Investigation of killer cell immunoglobulin-like receptor gene diversity in KIR3DL1 and KIR3DS1 in a transplant population.

Several overlapping amplicons were used to obtain the sequence of genomic DNA covering most of the coding regions of KIR3DL1 and KIR3DS1 from a family and 77 bone marrow transplant patients and their unrelated donors. Alleles 3DL1*00101 and *002 were most frequently observed in addition to 12 other known 3DL1 alleles. A single 3DS1 allele, 3DS1*01301, was identified in the 31 of 32 individuals carrying this gene. Two new alleles, 3DL1*01702 and 3DS1*058, were characterized. Three samples appeared to carry the duplicated killer cell immunoglobulin-like receptor (KIR) haplotype observed in other studies based on the presence of 3DS1 and two 3DL1 alleles. Additionally, one sample appeared to carry a novel KIR haplotype containing one 3DL1 and two 3DS1 alleles.

Seventeen novel alleles add to the already extensive KIR3DL3 diversity.

Exons 2-9 of KIR3DL3 alleles were characterized by genomic DNA sequencing in two families and in 78 bone marrow transplant samples. Several strategies were used to isolate single alleles for characterization and to resolve alternative allele combinations. We describe 17 novel 3DL3 alleles carried by 30 individuals. Compared with the most closely matched alleles, the new alleles differ by from one to three nucleotides and from zero to three amino acids. The majority of the substitutions were shared with other 3DL3 alleles although three novel polymorphic codons, 151 (exon 4), 327 (exon 7) and 352 (exon 9), are described. Of the 36 different 3DL3 alleles detected in the transplant population, the three most common alleles accounting for 47% of the total were KIR3DL3*00101 (13.5%), KIR3DL3*003 (21.2%) and KIR3DL3*00901 (12.2%).

KIR3DL2: diversity in a hematopoietic stem cell transplant population.

Exons 2 through 9 of KIR3DL2 were amplified from genomic DNA from 79 bone marrow transplantation patients and their unrelated donors. Sequencing of heterozygotes and isolated alleles identified 9 of the 17 known alleles. The alleles provide confirmation of previously submitted sequences and are carried by transformed B-cell lines that can be used as references for assay development. Alleles 3DL2*001, *002, *007 and *009 accounted for 111 of the total 144 possible alleles and were the only ones found in a homozygous state. New alleles (3DL2*017, *018, *019, *020, and *021) were found in seven transplant samples and one workshop cell. This study describes the development of reagents and protocols for sequencing of KIR3DL2 alleles from genomic DNA.

Allelic diversity in KIR2DL4 in a bone marrow transplant population: description of three novel alleles.

Genomic DNA sequencing was used to identify alleles of KIR2DL4 from 78 unrelated individuals involved in hematopoietic stem cell transplants. Eight known alleles were observed. Three new alleles, KIR2DL4*00203, *00502, *0080104, which differ from known alleles at the nucleotide but not at the protein sequence level, were also identified.

KIR2DL1 allelic diversity: four new alleles characterized in a bone marrow transplant population and three families.

Sequencing of PCR amplified genomic DNA including most of the coding region was used to identify killer cell immunoglobulin-like receptor 2DL1 alleles from three families and 77 bone marrow transplant patients and donors. Alleles 2DL1*00302 and *002 were frequently observed in addition to two other known alleles and four new alleles, 2DL1*00402, 2DL1*007, 2DL1*008, and 2DL1*009.

Searching for HLA-DRB1*1206 in volunteer marrow donors in four US population groups.

The frequencies of DRB1*12 alleles were determined in four US population groups by DNA sequencing. Only DRB1*120101 (or DRB1*1206 or *1210) and DRB1*120201 alleles were identified, the latter primarily in the Asian American population. Additional testing of a subset of samples to detect the presence of DRB1*1206 found all of the alleles to be DRB1*120101 (or DRB1*1210). Retesting of six samples previously typed as DRB1*1206 found only DRB1*120101 (or DRB1*1210).

Twenty-three novel HLA-B alleles identified during intermediate-resolution testing.

