Identification by sequencing based typing and complete coding region analysis of three new HLA class II alleles: DRB3*0210, DRB3*0211 and DQB1*0310

The study of HLA class II polymorphism by direct exon 2 DNA sequencing analysis has been established to be a reliable and accurate high-resolution typing procedure. This approach shows some advantages in relation to previous methods, polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO) and sequence-specific primers (PCR-SSP), basically due to the capability of analysis for the complete sequenced genomic region, including non-polymorphic motifs. DRB3 and DQB1 sequencing based typing (SBT) in unrelated bone marrow donor searching allowed us to detect three new alleles. The complete coding region sequences were characterised from cDNA. Two new DRB3 alleles, DRB3*0210 and DRB3*0211, were described in two Caucasian bone marrow donors. Both sequences showed single point mutations regarding DRB3*0202, producing amino acid replacements at positions 51 (Asp to Thr) and 67 (Leu to Ile), respectively. These two point mutations can be found in other DRB alleles, and suggest that gene conversion would be involved in the origin of both alleles. A new DQB1 sequence was found in a Spanish patient that showed two nucleotide differences, positions 134 and 141, with regard to its close similar DQB1*03011 allele. Only substitution at position 134 provoked amino acid replacement at residue 45, Glu to Gly. This single amino acid change would be involved in the lack of serologic recognition of this new molecule by DQ7-specific reagents.     

Identification of two novel HLA class II alleles, DQB1*0311 and DQB1*0620

Two novel HLA class II alleles have been identified in routine typing of a kidney transplant patient and a cord blood unit from the Australian Cord Blood Bank in Sydney. Sequence analysis of exon 2 of the DQB1 genes revealed the novel polymorphism. A substitution of A to C at nucleotide position 136 has been identified for the DQB1*0311 allele when compared to the closest-matched allele, DQB1*030201. An identical substitution has also been identified for the DQB1*0620 allele when compared to the closest-matched allele, DQB1*0602. The substitution results in an amino acid change from methionine to leucine at position 46 implicating different specificity and affinity of antigen binding.     

Identification of two novel HLA class II alleles, DQB1*030503 and DRB1*0447

Two novel human leukocyte antigen (HLA) class II alleles have been identified in routine typing of bone marrow donors for the Australian Bone Marrow Donor Registry in Sydney, Australia. Sequence analysis of exon 2 of both the DQB1 and DRB1 genes revealed the novel polymorphism. A silent substitution of G to A at nucleotide position 210 has been identified for the DQB1*030503 allele when compared to the closest matched allele, DQB1*030501. There is no associated amino acid difference between the translated products of the two alleles. The second new allele is a variant of the DRB1 gene. The DRB1*0447 allele was identified with three nucleotide substitutions compared to the closest matched allele DRB1*0436. There is a silent mutation at nucleotide position 303, G to C and two substitutions at adjacent nucleotide positions 344 and 345, T to G and G to T, respectively. The latter two substitutions result in an amino acid change from valine to glycine at position 86, implicating a different specificity and affinity of antigen binding.     

A novel DRB3 allele (DRB3*0208), a new allelic variant of DRB1*1502 (DRB1*15023) and two new DQB1 (DQBl*03012 and DQB1*0614) alleJes

Abstract: Four novel HLA Class II alleles were identified using CANTYPE reverse hybridization assay. The initial unusual SSO hybridization patterns were confirmed by cloning and sequencing analysis. DRB3*0208 allele is identical to DRB3*0202 except for three nucleotide substitutions (GAT→ AGC) changing codon 57 from Asp to Ser. This polymorphism has so far been undetected in DRB3 alleles. DRB1*15023 differs from DRB1*15021 by a single silent nucleotide substitution (AAC→AAT, both encoding for Asn) at codon 33. This polymorphism has not, until now, been identified in DRB alleles. Compared with DQB1*03011, the novel DQB1*03012 contains a single silent nucleotide substitution (GCA→GCG, both encoding for Ala) at codon 38. Finally, DQB1*0614 allele is identical to DQB1*0603 except for a single nucleotide substitution (TAC→ TTC), changing codon 9 from Tyr to Phe. Polymorphisms observed here in the DQB1*03012 and DQB1*0614 alleles are present in several of the known DQB1 alleles. DRB3*0208, DQB1*03012 and DQB1*0614 may have arisen from gene conversion, but the DRB1*15023 most likely was generated by a point mutation event. DQB1*0614 was detected in three related subjects, while each of the other three new alleles has only been detected once.     

