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

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

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

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

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

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

(www)

HLA-DRB3 and -DRB4 haplotypes represent ancestral lineages of contemporary MHC haplotypes See Kennedy, Singh & Dorak, 2012 for DRB4 lineage-specific SNP rs2395185: http://jnci.oxfordjournals.org/content/104/11/884.long

Unintentional Evolutionary-Based Haplotype Analysis

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

Why Leukemia?

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

Spontaneous leukemia shows the same association

Dorak et al, 1994 (www)

Conventional analysis shows no association even with homozygosity

Stratification by age revealed associations

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)

Dorak et al, 1996 (www)

Reminder: Mouse H-2 Studies

Dorak et al, 1999 (www)

Globocan 2002, IARC (www)

Globocan 2002, IARC (www)

Leukemia is more frequent in males and in developed countries

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

xMHC Loci Analysed in Childhood ALL 6p21.3 - 24.1 HSPA1B LTA EDN1 HLA-DRB1 HLA-DRB3/4/5 HLA-Bw HFE BF TNF HLA CLASS III REGION

Conventional Analysis

Conventional Analysis

HLA-DRB4 Association in Childhood ALL Homozygosity for HLA-DRB4 family is associated with susceptibility to childhood ALL in boys only (P < 0.0001, 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 = 0.002 (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)

HLA-DRB4 ASSOCIATION ADDITIVE MODEL Linear Model Logit estimates Number of obs = 265 LR chi2(1) = 14.24 Prob > chi2 = 0.0002 Log likelihood = -139.37794 Pseudo R2 = 0.0486 ------------------------------------------------------------------------------ caco | Common Odds Ratio Std. Err. z P>|z| [95% CI] -------------+---------------------------------------------------------------- drb4add | 2.208651 .4734163 3.70 0.000 1.45103 - 3.36186 Heterozygosity and Homozygosity LR chi2(2) = 22.00 Prob > chi2 = 0.0000 Log likelihood = -135.49623 Pseudo R2 = 0.0751 caco | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval] Wild-type | 1.00 (ref) Heterozygosity | 1.060652 .3557426 0.18 0.861 .549642 2.04676 Homozygosity | 6.258503 2.65464 4.32 0.000 2.72534 14.37211

HLA-DRB4 - HSPA1B HAPLOTYPE ASSOCIATION EFFECT MODIFICATION Logit estimates Number of obs = 532 LR chi2(3) = 23.97 Prob > chi2 = 0.0000 Log likelihood = -268.27826 Pseudo R2 = 0.0428 ------------------------------------------------------------------------------ caco | Coef. Std. Err. z P>|z| [95% Conf. Interval] -------------+---------------------------------------------------------------- sex | -.0299037 .2229554 -0.13 0.893 -.4668883 .4070808 hsp53 | 2.530033 .5929603 4.27 0.000 1.367852 3.692214 _IsexXhsp5~2 | -2.758189 .8812645 -3.13 0.002 -4.485436 -1.030943 _cons | -1.321474 .3517969 -3.76 0.000 -2.010984 -.6319651 CONFOUNDING BY SEX LR chi2(2) = 11.99 Prob > chi2 = 0.0025 Log likelihood = -274.26995 Pseudo R2 = 0.0214 caco | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval] hsp53 | 3.32777 1.191429 3.36 0.001 1.649684 6.712832 sex | .7693041 .1636106 -1.23 0.218 .5070725 1.167148 Adjusted for sex?

HLA-DRB4 - HSPA1B HAPLOTYPE ASSOCIATION BOYS ONLY Logit estimates Number of obs = 265 LR chi2(1) = 22.41 Prob > chi2 = 0.0000 Log likelihood = -135.29119 Pseudo R2 = 0.0765 ------------------------------------------------------------------------------ caco | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval] -------------+---------------------------------------------------------------- hsp53 | 12.55392 7.444028 4.27 0.000 3.926876 40.13392 GIRLS ONLY Logit estimates Number of obs = 267 LR chi2(1) = 0.13 Prob > chi2 = 0.7205 Log likelihood = -132.98706 Pseudo R2 = 0.0005 hsp53 | 0.796 .5189439 -0.35 0.726 .22181 2.856571 The association is modified by sex…

HFE-C282Y Association in Childhood ALL % % WELSH GROUP 117 patients - 415 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 - 238 newborns P = 0.0004; OR = 3.0 (1.7 to 5.4) In cALL: P < 0.0001; OR = 4.7 (2.5 to 8.9) Dorak et al, 1999 (www)

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)

Final Multivariable Logistic Regression Model (boys) Logit estimates Number of obs = 265 LR chi2(2) = 26.68 Prob > chi2 = 0.0000 Log likelihood = -133.15366 Pseudo R2 = 0.0911 ------------------------------------------------------------------------------ caco | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval] -------------+---------------------------------------------------------------- hsp53 | 12.90181 7.713223 4.28 0.000 3.99731 41.64217 hfe-c282y | 2.25126 .860627 2.12 0.034 1.064196 4.762431 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

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!

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

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)

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

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

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

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

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

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)

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)

Dorak et al, 1996 (www)

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

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

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

(www)

Major Study of MHC Haplotypes Dorak et al. 1992-2006 CYP21A2 HSPA1A/B NCR3 LTA DQA1 HLA-B DRB1 MICA NOTCH4 BF AIF1 TNF NFKBIL1 MHC CLASS III REGION Dorak et al, 2006 (www)

(www) Dorak et al, 2006 (www)

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)

Dorak et al, 2006 (www)

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)

HLA-DR Haplotypes & PRKRAP IMGT (www)

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?

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 "

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?

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

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

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

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

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

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

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 '

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?

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

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

Prenatal Selection Against Males One determinant is HLA-DRB3/4 homozygosity Dorak et al. Genes & Immunity 2002;3:263-9 (www) % P = 0.007

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

Hviid, 2005 (www)

POU5F1 POU domain, class 5, transcription factor 1 (OCT3, OCT4, OTF3, OTF4) Expressed in preimplantation embryos, stem cells and cancer GeneID: 282316 (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

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

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

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