- Email: [email protected]
P025 HLA antibody targets: Pre-transplant, post-transplant, post-graft loss
Abstracts / Human Immunology 78 (2017) 51–254
P024
71
LARGE SCALE WHOLE GENE SEQUENCING OF HLA CLASS I GENES REVEALS LOCUS-SPECIFIC CONSERVATION OF INTRONIC SEQUENCES Nezih Cereb a, Chase W. Nelson b, Soo Young Yang a. aHistogenetics, Ossining, NY, United States ; bAmerican Museum of Natural History, New York, NY, United States. Aim: We have been sequencing whole gene HLA Class I at large scale at Histogenetics on PacBio RSIIÒ platform since the beginning of July of 2016. To date we have sequenced around 185,000 samples for whole gene Class I. We have found 1645, 1697 and 1664 unique sequences for A, B and C genes, respectively. Most of the intronic sequence information comes from the introns flanking Antigen Recognition Site (ARS) encoding introns. In this study we wanted to analyze all intronic sequences to determine their evolutionary process. Methods: We performed whole gene HLA Class I gene sequencing as described earlier (Cereb et al. Hum Immunol. 2015 Dec;76(12):963–74). Exons were translated, aligned at the codon level using the ClustalW algorithm in MEGA7 (default settings), and the alignment was back-imposed on the nucleotide sequence. Intronic sequence diversity (d), and exonic nonsynonymous (dN) and synonymous (dS) sequence diversity, were calculated based on all pairwise comparisons of unique alleles using SNPGenie (adaptation of NeiGojobori method). Results: For HLA-A, dS-exons = 0.0404 while d-introns = 0.0236; for HLA-B, dS-exons = 0.0347 while dintrons = 0.0094; and for HLA-C, dS-exons = 0.0305 while d-introns = 0.0186. Conclusions: Our results are consistent with the hypothesis that diversity at synonymous coding sites has been elevated because of linkage to nonsynonymous sites, which experience overdominant (positive balancing) selection. In contrast, intronic sites are less closely linked to nonsynonymous coding sites than are synonymous coding sites, making them more likely to experience recombinational separation from coding sites and subsequent drift, which has acted to homogenize the sequences over evolutionary time thus resulting in locus-specific sequences un the introns.
N. Cereb: 5. Employee; Company/Organization; Histogenetics. 6. Stock Shareholder; Company/Organization; PacBio, Histogenetics. C.W. Nelson: 2. Consultant; Company/Organization; Histogenetics. S. Yang: 5. Employee; Company/ Organization; Histogenetics. 6. Stock Shareholder; Company/Organization; PacBio, Histogenetics.
P025
HLA ANTIBODY TARGETS: PRE-TRANSPLANT, POST-TRANSPLANT, POST-GRAFT LOSS Danny Youngs, Paul R. Warner, Idoia Gimferrer. Bloodworks Northwest, Seattle, WA, United States. Aim: It has often been noticed that the most frequent target of donor-specific HLA antibodies (DSA) in posttransplant monitoring are DQ antigens, at a rate significantly higher than that observed in patients awaiting transplant. This study is an attempt to quantify that observation. Methods: From 2012 to 2017 sera from patients who had received or were waiting to receive a 1st or 2nd second kidney transplant were tested using the LABScreen Single Antigen kit. To avoid counting patients twice within a stage (pre-1st transplant, post-transplant, relisted for 2nd transplant) patients were evaluated for this study only on their most recent serum within each stage. 5896 patients pre-1st transplant (3458 males, 2438 females), 2626 patients who received post-transplant testing for DSA, and 754 patients who were relisted for a 2nd transplant were evaluated. As reliable transfusion and pregnancy information was unavailable for most pre-transplant patients, the difference between male and female sensitization rates was used as a surrogate for the different sensitizing effects of transfusion versus pregnancy in this stage. Results: Sensitization to any HLA was 14% in pre-transplant males, 38% in pre-transplant females, 15% in post-transplant monitoring (but only evaluated for DSA), and 71% in patients relisted for a second transplant. Males and females pre-first transplant show significant differences in antibodies against class II, and also against HLA-B and to a lesser extent HLA-C. Post-transplant DSA shows a very high rate of antibodies against HLA-DQ, and relatively little against class I. Patients relisted for a second transplant show extensive antibody production against antigens of all loci, especially HLA-A, B, DRB1 and DQ. Conclusions: The three major sources of sensitization to HLA, transfusion, pregnancy and transplantation, show different rates of sensitization to different HLA loci. Among HLA antibody producers, antibodies to DQ are 2–3 times more frequent as DSA than they are as pre-transplant antibody targets.
