Transcript anita-nadkarni-national-institute-of

Effect of group of genetic markers
on induction of fetal Hemoglobin
and disease severity in
Hemoglobinopathies.
Dr. Anita Nadkarni , Deputy Director
National Institute of Immunohematology (ICMR)
Mumbai. INDIA
3rd International Conference on
Hematology & Blood Disorders
November 02-04, 2015 Atlanta, USA
• Thalassemias or sickle cell anemia are
monogenic disorders
• Mutation resides in the globin genes.
• This may lead to expectation that their
presentations will be similar.
• But it is not so.
• This because there are many other gene
(modifiers ) influencing the clinical
presentations of these patients
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What is a
Hemoglobinopathy?
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Hemoglobinopathies
(Disorders of Hemoglobin)
Quantitative defects
Thalassemia
Characterized by Reduced
or Absence of Synthesis of
Globin Chains
Qualitative defects
Structural Hemoglobin
Variants
Formation of Abnormal Hemoglobin is
due to Substitution of an Amino Acid
b thalassemia
a thalassemia
Hb E, HbD , HbS
a0
a+
b0
b+
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Indian Scenario
of
Hemoglobinopathies.
Burden of Hemoglobinopathies in India
Major Hb.
Disorder
b-thalassemia ,Sickle Cell
Anemia, HbE-Thal, HbS-Thal
Present in most ethnic groups
b-Thalassemia
Overall prevalence - 3-4%
30 to 40 million carriers
Estimates
High risk
groups
10000-12000 affected
births/year
1,500 new cases born each year
Punjabis, Sindhis, Lohanas,
Bohri
Prevalence – 5 – 15 %.
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HbS
Restricted to tribal and Scheduled
Caste Communities.
Central, Western and Southern India.
- Prevalence- 1 to 40 %
HbE
North East and Eastern Region
Prevalence – 4 to 30 %
Other
clinically
Significant
Disorders
db-b thalassemia, Hb SD disease, Hb
Lepore–b thal, Hb H disease, Unstable
and hyper-unstable variants
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CONDITIONS THAT LEAD TO
DETECTION OF HEMOGLOBINOPATHIES
•
•
•
•
•
•
•
Persistent anemia
Microcytosis
Hemolytic anemia
Polycythemia
Cyanosis
Population screening
Family work up
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Classical b-thalassemia heterozygotes

RBC Morphology
- Microcytosis, hypochromia,
anisopoikilocytosis,
target cells, basophilic
stippling.

Nestroft
- Positive

Red Cell Indices
- MCV (60-70fl) MCH (19-23pg)
RBC ( > 4.5 x 106/ml )

HbA2

HbF
-
4 - 7%
- 1 - 3%
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Increased Hb A2 level is
the characteristic diagnostic
feature of β thalassaemia trait
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Inheritance of b-thalassemia
BTT
BTT
NORMAL
25%
BTT
BTT
50%
b-Thal.
Homozygous
25%
PREVENTION IS THE ONLY CURE
b thalassemia homozygous
Severe
Thalassemia Major
Present clinically
before the age of
2 yrs
Milder
Thalassemia
intermedia
Present clinically
after the age of 2 yrs
• The clinical severity of b thalassemia is influenced
by many genetic factors
• There are some linked and unlinked genetic
determinants that are proposed to modify the
severity of b thalassemia.
• They are grouped as primary, secondary and
tertiary
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Primary, secondary, and tertiary modifiers
of b thalassemia
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Effect of Genotypic interaction on Phenotypic Variations
Genotype
Phenotype
Asymptomatic
b Thal Heterozygote
Genetic interaction
a gene triplication
aa/aaa
Thal. Intermedia
Dominant b thal.
Severe : Thal Major
Mild b thal mutation
Associated
a thal.
b Thal Homozygote
Milder: Thal Int.
AHSP
High Hb F
determinant
XmnI poly
(AT)x(T)y
Ag-d intergenic
region
PreG g globin
haplotypes
QTL: 2,6,8 ,X
• Higher HbF levels in patients with β thalassemia or
sickle cell anemia are associated with milder
phenotypes
• Recently, there has been considerable progress
made in defining the loci or genomic regions that
may often be involved in the regulation of the
switch from fetal  adult hemoglobin.
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Data from NIIH :
Genotype to Phenotype
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• Our objective was to study the effect of
cis-DNA haplotypes, motifs, or polymorphisms
that may contribute to higher HbF levels and a
milder clinical course.
• In our study we enrolled
 79 β thalassemia homozygous,
11 sickle cell anemia
 14 sickle-β thalassemia individuals.
 50 age - sex matched healthy individuals
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Clinical classification of b thal. homozygotes
79 b
thalassemia
homozygotes
40 b
thalassemia
major
39 b
thalassemia
intermedia
16 early
presenters
(2-4 years)
23 late
presenters (4
years onwards)
• 92.5% TM required regular blood transfusion (mean-11.94 units/year)
• 69.23% TI were untransfused (mean-2.30 units/year)
• 54 % of the TI showed a spleen bigger than 6 cm.
Genetic modifiers linked to
raised HbF




