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A new DRB1 allele (DRB1∗1311) identified by PCR-SSP and family study
Abstracts
139 A B S T R A C T S SELECTED AS POSTERS: A B S T R A C T S 76--144 Abstracts 76-78 N e w alleles and n e w genes
76
77
HLA-A*8001 IS A MEMBER OF A NEW FAMILY OF HLA-A ALLELES A. Wagner I, A. Hughes 2, M. Iandoli I, D. Stewart 3, S. Herbert 3, D. Watkins 4, C. Hurley I, S. Rosen-Bronson I ~Departments of Pediatrics and Microbiology, Georgetown University Medical Center, Washington, D.C., aDepartment of Biology~ The Pennsylvania State University, University Park, PA, ~Department of Transplantation, Ochsner Transplant Center, New Orleans, LA, 4Department of Pathology and Wisconsin Regional Primate Research Center, University o f Wisconsin-Madison, Madison, WI, USA A member of a new HLA-A family, HLA-A*8001, has been characterized from 3 African-American individuals expressing a u n i q u e HLA-A serologic specificity. The H L A A*8001 sequence i s most closely related to the alleles of the HLA-AI/3/ll family, although it also contains residues characteristic of the H L A - A 2 / 2 8 , -Ag, -AIO, and -A19 families. The HLA-A*8001 sequence contains unique nonsynonymous nucleotide substitutions absent from a l l other primate A locus alleles, as well as several nucleotide substitutions observed only in non-human primate A locus a l l e l e s . Neighbor-joining tree analysis
A NEWDRB1 ALLELE (DRB1*1311) IDENTIFIED BY PCR-SSP AND FAMILY STUDy
of HLA-A*8001 allele is a
supports
the
notion
that
the
HLA-A*8001
member of a new HLA-A locus family which was derived from the ancestral A3 lineage but diverged early in
the
evolution
of
the
HLA-A
locus.
78 A PERFECT TANDEM DUPLICATION 1N THE TNF BETA PROMOTER REGION IS A MARKER OF AN HLA-B60 HAPLOTYPE. S. D'Alfonso 1, A.M. Dall'Omo 2, L. Pratic62, I. Borelli 3, M. Berrino 2, P. Momigliano Richiardi 1. 1Dip. Scienze Mediche, Novara, Universita' Torino. 2Servizio lmunologia dei Trapianti, Ospedale Molinette, USSL 8 Torino 3Dip. Genetica Biologia e Chimica Medica, Universita' Torino, Italy The region between TNF and B loci is polymorphie and characterized by duplications which are retained within the same extended HLA haplotype. Moreover Alu and Met repeat sequences have been found telomerically to TNF beta gene. We describe here a perfect tandem duplication of a 145bp sequence in the TNF beta promoter region, newly detected in 2/20 XW homozygous cell lines. The molecular mechanism at the basis of this duplication is not clear. The sequences flanking the duplicated fragment show only a moderate homology. The duplicated sequence does not show any signifieative homology with sequences reported in data banks. The 2 cell lines with the duplication share the HLA-B60 allele. The duplicated fragment was never found in a panel of 88 random individuals, whereas 5 out of 39 HLA-B60 positive individuals showed exactly the same duplication indicating that this polymorphism is a conserved marker of a particular haplotype carrying HLA-B60. The duplication is stably inherited and segregates as a Mendelian character. The 7 haplotypes carrying the duplication are conserved from TNF alpha (eentromefically) to HLA-C (telomerically) loci and characterized by the following markers: HLA-Cw3, B60, TNFa2, TNFB*2, TNFc2, TNFA-G. On the contrary they do not share the same allele at HLA-DR, HSP70-1 and HLA-A loci. Notably none of these haplotypes carries the HLA-DR4 allele typically associated with B60 in Caucasoids. The characterization of the associated complotype is in progress.
P. Cracco, D. Becuwe, V. Lemaitre ~ , J.J. Huart, F. Dufesse. L a b o r a t o i r e d'Histocompatibilite, C.~.T.S., Zl rue C. Gu~ri~, ~ 0 Lille, France ; °Service de Nephrologie, CHU Valenciennes, France Up to now more than 60 HLA DRBI alleles have been characterJsed. Here we report a new DRBI allele (DRBI*I311) identified in a c a u c a s o i d renal patient. Using PCR-SSP assay, the patient DNA appears to be : DRBI*01 homozygous; DQBI*05, DQBI*0301. Indirect positive control consisting in specific amplification of the second DRB gene showed two PCR products in DRB3 and DRB6 specific amplification. This result suggests the presence of a new allele in the DR52 associated DRBI group. Additional techniques using modified ~CR-RFL? combined with group specific amplification of D R 5 2 - a s s o c i a t e d DRBI alleles were performed. The patterns observed were different frDm those expected for the known alleles. The analyses of the sequencing data show that the sequence is identical to DRBI*II041 with the exception of coden 58 "GCC" which is s u b s t i t u t e d for "GAG". The family study was performed among patient siblings and p a t i e n t children in order to characterise the haplotype : A1 B37 DRBI*I3!I DRB3*0202 DQBI*0301 DQAI*050i DPBI+I501. Our primers as well as Olerup's ones failed to amplify this allele. As a consequence, we report a new pair of primers which s p e c i f i c a l l y amplifies the following group of alleles : DRBI*I202 + 1103 + 1104 + 13ii. This finding emphasizes the impcrtance of PCR SSP control consisting in specific amplification of a second DRB gene and DQBI allele. The observation of linkage disequilibrium between DRBI, second DRB gone and DQBI has allowed us to described this novel allele.
