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Thalassemia Intermedia in India:
genotypic phenotypic correlations
Dr. R Saxena
Professor & Head, Department of Haematology
All India Institute of Medical Sciences, New Delhi, India
Beta Thalassemia -Distribution
Thalassemia Intermedia
A state of thalassemia syndrome:
Manifests later in life (> 2 years)
Less severe than thalassemia major
May require BT occasionally under stress
Patient may develop complications of anemia
Clinical phenotype Number (%)
Thalassemia major 293(76.3)
Thalassemia intermedia 91 (23.7)
Prevalence of Thalassemia Intermedia:
AIIMS (n=384)
Genotypes of Thalassemia Intermedia in
India (n=251)
• Clinico-Haematological Profile of Thalassemia Intermedia Patients. S Tyagi1, M. Kabra, N Tandon, R Saxena, H. P. Pati and V. P.
Choudhry Int J Hum Genet, 3(4): 251-258 (2003)
• Molecular Characterization Of Thalassemia Intermedia In in India . I. Panigrahi, S. Agarwal M, Pradhan, DR Choudhry, VP
Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.
Genotypes of TI
Beta Thalassemia
HbE Beta Thal
HbS Beta Thal
HbD Pb Beta Thal
HbE Lepore
HPFH Beta
Delta Beta Beta Thal
Phenotype in Thalassemia Intermedia
Phenotype in Beta Thalassemia
Depends on Chain imbalance of Beta and Alpha globin
chains
Excess alpha chain cause manifestations of Beta
Thalassaemia
Phenotypic heterogeneity within the various groups
Genotype Phenotype Correlation
What causes heterogeneity of phenotype of
thalassemia intermedia?
Primary Modulators: Mutations which affect β globin
gene
Secondary Modulators: Genetic Alteration that affect α
and γ globin genes
Tertiary Modulators: Genetic Alterations that don’t
affect globin gene directly
Taher A. Blood Cells Mol Dis. 2006;37:12.
Thalassemia intermedia: Beta thalassemia
Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S Agarwal, M. Pradhan, DR Choudhry, VP
Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.
0
20
40
60
80
Beta Thalassemia
75.9
24.1 Homozygous
Heterozygous
Pre
val
ence
(%
)
Types of beta thalassemia
Beta Thalassaemia Beta Zero Beta Plus
Beta globin Chain Synthesis Absent Partial
HbA Absent 10-30%
HbF 95-98% 70-90%
HbA2 2-5% 2-5%
phenotype severe mild
S Europe
Cd 39 C/T
IVS1-110 G/A
IVS1-6 T/C
IVS1-1 G/A
W. Africa
-29 A/G
-88 C/T
Others
S.E. Asia
Cd 41/42
IVS 1-5 G/C
Cd 17 A/T
-28 A/G
S Asia
IVS1-5 G/C
IVS1-1 G/T
Cd 8/9
Cd41/42
619 bp del
Most common beta-thalassemia
mutations in different countries
Beta thalassemia mutations in TI in India
Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S Agarwal, M. Pradhan, DR Choudhry, VP
Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.
Mutations in beta thalassemia
Severe phenotype
IVS-I-5 (G->T) beta+/beta0 (severe)
IVS-I-1 (G->T); beta0
Codons 8/9 (+G); beta0
Codons 41/42 (-TTCT); TT beta0
619 bp deletion beta0
Mild Phenotype:
Capsite +1 ( A-C): 5 (6.7%)
-88 (C-T): 5 (6.7%) Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S Agarwal, M. Pradhan, DR Choudhry, VP
Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.
Hb E
Epidemiology
Hemoglobin E
disease
HbE syndromes
M 1 2 3
1,2 are Hom. 3 is Het.
