Diagnostic difficulties in prevention and control program for thalassemia in Thailand: atypical thalassemia carriers

Transcription

1 Diagnostic difficulties in prevention and control program for thalassemia in Thailand: atypical thalassemia carriers Pranee Winichagoon Fucharoen Thalassemia Research Center Institute of Molecular Biosciences Mahidol University Thailand Nov

2 Typical Thalassemia Carriers β-thalassemia trait Normal Hb level Low MCV/MCH Hb type A 2 A Increased Hb A 2 >3.8% α-thalassemia 1 trait Normal Hb level Low MCV/MCH Hb type A 2 A Normal or slightly decreased Hb A 2 Obligatory case of Hb Bart s hydrops fetalis

3 Atypical Thalassemia Carriers Double heterozygous α- and β-thalassemia Normal Hb A 2 β-thalassemia trait Dominant β-thalassemia trait β-thalassemia with triplicated α-globin gene α-globin variant linked on α-thalassemia gene

4 α- Thalassemia 1 Trait Normal Hb level Low MCV (~ 70 fl) Hb type A 2 +A Normal or sl. decreased Hb A 2 DNA analysis Obligatory cases of Hb Bart s hydrops fetalis

5 Homozygous α-thalassemia 2 Normal allele ζ2 α2 α1 Heterozygous α-thalassemia 1 ζ2 ζ2 α2 α1 α2 α1 X X Homozygous α-thalassemia 2 ζ2 ζ2 α2 α1 X α2 α1 X Hematological parameters of α -thalassemia 2 homozygote are similar to α -thalassemia 1 heterozygote

6 Hb Bart s Hydrops Fetalis is homozygous α-thal 1 α2 α1

7 β-globin gene cluster α-globin gene cluster

8 αβ-thalassemia double heterozygote β-thal Mutation α-globin Genotype MCV (fl) MCH (pg) RDW (%) Hb A 2 (%) Hb F (%) β 0 -Thal trait αα/αα α/αα α Τ α/αα /αα β + -Thal trait αα/αα α/αα α Τ α/αα /αα Blood Cells, Molecules, and Diseases 2011; 47: 120

9 ffects of α-thalassemia to Hb levels Hb trait 25-30% Hb α-thal % Hb α-thal % Hb + Hb H 13-18% (A Bart s disease)

10 Study in 131 from Northeast, Thailand No. % αα/αα α 3.7 /αα α 4.2 /αα α CS α/αα α 3.7 / α 3.7 /αα Ann Hematol 2006; 85:450

11 Hb 13.2 g/dl MCV 77.4 fl MCH 27 pg Hb A 2 () 33.2 % Hb 12.7 g/dl MCV 68.6 fl MCH 22.8 pg HbA 2 () 21 % Hb F 0.5 % Hb Type A Hb F 0.6 % Hb Type A

12 Hb 13.2 g/dl Hb 12.7 g/dl MCV 84 fl MCH 27 pg MCV 68.6 fl MCH 22.8 pg Hb A 2 () 33.2 % HbA 2 () 21 % Hb F 0.3 % Hb F 0.6 % Hb Type A Hb Type A Hb 9.0 g/dl MCV 59.3 fl MCH 17.6 pg HbA 2 () 1.6 % Hb F 0.2 % Hb Type CS A 2 A Bart s H

13 Hb 13.2 g/dl Hb 12.7 g/dl MCV 77.4 fl MCH 27 pg MCV 68.6 fl MCH 22.8 pg Hb A 2 () 33.2 % HbA 2 () 21 % Hb F 0.5 % Hb F 0.6 % Hb Type A Hb Type A Hb 9.0 g/dl Hb 9.0 g/dl Hb 6.9 g/dl MCV 45.2 fl MCV 59.3 fl MCV 56.9 fl MCH 14.3 pg MCH 17.6 pg MCH 15.1 pg HbA 2 () 16.6 % HbA 2 () 1.6 % HbA 2 () 66.7 % Hb F 1.1 % Hb F 0.2 % Hb F 14.2 % Hb Type CSA Bart s Hb Type CSA 2 A Bart s H Hb Type CSF Bart s

14 Hb 12.7 g/dl Hb 10.2 g/dl MCV 67 fl MCH 22 pg MCV 68.8 fl MCH 19.9 pg HbA 2 () 83 % HbA 2 () 67.1 % Hb F 2.5 % Hb F 26.4 % Hb Type Hb Type F? Hb 7.6 g/dl MCV fl Hydrops fetalis MCH 26.3 pg HbA 2 () 0 % Hb F 0 % Hb Type Bart s Hb Bart s Hydrops fetalis

15 β-thalassemia/hb Disease MILD INTRMDIAT SVR - α/αα β + /β No. of cases Hb (g/dl) Hb F (g/dl ) Hb F (%) Hb (%)

16 INTRPRTATION Clinical data Laboratory data Family study History : Previous blood transfusion Type of specimens (cord/fetal blood, child/adult blood) DNA analysis

