thalassemia: from dna to clinical practice¸ªาม hb 12.9 g/dl, mcv 60 fl, of test positive, hb...

Thalassemia: From DNA to Clinical Practice

Noppadol Siritanaratkul, MD

Division of HematologyDepartment of MedicineFaculty of Medicine Siriraj Hospital

2 October 2009

Hematology-Siriraj HospitalThalassemia: From DNA to Clinical Practice

Check up at the Private hospital: claimed to be the most expensive hospital in Thailand

Hb 11.3 g/dL Hct 35%

Hb analysis: Hb A = 97%Hb A2 = 2.2 %Hb F 0.1%

Married and pregnant ~ 7 months


โรงพยาบาลเอกชนแห่งหนึง

เรียนอาจารย์ทีเคารพ

หนูขอเรียนปรึกษาเรืองผลเลือดของคู่สามีภรรยาดังนี

สามี Hb 12.9 g/dL, MCV 60 fL, OF test POSITIVE, Hb typing ปกติ

PCR for αthal (SEA) negative

ได้กิน FBC และ Folic acid มาตลอด 1 ปี

ภรรยา Hb 13.2 g/dL, MCV 92 fL, DCIP negative, Hb typing A2A, A2 = 2.3%

อาจารย์กรุณาช่วยให้คําแนะนําและ/หรือการตรวจเพิมเติมแก่คู่สมรสนีด้วยนะคะ

ขอแสดงความนับถือ

พญ. Anonymous Doctor


β globin locus: chromosome 11


IVS1 IVS2

1

A schematic representation of and globin genes

141

146

AAA

1

Exon1

Exon2 Exon3


Thalassemia (ธาลัสซีเมีย)

Inherited disorders of globin synthesis

in which the production of globin

chains is partially or completely

suppressed.


Excess chains

Heme

AHSP

precipitationα-Inclusion

bodies

Hb A

Chromosome 16

α-Gene cluster

Chromosome 11

-Gene cluster


Molecular mechanisms of -thalassemia

Point mutations:– ATA Box: -28 AG– CACCC box: -86 CG– Cap site: +1 AC– Initiation codon: ATGAGG– Splice site: IVS1-1 GT– Cleavage and Polyadenylation: AATAAA AATAGA– Consensus sequence: IVS1-5 GC

Mutations creating alternative splice sites– In introns: IVS2-654 CT– In exons: Codon19 AG Hb Malay

Codon26 GA Hb E


Molecular mechanisms of -thalassemia

Mutations producing nonfunctional mRNA

– Nonsense mutation: Codon 17 ATCodon 26 GT

– Frameshift muations: Codon 41/42 (-TTCT)Codon 71/72 +A

Deletions: 0.6 kb deletions, 27 kb deletion


Parenchyma Reticuloendothelial macrophages

Gut

Transferrin

NTBI

TransfusionsErythron

Iron distribution and turnover with transfusion therapy

Hershko C et al. Ann NY Acad Sci 1998;850:191–201, permission pending

NTBI=non-transferrin bound iron


Andrews NC. N Engl J Med 1999;341:1986–1995

Body iron dynamics during iron overload

NTBI is produced when serum transferrin is saturated

– LPI is a component of NTBI that is redox active

LPI is toxic:

– Haber Weiss reaction

O2. - + H2O2 O2 + OH- + HO

– Catalyzed by iron in two steps (Fenton reaction)

Fe3+ + O2·- Fe2+ + O2

Fe2+ + H2O2 Fe3+ + OH- + HO·

NTBI=non-transferrin bound iron; LPI=labile plasma iron


Consequences of Iron-Mediated ToxicityIncreased free iron

Hydroxyl radical generation

Lipid peroxidation

TGF-b1

Collagensynthesis

Fibrosis

Organelle damage

Lysosomal fragilityEnzyme leakage

Cell death

Porter J. Hematol/Oncol Clinics. 2005;19:S7. Cohen AR. In: Disorders of Hemoglobin: Genetics, Pathophysiology & Clinical Management. Cambridge University Press, 2001:979-1027.


Complications of iron overload

Liver cirrhosis/ fibrosis/cancer

Non-transferrin-bound iron circulates in the plasma

Excess iron promotes the generation of free

hydroxyl radicals, propagators of oxygen-related tissue damage

Insoluble iron complexes are deposited in body tissues and end-organ

toxicity occurs

Diabetes mellitus

Growth failure

Capacity of serum transferrin to bind iron is exceeded

Iron overload

Cardiac failure

Infertility


Iron overload oxidative damage

Cardinal features of thalassemia

Accumulation of unpaired globin chains Rbc membrane injury

Overall accelerated production of ROS deplete cellular antioxidants

Unpaired globin chains

DegradationDenaturation

Methemoglobin

Hemichromes

Inclusion body

Band 3clustering

IgG , C binding

Immune removal

Fe

Heme

Phosphatidyl-serine exposure

Activation of prothrombinase

complex

Hemin

Sp. band3crosslinking

Globin chains

Abnormal spectrin

association

Fragmentationdeformability

Mechanical removal

Lipid peroxidation

H2O2 O2- OH˙

High oxygenaffinity of RBCs

Selective survival ofHb F-containing cells

Fetal hemoglobin

(tissue hypoxia)(tissue hypoxia) Transfusion

DeathDeathCardiac failureCardiac failureCirrhosisCirrhosisEndocrine deficienciesEndocrine deficienciesNEJ Med 1999;341(2):99


