New technologies for the diagnosis of thalassemia:

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1 New technologies for the diagnosis of thalassemia: Cornelis Harteveld Leiden University Medical Center, The Netherlands ENERCA Ferrara 16 th of November 2013

2 Introduction to hemoglobinopathies Hb molecule HbA 98% HbA 2 2% Hemoglobinopathies: -/ -Thalassemia: Sickle Cell Disease: Hemoglobin variants: Thalassemia Abnormal hemoglobins microcytic hypochromic anemia reduced production - or -globin chains HbS/S, S/C, S/D, S/E, S/O-Arab hemolysis, polycythemia C.L.Harteveld, LUMC

Carriers unaffected, but recognized at hematologic

3 Hemoglobinopathy (HbP) Autosomal recessive High incidence (increasing in N-European countries) Carriers unaffected, but recognized at hematologic level geographical distribution: co-incides with malaria tropica

4 Laboratory diagnosis for HbP Hematology HPLC and Hb electroforesis normal Microcytic Hypochromic Normal iron HbA 2 Hb variant HbF Gap-PCR for 7 most common -thal deletions ~80% of all -thal > Sequencing -gene Sequencing -genes Additional ~15% of all -thal ~95% of all -thal > MLPA acgh Remaining ~4-5% - and -thal C.L.Harteveld, LUMC

$Biochemic separation Hb fractions Abnormal patterns of$ => Beta-thalassemia carrier -HbF elevated?

5 Biochemic separation Hb fractions Abnormal patterns of HPLC: HbF 33% HbA 2 5.6% HbA 2 normal -HbA 2 elevated? => Beta-thalassemia carrier -HbF elevated? => (A-gamma)delta-beta-thal deletion carrier C.L.Harteveld, Hemoglobinopathies Lab

6 BETA-THALASSEMIA CARRIERS Biochemic separation Hb fractions.. or Hb-electrophoresis (CE): -HbA 2 elevated? => Beta-thalassemia carrier -HbF elevated? => (A-gamma)delta-beta-thal deletion carrier C.L.Harteveld, Hemoglobinopathies Lab

Breakpoint info for primer design: gap-pcr for common beta-deletions in Thailand ε G γ A γ δ β Representative 1.2% agarose gel electrophoresis of the PCR product.

7 Breakpoint info for primer design: gap-pcr for common beta-deletions in Thailand ε G γ A γ δ β Representative 1.2% agarose gel electrophoresis of the PCR product. Lane 1: Positive for G (A ) 0 -thalassemia (Siriraj I deletion) Lane 2: Positive for 0 -thalassemia (3.5 kb deletion) Lane 3: Positive for 0 -thalassemia (Filipino deletion) Lane 4: Positive for 0 -thalassemia (SEA deletion) Lane 5: Positive for HPFH-6 Lane 6: Positive for Hb Lepore Lane 7: Positive for 0 -thalassemia (619 bp deletion) Lane 8: Positive for Thai ( ) 0 -thalassemia Lanes 9 and 10: Negative control Lane 11: Blank 620 bp 688 bp 743 bp 836 bp 974 bp 1159 bp 1270 bp 1447 bp ( bp) Collaboration Supan Fucharoen, Khon Kaen University Thailand (Tritipsombut et a. Hemoglobin 2012)

Gap-PCR (screening 7 most common α-thal deletions) 2 1

7 Med II 3000 2500 2000 1500 1200 1000 500 Norm HetRW

8 Gap-PCR (screening 7 most common α-thal deletions) Dutch I Thai Fil -( ) Med I SEA 3.7 Med II Norm HetRW HomRW -tripl. Norm HetLW Het SEA Het MedI Het20.5 Het FIL

9 Direct sequencing HBLBF1 HBLBF2 1 HBLBF3 2 HBLBR1 3 HBLBF4 HBLBR2 HBLBR3 4 HBLBR4 PCR using tagged primers generates overlapping fragments for automated direct sequencing About 95% of betathal. mutations All Hb variants of the beta-globin chain

