Effect of group of genetic markers on induction of fetal He - PowerPoint Presentation

Slide1

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, USASlide2

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)

ThalassemiaCharacterized by Reduced or Absence of Synthesis of Globin Chains

Structural Hemoglobin

VariantsFormation of Abnormal Hemoglobin is due to Substitution of an Amino Acid

a thalassemia

b

thalassemia

Hb

E,

HbD

,

HbS

a

0

a

+

b

0

b

+

Quantitative defects

Qualitative defects

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Indian Scenario of

Hemoglobinopathies.Slide6

Major

Hb. Disorder

b-thalassemia ,Sickle Cell Anemia, HbE-Thal,

HbS-Thal b-Thalassemia

Present in most ethnic groups

Overall prevalence - 3-4%Estimates

30 to 40 million carriers

10000-12000 affected births/year

1,500 new cases born each year

High risk groups

Punjabis,

Sindhis

,

Lohanas

,

Bohri

Prevalence – 5 – 15 %.

Burden of

Hemoglobinopathies

in India

anitahnadkarni@yahoo.comSlide7

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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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 )

HbA

2

-

4 - 7%

HbF

-

1 - 3%

Classical

b

-thalassemia heterozygotes

anitahnadkarni@yahoo.comSlide10

Increased Hb

A2 level is the characteristic diagnostic feature of β thalassaemia trait

anitahnadkarni@yahoo.comSlide11

PREVENTION IS THE ONLY CURE

BTT

BTT

BTT

BTT

50%

NORMAL

b

-

Thal

. Homozygous

25%

25%

Inheritance of

b

-thalassemiaSlide12

b

thalassemia homozygous

MilderThalassemia intermediaPresent 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

Severe

Thalassemia Major

Present clinically before the age of

2 yrs

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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

Genetic interaction

b

Thal Heterozygote

Thal

. Intermedia

Asymptomatic

b

Thal

Homozygote

Severe :

Thal

Major

Milder:

Thal

Int.

Mild

b

thal

mutation

Associated

a

thal

.

gene triplication

aa/aaa

High Hb Fdeterminant

XmnI

poly

(AT)

x

(T)

y

PreG

g

globin haplotypes

A

g-d

intergenic

region

Dominant

b

thal

.

QTL: 2,6,8 ,X

AHSPSlide15

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. anitahnadkarni@yahoo.comSlide16

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Data from NIIH : Genotype to Phenotype Slide17

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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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. Slide18

Clinical classification of b

thal. homozygotes

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.Slide19

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

anitahnadkarni@yahoo.comSlide20

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

. Slide21

All the SCA patients showed the homozygosity [+/+]

b

Thal Intermedia cases predominantly showed the presence of

+/+ [T/T] alleles

anitahnadkarni@yahoo.comSlide22

Presence of XMn1 + allele raises the

HbF

levelsXmnI

polymorphism and HbF levels in thalassemia intermedia casesanitahnadkarni@yahoo.comSlide23

(

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.

anitahnadkarni@yahoo.comSlide24

(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 S

b thal SCA Eb thal

db b

thal

NC Slide25

Coinheritance of both,

XmnI

+ allele and (AT)9(T)5 motif was present among the thalassemia intermedia patients

Combined effect XmnI poly

morphism and (AT)9(T)5 motif on HbF levels in severe and milder thalassemia cases

Among these patients, even the absence of (AT)9(T)5 motif did not lower the

HbF

levelSlide26

PreG

g globin haplotypes

TAGThe promoter region of the G

 globin gene has binding sites for TFs involved in the G 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 G globin haplotypes

TAG, TGG, TGA and TAASlide27

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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PreG

g

globin haplotypes

TAGSlide28

Type (no. of chromosomes)

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

%)

b

thalassemia

inter (n=78

)

44(56.4%)

15(19.2%)

10(12.8%)

9 (

11.5%)

Sickle cell anemia (n=22)

