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P106 Whole gene, whole haplotype and whole genome sequencing in human
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P105
Abstracts / Human Immunology 78 (2017) 51–254
SINGLE-CENTER EXPERIENCE IN ROUTINE POST-TRANSPLANT ANTIBODY TESTING OF RISK-STRATIFIED KIDNEY TRANSPLANT RECIPIENTS Anh Dinh, Francesca Cardarelli, Eliyahu Khankin, Martha Pavlakis, J. Ryan Peña. Beth Israel Deaconess Medical Center, Boston, MA, United States. Aim: Preformed (pf) and de novo(dn) donor-specific antibodies (DSA) are associated with antibody mediated rejection (AMR). We previously showed that 6% of low-risk renal transplant (tx) patients developed dn DSA at 1 year (y) post-tx (ASHI, 2016). This study extends our findings over 2y post-tx for low-risk, as well as for intermediate- and high-risk groups. Methods: Our Tx Center instituted routine post-tx antibody (Ab) testing by bead array in 2014. Frequency of post-tx monitoring was based on perceived risk for AMR due to DSA in a pre-tx sample: low (no DSA ever) at 1y; intermediate (historic pfDSA, flow crossmatch [FCXM] negative) at 1, 3, 6, and 12 months (m); high (current pfDSA, FCXM negative) at 2 weeks, 1, 3, 6, and 12 m. All groups with functioning grafts were monitored annually thereafter. Additional testing was performed due to planned or unplanned change in immunosuppression (IS) regimen. Results: From May 2014-June 2015, 73 patients received a kidney only tx with HLA immunologic risk as follows: 61 low (84%), 2 intermediate (3%), and 10 high (14%). Of low-risk patients, 54 were tested at 1y post-tx while 30/54 low-risk patients were tested at 2y post-tx (at time of analysis). In addition to 4 previously described patients with dnDSA at 1y post-tx, 3 patients developed dnDSA just beyond the 1y post-tx: 2 were tested due to IS change (decreased due to BK virus infection, another, to non-adherence) while 1 was due to graft dysfunction. On biopsy (bx), both low-risk patients tested for IS change had chronic changes while the patient with acute dysfunction had chronic Ab mediated rejection. Of 2 intermediate-risk patients, 1 developed dnDSA by 1 m post-tx with chronic changes on bx. Finally, 1/10 high-risk patients had increased strength of pfDSA and suffered acute AMR while another developed dnDSA (no increase in pfDSA) following IS change. The patients with dnDSA are being monitored more closely. Conclusions: In patients with routine post-tx Ab testing (n = 66), dnDSA was detected in 15% (7 low- 1 intermediate- and 2 high-risk); 1 high-risk patient developed increasing pfDSA. Strikingly, changes in IS (decrease due to infection or non-adherence) led to development of dnDSA. This suggests that routine post-tx DSA testing may be help identify evolving alloresponses and guide management but further analysis is needed.
P106
WHOLE GENE, WHOLE HAPLOTYPE AND WHOLE GENOME SEQUENCING IN HUMAN Nezih Cereb, HwaRan Kim, JeongIl Lee, Soo Young Yang. Histogenetics, Ossining, NY, United States. Aim: Recent advances in sequencing, sample and library preparation technologies made it possible to sequence entire genes, haplotypes and genome in human. In this study, we used four approaches; amplicon based whole gene sequencing, capture probe based MHC haplotype sequencing, PacBio Sequel system based whole genome sequencing and whole genome mapping based on Bionanogenomics’ Technology to study one individual’s MHC. We wanted to compare the information that we have obtained from each technology and confirm the haplotype predictions. Methods: We performed whole gene HLA Class I gene sequencing as described earlier (Cereb et al., Hum Immunol. 2015 Dec;76(12):963–74). We performed MHC region target sequence capture using RocheNimbleGen SeqCap EZ Library. Whole genome sequencing We prepared 40 to 50 kb libraries with 15 to 20 kb cut off size selection and followed manufacturer’s protocol thereafter. Sequencing performed on PacBio SequelÒ platform using v2 chemistry and v4 software. Movie time for these large fragments were set to 600 min and secondary analysis is done with SMRTLink’s (v4.0.0) SMRT Analysis Application: de novo Assembly (HGAP 4). To get 85 coverage, we have sequenced the same library in 62 different SMRTÒ Cell 1 M v2. Whole genome de novo assembly of PacBio sequencing is being performed by DNANexus. BioNano genome mapping was performed by Bio Nano genomics. Results: Haplotype determination of the individual tested with whole gene amplicon sequencing performed based on the family studies and is being compared to haplotypes determined by other methods. Conclusions: Now thanks to novel technologies we can decipher the complex genetic structure of MHC and other immune response genes with higher precision. This in turn will increase our ability to understand the genetic basis of the complex diseases.
N. Cereb: 6. Stock Shareholder; Company/Organization; Pacific Bio, Histogenetics. H. Kim: 5. Employee; Company/ Organization; Histogenetics. J. Lee: 5. Employee; Company/Organization; Histogenetics. S. Yang: 6. Stock Shareholder; Company/Organization; PacBio, Histogenetics.
