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Today’s agenda

Everything you always wanted to know about globin disorders IN ISRAEL but is not written in the textbook. Today’s agenda. 1. What are the different globin disorders in Israel? alpha and beta ? 2. What ethnic groups have them? 3. How to diagnose them?.

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Today’s agenda

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  1. Everything you always wanted to know about globin disorders IN ISRAEL but is not written in the textbook

  2. Today’s agenda 1. What are the different globin disorders in Israel? alpha and beta? 2. What ethnic groups have them? 3. How to diagnose them?

  3. פתגם רפואי אמריקאי:“When you hear hoofbeats……

  4. ...think of horses, not zebras.”

  5. a-thalassemia: molecular pathology GENOTYPE Hematology Designation 4 genes (2 on each Chr 16) MCV ~85 Normal state 3 genes (1 deleted) MCV 78-84 Silent carrier POINT MUTATIONS VARIABLE 2 genes (2 deleted MCV 69-72 a-thal trait in cis or trans) Mild anemia 1 gene present MCV 55-60 Hb H disease (3 genes deleted) Anemia, Hb 6-10 0 genes (4 genes deleted) Hydrops fetalis (incompatible withlife)

  6. Alpha globin mutations in Israel • Alpha globin gene deletions (5 types, both single and double gene deletions) • Multiplicities, extra alpha genes (2 types: 3 and 4 on a chromosome) (worsens beta thal) • Point mutations (8) • Variants (8): with either thal or unstable Hb phenotype (or NO phenotype) • Hasharon • Petak Tikvah • Setif • Shaare Zedek • Taybe • Rambam • Galveston • Pyrgos

  7. D 16 bp Poly A -a3.7 D T 39 aaa --Med -a4.2 D 5 bp a-thalassemia mutations in Israeli Arabs Over half are point mutations!

  8. a-globin mutations in Jewish ethnic groups -a3.7 Others --Med -a4.2 Moroccan --Yem Georgian aaaanti 3.7 a2 Poly A (A-G) Iraqi a2 IVS-I D 5 a2 Hb Hasharon Kurd Yemenite Ashkenazi 0 10 20 30 40 50 60

  9. Alpha globin/thal abnormalities in Israel

  10. Beta globin/thal mutations in Israel • Beta deletions: yes, but very rare • Point mutations: 31!! • Variants (10): With many phenotypes, mostly either sickling or thal (or no phenotype) • Sickle, C, O-Arab, D-Punjab • Lepore • E • Pyrgos • Rambam • Koln • Galveston

  11. Beta thal Mutations In Israeli Arabs By Region

  12. Beta globin/thal abnormalities in Israel

  13. Patient A: Hb Taybe 30 year old man: suspected b-thal intermedia • Family history: parents are first cousins • Son has microcytic anemia • Physical examination: high forehead with frontal bossing, mildly jaundiced, mild hepatomegaly, moderate splenomegaly (6 cm below costal margin) Laboratory Data • WBC 7,000, Hb 11.0, MCV 77.3, MCH 25, RDW 17, Retics 4%, Plts 165,000 • Peripheral smear: severe pathology: hypochromia, targets, teardrops, basophilic stippling, NRBC • Hb electrophoresis: Hb A2 2%, HbF 6% • No Hb H

  14. Patient A: DNA analysis • Sequence of b-globin genes and a-2 genes: Normal • Sequence of the a-1 globin genes: HOMOZYGOUS for Hb Taybe Hb Taybe is caused by a deletion of 3 nucleotides at codon 39 of the a-1 gene, resulting in an in-frame deletion of threonine Hyper-unstable hemoglobin, therefore not detected on electrophoresis Causes a thalassemia-like clinical picture TAYBE is common! In Arabs in north.

  15. FAMILY L b-thal intermedia ? IVS1-5/-101 Hb 9.3 MCV 61.5 MCH 18.5 A2 3.6% F 16.7% Hb 15.2 MCV 76.1 MCH 24.4

  16. FAMILY L b-thal intermedia a-thal IVS1-5/-101 Hb 9.3 MCV 61.5 MCH 18.5 A2 3.6% F 16.7% Hb 15.2 MCV 76.1 MCH 24.4 A2 1.5% F 0.4% Hasharon 20%

  17. FAMILY L -a 4.2/aa aHasharona/aa Hb 12.9 MCV 79.5 MCH 27.1 A2 2.1% F 0.4% Hb 15.6 MCV 82.4 MCH 28.4 A2 2.6% F 0.3% Hasharon 17% -a 4.2/ aHasharona IVS1-5/-101

