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