Recent Advances in Molecular
Understanding of NTDT: 2012
124 yrs of experience in medical care in Thailand
Vip Viprakasit, M.D., D.Phil.(Oxon)
Department of Pediatrics and thalassaemia Center
Faculty of Medicine, Siriraj Hospital
Mahidol University, THAILAND
Overview:
• Globin gene regulation and associated disorders
• Molecular basis of NTDT in a and b thalassaemia-haemoglobinopathies
- Conventional causes
- Unconventional causes
• Roles of genetic modifiers in NTDT
Structure and Expression of the Globin Genes
-40
-20
HS-40
0
20
z
a2 a1
40
Chromosome 16
FETUS
ADULT
a2g2
a2b2
EMBRYO
z2e2
b -LCR
b HS 4 3 2 1
e
g g
Haem
b
Chromosome 11
-40
-20
0
20
40
60
Structure and Expression of the Globin Genes
Hb A
Hb A2
Hb F
a2b2
a2d2
a2g2
Newborn
25%
<1%
75%
Adult
96%
3%
1%
The Molecular Basis for a and b thalassaemia
-40
-20
0
20
40
z
a2 a1
Chromosome 16
HS-40
Deletions
Deletions
Point Mutations
Chromosome 11
b-LCR
HS 4 3 2 1
-20
e
0
g g
20
b
40
60
Overview:
• Globin gene regulation and associated disorders
• Molecular basis of NTDT in a and b thalassaemia-haemoglobinopathies
- Conventional causes
- Unconventional causes
• Roles of genetic modifiers in NTDT
Molecular basis of a0 and a+ thalassaemias:
deletions & non-deletions
aTa /aa
aaT /aa
--/aa
-a/aa
(modified from Higgs, Disorder of Hemoglobin, 2001 with permission)
Hb H disease is the most common form of NTDT
• Patients with non-deletional type of Hb H disease
(--/aTa) had more severe clinical symptom than those with
deletional type of Hb H (--/-a)
--SEA/aCSa
--SEA/-a3.7
Review in Chui DH, Chan V. & Fucharoen S. Blood 2004
Hb H Hydrops Fetalis
Hydrops fetalis due to an unusual form of Hb H disease.
By Chan V et al, Blood. 1985, 66(1):224-8.
Hydropic fetus with 20-25% of Hb Bart’s
Molecular pathology:
1. - - TOT/aCD30a, del. GAG
2. - - SEA/aCD35a, TCCCCC
3. - - SEA/aCD59a, GGCGAC
4. - - SEA/aCD66a, CTGCCG
5.
apolyAa /apolyAa*
6. --SEA/aQSa
7. aCSa/aCSa and --SEA/aCSa
Viprakasit V. et al, Br J Haematol. 2002;117(3):759-62.
Fucharoen S. & Viprakasit V. Hematology (ASH) 2009
Common causes of NTD-b thalassaemia
b-Thalassaemia intermedia
b0 thal / b+ thal
b+ thal / b+ thal
Hb E / b thal.
b0 thal / HPFH
b+ thal / HPFH
b0 thal / db thal
b+ thal / db thal
Courtesy of Prof. V. S.Tanphaichitr
A wide spectrum of b globin mutations in Thailand
Summary of known b-globin genes
mutations identified in Thailand: 2010
• Each mutation can be classified as b0 or b+ thalassaemia based on the
output of b globin transcripts
11
Suchada Riolueang et al.