10 Direct sequencing 2 1 S13F S6R S13F S8R 2A 1A F S18R F S18R S3F 2B R S3F 1B R About 10% of alphathal. mutations All Hb variants of the alpha-globin chain

with tag primers tag1 tag2 5 3 primer hybridisation stuffer sequence primer sequence

11 MLPA for known and novel α-, β-thal deletions 3 3 Probe hybridisation 3 5 tag1 tag2 Target A Target B Probe ligation Target A Target B 5 5 PCR of ligated probes with tag primers tag1 tag2 5 3 primer hybridisation stuffer sequence primer sequence sequence sequence target A B C D E F G H I J A B C D E F G H I J C.L.Harteveld, LUMC

12 MLPA for known and novel α-, β-thal deletions 4 different MLPA kits on the market for α- and β-cluster, together telling the complete story: - MRC-Holland (P140B, P102B) - LGTC (HBA, HBB) Harteveld et al. JMG 2005 MRC-Holland P140B HS-40 HBZ HBZP HBA2 P HBA1 P HBA2 HBA1 HBQ tel #16p LGTC HBA C.L.Harteveld, LUMC

13 MLPA for known and novel α-, β-thal deletions Mb LGTC HBB G A MRC-Holland P102B

14 array CGH (to characterize breakpoints) Data Analysis NimbleScan: grid alignment Pat A Pat B - - SA Pat C - - JB - - SA

15 Examples of Genotype Phenotype discordance when new technologies help out: 3 cases

16 Case 1: Hb separation doesn t always tell you the complete story. Case F90467 Mrs S. 35 yrs microcytic hypochromic anemia: Hb 11.8 g/dl (normal 12-16) RBC 5.97 x10 12 /l (normal ) MCV 75 fl (normal 80-96) MCH 19.7 pg (normal ) C.L.Harteveld, LUMC 16 Sunday, December 15, 2013

17 Case 1: Hb separation doesn t always tell you the complete story. 2 1 HbA HbJP HbA % 20-25% 3% Sequence analysis -genes: 2 cd12 Ala>Asp; Hb J-Paris-I she has microcytic hypochromic anemia.. is there anything more??? C.L.Harteveld, LUMC 17 Sunday, December 15, 2013

18 Case 1: Hb separation doesn t always tell you the complete story. HbA HbA Hb JP Hb A 2 JP HbA HbJP HbA 2 HbA 2 P 70.3% 25.7% 3.3% 0.7% Elevated HbA 2 4% 2 cd12 Ala>Asp; Hb J-Paris-I Sequence analysis -gene: cd30 AGG>ACG, Arg>Thr; Hb Monroe disrupting splice site: IVS-1(-1) beta-thalassemic phenotype C.L.Harteveld, LUMC 18 Sunday, December 15, 2013

19 How technology comes together: A few interesting cases of beta-thal carriers Carriers are supposed to be clinically silent, However, sometimes simple carriers can present with beta-thalassemia intermedia

20 Case 2: beta-thal carrier acting like severe intermedia patient Hematological, biochemical and molecular data of the patients in 2005 * not measured or relevant because of transfusion therapy Age / Gender 54/F Patient FD Reference values Hb g/dl 8.0 [ ] Ht % 20.7 [ ] RBC x /l 3.13 [ ] MCV fl 66.1 [ ] MCH pg 25.0 [ ] Hb A % * [ ] HbA 2 % * [ ] HbF % * [< 1] gene sequencing β Cd 39 (C T) /β / α gap-pcr ααα/αα αα/αα Transfusion Every three weeks

21 Case 2: beta-thal carrier acting like severe intermedia patient MLPA analysis α-globin gene cluster: duplication α-cluster F.D. 2,5 2 1,5 1 green blu 0, F.D. HS-40 α 2 α 1 HS-40 α 2 α 1 #16p HS-40 α 2 α 1 β #11p

