B*4403 differing by a substitution of three nucleotides at codon 44, 45, and 50. Thus, low-resolution HLA typing using sequence-specific oligonucleotide hybridization or amplification using sequence-specific primers gave inconclusive results. DNA sequencing confirmed a variation of codons 44 and 45 (AGG AAG-->AGA GAG) and codon 50 (CCA-->CCG), resulting in an amino acid substitution Lys-->Glu at codon 45.     

Identification of a new HLA-B*08 variant, HLA-B*0804

The HLA-B locus is the most polymorphic locus known with currently over 100 different alleles described. Many of these alleles encode variants of the serologically-defined tissue transplantation antigens. This high level of diversity makes accurate tissue typing difficult. Here we present the sequence of a new HLA-B *08 variant, HLA-B *0804. found in Caucasian siblings JH and PF serologically typed as HLA-B51/B59 and HLA-B59/B60, respectively. Additionally, DNA-based typing by the polymerase chain reaction using sequence-specific primers (PCR-SSP) identified HLA-B *51 in JH and HLA-B *4001 in PF. However, PCR-SSP failed to identify a second allele in either of these individuals. The unusual finding of a B59 antigen in a Caucasian and the discrepant molecular typing results suggested that these individuals might express novel HLA molecules. Using denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we characterized a novel HLA-B *08 variant, HLA-B *0804. The presence of this allele was confirmed by cloning and sequencing. HLA-B *0804 differed from HLA-B *0801 by only one nucleotide substitution resulting in an amino acid replacement of phenylalanine by serine at position 67. Incidentally, this single nucleotide difference was sufficient to prevent amplification by PCR-SSP. This striking difference between both the serologically typed antigen and the PCR-SSP-identified allele compared to the sequenced allele supports the use of sequence-based typing for the analysis of HLA class I locus alleles.     

A single amino acid exchange shifts the serological reactivity of the novel HLA-B*4442 allele product from HLA-B44 to HLA-B21

A novel HLA-B (human leukocyte antigen-B) allele, HLA-B*4442, was identified both in a Czech patient with leukaemia and in his mother. The presence of a novel allele was initially suspected because conflicting results were obtained by serological and DNA typing techniques. The HLA typing using the polymerase chain reaction-sequence-specific primers (PCR-SSP) at the two-digit level indicated an allele belonging to the HLA-B*44 group, whereas serological typing indicated HLA-B21. Typing with PCR-sequence-specific oligonucleotides (PCR-SSO) resulted in a unique reaction pattern that could not be assigned to a known allele, PCR-SSP typing at the four-digit level did not match any known B*44 allele, either. The sequencing-based typing of the HLA-B locus then revealed the novel B*4442 allele that is identical with B*4405 except a single C-->G nucleotide exchange at position 572. This exchange results in an amino acid substitution from serine to tryptophan at position 167 of the expressed HLA-B protein. The B21 serological reactivity of the novel B*4442 allele product was confirmed by employing an additional serological panel of typing sera. Our findings support previous reports claiming that serine at the position 167 in the alpha-2 domain of the HLA-B protein is a major determinant of the HLA-B44(12) serological epitope.     

Identification of an HLA-B7 serological variant and its characterization by sequencing based typing

We have identified an HLA-B*07 variant allele, B*0716, in a Caucasoid cadaver kidney donor. The HLA class I type by polymerase chain reaction using sequence-specific primers (PCR-SSP) was A*01, 32; B*07, 08; Cw*07. Serological typing, using monoclonal and polyclonal anti-HLA antisera, gave disparate results for the B antigens. Monoclonal antibodies identified B7 and B8 antigens but polyclonal antisera recognised only the B8 antigen. PCR using sequencing based typing (PCR-SBT) confirmed the presence of both B*0703 and B*0801 alleles but with a mutation in one of the alleles. The HLA-B*07 allele was isolated by allele-specific PCR and was shown to have a mutation, G-->T, at 292 in exon 2. This mutation changes codon 74, which encodes aspartic acid (GAC) present in all previously identified B*07 alleles, to tyrosine (TAC) in the variant. The serological results suggest that codon 74 is a crucial part of a B7 antigen-specific epitope recognised by tissue typing polyclonal antisera.     

An HLB-B null allele (B*0808N) caused by a nucleotide deletion in exon 3, found in the family of a bone marrow transplant recipient

We have identified a variant HLA-B allele, B*0808N, segregating through two generations of healthy individuals, whilst HLA typing the family of a bone marrow patient. Serological typing identified a disparity between the father (A1, A3 B7 DR7) and the brother (A1, A2 B56 DR1, DR7) of the patient. Low/medium resolution polymerase chain reaction using sequence-specific primers (PCR-SSP) revealed a B*08 allele undetectable by serological methods. High resolution DNA typing by polymerase chain reaction-sequencing based typing (PCR-SBT), revealed a nucleotide deletion at position 131 (C) in exon 3, the only difference between the new allele and B*0801. The deletion results in a frame shift in the protein coding sequence, introducing a premature termination codon (TGA) in exon 4. Although a B*08 allele is present in these individuals, the deletion prevents correct expression of the antigen on the cell surface.     

