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88-P: Novel HLA-Cw*08 allele identified
Abstracts
S51
88-P
NOVEL HLA-CW*08 ALLELE IDENTIFIED. H. Redman,1 M. Marlowe,1 A. Hoffman,1 R. Medis,1 P. Hanavan,2 D.F. Lake,2 R.O. Endres.1 1HLA, Blood Systems Laboratories, Tempe, AZ, USA; 2Biodesign Institute, Arizona State University, Tempe, AZ, USA. Aim: Genomic DNA was tested in our laboratory using Sequence Based Typing (SBT), with Atria AlleleSEQR reagents and Conexio Assign analysis software. Results from this testing indicated Cw *0401 and Cw*0802 alleles with a single nucleotide mismatch in exon 2, base pair 205 (codon 69). The base pair change for one of the Cw alleles at this position was C to T. Methods: A sequencing reaction was set up with a Heterozygous Ambiguity Resolution Primer (HARP), using the initial HLA Cw Locus PCR product and HARP A2F98A. This primer was selected to amplify only the Cw*08 allele. Subsequently, a Qiagen HaploPrep probe (Cw312C) was used to separate the Cw*08 variant from the Cw*04 allele. This HaploPrep DNA sample was tested with Cw locus SBT reagents. Results: Results from the SBT HARP reaction provided evidence that the nucleotide substitution was located on the Cw*08 allele, with a single mismatch at base pair 205 (T nucleotide). This information was beneficial in selecting a proper HaploPrep probe. The SBT data from the HaploPrep DNA reaction confirmed that the nucleotide substitution in exon 2 of this DNA sample could be attributed to the Cw*08 allele. Sequencing of the B locus with the HaploPrep DNA suggests a HLA class I haplotype of A*0201, B*1402, Cw*08 variant (sample is homozygous for A*0201 allele). Conclusions: The nucleotide substitution, from C to T, at base pair 205 (codon 69) for this novel Cw*08 variant allele, changes the amino acid at this location from an arginine (R) to a cysteine (C) residue. Codon 69 is located on the top, center of the upper alpha helix, next to the binding groove of the HLA C molecule. The consequences of this change could strongly affect antibody and T cell receptor recognition, as well as peptide binding.
89-P
MANNOSE BINDING LECTIN 2 POLYMORPHISMS IN TRANSPLANT PATIENTS. Barbara Sotolongo, Brianna Ruiz, Manuel Carreno, Tzakis G. Andreas, Phillip Ruiz. Surgery, Transplant Laboratories, Leonard M. Miller School of Medicine, Miami, FL, USA; Pathology, Leonard M. Miller School of Medicine, Miami, FL, USA; Surgery, Division of Transplantation, Leonard M. Miller School of Medicine, Miami, FL, USA. Aim: The MBL2 gene encodes the mannose binding lectin (MBL) serum protein. MBL has a major role in innate immunity by activating the lectin complement pathway. In our study, we concentrated on genotyping six polymorphisms in the MBL2 gene that result in significant variations in the levels of the MBL protein of transplant patients. Low MBL protein levels can predispose transplant patients to recurrent infections and possible future graft rejection. Methods: Applied Biosystems’ Taqman SNP genotyping assays were used on whole blood to differentiate homozygotic from heterozygotic alleles (MBL2 SNPs). The MBL elisa assay for quantitative measurement of MBL in serum was used. Results: SNP genotyping assays were initially done on 100 transplant patients with a distribution of 59 kidney, 24 liver, and 18 other multiple GI transplant patients and MBL protein elisa measurements were also taken. The results indicate that multiple combinations of these six SNP genotypes result in reduced MBL protein levels and this could be a promising early marker of infection and graft rejection episodes in transplant patients. Conclusions: Transplant patients with these SNP mutations exhibited genotypes that estimate low MBL protein production in about 10% of the cases. The SNP assay in combination with the elisa will elucidate this connection to infectious disease states. We are in the process of determining the relationship between distribution of the MBL2 SNP genotypes, protein levels and infections in our transplant population.
