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Evolutionary pathways of DR4 haplotypes are marked by DQA1∗03 variants: DQA1∗0301 and DQA1∗0302
Abstracts
15
B 2.2.17
DNA SEQUENCING OF A NEW HLA-A ALLELE USING LOCUS SPECIFIC PCR AMPLIFICATION S Rosen-Bronson, AG Wagner, D Stewart, S Herbert, CK Hurley, Georgetown University School of Medicine, Washington DC, and The Ochsner Foundation Hospital, New Orleans, LA. Human populations express a diverse repertoire of HLA alleles. While class II alleles have become increasingly well characterized, a tally of the number and extent of polymorphism of the class I alleles has not been fully explored. We are studying an African American family which appears express a new HLA-A allele based on serologic data. The goal of our study was to sequence this new HLA-A allele. Total cellular RNA was isolated from B-LCL derived from family members who appear to express the new allele, cDNA was ~ynthesized from the RNA using an oligo(dT) primer and was subsequently amplified using HLA class I specific primers. In order to rapidly isolate and identify the alleles encoded by the individual class I loci, we have designed three sets of PCR primers. The HLA-A specific forward primer includes a unique GCC sequence located at the 5' end of all HLA-A alleles. A generic class I reverse primer (Ennis et al., 1990) was used together with our HLA-A specific forward primer to amplify the HLA-A alleles. The HLA-B locus alleles were amplified using the generic class I forward primer described by Ennis et al. and an HLA-B locus reverse primer which incorporates an HLA-B locus specific C located at position 972. Similarly, the HLA-C locus alleles were amplified using the generic class I forward primer and an HLA-C locus primer which incorporates the HLA-C locus specific G located at position 1034. The HLA-A, -B, and -C specific primers amplify products which are approximately i.i, 0.9, and I kb respectively. The size differences between the PCR products from each class I locus was used to verify the locus specific amplifications. The amplified products from the HLA-A locus specific PCR have been cloned into the Ml3mpl8 vector and are currently being sequenced.
B 2.3.18
EVOLUTIONARY P A T H W A Y S O F DR4 H A P L O T Y P E S ARE M A R K E D BY D Q A I * 0 3 VARIANTS: D Q A I * 0 3 0 1 A N D DQAI*0302. M A F e r n a n d e z - V i n a , M Falco, M Cerna, Y Sun, E Raimondi and P Stastny, Department of Internal Medicine, UT S o u t h w e s t e r n M e d i c a l Center, Dallas, Texas.
Two subtypes of DQAI*03 differing at residue 160 encoded in exon-3 have b e e n d e s c r i b e d and t h e s e could have distinctive associations with alleles at other loci. We have performed complete HLA-Class II typing of 84 homozygous B cell lines and 960 healthy individuals from 11 different ethnic groups and amplification of exon-3 of DQA1 followed by hybridization with SSOP to define DQAI*0301 and 0302. The haplotypes found in unrelated individuals werez DQAI*0301 was seen only in association with DQBI*0302, and s a m p l e s carrying DQBI*0201, 0301, 0303 and 0401 were always positive for DQAI*0302. Few haplotypes carrying DQBI*0302 had DQAI*0302. The fact that DQAI*0301 is completely included in DQBI*0302 and not vice v e r s a s u g g e s t s that DQAI*0301 may have arisen from a mutation in DQAI*0302 in a haplotype carrying DQBI*0302. Since DQBI*0302 is almost exclusively found in association with DR4 and with almost all DR4 subtypes, the current variants of DRBI of DR4 may be more recent than the mutation in DQAI*0302. One branch of DR4 haplotypes could contain DRBI*0405 separated from the remaining DR4 subtypes; this is e x c l u s i v e l y associated with DQAI*0302 and with multiple DQB1. The o t h e r DR4 subtypes carrying DQAI*0301 may have originated from e i t h e r DRBI*0401 or DRBI*0404. Study of microvariations in regions not subject to hypervariation may shed light on the evolution of HLA-D region haplotypes and help mark yet undetected genes involved in d i s e a s e susceptibiliy or resistance.
