1. HLA typing disease association and transplantation
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
β-chain
α-chain DP DQ DR
# of different alleles

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: 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 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

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 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)

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 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

12. 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)

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. HLA-A low resolution PCR-SSP
Patient with suspicion BSCR
HLA-A low resolution PCR-SSP
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. HLA-B low resolution PCR-SSP
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 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

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:

34. 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

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:
Illing PT et al. Current Opinion in Immunology 2013;25:81-89

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

40. 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

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

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

45. PCR-SSOP with Luminex technology

46. 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

47. 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

49. ??