1. HLA & Leukemia Associations:
What do they mean?
M. Tevfik DORAK
Environmental & Occupational Health
College of Public Health
Florida International University
Miami, USA

2. - Unravelling HLA associations?
- Why leukamia, why HLA?
- Unravelling HLA associations?
- What do they mean?
- HLA or MHC - LD?

3. Cooke & Hill. Nature Rev Genet 2001 (www)
Human Major Histocompatibility Complex
- HLA Complex -
Cooke & Hill. Nature Rev Genet 2001 (www)

4. Expressed HLA-DRB gene content of HLA class II haplotypes
(second DRB gene determines the ancestral lineage)
DRB1
DRB5
DRB3
DRB4
DRB1*10
DRB1*01
DRB1*15/16
DRB1*03
DRB1*04
DRB1*11/12
DRB1*13/14
DRB1*07
DRB1*08
DRB1*09

5. DRB4 / DRB5 / DRB1*01/10
DRB3 / DRB1*08
Klein, 1990 (www)

6. DRB3 / DRB1*08
DRB4 / DRB5 / DRB1*01/10
Ayala, 1994 (www)

7. (www)

8. HLA-DRB3 and -DRB4 haplotypes represent ancestral lineages of contemporary MHC haplotypes
See Kennedy, Singh & Dorak, 2012 for DRB4 lineage-specific SNP rs :

9. Unintentional Evolutionary-Based Haplotype Analysis

10. Search for a Leukemia Susceptibility Gene in the HLA Complex
Chronic Myeloid Leukemia (CML): Scotland and Turkey
Chronic Lymphoid Leukemia (CLL): Germany
Childhood Acute Lymphoblastic Leukemia (ALL):
Wales, Scotland and Turkey
All showed some form of HLA-DRB4 (DR53) association

11. Why Leukemia?

12. Inbred mouse strains are homozygous for H-2 haplotypes
Heterozygosity for the susceptibility haplotype
did not have an effect

13. Spontaneous leukemia shows the same association

14. Dorak et al, 1994 (www)

15. Conventional analysis shows no association even with homozygosity

16. Stratification by age revealed associations

19. P = 0.02
DR53 homozygosity was a risk marker (P = 0.01; OR = 3.36) and DR52 homozygosity was protective (P = 0.007; OR = 0.51).
Oguz et al, 2003 (www) (PDF)

20. Dorak et al, 1996 (www)

21. Reminder: Mouse H-2 Studies

22. Dorak et al, 1999 (www)

23. Globocan 2002, IARC (www)

24. Globocan 2002, IARC (www)

25. Leukemia is more frequent in males and in developed countries

26. Cartwright RA et al. Sex ratios and the risks of haematological malignancies. BJH 2002 (www)

27. xMHC Loci Analysed in Childhood ALL6p
HSPA1B
LTA
EDN1
HLA-DRB1
HLA-DRB3/4/5
HLA-Bw
HFE BF
TNF
HLA CLASS III REGION

28. Conventional Analysis

29. Conventional Analysis

30. HLA-DRB4 Association in Childhood ALL
Homozygosity for HLA-DRB4 family is associated with susceptibility to childhood ALL
in boys only (P < , OR = 6.1, 95% CI = 2.9 to 12.6 )
Controls are an unselected group of local newborns (201 boys & 214 girls)
* Case-only analysis P = (OR = 5.6; 95% CI = 1.8 to 17.6)
This association extends to a DRB4-HSP70 haplotype (OR = 8.3; 95% CI = 3.0 to 22.9)
This association has been replicated in Scotland and Turkey % % *
Boys, n=64
Girls, n=53 *
Dorak et al, 1999 (www)

31. HLA-DRB4 ASSOCIATION ADDITIVE MODEL Linear Model
Logit estimates Number of obs =
LR chi2(1) =
Prob > chi2 =
Log likelihood = Pseudo R =
caco | Common Odds Ratio Std. Err. z P>|z| [95% CI]
drb4add |
Heterozygosity and Homozygosity
LR chi2(2) =
Prob > chi2 =
Log likelihood = Pseudo R =
caco | Odds Ratio Std. Err z P>|z| [95% Conf. Interval]
Wild-type | (ref)
Heterozygosity |
Homozygosity |

