Genotypes/phenotypes Thalassemia in Asia - by Suthat Fucharoen MD of the Thalassemia Research Center Institute of Molecular Biosciences, Mahidol University, Thailand.

1. Genotypes/phenotypes
Thalassemia in Asia
Suthat Fucharoen M.D.
Thalassemia R
Th l i Research C t
h Center
Institute of Molecular Biosciences
Mahidol University, Thailand

2.    
 
  
  
Normal -Thalassemia
Thalassemia -Thalassemia
Thalassemia
Hb A = 22 (97%) 4 = HbH Increased HbA2
HbA2 = 22 (2.5%) 4 = HbBart’s (4-6%)
Hb F = 22 (0 5%)
(0.5%)
Normal Thalassemic
RBC RBC

3. Pathophysiology of
–Thalassemia/Hb E Disease

5. Interstitial deletional -thalassemias
--/
-thalassemia1 (--SEA, --THAI & --FIL)
-thalassemia2 (-3.7 & -4.2)
Hb Constant Spring -/
Hb Paksé
Range 10-25%
(modified from Higgs DR, Disorder of Hemoglobin, 2009)

6. Types of Mutations Resulting in -Thalassemia
yp g 
> 200 mutations

7. Thalassemia in Thailand
‐Thalassemia (‐thal1 and ‐thal2) 20 ‐ 30%
Hb Constant Spring ( -thal 2 like effect ) 1 ‐ 8%
-Thalassemia
Th l i 3 ‐
3 9%
Hemoglobin E 10 ‐ 53%
Total number of thalassemic patients and the number of
births per year (total births = 800,000/year)
Diseases Couple at risk Birth Living
(per year) (per year) patients
Homozygous -thalassemia
thalassemia 828 207 2,070
2 070
-Thalassemia/Hb E 12,852 3,213 96,390
,
Hb Bart s hydrops fetalis 3332 833 0
Hb H disease 22,400 5,600 336,000
Total ,
39,412 9,853
, 434,460
,
Fucharoen S. and Winichagoon P., 1988

8. Alpha Thalassemia in Thailand (2008)
Central South Northeast North
Mutations
No. % No. % No % No. %
Deletional Hb H 14 26.9
1. –SEA/‐α3.7 199 39.8 75 50 34 33.3
2. –SEA/‐α4.2 9 1.8 7 4.7 10 9.8
3 THAI/ α3.7
3. – /‐α 4 0.8
08 1 0.7
07 ‐ ‐
4. etc 1 0.2 ‐ ‐ ‐ ‐
Non‐Deletional Hb H
1. –SEA/αCSα 247 49.4 55 36.9 35 67.3 54 53
2. –SEA/αPSα 28 5.6 4 2.7 3 5.8 3 3
3. –THAI/αCSα 2 0.4 2 1.3 1 1
4. etc 10 2 5 3.3 ‐ ‐
Total
l 500
00 100
00 149
9 100
00 52
2 100
00 102
02 100
00

9. -Beta Thalassemia in Thailand (2008)
Central South Northeast North
Mutations
No. % No. % No % No. %
1. codon 41/42 (‐TCTT) 50 36.3 109 15.1 19 31.6 108 49.5
2. codon 17 (A‐>T) 24 17.4 41 5.7 13 21.7 75 34.4
3. codon 35 (C‐>A) 2 1.4 4 0.6 0 0 ‐ ‐
4. IVS1‐1 (G‐>T)
4 IVS1 1 (G T) 3 2.2
22 35 4.8
48 1 1.7
17 15 6.9
69
5. codon 71/72 6 4.3 4 0.6 8 13.3 13 6
6. etc. 5 3.5 35 4.8 1 1.7 ‐ ‐
7. IVS1‐5 (G‐>C) 7 5.1 98 13.6 0 0 ‐ ‐
8. IVS2‐654 (C‐>T) 8 5.8 16 2.2 5 8.3 1 0.4
9. codon 19 (A‐>G) 3 2.2 57 7.9 0 0 ‐ ‐
10. ‐28 (A‐>G) 20 14.5 20 2.8 1 1.7 3 1.4
( )
11. codon 26 (Hb E) ‐ ‐ 243 33.7 9 15 ‐ ‐
12. etc. 10 7.3 9 1.2 ‐ ‐ 3 1.4
13. δβ thalassemia ‐ ‐ 14 1.9 ‐ ‐ ‐ ‐
14. Unknown ‐ ‐ 37 5.1 3 5 ‐ ‐
Total 138 100 722 100 60 100 218 100

