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

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Presentation on theme: "HLA & Leukemia Associations: What do they mean? M. Tevfik DORAK Environmental & Occupational Health College of Public Health Florida International University."— Presentation transcript:

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

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

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

4 Expressed HLA-DRB gene content of HLA class II haplotypes (second DRB gene determines the ancestral lineage) DRB1DRB5DRB3DRB4 DRB1*10 DRB1*01 DRB1*15/16 DRB1*03 DRB1*04 DRB1*11/12 DRB1*13/14 DRB1*07 DRB1*08 DRB1*09

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

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

7 (www)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 Heterozygosity for the susceptibility haplotype did not have an effect Inbred mouse strains are homozygous for H-2 haplotypes

13 Spontaneous leukemia shows the same association

14 Dorak et al, 1994 (www)www

15 Conventional analysis shows no association even with homozygosity

16 Stratification by age revealed associations

17

18

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

20 Dorak et al, 1996 (www)www

21 Reminder: Mouse H-2 Studies

22 Dorak et al, 1999 (www)www

23 Globocan 2002, IARC (www)www

24 Globocan 2002, IARC (www)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)www

27 BF HSPA1BLTA TNF HLA CLASS III REGION HLA-DRB3/4/5 HLA-DRB1 EDN1 HFEHLA-Bw xMHC Loci Analysed in Childhood ALL 6p

28 Conventional Analysis

29

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)www

31 ADDITIVE MODEL Linear Model Logit estimates Number of obs = 265 LR chi2(1) = Prob > chi2 = Log likelihood = Pseudo R2 = caco | Common Odds Ratio Std. Err. z P>|z| [95% CI] drb4add | Heterozygosity and Homozygosity Logit estimates Number of obs = 265 LR chi2(2) = Prob > chi2 = Log likelihood = Pseudo R2 = caco | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval] Wild-type | 1.00 (ref) Heterozygosity | Homozygosity | HLA-DRB4 ASSOCIATION

32 EFFECT MODIFICATION Logit estimates Number of obs = 532 LR chi2(3) = Prob > chi2 = Log likelihood = Pseudo R2 = caco | Coef. Std. Err. z P>|z| [95% Conf. Interval] sex | hsp53 | _IsexXhsp5~2 | _cons | CONFOUNDING BY SEX Logit estimates Number of obs = 532 LR chi2(2) = Prob > chi2 = Log likelihood = Pseudo R2 = caco | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval] hsp53 | sex | Adjusted for sex? HLA-DRB4 - HSPA1B HAPLOTYPE ASSOCIATION

33 BOYS ONLY Logit estimates Number of obs = 265 LR chi2(1) = Prob > chi2 = Log likelihood = Pseudo R2 = caco | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval] hsp53 | GIRLS ONLY Logit estimates Number of obs = 267 LR chi2(1) = 0.13 Prob > chi2 = Log likelihood = Pseudo R2 = caco | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval] hsp53 | The association is modified by sex… HLA-DRB4 - HSPA1B HAPLOTYPE ASSOCIATION

34 HFE - C282Y Association in Childhood ALL 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) 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) % % Dorak et al, 1999 (www)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 = ; OR = 4.5, 95% CI = 1.6 to 13.0)

36 Final Multivariable Logistic Regression Model (boys) Logit estimates Number of obs = 265 LR chi2(2) = Prob > chi2 = Log likelihood = Pseudo R2 = 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 Supported by association with homozygosity and association in boys Immune nonresponsiveness is a recessive trait

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

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

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 DR chain HLA-DRB4 residue 81 is naturally different from that of all other DRB1/3/5 molecules Chervonsky, 1994 (www)www

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)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)www

47 Dorak et al, 1996 (www)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)www Human Major Histocompatibility Complex - simple map -

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

51 (www)www

52 NOTCH4 CYP21A2 BF NCR3HSPA1A/B AIF1 LTA TNFNFKBIL1 MHC CLASS III REGION DRB1 DQA1HLA-B MICA Major Study of MHC Haplotypes Dorak et al Dorak et al, 2006 (www)www

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

54

55

56

57 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)www

58 Dorak et al, 2006 (www)www

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

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

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 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 " Example: Linkage Disequilibrium

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

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

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

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

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

69 Post-translational modifications (www)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;3:263-9 (www)www P = %

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)www

76 Hviid, 2005 (www)www

77 POU5F1 POU domain, class 5, transcription factor 1 (OCT3, OCT4, OTF3, OTF4) Expressed in preimplantation embryos, stem cells and cancer GeneID: (www)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 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 Childrens 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)www (www)www


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