32. HLA-DRB4 - HSPA1B HAPLOTYPE ASSOCIATION
EFFECT MODIFICATION
Logit estimates Number of obs =
LR chi2(3) =
Prob > chi2 =
Log likelihood = Pseudo R =
caco | Coef. Std. Err z P>|z| [95% Conf. Interval]
sex |
hsp53 |
_IsexXhsp5~2 |
_cons |
CONFOUNDING BY SEX
LR chi2(2) =
Prob > chi2 =
Log likelihood = Pseudo R =
caco | Odds Ratio Std. Err z P>|z| [95% Conf. Interval]
hsp53 |
sex |
Adjusted for sex?

33. HLA-DRB4 - HSPA1B HAPLOTYPE ASSOCIATION
BOYS ONLY
Logit estimates Number of obs =
LR chi2(1) =
Prob > chi2 =
Log likelihood = Pseudo R =
caco | Odds Ratio Std. Err z P>|z| [95% Conf. Interval]
hsp53 |
GIRLS ONLY
Logit estimates Number of obs =
LR chi2(1) =
Prob > chi2 =
Log likelihood = Pseudo R =
hsp53 |
The association is modified by sex…

34. HFE-C282Y Association in Childhood ALL% %
WELSH GROUP
117 patients newborns
P = 0.005; OR = 2.8 (1.4 to 5.4)
In cALL: P = 0.02; OR = 2.9 (1.4 to 6.4)
SCOTTISH GROUP
135 patients newborns
P = ; OR = 3.0 (1.7 to 5.4)
In cALL: P < ; OR = 4.7 (2.5 to 8.9)
Dorak et al, 1999 (www)

35. EDN1 Association in Childhood ALL
Preliminary findings %
In a preliminary study in childhood ALL, EDN1 STR
207 bp allele (A6) frequency is increased in males
(P = 0.004; OR = 4.5, 95% CI = 1.6 to 13.0)

36. Final Multivariable Logistic Regression Model (boys)
Logit estimates Number of obs =
LR chi2(2) =
Prob > chi2 =
Log likelihood = Pseudo R =
caco | Odds Ratio Std. Err z P>|z| [95% Conf. Interval]
hsp53 |
hfe-c282y |
Attributable fraction for HLA-DRB4 - HSPA1B association: 15.2% (95% CI = 8.6 to 21.3)
Attributable fraction for HFE - C282Y association : 8.8% (95% CI = 0.0 to 17.4)
1 in 3 boys with ALL are homozygote for the ancestral DRB4 haplotype and 1 in 4 boys are positive for the HFE-C282Y mutation

37. Despite the obvious effect modification by sex, and recessive nature of most MHC associations in childhood leukemia, old habits are maintained and most studies only compare all cases with all controls, and using only one genetic model.
And, find nothing!

38. Possible Mechanisms of HLA-DRB4 Associations in Childhood ALL
Spurious
Population stratification, bias, chance
Causal I (immunological)
HLA-DRB4 family of haplotypes are functionally suboptimal in their antigen presentation role
Causal I (genetical)
Linkage disequilibrium with non-HLA genes has to be ruled out

39. Possible Mechanisms of HLA-DRB4 Associations in Childhood ALL
Spurious
Population stratification, bias, chance
Current Childhood ALL Case-Control Set
Genomic Control
Replication
Gene x Gene Interactions
(Tyneside Leukaemia Research Association funding secured)

40. Possible Mechanisms of HLA-DRB4 Association in Childhood ALL
HLA-DRB4 family of haplotypes are functionally suboptimal in their antigen presentation role

41. HLA-DRB4 family of haplotypes are functionally suboptimal in their antigen presentation role
Immune nonresponsiveness is a recessive trait
Supported by association with homozygosity and association in boys