  18. FAMILY L -a 4.2/ aHasharona IVS1-5/-101 -101/A -a 4.2/aa IVS1-5/A aHasharona /aa

  19. But sometimes it IS a zebra…in Israel

  20. Hb Koln • Hb Koln • Causes mild hemolytic anemia in heterozygote • Seen on Hb electrophoresis, HPLC • Is most common unstable Hb and can be seen as de novo mutation

  21. Hb Petak Tikvah • Leu-Asp 110 alpha 1 • Unstable; causes HbH in combination with deletional alpha thal

  22. Hb Setif • Alpha 94 Asp to Tyr • Unstable • Amount in heterozygotes: 12-15% • Hematology of carriers is normal • RBC “sickle” due to decreased solubility of hemoglobin • Causes HbH in combination with deletional alpha thal

  23. Hb Pyrgos • Beta chain codon 83 gly-asp • Totally normal hematology in carriers • 50% of total Hb

  24. Hb Galveston • Codon 117 CAC-CGC of beta globin • 45-50% in carriers • No clinical consequence

  25. Hb Rambam • Codon 69 of beta globin chain GGT-GAT (gly-asp) • Low oxygen affinity • Causes misidentification of HbA1C in diabetics

  26. Hb Petak Tikvah • Leu-Asp 110 alpha 1 • Unstable; causes HbH in combination with deletional alpha thal

  27. Diagnostic Methods • Automated CBC • Hb Electrophoresis • HPLC • Isoelectric focusing • DNA diagnosis • Need depends on goal: screening, or diagnosis or prenatal diagnosis

  28. Remember the MCV/MCH • This is your first clue that there is a problem, so use it! • European Molecular Genetics Quality Network recommends cutoffs: MCV<78 MCH<27 on fresh blood

  29. b-Thalassemia trait: RBC indices b-thal has large range of MCV/MCH, from very low (MCV ~60) to near normal (silent carrier). For screening, MCH is more sensitive.

  30. Level of b-globin determined by severity of the mutations b+ bo -101 -88 Poly A -28 N39 FS37 IVS2-1 FS44 Etc. IVS1-6 IVS1-110 IVS1-5

  31. MCV of thal trait varies with b-globin mutation

  32. MCH varies with b-globin mutation For screening in Israel: MCH <26

  33. It is always helpful to see hematological parameters of other family members

  34. Hb electrophoresis • Cellulose acetate, alkaline pH: Detects many variants but migration of C, E, A2, and O is similar and S, D, G co-migrate. • Agar at acid pH: distinguishes these from each other • Most important thing is to be familiar with your lab and know the pitfalls, mostly that there ARE pitfalls!

  35. HbA2 in thal trait: pitfalls • Many errors in HbA2: too high or too low • Different methods yield different normal values: column or electrophoresis; electrophoresis using densitometry versus elution (densitometry not sensitive) • Column method is not sensitive for mildly elevated levels • It is often worth repeating HbA2 in a different lab if it is borderline elevated • It is not uncommon to see LOW HbA2 with alpha thal trait but it should not be elevated

  36. HbA2: Pointers and Pitfalls • In a patient with b-thal trait, Hb A2 elevation can be masked by: • Delta thalassemia: we do not know if this exists in Israel! • Concurrent iron deficiency: must identify and treat first! (Remember there is a problem when analyzing soluble transferrin receptor which is elevated in thalassemia trait).

  37. HbA2: How high is it? • Usually elevated in b-thal trait • However, it can be elevated to various degrees: from just over normal limit (2.8% to 3.0% in some labs) very rarely as high as 6-7% • HbA2 runs like Lepore, if you see a patient with 20% A2 it is probably Lepore • Loose correlation with severity of mutation: Nearly always only mildly elevated in mild beta thal trait.

  38. HPLC for Hb A2 and Hb F

  39. HPLC for globin disorders

  40. HPLC Pointers and Pitfalls • Co-elution of different hemoglobins • Therefore the results are not considered to be diagnostic, need confirmation using another method. • For instance: HbS and A2 may co-elute, E, G and A2 co-elute.

  41. Definitive diagnosis of globin abnormalities Definitive diagnosis at DNA level: gene mapping and/or sequence analysis. Can be done for both alpha globin genes and beta globin gene.

  42. PCR analysis for alpha globin deletions

  43. “When you hear hoofbeats……

  44. ...think of horses, not zebras.”

  45. “This IS a second opinion. At first I thought you had something else.”

  46. THANKS! • Prof Eliezer Rachmilewitz • Prof Ariella Oppenheim • Dr Dori Filon • The staff of the hematology lab • All the referring doctors all over Israel especially: HaEmek Hospital

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