Analysis of Ambiguities of HLA-B Typing using the Dynal RELI(TM) SSO HLA-B Test and Development of an`Interpretation Program for Koreans' / 대한진단검사의학회지

BACKGROUND: In recent years, DNA typing has been increasingly used in HLA-A and B typing, and commercial kits based on the PCR-SSO method are most commonly used in Korea. However, SSO typing kits show ambiguities to some extent in the generic level typing of HLA-B alleles. We analyzed the ambiguities in the Dynal RELI(TM) SSO HLA-B test (Dynal B test) with confirmatory typing results, and developed and evaluated the accuracy and efficacy of an `Interpretation Program for Koreans'. METHODS: A total of 2, 169 Korean marrow donor registry samples were typed for HLA-B alleles using the Dynal B test (56 probes) and all of the 222 cases showing ambiguities were subjected to confirmatory typing. We have developed an `Interpretation Program for Koreans' for the Dynal B test on the basis of the allele frequencies of Korean, Japanese and Asian populations. The samples showing ambiguities in the Dynal B test were interpreted using the `Interpretation Program for Koreans' and the results were compared with confirmatory typing results. RESULTS: The Dynal B test showed 10.2% (222/2, 169) of ambiguities and these ambiguities were classified into 47 different band patterns. These ambiguity patterns were interpreted using the `Interpretation Program for Koreans', which showed ambiguities in 14 band patterns and 3.4% (73/2, 169) of the total samples. Among these ambiguities, 4 band patterns (55 samples) arose from those alleles which are not found in Koreans and rarely found in Japanese or Asians (B*1522, *3521, *7802). Thus, excluding these rarities, only less than 1% (18/2, 169) of samples resulted in ambiguities, and most (16/18) of these were B55 vs. B56 ambiguities. The results from the `Interpretation Program for Koreans' were fully concordant with the confirmatory typing results. CONCLUSIONS: The Dynal B test showed around 10% ambiguities and the `Interpretation Program for Koreans' showed 3.4% of ambiguities. Excluding the ambiguities with extremely low probabilities arising from rare alleles in Japanese or Asians, actually >S99% of the samples could be typed accurately using the program without additional confirmatory tests.

HLA-A, -B, -DR Allele Frequencies and Haplotypic Associations in Koreans Defined by Generic-Level DNA Typing / 대한진단검사의학회지

BACKGROUND: HLA allele and haplotype distribution varies widely among different ethnic groups. For organ transplantation, anthropology and disease association studies, reliable data on the HLA distribution in each ethnic group is needed. In recent years, more accurate DNA typing methods are increasingly used in place of the serologic typing method. METHODS: We examined HLA-A, -B, and -DR alleles at the generic (serologic) level in 1, 600 Koreans registered for the Korea Marrow Donor Program (KMDP) using the PCR-sequence specific oligonucleotide (SSO) method (Dynal RELI(TM) kit). Allele and haplotype frequencies were estimated by the maximum likelihood method using the computer program developed for the 11th International Histocompatibility Workshop. RESULTS: HLA alleles found in Koreans were 13 in A, 31 in B, and 13 in DR locus. Most frequent alleles with frequencies > or =10% were: A2, A24, A33, A11; B62, B44, B51; DR4, DR15, DR13, and DR8 in each locus in decreasing order of frequency. Subtype frequencies of B61 and B75 serologic specificities were identified: B*4002 (51.1%), *4003 (7.6%) and *4006 (41.3%) for B61, and B*1502 (9.5%) and *1511 (90.5%) for B75. Two-locus haplotypes with frequencies> or =0.1% were presented (99 A-B, 115 B-DR), among which those with frequencies> or =1.0% showing significant positive linkage disequilibrium (P or =0.1% were identified in Koreans, among which 38 haplotypes showed frequencies> or =0.5%. We compared the results of this study with those of our previous study of serologically typed HLA-A, -B and DNA typed HLA-DR in 2, 000 Koreans. Results from the two studies were similar, but blank frequencies were decreased to 0% for HLA-A, -B, and -DR locus compared with the frequencies of 0.3-0.8% in the previous study (A, 0.3%; B, 0.8%; DR, 0.3%) and all of the serologic splits could be assigned in this study. CONCLUSIONS: In this study, we provided the allele and haplotype frequencies of HLA-A, -B, and -DR in Koreans defined by a DNA typing method, which can be used as basic data on Koreans for organ transplantation and disease association studies.

Analysis of Ambiguities of HLA-DR Typing using the Dynal RELI(TM) SSO HLA-DRB Kit and Development of an 'Interpretation Program for Koreans' / 대한진단검사의학회지

BACKGROUND: HLA-DR typing kits using reverse-SSO (sequence specific oligonucleotide) method show considerable ambiguities in HLA-DRB1 generic typing. We analyzed the ambiguities of the Dynal RELI(TM) SSO HLA-DRB test (Dynal DRB test) and developed an 'Interpretation Program for Koreans'. METHODS: A total of 3,000 Koreans were typed for HLA-DRB1/B3/B4/B5 using the 36 probe Dynal DRB test and all of the cases showing ambiguities in HLA-DRB1 generic typing were subjected to confirmatory typing using the PCR-single strand conformation polymorphism (SSCP) method. On the basis of these results, an 'Interpretation Program for Koreans'was developed for the 45 probe Dynal DRB test. RESULTS: Among 3,000 Koreans tested by the 36 probe Dynal DRB test, 456 cases (15.2%) showed ambiguities. In 95% of the ambiguity cases (433/456) and 99.2% of the total cases tested (433/3,000), the'most probable type'could be expected from the DRB1 gene frequencies and DRB1-B3/B4/B5 associations in Koreans and these results were in accordance with the confirmatory typing results as well as the results given by the 'Interpretation Program for Koreans'. Similarly, the 'Most Probable'could be assigned by the program in 99.4% (348/350) of the cases tested with the 45 probe Dynal DRB test. CONCLUSIONS: Ambiguity in the Dynal DRB test was observed in >15% of the Korean samples tested. The majority (95%) of the ambiguities could be resolved on the basis of HLA-DRB1 gene frequencies and DRB1-B3/B4/B5 associations in Koreans. Furthermore, using the program developed in this study, the correct assignment of DRB1 generic types was possible without additional typing in the majority (>99%) of the cases tested.