42. HLA-DRB4 family of haplotypes are functionally suboptimal in their antigen presentation role
HLA-DRB4 family of haplotypes express DRB1 and DRB4 genes at a lower level
Louis, 1994 (www); Vincent, 1996 (www) & 1997 (www)
The lowest cumulative expression of HLA-DRB genes (even lower in homozygotes) may result in:
- DRa-DQb mixed isotype heterodimer formation
- Aberrant T-cell response and autoimmune reactions
Charron, 1984 (www); Lotteau, 1987 (www) & 1989 (www); Matsunaga, 1990 (www); Spencer 1989 (www) & 1992 (www) & 1993 (www)
Supported by association with homozygosity

43. HLA-DRB4 family of haplotypes are functionally suboptimal in their antigen presentation role
HLA-DR53 interacts poorly with CD4
(www)
Supported by association with homozygosity

44. HLA-DRB4 family of haplotypes are functionally suboptimal in their antigen presentation role
HLA-DRB1 residue 81 substitution severely affects intracellular transport of the mutant DRb chain
Chervonsky, 1994 (www)
HLA-DRB4 residue 81 is naturally different from that of all other DRB1/3/5 molecules

45. HLA-DRB4 family of haplotypes may be involved in susceptibility through molecular mimicry
HLA-DRB4 HVR3 epitope (LLERRRAE) is mimicked in its entirety by adenovirus and EBV
Dorak et al, 1994 (www)

46. HLA-DRB4 family of haplotypes may be involved in susceptibility through molecular mimicry
Anti-HLA autoantibodies are present in other infectious diseases due to molecular mimicry
Dorak et al, 1996 (www)

47. Dorak et al, 1996 (www)

48. Possible Mechanisms of HLA-DRB4 Associations in Childhood ALL
Spurious
Population stratification, bias, chance
Causal I (immunological)
HLA-DRB4 family of haplotypes are functionally suboptimal in their antigen presentation role
Causal II (genetical)
Linkage disequilibrium with non-HLA genes has to be ruled out

49. Cooke & Hill. Nature Rev Genet 2001 (www)
Human Major Histocompatibility Complex
- simple map -
Cooke & Hill. Nature Rev Genet 2001 (www)

50. Most gene-dense region in the genome
Human Major Histocompatibility Complex
Most gene-dense region in the genome
Xie, 2003 (www)

51. (www)

52. Major Study of MHC Haplotypes
Dorak et al
CYP21A2
HSPA1A/B
NCR3
LTA
DQA1
HLA-B
DRB1
MICA
NOTCH4 BF
AIF1
TNF
NFKBIL1
MHC CLASS III REGION
Dorak et al, 2006 (www)

53. (www)
Dorak et al, 2006 (www)

57. Wenshuo Shao, Richard A. Kaslow, M. Tevfik Dorak
TWO SNPs at HSPA1B and HLA-DQA1 IDENTIFY ANCESTRAL MHC CLASS II LINEAGES
Wenshuo Shao, Richard A. Kaslow, M. Tevfik Dorak
Department of Epidemiology, University of Alabama at Birmingham, Birmingham, Alabama, United States
ASHI 2004 Poster
Dorak et al, 2006 (www)

58. Dorak et al, 2006 (www)

59. Possibilities PRKRAP (Chida et al, 2001 (www)) RXRB and HLA-DPB1
HLA-G and HLA-DRB
POU5F1, TCF19, PBX2, NOTCH4, BRD2, KIFC1, ZNRD1
See also Shiina, 2004 (www)

60. HLA-DR Haplotypes & PRKRAP
IMGT (www)

61. Example: Linkage Disequilibrium
HLA-B47 association with congenital adrenal hyperplasia (Dupont et al, Lancet 1977)
HLA-B14 association with late-onset adrenal hyperplasia (Pollack et al, Am J Hum Genet 1981)
Is congenital adrenal hyperplasia an immune system-mediated disease?