139 A B S T R A C T S SELECTED AS POSTERS: A B S T R A C T S 76--144 Abstracts 76-78 N e w alleles and n e w genes
76
77
HLA-A*8001 IS A MEMBER OF A NEW FAMILY OF HLA-A ALLELES A. Wagner I, A. Hughes 2, M. Iandoli I, D. Stewart 3, S. Herbert 3, D. Watkins 4, C. Hurley I, S. Rosen-Bronson I ~Departments of Pediatrics and Microbiology, Georgetown University Medical Center, Washington, D.C., aDepartment of Biology~ The Pennsylvania State University, University Park, PA, ~Department of Transplantation, Ochsner Transplant Center, New Orleans, LA, 4Department of Pathology and Wisconsin Regional Primate Research Center, University o f Wisconsin-Madison, Madison, WI, USA A member of a new HLA-A family, HLA-A*8001, has been characterized from 3 African-American individuals expressing a u n i q u e HLA-A serologic specificity. The H L A A*8001 sequence i s most closely related to the alleles of the HLA-AI/3/ll family, although it also contains residues characteristic of the H L A - A 2 / 2 8 , -Ag, -AIO, and -A19 families. The HLA-A*8001 sequence contains unique nonsynonymous nucleotide substitutions absent from a l l other primate A locus alleles, as well as several nucleotide substitutions observed only in non-human primate A locus a l l e l e s . Neighbor-joining tree analysis
A NEWDRB1 ALLELE (DRB1*1311) IDENTIFIED BY PCR-SSP AND FAMILY STUDy
of HLA-A*8001 allele is a
supports
the
notion
that
the
HLA-A*8001
member of a new HLA-A locus family which was derived from the ancestral A3 lineage but diverged early in
the
evolution
of
the
HLA-A
locus.
78 A PERFECT TANDEM DUPLICATION 1N THE TNF BETA PROMOTER REGION IS A MARKER OF AN HLA-B60 HAPLOTYPE. S. D'Alfonso 1, A.M. Dall'Omo 2, L. Pratic62, I. Borelli 3, M. Berrino 2, P. Momigliano Richiardi 1. 1Dip. Scienze Mediche, Novara, Universita' Torino. 2Servizio lmunologia dei Trapianti, Ospedale Molinette, USSL 8 Torino 3Dip. Genetica Biologia e Chimica Medica, Universita' Torino, Italy The region between TNF and B loci is polymorphie and characterized by duplications which are retained within the same extended HLA haplotype. Moreover Alu and Met repeat sequences have been found telomerically to TNF beta gene. We describe here a perfect tandem duplication of a 145bp sequence in the TNF beta promoter region, newly detected in 2/20 XW homozygous cell lines. The molecular mechanism at the basis of this duplication is not clear. The sequences flanking the duplicated fragment show only a moderate homology. The duplicated sequence does not show any signifieative homology with sequences reported in data banks. The 2 cell lines with the duplication share the HLA-B60 allele. The duplicated fragment was never found in a panel of 88 random individuals, whereas 5 out of 39 HLA-B60 positive individuals showed exactly the same duplication indicating that this polymorphism is a conserved marker of a particular haplotype carrying HLA-B60. The duplication is stably inherited and segregates as a Mendelian character. The 7 haplotypes carrying the duplication are conserved from TNF alpha (eentromefically) to HLA-C (telomerically) loci and characterized by the following markers: HLA-Cw3, B60, TNFa2, TNFB*2, TNFc2, TNFA-G. On the contrary they do not share the same allele at HLA-DR, HSP70-1 and HLA-A loci. Notably none of these haplotypes carries the HLA-DR4 allele typically associated with B60 in Caucasoids. The characterization of the associated complotype is in progress.
P. Cracco, D. Becuwe, V. Lemaitre ~ , J.J. Huart, F. Dufesse. L a b o r a t o i r e d'Histocompatibilite, C.~.T.S., Zl rue C. Gu~ri~, ~ 0 Lille, France ; °Service de Nephrologie, CHU Valenciennes, France Up to now more than 60 HLA DRBI alleles have been characterJsed. Here we report a new DRBI allele (DRBI*I311) identified in a c a u c a s o i d renal patient. Using PCR-SSP assay, the patient DNA appears to be : DRBI*01 homozygous; DQBI*05, DQBI*0301. Indirect positive control consisting in specific amplification of the second DRB gene showed two PCR products in DRB3 and DRB6 specific amplification. This result suggests the presence of a new allele in the DR52 associated DRBI group. Additional techniques using modified ~CR-RFL? combined with group specific amplification of D R 5 2 - a s s o c i a t e d DRBI alleles were performed. The patterns observed were different frDm those expected for the known alleles. The analyses of the sequencing data show that the sequence is identical to DRBI*II041 with the exception of coden 58 "GCC" which is s u b s t i t u t e d for "GAG". The family study was performed among patient siblings and p a t i e n t children in order to characterise the haplotype : A1 B37 DRBI*I3!I DRB3*0202 DQBI*0301 DQAI*050i DPBI+I501. Our primers as well as Olerup's ones failed to amplify this allele. As a consequence, we report a new pair of primers which s p e c i f i c a l l y amplifies the following group of alleles : DRBI*I202 + 1103 + 1104 + 13ii. This finding emphasizes the impcrtance of PCR SSP control consisting in specific amplification of a second DRB gene and DQBI allele. The observation of linkage disequilibrium between DRBI, second DRB gone and DQBI has allowed us to described this novel allele.