Molecular Haematology IGlobin Disorders

Dr Edmond S K MaDivision of HaematologyDepartment of Pathology

The University of Hong Kong

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

Prevalence of thalassaemia in Hong Kong Chinese

-thalassaemia 5%

-thalassaemia 3.1%

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%

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

Detection of thalassaemia

• Red cell indices (MCV, MCH)

• Determine iron status

• HPLC analysis

• Hb and globin chain electrophoresis

• Detection of HbH inclusion bodies

Laboratory diagnosis of thalassaemia by HPLC

Haemoglobin electrophoresis

Detection of HbH inclusion bodies

New approaches in diagnosis of SEA deletion:

gap-PCR for SEA deletion

New approaches in diagnosis of SEA

deletion: detection of -globin chains in adults

-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

Prevalence of thalassaemia in Hong Kong Chinese (MCV < 80 fL)

-thalassaemia(--SEA) -thalassaemia deletion 90%

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

Single -globin gene deletion (-) and triplicated -globin gene () configuration

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

Multiplex PCR for 3 commonest -thalassaemia deletion

LIS1 control -2 gene

SEA deletion

3.7 kb deletion4.2 kb deletion

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

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

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

16kb

10.5 kb

14.5kb

12.6 kb

7.0 kb

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)

Reverse dot blot

Chan V et al, BJH 104: 513-5, 1999

Multiplex mini-sequencing screen

Wang W et al, Clin Chem 49: 800 – 803, 2003

Molecular screening of non-deletional -globin gene mutations by denaturing HPLC

Guida V et al, Clin Chem 50: 1242 – 1245, 2004

Thalassaemia array

Chan K et al, BJH 124: 232 – 239, 2004

Thalassaemia array

-thalassaemia phenotypes-thalassaemia trait• Aymptomatic• Hypochromic

microcytic red cells

• High HbA2

• Variable HbF• Genotype: simple

heterozygotes for -thalassaemia alleles

-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

-thalassaemia syndromes

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

Why study genotype phenotype relationship?

• Genetic counselling

• Management decisions

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

Mutation detection by dot blot hybridization

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

Southern blot hybridization with -probe

PCR-based mutation detection

-multiplex PCR

-thalassaemia PCR

The spectrum of -thalassaemia alleles in Chinese

Genotype phenotype correlation in 0/0 thalassaemia

Genotype phenotype correlation in 0/+ thalassaemia

Homozygous 0/0 and compound heterozygous 0/+ thalassaemia

Clinical phenotype of +/+ thalassaemia

Clinical phenotype of HbE / -thalassemia

Molecular pathology of -thalassaemia

Thalassaemia intermedia: family study 1

Thalassaemia intermedia: family study 2

Thalassaemia screening using MCV and MCH cutoff

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

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)

Co-inheritance of -thalassaemia in severe -thalassaemia

Co-inheritance of -thalassaemia in severe -thalassaemia

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

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

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

Triplicated -globin gene in -thalassaemia heterozygotes

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

Triplicated -globin gene in -thalassaemia heterozygotes

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

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

Effect of XMnI G-promoter polymorphism

Genotype phenotype correlation in 0/0 thalassaemia

Genetic determinants of high HbF

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

Genetic modifiers of single gene disorders

Primary modifiers

Secondary modifiers

Tertiary modifiers

Hyperbilirubinaemia

Jaundice

Gall stones

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

UGT1A1 alleles in Chinese

Hsieh S-Y et al, Am J Gastroenterol 96: 1188 - 1193, 2001

Detection of UGT1A1 polymorphisms

• UGT1A1 promoter genotype– direct sequencing of PCR product

• Gly71Arg mutation at exon 1– PCR restriction analysis of MspI cleavage site

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

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)

Predictors of bilirubin level

Predictors of gall stones

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%

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

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

TFR2 polymorphism and iron overload in transfusion independent -thalassaemia intermedia

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

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)