HLA-A*3306, a novel variant in the Indian population.

We describe a new HLA-A allele, A*3306, which was identified by sequencing based typing (SBT) in an individual of Indian origin. A*3306 is similar to A*3303, with a difference at position 228 (A to G). This difference leads to an amino-acid change at codon 52 from Ile (ATA) to Met (ATG). Until now this position has been considered conserved.

DPB1*8501, a novel DPB1 variant in the US Black population.

We describe a new DPB1 allele, DPB1*8501, which was identified by sequencing-based typing (SBT) in the UCLA exchange. DPB1*8501 is similar to DPB1*2701 with a difference at position 272, (G to A). This difference leads to an amino-acid change of codon 91 from arginine (CGC) to histidine (CAC). Until now this position has been considered conserved. This substitution is located at the 3' site of exon 2, and may interfere with typing strategies using primers or probes located in this region.

Criteria to define HLA haplotype loss in human solid tumors.

Short tandem repeat (STR) markers are currently used to define loss of heterozygosity (LOH) of genes and chromosomes in tumors. Chromosome 6 and chromosome 15 STR markers are applied to define loss of HLA and related genes (e.g. TAP and beta2m). The number of STR identified in the HLA region is still increasing. In this study, seven representative STR markers covering the 6p/6q arms of chromosome 6 including the HLA region and two for chromosome 15 flanking the beta2m gene, were selected as minimally required for reliable LOH studies. A multiplex polymerase chain reaction (PCR) strategy is proposed when small number of cells are available in microdissected tumor samples.

Microsatellite analysis of microdissected tumor cells and 6p high density microsatellite analysis in head and neck squamous cell carcinomas with down-regulated human leukocyte antigen class I expression.

Down-regulated human leukocyte antigen (HLA) class I expression is frequently correlated with allelic loss at 6p21.3, which is the location of the HLA coding sequence, in head and neck squamous cell carcinomas (HNSCCs). Previously, we have demonstrated loss of heterozygosity (LOH) at 6p21.3 for at least one locus in 49% of the HNSCCs using 5 microsatellite markers spanning the 4 megabase HLA region. In the present study, the detection threshold (25%) to assign LOH was addressed by laser-assisted microdissection of tumor cells from tumors containing marginal loss. In addition, we describe high density microsatellite analysis of chromosome 6p21.3 in HNSCC with down-regulated HLA class I expression. The purpose of this study was to refine the identification of genetic alterations at 6p21.3 and to pinpoint allelic loss to individual HLA class I genes, using additional markers closely located to the HLA-A, -B, and -C loci and the transporter associated with antigen processing (TAP) genes. LOH analysis by amplification of microsatellite markers and subsequent fluorescent detection is a rapid and sensitive technique to predict HLA class I loss phenotypes in tumors. LOH can be identified at 25% relative signal reduction. Analysis of heterogeneous tumor samples and samples containing a small amount of tumor cells is facilitated by laser-assisted microdissection of tumor cells. In addition, we showed that accurate HLA LOH analysis requires application of microsatellite markers in close proximity to HLA class I and TAP genes.

A novel HLA-A24 (A*2420) allele identified in the Atayal tribe of Taiwan.

The Taiwan indigenous population groups are classified into different tribes according their linguistic classification and cultural anthropology. One of the tribes, the Atayal, showed a high frequency of A24 alleles by SSOP analysis. High-resolution sequencing based typing identified a A*2402 variant "A*2420" which was found in 6 unrelated individuals. High-resolution typing is required to identify HLA polymorphism in the Taiwanese minority groups.

Characterization of a novel HLA-A2 variant, A*02012, in the Southern Thai-Muslim population.

Southern Thai Muslims (STM)--from Nakon Si Thammarat, whose ancestors come mainly from Malaysia--constitute more than half of all Thai Muslims which, in total, represent approximately 10% of the country's population. The most common A2 subtypes in STM were A*0203 (n=15), A*0201 (n=8) and A*0207 (n=7). In this study, samples with unexpected amplification patterns were sequenced. Three individuals were indicative of a novel A2 allele, now known as A*02012.

