Thalassemia as one of human genetic disorders

Genes and Human Disease

CF PKD HC
High penetrance, Single gene

Polygenic, Reduced penetrance

Snakebite Infectious disease

T1DM IBD Asthma

T2DM Pure environment HT Osteoporosis Schizophrenia

Genetic disorders

Purely acquired (environmental)

Multifactorial (environmental and genetic)

Purely genetic

Chromosomal
Infections Nutrition deficiencies Congenital abnormalities

Single-gene disorders

Common chronic disorders of adult life

Single gene disorders

Autosomal Sex linked

Recessive Sickle cell anemia (rare 1/400) -Thalassemia (rare 1/3) -Thalassemia (rare 1/4) 1 Antitrypsin deficiency Adenosine deaminase deficiency Cystic fibrosis (1/2000 Caucasians) Phenylketonuria (1/5000 1/200.000)

Dominant
Familial hypercholesterolemia

(1/500 heterozygous) Huntington disease (4-8/100.000) Myotonic dystrophy (1/1000 10.000) Neurofibromatosis (1/3000 1/5000) Osteogenesis imperfecta (1/15.000)

Single gene disorders

Autosomal Sex linked

Recessive
G-6PD deficiency

Dominant
Vitamin D resistant rickets

(rare -1/20 males) Haemophilia A (1/10.000 males) Fragile X syndrome (1/1500 males) (1/2000 1/3000 females) Duchene muscular dystrophy (1/3000 males)

Thalassemia
a group of inherited disorders of hemoglobin synthesis affecting the globin chain that are characterized usually by mild to severe hemolytic anemia
(http://www.merriam-webster.com/dictionary/thalassemia).

a group of inherited disorders characterized by reduced or absent amounts of hemoglobin, the oxygencarrying protein inside the red blood cells
(http://medical-dictionary.thefreedictionary.com/thalassemia).

Hemoglobinopathies (Hemoglobin Disorders)

Structural defects in the hemoglobin molecule. Alterations in the gene for one of the two hemoglobin subunit chains, alpha () or beta (), are called mutations.

Hb S (sickle cell hemoglobin) Hb E Hb D Hb O (Hb O Arab, Hb O Indonesia)

Diminished production of one of the two subunits of the hemoglobin molecule. Mutations that produce this condition are termed thalassemias.

-thal -thal ,-thal

Hemoglobin structure

Hemoglobin structure

Functional Hemoglobin
Heme & Globin Heme & Globin

Heme & Globin

Heme & Globin

Heme & Globin Heme & Globin

Heme & Globin

Heme & Globin

Heme & Globin Heme & Globin

Heme & Globin Heme & Globin

Dalam tiap lingkaran merah atau hijau (tiap monomer Hb) terdapat molekul heme & globin

Hemoglobin synthesis

 The Molecular structure is similar to

Myoglobin :
MW 17,000 ; a monomer of protein with 153

AA residues stores oxygen in red muscle tissue will be released under condition of oxygen deprivation (e.g. Severe exercise) and used by muscle mitochondria for ATP synthesis

 75% of the AA residues are present in 8

-helix (helix A to H) Histidin F8 and E7 perform unique roles in oxygen binding Oxygen-binding curve for myoglobin is hyperbolic

 Hemoglobin:

Transports oxygen, CO2 & protons Its allosteric properties results from its

quaternary structures A tetramer composed of pairs of different polypeptides/subunits (, , , etc. globin chains) a pair of globin chain product of gene cluster in chromosome 11 & a pair of globin chain product of gene cluster in chromosome 16

Hb binds 2 protons for every 4 oxygen molecules released & thus contributes significantly to buffering capacity of blood increase in proton concentration promotes oxygen release, while increase in PO2 promotes proton release. At the lungs, oxygenation of Hb is accompanied by expulsion and subsequent expiration of CO2 Bohrs effect (a reversible phenomenon with that in the peripheral tissues)

2,3-Bisphosphoglycerate (BPG) in Hb Formed from glycolytic intermediate 1,3-

bisphosphoglycerate One molecule of BPG is bound per Hb tetramer in the central cavity the space is wide enough when Hb is in the T form (deoxygenated)

