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Highlights on globin gene switch: new target for therapeutics
Renzo Galanello Pediatric Clinic 2 and Thalassemia Unit–
University of Cagliari (Italy) Ospedale Regionale Microcitemie ASL8
6th International Scientific Meeting New biologci and analytic issue on Hemoglobin A2 and other minor Hemoglobins
ORGANIZATION AND DEVELOPMENTAL EXPRESSION OF GLOBIN GENES
birth
α β
γ
γ
β ζ ε
1 year 4 weeks
δ
LCR
ε β δ Gγ Aγ CHROMOSOME 11
CHROMOSOME 16 ζ α α
HS 40
Moi P.
3
Type And Distribution Of β-Thalassemia Mutations
Thein et al. 2004.
Promoter
RNA Processing
5’ & 3’ UTR
ATG Nonsense codons Frameshifts
Β+
Β+
Β+, βº
βº
βº
βº
1 2 3
CCAAT
CACCC
CACCC
ATAAA ATAA
3’ 3’ UTR 105 146
AG TAA
IVS 2
GT AG GT CATG
IVS 1 5’ UTR 30 104 30 1
5’
Globin chain synthesis with β-thalassemia mutations
Brith
α
γ
γ
β 1year 4 weeks
α
HbA
HbF
Moi P.
Pathophysiology of β-thalassemia
Normal production alpha chains
Absent or decreased beta chains
Excess unstable alpha tetramers
Absent or decreased haemoglobin A
Ineffective erythropoiesis
Hemolysis
ANEMIA
HbA alpha 2/beta 2
6
a globin
β + γ globin
Mild Phenotype
α thalassemia Mild/silent β alleles Increased γ
Severe Phenotype
a globin
β + γ globin
Mechanisms Of β-Thalassemia Intermedia
7
HOMOZYGOUS SARDINIAN δβ°-THALASSEMIA
MCV fl 72.7 MCH pg 24.2 F 18.8 A2 % 2.8
Hb g/dl 11.9 MCV pg 63.4 MCH pg 21.6 F % 99.7 A2% 0.3 Aγ % 72.0
δβ°-thalassemia
DNA analysis: β39 C→T homozygous Aγ -196 C→T homozygous
Age 3.5 y
Galanello et al,Blood 2002
Why interest in fetal hemoglobin and hemoglobin switching?
ü Hb F is a strong modifier of hemoglobinopathies’ severity
ü level of HbF is a variable and inducible quantitative trait in humans
ü to understand the mechanisms of thalassemia intermedia
ü to understand the general mechanisms of gene expression and of developmental gene regulation
ü to develop targeted therapeutic approaches for ameliorating the severity of the beta-hemoglobinopathies
.
.
Population distribution of fetal hemoglobin
Thein SL,2009
• interindividual HbF variation is highly heritable
• genetic investigation is expected to identify genetic factors that controls HbF production
11346–11351 PNAS July 3, 2007 vol. 104 no. 27
QTLs map to HBS1L-MYB and BCL11A
Copyright ©2008 by the National Academy of Sciences
Uda M.,Galanello R et al, Proc. Natl. Acad. Sci. USA 105, 1620-1625 (2008)
GWAS results for HbF
Hb F variation associated SNPs
Chr 2
2p16 BCL11A
rs766432
rs1427407
rs11886868
rs4671393
Chr 6
6q23 MYB
rs9399137
rs4895441
Chr 11
11p15 HBB
rs4910742
rs7482144
p= 9.14e-08
p= 0.018 p= 0.446
Bcl11A and HbF v genetic association detected by G-WAS
v SNPs in IVS2 described in different populations, in HPFH, beta thalassemia, HbE, SCD
v High HbF is associated with low Bcl11A expression
v Bcl11A expression varies at different developmental stages
v Bcl11A is down-regulated by KLF1 gene
VOLUME 42 | NUMBER 9 | SEPTEMBER 2010 Nature Genetics
Schematic mechanism of KLF1 action
KLF1 direct binding direct binding
β-promoter BCL11A promoter
induces β-globin gene expression
inhibition of γ globin gene expression
γ-‐globin gene expression in β-‐talassemia and in HPFH
Birth
α
γ
γ
β 1 year 4 weeks
δ
α
γ-HPFH
Moi P.
