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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
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)
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
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)
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)
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