vip viprakasit, m.d., d.phil.(oxon) department of pediatrics and thalassaemia center
DESCRIPTION
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 :. - PowerPoint PPT PresentationTRANSCRIPT
Vip Viprakasit, M.D., D.Phil.(Oxon)Vip Viprakasit, M.D., D.Phil.(Oxon)Department of Pediatrics and thalassaemia CenterDepartment of Pediatrics and thalassaemia Center
Faculty of Medicine, Siriraj HospitalFaculty of Medicine, Siriraj HospitalMahidol University, THAILANDMahidol University, THAILAND
124 yrs of experience in medical care in Thailand
Recent Advances in Molecular Understanding of NTDT: 2012
Overview:
• Globin gene regulation and associated disorders
• Molecular basis of NTDT in and thalassaemia-haemoglobinopathies
- Conventional causes
- Unconventional causes
• Roles of genetic modifiers in NTDT
Structure and Expression of the Globin Genes
Chromosome 16
Chromosome 11
EMBRYO FETUS ADULT
Haem
HS-40
-LCR123 HS 4
-40 -20 0 20 40
-40 -20 0 20 40 60
Newborn AdultHb A 22 25% 96%Hb A2 22 <1% 3% Hb F 22 75% 1%
Structure and Expression of the Globin Genes
Deletions
Point MutationsDeletions
The Molecular Basis for and thalassaemia
Chromosome 16
Chromosome 11
0 20 40-20
HS-40
-LCR
0 20 40 60
123 HS 4
-40
-20
Overview:
• Globin gene regulation and associated disorders
• Molecular basis of NTDT in and thalassaemia-haemoglobinopathies
- Conventional causes
- Unconventional causes
• Roles of genetic modifiers in NTDT
(modified from Higgs, Disorder of Hemoglobin, 2001 with permission)
Molecular basis of 0 and + thalassaemias: deletions & non-deletions
--/
-/
T/T/
• Patients with non-deletional type of Hb H disease (--/T) had more severe clinical symptom than those with deletional type of Hb H (--/-)
--SEA/CS --SEA/-3.7
Review in Chui DH, Chan V. & Fucharoen S. Blood 2004
Hb H disease is the most common form of NTDT
Hb H Hydrops FetalisHb 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/CD30del. GAG2. - - SEA/CD35, TCCCCC3. - - SEA/CD59GGCGAC4. - - SEA/CD66CTGCCG5. polyA /polyA6. --SEA/QS7. CS/CSand --SEA/CS
Viprakasit V. et al, Br J Haematol. 2002;117(3):759-62. Fucharoen S. & Viprakasit V. Hematology (ASH) 2009
Common causes of NTD- thalassaemia
0 thal / + thal+ thal / + thalHb E / thal.0 thal / HPFH+ thal / HPFH0 thal / thal+ thal / thal
-Thalassaemia intermedia
Courtesy of Prof. V. S.Tanphaichitr
11Suchada Riolueang et al. 16th National Thalassaemia Conference, Bangkok 2010
Summary of known Summary of known -globin genes -globin genes mutations identified in Thailand: 2010mutations identified in Thailand: 2010
A wide spectrum of globin mutations in Thailand
• Each mutation can be classified as 0 or + thalassaemia based on the output of globin transcripts
thalassaemia intermedia (Hb E/ thalassaemia)
Courtesy of Prof. V. S.Tanphaichitr
0/ + + E
Most common 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 & thalassemia interactions with NTDT and TDT phenotype
Genotype N M(%) Clinical phenotype
--HPFH-6/ 6 3 (50) High Hb F (Hb F26-34%)
--HPFH-6/E 3 3 (100) Mild thal/Hb E disease
--HPFH-6/-31 1 1(100) thalassemia intermedia
--()/ 3 1 (33) High Hb F (Hb F 29-32%)
--()/Cd41-42 1 1 (100) thal major (splenectomy)
-619/E 1 1(100) thal major (SCT)
--Chinese/ 1 1 (100) High Hb F (Hb F12.8%)
--Chinese/E 1 1 (100) Mild thal/Hb E disease
--Lepore/E 1 1 (100) Hb Lepore 12.5%
--Lepore/E 2 2 (100) Mild 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 and thalassaemia-haemoglobinopathies
- Conventional causes
- Unconventional causes/novel mechanism/trans acting mutations
• Roles of genetic modifiers in NTDT
Upstream deletional 0-thalassaemia
Fucharoen S & Viprakasit V, Hematology (ASH) 2009
Viprakasit V. et al, British Journal of Haematology, 2003 120(5): 867-75
Unusual deletions causing 0 thalassaemia and Clinical Hb H disease
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
Gibbons RJ, Picketts DJ, Villard L, Higgs DR. Mutations in a putative global transcriptional
regulator cause X-linked mental retardation with -thalassemia (ATR-X syndrome).
Cell 80: 837-845.