Xmn1 polymorphism : −158Gg(CT)
(AT)x(T)y polymorphism : (AT)9(T)5
PreG g globin haplotypes : TAG
Ag-d intergenic region haplotype : T
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Xmn1 polymorphism
−158Gg(CT)
T allele associated with increased Hb F
* Explains 2-10% of variance in Hb F
* Ameliorates the severity of b thalassemia and sickle
cell disease
Presence of the T allele in Indian patients
* b-Thalassemia Intermedia - 68 %
* b-Thalassemia Major - 29 %
Nadkarni et. al. Am. J. Hematol 2001; 68:75-80.
Italia et. al. Clin. Chim. Acta. 2009; 407: 10-15.
• All the SCA patients showed the homozygosity [+/+]
• b Thal Intermedia cases predominantly showed the
presence of +/+ [T/T] alleles
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XmnI polymorphism and HbF levels
in thalassemia intermedia cases
Presence of XMn1 + allele raises the HbF levels
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(AT)x(T)y polymorphism
(AT)9(T)5
• This motif is a putative binding site for a repressor
protein, beta protein 1 (BP1).
• Variations in the (AT)x (T)y repeats affect the binding
affinity of BP1, thereby alter the expression of the
beta-globin gene.
• The sequence polymorphism (AT )9 ( T)5 at–540 of
globin gene is associated with decreased b globin gene
and high HbF expression.
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(AT)X(T)Y POLYMORPHISM : (AT)9(T)5
3 different motifs- (AT)7(T)7, (AT)8(T)9 and (AT)9(T)5
TM
TI Sb thal SCA Eb thal db b thal NC
Combined effect XmnI poly morphism and (AT)9(T)5 motif on
HbF levels in severe and milder thalassemia cases
• Coinheritance of both, XmnI + allele and (AT)9(T)5 motif
was present among the thalassemia intermedia patients
• Among these patients, even the absence of (AT)9(T)5
motif did not lower the HbF level
PreG g globin haplotypes TAG
•The promoter region of the G g globin gene has binding
sites for TFs involved in the Gg globin gene expression.
• 3 polymorphic sites 
-1450 (T/G), -1280 (A/G) and -1225 (A/G)
• The combination of the these give rise to the Pre Gg
globin haplotypes TAG, TGG, TGA and TAA
PreG g globin haplotypes TAG
DNA sequencing of promoter region of Pre G g globin
gene showing the presence of TAG haplotype
-1450 T/T
-1280 A/A
-1225 G/G
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Distribution of Pre G g globin gene haplotypes
Pre G g globin gene haplotypes
No. (%)
TAG
TGG
TGA
TAA
b thalassemia major (n=80) 12 (15%) 23 (28.7%) 35 (43.7%) 10(12.5%)
Type (no. of chromosomes)
b thalassemia inter (n=78)
44(56.4%) 15(19.2%) 10(12.8%) 9 (11.5%)
Sickle cell anemia (n=22)
Sickle-b thalassemia (n=28)
22 (100%)
17(60.7%) 8 (28.5%)
-
3 (10.7%)
HbE-b thalassemia (n=32)
6 (18.7%) 13(40.6%)
2(6.25%)
11(34.3%)
b thal -db/HPFH (n=16)
9 (56.2%)
5 (31.2%)
-
2 (12.5%)
Normal Controls (n=100)
15 (15%)
64 (64%)
13 (13%)
8 (8%)
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The effect of TAG haplotype on HbF levels
Pr of TAG
120
Ab of TAG
Mean HbF (%)
100
80
60
40
20
0
TM (n = 40) TI (n = 39) SCA (n = 11) ST (n = 14) ET (n = 16 ) HF (n = 8)
• Presence of TAG haplotype  higher HbF levels
• The mean HbF level TI patients :
 presence of TAG ( 81.42±24.52% )
 absence of TAG (65.61±30.29%).
Distribution of TAG haplotype and XmnI
polymorphism among homozygotes
• Presence of both the alleles : TI- 32%, TM - 9% (p<0.005).
• Absence of both the alleles
: TI – 5%, TM- 60% (p<0.005).
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Ag-d intergenic region haplotype: T
•The intergenic region between Ag and d globin genes
has been reported to be implicated in the intergenic
transcription
• It may contribute to the switched off status of g gene
• Switched on status of d and b globin genes in adults
• Any disturbance in this function may affect the
transcription rate of g , d and b globin genes
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Ag-d intergenic region haplotype: T
1
2
3
4
5
6
7
228 229 416
