The Human Major Histocompatibility Complex
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1 The Human Major Histocompatibility Complex 1
2 Location and Organization of the HLA Complex on Chromosome 6 NEJM 343(10):
3 Inheritance of the HLA Complex Haplotype Inheritance (Family Study) 3
4 Structure of HLA Class I and Class II Molecules NEJM 343(10):
5 Cellular Distribution of HLA Class I and Class II Molecules HLA Class I: Expressed on the surface of nearly all nucleated cells The highest level of Class I molecules are expressed on lymphocytes Fibroblasts, muscle cells, hepatocytes and neural cells express very low levels of Class I molecules HLA Class II: Expressed primarily on Ag-presenting cells (macrophages, dendritic cells and B cells) 5
6 Antigen Processing and Presentation HLA Class I HLA Class II NEJM 343(10):
7 Interaction Between T-Cell Receptor and HLA - Peptide Complex 7
8 HLA Class I Molecule 8
9 HLA Class I Molecule Peptide-binding cleft binds 8-10 amino acids in length 9
10 HLA Class II Molecule Peptide-binding cleft binds amino acids in length 10
11 Polymorphism in HLA Class I Genes In the human population, over 11,000 HLA Class I alleles have been identified some are null alleles, synonymous or differ in intronic region Number of polymorphisms (alleles) HLA Class I 11,859 Alleles (Dec. 2016) 0 A B C 11
12 Polymorphism in HLA Class II Genes In the human population, over 3,800 HLA Class II alleles have been identified some are null alleles, synonymous or differ in intronic region Number of polymorphisms (alleles) HLA Class II 3,805 Alleles (Dec. 2016) 0 DR DQ DP 12
13 HLA Nomenclature 13
14 Additional Suffixes Added to the Allele Number to Indicate Expression Status: N L Null alleles that have been shown not to be expressed i.e. B*15:190N Low cell surface expression i.e. A*24:02:01:02L S Q Secreted molecule but not expressed on cell surface i.e. B*44:02:01:02S Questionable allele expression on cell surface i.e. A*23:19Q 14
15 Our Laboratory s Current HLA Typing Approach Sequence Specific Primers (SSP) Low / Intermediate Resolution Typing Resolve Ambiguities Reverse Sequence Specific Oligonucleotide (rsso) Luminex Low / Intermediate Resolution Typing High-throughput Typing Sequence Based Typing (SBT) High Resolution Typing Low / Intermediate Resolution Typing High-throughput Typing 15
16 SSP - Agarose Gel Electrophoresis 16
17 Intermediate Resolution HLA Typing (rsso) rsso: Reverse Sequence Specific Oligonucleotide Genomic DNA is PCR amplified by locus specific primers The PCR product is hybridized with a panel of probes (bound to fluorescently coded microbeads) directed against known polymorphisms The assignment of the HLA typing is determined by analysis software 17
18 rsso Luminex 18
19 High Resolution HLA Typing (SBT) SBT: Sequence Based Typing Genomic DNA is PCR amplified Each HLA locus is amplified with either generic primers or allele specific primers Amplified DNA is used as a template for sequencing reactions using fluorescent dye-labeled dideoxynucleotides (ddatp, ddttp, ddctp, ddgtp) Fluorescently labeled sequence fragments are analyzed by a capillary DNA sequencer for laser detection The assignment of the HLA typing is determined by analysis software 19
20 Sequence Based Typing (SBT) 20
21 HLA Typing Ambiguities Due to Heterozygous Sequence HLA Class I sequencing of exons 2 and 3 may give: HLA-B*44:02/*44:19N/*44:27 HLA-B*44:02 is the most common allele HLA-B*44:19N has a single base substitution in exon 1 (not analyzed) HLA-B*44:27 has a single base substitution in exon 4 (not analyzed) HLA-C*04:01/*04:09N HLA-C*04:01 is the most common allele HLA-C*04:09N has a single base deletion in exon 7 (not analyzed) HLA-DRB1 sequencing of exon 2 may give: HLA-DRB1*14:01/*14:54 HLA-DRB1*14:54 is the common allele HLA-DRB1*14:54 has a single base substitution in exon 3 (not analyzed) 21
22 Reporting of Ambiguous Heterozygous HLA Typing: Ambiguous HLA Typing Exon Identity A*02:16/02:131 A*02:03:01/02:03:06/02:253/02:264/02:370/02:480/02:505/ 02:557 B*13:02:01:01/13:02:01:02/13:02:01:03/13:02:05/13:02:09/ 13:02:16/13:38/13:69/13:96/13:99 C*16:01:01:01/16:01:01:02/16:01:01:03/16:01:01:04/16:58/ 16:97/16:100 DRB1*12:01:01:01/12:01:01:02/12:01:01:03/12:06/12:10/ 12:17 A*02:16P A*02:03:01G B*13:02:01G C*16:01:01G DRB1*12:01:01G Class I Exons 2 and 3 Class II Exon 2 22
23 CASE STUDY: HLA Typing Discrepancy Method Allele 1 Allele 2 rsso DRB1*14:01 DRB1*16:02 SBT (Typing Kit # 1) DRB1*14:01/ SBT (Typing Kit # 2) DRB1*14:01/54/113 DRB1*16 Novel 23
24 CASE STUDY: HLA Typing Discrepancy Codon 6, Position 16 24
25 Clinical Applications Solid Organ Transplantation Hematopoietic Stem Cell Transplantation Bone Marrow Cord Blood Non-transplant Applications Population Studies Disease Association Drug Sensitivity Platelet /Transfusion Medicine 25
26 HLA and Hematopoietic Cell Transplantation GvH HLA Mismatch Patient has HLA alleles not present in the donor Patient Donor A*02:01:01 A*23:01:01 A*02:01:01 A*02:01:01 B*08:01:01 B*07:01:01 B*08:01:01 B*07:01:01 C*03:03:01 C*07:01:01 C*03:03:01 C*07:01:01 DRB1*01:02:01 DRB1*04:01:01 DRB1*01:02:01 DRB1*01:02:01 DQB1*03:02:01 DQB1*05:01 DQB1*03:02:01 DQB1*05:01 26
27 HLA and Hematopoietic Cell Transplantation Rejection HLA Mismatch Donor has HLA alleles not present in the patient Patient Donor A*26:01:01 A*33:03:01 A*26:01:01 A*33:03:01 B*40:02:01 B*40:02:01 B*40:02:01 B*48:01:01 C*03:03:01 C*08:01:01 C*03:03:01 C*08:01:01 DRB1*03:01:01 DRB1*15:01:01 DRB1*03:01:01 DRB1*15:01:01 DQB1*02:01:01 DQB1*06:02:01 DQB1*02:01:01 DQB1*06:02:01 27
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