 Binds more weakly to fetal Hb than to

adult Hb Increase concentration of BPG lowers the affinity of Hb for oxygen (decreases P50) increasing the ability of Hb to release oxygen at the tissues

As CO2 is absorbed in the blood, the carbonic anhydrase (CA) in erythrocyte catalyzes the formation of carbonic acid, which in turn rapidly dissociate into bicarbonate and a proton. To avoid increasing the acidity of blood, a buffering system must absorb these excess protons this is carried out by Hb

CO2 + H2O H2CO3 HCO3- + H+

CA

spontaneous

ALA PBG Protoporphyrin IX

Fe

COO CH2 CH2 C CH2 NH3+ O +

COO CH2 CH2 C CH2 NH3+ O

PBG Synthase
COO

COO CH2 CH2 C CH N H

2 H 2O

CH2 C

H2C

NH3+

HEME

2 -aminolevulinate (ALA)

porphobilinogen (PBG)

Chromosome #16
5' Globin Genes : Chains Synthesized Hb types : 2 2 (Gower-I) 2 2 (Portland) Embryo 2 2 (Gower-II) 2 1 2 1 1 3'

Chromosome #11
5' Globin Genes : Chains Synthesized Hb types : G G A 2 2 2 2 (Hb-F) Fetus A 2 2 2 2 (Hb-A2) (Hb-A) Adult G A 3'

% of total globin 50 synthesis

30

10 6 18 30
birth

18

30

42

prenatal age (wks)

postnatal age (wks)

Types of human hemoglobin from embryonal to adult life

Hb Gower 1 Hb Portland Hb Gower 2 Hb Fetal (HbF) Hb Adult (HbA) Hb Adult minor (HbA2)

= 2 2 = 2 2 = 22 = 22 = 22 = 22

Mutant human Hb
Causes hemoglobinopathy (when biologic

function is altered) Due to mutations in the gene that code for globin chains: Structurally abnormal Hb (HbM, HbS, HbE, HbC etc) Reduced synthesis of Hb (thalassemias) Diagnosed by special method (e.g. molecular diagnosis)

Thalassemia in Indonesia

Tabel 5.1. Urutan basa primer ARMS dan kontrol tujuh mutan umum di Asia-Pasifik menurut Old et al. (1990)

Primer**
Kontrol gena globin- Control A Control B Common C Common D Common E (R) (F) (F) (R) (R)

Urutan Basa Primer * ARMS ( 5- 3)

Pasangan primer ARMS

Panjang Produk PCR (bp)

CAATGTATCATGCCTCCTTTGCACC GAGTCAAGGCTGAGAGATGCAGGA ACCTCACCCTGTGGAGCCAC CCCCTTCCTATGACATGAACTTA CTTGTGGGCCAGGGCATTAGCC

Control B Control A Common D Common C

861 861 676 676

Mutasi gena globin- Cd 15 normal ( F) Cd 15 mutant (GA) ( F) Cd 8/9 normal ( R) Cd 8/9 mutant (+G) ( R) Cd 26 normal ( R) Cd 26 mutant (GA) ( R) Cd 41/42 normal ( R) Cd 41/42 mutant (-CTTT) ( R) IVS I-1 normal ( F) IVS I-1 mutant (GT) ( R) IVS I-5 normal ( R) IVS I-5 mutant (GC) ( R) IVS II-654 normal ( F) IVS II-654 mutant (CT) ( F) TGAGGAGAAGTCTGCCGTTACTGGCCAGTG TGAGGAGAAGTCTGCCGTTACTGGCCAGTA CTTTGCCCCACAGGGCAGTAACGGCACACT CTTTGCCCCACAGGGCAGTAACGGCACACC TAACCTTGATACCAACCTGCCCAGGGCGTC TAACCTTGATACCAACCTGCCCAGGGCGTT GAGTGGACAGATCCCCAAAGGACTCAAAGA GAGTGGACAGATCCCCAAAGGACTC AACCT GATGAAGTTGGTGGTGAGGCCCTGGGTAGG TTAAACCTGTCTTGTAACCTTGATACGAAA CTCCTTAAACCTGTCTTGTAACCTTGTTAC CTCCTTAAACCTGTCTTGTAACCTTGTTAG GAATAACAGTGATAATTTCTGGGTTAACGC GAATAACAGTGATAATTTCTGGGTTAACGT Common D Common D Common C Common C Common C Common C Common C Common C Common D Common C Common C Common C Common E Common E 500 500 214 214 267 267 443 439 454 281 285 285 346 346

