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Identification of two novel HLA-B null alleles using next generation sequencing
108
P102
Abstracts / Human Immunology 76 (2015) 38–167
IDENTIFICATION OF TWO NOVEL HLA-B NULL ALLELES USING NEXT GENERATION SEQUENCING. Jean Garcia-Gomez, Ketevan Gendzekhadze, Michiko Taniguchi, David Senitzer. City of Hope National Medical Center, Duarte, CA, United States. Aim: Typing of two patients (one of Asian and one of Hispanic descent) using SSOP and SBT resulted in the detection of two new HLA-B null alleles. Detailed analysis of the probe and sequence data showed the new null alleles to be generated by single nucleotide point mutations. To confirm these results, the DNA samples were tested by next generation sequencing (NGS). Methods: DNA was extracted from whole blood using the Promega Maxwell 16 Blood DNA Purification kit and Promega Maxwell 16 IVD instrument. Low resolution HLA typing was obtained by performing DNA amplification and probe hybridization using the One Lambda LABType SSO HLA-B Locus kit. High resolution HLA typing was achieved by DNA amplification and subsequent sequencing of the amplified product using the Conexio Genomics SBT Resolver kits. Confirmation testing of the novel HLA-B null alleles was performed by amplifying the DNA samples using the Scisco Genetics NGS HLA kit. Results: Results from SSOP, SBT and NGS testing confirmed the presence of two new null alleles. The first null allele from the Hispanic individual is similar to B⁄35:01:01:01 with a mutation at position 295 (codon 75) in exon 2. This position appears to be a common polymorphic site for HLA Class I null alleles: A⁄02:366N, A⁄03:178N, A⁄24:60N, B⁄08:72N, B⁄15:94N, B⁄15:294N, B⁄44:58N, B⁄51:41N, C⁄01:69N, C⁄03:277N, C⁄07:152N, C⁄12:84N and C⁄17:27N. The nucleotide change (GAG ? TAG) results in the creation of a stop codon. The second null allele from the Asian individual is similar to B⁄15:01:01:01 with a mutation at position 862 (codon 264) in exon 4. The nucleotide change (GGA ? TGA) results in the creation of a stop codon. There are no documented polymorphisms at this position for any other allele. However, A⁄02:43N has an insertion at codon 236 that causes a frameshift and premature stop at codon 264. Conclusions: Identification of null alleles is important in determining what donors should be evaluated and selected for hematopoietic stem cell transplants. Misidentification could have an impact on graft outcome and lead to complications such as graft-versus-host disease. NGS has shown to be a valuable method for the identification of these kinds of new variants. Furthermore, the variant in exon 4 may suggest that typing outside of the HLA Class I peptide binding domains (exon 2 and 3) should be performed.
P103
HIGH THROUGHPUT, LOW COST DETECTION OF GAMMA BLOCK SNPS USING A CAPTURE PROBE TARGET ENRICHMENT NEXT GENERATION SEQUENCING ASSAY. Hayley Hogan, Rhys Cransberg, Megan Jordan, Bin Jiao, David Sayer. Conexio, Perth, Australia. Aim: Previous studies have demonstrated that mismatching for SNPs in the Gamma block of the central MHC increases the risk of severe acute GvHD following unrelated HSCT. These studies were performed using PCR-SSP, a protocol that is not amenable to high throughput testing. In order to facilitate high throughput testing for retrospective studies we have developed a capture probe based next generation sequencing assay for the detection of Gamma block SNPs. Methods: We designed 120 mer capture probes tiling the genetic region of interest. 200 ng of DNA from 20 samples that had previously been Gamma-Type™ tested by PCR-SSP were fragmented using a Covaris M220 Ultrasonicator™ using the following conditions: duty factor 20%, peak incident power 50 W, 200 cycles per burst, for 45 s at a temperature of 20 °C – to give a peak distribution of fragments approximately 550 bp in length. The fragments were repaired, size selected by a dual-bead based protocol, adenylated and adapters were ligated. The fragments were enriched and capture probes were used to isolate fragments containing Gamma block specific sequences. The Gamma block specific fragments were sequenced using a MiSeqÒ Next Generation DNA Sequencer, generating 300 bp paired-end reads. The Gamma-Type™ variant positions were annotated within Assign™ MPS resulting in automated reporting of Gamma block SNPs enabling direct comparison between donors and patients. Results: Even coverage of >1000 calls were obtained for all samples across all positions. There was complete concordance between the PCR-SSP results and the capture probe assay, with additional polymorphisms seen for two of the samples. The additional polymorphisms did not affect the matching/mismatching outcome with the donor/patient pairs. Conclusion: The capture probe approach is amenable to low cost and high throughput testing with the added advantage that the precise sequence is reported, whereas by PCR-SSP, only the PCR target sequence is detected. Furthermore the Gamma block capture probes can be included with HLA probes to provide complete HLA and Gamma block SNP detection in one assay. Large retrospective studies have the potential to identify the non-HLA regions within the MHC that influence outcomes in unrelated HSCT.