haplotype
T
A
GT
R
A
G
--
T
8
755BP
435BP
320BP
T/T genotype - lanes 4, 6,
R/T genotype - lanes 3, 5, 7, 8,
MW marker VIII- lane 1
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Distribution of the Ag-d intergenic region haplotype
Type (no. of cases)
b TM (n=40)
b TI (n=39)
Sickle cell anemia (n=11)
Sickle-b thalassemia (n=14)
HbE-b thal. (n=16)
b thal. -db/HPFH (n=8)
Normal controls (n=50)
Ag-d intergenic region haplotypes
No. (%)
R/R
28 (70%)
10(25.6%)
6 (37.5%)
2 (25%)
21 (42%)
R/T
4 (10%)
7(17.9%)
9 (64.2%)
8(50%)
2 (25%)
12 (24%)
T/T
8 (20%)
22(56.4%)
11 (100%)
5 (35.7%)
2(12.5%)
4 (50%)
17 (34%)
Mutant haplotype (T) – 75% clinically milder patients
Ref . haplotype (R)
– 80% clinically severe patients
T I  56.41% of the patients were homozygous for the T
haplotype T/T
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The effect of T haplotype on the HbF level in patient
• Higher levels of HbF confined to homozygosity
(T/T)
• Haplotype T even in heterozygosity was sufficient
in elevating the mean HbF levels.
Combined effect of XmnI poly morphism , Pre G g globin gene
and Ag-d intergenic region haplotype
on HbF levels in severe and milder thalassemia cases
Pres. of both the alleles : TI-39% ,TM- 5% Absence of both the alleles : TM -42%
Presence of both the alleles : TI-50% Absence of both the alleles : TM -50%
The association of the raised HbF determinant
and HbF levels on clinical severity of the disease
PreGg globin
haplotype (no.)
HbF (Mean±SD)
Ag-dintergenic
region haplotype
(no.)
HbF (Mean±SD)
Presence Absence
T
T
XmnIpolymorphism
(no.)
HbF (Mean±SD)
(AT)x(T)y
polymorphism
(no.)
Hb (Mean±SD)
Presence Absence T Presence Absence
T
(AT)9(T)5 (AT)9(T)5
Presence
TAG
Absence
TAG
b
TI
(n=39)
(22)
81.4±
24.52*
(17)
65.61±
30.29*
(29)
76.52±
27.36#
(10)
60.10±
37.49
(25)
74.52±
8.98
(14)
51.2±
6.22
(10)
82.73±
23.11
(29)
71.31±
32.5
b
TM
(n=40)
(6)
54.47±
31.01#
(34)
22.89±
35.54#
(12)
37.15±
35.54
(28)
28.11±
31.01
(11)
28.59±
35.75
(29)
26.44±
38.34
(6)
21.62±
38.19
(34)
29.26±
37.29
Sickle
chrom
(n=36)
(36)
23.53±
10.82
-
(36)
23.53±
10.82
-
(36)
25.53±
10.82
-
(23)
25.06±
9.53
(13)
19.8±
13.55
*p<0.01, # p<0.05
Presence of allele led to increase in HbF levels.
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Concomitant effect of factors contributing
in raised HbF levels
Interaction Plot for HbF(%)
Data Means
7
5
7
9
5
5
7/
9/
7/ / 8/
9/
9/
7/
7/ /9/
9
9/ / 5/
/
/
/
/
7
7
8
8
8
9
R
R/
T
R/
T
T/
90
60
Xmn1
Xmn1
-/+/+/+
30
90
60
(AT)x(T)y
30
90
60
Pre Gg
30
90
60
Ag-d
30
- /-
+/
+
+/
AG
GG TA G TGG T GA
/T
/T
A
A
G/
G/
A/
TA
TA
TA
TA
TG
Matrix Interaction Plots for Xmn1, (AT)x(T)y, Pre Gg& Ag-d
(AT)x(T)y
7/7/7/7
7/7/9/5
8/9/7/7
8/9/8/9
8/9/9/5
9/5/9/5
Pre Gg
TAA/TAG
TAA/TGG
TAG/TAG
TAG/TGG
TGA/TGA
Ag-d
R/R
R/T
T/T
Conclusion
 The genotype-phenotype correlation observed
highlights the possible cooperative role of some
polymorphisms in cis to the b-globin gene.
 It seems that a combination of the TAG -Pre G g
Globin, T -Ag -d intergenic region haplotype and
the (AT)9 (T)5 configuration constitute a
topography that co-relates with raised HbF levels.
 This combination might have helped our patients in
decreasing the disease severity.
 Despite numerous observations, it remains unclear
whether only some polymorphisms or the entire
configuration are involved in the Hb F induction.
Take home massage
 The HbF-associated SNPs, increasing the
production of HbF over the lifetime of a
patient, may be considered as an innate
therapy for several hemoglobin disorders.
Genotyping of these variants may
eventually help to predict the severity
risk in newborns and, accordingly,
improve genetic counseling.
 Some patients may have additional
modifying factors, either environmental or
genetic, contributing to the unexplained
variability.
Dept. of Hematogenetics
THANK YOU !
Fight for thalassemia
prevention
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