330 bp
310 bp
Status Occurrence Frequency phenotype
Heterozygous E 52/ 339 15.33% asymptomatic
Homozygous E 41/339 12.1% asymptomatic
E beta 246 /339 72.56 TI
HbE Mutation (PCR-RFLP) variant Hb caused by Glutamic acid to Lysine (G-A) at
26th position of β globin chain
E beta thalassemia:Phenotypes
Clinical Characteristics Non Severe Severe
Age of presentation >3yrs ≤3yrs
Transfusion Requirements None or < 4units/year, frequent
Age of transfusion started at least after three years of age <3yrs
Spleen Size ≤3cm 3—10 cm
Hb >5g/dl 3-5g/dl
Growth & development >5percentile 3-5percentile
β mutations in HbE Beta thalassemia
Genotype Total (240) Severe (132) Non severe (108) P value
β globin gene mutations
IVS1–5 (G C) 102(42.5%) 53(40.1%) 49(45.3%) NS
CD 41/42 (-CTTT) 35 (14.6%) 18(13.6%) 17(15.7%) NS
CD 8/9 (+G) 30 912.5%) 20(15.1%) 10(9.2%) NS
CD 30(G-C) 18 (7.5%) 11(8.3%) 7(6.5%) NS
CD 15 (G-A) 24 (10%) 12(9.1%) 12(11.1%) NS
Cap site +1(A-C) 4(1.7%) 0 4(3.7%) 0.026
619 bp del. 9 (3.75%) 6(4.5%) 3(2.8%) NS
-88(C-T) 3(1.25%) 0 3(2.8%) 0.05
CD 16(-C) 2(0.83%) 2(1.5%) NS
IVS1-1 (G-T) 8(3.3%) 6(4.5%) 2(1.9%) NS
CD 39(C-T) 1(0.42%) 1(.76%) -
IVS-2 position 1(G-A) 1 (0.42% 1(.76) -
IVSII-613(C-T) 1 (0.42%) 1(.76%) -
initiation codon (ATG-ACG) 2 (0.84%) 1(.76%) 1(.92%) NS
Total 240 132 108
HS5 HS4 HS3 HS2 HS1
ε Gγ Aγ θβ δ β 5 4 3 2 1
Promoter Exon 1 IVSI Exon 2 IVSII Exon 3
3’ enhancer region 5’ silencer region
(AT)x(T)y
LCR Locus control region in beta globin gene
Locus control region (LCR)is located upstream of the β globin complex and regulates beta globin
gene.
Polymorphisms in Beta LCR in India (by
sequencing)
Region Status in our Population
HS1 : (CA)x(TA)y Reported Alteration Found
HS2 : (AT)x(N)y(AT)z Novel Alteration found
HS3 : sequencing , no repeats No Alteration found
HS4 : TGGGG(A/G)CCCCA Reported Alteration found
HS5 : sequencing, no repeats No Alteration found
5’Silencer Region: (AT)x (T)y Reported Alteration Found
3’Enhancer Region: sequencing, no repeats No Alteration found
LCR genotypes and phenotypes in Hb E thalassemia Gene variation Non severe (108) Severe (132) P value
HS1 (CA)x(TA)y
(CA)12(TA)6(Wild)
(CA)10(TA)8
(CA)12(TA)7
(CA)11(TA)7
41(37.9%)
24(22.2%)
22(20.3%)
21(19.4%)
85(64.3%)
20(15.1%)
17(12.8%)
10(7.5%)
.001
NS
NS
.01
HS2 (AT)xNy(TA)z
(AT)9N12(AT)10
(AT)10N12(AT)11(Wild)
(AT)9N12(AT)11
(AT)8N12(AT)11
31(28.7%)
30(27.7%)
31(28.7%)
16(14.8%)
23(17.4%)
45(34%)
47(35.6%)
17(12.9%)
.025
NS
NS
NS
5’ Silencer region (AT)x(T)y
(AT)9(T)5
(AT)8(T)4
(AT)8(T)7
(AT)7(T)7(Wild)
(AT)8(T)5
(AT)8(T)6
48(44.4%)
6(5.5%)
12(1.8%)
3(2.8%)
32(29.6%)
7(6.5%)
51(38.6%)
6(4.5%)
21(15.9%)
11(8.3%)
32(24.2%)
11(8.3%)
NS
NS
.001
.06
NS
NS
HS4 (TGGGG(A/G)CCCCA)
A(Wild)
G
79(73.1%)
29(19.4%)
109(82.5%)
23(17.4%)
.001
NS
Hb S
Epidemiology:
Hemoglobin S
disease
S Beta thalassemia
AIIMS
(n=349)
Phenotype West
SA 99 (28.3%) mild 23%
SS 175 (50.2%) severe 65.5%
Sβ
Sβ+
Sβ0
75 (21.5%)
TI 11.5% 29 (39%)
46(61%)
Comparative clinical parameters of HbSβ0-thalassemia and
HbSβ+-thalassemia patients(N=75)
Beta Thalassemia mutations in HbSβ patients (n= 75)
HbSβ0 : Common mut.