10. ANEMIA IN THALASSEMIA
Concordance and Discordance Among Sib Pairs
Fucharoen S et al. Am J Med Genet 1984; 19: 39-44

11. Variable severity in -thalassemia disorder
Intermediate Normal Hemoglobin value
12 to 14 g/dl
tients
Numb of Pat
ber
Severe Mild
Fucharoen S et al Birth Defects 1988; 23(5A): 241-8

12. Thalassemia:
Genotype-phenotype Interaction
G t h t I t ti
1. Better d t di
1 B tt understanding of
f
gene-gene interaction,
natural history, prognosis
2. Decision of therapeutic
p
intervention eg. BM transplantation
3.
3 Prenatal diagnosis and
induced abortion
4. New h
4 N therapeutic i
i intervention
i

13. Factors affecting severity of -thalassemia disease
Heterogeneity of Primary Mutations:
H t it f P i M t ti

-Thalassemia mutations: -Thal, -Thal
,
Genetic Modifiers:
Coinheritance of -thalassemia
Stimulation of Hb F production
1. Concomitant inheritance of -thalassemia in -thalassemia/Hb E disease.
Am J Hematol 1985; 20:217.
;
2. Severity differences in -thalassemia/hemoglobin E syndromes; implication of
genetic factor. Br J Haematol 1993; 83; 633.
3. Genetic factors affecting clinical severity in beta-thalassemia syndromes.
J Pediatrics Hematol/Oncol 2000; 22: 573.

14. Association Studies
• Detect association between genetic variants and phenotype
across families
– Case-Control designs
– Cohort designs
Single Nucleotide Polymorphisms: SNPs
• Biallelic polymorphism
• Accounting for ~90% of human genetic variants
• Occurring on coding and non-coding chromosome region
SNP markers associated with modifier alleles
GENE
SNP modifier SNP SNP SNP
marker allele marker marker marker

15. SCORING SYSTEM FOR CLASSIFYING THE DISEASE
SEVERITY IN  THALASSEMIA PATIENTS
-THALASSEMIA
Clinical criteria Points scored
0 0.5 1 2
Hb at steady state (g/dL) > 7.5 - 6-7.5 <6
Age at first transfusion (yr) >10 - 5-10 ≤4
Requirement for transfusion None/Rare - Occasional Regular
Size of spleen (cm) <3 - 3-10 >10 or
splenectomized
Age at presentation (y )
g p (yr) >10 3-10 ≤2
Growth development >25th percentile 3rd-25th percentile <3rd percentile
Severity grouping Total score range
Mildly affected 0-3.5
Moderately affected 4-7
Severely affected 7.5-10
(Sripichai O et al, 2008)
15

16. Distribution of 0-thalassemia/Hb E patients based on -globin gene mutations.
50.0
00
45.0
40.0
entage of cases
35.0
30.0
f
25.0
20.0
15.0
50
Perce
10.0
5.0
0.0
Cod41/42 Cod17 IVS II-654 IVS I-1 IVS I-5 Cod35 Cod71/72
Rare Unchar
(-TTCT) (A>T) (C>T) (G>T) (G>C) (C>A) (+A)
All samples (n=517) 44.0 24.1 9.9 4.6 6.2 1.5 2.5 4.4 2.7
Pooled mild (n=197) 42.1 24.9 9.6 8.6 4.1 2.5 0.5 7.6 0.0
Pooled severe (n=198) 45.5 24.2 14.6 1.5 7.1 1.0 4.0 2.0 0.0