S51
88-P
NOVEL HLA-CW*08 ALLELE IDENTIFIED. H. Redman,1 M. Marlowe,1 A. Hoffman,1 R. Medis,1 P. Hanavan,2 D.F. Lake,2 R.O. Endres.1 1HLA, Blood Systems Laboratories, Tempe, AZ, USA; 2Biodesign Institute, Arizona State University, Tempe, AZ, USA. Aim: Genomic DNA was tested in our laboratory using Sequence Based Typing (SBT), with Atria AlleleSEQR reagents and Conexio Assign analysis software. Results from this testing indicated Cw *0401 and Cw*0802 alleles with a single nucleotide mismatch in exon 2, base pair 205 (codon 69). The base pair change for one of the Cw alleles at this position was C to T. Methods: A sequencing reaction was set up with a Heterozygous Ambiguity Resolution Primer (HARP), using the initial HLA Cw Locus PCR product and HARP A2F98A. This primer was selected to amplify only the Cw*08 allele. Subsequently, a Qiagen HaploPrep probe (Cw312C) was used to separate the Cw*08 variant from the Cw*04 allele. This HaploPrep DNA sample was tested with Cw locus SBT reagents. Results: Results from the SBT HARP reaction provided evidence that the nucleotide substitution was located on the Cw*08 allele, with a single mismatch at base pair 205 (T nucleotide). This information was beneficial in selecting a proper HaploPrep probe. The SBT data from the HaploPrep DNA reaction confirmed that the nucleotide substitution in exon 2 of this DNA sample could be attributed to the Cw*08 allele. Sequencing of the B locus with the HaploPrep DNA suggests a HLA class I haplotype of A*0201, B*1402, Cw*08 variant (sample is homozygous for A*0201 allele). Conclusions: The nucleotide substitution, from C to T, at base pair 205 (codon 69) for this novel Cw*08 variant allele, changes the amino acid at this location from an arginine (R) to a cysteine (C) residue. Codon 69 is located on the top, center of the upper alpha helix, next to the binding groove of the HLA C molecule. The consequences of this change could strongly affect antibody and T cell receptor recognition, as well as peptide binding.
89-P
MANNOSE BINDING LECTIN 2 POLYMORPHISMS IN TRANSPLANT PATIENTS. Barbara Sotolongo, Brianna Ruiz, Manuel Carreno, Tzakis G. Andreas, Phillip Ruiz. Surgery, Transplant Laboratories, Leonard M. Miller School of Medicine, Miami, FL, USA; Pathology, Leonard M. Miller School of Medicine, Miami, FL, USA; Surgery, Division of Transplantation, Leonard M. Miller School of Medicine, Miami, FL, USA. Aim: The MBL2 gene encodes the mannose binding lectin (MBL) serum protein. MBL has a major role in innate immunity by activating the lectin complement pathway. In our study, we concentrated on genotyping six polymorphisms in the MBL2 gene that result in significant variations in the levels of the MBL protein of transplant patients. Low MBL protein levels can predispose transplant patients to recurrent infections and possible future graft rejection. Methods: Applied Biosystems’ Taqman SNP genotyping assays were used on whole blood to differentiate homozygotic from heterozygotic alleles (MBL2 SNPs). The MBL elisa assay for quantitative measurement of MBL in serum was used. Results: SNP genotyping assays were initially done on 100 transplant patients with a distribution of 59 kidney, 24 liver, and 18 other multiple GI transplant patients and MBL protein elisa measurements were also taken. The results indicate that multiple combinations of these six SNP genotypes result in reduced MBL protein levels and this could be a promising early marker of infection and graft rejection episodes in transplant patients. Conclusions: Transplant patients with these SNP mutations exhibited genotypes that estimate low MBL protein production in about 10% of the cases. The SNP assay in combination with the elisa will elucidate this connection to infectious disease states. We are in the process of determining the relationship between distribution of the MBL2 SNP genotypes, protein levels and infections in our transplant population.