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immobilized SSO probe HLA- A test reveals new HLA-A alleles
Abstracts
67
B-2.1 #17
STUDY OF H L A - B 3 9 A L L E L E S I N J A P A N E S E AND T H E D E S C R I P T I O N OF A N O V E L B39 A L L E L E , B ' 3 9 0 4 . A O g a w a x'2, K T o k u n a g a 2, F N a k a j i m a ~ , A K i k u c h i 1, S K a r a k i 1, K K a s h i w a s e 2, T J u j i 2 , a n d M T a k i g u c h i 1, 1Department o f T u m o r Biology, U n i v e r s i t y o f Tokyo, Tokyo, JPN. 2Japanese Red Cross Central Blood Center, Tokyo, JPN. 3Kanagawa Red Cross Blood Center, Y o k o h a m a , JPN. HLA-B39 was found in 7.9% of the Japanese population by serological analysis using 2,650 panel cells, and the antigen was subdivided into three subtypes: 6.4% of B39.1 (B'3901), 0.9% of B39.2 (B'3902) and 0.6% of a novel subtype, B39N. To identify this novel allele, a gene encoding HLA-B39N was cloned and the 7 exons were sequenced. This gene encoding HLA-B39N (B'3904) and the one encording B'39011 differ by two nucleotide substitutions at codons 11 and 12, while B'3904 and B'39013 differ by three nucleotide substitutions at codons 11, 12 and 312. The nucleotide difference at codon 11 produces a change from serine in B'3901 to alanine in B'3904, while another difference at codon 12 changes valine in B'3901 to methionine in B'3904. The residues 11 and 12 are located on a 13-sheet out of the peptide binding floor and completely buried in the molecule. These results suggest that the substitutions at these residues alter the conformation of the B39 molecule resulting in the formation of the cpitope of alloantibodies. Analysis of HLA-B*3901 genes showed that both B'39011 and B'39013 occur in the Japanese population. The present study suggests that B'3904 may have evolved from B'39011 rather than from B'39013.
B-2.1 #18
POPULATION SCREENING WITH A PCR/IMMOBILIZED SSO PROBE HLAA TEST REVEALS NEW HLA-A ALLELES. ~ , R Apple, HA Erlich. Roche Molecular Systems, Inc., Alameda, CA. We have used PCR -DNA amplification and an immobilized SSO probe method (either as dot blot or line blot) to type the HLA-A locus in clinical samples, in population studies, and in 10 th HLA workshop cell lines. This HLA-A typing system uses one locus specific primer pair to amplify a 990 bp fragment of exons 1, 2 and 3, and a set of 50 SSO probes plus a control probe, immobilized on a single nylon strip in a single hybridization reaction. The set of probes can identify 36 homozygous HLA-A alleles; 594 heterozygous types out of 611 (97.2%) possible heterozygous probe patterns can be detected as a unique probe reactivity pattern. This typing system, which takes only 4-5 hours, including sample preparation and genotype interpretation by computer program, has revealed new alleles of A68 and A30.
67
B-2.1 #17
STUDY OF H L A - B 3 9 A L L E L E S I N J A P A N E S E AND T H E D E S C R I P T I O N OF A N O V E L B39 A L L E L E , B ' 3 9 0 4 . A O g a w a x'2, K T o k u n a g a 2, F N a k a j i m a ~ , A K i k u c h i 1, S K a r a k i 1, K K a s h i w a s e 2, T J u j i 2 , a n d M T a k i g u c h i 1, 1Department o f T u m o r Biology, U n i v e r s i t y o f Tokyo, Tokyo, JPN. 2Japanese Red Cross Central Blood Center, Tokyo, JPN. 3Kanagawa Red Cross Blood Center, Y o k o h a m a , JPN. HLA-B39 was found in 7.9% of the Japanese population by serological analysis using 2,650 panel cells, and the antigen was subdivided into three subtypes: 6.4% of B39.1 (B'3901), 0.9% of B39.2 (B'3902) and 0.6% of a novel subtype, B39N. To identify this novel allele, a gene encoding HLA-B39N was cloned and the 7 exons were sequenced. This gene encoding HLA-B39N (B'3904) and the one encording B'39011 differ by two nucleotide substitutions at codons 11 and 12, while B'3904 and B'39013 differ by three nucleotide substitutions at codons 11, 12 and 312. The nucleotide difference at codon 11 produces a change from serine in B'3901 to alanine in B'3904, while another difference at codon 12 changes valine in B'3901 to methionine in B'3904. The residues 11 and 12 are located on a 13-sheet out of the peptide binding floor and completely buried in the molecule. These results suggest that the substitutions at these residues alter the conformation of the B39 molecule resulting in the formation of the cpitope of alloantibodies. Analysis of HLA-B*3901 genes showed that both B'39011 and B'39013 occur in the Japanese population. The present study suggests that B'3904 may have evolved from B'39011 rather than from B'39013.
B-2.1 #18
POPULATION SCREENING WITH A PCR/IMMOBILIZED SSO PROBE HLAA TEST REVEALS NEW HLA-A ALLELES. ~ , R Apple, HA Erlich. Roche Molecular Systems, Inc., Alameda, CA. We have used PCR -DNA amplification and an immobilized SSO probe method (either as dot blot or line blot) to type the HLA-A locus in clinical samples, in population studies, and in 10 th HLA workshop cell lines. This HLA-A typing system uses one locus specific primer pair to amplify a 990 bp fragment of exons 1, 2 and 3, and a set of 50 SSO probes plus a control probe, immobilized on a single nylon strip in a single hybridization reaction. The set of probes can identify 36 homozygous HLA-A alleles; 594 heterozygous types out of 611 (97.2%) possible heterozygous probe patterns can be detected as a unique probe reactivity pattern. This typing system, which takes only 4-5 hours, including sample preparation and genotype interpretation by computer program, has revealed new alleles of A68 and A30.