22 Case 2: beta-thal carrier acting like severe intermedia patient (kb) probe nr p13.3 MSLN NARFL C160rf23-25 C16orf20-22 RHBDL C16orf39 C16orf15-19 WFIKKN1 RAB40C PIGQ C16orf11 SOLH KIAA0665 DECR2 C16orf43 AXIN PDIP RGS11 C16orf9 LUC71 Alpha cluster C16orf35 MPG C16orf8 C16orf33 F.D. B.S. F.K. H. N.R. duplicated region MCS-R / ; / 0 Multiple α-genes might be a more common modulator in β-thal carriers showing intermedia phenotype

23 Case 3: mosaic for beta-thalassaemia mutation Propositus behaves like beta-thal Major, but was born as a carrier HBBc.230delCfs (cd76(-c)) Mosaic?? => deletions were excluded by MLPA

24 Case 3: mosaic for beta-thalassaemia mutation SNP s can be AA, BB or AB equally distributed along chromosome ~730,000 SNPs all over the genome A/A A/B B/B

25 Normal Case 3: mosaic for beta-thalassaemia mutation AA AB BB short arm long arm Paternal #11 Maternal #11 SNP s along chromosome 11 Chromosome 11

26 Case 3: mosaic for beta-thalassaemia mutation Father FP CG + Mother mosaic for UPD of almost entire 11p in patient CG Paternal allele contains beta-mutation Cells with UPD overgrow the normal ones over time

27 Case 3: mosaic for beta-thalassaemia mutation Mechanism giving growth advantage to 11p isodisomic cells: Loss of imprinted growth regulatory genes IGF2 and H19 (on 11p15.5) M P IGF2 IGF2 H19 methylated H19

28 Case 3: mosaic for beta-thalassaemia mutation Mechanism giving growth advantage to 11p isodisomic cells: Loss of imprinted growth regulatory genes IGF2 and H19 (on 11p15.5) P P IGF2 IGF2 methylated H19 methylated H19 Growth advantage red cells having paternal beta-mutation Paternally inherited allele expresses only IGF-2 because of methylated H19 (tumor suppressor)

29 seen in 3 unrelated patients with late-onset beta-thal intermed: normal

30 Summary Standard approach in diagnostics for haemoglobinopathies: Haematological parameters (Hb, Ht, RBC, MCV, MCH) Biochemical analysis (HPLC, Hb-electrophoresis) DNA analysis: Screening for common deletions (gap-pcr) Detection point mutations (direct sequencing) Screening less common deletions (MLPA) More advanced approaches in DNA diagnostics: acgh in custom made fine-tiling array Illumina array technology for detection of mosaicisms

31 Acknowledgements LUMC Marion Phylipsen Piero Giordano Peter van Delft Greet Bakker-Verwey Lisette Hoogendijk Sandra Arkesteijn Rianne Schaap Johan den Dunnen Yavuz Ariyurek Bert Bakker Christi van Asperen Athens University of Medicine Jan Traeger-Synodinos Cyprus Institute of Genetics Marina Kleanthous Erasmus MC George Patrinos Khon Kaen University, Thailand Supan Fucharoen Radcliff Hospital, Oxford Douglas Higgs Vip Viprakasit Richard Gibbons Ospedale Maggiore, Milan Nica Capellini Sir Charles Gairdner Hosp., Nedlands Jill Finlayson Jewish General Hospital, Quebec Marc Tischkovitz Hadassah Hospital, Jerusalem Deborah Rund Inst fur Med & molec Diagn, Zurich Hannes Frischknecht King s College Londen School of Med Swee Lay Thein C.L.Harteveld, LUMC

Next Generation Sequencing as a tool for breakpoint analysis in rearrangements of the globin-gene clusters

Next Generation Sequencing as a tool for breakpoint analysis in rearrangements of the globin-gene clusters XXXth International Symposium on Technical Innovations in Laboratory Hematology Honolulu, Hawaii