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HLA typing d isease association and transplantation

HLA typing d isease association and transplantation. 03-11-2017 Course Molecular Diagnostics Wim Dik Immunology, Erasmus MC. HLA (MHC) class I and II. HLA: human leukocyte antigen major histocompatibility complex (MHC) molecules. Function:

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HLA typing d isease association and transplantation

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  1. HLA typingdisease association and transplantation 03-11-2017 Course Molecular Diagnostics Wim Dik Immunology, Erasmus MC

  2. HLA (MHC) class I and II HLA: human leukocyte antigen major histocompatibility complex (MHC) molecules Function: Presentation of peptide antigens (epitopes) to the immune system Classical HLA class I genes: HLA-A, -B, -C => ligand for TCR on CD8+ T cell Classical HLA class II genes: HLA-DR, -DQ, -DP => ligand for TCR on CD4+ T cell

  3. Structure of MHC class I and class II 6p21.31 Peptide binding-cleft heavy (α) glycoprotein chain ~44 kDa heavy (α) glycoprotein chain ~34 kDa light (β) glycoprotein chain ~28 kDa β2-microglobulin light chain ~12 kDa (chromosome 15) HLA-DP A/B HLA-DQ A/B HLA-DR A/B HLA-A HLA-B HLA-C

  4. HLA polymorphism HLA class I and II are the most polymorphic molecules/genes of the human genome: they thus show the highest diversity in allelic variants within the human population DR β-chain α-chain # of different alleles DQ DP β-chain α-chain β-chain α-chain

  5. HLA polymorphism B*07:02 GC TCC CAC TCC ATG AGG TAT TTC TAC ACC TCC GTG TCC CGG CCC GGC CGC GGG GAG CCC CGC TTC ATC TCA GTG B*27:01 -- --- --- --- --- --- --- --- C - - --- --- --- --- --- --- --- --- --- --- --- --- --- --- A- C --- Polymporphisms contribute to amino acid substitution(s) in the peptide binding cleft of the HLA-molecule

  6. HLA polymorphism: variation peptide binding cleft HLA-class II DR: only variation in β-chain DP and DQ: variation in β- and α-chain 1 2 2 3 3 4 4 5 5 6 7 6 8 Exon Exon Protein domain β1 β2 Tm cyt 3’UT Protein domain L α1 α2 α3 Tm 3’UT cyt HLA-I α-chain gene HLA-II β-chain gene 1

  7. HLA polymorphism and polygeny Expression of HLA alleles is codominant: proteins of both alleles are expressed equally by the cell, and both proteins can present antigens to T-cells ...BOTH contribute to the diversity of MHC molecules expressed by an individual

  8. HLA polymorphism and polygeny At the population level as many alleles are present at significant frequencies The diversity of HLA antigens endows populations with the increased capacity to deal with and respond to a wide variety of foreign antigens and pathogens that may change in time and place

  9. HLA Antigens Alleles vs serological HLA specificities

  10. HLA-DNA nomenclature • A* • A*24 • A*24:02 • A*24:02:01 • A*24:02:01:01 • N • L

  11. HLA-DNA nomenclature • A* * = DNA-typing • A*24 exonvariation, A*24 group, serologic A24 • A*24:02variation in DNA-sequenceand in protein • A*24:02:01variation in DNA-sequence, NOT in protein • A*24:02:01:01variation in intron • N Nullallele (no expression) exp. A*24:09:N • L Low expressionexp. A*24:02:L

  12. HLA terminology DRB1*04:01 Allele: (combination of HLA alleles on a single chromosome) Haplotype: DRB1*04:01 DQB1*03:02 DRB1*04:01 DQB1*03:02 Genotype: DRB1*03:01 DRB1*02 DQB1*02

  13. Diversity of HLA Consequence: diversity in adaptive immune response But also: predisposition to (auto-)immune disease

  14. HLA and auto-immune disease etc. !

  15. Bechterew’s disease Ankylosing spondylitis (AS) Spondylitis ankylopoëtica (SpA) Inflammation of joints in spine and pelvis X-ray Grade 0: normal Grade 4: severe sacroiliitis

  16. HLA-B27 allelic variation 2017: HLA-B*27:01 to HLA-B*27:164 Strong association with Bechterew’s disease: B*27:05 (Caucasian) B*27:04 (Asian) B*27:02 (Mediterranean) Also: B*27:01, B*27:03, B*27:07, B*27:08, B*27:10, B*27:13, B*27:14, B*27:15, B*27:19, B*27:25, etc… Weak association: B*27:06 en B*27:09

  17. HLA-B*27 molecular typing DNA isolation

  18. HLA-B*27 molecular typing PCR-SSP

  19. HLA-B*27 PCR-SSP PCR-SSP Olerup method (exon 2) Olerup O. HLA-B27 typing by a group-specific PCR amplification. Tissue Antigens 1994;43:253–6 PCR-SSP Dominguez method (exon 3) Dominguez O, Coto E, Martinez-Naves E, Choo SY, Lopez-Larrea C Molecular typing of HLA-B27 alleles. Immunogenetics 1992;36:277–82

