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![Page 1: Molecular Haematology I Globin Disorders Dr Edmond S K Ma Division of Haematology Department of Pathology The University of Hong Kong.](https://reader035.fdocuments.net/reader035/viewer/2022062421/56649ddd5503460f94ad57a2/html5/thumbnails/1.jpg)
Molecular Haematology IGlobin Disorders
Dr Edmond S K MaDivision of HaematologyDepartment of Pathology
The University of Hong Kong
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Thalassaemia
• First described by Thomas B. Cooley in 1925
• The term thalassaemia was first coined in 1932 based on the Greek word (thalassa) meaning the sea
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Prevalence of thalassaemia in Hong Kong Chinese
-thalassaemia 5%
-thalassaemia 3.1%
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Prevalence of thalassaemia in Hong Kong Chinese
-thalassaemia
(--SEA) -thalassaemia deletion 90%
-thalassaemia
codons 41-42 (-CTTT) 0 45%
IVSII-654 (CT) 0 20%
nt-28 (AG) + 16%
codon 17 (AT) 0 8%
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Carrier detection
• Antenatal screening– Obstetrical Units of the Hospital Authority– Maternal and Child Health Centres– Private sector
• Pre-marital and pre-pregnancy testing– Family Planning Association
• Community based thalassaemia screening– Children’s Thalassaemia Foundation
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Detection of thalassaemia
• Red cell indices (MCV, MCH)
• Determine iron status
• HPLC analysis
• Hb and globin chain electrophoresis
• Detection of HbH inclusion bodies
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Laboratory diagnosis of thalassaemia by HPLC
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Haemoglobin electrophoresis
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Detection of HbH inclusion bodies
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New approaches in diagnosis of SEA deletion:
gap-PCR for SEA deletion
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New approaches in diagnosis of SEA
deletion: detection of -globin chains in adults
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-globin gene mutations
• Deletional (common)
--SEA
-3.7
-4.2
• Non-deletional (rare)
Hb CS
Hb QS
codon 30 deletion
Hb Q-Thailand
Hb Westmead
2 codon 31
2 codon 59
Others
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Prevalence of thalassaemia in Hong Kong Chinese (MCV < 80 fL)
-thalassaemia(--SEA) -thalassaemia deletion 90%
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Single-globin gene deletion and triplicated -globin gene
• Prevalence– 6% for –3.7 and –4.2
• Hb 13.6 ± 0.12 g/dL (11.8 – 15.6)
• MCV 83.0 ± 0.33 fL (77.9 – 88.1)
• MCH 27.2 ± 0.16 pg (24.1 – 29.7)
– 1.5% for anti-3.7 and anti-4.2
• Hb 13.5 g/dL, MCV 85.5 fL, MCH 28.7 pg
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Single -globin gene deletion (-) and triplicated -globin gene () configuration
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Molecular diagnosis of -thalassaemiaClark & Thein, Clin Lab Haematol 26: 159-76; 2004
• Deletions– Gap PCR
– Southern blotting
• Non-deletional mutation: on specifically amplified 2 or 1 genes– Restriction digest
– ARMS-PCR
– ASO
– Direct sequence analysis
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Multiplex PCR for 3 commonest -thalassaemia deletion
LIS1 control -2 gene
SEA deletion
3.7 kb deletion4.2 kb deletion
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LIS internal control (2350 bp)-α3.7 (2022/2029 bp)
α2 (1800 bp)
-α4.2 (1628 bp)
--SEA (1349 bp)
water αα/--SEA ladder -α3.7/--SEA -α4.2/--SEA αα/αα blank
Multiplex PCR for 3 commonest -thalassaemia deletion
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Restriction fragment length polymorphism (RFLP)
The principle of RFLP as shown is used to diagnose the different types of -globin genotypes relevant to -thalassaemia.
GelSmallerfragment
Largerfragment
Key
restriction enzyme sites
probe region
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A typical RFLP result of different -thal genotypes:
Genotypes
/
/- 3.7 - 3.7/-3.7 / - 4.2 - 4.2/ - 4.2
Bam HI probes with -globin
14.5 kb 14.5 kb; 10.5 kb
10.5 kb 14.5 kb; 10.5
10.5
Bgl II probed with -globin
12.6 kb; 7.0
16.0 kb 12.6; 7.0
16.0 12.6; 7.0
7.0
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16kb
10.5 kb
14.5kb
12.6 kb
7.0 kb
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Multiplex ARMS for the 3 commonest non-deletional 2-globin gene mutations
Internal control (930 bp)
cd30(ΔGAG) (772 bp)
HbQS (234 bp)
HbCS (184 bp)
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Reverse dot blot
Chan V et al, BJH 104: 513-5, 1999
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Multiplex mini-sequencing screen
Wang W et al, Clin Chem 49: 800 – 803, 2003
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Molecular screening of non-deletional -globin gene mutations by denaturing HPLC
Guida V et al, Clin Chem 50: 1242 – 1245, 2004
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Thalassaemia array
Chan K et al, BJH 124: 232 – 239, 2004
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Thalassaemia array
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-thalassaemia phenotypes-thalassaemia trait• Aymptomatic• Hypochromic
microcytic red cells
• High HbA2
• Variable HbF• Genotype: simple
heterozygotes for -thalassaemia alleles
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-thalassaemia phenotypes-thalassaemia major• Onset < 1 year
• Transfusion dependent
• Many complications
• Markedly HcMc RBC
• Nucleated reds
• Majority HbF
• Genotypes: homozygous or compound heterozygous for -thalassaemia alleles
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-thalassaemia syndromes
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Defining disease severity
• Age at diagnosis
• Steady state or lowest haemoglobin level
• Age at first transfusion
• Frequency of transfusion
• Splenomegaly or age at splenectomy
• Height and weight in percentile
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Why study genotype phenotype relationship?