17. N -thalassemia
No th l i With -thalassemia
th l i
7.0 7.0
Mild with alpha-
6.0
60 Mild 6.0
60 thalassemia
oglobin F (g/dl)
oglobin F (g/dl)
Severe
5.0 5.0 Mild without alpha-
thalassemia
4.0 4.0
3.0 3.0
Hemo
Hemo
2.0 2.0
1.0 1.0
0.0 0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0
Hemoglobin ( /dl)
H l bi (g/dl) Hemoglobin (g/dl)

18. SNP map, Beta Globin Gene Cluster
map
. .. Confirmed Thai polymorphisms (n = 503):
.
.. .. 10 C
Common RFLPs
RFLP
.
54 Reference SNPs
. . 3 Novel SNPs
. . . Median Intermarker = 514 bp
. . . ...
. . . ..
. .
. . .
....
.
.. . .
... . . . . . ..
. .. . ..
.. .. . .
...
LCR

19. XmnI–Gγ POLYMORPHISM AS A MODIFYING FACTOR IN β-THALASSEMIA
β
Allele distribution of XmnI in Mild &
Association between Xmn I genotype and
Severe β0-thalassemia/Hb E patients.
Hb F level stratified by severity group (n=517).
100%
90% All subjects Mild
MILD SEVERE
Severe
80% 74%
70% 64%
60%
% patients
53%
50%
40% 34% 34%
30%
20% 16%
10%
10%
2%
0%
/ / /
XmnI +/+ XmnI +/ X
X I X I +/- XmnI -/-
I / XmnI +
X I
-/- +/- +/+ -/- +/- +/+
Xmn I-Gγ site Xmn I-Gγ site
β-Thalassemia/Hb E patients with XmnI -/- are 35 times more likely to have
severe disease than patients with XmnI +/+.
β-Thalassemia/Hb E patients with XmnI +/+ and +/- h
h l i / b i ih / d / have hi h Hb F level than
higher b l l h
patients with XmnI -/- (p-value < 0.001).

20. High correlation r2, value in β-globin cluster
correlation,
46 kb
LCR ε Gγ Aγ δ β
3 TagSNPs cover 88% of the genotypes in large LD block

21. Genotype of 3 TagSNPs
Genotypes 95% CI
bg2 bg11 bg200 Mild Severe Total odd ratio
frequency for odd ratio
AA AA CA 1 1 0.21
AA AA CC 14 64 78 16.02 3.55 1.93 - 6.55
AA AG CA 4 4 0.82
AA AG CC 2 2 0.41
AC AA CA 1 1 0.21
AC AA CC 2 19 21 4.31 6.59 1.51 - 28.65
AC AG CA 119 152 271 55.65
55 65 0.66
0 66 0.46 0.96
0 46 - 0 96
AC AG CC 4 10 14 2.87 1.66 0.51 - 5.38
AC GG AA 6 1 7 1.44 0.10 0.01 - 0.89
CC AA CC 3 3 0.62
0 62
CC AG CA 6 13 19 3.90 1.44 0.53 - 3.86
CC AG CC 4 4 0.82
CC GG AA 27 3 30 6.16 0.06 0.01 - 0.21
CC GG CA 11 20 31 6.37 1.20 0.56 - 2.58
CC GG CC 1 1 0.21

22. 120
Al cases (n=1081)
l
100 Pooled cases (n=395)
80
- Unrelated patients
l t d ti t
N me o c s s
u b r f ae
Regionally-Matched 60
- Extremely Severity
DNA Pools
40
20 - Excluded known
Construction 0
modifying factors
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
Disease Severity Score
DNA1, DNA2 … DNAnMild DNA1, DNA2 … DNAnSevere
Mild Severe
DNA pool DNA pool
(n=197) (n=198)
110,000 SNPs genotype using MassARRAY system
, g yp g y
SNPs p-value
<0.05
<0 05
854 SNPs were genotype individually in 198 mild, 305 severe patients
GENOMEWIDE SEARCH FOR DISEASE MODIFIER GENES IN β0-THALASSEMIA/HBE