Identification of new DRB1*07 (DRBl*0703), DRB1*08 (DRB1*0817) and two DRB3* (DRB3*0302 and DRB3* 01014) alleles

Abstract: We report here the identification of four novel DRB alleles using a reverse hybridization (CANTYPE) assay. Molecular cloning and sequencing confirmed the initial unusual hybridization patterns. All four new alleles were detected during routine HLA typing for the Canadian Unrelated Bone Marrow Donor Registry. DRBl*0703 is identical to DRB1*0701 except for a single nudeotide substitution (AGA→AGT), changing codon 29 from Arg to Ser, a so far undetected DRB polymorphism. DRB1*0817 differs from DRBl*0801 by a single nucleotide substitution (TAC→TTC), changing codon 47 from Tyr to Phe. This polymorphism has not, until now, been identified in DRB1*08 alleles. Compared with DRB3*0301, DRB3*0302 contains a single nucleotide substitution (TAC→CAC) at codon 30, changing the encoded Tyr to His. This polymorphism is typical for DRB3*02 alleles. DRB3*01014 is identical to DRB3*0101 except for a single silent nucleotide substitution (GGG→GGA) at codon 84. This polymorphism has previously only been described for the DRB1*15012 allele. DRB1*0817, DRB3*0302 and DRB3*01014 may have arisen from gene conversion, but DRB1*0703 most likely was generated by a point mutation event. The DRB3*0302 allele was detected in two unrelated subjects, while the other three have each only been detected once.     

Identification of two new HLA-DRB1 alleles,DRB1*1138 and DRB1*1344

Two new HLA-DRB1 alleles have been identified by sequencing based typing (SBT). HLA-DRB1*1138 and DRB1*1344 were discovered after following up ambiguous results involving unusual alleles after DRB1 generic typing.     

Novel HLA-DRB3 alleles discovered during routine sequencing based typing,DRB3*02023, DRB3*0212, DRB3*0213 and DRB3*03012

We report the discovery of four HLA-DRB3 alleles during routine sequencing based typing (SBT); DRB3*02023, DRB3*0212, DRB3*0213 and DRB3*03012. These alleles differ from other HLA-DRB3 alleles by previously undescribed single nucleotide polymorphisms.     

Novel HLA-DRB1 alleles discovered during routine sequencing based typing,DRB1*03052, DRB1*04032, DRB1*1139 and DRB1*1346

We report the discovery of four HLA-DRB1 alleles during routine sequencing based typing (SBT). These alleles--DRB1*03052, DRB1*04032, DRB1*1139 and DRB1*1346--differ from previously identified DRB1 alleles by known nucleotide polymorphisms.     

Identification of three novel alleles: DRB3*0110, DRB1*1140, and DRB1*140102

Three novel human leukocyte antigen class II alleles (DRB3*0110, DRB1*1140, and DRB1*140102) are described here. The three novel alleles were initially detected as previously unidentified SSO hybridization patterns using CANTYPE((R)) reverse hybridization assay. Sequences were determined by cloning/sequencing. DRB3*0110 allele is identical to DRB3*010101, except for a single nucleotide substitution (CGC-->AGC) changing codon 39 from Arg to Ser. This polymorphism has not, until now, been identified in DRB allele. Thus, this is an unusual mutation as the codon 39 is a fairly conserved region. The new DRB1*1140 is identical to DRB1*1116, except for a single nucleotide substitution at codon 67 from ATC (encoding for isoleucine) to TTC (encoding for phenylalanine). This polymorphism is commonly found in DRB1*11 alleles. Compared with DRB1*140101, DRB1*140102 contains a single silent nucleotide substitution (TAT-->TAC, both encoding for tyrosine) at codon 78. This polymorphism is commonly found in DRB1*14 alleles. The three new DRB alleles may have been generated by a point mutation event. The DRB3*0110 and DRB1*140102 were identified in Caucasoid individuals. The ethnic origin of the subject carrying the DRB1*1140 allele is Egyptian. The DRB1*140102 was detected in two unrelated individuals; the DRB3*0110 and DRB1*1140 were only identified once, in a total population of 80,000.     

Identification of three new DRB3* (DRB3*0106, DRB3*0107 and DRB3*02022) alleles

Three novel DRB3* alleles were identified using CANTYPE reverse hybridization assay. The initial unusual hybridization patterns of DRB3-specific polymerase chain reaction (PCR)-amplified DNA from each subject were confirmed by cloning and sequencing analysis. DRB3*0106 allele is identical to DRB3*0101 except for a single nucleotide substitution (CTG-->GTG) changing codon 38 from Leu to Val. This polymorphism is commonly found in DRB3*03 alleles. Compared with DRB3*0202, DRB3*02022 contains a single silent nucleotide substitution (AAT-->AAC, both encoding for Asn) at codon 77. This polymorphism is also present in DRB3*0204 allele. The new DRB3*0107 allele has a sequence unique to DRB3 alleles. From codon 5 to codon 36 the sequence is identical to that of DRB3*0101 allele. From codon 37 to codon 87 the sequence of DRB1*0107 allele is identical to that of DRB3*0202. This sequence would thus explain the CANTYPE(R) DRB3-specific unusual pattern of reactions. The new DRB3*0107 could have arisen from a gene conversion between DRB3*0101 and DRB3*0202 alleles, but the DRB3*0106 and the DRB3*02022 may have been generated by a point mutation event. The DRB3*0107 allele was identified in a Caucasoid individual. The ethnic origin of the subjects carrying the other two alleles are unknown. The three alleles presented here were only identified once, in a total population of 49,000.     

