A nonsense mutation in exon 3 results in the HLA-B null allele B*5127N

A new HLA-B null allele has been identified within the B*51 group by combined serological and molecular typing of an Italian Caucasoid family. Serological data indicated that the proband typed homozygous for A2 and B60. Confirmatory typing using sequence specific oligonucleotide hybridization (SSPOH) detected a second B allele within the B*51 group. Allele specific typing (SSP) for B*51 subtypes, including the known B*5111N allele, was performed, and typing results were consistent with B*5101, suggesting the presence of a new null variant. Cloning and sequencing of this allele identified a B*5101 variant with a nonsense mutation in exon 3. This new null allele has been designated B*5127N. The combined use of serologic and DNA-based typing methods facilitates the identification of null and low-expression alleles. An overview of null alleles of class I HLA is presented.     

A deletion in exon 3 results in a new HLA-A*01 null allele (A*0115N) in a Martinican woman

We report the identification of a new HLA-A null allele, HLA-A*0115N. This null allele has been identified within the A*01 group by a combination of serological and molecular typing [Polymerase chain reaction (PCR) sequence-specific primers, PCR sequence-specific oligoprobes and sequence-based typing (SBT)] in a potential intrafamilial bone marrow donor from Martinique (French West Indies). To characterize this A*01 null allele, we performed DNA typing by PCR-SBT on genomic DNA from the beginning of exon 2 (position 84) through the end of the exon 4 (position 895) and revealed a nucleotide deletion at the end of the exon 3. This sole difference between the new allele and the HLA-A*0101 generates a premature stop codon (TGA) in the beginning of exon 4. This deletion most likely explains the lack of cell surface expression of the encoded protein despite the presence of A*01 allele. The absence of correct expression of the antigen on the cell surface was confirmed by one-dimensional isoelectric focusing (1D-IEF). To date, this is the fourth null allele described within the A*01 group.     

Identification of an HLA-B*07 allele variant (B*0740) in the Chinese Han population

A novel HLA-B*07 allele, B*0740, has been identified by sequence-based typing (SBT) in the Chinese Han population. This new allele is identical to B*0705 and B*0706 for exons 2, 3, and 4, except for a single nucleotide at position 605 of codon 202 in exon 3 (AAG-->ATG) leading to an amino acid change from lysine to methionine. SBT was performed following allele separation using the Haploprep method. The serological equivalence of B*0740 to the B7 antigen did not change.     

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.     

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.     

应用顺序特异性引物聚合酶链反应检测华南人群HLA-B座位第4外显子变异

目的：为探明中国华南人群中因HLA-B座位发生基因突变而引起表达变异存在的可能性。方法：用顺序特异性引物聚合酶链反应（PCR-SSP）对75例经血清血分型为纯合型样本再次分型，对有差异的结果使用PCR-SSP方法检测第四外显子突变情况。结果：75例血清学指定为纯合型标本经DNA分型证实有12例为杂合型。12例结果有差异的样本经PCR-SSP法表达变异的筛查发现有1例存在第四外显子额外胞嘧啶插入，经DNA分型发现的第二基因为沉默基因。结论：应用PCR-SSP方法可检测HLA-B等位基因突变，该突变导致HLA-B基因表达无效，进行HLA基因表达变异的检测可提高HLA分型的准确性，更加有效的指导临床进行器官和骨髓的选配。     

Second HLA-A*68 null allele,A*6818 N,identified

A second HLA-A*68 null allele, HLA-A*6818 N, was identified in our laboratory after discrepant results were obtained between class I serological and molecular typing in a male patient suffering from narcolepsy. HLA-A*6818 N displays a sequence identical to that of the HLA-A*6802 allele, except in exon 2 where 20 nucleotides inserted at codon position 48 are a repeat of the 20 preceding nucleotides. This duplication creates a shift of the reading frame, which leads to a premature non-sense codon at position 59 of the null allele.     

Identification of a novel allele HLA-B*7805 in a Japanese female

A new HLA-B*78 allele, B*7805, was identified in a healthy Japanese female. The results of her serological HLA class I typing showed an unusual Bw4/Bw6 pattern with strongly positive reactivity to anti-Bw6, i.e. A24, -, B52, -, Bw4, Bw6. In DNA typing, she was typed as A*24, -, B*52, B*78-like, Cw1202, -, (Bw4, Bw6). Cloning and sequencing of exon 2 and exon 3 of her B locus genes revealed a new allele B*7805. The cloned B*7805 differed from B*78021 by three nucleotide substitutions in exon 2 at position 259 (A to G), 261 (C to G) and 272 (A to C), and contained sequences defining Bw6 motif in the region of codon 77 to 83.     

HLA-B*0749N: the first HLA-B*07 null allele

In the present article, we report the identification of the first HLA-B*07 null allele found in a Polish patient awaiting a kidney allograft. A discrepant result obtained between serological typing (HLA-B "blank") and high-resolution molecular typing using PCR-SSP method (HLA-B*070201 allele) suggested the presence of a null allele. Genomic DNA sequencing of the HLA-B*07 allele revealed a single nucleotide substitution at the 3' end of the exon 4 leading to a premature stop codon.     