HbSβ+ : Cap site, -88 CT
0% 5%
10% 15% 20% 25% 30%
24.1%
13.8% 10.3%
13.0% 8.7% 6.9%
17.2%
26.1%
19.6% 17.4% 17.2%
10.3%
6.5% 8.7%
Hb… Hb…
Secondary modifiers
Alpha Globin Gene defects:
Alpha gene deletions and triplications
Gamma globin gene defects:
Xmn-1 polymorphisms
HPFH mutation
Delta Beta thalassemia
Alpha Thalassemia Prevalence in India:1% to 18%
Genetic Alterations
Deletions
Single Gene Deletions: Alpha 3.7 Kb Deletion (α3.7 ) ,
Alpha 4.2 Kb Deletion (α4.2 )
Double Gene Deletions: South African (--SA ) ,
South East Asian (--SEA) Point Mutations
Constant Spring Mutation (ααCS )
Hb Pakse Mutation (ααPS )
Triplication : (ααα3.7)
A : Mutant tube 1.8Kb
B : Normal tube 1.8 Kb
1 & 3 : Homozygous Mutant
2 & 4 : Heterozygous
t
1 & 3 : Homozygous Mutant
2 & 4 : Heterozygous
Gap PCR Alpha thalassemia 3.7 del
Alpha Mutations in Beta thalassemia
presenting as TI
0%
20%
40%
60%
80%
Beta Homozygous
Beta Heterozygous
36%
4% 0
60%
Alpha Deletion
Alpha Triplication
Alpha del:α3.7 =90% α4.2 =5% --SA =5%
--SEA not seen in India
Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S. Agarwal, M. Pradhan, DR
Choudhry, VP Choudhyry, R. Saxena. Haematologica 2006;91(9):12790-1280.
Alpha mutations in E beta thalassemia Mutations Total (240) Severe (132) Non severe (108) P value
Deletions 19 (7.9%) 10 (7.5%) 9 (8.3%) NS
Single Gene Deletion (α3.7)
Heterozygous
Homozygous
18 (7.4%)
14 (5.8%)
4 (1.6%)
10 (7.5%)
10 (7.5%)
0 (0%)
8 (7.4%)
4 (3.7%)
4 (3.7%)
NS
NS
0.03
Double Gene Deletions (--SA ) 1 (0.4%) 0 (0%) 1 (0.9%) NS
Point Mutations 9 (3.8%) 0 (0%) 9(8.3%) 0.02
ααCS 6 (2.1%) 0(0%) 6(4.6%) 0.03
ααPS 3 (1.3%) 0(0%) 3(2.8%) 0.04
Triplication 11(4.6%) 8(6.1%) 3(2.8%) 0.002
Poly A (AATAAA-AATA--)^ 2 (0.8%) 0(0%) 2(1.8%) NS
Total 41 (17.1%) 18 (16.7%) 23(21.3%) NS
α4.2 ; --SEA not present
Effect of alpha gene numbers on Phenotype of HbS-β (n=75)
0% 10% 20% 30% 40% 50% 60% 70% 80% 90%
100%
Sb without alpha Del
Sb with alpha Del
Sb with Alpha Trip
Alpha 3.7 del had milder phenotype
Alpha triplication had severe phenotype
Phenotypic effect of α-globin gene numbers on Indian sickle β-thalassemia patients. Pandey SK, Pandey S, Ranjan R, Shah V,
Mishra RM, Sharma M, Saxena R. J Clin Lab Anal. 2014, 28(2):110-3.
α3.7 =89% ; α4.2 =11%
--SA ; --SEA not present
Hb D
Epidemiology
Hemoglobin D
Punjab
disease
SD hemoglobinopathy
Homozygous HbD Punjab: mild hemolytic anemia
Heterozyg D: clinically silent
Double hetero D and S:
Pakistan, Iran, UAE and Mexico: clinical presentation of Hb SD-disease
similar to severe form of sickle cell anemia
AIIMS study on 15 pts of HbD-S showed the severity of disease with
thalassemia intermedia.
