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

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slide1

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

slide2

- Why leukamia, why HLA?

- Unravelling HLA associations?

- What do they mean?

- HLA or MHC - LD?

slide3

Human Major Histocompatibility Complex

- HLA Complex -

Cooke & Hill. Nature Rev Genet2001 (www)

slide4

DRB1*01

DRB1*15/16

DRB1*03

DRB1*04

DRB1*11/12

DRB1*13/14

DRB1*07

DRB1*08

DRB1*09

DRB1*10

DRB1

DRB5

DRB3

DRB4

Expressed HLA-DRB gene content of HLA class II haplotypes

(second DRB gene determines the ancestral lineage)

slide5

DRB4 / DRB5 / DRB1*01/10

DRB3 / DRB1*08

Klein, 1990 (www)

slide6

DRB3 / DRB1*08

DRB4 / DRB5 / DRB1*01/10

Ayala, 1994 (www)

slide8

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

slide10

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

slide12

Inbred mouse strains are homozygous for H-2 haplotypes

Heterozygosity for the susceptibility haplotype

did not have an effect

slide19

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)

slide25

Leukemia is more frequent in males

and in developed countries

slide26

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

slide27

xMHC Loci Analysed in Childhood ALL

6p21.3 - 24.1

EDN1

HSPA1B

LTA

HLA-DRB1

HLA-DRB3/4/5

HLA-Bw

HFE

BF

TNF

HLA CLASS III REGION

slide30

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

%

%

*

Girls, n=53

Boys, n=64

*

Dorak et al, 1999 (www)

slide31

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

Logit estimates Number of obs = 265

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

------------------------------------------------------------------------------

slide32

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

Logit estimates Number of obs = 532

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?

slide33

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

------------------------------------------------------------------------------

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…

slide34

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)

slide35

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)

slide36

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

slide37

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!

slide38

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

slide39

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)

slide40

Possible Mechanisms of HLA-DRB4 Association in Childhood ALL

HLA-DRB4 family of haplotypes are functionally suboptimal in their antigen presentation role

slide41

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

slide42

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

slide43

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

slide44

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

slide45

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)

slide46

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)

slide48

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

slide49

Human Major Histocompatibility Complex

- simple map -

Cooke & Hill. Nature Rev Genet2001 (www)

slide50

Human Major Histocompatibility Complex

Most gene-dense region in the genome

Xie, 2003(www)

slide52

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)

slide53

(www)

Dorak et al, 2006 (www)

slide57

TWO SNPs atHSPA1BandHLA-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)

slide59

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)

slide61

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?

slide62

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 "

slide63

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?

slide64

Rajsbaum, 2002 (www)

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

any genetic association

slide65

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

33,100M to 34,480M

Map Viewer (www)

slide68

HLA association studies need to be extended to:

Epigenetics

Regulatory region polymorphisms

Allelic expression differences

Epistatic interactions

Haplotypes

Post-translational modifications

Proteomics

slide69

HLA association studies need to be extended to:

Post-translational modifications

(www)

slide70

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 '

slide71

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?

slide72

Prenatal Selection Against Males

For each 100 females, 54 males are lost during pregnancy

M/F

slide73

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

slide74

Prenatal Selection Against Males

One determinant is HLA-DRB3/4 homozygosity

Dorak et al. Genes & Immunity 2002;3:263-9 (www)

%

P = 0.007

slide75

HLA-G

Human homologue of the mouse Ped (preimplantation embryo development) gene

Preimplantation embryonic expression

Associations with birth weight and miscarriages

GeneID: 3135 (www)

slide77

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 cancerBiochim Biophys Acta 1984;738(3):93-102

slide78

MHC > HLA

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

slide79

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 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