Twenty-three novel human leukocyte antigen-B alleles are described: B*070204, *0738, *0742, *0821, *130202, *1312, *1575, *1598, *1599, *270507, *2728, *350104, *3558, *3811, *3931, *3932, *4045, *4107, *420501, *4812, *510106, *5520, and *5616. Thirteen of the variants are single-nucleotide substitutions from their most homologous allele, eight resulting in amino acid changes (B*0742, *1312, *1598, *1599, *3558, *3931, *4107, and *5616) and five with silent substitutions (B*070204, *130202, *270507, *350104, and *510106). Three alleles (B*0738, *4812, and *5520) differ by five nucleotide changes, altering four amino acids. The remaining seven alleles differ from their most similar alleles by two to three nucleotides, altering from one to two amino acids.

KIR3DL3 allelic diversity: six new alleles exhibit both conservative and non-conservative substitutions.

KIR3DL3 alleles were characterized in two families and one unrelated individual. Based on exon 2-9 nucleotide sequences, six novel alleles, 3DL3*00402, *005, *006, *007, *00801, *00802, were identified bringing the total number of known alleles to 11. Compared with 3DL3*001, the six new alleles differ by from three to nine nucleotides and from three to four amino acids. The new alleles double the number of known polymorphic positions to 18 with variation in exons encoding the extracellular domains, transmembrane region, and a portion of the cytoplasmic tail. Many of the nucleotide substitutions are shared among alleles of 3DL3 or other KIR loci, but five were found only in single 3DL3 alleles. Comparison of intron sequences among individuals carrying the same allele showed a modest number of substitutions with the exception of 3DL3*001 which differed substantially in its intron sequences. Two alleles sharing coding region sequences, 3DL3*00201 and 3DL3*00202, were also substantially different in intron sequences.

Strategies for evaluating B*18 allelic diversity by sequence-based typing applied to studies of a population from Singapore and African-Americans.

Strategies to resolve B*18 alleles which carry a deletion in intron 1 close to the 5' end of exon 2 relative to other HLA-B alleles or a null allele mutation in exon 1 and to resolve ambiguities among allele combinations including B*18 are described. B*18 allele frequencies from volunteer donors recruited for two hematopoietic stem cell registries show the presence of two alleles, B*180101 and B*1802, in a population from Singapore and only B*180101 in African-Americans.

Ten novel HLA-DRB1 alleles and one novel DRB3 allele.

Ten novel HLA-DRB1 and one DRB3 alleles are described. Eight of the variants are single-nucleotide substitutions, four resulting in an amino acid change (DRB1*1145, *1148, *0828 and *1514) and four with silent substitutions (DRB1*040504, *130103, *160502 and DRB3*020204). Two alleles differ by two nucleotide changes altering one (DRB1*1447 and *1361) amino acid and one allele alters three nucleotides and two amino acids.

Genomic characterization of KIR2DL4 in families and unrelated individuals reveals extensive diversity in exon and intron sequences including a common frameshift variation occurring in several alleles.

The KIR2DL4 gene including a portion of exon 1 through exon 9 was sequenced from two families and eight cell lines from the International Histocompatibility Workshop (IHWS). Two known alleles and eight variants were detected. Overall, there were five synonymous and three non-synonymous changes when the variants were compared to the coding sequences of the most closely related known alleles plus a common frameshift change in five of the variant alleles. Alignment of the new variants with all known alleles showed that the regions encoding the extracellular region and the cytoplasmic tail were the most polymorphic. Two non-synonymous changes, P146H and L161V, occurred in an extracellular immunoglobulin-like domain. Five of the eight variants had a single adenine deletion in the exon encoding the transmembrane region, potentially resulting in a truncated protein lacking the cytoplasmic tail. The distribution of the deletion variant among many KIR2DL4 alleles may explain the high frequency of this variation in the population. Four of the eight consanguineous IHWS cell lines were found to be heterozygous for KIR2DL4 carrying two alleles that differed from one another by a few nucleotide substitutions. Analysis of intron sequences in the families revealed the nature and distribution of interspersed repeat elements which comprise 46% of the KIR2DL4 nucleotide sequence and consist of 12 elements including six SINEs (13.73% of the total length), one LINE (12.41%), and five LTR elements (19.51%). The results revealed the presence of extensive diversity in the KIR2DL4 gene. This is the first extensive report providing both exon and intron data in related individuals.

Seventeen novel HLA-A alleles.

This paper describes 17 novel HLA-A alleles: A*0244, A*0251, A*0254, A*0309, A*1111, A*2432, A*2434, A*2504, A*2618, A*2905, A*2906, A*3106, A*3107, A*3207, A*6820, A*7407, and A*7408. Most substitutions are found in other alleles.

Twenty-five novel HLA-B alleles.