% of Antibody Producers Making Antibodies to Locus HLA-A HLA-B HLA-C HLA-DRB1 DRB345 HLA-DQ HLA-DP Males PreTxp
46%
43%
14%
16%
7%
20%
2%
Females PreTxp
48%
63%
23%
41%
21%
27%
8%
Post-Txp (DSA)
11%
9%
7%
15%
23%
68%
4%
64%
38%
54%
39%
71%
26%
Relist for 2nd Txpl 64%
P024
71
LARGE SCALE WHOLE GENE SEQUENCING OF HLA CLASS I GENES REVEALS LOCUS-SPECIFIC CONSERVATION OF INTRONIC SEQUENCES Nezih Cereb a, Chase W. Nelson b, Soo Young Yang a. aHistogenetics, Ossining, NY, United States ; bAmerican Museum of Natural History, New York, NY, United States. Aim: We have been sequencing whole gene HLA Class I at large scale at Histogenetics on PacBio RSIIÒ platform since the beginning of July of 2016. To date we have sequenced around 185,000 samples for whole gene Class I. We have found 1645, 1697 and 1664 unique sequences for A, B and C genes, respectively. Most of the intronic sequence information comes from the introns flanking Antigen Recognition Site (ARS) encoding introns. In this study we wanted to analyze all intronic sequences to determine their evolutionary process. Methods: We performed whole gene HLA Class I gene sequencing as described earlier (Cereb et al. Hum Immunol. 2015 Dec;76(12):963–74). Exons were translated, aligned at the codon level using the ClustalW algorithm in MEGA7 (default settings), and the alignment was back-imposed on the nucleotide sequence. Intronic sequence diversity (d), and exonic nonsynonymous (dN) and synonymous (dS) sequence diversity, were calculated based on all pairwise comparisons of unique alleles using SNPGenie (adaptation of NeiGojobori method). Results: For HLA-A, dS-exons = 0.0404 while d-introns = 0.0236; for HLA-B, dS-exons = 0.0347 while dintrons = 0.0094; and for HLA-C, dS-exons = 0.0305 while d-introns = 0.0186. Conclusions: Our results are consistent with the hypothesis that diversity at synonymous coding sites has been elevated because of linkage to nonsynonymous sites, which experience overdominant (positive balancing) selection. In contrast, intronic sites are less closely linked to nonsynonymous coding sites than are synonymous coding sites, making them more likely to experience recombinational separation from coding sites and subsequent drift, which has acted to homogenize the sequences over evolutionary time thus resulting in locus-specific sequences un the introns.
N. Cereb: 5. Employee; Company/Organization; Histogenetics. 6. Stock Shareholder; Company/Organization; PacBio, Histogenetics. C.W. Nelson: 2. Consultant; Company/Organization; Histogenetics. S. Yang: 5. Employee; Company/ Organization; Histogenetics. 6. Stock Shareholder; Company/Organization; PacBio, Histogenetics.
P025
HLA ANTIBODY TARGETS: PRE-TRANSPLANT, POST-TRANSPLANT, POST-GRAFT LOSS Danny Youngs, Paul R. Warner, Idoia Gimferrer. Bloodworks Northwest, Seattle, WA, United States. Aim: It has often been noticed that the most frequent target of donor-specific HLA antibodies (DSA) in posttransplant monitoring are DQ antigens, at a rate significantly higher than that observed in patients awaiting transplant. This study is an attempt to quantify that observation. Methods: From 2012 to 2017 sera from patients who had received or were waiting to receive a 1st or 2nd second kidney transplant were tested using the LABScreen Single Antigen kit. To avoid counting patients twice within a stage (pre-1st transplant, post-transplant, relisted for 2nd transplant) patients were evaluated for this study only on their most recent serum within each stage. 5896 patients pre-1st transplant (3458 males, 2438 females), 2626 patients who received post-transplant testing for DSA, and 754 patients who were relisted for a 2nd transplant were evaluated. As reliable transfusion and pregnancy information was unavailable for most pre-transplant patients, the difference between male and female sensitization rates was used as a surrogate for the different sensitizing effects of transfusion versus pregnancy in this stage. Results: Sensitization to any HLA was 14% in pre-transplant males, 38% in pre-transplant females, 15% in post-transplant monitoring (but only evaluated for DSA), and 71% in patients relisted for a second transplant. Males and females pre-first transplant show significant differences in antibodies against class II, and also against HLA-B and to a lesser extent HLA-C. Post-transplant DSA shows a very high rate of antibodies against HLA-DQ, and relatively little against class I. Patients relisted for a second transplant show extensive antibody production against antigens of all loci, especially HLA-A, B, DRB1 and DQ. Conclusions: The three major sources of sensitization to HLA, transfusion, pregnancy and transplantation, show different rates of sensitization to different HLA loci. Among HLA antibody producers, antibodies to DQ are 2–3 times more frequent as DSA than they are as pre-transplant antibody targets.
% of Antibody Producers Making Antibodies to Locus HLA-A HLA-B HLA-C HLA-DRB1 DRB345 HLA-DQ HLA-DP Males PreTxp
46%
43%
14%
16%
7%
20%
2%
Females PreTxp
48%
63%
23%
41%
21%
27%
8%
Post-Txp (DSA)
11%
9%
7%
15%
23%
68%
4%
64%
38%
54%
39%
71%
26%
Relist for 2nd Txpl 64%