Molecular Haematology I Globin Disorders Dr Edmond S K Ma Division of Haematology Department 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  -thalassaemia5%  -thalassaemia3.1%

Prevalence of thalassaemia in Hong Kong Chinese  -thalassaemia (-- SEA )  -thalassaemia deletion90%  -thalassaemia codons (-CTTT)  0 45% IVSII-654 (C  T)  0 20% nt-28 (A  G)  + 16% codon 17 (A  T)  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 -   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 deletion90%

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  -thalassaemia Clark & Thein, Clin Lab Haematol 26: ; 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 deletion 4.2 kb deletion

LIS internal control (2350 bp) -α 3.7 (2022/2029 bp ) α2 (1800 bp) -α 4.2 (1628 bp) -- SEA (1349 bp) water αα/-- SEA ladder -α 3.7 /-- SEA -α 4.2 /-- 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. Gel Smaller fragment Larger fragment Key restriction enzyme sites probe region

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 HbA 2 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 Panel 1: 1-6 1:-28 Heterozygote 2:-28/71-72 Compound Heterozygote 3:Codon 17 Heterozygote 4:Codon 43 Heterozygote 5:100 bp DNA Ladder 6:Reagent Blank Control Panel 2:7-8 7:IVS Heterozygote 8:Reagent Blank Control Internal control 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 Yes  0 /  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

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

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 mutation  0 /  0 severe  0 /  + 2/3 severe; 1/3 intermedia  0 /  +++ intermedia   /  + intermedia (mild) Concurrent  -thalassaemia SEA deletion ameliorate  0 /  + only but not necessarily  0 /  0 Triplicated  -globin gene in  -thalassaemia heterozygotes Often 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

A   -HPFH: nt -196 C→T SubjectSex/AgeHb (g/dL) MCV (fL) MCH (pg) HbA 2 (%) HbF (%) HbH bodies α-genotypeβ-genotype IndexF/ Negativeζζζαα/ζζααβ 41/42(-CTTT) /β A Elder brother 1 M/ Negativeζζζαα/ζζααβ 41/42(-CTTT) /β A Elder brother 2 M/ Negativeζζζαα/ζζααβ 41/42(-CTTT) /β A Elder Sister F/ Negativeζζαα/ζζααβ A / β A DaugtherF/ Negativeζζαα/ζζααβ 41/42(-CTTT) /β A Son of elder brother 2 M/ Negativeζζαα/ζζααβ 41/42(-CTTT) /β A Note: All subjects are negative for XmnI G γ-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: , 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%) MajorIntermedia (TA) 7 homozygous 02 (TA) 7 heterozygous14 (2)9 (1) G71R homozygous42 G71R heterozygous24 (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 cohort AlleleFrequency C282Y0% H63D1.3% S65C0%

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 PolymorphismPatientsControlp-value exon 5 I238M 7.1%4.7%0.24 IVS CA24.5%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)