新等位基因HLA-B*9526的确认和序列分析

目的 鉴定中国人群中人类白细胞抗原(human leukocyte antigen,HLA)新等位基因HLA-B*9526,并进行核苷酸序列分析.方法 使用序列特异性寡核苷酸PCR技术进行HLA基因分型,发现1个反应格局异常的等位基因,应用分子克隆和DNA双向测序技术测定新等位基因的核苷酸序列,并与已知等位基因进行序列比对分析.结果 检出反应格局异常的DNA样本,经过克隆测序得到两个等位基因,分型结果一个为B*5403,另一个的核苷酸序列与已知的HLA等位基因均不同,该基因序列与同源性最高的HLA-B*1507基因序列相比在第3外显子区域中425位碱基发生A→G突变,导致142位编码氨基酸由酪氨酸变成半胱氨酸.结论 一个新的HLA-B等位基因被确认,并被世界卫生组织HLA因子命名委员会正式命名为HLA-B*9526.     

Identification of a novel allele HLA-B*5610 in a Chinese potential bone marrow donor

A novel HLA-B allele, B*5610, has been identified in a potential bone marrow donor, his mother and brother using DNA-based typing and molecular cloning methods. The B*5610 allele differs from the closest matching HLA sequence of B*5602 by two nucleotide substitutions in exon 3, 559 C-->A and 560 T-->C, resulting in an amino acid change from Leu (CTG) to Thr (ACG) at codon 187. This new allele was segregated together with A*24020101 and DRB1*140101 in the proband's family. Serology study revealed that B*5610 is associated with B22 specificity. A PCR-SSP method was developed to distinguish B*5610 from other B*56 alleles. No further individuals with B*5610 were detected in 5000 Chinese bone marrow blood donors.     

Sequence, genetics and serology of a new HLA-B allele: B*4903

A new HLA-B allele - B*4903 - was detected by the polymerase chain reaction using sequence-specific priming (PCR-SSP), in a Caucasoid bone marrow panel donor, that differs from B*4901 by 8 nucleotides at positions 141, 142, 144, 165, 167, 193, 206 and 213 in exon 2. These substitutions all occur in HLA-B*51 and B*52 alleles and encode 4 amino acid substitutions at positions 24 (Thr to Ala), 32 (Leu to Gln), 41 (Thr to Ala) and 45 (Lys to Thr). This suggests that B*4903 occurred following a gene conversion-like event involving B*4901 and probably a B*51 allele. HLA-B*4903 was identified on a haplotype with: HLA-A*0201; Cw*07; DRB1*1302/34; DRB3*0301; DQA1*0102; DQB1*0604; BfS; C4A3; C4BQ0 and encodes a unique serological specificity which was characterised by the reactivity of 55 antisera directed towards at least four predicted epitopes. No further examples of B*4903 were found in 15,796 consecutive HLA PCR-SSP typed donors from the Welsh Bone Marrow Donor Registry, indicating that this allele has a phenotype frequency of <0.01% and a gene frequency of <0.00004.     

Immunogenetics of a new HLA-B null allele, HLA-B*4423N.

The second example of an HLA-B*44 null allele (B*4423N) was identified by discrepancies between serological and polymerase chain reaction-sequence-specific primer (PCR-SSP) typing in two north-western European Caucasoid unrelated stem cell donor volunteers. HLA-B*4423N was identical to B*440201 except for a single nucleotide substitution at position 493 in exon 3, resulting in a premature stop codon at bases 493-495 (TAG rather than CAG at codon 141). As expected, comprehensive serological testing using 54 antisera, directed towards B44 or Bw4, failed to identify the HLA-B44 (Bw4) specificity. The B*4423N-bearing haplotype was identified as A*0201, Cw*0501, DRB1*0408, DRB4*01, DQA1*03, DQB1*0304 and the frequency of B*4423N estimated as 0.00006 (carriage frequency 0.0121%) in 16 533 subjects resident in Wales.     

Identification of a new HLA-B*40 allele, HLA-B*4081, in a Chinese individual

A novel human leukocyte antigen (HLA)-B*40 allele, officially named B*4081, was identified during routine high-resolution sequence-based typing in a Chinese potential hematopoietic stem cell transplantation donor. The HLA-B*4081 allele shows one nucleotide difference from B*400101 in exon 2 at nucleotide position 124 where G→C (codon 18 GGG→CGG) resulting in a coding change, 18Gly is changed to Arg, this is a unique nucleotide change among the HLA class I alleles, suggesting a point mutation mechanism.     

