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Discrepancies between serology and linked RFLP HLA-DR typing
38
Abstracts
#59 5.4
#60 4.2
DISCREPANCIES BETWEEN SEROLOGYAND LINKED RFLP HLA-DR TYPING. PJ S i n n o t t , AJ Ivinson and PA Dyer. NW Regional Tissue Typing Laboratory, HANCHESTER UK. We have compared HLA-DR types obtained by conventional serology w i t h a linked RFLP techniaue in 139 i n d i v i d u a l s . Serology was performed w i t h Class I I Dynabeads and a local c y t o t o x i c t y p i n g t r a y . Linked RFLP t y p i n g was by probing o f Southern b l o t s c a r r y i n g Taq I digested DNA w i t h the HLA-DRB probe pRTV1. The o v e r a l l discrepancy rate was 14.4~. Of these 13/20 involved HLA-DRw6. Serology f a i l e d t o i d e n t i f y the DR 1,Br phenotyl:e in s i x cases. The a l l e l e and gene frequences obtained by both techniques demonstrate an over representation o f DRw6 in serology (26.6~) compared w i t h RFLP t y p i n g ( 2 2 . 2 t ) . Both serology (13.7X) and RFLP (12.2~) revealed s i m i l a r l e v e l s o f homozygosity.
BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF A SEROLOGICALLYUNDEFINED HLA-A ALLELE. ~ , SY Choo and JA Hansen, Fred Hutchinson Cancer Research Center and Department of Medicine, University of Washington, Seattle, WA. Serological typing of an African-Americanfamily was unable to define one HLA-A allele (blank A')C) on cells from a patient (A34,Bw53,DR2; A'X',Bw56,DRw6) with chronic myeicgenous leukemia, her mother (A3,Bw53,DRw6; A'X',Bw56,DRw6), and her brother (Aw68,B7,DR2; A'X',Bw56,DRw6), who share the maternal haplotype A'X',Bw56,DRw6. Isoelectdc focusing studies on immunopracipitatedclass I antigens indicated that the A'X' protein was bound to ~2-microglobulinand expressed on the sudace of EBV-transformedB cells from the three individualsdescribed above. The polyme~asechain reaction (PCR) was used to amplify full-length 1.1kb HLA-A cDNA frag~nts from the mother and brother using primers derived from exon 1 and an HLA-A specific S ~ e n c e of the 3' untranslated region. AmplifiedeDNA productswere cloned and the DNA ~ e n c e of multiple clones determined. Clones encoding for Aw68 (shown to be A'6802) andA3 (A*0301) were identified from the brother and mother respectively. A third unique s o q u e ~ shared by the mother and brother was confirmed in rr,ultipleclones dedved from several i ~ p e ~ n t PCR reactions, and thus assumedto representthe A'X' gene. In general, A'X' has an HLA-A-like sequence, including 138-Met. However,NX' has nine unique amino acid residues in the three extraceliolar domains. Three substitutions are located at positions 31, 35, and 255 where all other HLA alleles have conserved amino acid residues. Six new amino acid substitutions are found at polymorphiopositions 56, 74,152, 163, 253, and 268. The unusually large number of allelespecific residues in A'X' may cause the lack of reactivity of this ~lecule with conventional typing antisera. The large degree of new polyrnorphismfound in the A'X' allele is evidence that there might exist more divereity in non-Caucasoidpopulations, which is currently not identifiedbecause of the lack of informativeseroiogicalreagents. It is likely that these alleles would be functional, thus molacu~ar characterization of these new alleles and future development of a molecular typing strategy will facilitate precise HLA matching for transplantation.
Abstracts
#59 5.4
#60 4.2
DISCREPANCIES BETWEEN SEROLOGYAND LINKED RFLP HLA-DR TYPING. PJ S i n n o t t , AJ Ivinson and PA Dyer. NW Regional Tissue Typing Laboratory, HANCHESTER UK. We have compared HLA-DR types obtained by conventional serology w i t h a linked RFLP techniaue in 139 i n d i v i d u a l s . Serology was performed w i t h Class I I Dynabeads and a local c y t o t o x i c t y p i n g t r a y . Linked RFLP t y p i n g was by probing o f Southern b l o t s c a r r y i n g Taq I digested DNA w i t h the HLA-DRB probe pRTV1. The o v e r a l l discrepancy rate was 14.4~. Of these 13/20 involved HLA-DRw6. Serology f a i l e d t o i d e n t i f y the DR 1,Br phenotyl:e in s i x cases. The a l l e l e and gene frequences obtained by both techniques demonstrate an over representation o f DRw6 in serology (26.6~) compared w i t h RFLP t y p i n g ( 2 2 . 2 t ) . Both serology (13.7X) and RFLP (12.2~) revealed s i m i l a r l e v e l s o f homozygosity.
BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF A SEROLOGICALLYUNDEFINED HLA-A ALLELE. ~ , SY Choo and JA Hansen, Fred Hutchinson Cancer Research Center and Department of Medicine, University of Washington, Seattle, WA. Serological typing of an African-Americanfamily was unable to define one HLA-A allele (blank A')C) on cells from a patient (A34,Bw53,DR2; A'X',Bw56,DRw6) with chronic myeicgenous leukemia, her mother (A3,Bw53,DRw6; A'X',Bw56,DRw6), and her brother (Aw68,B7,DR2; A'X',Bw56,DRw6), who share the maternal haplotype A'X',Bw56,DRw6. Isoelectdc focusing studies on immunopracipitatedclass I antigens indicated that the A'X' protein was bound to ~2-microglobulinand expressed on the sudace of EBV-transformedB cells from the three individualsdescribed above. The polyme~asechain reaction (PCR) was used to amplify full-length 1.1kb HLA-A cDNA frag~nts from the mother and brother using primers derived from exon 1 and an HLA-A specific S ~ e n c e of the 3' untranslated region. AmplifiedeDNA productswere cloned and the DNA ~ e n c e of multiple clones determined. Clones encoding for Aw68 (shown to be A'6802) andA3 (A*0301) were identified from the brother and mother respectively. A third unique s o q u e ~ shared by the mother and brother was confirmed in rr,ultipleclones dedved from several i ~ p e ~ n t PCR reactions, and thus assumedto representthe A'X' gene. In general, A'X' has an HLA-A-like sequence, including 138-Met. However,NX' has nine unique amino acid residues in the three extraceliolar domains. Three substitutions are located at positions 31, 35, and 255 where all other HLA alleles have conserved amino acid residues. Six new amino acid substitutions are found at polymorphiopositions 56, 74,152, 163, 253, and 268. The unusually large number of allelespecific residues in A'X' may cause the lack of reactivity of this ~lecule with conventional typing antisera. The large degree of new polyrnorphismfound in the A'X' allele is evidence that there might exist more divereity in non-Caucasoidpopulations, which is currently not identifiedbecause of the lack of informativeseroiogicalreagents. It is likely that these alleles would be functional, thus molacu~ar characterization of these new alleles and future development of a molecular typing strategy will facilitate precise HLA matching for transplantation.