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HLA typing of class I alleles using PCR and hybridization with oligonucleotides: HLA-B27 and an African B27 subtype
Abstracts
15 HLA TYPING OF CLASS I ALLELES USING PCR AND HYBRIDIZATION WITH OLIGONUCLEOTIDES: HLA-B27 AND AN AFRICAN B27 SUBTYPE. AVS Hill, CEM Allsopp, D K w l a t k o w s k l , BM Greenwood*, AJMcMichael, I n s t i t u t e of M o l e c u l a r M e d i c i n e , O x f o r d , UK; M R C L a b o r a t o r i e s , F a j a r a , T h e G a m b i a . Oligonucleotide hybridization following PCR amplification offers the possibility of H L A c l a s s I typing with increased precision using very small samples. E x o n 2 ( e n c o d i n g t h e ul d o m a i n ) of H L A c l a s s I a l l e l e s was amplified using class I specific primers. An 18-met oligonucleotide probe spanning nucleotides encoding amino a c i d s 6 6 - 7 1 of t h i s d o m a i n d e t e c t e d H L A - B 2 7 b u t n o n e of 42 o t h e r class I alleles tested. An 18-mer probe spanning nucleotides encoding amino acids 57-61 (with h i s t i d i n e at p o s i t i o n 59) d e t e c t e d t h e H L A - B * 2 7 0 3 s u b t y p e in ii of 18 H L A - B 2 7 p o s i t i v e West African (Gambian) individuals. Hence, HLA-B*2703 which was first reported in an A m e r i c a n B l a c k a n d a p p e a r s to b e e x t r e m e l y r a r e or a b s e n t in C a u c a s i a n s a n d O r i e n t a l s is c o m m o n a m o n g s t H L A B27 p o s i t i v e i n d i v i d u a l s in W e s t A f r i c a . More complex strategies will be required for other a l l e l e s c o m p o s e d of " p a t c h w o r k " s e q u e n c e s . For example, o u r s e q u e n c e a n a l y s i s of H L A - B w 5 3 s h o w s t h a t it d i f f e r s f r o m H L A - B 3 5 o n l y at t h e s e q u e n c e e n c o d i n g t h e B w 4 / B w 6 epitope. However, initial experience with allele-group s p e c i f i c a m p l i f i c a t i o n to t y p e t h e s e t w o c o m m o n G a m b i a n a l l e l e s s u p p o r t s t h e b e l i e f t h a t t h i s a p p r o a c h s h o u l d be e x t e n d a b l e to a l l o w a c o m p l e t e P e R - b a s e d t y p i n g s y s t e m for c l a s s I a l l e l e s .
MOLECULAR ANALYSIS OF A HLA-A ALLELE SPECIFIC EXPRESSION DEFECT SEGREGATING IN A HEALTHY DUTCH FAMILY. N.M. Lardy, A.R. van der Horst, R.E. Bontrop* and L.P. de Waal. Central Lab. Netherl. Red Cross Blood Transfusion Service and the Lab. of Exp. and Clin. Immunol. of the University of Amsterdam, Amsterdam, *TNO, Rijswijk, The Netherlands HLA typing of a healthy Dutch individual in the standard NIH microcytotoxicity test failed to detect a second HLA-A locus antigen. Family analysis demonstrated that the absence of a second HLA-A allele was not due to homozygosity and that the HLA-A "blanc" allele segregated as a normal HLA-A locus antigen. The haplotype was: HLA-A "blanc", B8, Cw7, DR3, DRw52, DQw2. Precipitation studies were performed using monoclonal antibodies directed against the heavy chain of HLA class I antigens associated with beta-2-microglobulin and against the free heavy chain of HLA class I antigens. The experiments failed to detect a HLA-A "blanc" product. Mitogen stimulation, EBV transformation or treatment with r-IFN-gamma and r-TNF-alpha did not result in the expression of a second HLA-A antigen. Pedigree analysis suggested a segregation distortion in that more family members than expected inherited the HLA-A "blanc" allele. To our knowledge this is the first description of a HLA allele specific expression defect segregating in a healthy family.