* huruf berwarna biru menunjukkan basa mutan , huruf merah menunjukkan basa mismatch dan ** F = primer forward, R = primer reverse.

adalah delesi.

Cm C 62028 Cm C 676 bp 62470

62703 Cm D

62028
Cm C

442 bp Cd 41-42 N 62466

62028
Cm C 62028

439 bp Cd 41-42 M 62294 267 bp Cd 26 M/N IVSII-564 M/N 63601 Cm E 63610 74086 861 bp Cont A

Cm C 214 bp 62241

62028 Cd 8-9 M/N

Gena globin-
IVS-I
62408

63224 346 bp

EXON-1
62187 Cm C 62028 285 bp 62278 62312

EXON-2
62631

IVS-II
Cont B 63224

EXON-3
63482

IVSI-5 M/N IVSI-1N 62703 62250 454 bp

Cm C 62308 62028 281 bp IVSI-1M Cd15 M/N

Cm D

62703

62204

500 bp

Cm D

Gambar . Skema posisi primers ARMS untuk mutasi umum Asia Pasifik pada gena globin-. Angka merah menunjukkan nomor urutan basa pada gene bank, huruf di atas tanda panah menunjukkan nama primer, bulatan merah pada primer menunjukkan posisi mutasi dan angka biru menunjukkan panjang fragmen DNA yang teramplifikasi.

Distribution of -thalassemia, Hb-E and Hb-OIna carrier and HbE homozygote in some Indonesian populations
Number of subject tested 135 115 134 108 111 125 104 130 113 116 162 127 82 138 118 120 121 2 059 Abnormal Hemoglobin (%) * --------------------------------------------thal : Hb-E : HbE Homo : Hb-OIna 2 (1,5) 6 (5,2) 5 (3,7) 10 (9,3) 6 (5,4) 4 (3,2) 0 4 (3,1) 0 0 2 (1,2) 0 1 (1,2) 4 (2,9) 6 (5,1) 3 (2,5) 0 53 0 5 (4,3) 4 (3,0) 7 (6,5) 5 (4,5) 6 (4,8) 11(10,6) 2 (1,5) 0 2 (1,7) 6 (3,7) 0 5 (6,1) 6 (4,3) 8 (6,8) 36(30,0) 0 103 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 (3,3) 0 4 0 0 0 0 0 0 0 0 0 0 0 5 (4,0) 0 0 0 0 0 5

Population 1. Batak 2. Melayu 3. Minangkabau 4. Palembang 5. Bangka 6. Jawa 7. Tengger 8. Dayak 9. Banjar 10. Minahasa 11. Palu-Kaili 12. Toraja 13. Bali 14. Sasak 15. Sumbawa 16. Sumba 17. Alor Total

* Figures in brackets are in %.

Batak
1,5 0

Minahasa Melayu
5,2 4,3

Dayak Bangka
5,4 4,5
3,1 1,5

0 1,7

Minang
3,7 2,9

Palu
1,2 3,7

Banjar
0 0

Toraja
0 4*

Palembang
9,2 6,5

Tengger 0 10,6 Bali Jawa

3,2 4,8 2,9

Sumbawa
5,1 6,8

Alor
0 0

1,2 6,1

= trait thalassemia- = trait hemoglobin-E

Sasak 4,3 Sumba

2,5

36,6

Gambar . Pola distribusi dan prevalensi trait thalassemia- dan hemoglobin-E pada berbagai populasi di Indonesia. * adalah hemoglobin OIna.