S.Pandey , R.Ranjan , T.Seth , V.Shah , R.M.Mishra , S.W.Pandey , R.Saxena Clinical-hematological profile of
Indian hemoglobin sickle cell D Punjab patients RRBS, 6(2), 2012 [41-43]
Effect of Alpha deletions in HbSD
N=10 N=5
S.Pandey , R.Ranjan , T.Seth , V.Shah , R.M.Mishra , S.W.Pandey , R.Saxena Clinical-hematological profile of
Indian hemoglobin sickle cell D Punjab patients RRBS, 6(2), 2012 [41-43]
α3.7 =100% α4.2 ; --SA ; --SEA not present
Xmn-1 polymorphism Xmn-1 polymorphism :C-T variation at position -158 upstream of G gamma gene which is detected by restriction enzyme Xmn-1
650 bp
450 bp
200 bp
1 2 3 4 5 6 25bp 7 8 9 10 11 12
PCR RFLP: Lane 1,4,5,8,9,10,11 heterozygous; 2,3,6,12 homozygous & lane 7 is normal
Xmn-1 (+/+) Xmn-1 (+/-) Xmn-1 (-/-) P value
HbF 36.2+4.7 32.1+5.2 17.6+4.8 <0.001
JK Science Vol. 8 No. 3, July-September 2006
HPFH and Delta Beta deletions based on origin
HPFH types
HPFH-1 – African
HPFH-2 – Ghanaian
HPFH-3 – Asian Indian
HPFH-4 – Southern Italian
HPFH-5 – Italian
HPFH-6 – Vietnamese
HPFH-7 – Kenyan
SEA-HPFH – Southeast Asian
Rapid detection of deletions causing delta beta thalassemia and hereditary persistence of fetal hemoglobin by
enzymatic amplification JE Craig, RA Barnetson, J Prior, JL Raven and SL Thein Blood 1994 83:1673-1682
DELTA BETA Types
Spanish (δβ)o
Sicilian (δβ)o
Chinese (δβ)o
Asian Indian Inv. Deletion Gγ(Aγδβ)0
Turkish (δβ)o
Japanese (δβ)o
Black (δβ)o
HbE beta thalassemia: Prevalence of HbF modifiers
Genotype Total Non severe (108) Severe (132) P value
#c.211 C-T(XmnI) +/+ 11(4.6%) 5(4.6%) 6(4.7%) NS
#c.211 C-T (XmnI)+/- 94(39.2%) 37(34.2%) 57(43%) NS
HPFH-3 (HPFH) 6(2.5%) 6(5.6%) 0(0%) <.001
Asian Indian Inv/Del
Gγ(Aγδβ)0 [delta beta)
2(.83%) 2(1.9%) 0(0%) NS
More than one modulator 7(2.9%) 7(6.5%) 0(0%) <.001
Negative for modulator 121(50.4%) 54(50%) 67(50.7%) NS
β-Globin gene cluster polymorphisms are strongly associated with severity of HbE/β0-thalassemia. R Saxena et al Clin Genet 2007: 72:
497–505
*2 Asian Indian Inv/Del Gγ(Aγδβ)0 had +/+, 3 HPFH-3 had +/-, 2 HPFH3 had +/+ Xmn-I pol.
Note: A study from Thialand in 207 mild and 307 severe patients concluded that T allele of XmnI pol. is more likely
associated with mild disease.
Effect of Xmn-1 on Phenotype of HbS beta(n=75)
Modulating Effect of the −158 Gγ (C→T) Xmn1 Polymorphism in Indian Sickle Cell Patients. S. Pandey, Sweta
Pandey, Rahasya MM and R Saxena. Mediterr J Hematol Infect Dis. 2012; 4(1): e2012001.
Tertiary modifiers a: HFE mutations Iron Overload
C282 Y
H63D
S65C
b: Uridine diphosphoglucuronate glucuronosyltransferase (UGT)
polymorphisms Hyperbilirubinemia
TA7TAA
TA8TAA
211(G-A)
1456 (T-G)
1091(C-T)
686(C-A)
Iron Overload in Thalassemia Intermedia
A major problem
Does not correlate with number of BT
Role of modifier genes speculated
HFE gene: commonest gene implicated:HFE gene protein binds β2-
microglobulin and decreases affinity of TFR receptor
Transferrin receptor gene
Tapasree Goswami, and Nancy C. Andrews J. Biol. Chem. 2006;281:28494-28498
HFE mechanism
Thal Intermedia
Prevalence of HFE mutation H-63D (AIIMS)
0
2
4
6
8
10
12
14
16
18
%
HFE mutation H63D predicts risk of iron over load in thalassemia intermedia irrespective of blood transfusions. Sharma V, Panigrahi
I, Dutta P, Tyagi S, Choudhry VP, Saxena R. IJPM 2007 Jan;50(1):82-5
Thalassemia Intermedia: HFE and Ferritin
0
10
20
30
40
50
60
70
80
90
100
HFE +VE HFE-VE
<500 ng/ml
>500 ng/ml%
Mean BT/ Pt: 3.83 4.52
Mean Age (yrs): 11.3 10.27 HFE mutation H63D predicts risk of iron over load in thalassemia intermedia irrespective of blood transfusions. Sharma V, Panigrahi
I, Dutta P, Tyagi S, Choudhry VP, Saxena R. IJPM 2007 Jan;50(1):82-5
Genotype No. of patients with >1000ng/ml/Total no. of patients
H63D/H63D 16/18(88.8%)
wt/wt 65/189(34.4%)
Incidence of higher ferritin levels due to H63D
mutation
Thalassemia Intermedia: Jaundice
homozygous (n=76) Heterozygous (n=17)
Pallor 69 (90.8%) 17 (100%)
Fatigue 35 (46%) 12 (70.5%)
Jaundice 32 (42.1%) 12 (70.5%)
Failure to gain wt. 16 (21.1%) 05(29.4%)
Spleen NP < 6cm 6 cm
05 (6.6%) 52 (68.4%) 19 (25%)
02 (11.8%) 07 (41.2%) 08 (47%)
Liver NP < 6 cm 6 cm
05 (6.6%) 62 (81.5%) 09 (11.8%)
01 (5.9%) 15 (88.2%) 01 (5.9%)
Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S Agarwal, M. Pradhan, DR Choudhry, VP
Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.