X-linked thalassemia/mental retardation (ATR-X) syndrome
ATRX-gene or XNP2 or XH2
Other syndromes with ATRX mutations:Carpenter syndrome, Juberg-Marsidi syndrome, Smith-Fineman-Myers syndrome, X-linked mental retardation with spastic paraplegia
Gibbons RJ, Higgs DR., Am J Med Genetics, 2000
0 thal /0 thal /0 thal /+ thal /+ thal /+ thal /
Interaction of thal alleles with extra copies of globin genes
• 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/L, MCV 62 fL, MCH 24 pg MCHC 33 pg/dL, RDW 27 % Retic. 7%
• Hemoglobin typing: A, A2 (7%), F (10%)
• DNA analysis: / and /41/42
Patient APatient A• 42 yrs-male with Hb 7-9 g/dL• Clinical diagnosis with thal. Intermedia• S/P splenectomy• Hb typing: A 81% A2 4.8% F 14.2%• /+ globin synthesis: 1.79
Patient BPatient B• 23 yrs-female with Hb 7-9 g/dL• Clinical diagnosis with thal. Intermedia• Marked splenomegaly (10 cm BLCM)• Hb typing: A 87% A2 4.3% F 8.6%• /+ globin synthesis: 1.65
39CT/39CT39CT/---
Baden C et al, Lancet 2002: 359; 131-2, Galanello R et al. BJH 2004: 127: 604-6
A594P
XPD mutations in Patients with Trichothiodystrophy Causes thalassaemia phenotype
Hb 11.5 g/dL Hct 34%MCV 65 fL MCH 24 pgHb A A2 (A2 4.5%)
Haematological phenotype mimic that of mild 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
Viprakasit, et al. Human Molecular Genetics 2001:2797-802 (2001)
Null and missense XP specific mutationsNull and missense TTD specific mutations
I Ia II III IV V VI
Fs 730
R722W
A594P
R487G
R112H
R683W
del 488-93 Intron 7 splice donor
R616P del 716/L461V
Fs 609
Further Example of Trans Acting Mutations Causing Thalassaemia
Nichols KE et al. Nature Genetics 24, 266 - 270 (2000)
Patients Age Hb (g/dl)
Hct (%) RBC x106/l
MCV (fl) MCH (pg)
Retic (%)
RDW (%)
Plt x103/l
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 : chain synthesis ratios were 0.55 to 0.67 in affected individuals
Further Example of Trans Acting Mutations Causing Thalassaemia
Familial dyserythropoietic anaemia and thrombocytopenia due to an inherited mutation in in GATA1GATA1
Nichols KE et al. Nature Genetics 24, 266 - 270 (2000)
Thalassaemia in the South Pacific
thalassaemia is common in the South PacificSome Individuals with thalassaemia have an apparently normal genotype ()
We identified three Families one from PNG and two from Vanuatu with HbH disease
HbH Disease In a Family From Papua New Guinea
Is this really HbH disease?
Haematological Phenotypes
/ Globin mRNA Ratio ~0.5 based on qPCR
/ Globin Synthesis Ratio 0.39-0.52 NR 1.06 ± 0.11
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
17 Differences in the region underlying the new peak of expression 7 After Removal of known SNPs
Identifying a Candidate SNP
50 100 150 200 250 300
D
141 T/T 31 T/C 5 C/C
1 In linkage with the disease
Overview:
• Globin gene regulation and associated disorders
• Molecular basis of NTDT in and thalassaemia-haemoglobinopathies
- Conventional causes
- Unconventional causes
• Roles of genetic modifiers in NTDT
Thein SL, Brit J Haematol 2004, p 264-274)
Roles of genetic modifiers on -thal intermedia
genes genes
Excess globin chainsHb Bart’s
Thein SL, Brit J Haematol 2004, p 264-274)
Roles of genetic modifiers on -thal syndromes
Summary:Summary:
• Molecular basis of NTDT either and 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
AcknowledgementsAcknowledgements
Nipon Chalaow Suchada Ruengleung Nuntawut Chat-Utai Rapeepun Shompoopuong Punyanuch Pornpanich Kalaya Tachavanich Warapron Glomglao Supachai Ekwattanakit
Faculty of Medicine, Siriraj HospitalMahidol University, THAILAND
Chompunut KanjanakornDr. Chanin Limwongse
Prof. Suthat FucharoenProf. Voravarn S. Tanphaichitr
Thailand Research FundBIOTEC, Thailand
Vip’s LaboratoryVip’s Laboratory
Disorders of Globin Chain Production
1. Quantitative defects: Thalassaemia syndromecausing imbalance of globin chain production
2. Qualitative defects: Haemoglobinopathiesresult from substitution, deletion or insertion of one or more amino acid
3. Hereditary of Persistence of Fetal hemoglobin
50 100 150 200 250 300
Excluded deletions and rearrangementsNo point mutationsNo other abnormalities
Genetic Analysis of the Globin Locus
D
De Gobbi M* & Viprakasit V* et al. Science 2006; 1215-7