  20. HLA-B*27 PCR-SSP 2% agarose, EtBr stained control gene B*27

  21. HLA-B*27 TaqMan PCR

  22. HLA-B*27 PCR-SBT Highest resolution: sequence-based typing (SBT) Advantage: specificity for disease association

  23. HLA-A29 and Birdschot chorioretinopathy BSCR: (auto)immune disorder of the eye 2017: HLA-A*29:01 to HLA-A*29:104 ~ 100% of BSCR is HLA-A29 positive HLA-A*29:02 HLA-A*29:01 HLA-A*29:10

  24. HLA-A low resolution PCR-SSP

  25. Patient with suspicion BSCR HLA-A low resolution PCR-SSP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 HLA-A*03 A*29

  26. HLA-B51 and Behçets disease Behçet’s disease (BD): systemic auto-inflammatory occlusive vasculitis presenting with oral and genital aphthous ulcers, skin lesions and ocular inflammaiton de Menthon et al, Arthritis & Rheumatism 2009:61;1287–1296

  27. Patient with suspicion Behçets disease HLA-B low resolution PCR-SSP 1 47 HLA-B*44 B*51

  28. Coeliac disease => small intestinal inflammation and malabsorption develops after dietary exposure of the small intestine to gluten peptides derived from wheat, barley or rye H&E CD3 H&E CD3 3A 0 1 3B 2 3C Grade 3 villous atrophy A: mild B: marked C: flat mucosa Michael Marsh et al. Gastroenterology 1992 Grade 1 >25IEL/100 EC Grade 2 also crypt hyperplasia

  29. HLA and coeliac disease ~30-40% general population is HLA-DQ2 or -DQ8 positive ~98% of CD patients is HLA-DQ2 and/or -DQ8 positive HLA-DQ typing: clinical relevance >> high negative predictive value

  30. HLA-DQ haplotypes in coeliacs DQ2.5: DQA1*0501-DQB1*02 DQ2.2: DQA1*0201-DQB1*02 DQ8: DQA1*0301-DQB1*0302

  31. DQA and DQB PCR PCR: exon 2 derived fragment 2x DQA DNA 2x DQB

  32. Multiplex ligation-dependent probe amplification (MLPA) All ligated probes have indentical end sequences, permitting simultaneous PCR amplification using one primer pair Schouten et al Nucleic Acids Research 2002;30:e57

  33. HLA-DQ2/8 MLPA X Van Beek et al Clin Chem Lab Med 2013;51:1191-1198

  34. HLA-DQ2/8 PCR-Sequence Specific Oligonucleotide Probe Typing (PCR-SSOP) Novel development, array based, in use @ Immunology EMC Sensitive, specific and fast Yellow: HLA-DQA1 alleles Green: HLA-DQB1 alleles

  35. HLA-DQ2/8 Array based

  36. Diversity of HLA Consequence: diversity in adaptive immune response But also: predisposition to (auto-)immune disease Adverse reactions to medication

  37. HLA and drug hypersensitivity Illing PT et al. Current Opinion in Immunology 2013;25:81-89

  38. HLA-B*57:01 and Abacavir hypersensitivity syndrome Abacavir: nucleoside reverse transcriptase inhibitor used for treatment of HIV infection Pharmacological interaction with immune receptor concept (p-i concept) Chaponda M. and Pirmohamed M. Br J Clin Pharmacol 2011;71:659-671 Illing PT et al. Current Opinion in Immunology 2013;25:81-89

  39. HLA-B*57:01 genotyping (PCR-SSP) 4 2 16 10 1 5 6 7 8 9 3 11 12 13 14 15

  40. HLA-DNA nomenclatureandtyping in diseaseassociation/drug reaction • A* * = DNA-typing • A*24 exonvariation, A*24 group, serologic A24 • A*24:02variation in DNA-sequenceand in protein • A*24:02:01variation in DNA-sequence, NOT in protein • A*24:02:01:01variation in intron • N Nullallele (no expression) exp. A*24:09:N • L Low expressionexp. A*24:02:L

  41. Diversity of HLA Consequence: diversity in adaptive immune response But also: predisposition to (auto-)immune disease Adverse reactions to medication transplant rejection

  42. 4 days post transplant 12 days post transplant

  43. 4 days post transplant 12 days post transplant HLA I&II match HLA I mismatch HLA II mismatch

  44. Transplantation and matching Solid organs Kidney: HLA-A ,-B, -DRB1 Hematological more extensive: HLA-A, -B, -C, -DRB1, -DQB1 –DPB1

  45. PCR-SSOPwith Luminex technology

  46. HLA-DNA nomenclatureandtypingin transplantation • A* * = DNA-typing • A*24 exonvariation, A*24 group, serologic A24 • A*24:02variation in DNA-sequenceand in protein • A*24:02:01variation in DNA-sequence, NOT in protein • A*24:02:01:01variation in intron • N Nullallele (no expression) exp. A*24:09:N • L Low expressionexp. A*24:02:L

  47. HLA-DNA nomenclatureandtypingin transplantation • A* * = DNA-typing • A*24 exonvariation, A*24 group, serologic A24 • A*24:02variation in DNA-sequenceand in protein • A*24:02:01variation in DNA-sequence, NOT in protein • A*24:02:01:01variation in intron • N Nullallele (no expression) exp. A*24:09:N • L Low expressionexp. A*24:02:L

  48. ?? w.dik@erasmusmc.nl

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