UGT (Uridine Diphosphate Glucuronosyl
Transferase-1) Polymorphism
Gene for UDP-glucuronosyltransferase-1 (UGT1*1):
enzyme responsible for hepatic glucuronidation of
bilirubin
The increased level of bilirubin has been related to a
polymorphism of the promoter of the UGT1*1 gene
Frequency of UGT polymorphisms in Patients
with E beta & controls
Genotype Patients Controls P value
A(TA)6TAA/A(TA)6TAA 42(17.5%) 61(61%) .001
A(TA)6TAA/A(TA)7TAA 55(22.9%) 20(20%) NS
A(TA)7TAA/A(TA)7TAA 23(9.5%) 1(1%) .001
A(TA)6TAA/A(TA)8TAA 4(1.6%) 0(0%) NS
A(TA)7TAA/211(G-A) 44(18.3%) 0(0%) .001
A(TA)6TAA/211(G-A) 43(17.9%) 13(13%) NS
211(G-A)/211(G-A) 15(6.3%) 0(0%) .001
Genotype T. Bilirubin Ind. Bilirubin
(TA)6/(TA)6 3.6±1.5 2.5±1.5
(TA)6/(TA)7 3.7±1.1 2.5±1.0
(TA)6/(TA)8 4.5±1.8 3.4±1.7
211(G-A)/(TA)6 3.6±1.0 2.6±1.0
(TA)7/(TA)7* 6.1±1.6 5.3±1.7
211(G-A)/211(G-A)* 6±1.1 5.1±1.0
211(G-A)/(TA)7* 8.0±2.4 7.5±2.3
Effect of UGT1A1 pol. on serum bilirubin levels
in HbE/β thalasssemia patients
*P value = <.05
Hb E/beta-thalassaemia: a common & clinically diverse disorder. Nancy F. Olivieri et al Indian J Med
Res 134, October 2011, pp 522-531
Summary Coexistence of hemoglobinopathy with beta thalassemia gene
makes the phenotype :
severer than the homozygous hemoglobinopathy
milder than thalassemia major
Thalassemia intermedia itself shows marked phenotypic variability:
Severity is increased by:
5 common beta thal mutations: IVS1-5,IVS1-1, Cd8/9, Cd41/42,619bp
del
Alpha triplications: ααα3.7
Beta LCR Polymorphisms: 5’ Silencer –
(AT)8(T)7
(AT)7(T)7
HS4- A Allele
Summary(contd.)
Mild phenotype by coexistence of :
Alpha deletions: -α 3.7, -α 4.2, --SA
Xmn-1 polymorphism
Beta Mutations: Cap+1 A/C; -88 C/T
HPFH: HPFH-3
Beta LCR Polymorphism: HS1- (CA)11(TA)7
HS2-(AT)9N12(AT)10
Summary(contd.)
Iron overload is high in HFE gene mutation (H63D)
Chance of occurance of jaundice is
higher in presence following polymorphisms;
A(TA)7TAA/A(TA)7TAA
A(TA)7TAA/211(G-A)
211(G-A)/211(G-A)
It is lower in following polymorphism
A(TA)6TAA/A(TA)6TAA
Acknowledgements
• Department of Science and Technology
• Indian Council of Medical Research