Transcript 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.

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?
- HLA or MHC - LD?
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*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
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
F ig ure 1. Hom oz yg osity ra te s for DR52 a nd DR53 in
a g e gro up s o f CM L p a tie n ts
30.0
DR52
25.0
DR53
20.0
P = 0.02
15.0
10.0
5.0
0.0
18-32 yr
33-42 yr
43-60 yr
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
EDN1
LTA
HLA-DRB1
HLA-DRB3/4/5
BF
TNF
HLA CLASS III REGION
HLA-Bw
HFE
Conventional Analysis
Conventional Analysis
HLA-DRB4 Association in Childhood ALL
40
25
%
%
*
Patients
30
Controls
Boys, n=64
Patients
20
15
Controls
Girls, n=53
20
10
*
10
5
0
0
DRB5
DRB3
DRB4
DRB5
DRB3
DRB4
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
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
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
------------------------------------------------------------------------------
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?
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…
HFE-C282Y Association in Childhood ALL
30
%
25
%
Patients
Controls
50
45
40
Patients
Controls
35
20
30
15
25
20
10
15
10
5
5
0
0
All patients
Males Only
Males (cALL)
All patients
Males Only
Males (cALL)
WELSH GROUP
SCOTTISH 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)
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
%
35
Males
30
Females
25
20
15
10
5
0
Allele 6
Allele 8
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
Log likelihood = -133.15366
Number of obs
LR chi2(2)
Prob > chi2
Pseudo R2
=
=
=
=
265
26.68
0.0000
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
Human Major Histocompatibility Complex
- simple map -
Cooke & Hill. Nature Rev Genet 2001 (www)
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
DRB1
HLA-B
NOTCH4
BF
AIF1
TNF
NFKBIL1
MICA
MHC CLASS III REGION
Dorak et al, 2006 (www)
(www)
Dorak et al, 2006 (www)
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 HLADPB1*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?
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
1.6
MALES
1.4
FEMALES
1.2
M/F
1
0.8
0.6
0.4
0.2
0
CONCEPTION
BIRTH
For each 100 females, 54 males are lost during pregnancy
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
35
%
Males
Females
30
25
P = 0.007
20
15
10
5
0
DRB3/DRB3
DRB3/DRB4
DRB4/DRB4
Dorak et al. Genes & Immunity 2002;3:263-9 (www)
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
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
(www)
MHC > HLA
Many non-HLA genes within the MHC may be
responsible for HLA associations observed in many
diseases including childhood ALL
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
Thanks for the support to:
(www)
(www)
(www)