17 Multiplex PCR for α-thalassemia THAI-F SA-F 4.2-F 3.7-F α2-r 4.2-R 3.7-R THAI-R SA-R ξ2 ψξ1 ψα2 ψα1 α2 α1 θ1 -α 3.7 -α SA --THAI Normal fragment; 3.7-F: α2-r = 1,800 bp - α 3.7 fragment ; 3.7-F: 3.7-R = 2,022 bp - α 4.2 fragment ; 4.2-F/4.2-R = 1,628 bp - - SA fragment ; SA-F/SA-R = 1,349 bp - - THAI fragment ; THAI-F/THAI-R = 1,153 bp SA N N 1.5% agarose gel electrophoresis

18 -- SA /αα α2-globin -- SA Normal α-thal 1 α2-globin -- SA Normal α-thal 1 -- THAI -α 3.7 /-α 3.7 α2-globin αα/αα Normal α-thal 1 Munkongdee T et al. Rapid diagnosis of α-thal by melting curve analysis. J Mol Diag 2010; 12:

19 Immunodetection of Hb Bart s Screening for α-thalassemia Positive for: α-thalassemia 1 heterozygote, α-thalassemia 2 homozygote, Hb H disease, A Bart s disease and Hb Bart s hydrops fetalis

20 Immunodetection of Hb Bart s Sensitivity and specificity for heterozygous α-thal1, homo α-thal 2 and Hb CS from 308 samples Total Negative Weak Positive Positive αα/αα * -α/αα * α T α/αα (CS) -α/-α /αα 45 2** /-α or /α CS α Sensitivity 97% Specificity 93%

21 Immunochromatographic strip test for α-thalassemia

22 Normal Hb A 2 β-thalassemia trait Hb A 2 reference intervals Normal: % β-thal trait: 3.8-7% A 2 borderline: % The prevalence of borderline A 2 carriers in populations with high frequency of β-thalassemia has been reported in 2.2% to 3.0% in one study and up to 16.7% in another study (Maggio A,Haematologica 2008). Andrea Mosca, Università degli Studi di Milano

23 Normal Hb A 2 β-thalassemia trait Hemoglobin, 2013; 37(2): 201

24 Borderline HbA 2 levels associated with reduced MCV and MCH Mild β + -thalassemia mutations: IVSI-6 (HBB c T->C) Coinherited δ- and β-thalassemia (in cis or in trans) β-promoter mutations: -92 (HBB c.-142 C->T) Coexisting iron deficiency anemia MCV A 2 Borderline HbA 2 with normal MCV and MCH may be an outlier value of the normal HbA 2 distribution in the non carriers or the effect of genetic determinants able to increase HbA 2 levels. Triplication of the α-globin genes β-promoter mutations: -101 (HBB c.-151 C->T) Some HBD and HBB gene variants

25 Andrea Mosca, Università degli Studi di Milano In ast Indians, β 0 -Thal, codon 15 G>A (HBB: c.47g>a) in cis with missense mutation of the δ-globin gene (Hb A 2 -India, HBD: c.350g>t).

26 β-globin mrna stability Normal β globin mrna T 1/2 : 60 hours Unstable β globin mrna => β globin protein => β thalassemia phenotype Nonsense Mediated Decay (Premature termination codon) Other stability determinants

27 3 Premature Termination codon leading to dominant β-thalassemia β globin mrna 5 3 Location of PTC (Nonsense mediated decay) 50 nt mrna instability β mrna level Maximal Nil / Minimal Normal Thal Phenotype β 0 Thal intermedia Inheritance Recessive Dominant

28 Dominant β thalassemias due to 3 PTCs Thal intermedia in heterozyotes Persistence of mrna Unstable β globin variant PTC N Undegraded PTC (β121) - bearing β globin cdna (Hall & Thein, Blood 83:2031-7) rythroblastic inclusions with β-globin precipitates (Ho et al, Blood 89:322-28)

29 Heterozygote β-thalassemia + xcess of α-globin gene (Triplicated α-globin gene ) Heterozygote Thal Minor Heterozygote Thal Intermedia Normal ζ2 α2 α1 α β α β Triplication ζ2 α2 α1 α1/ α2 αα/αα δ γ αα/ααα δ γ

30 β-thalassemia Trait with ααα/αα S.T. R.M. Hb (g/dl) MCV (fl) MCH (pg) MCHC (g/dl) Hb A Hb F β-thal mutation codons 41/42, IVS-I nt 5, -TCTT G -> C

31 Hb Q (α 74 Asp-His)-H Disease Hb: 9.5 g/dl RBC: 4.83X10 12 /L HCT: 30.5 % MCV: 63 fl MCH: 19.6 pg MCHC: 31 g/dl Bart s+h Hb A 2 : 1.6% Hb Q (RT 4.66): 96.4% Hb Bart s: positive DNA studies : --/- 4.2 α Q Hb Q

32 Hb Q-H disease Q Q Normal Hb Q trait Hb Q-H disease Hb Q mutation (α1 CD74 Asp-His) occurs on the 4.2 kb α- thalassemia 2 deletion gene (- 4.2 α Q ), resulting in an α-thalassemia 2 phenotype

33 Acknowledgement