Polymerase Chain Reaction Using Sequence-specific Oligonucleotide Probes Research Articles

HLA associations in South Asian multiple sclerosis

Background: Previous efforts to identify Human Leukocyte Antigen (HLA) gene associations with multiple sclerosis (MS) in the South Asian population have been underpowered. Aim: To identify the primary HLA class II alleles associated with MS in Indians. Methods: We typed HLA-DRB1, -DQA1 and -DQB1 in 419 patients and 451 unrelated controls by polymerase chain reaction using sequence specific oligonucleotide probes (PCR-SSOP). Results: At the gene level DRB1 showed significant evidence of association (p=0.0000012), DQA1 showed only marginal evidence of association (p=0.04) and there was no evidence for association at DQB1 (p=0.26). At the DRB1 locus association is confirmed with the *15:01 (p=0.00002) and the *03 (p=0.00005) alleles. Conclusion: Our study confirms that the risk effects attributable to the HLA- DRB1*15:01and DRB1*03 alleles seen in Europeans are also seen in Indians. The absence of any evidence of association with DQB1 alleles reflects the lower linkage disequilibrium between DQB1 alleles and DRB1 risk alleles present in this population, and illustrates the potential value of fine mapping signals of association in different ethnic groups.

P52 Characteristics of HLA Class I and Class II Antigens of the Somali Population

HLA antigens of the Somali population are not categorised as well as those of other international ethnic groups. We analysed the HLA antigens of 76 unrelated Somalis who lived in the west of England. HLA ‐A, ‐B, ‐C and DRB1 typing was performed by polymerase chain reaction using sequence‐specific oligonucleotide probes (PCR‐SSOP) at a low‐intermediate resolution level. Phenotype frequency, gene frequency and haplotype frequency were used to study the relationship between Somalis and other relevant populations. The antigens with highest frequencies were HLA ‐A1, A2, and A30; B7, B51 and B39; Cw7, Cw16, Cw17, Cw15 and Cw18; DR 13, DR17, DR8 and DR1. HLA haplotypes with high significance and characteristics of the Somali population are B7‐Cw7, B39‐Cw12, B51‐Cw16, B57‐Cw18. The result of HLA class I and class II antigen frequencies show that the Somali population appear more similar to Arab or Caucasoid than to African populations. The results are consistent with hypothesis, supported by cultural and historical evidence, of common origin of the Somali population. This study will serve as a reference for further anthropological studies, as well as studies of associations between HLA and disease.

Genotyping BoLA-DRB3 alleles in Brazilian Dairy Gir cattle (Bos indicus) by temperature-gradient gel electrophoresis (TGGE) and direct sequencing.

BoLA-DRB3 is a gene of the major histocompatibility complex (MHC) in cattle. The product of the BoLA-DRB3 gene is a beta chain of an MHC class II molecule, a glycoprotein expressed on the surface of antigen-presenting cells (APCs). Responses of CD4+ T lymphocytes to peptides are dependent on the presentation of peptide ligands bound to class II molecules on APCs. Genotyping of the BoLA-DRB3 gene is relatively complex due to the extensive polymorphism of this locus. Current techniques for assignment of genotypes are polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), direct sequencing of PCR products, cloning-sequencing, polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP), and denaturant-gradient gel electrophoresis. These techniques are time-consuming, do not discriminate all possible alleles, or are not readily reproducible. The objective of this study was to genotype BoLA-DRB3 using temperature-gradient gel electrophoresis (TGGE) to separate alleles before sequencing. PCRs using 28 DNA samples from Gir Dairy cattle (a Brazilian breed of Bos indicus) were submitted to TGGE. New PCR products were generated from separated alleles, purified, and sequenced. Allele separation was possible in 21 out of 26 heterozygote samples (81%). Results indicate that two sequence reads (forward and reverse) were sufficient for accurate genotyping of BoLA-DRB3 alleles. Separation of alleles by TGGE provides high-throughput, reliable typing of BoLA-DRB3, which is critical in disease association studies in cattle.

HLA-DR and -DQ polymorphism in Cameroon.