23. 160 Candidate Modifier Genes/Regions
from Genomewide Association Study
APOPTOSIS & CELL CYCLE REGULATION
Cell cycle
regulation, Associated PROTEASE & UBIQUITINATION
Fas apoptotic inhibitory molecule 3
Apoptosis Function REGULATORY FACTORS
NIMA (never in mitosis gene a)- related kinase 11
calpain 1 (mu/I) large subunit
1,
Proteolysis SIGNAL2TRANSDUCTION 1
sestrin
high-mobility group box
NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5,
T-box 2
INTERESTING BIOLOGICAL FUNCTION
NACHT, leucine rich repeat and PYD containing 14
16kDa beta 4 binding protein
mitogen-activated protein kinase kinase kinase 9
integrin
leukemia sarcoma(TEL1 oncogene homolog 1 (avian)
v-ros UR2 inhibitory virus receptor
HtrA serine gene 6 factor
ets variant assembly factor 1, subunit A (p150)
chromatin peptidase oncogene)
p rich interactive domain 5B (
protein-coupled receptor 22 (MRF1-like)
AT p p )
oncoprotein induced transcript 3 2
phosphoinositide-3-kinase, thrombospondin type 1 motif, 6
phosphoinositide 3 kinase class 2, alpha polypeptide
ADAM metallopeptidase withsuppressor of hairless
adenylate kinase 5 protein
recombining binding 2 (juvenile)
hemochromatosis type
FERM, RhoGEF and III (DNA directed) polypeptide D, 44kDa
polymerase (RNA) pleckstrin domain protein
(Drosophila)
frataxin
protein tyrosine kinaseinitiation factor 2C, 4
eukaryotic translation 2 beta
gastric inhibitory polypeptide
DnaJ (Hsp40) related, subfamily B, member 13
neurolysin (metallopeptidase M3 family)
TAO kinaseconverting enzyme 1
endothelin 3 beta, acid 3 (cytosolic)
glucosidase,
receptor determining region Y)-box 5receptor
axin(sex tyrosine inhibitor,M5 orphan
kinase-like
SRY p p S-transferase , clade B, member 11
g p
glutathione
serpin peptidase ,
Non-Associated
i t d membrane associated guanylatefactor 3 WW and PDZ
pre-B-cell l k
lipase ll leukemia t
B i transcription f kinase,
g an ti
Wilms tumor associated protein
i late kinase
t
Regulatory carboxypeptidase E
domain containing activity-1
transcription factor
glucose transporter
Function protein kinase, cGMP-dependent, type I
interleukin protein 152 transposase fusion gene
ring finger 24 transporting
SET domain and mariner
ATPase, Ca++
Factor src family associatedhomolog 1 (Drosophila)
sex comb on midleg phosphoprotein 1
ATPase, H+ transporting
F-box and WD-40 domain protein 11 2C, 3
Rho guanine nucleotide exchange factor (GEF)
eukaryoticvoltage-gated channel
potassium translation initiation factor
12 HSA275986
transcriptiond alpha-linkedd idi ddipeptidase-like 1
calcium h factor activity acidic di t
N-acetylated l l activity-2
lt k d 2
N l i t lchannel,hvoltage-dependent tid
t li lik
transcription factor659
zinc finger protein SMIF
Unknown Function chloride intracellular channel 5
vascular cell adhesion molecule 1
zinc adhesion
cell finger protein 214
regulator of G-protein signalling like 2
zinc finger protein 609
Golgi function
tumor necrosis factor receptor superfamily, member 11,
mitochondrial protein
activator of NFKB
Hypothetical Protein

24. 2nd Genome‐wide Association Study (GWAS)
2nd Genome‐wide Association Study (GWAS)
β0‐Thalassemia/Hb E patients
383 SEVERE CASES 235 MILD CASES
GWAS analysis
• QC data Quality Controls : MAF > 0.05, call rate > 0.99
Q
• Significant levels
Statistical Analysis using chi‐square test for general model and Fisher exact test for dominant recessive and allelic model