• Genetic counselling
• Management decisions
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Genetic factors affecting disease severity
• Nature and severity of -globin mutation
• Co-inheritance of -thalassaemia or triplicated -globin genes
• Genetic determinant(s) for enhanced -globin chain production
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Mutation detection by dot blot hybridization
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Detection of five -thalassaemia mutations by ARMS
1 2 3 4 5 6 7 8
Panel 1: 1-6
1: -28 Heterozygote2: -28/71-72 Compound Heterozygote3: Codon 17 Heterozygote4: Codon 43 Heterozygote5: 100 bp DNA Ladder6: Reagent Blank Control
Panel 2: 7-8
7: IVS 2-654 Heterozygote
8: Reagent Blank Control
Internalcontrol
-28
17
43
71-72
654
Internal control
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Southern blot hybridization with -probe
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PCR-based mutation detection
-multiplex PCR
-thalassaemia PCR
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The spectrum of -thalassaemia alleles in Chinese
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Genotype phenotype correlation in 0/0 thalassaemia
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Genotype phenotype correlation in 0/+ thalassaemia
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Homozygous 0/0 and compound heterozygous 0/+ thalassaemia
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Clinical phenotype of +/+ thalassaemia
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Clinical phenotype of HbE / -thalassemia
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Molecular pathology of -thalassaemia
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Thalassaemia intermedia: family study 1
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Thalassaemia intermedia: family study 2
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Thalassaemia screening using MCV and MCH cutoff
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Co-inheritance of -thalassaemia determinants significantly ameliorates the
phenotype of severe -thalassaemia
Yes0/0 homozygotes + two -globin gene deletion or
non-deletional -globin gene mutation
+-thalassaemia homozygotes or compound
heterozygotes single -globin gene deletion
No 0/0 homozygotes + single -globin gene deletion
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Co-inheritance of -thalassaemia determinants significantly ameliorates the phenotype of
severe -thalassaemia Points to note:
• Molecular heterogeneity of -thalassaemia and -thalassaemia alleles results in wide range of clinical outcomes
• Small numbers of patients in each category
• Variations among different populations (e.g. in Thai patients -thalassaemia ameliorates severe -thalassaemia only in the presence of
at least one -thalassaemia allele)
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Co-inheritance of -thalassaemia in severe -thalassaemia
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Co-inheritance of -thalassaemia in severe -thalassaemia
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Co-inheritance of -thalassaemia in severe -thalassaemia
Conclusion
The co-inheritance of (--SEA) -thalassaemia (SEA)
deletion ameliorates the clinical phenotype of 0/+
but not necessarily 0/0-thalassaemia in Chinese
patients
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Co-inheritance of -thalassaemia in severe -thalassaemia
Implications
1. Detection of SEA deletion in couples at risk of offspring affected by 0/+-thalassaemia (~ 8 / year)
2. At prenatal diagnosis, a genotype of 0/+ -thalassaemia + SEA deletion is predictive of thalassaemia intermedia, but the same cannot be said for 0/+-thalassaemia alone or 0/0-thalassaemia + SEA deletion
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Triplicated -globin gene in -thalassaemia heterozygotes
• Observed in 15% of thalassaemia intermedia, not seen in thalassaemia major
• Presentation in adulthood
• May also be associated with a phenotype of thalassaemia trait
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Triplicated -globin gene in -thalassaemia heterozygotes
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Triplicated -globin gene in -thalassaemia heterozygotes
• Distinction from simple -thalassaemia heterozygotes– Presence of red cell abnormalities– Circulating normoblasts– More anaemic– Higher HbF levels
• Explain the inheritance of families in which only one parent is thalassaemic
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Triplicated -globin gene in -thalassaemia heterozygotes
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Genetic basis for phenotypic variation in the Chinese
