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HLA typing disease association and transplantation
Course Molecular Diagnostics Wim Dik Immunology, Erasmus MC
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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
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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
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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 β-chain α-chain DP DQ DR # of different alleles
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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: C A- C --- Polymporphisms contribute to amino acid substitution(s) in the peptide binding cleft of the HLA-molecule
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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 3 4 5 6 7 8 L α1 Tm cyt 3’UT α2 α3 Exon Protein domain HLA-I α-chain gene β1 β2 HLA-II β-chain gene
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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
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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
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HLA Antigens Alleles vs serological HLA specificities A B C DR DQ DP
Cw1 DR1 DQ1 DPw1 A2 B7 B51(5) Cw2 DR103 DQ2 DPw2 A203 B703 B5102 Cw3 DR2 DQ3 DPw3 A210 B8 B5103 Cw4 DR3 DQ4 DPw4 A3 B12 B52(5) Cw5 DR4 DQ5(1) DPw5 A9 B13 B53 Cw6 DR5 DQ6(1) DPw6 A10 B14 B54(22) Cw7 DR6 DQ7(3) A11 B15 B55(22) Cw8 DR7 DQ8(3) A19 B16 B56(22) Cw9(w3) DR8 DQ9(3) A23(9) B17 B57(17) Cw10(w3) DR9 A24(9) B18 B58(17) DR10 A2403 B21 B59 DR11(5) A25(10) B22 B60(40) DR12(5) A26(10) B27 B61(40) DR13(6) A28 B2708 B62(15) DR14(6) A29(19) B35 B63(15) DR1403 A30(19) B37 B64(14) DR1404 A31(19) B38(16) B65(14) DR15(2) A32(19) B39(16) B67 DR16(2) A33(19) B3901 B70 DR17(3) A34(10) B3902 B71(70) DR18(3) A36 B40 B72(70) A43 B4005 B73 DR51 A66(10) B41 B75(15) DR52 A68(28) B42 B76(15) DR53 A69(28) B44(12) B77(15) A74(19) B45(12) B78 A80 B46 B81 B47 B82 B48 B49(21)
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HLA-DNA nomenclature A* A*24 A*24:02 A*24:02:01 A*24:02:01:01 N L
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HLA-DNA nomenclature A* * = DNA-typing
A*24 exon variation, A*24 group, serologic A24 A*24:02 variation in DNA-sequence and in protein A*24:02:01 variation in DNA-sequence, NOT in protein A*24:02:01:01 variation in intron N Null allele (no expression) exp. A*24:09:N L Low expression exp. A*24:02:L
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HLA terminology DRB1*02 DQB1*03:02 DRB1*04:01 DRB1*03:01 DQB1*02
Allele: Haplotype: Genotype: (combination of HLA alleles on a single chromosome)
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Diversity of HLA Consequence: diversity in adaptive immune response
But also: predisposition to (auto-)immune disease
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HLA and auto-immune disease
etc. !
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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
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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
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HLA-B*27 molecular typing
DNA isolation
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HLA-B*27 molecular typing
PCR-SSP
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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
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HLA-B*27 PCR-SSP 2% agarose, EtBr stained control gene B*27
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HLA-B*27 TaqMan PCR
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HLA-B*27 PCR-SBT Highest resolution: sequence-based typing (SBT)
Advantage: specificity for disease association
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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
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HLA-A low resolution PCR-SSP
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HLA-A low resolution PCR-SSP
Patient with suspicion BSCR HLA-A low resolution PCR-SSP HLA-A*03 A*29
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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
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HLA-B low resolution PCR-SSP
Patient with suspicion Behçets disease HLA-B low resolution PCR-SSP 1 47 HLA-B*44 B*51
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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 1 2 3A 3C 3B Grade 1 >25IEL/100 EC Grade 2 also crypt hyperplasia Michael Marsh et al. Gastroenterology 1992 Grade 3 villous atrophy A: mild B: marked C: flat mucosa
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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
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HLA-DQ haplotypes in coeliacs
DQ2.5: DQA1*0501-DQB1*02 DQ2.2: DQA1*0201-DQB1*02 DQ8: DQA1*0301-DQB1*0302
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DQA and DQB PCR PCR: exon 2 derived fragment 2x DQA DNA 2x DQB
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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
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HLA-DQ2/8 MLPA X Van Beek et al Clin Chem Lab Med 2013;51:
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HLA-DQ2/8 PCR-Sequence Specific Oligonucleotide
Probe Typing (PCR-SSOP) Novel development, array based, in Immunology EMC Sensitive, specific and fast Yellow: HLA-DQA1 alleles Green: HLA-DQB1 alleles
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HLA-DQ2/8 Array based
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Diversity of HLA Consequence: diversity in adaptive immune response
But also: predisposition to (auto-)immune disease Adverse reactions to medication
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HLA and drug hypersensitivity
Illing PT et al. Current Opinion in Immunology 2013;25:81-89
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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: Illing PT et al. Current Opinion in Immunology 2013;25:81-89
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HLA-B*57:01 genotyping (PCR-SSP)
2 4 3 5 6 8 7 9 10 11 12 13 14 15 16
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HLA-DNA nomenclature and typing in disease association/drug reaction
A* * = DNA-typing A*24 exon variation, A*24 group, serologic A24 A*24:02 variation in DNA-sequence and in protein A*24:02:01 variation in DNA-sequence, NOT in protein A*24:02:01:01 variation in intron N Null allele (no expression) exp. A*24:09:N L Low expression exp. A*24:02:L
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Diversity of HLA Consequence: diversity in adaptive immune response
But also: predisposition to (auto-)immune disease Adverse reactions to medication transplant rejection
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4 days post transplant 12 days post transplant
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HLA I&II match HLA I mismatch HLA II mismatch 4 days post transplant
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Transplantation and matching
Solid organs Kidney: HLA-A ,-B, -DRB1 Hematological more extensive: HLA-A, -B, -C, -DRB1, -DQB1 –DPB1
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PCR-SSOP with Luminex technology
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HLA-DNA nomenclature and typing in transplantation
A* * = DNA-typing A*24 exon variation, A*24 group, serologic A24 A*24:02 variation in DNA-sequence and in protein A*24:02:01 variation in DNA-sequence, NOT in protein A*24:02:01:01 variation in intron N Null allele (no expression) exp. A*24:09:N L Low expression exp. A*24:02:L
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HLA-DNA nomenclature and typing in transplantation
A* * = DNA-typing A*24 exon variation, A*24 group, serologic A24 A*24:02 variation in DNA-sequence and in protein A*24:02:01 variation in DNA-sequence, NOT in protein A*24:02:01:01 variation in intron N Null allele (no expression) exp. A*24:09:N L Low expression exp. A*24:02:L
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