P105
Abstracts / Human Immunology 78 (2017) 51–254
SINGLE-CENTER EXPERIENCE IN ROUTINE POST-TRANSPLANT ANTIBODY TESTING OF RISK-STRATIFIED KIDNEY TRANSPLANT RECIPIENTS Anh Dinh, Francesca Cardarelli, Eliyahu Khankin, Martha Pavlakis, J. Ryan Peña. Beth Israel Deaconess Medical Center, Boston, MA, United States. Aim: Preformed (pf) and de novo(dn) donor-specific antibodies (DSA) are associated with antibody mediated rejection (AMR). We previously showed that 6% of low-risk renal transplant (tx) patients developed dn DSA at 1 year (y) post-tx (ASHI, 2016). This study extends our findings over 2y post-tx for low-risk, as well as for intermediate- and high-risk groups. Methods: Our Tx Center instituted routine post-tx antibody (Ab) testing by bead array in 2014. Frequency of post-tx monitoring was based on perceived risk for AMR due to DSA in a pre-tx sample: low (no DSA ever) at 1y; intermediate (historic pfDSA, flow crossmatch [FCXM] negative) at 1, 3, 6, and 12 months (m); high (current pfDSA, FCXM negative) at 2 weeks, 1, 3, 6, and 12 m. All groups with functioning grafts were monitored annually thereafter. Additional testing was performed due to planned or unplanned change in immunosuppression (IS) regimen. Results: From May 2014-June 2015, 73 patients received a kidney only tx with HLA immunologic risk as follows: 61 low (84%), 2 intermediate (3%), and 10 high (14%). Of low-risk patients, 54 were tested at 1y post-tx while 30/54 low-risk patients were tested at 2y post-tx (at time of analysis). In addition to 4 previously described patients with dnDSA at 1y post-tx, 3 patients developed dnDSA just beyond the 1y post-tx: 2 were tested due to IS change (decreased due to BK virus infection, another, to non-adherence) while 1 was due to graft dysfunction. On biopsy (bx), both low-risk patients tested for IS change had chronic changes while the patient with acute dysfunction had chronic Ab mediated rejection. Of 2 intermediate-risk patients, 1 developed dnDSA by 1 m post-tx with chronic changes on bx. Finally, 1/10 high-risk patients had increased strength of pfDSA and suffered acute AMR while another developed dnDSA (no increase in pfDSA) following IS change. The patients with dnDSA are being monitored more closely. Conclusions: In patients with routine post-tx Ab testing (n = 66), dnDSA was detected in 15% (7 low- 1 intermediate- and 2 high-risk); 1 high-risk patient developed increasing pfDSA. Strikingly, changes in IS (decrease due to infection or non-adherence) led to development of dnDSA. This suggests that routine post-tx DSA testing may be help identify evolving alloresponses and guide management but further analysis is needed.
P106
WHOLE GENE, WHOLE HAPLOTYPE AND WHOLE GENOME SEQUENCING IN HUMAN Nezih Cereb, HwaRan Kim, JeongIl Lee, Soo Young Yang. Histogenetics, Ossining, NY, United States. Aim: Recent advances in sequencing, sample and library preparation technologies made it possible to sequence entire genes, haplotypes and genome in human. In this study, we used four approaches; amplicon based whole gene sequencing, capture probe based MHC haplotype sequencing, PacBio Sequel system based whole genome sequencing and whole genome mapping based on Bionanogenomics’ Technology to study one individual’s MHC. We wanted to compare the information that we have obtained from each technology and confirm the haplotype predictions. Methods: We performed whole gene HLA Class I gene sequencing as described earlier (Cereb et al., Hum Immunol. 2015 Dec;76(12):963–74). We performed MHC region target sequence capture using RocheNimbleGen SeqCap EZ Library. Whole genome sequencing We prepared 40 to 50 kb libraries with 15 to 20 kb cut off size selection and followed manufacturer’s protocol thereafter. Sequencing performed on PacBio SequelÒ platform using v2 chemistry and v4 software. Movie time for these large fragments were set to 600 min and secondary analysis is done with SMRTLink’s (v4.0.0) SMRT Analysis Application: de novo Assembly (HGAP 4). To get 85 coverage, we have sequenced the same library in 62 different SMRTÒ Cell 1 M v2. Whole genome de novo assembly of PacBio sequencing is being performed by DNANexus. BioNano genome mapping was performed by Bio Nano genomics. Results: Haplotype determination of the individual tested with whole gene amplicon sequencing performed based on the family studies and is being compared to haplotypes determined by other methods. Conclusions: Now thanks to novel technologies we can decipher the complex genetic structure of MHC and other immune response genes with higher precision. This in turn will increase our ability to understand the genetic basis of the complex diseases.
N. Cereb: 6. Stock Shareholder; Company/Organization; Pacific Bio, Histogenetics. H. Kim: 5. Employee; Company/ Organization; Histogenetics. J. Lee: 5. Employee; Company/Organization; Histogenetics. S. Yang: 6. Stock Shareholder; Company/Organization; PacBio, Histogenetics.