15
B 2.2.17
DNA SEQUENCING OF A NEW HLA-A ALLELE USING LOCUS SPECIFIC PCR AMPLIFICATION S Rosen-Bronson, AG Wagner, D Stewart, S Herbert, CK Hurley, Georgetown University School of Medicine, Washington DC, and The Ochsner Foundation Hospital, New Orleans, LA. Human populations express a diverse repertoire of HLA alleles. While class II alleles have become increasingly well characterized, a tally of the number and extent of polymorphism of the class I alleles has not been fully explored. We are studying an African American family which appears express a new HLA-A allele based on serologic data. The goal of our study was to sequence this new HLA-A allele. Total cellular RNA was isolated from B-LCL derived from family members who appear to express the new allele, cDNA was ~ynthesized from the RNA using an oligo(dT) primer and was subsequently amplified using HLA class I specific primers. In order to rapidly isolate and identify the alleles encoded by the individual class I loci, we have designed three sets of PCR primers. The HLA-A specific forward primer includes a unique GCC sequence located at the 5' end of all HLA-A alleles. A generic class I reverse primer (Ennis et al., 1990) was used together with our HLA-A specific forward primer to amplify the HLA-A alleles. The HLA-B locus alleles were amplified using the generic class I forward primer described by Ennis et al. and an HLA-B locus reverse primer which incorporates an HLA-B locus specific C located at position 972. Similarly, the HLA-C locus alleles were amplified using the generic class I forward primer and an HLA-C locus primer which incorporates the HLA-C locus specific G located at position 1034. The HLA-A, -B, and -C specific primers amplify products which are approximately i.i, 0.9, and I kb respectively. The size differences between the PCR products from each class I locus was used to verify the locus specific amplifications. The amplified products from the HLA-A locus specific PCR have been cloned into the Ml3mpl8 vector and are currently being sequenced.
B 2.3.18
EVOLUTIONARY P A T H W A Y S O F DR4 H A P L O T Y P E S ARE M A R K E D BY D Q A I * 0 3 VARIANTS: D Q A I * 0 3 0 1 A N D DQAI*0302. M A F e r n a n d e z - V i n a , M Falco, M Cerna, Y Sun, E Raimondi and P Stastny, Department of Internal Medicine, UT S o u t h w e s t e r n M e d i c a l Center, Dallas, Texas.
Two subtypes of DQAI*03 differing at residue 160 encoded in exon-3 have b e e n d e s c r i b e d and t h e s e could have distinctive associations with alleles at other loci. We have performed complete HLA-Class II typing of 84 homozygous B cell lines and 960 healthy individuals from 11 different ethnic groups and amplification of exon-3 of DQA1 followed by hybridization with SSOP to define DQAI*0301 and 0302. The haplotypes found in unrelated individuals werez DQAI*0301 was seen only in association with DQBI*0302, and s a m p l e s carrying DQBI*0201, 0301, 0303 and 0401 were always positive for DQAI*0302. Few haplotypes carrying DQBI*0302 had DQAI*0302. The fact that DQAI*0301 is completely included in DQBI*0302 and not vice v e r s a s u g g e s t s that DQAI*0301 may have arisen from a mutation in DQAI*0302 in a haplotype carrying DQBI*0302. Since DQBI*0302 is almost exclusively found in association with DR4 and with almost all DR4 subtypes, the current variants of DRBI of DR4 may be more recent than the mutation in DQAI*0302. One branch of DR4 haplotypes could contain DRBI*0405 separated from the remaining DR4 subtypes; this is e x c l u s i v e l y associated with DQAI*0302 and with multiple DQB1. The o t h e r DR4 subtypes carrying DQAI*0301 may have originated from e i t h e r DRBI*0401 or DRBI*0404. Study of microvariations in regions not subject to hypervariation may shed light on the evolution of HLA-D region haplotypes and help mark yet undetected genes involved in d i s e a s e susceptibiliy or resistance.