The high degree of polymorphism at HLA class I and class II loci makes high resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results due ...to incomplete genomic coverage and inability to set phase for HLA haplotype determination. The 454 Life Sciences GS FLX next generation sequencing system coupled with Conexio ATF software can provide very high resolution HLA genotyping. High throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, and DRB3, DRB4 and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs (bp) and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and Conexio ATF software allows reliable identification of HLA genotypes at high resolution.
Acknowledgments:
This work was supported in part by office of Naval Research cooperative agreement # N‐00014‐96‐2‐0016 to the National Marrow Donor Program and by funding from the E.B. Foerderer Fund ...of the Children’s Hospital of Philadelphia to D.M.
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The new allele HLA‐B*1559 described is of Hispanic origin. This allele carries the exon 2 of HLA‐B*35 alleles, and is identical to HLA‐B*3501 and the exon 3 of HLA‐B*1530. Serologically it is typed as B35. The most likely scenario for its generation seems to be a gene conversion event that involved the, frequent in Hispanic populations, B*1522 allele and a B*1530 allele. A segment of the B*1530 allele that included at least the sequences 412–419 would be transferred on a B*1522 background ( Note).
: A total of 42,160 individuals were typed for HLA‐A and HLA‐B by both serology and PCR‐based typing. The HLA assignments included all of the known serological equivalents. The majority of the ...individuals (99.9%) were from U.S. minority population groups. The serologic typing was performed between 1993 and 1997 at the time of recruitment for the National Bone Marrow Program (NMDP) registry. The polymerase chain reaction (PCR)‐based typing was carried out in two phases. In phase I, DNA typing was performed by PCR using sequence‐specific oligonucleotide probes (PCR‐SSOP) or PCR using sequence‐specific primers (PCR‐SSP) without knowledge of the serologic assignments. Discrepancies were identified between the serologic and DNA assignments in 24% of the volunteers (8% of volunteers differed for only HLA‐A assignments, 13% for HLA‐B, and 3% for both HLA‐A and ‐B) and a potential explanation was assigned each discrepant serology/DNA pair. In phase II, a random sampling scheme was used to select a statistically significant number of individuals for repeat DNA typing from each of these categories. The categories included antigens missed by serology, nonexpressed (null) alleles, PCR amplification failures, misassignment of antigens and nomenclature issues. Only a single individual was found to carry a null allele. DNA‐based testing correctly typed nearly 99% of the donors at HLA‐A, more than 98% at HLA‐B, and more than 97% at both HLA‐A and ‐B validating this methodology for registry typing.
Acknowledgments:
This work was supported in part by office of Naval Research cooperative agreement # N‐00014‐96‐2‐0016 to the National Marrow Donor Program and by funding from the E.B. Foerderer Fund ...of the Children’s Hospital of Philadelphia to D.M.
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The described alle HLA‐B*0811 is of Caucasoid origin. Most likely it derived from a point mutation of the B*0801 allele at position 559, where an adenine was converted to guanine. This position corresponds to codon 163 and resulted in an amino acid change from threonine to alanine. This substitution may influence both, T‐cell interactions and/or peptide binding ( Note).
DNA typing of HLA class II alleles of the DRB1/3/4/5 and DQB1 loci using sequence-specific oligonucleotide probes and polymerase chain reaction amplified DNA has been used for the large-scale typing ...of donors for the National Marrow Donor Program unrelated donor registry. The results of quality control analysis for the second year of the project (10/1/939/30/94) show the typing continues to be highly accurate, specific, and reliable. The average percent of correctly classified HLA oligotypes (groups of alleles defined by a hybridization pattern with a panel of sequence-specific oligonucleotide probes) based on 9,244 DRB1 and 7,244 DQB1 assignments was 99.8% (range 99.4%100.0%) for DRB1/DRB3/DRB4/DRB5 and 99.8% (range 99.6%100.0%) for DQB1. This level of accuracy is particularly remarkable because the 4,636 DRB quality control samples were tested blindly and could not be distinguished from 57,580 donor samples tested at the same time by the laboratories.
DNA‐based typing of HLA class I alleles of the HLA‐A and HLA‐B loci using sequence‐specific oligonucleotide primers and/or probes has been used for the large‐scale typing of individuals for the ...National Marrow Donor Program® unrelated donor registry. Typing was performed by 16 laboratories at a low level of resolution (e.g. A*01, B*07). The results of blinded quality control analysis for the first 12 months of the project show the typing to be highly accurate, specific and reliable. The total error rate based on 11,545 HLA‐A and 11,428 HLA‐B assignments was 1.1% for HLA‐A and 1.9% for HLA‐B. This level of accuracy is particularly remarkable because the quality control samples could not be distinguished from 64,180 donor samples tested at the same time by the laboratories.
Enzymatic glycogen regulation in mouse splenocytes cultured in vitro with and without LPS, was studied from 0-72 h. Increased 3Hglucose uptake and hexokinase activity demonstrated the activation of ...cells treated with LPS. There was a greater time-dependent increase of cellular glycogen content in LPS-stimulated cells as compared to control. Glycogen synthetase I in LPS-stimulated cells increased about 200% above control cells to a plateau at 48 h, while in unstimulated cells there was little increase throughout. Glycogen synthetase D increased continually to 72 h in both groups. In the stimulated cells, phosphorylase increased only 90% above control cells up to 48 h. It was concluded that the increased glycogen content of LPS-stimulated cells seen at 48 h may result from an increase in both glycogen synthetase I and D activity compared to lesser increase in hydrolysis. However, between 48 and 72 h, the period of RNA and DNA increased synthesis, the glycogen content of stimulated cells did not increase further, consistent with the observation that synthetase I activity remained constant and synthetase D decreased. Thus, following mitogenic stimulation, the net effect of the enzymatic regulation is to increase cellular glycogen, as an energy source for subsequent events.
DNA typing of HLA class II alleles of the DRB1/3/4 and DQB1 loci using sequence-specific oligonucleotide probes and polymerase chain reaction-amplified DNA was used for the large-scale typing of ...donors for the National Marrow Donor Program unrelated donor registry. The results of quality control analysis for the first 7 months of the project show the typing to be highly accurate, specific, and reliable. The percent of correctly classified HLA oligotypes based on 1652 DRB1 and 1652 DQB1 assignments was greater than 99% for DRB1/DRB3/DRB4 and greater than 98% for DQB1. This level of accuracy is particularly remarkable because the quality control samples could not be distinguished from 9011 donor samples tested at the same time by the laboratories.
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