15 HLA TYPING OF CLASS I ALLELES USING PCR AND HYBRIDIZATION WITH OLIGONUCLEOTIDES: HLA-B27 AND AN AFRICAN B27 SUBTYPE. AVS Hill, CEM Allsopp, D K w l a t k o w s k l , BM Greenwood*, AJMcMichael, I n s t i t u t e of M o l e c u l a r M e d i c i n e , O x f o r d , UK; M R C L a b o r a t o r i e s , F a j a r a , T h e G a m b i a . Oligonucleotide hybridization following PCR amplification offers the possibility of H L A c l a s s I typing with increased precision using very small samples. E x o n 2 ( e n c o d i n g t h e ul d o m a i n ) of H L A c l a s s I a l l e l e s was amplified using class I specific primers. An 18-met oligonucleotide probe spanning nucleotides encoding amino a c i d s 6 6 - 7 1 of t h i s d o m a i n d e t e c t e d H L A - B 2 7 b u t n o n e of 42 o t h e r class I alleles tested. An 18-mer probe spanning nucleotides encoding amino acids 57-61 (with h i s t i d i n e at p o s i t i o n 59) d e t e c t e d t h e H L A - B * 2 7 0 3 s u b t y p e in ii of 18 H L A - B 2 7 p o s i t i v e West African (Gambian) individuals. Hence, HLA-B*2703 which was first reported in an A m e r i c a n B l a c k a n d a p p e a r s to b e e x t r e m e l y r a r e or a b s e n t in C a u c a s i a n s a n d O r i e n t a l s is c o m m o n a m o n g s t H L A B27 p o s i t i v e i n d i v i d u a l s in W e s t A f r i c a . More complex strategies will be required for other a l l e l e s c o m p o s e d of " p a t c h w o r k " s e q u e n c e s . For example, o u r s e q u e n c e a n a l y s i s of H L A - B w 5 3 s h o w s t h a t it d i f f e r s f r o m H L A - B 3 5 o n l y at t h e s e q u e n c e e n c o d i n g t h e B w 4 / B w 6 epitope. However, initial experience with allele-group s p e c i f i c a m p l i f i c a t i o n to t y p e t h e s e t w o c o m m o n G a m b i a n a l l e l e s s u p p o r t s t h e b e l i e f t h a t t h i s a p p r o a c h s h o u l d be e x t e n d a b l e to a l l o w a c o m p l e t e P e R - b a s e d t y p i n g s y s t e m for c l a s s I a l l e l e s .
MOLECULAR ANALYSIS OF A HLA-A ALLELE SPECIFIC EXPRESSION DEFECT SEGREGATING IN A HEALTHY DUTCH FAMILY. N.M. Lardy, A.R. van der Horst, R.E. Bontrop* and L.P. de Waal. Central Lab. Netherl. Red Cross Blood Transfusion Service and the Lab. of Exp. and Clin. Immunol. of the University of Amsterdam, Amsterdam, *TNO, Rijswijk, The Netherlands HLA typing of a healthy Dutch individual in the standard NIH microcytotoxicity test failed to detect a second HLA-A locus antigen. Family analysis demonstrated that the absence of a second HLA-A allele was not due to homozygosity and that the HLA-A "blanc" allele segregated as a normal HLA-A locus antigen. The haplotype was: HLA-A "blanc", B8, Cw7, DR3, DRw52, DQw2. Precipitation studies were performed using monoclonal antibodies directed against the heavy chain of HLA class I antigens associated with beta-2-microglobulin and against the free heavy chain of HLA class I antigens. The experiments failed to detect a HLA-A "blanc" product. Mitogen stimulation, EBV transformation or treatment with r-IFN-gamma and r-TNF-alpha did not result in the expression of a second HLA-A antigen. Pedigree analysis suggested a segregation distortion in that more family members than expected inherited the HLA-A "blanc" allele. To our knowledge this is the first description of a HLA allele specific expression defect segregating in a healthy family.