HLA-DRB1, -DQA1 and -DQB1 allele frequencies were determined by high-resolution polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) and/or DNA sequencing in 126 healthy individuals in Cameroon. Eighteen DRB1, 11 DQA1, and 18 DQB1 alleles were observed. The most common alleles at each locus were DRB1*1503 (29%), DRB1*1301 (13%); DQA1*0102 (38%), DQA1*0302 (11%), DQA1*0401 (11%); and DQB1*0602 (31%), DQB1*0301 (13%), DQB1*0501 (12%). Forty-four different haplotypes were identified including 12 novel haplotypes demonstrating the HLA class II diversity resulting from allele combinations in this population. A single predominant DRB1*1503-DQA1*0102-DQB1*0602 haplotype was observed with a frequency of 27%. In summary, this study of HLA class II polymorphism in Cameroon demonstrates the extent of diversity in this population.

Identification of a novel allele, DRB1*1340, in two Brazilian individuals.

We identified a novel HLA-DRB1 allele, named DRB1*1340 by the WHO HLA Nomenclature Committee, in two Brazilian individuals. Typing by polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) showed a DRB1*13 allele with an unusual hybridization pattern. DNA sequencing of both strands and comparison of the sequence with previously described DRB1 alleles revealed that the most similar allele is DRB1*1301, from which DRB1*1340 differs by a single nucleotide (T-->A) in exon 2, at position 127, codon 47 (Phe-->Tyr). The sequence received accession number AJ237964 from the EMBL database.

DRB3 alleles with variations in the annealing sites of commonly used amplification primers.

New HLA alleles are often identified initially from observing uncommon patterns found in low-resolution typing performed via polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP). Recently, the HLA-DR oligotyping analysis of two Caucasian, one Caucasian/American Indian and two African American individuals resulted in the identification of three novel DRB3 alleles. Using DRB-specific primer sets commonly employed in amplification-based typing, all four individuals were originally characterized as DRB3 negative. Direct sequencing identified DRB3*0104 (variation at codon 8, TCG instead of TTG), and DRB3*0101202 (variation at intron (-13), G instead of C). One individual appeared to carry a DR52-associated DRB1 allele without an associated DRB3 allele. Lack of conservation at the junction of intron 1 and exon 2 of the DRB3 gene suggests that commonly used DRB-specific primer sets may need to be modified.

Characterisation of a novel HLA-A pseudogene, HLA-BEL, with significant sequence identity with a gorilla MHC class I gene.

During the development of an HLA-A polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) method for the identification of HLA-A*24 and -A*30 alleles, group amplification resulted in the formation of an unusual PCR product in certain individuals. This fragment was approximately 900 bp smaller than the expected product and was also detected in some non-HLA-A*24- and -A*30-positive individuals acting as negative controls for the group specific amplification. Nucleotide sequence analysis of this product identified it as a unique class I gene sequence displaying homology to both primate and human class I A-locus genes. The entire gene was amplified using PCR and the complete DNA sequence information from exon 1 to exon 8, including introns, was determined. A recombination event was identified which results in the fusion of intron 2 with intron 3, causing a deletion of the intervening exon 3 sequence. In addition, there are two cytosine insertions in the poly-cytosine stretch at the start of exon 4 which cause a frameshift and premature termination. The exon 1 and 2 sequences most closely align with the gorilla allele A*0501, displaying only five mismatches. PCR analysis has established that the gene is associated with the following HLA-A types: HLA-A*3001, -A*3301, -A*3303, -A*6802, -A*2901, -A*0203, -A*0205 and -A*31012. Reverse transcription (RT)-PCR analysis of individuals containing this gene failed to detect any mRNA transcription, suggesting that this is a previously undescribed non-expressed class I pseudogene which we have provisionally named HLA-BEL. Its unique gene structure gives a possible insight into the evolutionary pathway that created HLA class I genes.

The relative frequencies of HLA-A*10 alleles in five major United States ethnic populations.

The frequency of each A*10 allele was determined in 5 major United States ethnic populations randomly selected from a pool containing 82,979 unrelated individuals. The phenotype frequency of A10 was 10.5% in Caucasians, 14.0% in African-Americans, 21.1% in Asians/Pacific Islanders, 10.6% in Hispanics, and 9.8% in Native Americans. Fifty-nine individuals who had at least one A10 antigen were randomly chosen from each ethnic group for polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing. Thirteen of sixteen known A10 alleles were identified in this pool. The most common alleles observed were: A*2601 in Caucasians (55%), Hispanics (58%), and Native Americans (45%); A*3402 in African-Americans (34%); and A*3401 in Asians/Pacific Islanders (61%). The African-American and Asian/Pacific Islander populations differ from all other populations in the distribution of A*10 alleles, particularly, A*2601, A*3401, and A*3402.

DNA typing of HLA class II genes (HLA-DR, -DQ and -DP) in Japanese patients with histiocytic necrotizing lymphadenitis (Kikuchi's disease).