Description of a new DRB1*11 allele (DRB1*1132).

We describe a new DRB1*11 allele which is similar to DRB1*11011 except at codon 74, where a GCG is changed for a GTG leading to an alanine/valine substitution. This new allele was carried by a Caucasian patient suffering from rheumatoid arthritis and by her healthy daughter. The motif at codon 74 of the new DRB1*11 is not found in any other known DRB alleles, nor among the published DQA1, DQB1, DPA1 or DPB1 alleles, and therefore suggests a mechanism of point mutation.

DPB1*7601, a novel DPB1 variant in the Caucasoid population.

A new DPB1 allele has been identified in a Caucasoid individual, DPB1*7601. The sequence of the complete second exon has been confirmed by cloning and subsequent sequencing. This allele differs by one amino acid, at codon 36, from DPB1*1401, as indicated by SBT and PCR-SSP analysis. The amino-acid motif introduced by the change is shared by DPB1*0401 and some rare alleles. It remains unclear whether the change is due to interallelic microgen conversion or a single point mutation.

HLA-A towards a high-resolution DNA typing.

Sequencing-based typing (SBT) and sequence-specific oligonucleotide probing (PCR-SSOP) are DNA-based typing approaches to identify HLA-A alleles. In this study PCR-SSOP SBT have been evaluated and considered to reach a high-resolution typing. Based upon serological typing, 32 genomic samples were typed by SBT and PCR-SSOP Three main clusters of resolution could be defined. The advantage of the PCR-SSOP approach is the possibility to type numerous samples in a short time. SBT minimizes the number of ambiguous heterozygous combinations and often allows direct detection and identification of new alleles.

Comparison of DRB sequence-based typing using different strategies.

Sequence-based typing (SBT) has become an important tool in the identification of HLA alleles. In this study a comparison was made between SBT of DRB1/3/4/5 alleles performed in two laboratories each using a different strategy for SBT. The laboratories in Utrecht and in Maastricht performed direct sequencing of PCR amplified genomic DNA from 30 selected samples. Primers and conditions for PCR amplification were different. Sequencing was either performed with T7 polymerase, using internal sequencing primers, or with cycle sequencing using an M13 tailed system. Two different automated DNA sequencers were used; the ALFexpress from Pharmacia and Applied Biosystems 373A. We concluded that nor the method of sequencing nor the sequencing machine influences typing results. However the PCR reaction used for generating template DNA is the most critical step. Different primers and different conditions can lead to false negative reactions. The fact that these false negative reactions can occur with different alleles in different combinations but not in all, implicates that extensive quality control is needed to assure correct typing results.

A generic sequencing based typing approach for the identification of HLA-A diversity.

Sequencing Based Typing (SBT) is a generic approach for the identification of HLA-A polymorphism. This approach includes the high resolution typing of the HLA-A broad reacting groups, HLA-A subtypes and will identify new alleles directly. The SBT approach described here uses a locus specific amplification of DNA from exon 1 to exon 5. The resulting 2,022 bp PCR product serves as a template for the subsequent sequencing reactions. Amplification is followed by direct sequencing of exons 2, 3 and 4 in both orientations with fluorescently labeled primers to define all polymorphic positions leading to a high resolution typing result. In this study the sequence of exons 2 and 3 of a panel of 49 cell lines was determined. In addition, the exon 4 region of 35 cell lines was also sequenced to evaluate the exon 4 polymorphism. The HLA-A type of most of the cells could be identified by sequencing only exons 2 and 3. However, the sequence of exon 4 was required to discriminate A*0201 from A*0209 and A*0207 from A*0215N. In this panel, an identical new "HLA-A*0103" was identified in two Caucasian samples.