Identification of the null HLA-A2 allele, A*0232N

We have identified a null HLA-A*02 allele, HLA-A*0232N, by using a combination of serology, flow cytometry, polymerase chain reaction using sequence-specific primers (PCR-SSP) and full-length sequencing. The null HLA-A2 allele was identified in an Asian individual originally typed by serology as an apparently homozygous HLA-A3, B51. Subsequent genotyping by PCR-SSP identified the genotype as HLA-A*0201, *0301, B*51, Cw*1402. The serological type and lack of detectable HLA-A2 was confirmed using monoclonal antibody typing reagents. Flow cytometry studies failed to identify any cell surface HLA-A2 expression on the patient's peripheral blood lymphocytes. Genotyping using a PCR-SSP set designed to detect null alleles revealed the mutation had not been previously described. Full-length sequencing of the allele identified an allele which was subsequently named HLA-A*0232N. This allele is identical to HLA-A*0201 except for a novel point mutation (T for C) at position 493 which creates a premature stop codon. The sequencing enabled the development of a monospecific A*0232N PCR-SSP reaction which was used to screen 973 DNA samples: no further examples of A*0232N were identified.     

Identification of a novel allele HLA-A*1113 in a Japanese donor

Anew HLA-A*11 allele, A*1113, was identified in a healthy Japanese female. She was typed as HLA-A11?, A2, B46, B67, Cw1, Cw7 (Bw6) with unusual serological reactivity of A11, suggesting possible presence of a new A*11 allele. The novel A*1113 allele was identified by haplotypic group-specific allele amplification using A*11 allele-specific primer pairs and sequence-based typing. The A*1113 allele differs from A*11011 by one nucleotide substitution in exon 3 at position 503 (A --> G) which causes an amino acid change in the alfa2 domain at residue 144 (lysine : K --> arginine : R), thus resulting in the unusual serological reactivity.     

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.     

A new allele, HLA-B*1555, identified in an African patient awaiting bone marrow transplantation

Human HLA genes exhibit extreme polymorphism, the extent of which is emphasised by the identification of an ever increasing number of new alleles by DNA-based typing strategies. Here we describe a novel allele, belonging to the HLA-B*15 group, which was identified in an African patient awaiting a bone marrow transplant This individual was shown to exhibit two HLA-B alleles, B*5301 and a new allele which has been named B*1555. B*1555 differs from B*1531 in exon 3 by a single nucleotide substitution. This substitution results in a change in the amino acid residue at position 97, which is located within the beta-pleated sheet region of the HLA molecule.     

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.     

HLA-B*8202 identified in a Caucasoid potential bone marrow donor

Sequence analysis of HLA class I alleles has continued to reveal the true extent of polymorphism, particularly for B-locus alleles. This diversity can arise through reshuffling of polymorphic sequences generated by point mutation, resulting in interallelic recombination or intergenic recombination (1). Here we describe a new B-locus allele, B*8202, which is structurally most similar to B*8201, having only one nucleotide difference in exon 3 at nucleotide 557, resulting in an amino acid change of aspartic acid to glycine at residue 162. Glycine is the consensus amino acid for B-locus alleles, which suggests that B*8202 is older than B*8201 in evolutionary terms. B*8201 was found to be a hybrid of B*4501 and B*5602 that may have arisen through recombination events, explaining the serological patterns observed with these allotypes. The importance of high-resolution typing is emphasised here as routine typing suggested the presence of B*8201 and the new variant allele may have been missed had it not been typed further by sequence-based typing.     

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.     

HLA-B*4012: a new allele with unique serological features

We have defined the new allele HLA-B*4012, which had been isolated from a black individual. It was initially recognized as a serologically unique allele when typing her father for renal transplantation. The HLA class I phenotype was A*0201,*6602; B*4001,*4012; Bw6; Cw*0304,*1505. Sequencing from exon 1 through intron 3 of B*4012 was performed. B*4012 is identical to B*4001 and B*4010 in exon 3, and in the 3' part of exon 2, but it is unique in that exon 1 and the 5' part of exon 2 are identical to B*1503, B*1509, B*1510, B*1518, B*1523, and B*1529. The generation of this allele is best explained by a recombination event in exon 2 (break point between nucleotides 205 and 222 from the beginning of the coding region) of B*4001 or B*4010 with one of these B*15 variants as a donor allele. Its unique serological feature (B48, B60, B70, and B72 reactivity) is consistent with the sequence data of its donor alleles.     

Presence of the DRB4*0103102N null allele in different DRB1*04-positive individuals

The DRB4 gene encoding the DR53 antigen is present in DRB1*04-, DRB1*07- and DRB1*09-positive individuals. Eight allelic variants of DRB4 have been recognized, 5 resulting in an expressed DR53 antigen and 3 belonging to the null alleles. So far the DRB4*0103102N null allele had been found exclusively in individuals carrying the haplotype DR7,-DQ9. High-resolution typing of HLA class II by polymerase chain reaction using sequence-specific primers (PCR-SSP) and/or sequence-based typing of kidney patients and their families revealed the presence of the DRB4*0103102N null allele segregating with DRB1*04 and DQB1*03 in 4 different families. Three different haplotypes on which the null allele was located, were recognized by family studies: DRB1*0401, DQB1*0301; DRB1*0402, DQB1*0302 and DRB1*0404, DQB1*0302. Determination of the DR53 specificity of antisera reacting with DR53-positive individuals has always been difficult due to the simultaneous presence of DR4, 7 or 9. Identification of DR4-positive DR53-negative individuals as described here, provided the serological reactions with DR53-antisera and revealed the antibody specificities in the antisera used.