25. Results of genome-wide association
Nu
uinoon et
t al. Hum
m Geneti
ics 125: 3
303‐14, 2010

26. 618 Thai β0 thalassemia/Hb E patients
618 Thai β0‐thalassemia/Hb E patients
# Significant SNP from Sardinian population (Galanello et al, Blood 114: 3935‐7, 2009)
g p p ( , , )

27. Galanello et al. Blood 114: 3935‐7, 2009

28. Sardinians
Galanello et al. Blood 114: 3935‐7, 2009

29. All -globin gene mutations
618 0‐thalassemia/Hb E patients

30. Codon 41/42, -TCTT
297 0‐thalassemia/Hb E patients

31. The box plot shows the distribution of HbF (%) and absolute HbF (g/dl)

32. FETAL HEMOGLOBIN ANALYSIS
Linear regression analysis Number of
(Additive model, P value) high-HbF allele
Locus rs ID SNP allele High-HbF HbF (%) Abs HbF (g/dL) 0 1 2
allele
BCL11A rs766432 A/C C 2.33E-07 1.20E-09 AA AC CC
HBS1L-MYB rs9399137 A/G G 2.76E-18 5.93E-19 AA AG GG
HBG2(XmnI) rs7482144 C/T T 2.52E-19 1.90E-23 CC CT TT
32

33. Conclusion: 1
1. We have introduced a scoring system to classify
-thalassemia patients into mild, intermedia and severe
cases. Mild patients have hi h Hb F levels.
ti t h higher l l
2. β-Thalassemia/Hb E patients with XmnI -/- are 35
β p
times more likely to have severe disease than patients
with XmnI +/+.
3. Three “novel SNPs” on the -globin gene cluster show
novel SNPs  globin
strong association with disease severity and Hb F
levels.

34. Conclusion: 2
4. Genome wide scan for SNPs of the whole genome
revealed 150 SNP on 100 genes that showed strong
l d SNPs h h d
association with disease severity
5. 2nd GWS revealed SNPs in 3 major regions that
showed strong association with disease severity, namely
-globin gene cluster, intergenic region between Myb
cluster
and HBS1L and BCL11A. The function of these
regions in the modifying of disease severity need
further studies.
studies

35. pVal = 0.0381
pVal = 0.0158
GeneGo Display of the largest pVal = 0.0141
network of genes with direct
interactions
(6) pVal = 0.0013
pVal = 0.0834
(5) pVal = 0.0042
pVal = 0.00162
pVal = 0.0737
(2) pVal = 0.0123

36. Systems Biology & Thalassemia
Globin gene Red Cell Anemia
e a Iron Clinical
mutation Manifestation
Genotype Phenotype
Drug Organ
Erythropoiesis
Metabolism Pathology
Apoptosis
Iron Absorption
Ferrokinetic
Oxidative Stress
Antioxidants Heart
GDF 15 Liver
Endocrine glands
g
UGT
Cyt P450 CVS
High HbF Genes
Epo

38. Acknowledgement
Siriraj Hospital, Mahidol University: Noppadol Siritanaratanakul
Ramadhibodi Hospital, Mahidol University: Suporn Chancharunee,
Tanyachai Sura,
T h iS
Ampaiwan Chuansumrit
Chulalongkorn University: Issarang Nuchprayoon
Chiangrai P h
Chi i Pachanukraw H
k Hospital:
it l Saranya S
S Suwansings
i
Maharaj Nakornrajchasima Hospital: Somchai Intarasiripong,
Nittaya Visanuyothin
Lampang Hospital:
L H it l Ladda S ib ib
L dd Sriboriboonsini
Uttaradit Hospital: Chuchart Koosirirat,
Anong Benjakoranee
Chonburi H
Ch b i Hospital:
it l Kesada Ch
K d Chansawang
Saraburi Hospital: Soraya Thammarak,
Bang-orn Ubol
Maharaj Nakornsrithammarat:
M h jN k ith t Wanchai Kachonwattanakul
W h iK h tt k l
Nakhonpathom Hospital: Piatip Boonmongkol,
Navarat Chantrakul

39. Acknowledgement