Diabetes1Pancreas

Gonads

Cirrhosis, carcinoma1LiverCardiomyopathy1HeartHypoparathyoidism1ParathyroidHypothyroidism1Thyroid

Hypogonadotrophic hypogonadism1Pituitary

ConsequencesOrgan

Joints Arthropathy2

Skin Pigmentation2

Hypogonadotrophic hypogonadism1

1. Taher A, et al. Semin Hematol. 2007;44:S2.2. Brittenham G. In Hoffman R, et al, ed. Hematology: Basic Principles and Practice, 4th ed.

Philadelphia, PA: Churchill Livingstone, 2004.

Organ Systems Affected by Iron Overload


Extramedullary hematopoiesis


Hemoglobinopathies (ฮีโมโกลบินผิดปรกต)ิ

Hb Q – Mahidol (Thailand) 1: GAC CAC (- 4.2)

Hb Constant Spring 2 : CD142 TAA CAA

Hb Pakse 2 : CD142 TAA TAT

Hb Suan-Dok 2 : CD109 CTG CGG


Hemoglobinopathies (ฮีโมโกลบินผิดปรกต)ิ

Hb E :CD26 GAG AAG

Hb C :CD 6 TAA CAA

Hb Malay :CD19 AAC AAG

Hb J Bangkok (Korat) :CD56 GGC GAA

Hb Tak :CD147 +AC


Hemoglobin E

Mutation CD26 GAG AAG (GluLys)

Activate alternate splice site atCD25moderate reduction of normal spiced mRNA

Hb Malay CD19 AAC AGC (AsnSer) also activates cryptic splice site at CD17


Prevalence of Thalassemia & Hemoglobinopathies in Thailand

thal 1 Bangkok 3.5%

Chiangmai 14.7%

thal 2 Bangkok 16%

Chiangmai 19%

thal Bangkok 3%

Chiangmai 9%


Prevalence of Thalassemia & Hemoglobinopathies in Thailand

Hemoglobin E Bangkok 13%

North eastern 50%

Hemoglobin CS 4%


33

อุบัติการของโรคธาลัสซีเมียทีพบบ่อยในประเทศไทย

โรค ทารกทีคลอด(ต่อปี)จํานวนผู้ป่วยทีมีชีวิต

อยู่ทั งหมด

Hb Bart’sอาการรุนแรงทีสุด

1,250 0

ธาลัสซีเมียเบต้าอาการรุนแรงปานกลาง

3,875 103,750

ธาลัสซีเมียชนิดHอาการไม่รุนแรง

7,000 420,000


Thalassemia syndromes

thalassemia syndromes

thalassemia syndromes


Deletions in the -globin gene cluster


Prevalence of (- - SEA) -thal

Regions heterozygote(%)

Northern Thailand 14.0

Central Thailand 3.7

Hong Kong 4.5

Northern Taiwan 3.5

Southern China 5.0-8.8


Deletions cause – thal2

121

3.7

4.2


Clinical types of thalassemia

Asymptomatic : heterozygote

Symptomatic

mild - Hb H disease

- Homozygous CS

severe - Hb Bart’s hydrops fetalis


12

12

12

12

1CS

thal 1 trait

thal 2 trait

Hb H disease

Hb Bart’s hydrops fetalis

Normal

thal 2 homozygote


Hb Bart’s Hydrops Fetalis


Absence of all -globin genes

Decreased Hb synthesis Hb Bart’s 80-90% Hb Portland 10-20%

Anemia Tissue Hypoxia

Enlarged Placenta Heart Failure ExtramedullaryHematopoiesis

Maternal complications

Congenital anomalies

Hepatosplenomegaly


Maternal Complications

Mean gestation at delivery 31 wk

Malpresentation of fetus 37%

Cesarean section 14 – 17%

Postpartum complications:

life-threatening hypertension

retained placenta, hemorrhage


Hemoglobin H disease

Compound heterozygote

- thal1/ thal2 (- - - thal1/ CS (- -CS

mild anemia Hb 8 - 10 g/dL

hepatosplenomegaly

newborn : Hb Bart’s 20 - 40%


Hemoglobin H disease

Acute hemolysis following infections

Numerous Inclusion bodies ( 4) Hb analysis : Hb H, Hb Bart’s


Clinical types of thalassemia

Asymptomatic : thalassemia trait

Thalassemia intermedia

- HbE / thal

- + thal/ + thal

Thalassemia major

- 0 / 0 thalassemia


Type Position mutation

- thalassemia Codon 17 AT

IVSI-1 GT

Codon 35 CA

Codon 41 – C

Codon 41/42 – TCTT

Codon 71/72 + A

3.4 kb deletion

+- thalassemia – 87 CG

– 28 AG

– 29 AG

IVSI-5 GC

IVSII-654 CT

Codon 126 TG

thalassemia: from dna to clinical practice¸ªาม hb 12.9 g/dl, mcv 60 fl, of test positive, hb...

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