16th
National Thalassaemia Conference, Bangkok 2010
b thalassaemia intermedia (Hb E/b thalassaemia)
b 0/ b +
+ bE
Courtesy of Prof. V. S.Tanphaichitr
Most common b globin gene cluster deletions
found in Asia-Pacific
Nipon Chalaow, Suchada Riolueang, Chompunut Karnjanakorn, Chanin Limwongse & Vip Viprakasit;
To be presented at APIA 2012, Taiwan
Summary of HPFH & db thalassemia
interactions with NTDT and TDT phenotype
Genotype
N
M(%)
Clinical phenotype
--HPFH-6/b
6
3 (50)
High Hb F (Hb F26-34%)
--HPFH-6/bE
3
3 (100)
Mild b thal/Hb E disease
--HPFH-6/b-31
1
1(100)
thalassemia intermedia
--(db)/b
3
1 (33)
High Hb F (Hb F 29-32%)
--(db)/bCd41-42
1
1 (100)
b thal major (splenectomy)
b-619/bE
1
1(100)
b thal major (SCT)
--Chinese/b
1
1 (100)
High Hb F (Hb F12.8%)
--Chinese/bE
1
1 (100)
Mild b thal/Hb E disease
--Lepore/bE
1
1 (100)
Hb Lepore 12.5%
--Lepore/bE
2
2 (100)
Mild b thal/Hb E disease
Total
16
11 (68)
Vip Viprakasit: Unpublished data 2012
Overview:
• Globin gene regulation and associated disorders
• Molecular basis of NTDT in a and b thalassaemia-haemoglobinopathies
- Conventional causes
- Unconventional causes/novel mechanism/trans acting mutations
• Roles of genetic modifiers in NTDT
Upstream deletional a0-thalassaemia
Fucharoen S & Viprakasit V, Hematology (ASH) 2009
Unusual deletions causing a0 thalassaemia and Clinical
Hb H disease
Viprakasit V. et al, British Journal of Haematology, 2003 120(5): 867-75
Weatherall DJ, Higgs DR, Bunch C, et al,
Hemoglobin H disease and mental retardation.
A new syndrome or a remarkable coincidence?
N Engl J Med 305: 607-612. 1981
X-linked thalassemia/mental retardation (ATR-X) syndrome
ATRX-gene or XNP2 or XH2
Gibbons RJ, Picketts DJ, Villard L, Higgs DR.
Mutations in a putative global transcriptional
regulator cause X-linked mental retardation with athalassemia (ATR-X syndrome).
Cell 80: 837-845.
Gibbons RJ, Higgs DR., Am J Med Genetics, 2000
Other syndromes with ATRX mutations:
Carpenter syndrome, Juberg-Marsidi syndrome,
Smith-Fineman-Myers syndrome,
X-linked mental retardation with spastic paraplegia
Interaction of b thal alleles with extra copies
of a globin genes
b0 thal /aaa/aa
b0 thal /aaaa/aa
b0 thal /aaaaa/aa
b+ thal /aaa/aa
b+ thal /aaaa/aa
b+ thal /aaaaa/aa
• 24-year old Thai woman with underlying
anemia since 7 years old
• She received less than 7 blood transfusions in
her lifetime
• Hb 7.5 g/dL Hct 26% RBC 4.8 x106/mL, MCV 62
fL, MCH 24 pg MCHC 33 pg/dL, RDW 27 %
Retic. 7%
• Hemoglobin typing: A, A2 (7%), F (10%)
• DNA analysis:
aaa/aa and b/b41/42
39CT
b39CT
b39CT
/ b/--Patient A
• 42 yrs-male with Hb 7-9 g/dL
• Clinical diagnosis with b thal. Intermedia
• S/P splenectomy
• Hb typing: A 81% A2 4.8% F 14.2%
• a/b+g globin synthesis: 1.79
Patient B
• 23 yrs-female with Hb 7-9 g/dL
• Clinical diagnosis with b thal. Intermedia
• Marked splenomegaly (10 cm BLCM)
• Hb typing: A 87% A2 4.3% F 8.6%
• a/b+g globin synthesis: 1.65
Baden C et al, Lancet 2002: 359; 131-2, Galanello R et al. BJH 2004: 127: 604-6
XPD mutations in Patients with Trichothiodystrophy
Causes b thalassaemia phenotype
A594P
Hb 11.5 g/dL Hct 34%
MCV 65 fL MCH 24 pg
Hb A A2 (A2 4.5%)
Haematological phenotype mimic that of
mild b thalassaemia
Viprakasit, et al. Human Molecular Genetics 2001:2797-802 (2001)
Schematic Representation of Seven-Conserved Helicase Domains of
XPD Gene and Mutations in TTD and XP-D Patients
R722W
R112H
R683W
R487G
Fs 730
A594P
I
Ia
II
III
IV
V
VI
del 716/L461V
del 488-93 Intron 7 R616P
splice donor
Fs 609
Null and missense TTD specific mutations
