HLA & Leukemia Associations:HLA & Leukemia Associations:What do they mean?What do they mean?

M. Tevfik DORAKM. Tevfik DORAK

Environmental & Occupational HealthCollege of Public Health

Florida International UniversityMiami, USA

http://www.dorak.info

- 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 -

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

Klein, 1990 (www)

DRB4 / DRB5 / DRB1*01/10 DRB3 / DRB1*08

Ayala, 1994 (www)

DRB4 / DRB5 / DRB1*01/10

DRB3 / DRB1*08

(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?

Heterozygosity for the susceptibility haplotype did not have an effect

Inbred mouse strains are homozygous for H-2 haplotypes

Spontaneous leukemia shows the same association

Dorak et al, 1994 (www)

Conventional analysis shows no association even with homozygosity

Stratification by age revealed associations

Figure 1. Hom ozygosity ra te s for DR52 a nd DR53 in a ge groups of CM L pa tie nts

0.0

5.0

10.0

15.0

20.0

25.0

30.0

18-32 yr 33-42 yr 43-60 yr

DR52

DR53

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)

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)

BF

HSPA1B LTA

TNF

HLA CLASS III REGION

HLA-DRB3/4/5

HLA-DRB1EDN1

HFEHLA-Bw

xMHC Loci Analysed in Childhood ALL6p21.3 - 24.1

Conventional AnalysisConventional Analysis

Conventional AnalysisConventional 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)

ADDITIVE MODELADDITIVE MODEL

Linear ModelLinear Model

Logit estimates Number of obs = 265 LR chi2(1) = 14.24 Prob > chi2 = 0.0002Log 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 HomozygosityHeterozygosity and Homozygosity

Logit estimates Number of obs = 265 LR chi2(2) = 22.00 Prob > chi2 = 0.0000Log 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 2.04676Homozygosity | 6.258503 2.65464 4.32 0.000 2.72534 14.37211------------------------------------------------------------------------------

HLA-DRB4HLA-DRB4 ASSOCIATION ASSOCIATION

EFFECT MODIFICATIONEFFECT MODIFICATION

Logit estimates Number of obs = 532 LR chi2(3) = 23.97 Prob > chi2 = 0.0000Log 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 SEXCONFOUNDING BY SEX

Logit estimates Number of obs = 532 LR chi2(2) = 11.99 Prob > chi2 = 0.0025Log 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-DRB4HLA-DRB4 - - HSPA1BHSPA1B HAPLOTYPE ASSOCIATION HAPLOTYPE ASSOCIATION

BOYS ONLYBOYS ONLYLogit estimates Number of obs = 265 LR chi2(1) = 22.41 Prob > chi2 = 0.0000Log 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 ONLYGIRLS ONLYLogit estimates Number of obs = 267 LR chi2(1) = 0.13 Prob > chi2 = 0.7205Log likelihood = -132.98706 Pseudo R2 = 0.0005------------------------------------------------------------------------------ caco | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval]-------------+---------------------------------------------------------------- hsp53 | 0.796 .5189439 -0.35 0.726 .22181 2.856571------------------------------------------------------------------------------

The association is modified by sex…

HLA-DRB4HLA-DRB4 - - HSPA1BHSPA1B HAPLOTYPE ASSOCIATION HAPLOTYPE ASSOCIATION

HFE-C282Y Association in Childhood ALL

SCOTTISH GROUP 135 patients - 238 newbornsP = 0.0004; OR = 3.0 (1.7 to 5.4)In cALL: P < 0.0001; OR = 4.7 (2.5 to 8.9)

WELSH GROUP 117 patients - 415 newbornsP = 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)

EDN1 Association in Childhood ALLPreliminary 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 = 0.0000Log 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

SpuriousPopulation 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

SpuriousPopulation 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

Supported by association with homozygosity and association in boys

Immune nonresponsiveness is a recessive trait

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)

Charron, 1984 (www); Lotteau, 1987 (www) & 1989 (www); Matsunaga, 1990 (www); Spencer 1989 (www) & 1992 (www) & 1993 (www)

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)

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)

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

SpuriousPopulation 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 -

Xie, 2003 (www)

Human Major Histocompatibility Complex

Most gene-dense region in the genome

(www)

NOTCH4

CYP21A2

BF

NCR3HSPA1A/B

AIF1

LTA

TNF NFKBIL1

MHC CLASS III REGION

DRB1

DQA1 HLA-B

MICA

Major Study of MHC HaplotypesDorak et al. 1992-2006

Dorak et al, 2006 (www)

(www)

Dorak et al, 2006 (www)

TWO SNPs TWO SNPs atat HSPA1BHSPA1B andand HLA-DQA1HLA-DQA1 IDENTIFY ANCESTRAL MHC CLASS II LINEAGES 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

TWO SNPs TWO SNPs atat HSPA1BHSPA1B andand HLA-DQA1HLA-DQA1 IDENTIFY ANCESTRAL MHC CLASS II LINEAGES 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)

PossibilitiesPRKRAP (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)

IMGT (www)

HLA-DR Haplotypes & PRKRAP

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?

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

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? Why not LD?

Linkage disequilibrium 'confounding by locus' has to be ruled out before attributing a direct causal role to

any genetic association

Rajsbaum, 2002 (www)

Extended HLA Class II Region (HLA-DPB2 to KIFC1)

Map Viewer (www)

33,100M to 34,480M

Map Viewer (www)

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

Predicted and Experimental Binding of Peptides to Class I MHC Molecules

(www)

Epigenetics

Regulatory region polymorphisms

Allelic expression differences

Epistatic interactions

Haplotypes

Post-translational modifications

Proteomics

HLA association studies need to be extended to:

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 MalesA 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/FM/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 MalesOne 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 3rdThe borderland of embryogenesis and carcinogenesis. MHC-linked genes affecting development and their possible relationship to the development of cancerBiochim Biophys Acta 1984;738(3):93-102

MHC > HLAMany non-HLA genes within the MHC may be

responsible for HLA associations observed in many diseases including childhood ALL

ACKNOWLEDGEMENTSACKNOWLEDGEMENTSI am grateful to all who have taught, helped and supported in one

way or another at

Glasgow Leukaemia Research LaboratoryGlasgow Tissue Typing Laboratory

Glasgow Royal Hospital for Sick ChildrenUniversity of Wales College of Medicine

Welsh Transplantation & Immunogenetics LaboratoryHLA Lab, Martin Luther University, Halle, Germany

St Jude Children’s Hospital, HLA LaboratoryUniversity of Tennessee at MemphisUniversity 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:Thanks for the support to:

(www)

(www)

(www)