Twenty-five novel HLA-B alleles are described in this paper: B*0729, B*1309, B*1814, B*1815, B*2724, B*2725, B*3539, B*3926, B*4037, B*4040, B*4042, B*4043, B*4044, B*4204, B*440203, B*4428, B*4429, B*4430, B*4505, B*5308, B*5309, B*5510, B*5511, B*570102, and B*5709. Most of the variants are single nucleotide substitutions. Two involve variants at the Bw4/Bw6 epitope. Two alleles carry substitutions of conserved amino acids.

Limited diversity of HLA-DRB1*02 alleles and DRB1-DRB5 haplotype associations in four United States population groups.

At least 60 DRB1*02 positive individuals from each of four US population groups found within a hematopoietic stem cell volunteer donor registry - Caucasoids, African Americans, Asians/Pacific Islanders, and Hispanics - were randomly selected from a database of 82,979 individuals. DRB1*02 alleles were identified by DNA sequencing. A total of five of 23 known DRB1*02 alleles were detected. DRB1*15011 was the predominant DRB1*02 allele in Caucasoids and Hispanics. The most common DRB1*02 allele observed in African Americans was DRB1*1503, and DRB1*15021 in Asians/Pacific Islanders. Caucasoids were found to be the least diversified; only DRB1*15011 and DRB1*16011 were observed. A subset of individuals was also typed for DRB5 alleles by DNA sequencing. DRB5*01011, DRB5*0102, DRB5*0103, DRB5*0108N and DRB5*0202 were detected and nine DRB1-DRB5 haplotypes defined.

Development of informatics tools for complex gene systems: killer-cell immunoglobulin-like receptor genes.

Killer-cell immunoglobulin-like receptors (KIRs) are a newly described family of polymorphic and highly homologous genes that have been difficult to classify and characterize. Before comprehensive analyzes of the genes are completed, researchers must struggle with completing the task of classifying and characterizing what is currently known. A collection and alignment of all KIR sequences found in GenBank was created to facilitate oligonucleotide reagent development and to provide an overall picture of this complex gene system. Two methods, a direct measurement of homology and phylogenetic analysis, were used to categorize sequences previously not specifically identified as belonging to a particular locus. The two methods agreed for 64.2% of sequences. A further 22.6% of uncategorized sequences were specified by only one method, although the assignments were consistent. Some sequences (11.3%) could not be assigned to a locus by either method. For one sequence, the two methods disagreed as to the locus assignment (1.9%). The failure to categorize a sequence was usually related to the short length of the sequence and the similarity among KIR loci. The tools developed in this study have been valuable in the analyses of KIR sequences and can be used for any complex gene system.

Novel HLA-B alleles formed by an inter-locus recombination with HLA-C, HLA-B*0713 and B*6702.

This paper describes two novel HLA-B locus alleles. B*0713 appears to have resulted from an interlocus recombinational event which inserted a sequence from an HLA-C allele into B*0702. This event altered a significant portion of the alpha-1 domain alpha helix. B*6702 shares much of its exon 2 sequence with B*0713 but is identical in exon 3 to several HLA-B locus alleles including B*67012.

Twenty-nine new HLA-B alleles associated with antigens in the 5C CREG.

This paper describes 29 novel HLA-B locus alleles identified during low-resolution typing. The majority of the novel alleles carry new patterns of previously known polymorphic motifs or codons. Three alleles carry alterations in the Bw4/Bw6 epitope. Five alleles carry novel substitutions.

Novel HLA-B alleles associated with antigens in the 7C CREG.

This paper describes 9 novel HLA-B locus alleles. All of the alleles carry sequence motifs observed in other HLA-B alleles.

Novel HLA-B alleles associated with antigens in the 8C CREG.

This paper describes 13 novel HLA-B locus alleles, B*0809, B*0812, B*0813, B*0814, B*14062, B*3804, B*3806, B*3914, B*3915, B*3918, B*3919, B*3920, and B*3922 which represent new patterns of known polymorphic residues.

New alleles in the B44 family including B*44022, B*44032, B*4411, B*4420, B*4421, B*4424, and B*8301.

Seven new HLA-B locus alleles have been described. B*44022 and B*44032 are silent substitutions altering known alleles. B*4411 carries a unique Bw4-like epitope. B*4420, B*4421, and B*4424 carry new combinations of motifs previously observed in other alleles. B*8301 appears to be the result of the replacement of exon 2 from B*4402 with exon 2 from B*5603.