• Severity of -thalassaemia mutation0/0 severe
0/+ 2/3 severe; 1/3 intermedia
0/+++ intermedia
/+ intermedia (mild)
• Concurrent -thalassaemiaSEA deletion ameliorate 0/+ only but not necessarily 0/0
• Triplicated -globin gene in -thalassaemia heterozygotesOften associated with thalassaemia intermedia phenotype
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Genetic basis for phenotypic variation in the Chinese
• Determinants of HbF production– XMnI G-promoter polymorphism: inconsistent
effect– Familial determinants of high HbF remains to be
defined
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Effect of XMnI G-promoter polymorphism
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Genotype phenotype correlation in 0/0 thalassaemia
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Genetic determinants of high HbF
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Genetic determinants of high HbF
A-HPFH: nt -196 C→T
Subject Sex/Age Hb
(g/dL)
MCV
(fL)
MCH
(pg)
HbA2
(%)
HbF
(%)
HbH
bodies
α-genotype β-genotype
Index F/42 8.2 61.3 21.8 4.5 34.9 Negative ζζζαα/ζζαα β41/42(-CTTT)/βA
Elder brother 1
M/52 11.8 59.9 20.3 5.8 0.8 Negative ζζζαα/ζζαα β41/42(-CTTT)/βA
Elder brother 2
M/46 11.4 58.3 19.2 4.8 45.3 Negative ζζζαα/ζζαα β41/42(-CTTT)/βA
Elder Sister
F/48 12.6 91.5 29.9 2.2 13.3 Negative ζζαα/ζζαα βA/ βA
Daugther F/13 10.5 60.2 19.6 5.7 0.8 Negative ζζαα/ζζαα β41/42(-CTTT)/βA
Son of elder brother 2
M/12 12.3 62.3 18.3 5.6 1.5 Negative ζζαα/ζζαα β41/42(-CTTT)/βA
Note: All subjects are negative for XmnI Gγ-polymorphism
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Genetic modifiers of single gene disorders
Primary modifiers
Secondary modifiers
Tertiary modifiers
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Hyperbilirubinaemia
Jaundice
Gall stones
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UGT1A1 mutations and hyperbilirubinaemia
• Uridine-diphosphoglucuronate glucuronosyltransferase– UGT1 gene : 12 isoforms with alternative first exons
– UGT1A1 contributes most significantly to bilirubin glucuronidation
– Mutations in coding region and promoter
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UGT1A1 alleles in Chinese
Hsieh S-Y et al, Am J Gastroenterol 96: 1188 - 1193, 2001
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Detection of UGT1A1 polymorphisms
• UGT1A1 promoter genotype– direct sequencing of PCR product
• Gly71Arg mutation at exon 1– PCR restriction analysis of MspI cleavage site
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143bp
119bp
24bp
M W h W W W W H h h W W H
Homozgyous (TA)6
Homozgyous (TA)7
Heterozgyous (TA)6/(TA)7
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Prevalence of UGT1A1 polymorphisms(TA)7 = 25 cases (19.6%); G71R = 34 cases (26.8%)
Major Intermedia
(TA)7 homozygous 0 2
(TA)7 heterozygous 14 (2) 9 (1)
G71R homozygous 4 2
G71R heterozygous 24 (2) 4 (1)
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Predictors of bilirubin level
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Predictors of gall stones
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Genetic haemochromatosis and iron overload in -thalassaemia
• Homozygosity for HFE alleles C282Y and H63D – predisposes to iron overload in -thalassaemia
• Prevalence in Chinese patient cohortAllele Frequency
C282Y 0%
H63D 1.3%
S65C 0%
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Transferrin receptor-2 (TFR2) mutations and iron overload
• Homologue of transferrin receptor with 48% identity and 66% similarity
• Common affinity for diferric transferrin• Lack of affinity for HFE protein
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Transferrin receptor-2 (TFR2) polymorphisms
• Allelic frequency
Polymorphism Patients Control p-value
exon 5 I238M 7.1% 4.7% 0.24
IVS16+251 -CA 24.5% 22.2% 0.54
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TFR2 polymorphism and iron overload in transfusion independent -thalassaemia intermedia
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Genetics of osteoporosis in thalassaemia
• Heterozygous (Ss) or homozygous (ss) polymorphism of COLIA1 gene: ↓ BMD– Perrotta et al, Br J Haematol 111: 461, 2000
• VDR BB genotype: ↓ spine BMD than bb genotype– Dresner Pollak et al, Br J Haematol 111: 902, 2000
• VDR FF genotype: shorter stature and ↓ BMD– Ferrara et al, Br J Haematol 117: 436, 2002
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Conclusions
• Disease severity explainable by nature of -thalassaemia mutation and interacting -thalassaemia
• Problem of discordant phenotype in 0/+
• Genetic modifiers may play in role in modulating phenotype (especially complications)