P102
Abstracts / Human Immunology 76 (2015) 38–167
IDENTIFICATION OF TWO NOVEL HLA-B NULL ALLELES USING NEXT GENERATION SEQUENCING. Jean Garcia-Gomez, Ketevan Gendzekhadze, Michiko Taniguchi, David Senitzer. City of Hope National Medical Center, Duarte, CA, United States. Aim: Typing of two patients (one of Asian and one of Hispanic descent) using SSOP and SBT resulted in the detection of two new HLA-B null alleles. Detailed analysis of the probe and sequence data showed the new null alleles to be generated by single nucleotide point mutations. To confirm these results, the DNA samples were tested by next generation sequencing (NGS). Methods: DNA was extracted from whole blood using the Promega Maxwell 16 Blood DNA Purification kit and Promega Maxwell 16 IVD instrument. Low resolution HLA typing was obtained by performing DNA amplification and probe hybridization using the One Lambda LABType SSO HLA-B Locus kit. High resolution HLA typing was achieved by DNA amplification and subsequent sequencing of the amplified product using the Conexio Genomics SBT Resolver kits. Confirmation testing of the novel HLA-B null alleles was performed by amplifying the DNA samples using the Scisco Genetics NGS HLA kit. Results: Results from SSOP, SBT and NGS testing confirmed the presence of two new null alleles. The first null allele from the Hispanic individual is similar to B⁄35:01:01:01 with a mutation at position 295 (codon 75) in exon 2. This position appears to be a common polymorphic site for HLA Class I null alleles: A⁄02:366N, A⁄03:178N, A⁄24:60N, B⁄08:72N, B⁄15:94N, B⁄15:294N, B⁄44:58N, B⁄51:41N, C⁄01:69N, C⁄03:277N, C⁄07:152N, C⁄12:84N and C⁄17:27N. The nucleotide change (GAG ? TAG) results in the creation of a stop codon. The second null allele from the Asian individual is similar to B⁄15:01:01:01 with a mutation at position 862 (codon 264) in exon 4. The nucleotide change (GGA ? TGA) results in the creation of a stop codon. There are no documented polymorphisms at this position for any other allele. However, A⁄02:43N has an insertion at codon 236 that causes a frameshift and premature stop at codon 264. Conclusions: Identification of null alleles is important in determining what donors should be evaluated and selected for hematopoietic stem cell transplants. Misidentification could have an impact on graft outcome and lead to complications such as graft-versus-host disease. NGS has shown to be a valuable method for the identification of these kinds of new variants. Furthermore, the variant in exon 4 may suggest that typing outside of the HLA Class I peptide binding domains (exon 2 and 3) should be performed.
P103
HIGH THROUGHPUT, LOW COST DETECTION OF GAMMA BLOCK SNPS USING A CAPTURE PROBE TARGET ENRICHMENT NEXT GENERATION SEQUENCING ASSAY. Hayley Hogan, Rhys Cransberg, Megan Jordan, Bin Jiao, David Sayer. Conexio, Perth, Australia. Aim: Previous studies have demonstrated that mismatching for SNPs in the Gamma block of the central MHC increases the risk of severe acute GvHD following unrelated HSCT. These studies were performed using PCR-SSP, a protocol that is not amenable to high throughput testing. In order to facilitate high throughput testing for retrospective studies we have developed a capture probe based next generation sequencing assay for the detection of Gamma block SNPs. Methods: We designed 120 mer capture probes tiling the genetic region of interest. 200 ng of DNA from 20 samples that had previously been Gamma-Type™ tested by PCR-SSP were fragmented using a Covaris M220 Ultrasonicator™ using the following conditions: duty factor 20%, peak incident power 50 W, 200 cycles per burst, for 45 s at a temperature of 20 °C – to give a peak distribution of fragments approximately 550 bp in length. The fragments were repaired, size selected by a dual-bead based protocol, adenylated and adapters were ligated. The fragments were enriched and capture probes were used to isolate fragments containing Gamma block specific sequences. The Gamma block specific fragments were sequenced using a MiSeqÒ Next Generation DNA Sequencer, generating 300 bp paired-end reads. The Gamma-Type™ variant positions were annotated within Assign™ MPS resulting in automated reporting of Gamma block SNPs enabling direct comparison between donors and patients. Results: Even coverage of >1000 calls were obtained for all samples across all positions. There was complete concordance between the PCR-SSP results and the capture probe assay, with additional polymorphisms seen for two of the samples. The additional polymorphisms did not affect the matching/mismatching outcome with the donor/patient pairs. Conclusion: The capture probe approach is amenable to low cost and high throughput testing with the added advantage that the precise sequence is reported, whereas by PCR-SSP, only the PCR target sequence is detected. Furthermore the Gamma block capture probes can be included with HLA probes to provide complete HLA and Gamma block SNP detection in one assay. Large retrospective studies have the potential to identify the non-HLA regions within the MHC that influence outcomes in unrelated HSCT.