Dayak Batak
IVS I-5

Melayu
IVS I-5 IVS I-1 Cd 26

Bangka
IVSI-5 IVS I-1 Cd 8/9 Cd 41/42 Cd-26

IVSI-5 IVS I-1 Cd 41/42 Cd-26

Minahasa
Cd-26

Palu

Minang
IVS I-5 IVS I-1 Cd 8/9 Cd-26

Banjar
NA

IVS I-5

Toraja
NA

Palembang
IVS I-5 IVS I-1 Cd 41/42 IVS II-654 Cd-26

Tengger
Cd- 26

Sumbawa
IVS I-5 IVS I-1 Cd-26

Alor
NA

Jawa
IVS I-5 IVS I-1 IVS II-654 Cd-26

Bali
Cd 8/9 Cd-26

Sasak
IVS I-5 IVS I-1 Cd 8/9 Cd-26

Sumba
IVS I-5 Cd-26

Fig . Distribution of common mutants of -thalassemia and hemoglobin-E in some Indonesian populations.

Thalassemia, particularly -thalassemia are common in Indonesia It is found in almost all populations studied Mutants underlying the disorders are quite varied, but mostly similar to those found in Asian populations

Catabolism of Heme

Heme breakdown
During its 120 day life span the erythrocyte has traveled 200-300 miles. The process of aging is called senescence. Enzyme activity decreases (esp. glycolytic enzyme which helps break down glucose, the source of erythrocyte energy), and the cell looses its deformability.

MCHC (mean corpuscular hemoglobin concentration) increases, the cell becomes rounder, and the MCV mean corpuscular volume) decreases. 90% of destruction of senescent Erythrocytes occurs by extravascular hemolysis. Macrophages of the mononuclear phagocyte system remove them from circulation.

Macrophages of the spleen are especially active in removing aging, dead and abnormal erythrocytes (e.g. cells containing Heinz bodies or HowellJolly bodies, siderocytes, target cells, schistocytes, tear drop cells and antibody-coated erythrocytes).

Normally, senescent red blood cells and heme from other sources are engulfed by cells of the reticuloendothelial system. The globin is recycled or converted into amino acids, which in turn are recycled or catabolized as required.

Heme is oxidized, with the heme ring being opened by the endoplasmic reticulum enzyme, heme oxygenase. The oxidation step requires heme as a substrate, and any hemin (Fe3+) is reduced to heme (Fe2+) prior to oxidation by heme oxygenase

Pathway for the degradation of heme to bilirubin. Substituents: M = methyl, P = proprionic, V = vinyl

In individuals with abnormally high red cell lysis, or liver damage with obstruction of the bile duct, the bilirubin and its precursors accumulate in the circulation; the result is hyperbilirubinemia, the cause of the abnormal yellowish pigmentation of the eyes and tissues known as jaundice.

The protoporphyrin ring of heme is disassembled and from the body. Its alpha carbon is exhaled in the form of CO2. The opened tetrapyrrole, biliverdin, is converted to bilirubin which is then carried to the liver by the plasma protein, albumin.

In the liver bilirubin is conjugated to glucuronide to make it water soluble and excreted along with bile into the intestines. In the intestines it is converted by bacteria into stercobilinogen and excreted in the stool; some is eliminated as urobilinogen in the urine. Stercobilinogen and urobilinogen give feces and urine their color.

Unconjugated bilirubin (prehepatic) and conjugated bilirubin (posthepatic) are measured in serum as indirect (unconjugated) and direct (conjugated) bilirubin; used to monitor amount of hemolysis. Bilirubin and its catabolic products are collectively known as the bile pigments.

Intravascular hemolysis
About 10% of normal erythrocyte destruction occurs by intravascular hemolysis. In circulation the red cell is subjected to metabolic and mechanical stresses: turbulence, endothelial damage and fibrin deposition, incompatibility due to transfusion errors resulting in red cell fragmentation (schistocytes) and/or intravascular hemolysis.

When the erythrocyte ruptures, hemoglobin is released into the blood. The hemoglobin dissociates into alpha-beta dimers and is picked up haptoglobin, a protein carrier, to prevent renal excretion of hemoglobin. Haptoglobin carries the hemoglobin to the liver for further catabolism where the process proceeds as with extravascular hemolysis.