Novel HLA-DRB1 alleles revealed by sequencing based typing,DRB1*04053 and DRB1*1143

We report the discovery of two HLA-DRB1 alleles by sequencing based typing (SBT). DRB1*04053 differs from previously reported DRB1 alleles by a single synonymous nucleotide substitution, resulting in a unique polymorphism at codon 93. DRB1*1143 differs from previously identified DRB1 alleles by a single non-synonymous nucleotide substitution, resulting in a polymorphism observed in other DRB1 and DRB3 alleles1.     

Description of three novel HLA-DRB3 alleles: DRB3*010105, DRB3*0112 and DRB3*0113

This communication describes three novel DRB3 alleles whose exon 2 sequences are identical to that of DRB3*010102 except for a single nucleotide substitutions. Comparing with DRB3*010102, the sequence of DRB3*010105, DRB3*0112, and DRB3*0113 differ at codon 31 (TTC -> TTT), codon 84 (GGG -> CGG; Gly -> Arg), and codon 37 (TTC -> CTC; Phe -> Leu), respectively.     

Identification of three new DRB1 alleles,DRB1*0107, *0425 and *13012 and confirmation of DRB4*01033

The characterization of three novel DRB1 alleles is described, DRB1*0107, DRB1*0425 and DRB1*13012 as well as confirmation of DRB4*01033. Two alleles, DRB1*0107 and *0425, showed amino acid differences with previously identified HLA molecules. In DRB1*0107, the glutamine at position 10 was substituted by a glutamic acid. DRB1*0425 showed one amino acid difference with DRB1*0418 (I to F) at position 67, and five amino acid differences with DRB1*04011 at positions 67 (L to F), 70 (Q to D), 71 (K to R), 74 (A to L) and 86 (G to V). The alleles DRB1*13012 and DRB4*01033 had protein sequences identical to DRB1*13011 and DRB4*01031/01032, respectively. Nucleotide differences were present at position 306 for DRB1*13012 and at position 321 for DRB4*01033.     

Two new HLA-DQB alleles routinely identified by sequence-based typing: DQB1*030103 and DQB1*0505

Here, we report the identification of two novel human leukocyte antigen-DQB1 alleles, DQB1*030103 and DQB1*0505, found by routine typing using commercial kits.     

A new DRB1*15 allele (DRB1*1506) identified by sequence-based typing

A new DRB1*15 allele (DRB1*1506) was detected during the studies of the 12th International Histocompatibility Workshop within the Allele and Haplo-type Society #11 which studied the DR2 and DR51 antigens. The new allele was found in four unrelated Asian Indian individuals by sequence-based typing. It has a base substitution from T to C in codon 50 at a previously considered conserved position.     

Identification of novel DRBI*13 (DRB1*1333), DRB1*04(DRB1*0426) and DRB5* (DRB5*0109) alleles

Abstract: Three novel HLA class H alleles (DRB1*1333, DRB1*O426, DRB5*0109) are described here. The 3 novel alleles were initially detected as previously unidentified SSO hybridization patterns using the CANTYPE reverse hybridization assay. Sequences were determined by cloning/sequencing. DRB1*1333 is identical to DRB1*1303 except for a single nucleotide substitution (ACC→AAC), changing codon 77 from Thr to Asn. This polymorphism is typical for DRB1*03 alleles. DRB1*0426 is identical to DRB1*0401 except for a single nucleotide substitution (GCC→ACC) at codon 58, changing the encoded Ala to Thr. DRB5*0109 is identical to DRB5*0101, except for a single nucleotide substitution (GAC→AAC), changing codon 70 from Asp to Asn. Both latter polymorphisms were so far undetected in DRB alleles. DRB1*1333 could have arisen from a gene conversion event, but DRB1*0426 and DRB5*0109 most likely were generated by point mutation events. For all 3 alleles, the sequence was confirmed by the original hybridization pattern (DRB1*1333) or by hybridization to a newly designed probe (DRB1*0426 and DRB5*0109). Ethnic backgrounds were Lebanese for DRB1*1333 and Caucasian for DRB1*0426 and DRB5*0109.