62. Example: Linkage Disequilibrium
HLA-B47 association with congenital adrenal hyperplasia is due to deletion of CYP21A2 on HLA-B47DR7 haplotype
HLA-B14 association with late-onset adrenal hyperplasia is due to an exon 7 missense mutation (V281L) in CYP21A2 on HLA-B14DR1 haplotype
" increased sensitivity of haplotype analysis "

63. Preliminary evidence of an association between HLA-DPB1
Preliminary evidence of an association between HLA-DPB1*0201 and childhood common ALL supports an infectious aetiology
Leukemia 1995;9(3):440-3
Evidence that an HLA-DQA1-DQB1 haplotype influences susceptibility to childhood common ALL in boys provides further support for an infection-related aetiology
Br J Cancer 1998;78(5):561-5
Why not LD?

64. any genetic association
Rajsbaum, 2002 (www)
Linkage disequilibrium 'confounding by locus' has to be ruled out before attributing a direct causal role to
any genetic association

65. Extended HLA Class II Region (HLA-DPB2 to KIFC1)
33,100M to 34,480M
Map Viewer (www)

66. Further Centromeric Region (35,3M to 36,8M)
Map Viewer (www)

67. Predicted and Experimental Binding of Peptides to Class I MHC Molecules
(www)

68. HLA association studies need to be extended to:
Epigenetics
Regulatory region polymorphisms
Allelic expression differences
Epistatic interactions
Haplotypes
Post-translational modifications
Proteomics

69. HLA association studies need to be extended to:
Post-translational modifications
(www)

70. Why is there a gender effect in genetic susceptibility?
From the time of fertilization, males are
subject to selection
Newborn ' male disadvantage ' is well-known
Childhood cancers are more frequent in boys
Males live shorter than females: ' the fragile male '

71. Selection Against Males
A newborn boy has a 1 in 300 chance of developing a cancer by age 20 as opposed to 1 in 333 chance for a newborn girl
This corresponds to about 10% higher risk for boys!
The risk is higher for male zygotes for failure during embryogenesis too
For each 100 females, 54 males are lost during pregnancy!
Is there a link?

72. Prenatal Selection Against Males
For each 100 females, 54 males are lost during pregnancy
M/F

73. Prenatal Selection Against Males
Survivors of threatened abortions are at higher risk for childhood leukaemia
Parental HLA sharing is a risk marker for both recurrent miscarriage and childhood leukaemia
Miscarriage history is associated with higher risk for childhood leukaemia

74. Prenatal Selection Against Males
One determinant is HLA-DRB3/4 homozygosity
Dorak et al. Genes & Immunity 2002;3: (www) %
P = 0.007

75. HLA-G
Human homologue of the mouse Ped (preimplantation embryo development) gene
Preimplantation embryonic expression
Associations with birth weight and miscarriages
GeneID: 3135 (www)

76. Hviid, 2005 (www)

77. POU5F1 POU domain, class 5, transcription factor 1
(OCT3, OCT4, OTF3, OTF4)
Expressed in preimplantation embryos, stem cells and cancer
GeneID: (www)
Gill TJ 3rd
The borderland of embryogenesis and carcinogenesis.
MHC-linked genes affecting development and their possible relationship to the development of cancer Biochim Biophys Acta 1984;738(3):93-102

78. MHC > HLA
Many non-HLA genes within the MHC may be responsible for HLA associations observed in many diseases including childhood ALL

79. ACKNOWLEDGEMENTS Glasgow Leukaemia Research Laboratory
I am grateful to all who have taught, helped and supported in one way or another at
Glasgow Leukaemia Research Laboratory
Glasgow Tissue Typing Laboratory
Glasgow Royal Hospital for Sick Children
University of Wales College of Medicine
Welsh Transplantation & Immunogenetics Laboratory
HLA Lab, Martin Luther University, Halle, Germany
St Jude Children’s Hospital, HLA Laboratory
University of Tennessee at Memphis
University of Alabama at Birmingham
Newcastle University (U.K.)
School of Clinical Medical Sciences (Child Health)
HUMIGEN LLC, The Institute for Genetic Immunology, Hamilton, NJ

80. Thanks for the support to:
(www)
(www)
(www)