The recombinant HLA-B*5518 allele supports the evidence of conserved haplotype association of rare alleles

Allelic polymorphism of the major histocompatibility complex arises mostly from gene recombination. Intralocus gene recombination usually involves short fragments of DNA leading most commonly to single-nucleotide substitutions and rarely involves large fragments. Here, we report a new recombinant human leukocyte antigen (HLA)-B*5518 allele that has arisen via recombination of a large fragment of DNA spanning more than 70 nucleotides. During routine HLA typing of potential volunteer donors for the National Marrow Donor Program((R)), a new HLA-B allele was identified in two donors from Guam. The allele, B*5518, appears to be a product of recombination between B*5502 and B*40. Exons 1, 3, and 4 of the new allele belong to B*5502, whereas part of exon 2 belongs to one of B*40 alleles. Introns 1 and 2 appear to belong to B*55, suggesting that the recombination event may have occurred within the homologous parts of exon 2.     

Routine HLA-B genotyping with PCR-sequence-specific oligonucleotides detects a B*52 variant (B*5206)

A new human leukocyte antigen (HLA)-B allele was found during routine typing of samples for a German unrelated bone marrow donor registry, the "Aktion Knochenmarkspende Bayern". After first interpretation of data of two independent low-resolution sequence-specific oligonucleotide typing tests, a B*51 variant was suggested. Further analysis via sequence-based typing identified the sequence as new B*52 allele. This new allele officially assigned as B*5206 differs from HLA-B*520102 by one nucleotide exchange in exon 2. The mutation is located at nucleotide position 274, at which a cytosine is substituted by a thymine leading to an amino acid change at protein position 67 from serine (TCC) to phenylalanine (TTC).     

Identification of a novel HLA-B allele--HLA-B*4902

We report herein the identification of HLA-B*4902. This new allele was identified in a Caucasian individual serologically typed as B49. The allele codifying for this antigen was not clearly detectable with polymerase chain reaction using sequence-specific primers (PCR-SSP) because of an atypical amplification pattern. DNA sequencing demonstrated the presence of a new variant due to two nucleotide substitutions (from G to C and from T to C) in exon 2 at nucleotides 309 and 311 respectively. These substitutions would result in a silent mutation and in one amino acid substitution from Ile to Thr, respectively.     

Identification and nucleotide sequence of a new null allele,HLA B*3540N

We report the definition of an HLA class I null allele that has been identified within the B35 group by a combination of serological and molecular typing. This allele, which has been named B*3540N, was detected in a French, potential unrelated hematopoietic stem cell donor of unknown ethnic origin, selected as a probable match for an Irish patient. The presence of the null allele was initially determined by the absence of B35 reactivity by serological typing, in contrast to positive reactions by PCR-SSP and PCR-SSO typing. Subsequent sequencing of clones containing the full genomic sequence of the B*35 allele identified a single nucleotide deletion within exon 4 which resulted in the introduction of a stop codon downstream within exon 4.     

Identification of the novel allele B*4427 and a confirmatory sequence (B*44022)

This report presents a novel allele, HLA-B*4427, which was identified in a bone marrow donor of Caucasian origin, and a confirmatory sequence (B*44022). Sequence analysis revealed the new allele differs from B*44021 by a single nucleotide exchange at position 668 (C-->T), which is located in exon 4. At the protein level, it is the only B*44 variant to produce an Ala in place of a Val at codon 199. Its structure suggests that it may have originated from a point mutation in B*44021 or by gene conversion with a variety of HLA-B alleles. Cloning and sequencing of the allele B*44022 revealed a sequence identical to B*44021 and B*44 exon 4, with the codon GTC (Val) in position 199.     

Identification of a novel HLA-B allele, HLA-B*3713, in a Chinese individual

A novel human leukocyte antigen (HLA) class I allele, HLA-B*3713, has been identified in a Chinese individual. The HLA-B*3713 allele differs from the closest matching allele B*370101 by one nucleotide substitutions in exon 3 at nt 527(T-->A), resulting in an amino acid change from Val (GTG) to Glu (GAG) at codon 152.     

HLA-B*1832, a novel B allele was found through high-resolution HLA typing of a Spanish blood donor

In this paper, we describe the novel human leukocyte antigen (HLA)-B*1832 allele that we found in a female Spanish volunteer blood donor for clinical investigation during her high-resolution HLA typing. The HLA-B typing is B*1801 , 1832 , and the DNA sequence is homozygous with the exception characterized by a nucleotide exchange 'C' to 'A' at position 505, which, in consequence, replaced arginine at codon 169 (CGC) by serine in the new allele B*1832.