The pathogenesis of histiocytic necrotizing lymphadenitis (HNL), which was reported first by Kikuchi et al. and Fujimoto et al. in 1972, is as yet unknown. HNL is frequently reported in Asian countries including Japan, however it is rare in Europe and North America. To elucidate whether the human leukocyte antigen (HLA) alleles and haplotypes are associated with HNL, we performed DNA typing of HLA class II genes (HLA-DR, -DQ, and -DP) in 86 patients with HNL and 525 unrelated healthy Japanese controls with polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP). In this study, we found DPA1*01 and DPB1*0202 allele frequencies in HLA class II genes are significantly higher in HNL patients than in normal controls. It is known that the frequency of DPB1*0202 alleles is extremely low or absent in Caucasians (e.g., French 0.4%, Italian 0.8%) and Negroid (e.g., South African 0%, Hottentot 0%), but relatively frequent in Asians (e.g., Korean 9.9%, Japanese 4.5%). Previous reports have said the incidence of HNL is frequent in Asians but rare in other races. In light of this background, HLA class II genes of HNL and the incidence of HNL in Asian countries, including Japan, might have a positive relationship to DPA1*01 and DPB1*0202 allele.

Allele resolution of HLA-A using oligonucleotide probes in a two-stage typing strategy.

High-resolution polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing methods for HLA-A identification have been established. The four systems, which operate independently of each other, are intended for use as secondary typing systems following HLA-A identification with a medium-resolution PCR-SSOP technique. The systems, all using digoxigenin-labelled probes, are based on group specific amplifications for resolution of: i) HLA-A*29 & -A*33; ii) HLA-A*24 & -A*30; and iii) HLA-A*26, -A*25, -A*11, -A*34, -A*66 and -A*68 alleles, respectively. The fourth system, for the detection of HLA-A*02 alleles, is a modification of a previously reported PCR-SSOP subtyping system. The methods have been applied to individuals from the local bone marrow registry and HLA-A allele frequencies for the Northern Ireland population have been established.

High-resolution sequence typing of HLA-DQA1 and -DQB1 exon 2 DNA with taxonomy-based sequence analysis (TBSA) allele assignment.

High-resolution DNA sequencing of exon 2 of DQA1 and DQB1 genes that uses a taxonomy-based sequence analysis (TBSA) method to assign alleles was developed. The system uses fewer primers for polymerase chain reaction (PCR) amplification and sequencing than other methods and yields accurate DQA1 and DQB1 typing when either homozygous or heterozygous DNA samples are tested. The approach was initially corroborated by the correct typing of 10 blinded samples that had been previously typed by PCR using sequence-specific oligonucleotide probes (PCR-SSOP) or serology, and subsequently confirmed by sequencing of cloned PCR products. DNA from peripheral blood cell samples of 130 individuals enrolled in a case-control analysis of HLA determinants of abdominal aortic aneurysm were subsequently evaluated. Overall, 8 different DQA1 and 19 DQB1 alleles were identified. All 21 DQA1 heterozygous combinations and 45 of 49 DQB1 heterozygous combinations were successfully resolved with TBSA. The two pairs of heterozygous DQB1 combinations that were not unambiguously typed required sequence specific PCR amplification for correct allele identification. We conclude that the method provides precise analysis for HLA-DQ typing.

Molecular characterization of HLA class I in Colombians carrying HLA-A2: high allelic diversity and frequency of heterozygotes at the HLA-B locus.

Polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing was used to analyze HLA class I A, B and C alleles in three different Colombian populations. Fifty-nine samples were from Hispano-American Mestizos living in the urban areas of Cali (referred to here as Aso population). Forty-four and thirty samples were from the African Black populations of Zacarias (Zac) and Punta Soldado (PS), respectively. Samples were selected for expression of HLA-A2 by monoclonal antibody staining and allele-specific hybridization, and their HLA-A2 subtype distribution has been reported previously. Although only a limited number of samples was analyzed, the data suggest the existence of a remarkable degree of HLA class I polymorphism in the populations studied, with representatives of most serological classes. Despite their common African origin, the populations Zac and PS, both resident in malaria endemic regions, showed some striking differences in allelic distribution for all three class I loci. Furthermore, the samples from Aso and PS, but not Zac, showed a low percentage of blank alleles at the HLA-B locus (0 and 0.4%, respectively), suggesting the possibility of a heterozygote advantage for HLA-B alleles in Colombian populations.