Null and missense XP specific mutations
Viprakasit, et al. Human Molecular Genetics 2001:2797-802 (2001)
Further Example of Trans Acting Mutations
Causing Thalassaemia
Patients
Age
Hb
(g/dl)
Hct (%)
RBC
x106/ml
MCV (fl)
MCH
(pg)
Retic
(%)
RDW
(%)
Plt
x103/ml
II-2
23 mths
8.5
29.2
3.3
88.6
26.1
3
29
24
II-2
8 mths
8.2
27.4
2.9
96.2
28.8
3
19.1
11
I-2
28 yrs
11.3
33.5
3.6
94.0
31.6
1
13.1
53
HbA2 level was at 3.8%-4.4%
(normal range: 1.8%-3.4%)
The a : b chain synthesis ratios
were 0.55 to 0.67 in affected
individuals
Nichols KE et al. Nature Genetics 24, 266 - 270 (2000)
Further Example of Trans Acting Mutations
Causing Thalassaemia
Familial dyserythropoietic anaemia and thrombocytopenia due to an
inherited mutation in GATA1
Nichols KE et al. Nature Genetics 24, 266 - 270 (2000)
a Thalassaemia in the South Pacific
a thalassaemia is common in the South Pacific
Some Individuals with a thalassaemia have an apparently normal genotype (aa/aa)
We identified three Families one from PNG and two from Vanuatu
with HbH disease
HbH Disease In a Family From Papua New Guinea
aa/aa
aa/aa
aa/aa
Is this really HbH disease?
Haematological Phenotypes
a/b Globin Synthesis Ratio
0.39-0.52
NR 1.06 ± 0.11
a/b Globin mRNA Ratio
~0.5 based on qPCR
NR 1 ± 0.2
RNA FISH analysis confirmed a down regulation at the transcriptional
level
De Gobbi M* & Viprakasit V* et al. Science 2006; 1215-7
Identifying a Candidate SNP
50
100
150
200
250
z aDaa
17
Differences in the region underlying the new peak of expression
7
After Removal of known SNPs
1
In linkage with the disease
aa/aa
aa/aa
aa/aa
141
31
5
T/T
T/C
C/C
300
Overview:
• Globin gene regulation and associated disorders
• Molecular basis of NTDT in a and b thalassaemia-haemoglobinopathies
- Conventional causes
- Unconventional causes
• Roles of genetic modifiers in NTDT
Roles of genetic modifiers on b-thal intermedia
Thein SL, Brit J Haematol 2004, p 264-274)
Roles of genetic modifiers on a-thal syndromes
a genes
b genes
Hb Bart’s
Excess b globin chains
Thein SL, Brit J Haematol 2004, p 264-274)
Summary:
• Molecular basis of NTDT either a and b globin genes related are
heterogeneous with several novel molecular defects have been
increasingly found
• Identification of molecular pathology of NTDT patients will be useful
for management plan (early treatment and intervention including
transfusion programme), prevention & control measures
• Basic globin gene analyses should be first performed to identify
common molecular pathology, if not found, a further detail study is
highly recommended and this can provide new insights to understand
gene regulation and genome biology
Acknowledgements
Vip’s Laboratory
Nipon Chalaow
Suchada Ruengleung
Nuntawut Chat-Utai
Rapeepun Shompoopuong
Punyanuch Pornpanich
Kalaya Tachavanich
Warapron Glomglao
Supachai Ekwattanakit
Faculty of Medicine, Siriraj Hospital
Mahidol University, THAILAND
Chompunut Kanjanakorn
Dr. Chanin Limwongse
Prof. Suthat Fucharoen
Prof. Voravarn S. Tanphaichitr
Thailand Research Fund
BIOTEC, Thailand
Disorders of Globin Chain Production
1. Quantitative defects: Thalassaemia syndrome
causing imbalance of globin chain production
2. Qualitative defects: Haemoglobinopathies
result from substitution, deletion or insertion of one
or more amino acid
3. Hereditary of Persistence of Fetal hemoglobin
Genetic Analysis of the a Globin Locus
50
100
150
200
250
300
z aDaa
Excluded deletions and rearrangements
No point mutations
No other abnormalities
De Gobbi M* & Viprakasit V* et al. Science 2006; 1215-7