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4
Tal. Major
Tal. Inter
Numero di alleli positivi
Pro
porz
ione
paz
ient
i
CONTRIBUTO DI ALCUNI ALLELI NEL DETERMINARE IL FENOTIPO DI TALASSEMIA MAJOR E INTERMEDIA
Blood,2009
Locus p Hazards Ra<o Harrell’s C-‐index Predictor for later transfusion start
HBG2:g.-‐58C>T <0.001 0.081 0.54 +/-‐ α gene defects <0.001 0.514 0.61 class 2
BCL11A rs1427407 <0.001 2.391
0.63 T allele
rs10189857 0.005 1.312 G allele
HBS1L/MYB rs4895441 <0.001 1.979
0.57 G allele
rs6904897 0.020 0.697 TT genotype Gender 0.016 0.738 0.52 Male
First quar*le (pa*ents with more posi*ve predictors combina*ons)
Second quar*le
Third quar*le
Fourth quar*le (pa*ents with more nega*ve predictors combina*ons)
Survival curves for 316 pa<ents with different combina<ons of predictors for later and earlier <me to transfusion
Danjou F et al.,2012
Clinically relevant fetal hemoglobin modifiers
• Genetic: - in cis variants: - γ globin genes - β globin cluster - LCR
- in trans variants: - BCL11A - HBS1L-MYB - KLF1 mutations
- GATA1 - trisomy 13 • Epigenetic:
- DNA methylation
- histone modification (acetylation, phosphorylation, methylation)
- erythropoiesis expansion (perturbation of erythroid kinetics)
Cao A Moi P. Galanello R.: Pediatric Reports 2011
Current hemoglobin switching model
Variance components of the F-cell trait
Age ad sex 2% Chr.11p15.4 – b globin locus 10%
Chr.6q23.3 – HMIP 19%
Chr.2q16.1 – BCL11A 15%
Other, unknown, genes
Environment 9%
(MenzelS, 2009)
Rationale for HbF induction!
ü Gamma globin genes are intact"
ü HbF will functionally compensate for the absence of HbA (HPFH homozygotes, genetic compounds (β-thal/HPFH)"
"
ü HbF in post-natal life is influenced by physiological and genetic factors (pregnancy, acute erythroid expansion, haematological diseases, haemoglobinopathies)"
ü High cost of conventional treatment"
ü Problems wit blood safety and availability in several countries" HbA = adult haemoglobin; HbF = fetal haemoglobin.
HbF inducers
Class Compound
Hypomethylating/cytotoxic agents 5-azacytidine, decitabine
Cytotoxic agents hydroxyurea
Histone Deacetylase Inhibitors butyrate analogs, short chain fatty acids, adipicin, scriptaid, trichostatin A, valproic acid,
Antioxidants resveratrol, angelicin, curcumin………
Others
Erythropoietin, thalidomide
Atweh g eta l Hematol Incol Clin N Am 2010
• histone hyperacetylation
• p38-MAPK phosphorylation
• ERK MAPK dephosphorylation
• sGC/cGMP pathway activation
• increased γmRNA translation
• sGC/cGMP pathway activation
• increase of CAMP
• hypomethylation by inhibition of DNA
• hyperacetylation (?)
thalidomide
Mechanisms for γ-globin induction by pharmacologic agents
Summary of clinical trials with HU in β-thalassemia syndromes
ü 18 trials
ü 7 thalassemia major, 8 intermedia, 3 HbE/β-thal
ü treated patients 573
ü number of patients/trial: 2 to 163
ü variable doses 3 to 30 mg/kg/day
ü responding patients: 25 to 100%
ü total hemoglobin increase: <1 g up to 4g/dl
ü best response in thalassemia intermedia
ü response correlates with XmnI polymorphism
Thalidomide therapy in patients with thalassemia major.
A genotype –28A→C/cd39
B genotype IVS1-6/cd44C
100 mg/kg/d HbF=∼100%
75 mg/kg/d HbF=50 → 73%
Aguilar-Lopez et al Blood Cell Mol Dis 2008
Pharmacologic induction of HbF:concerns and limitations
Ø potential toxicity→ safety an long term
Ø unpredictable and variable response
Ø effectiveness and long term (loss of efficacy overtime)
Ø compliance
Future directions
Ø other safer and more effective compounds
Ø better understanding of molecular mechanisms
Ø combination therapies
Ø correlation with individual genetic determinants
Ø personalized therapies
Ø impact on natural history
Bianchi N et al
Rapamycin
Induction of HbF by rapamycin
Conclusions v second generation DNA technologies have improved the knowledge of
globin gene modifiers
v well phenotyped cohorts of patients are essential for genetic research:
v gene modifiers are useful in the follow-up of disease -related complications and in drug selection and dosing
v genetic studies of disease modifiers can identify new potential targets for therapy
v an integrated approach by clinicians, geneticists, clinical researchers and basic scientists is needed to further improve the knowledge of variability and the treatment of patients with hemoglobinopathies
your genetics is only as good as your phenotype
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