As haptoglobin is depleted, unbound hemoglobin dimers appear in the plasma (hemoglobinemia) and are reabsorbed by the kidney up to a certain level and converted to hemosiderin; beyond this level hemoglobin shows up in the urine (hemoglobinuria) Intravascular hemolysis results in pink, red or brown plasma (hemoglobinemia). Urine may also show red color (hemoglobinuria).

http://diaglab.vet.cornell.edu/clinpath/modules/chem/images/bilirubin%20metabolism.jpg

Clinical Aspect of Heme Metabolism

Clinical problems associated with heme metabolism are of two types.

Disorders that arise from defects in the

enzymes of heme biosynthesis are termed the porphyrias and cause elevations in the serum and urine content of intermediates in heme synthesis. Inherited disorders in bilirubin metabolism lead to hyperbilirubinemia

Porphyria

Enzyme Defect

Primary Symptom

Erythroid Class
X-linked sideroblastic anemia, XLSA Congenital erythropoietic porphyria, CEP -aminolevulinic acid synthase 2, ALAS2 progressive iron accumulation, fatal if not treated

uroporphyrinogen III cosynthase

photosensitivity

Erythropoietic protoporphyria, EPP

ferrochelatase

photosensitivity

Hepatic Class
ALA dehydratase deficient porphyria, ADP
Acute intermittent porphyria, AIP

ALA dehydratase: also called porphobilinogen synthase

PBG deaminase: also called hydroxymethylbilane synthase or rarely uroporphyrinogen I synthase coproporphyrinogen oxidase

neurovisceral

neurovisceral

Hereditary coproporphyria, HCP

neurovisceral, some photosensitivity

neurovisceral, some photosensitivity photosensitivity

Variegate porphyria, VP Porphyria cutanea tarda type I, PCT type I, also called the sporadic type PCT Porphyria cutanea tarda type II, PCT type II, also called the familial type PCT, may also be referred to as hepatoerythropoietic porphyria, HEP

protoporphyrinogen oxidase

hepatic uroporphyrinogen decarboxylase

uroporphyrinogen photosensitivity decarboxylase in non-hepatic , some tissues neurovisceral

Gene pool of the Indonesian populations

Ethnically very diverse and genetically can be grouped into two clusters
Mongoloid genes

Western cluster

Australomelanesian genes

Eastern cluster

Implication of research in thalassemia

Thalassemia research
For the sake of what ?
Science & technology? Scientist(s)? Government? Funding agency? Patients? Others?

Scale of the problem: Carrier estimation of haemoglobinopathies

Non-Java populations (approx. 90 million)

-thalassaemia HbE

: 4.3% : 2.4% : 3.0% : 3.0%

Java (approx. 110 million)

-thalassaemia HbE

Total Indonesia (approx. 200 million)

-thalassaemia HbE

: 3.55 ( 1.2%) : 2.75 ( 1.2%)

Estimation of cases

- Thalassaemia homozygotes:

1600 (range 750-2850) cases

- Thalassaemia /HbE compound heterozygotes:

2500 (range 1000-4750) cases Total > 4100 cases or approx. 1 per 1000 live birth (present birth rate in Indonesia is estimated 25.3 per 1000 or 5.06 million per annum)

Estimated number of cases requiring transfusion

- Thalassaemia homozygotes:
> 32,000 (1600 x 20; assuming they live to

age 20)

- Thalassaemia /HbE compound heterozygotes:

> 18,000 (2500 x 15; assuming they start

requiring blood transfusion at age 5 & live to age 20 and only half of them need transfusion)

Total: 50,000

Estimated unit of blood required for transfusion

- Thalassaemia homozygotes & Thalassaemia /HbE compound heterozygotes:

2-3 units per month (25 units annually)

Total annual requirement : 50,000 x 25 unit = 1.25 million units

Future Steps
Research

& application Gene therapy & alternative therapy Reduce blood transfusion interval Oxidant status and role of antioxidant in patients Reduce marriage among carriers Role of other globin genes with their product in substituting the defective globin gene

 Proposed

preventive measures with genetic counseling: Premarital carrier (trait) testing Prenatal diagnosis for carrier couples

Thank you