22 (100%)

-

-

-

Sickle-

b

thalassemia (n=28)

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%)

Distribution of Pre G

g

globin gene haplotypes

anitahnadkarni@yahoo.comSlide29

The effect of TAG haplotype on HbF levels

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%). Slide30

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). anitahnadkarni@yahoo.comSlide31

A

g-d intergenic region haplotype: T

The intergenic region between A

and  globin genes has been reported to be implicated in the intergenic transcription

Any disturbance in this function may affect the

transcription rate of  ,  and



globin genes

It

may contribute to the switched off status of





gene

Switched

on status of

 and  globin genes in adults

anitahnadkarni@yahoo.comSlide32

228

229

416haplotype

TAGTRAG

--T

1 2 3 4 5 6 7 8

755BP

435BP

320BP

T/T genotype -

lanes 4, 6

,

R/T genotype

-

lanes 3, 5, 7, 8,

MW marker VIII-

lane 1

A

g-d

intergenic region

haplotype: T

anitahnadkarni@yahoo.comSlide33

Type (no. of cases)

A

g-d

intergenic region haplotypes No. (%)

R/R

R/T

T/T

b

TM

(n=40

)

28 (70

%)

4 (10

%)

8 (

20%)

b

TI

(n=39

)

10(25.6%)

7(17.9%)

22(56.4%)

Sickle cell anemia (n=11)

-

-

11 (100

%)

Sickle-

b

thalassemia (n=14)

-

9 (64.2%)

5 (35.7%)

HbE

-

b

thal

.

(n=16)

6 (37.5

%)

8(50

%)

2(12.5

%)

b

thal

.

-

db

/HPFH (n=8)

2 (

25%)

2 (

25%)

4 (

50%)

Normal controls (n=50)

21 (

42%)

12 (

24%)

17 (

34%)

Distribution of the A

g-d

intergenic

region haplotype

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

anitahnadkarni@yahoo.comSlide34

Higher levels of HbF confined to homozygosity (T/T)

Haplotype T even in heterozygosity was sufficient in elevating the mean HbF levels.

The effect of T haplotype on the HbF level in patientSlide35

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%

Combined effect of

XmnI poly morphism ,

Pre G g globin gene and A

g-d

intergenic region haplotype

on HbF levels in severe and milder thalassemia casesSlide36

PreG

g

globin haplotype (no.)

HbF (

Mean±SD)

A

g-

d

intergenic

region haplotype

(no.)

HbF (

Mean±SD

)

XmnI

polymorphism (no.)

HbF (Mean±SD)

(AT)

x

(T)

y

polymorphism

(no.)

Hb (Mean±SD)

Presence

TAG

Absence

TAG

Presence

T

Absence

T

Presence

T

Absence

T

Presence

(AT)

9

(T)

5

Absence

(AT)

9

(T)

5

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

The association of the raised HbF determinant

and HbF levels on clinical severity of the disease

*p<0.01, # p<0.05

Presence of allele led to increase in HbF levels.

anitahnadkarni@yahoo.comSlide37

Concomitant effect of factors contributing

in raised HbF levels

Matrix Interaction Plots for Xmn1, (AT)x(T)y, Pre

G

g

&

Ag-dSlide38

This combination might have helped our patients in decreasing

the disease severity. The genotype-phenotype correlation

observed highlights the possible cooperative role of some polymorphisms

in cis to the -globin gene.Conclusion

It seems that a combination of the TAG -Pre G

g Globin, T -A

g -d

intergenic region

haplotype

and the

(AT)9 (T)5

configuration

constitute

a topography that co-relates with raised

HbF

levels.

Despite numerous observations, it remains unclear whether only some polymorphisms or the entire configuration are involved in the

Hb F induction.Slide39

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.

Take home massage

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.Slide40

Dept. of HematogeneticsSlide41

Thank you !

Fight for thalassemia prevention Slide42

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