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Principles of Molecular Disease : Lessons from the Hemoglobinopathies

Principles of Molecular Disease : Lessons from the Hemoglobinopathies. The effect of mutations on protein function. mutations resulting in a loss of function of the protein mutations resulting in a gain of function of the protein

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Principles of Molecular Disease : Lessons from the Hemoglobinopathies

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  1. Principles of MolecularDisease: Lessonsfrom the Hemoglobinopathies PostgraduatecourseHumanGenetics 13/12/2013 Bert Callewaert, MD, PhD Center forMedicalGenetics GhentUniversity Hospital

  2. The effect of mutationsonproteinfunction • mutationsresulting in a loss of functionof the protein • mutationsresulting in a gain of function of the protein • mutationsresulting in a novelpropertyby the protein • mutationsresulting in gene expression at the wrong time or place PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  3. Loss-of-functionmutations • deletion of the entire gene (and eventuallyalsocontiguousgenes) • examples: microdeletionsyndromes, monosomies (Turner), a-thalassemias • chromosomalrearrangements • premature stop codon (nonsenseor frameshift mutations) • missense mutationsmayabolishproteinfunction • e.g. Catshl syndrome: loss- of – function FGFR3 FGFR3 p.R621H Severity of disease ~ amount of function lost PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  4. Loss-of-function mutations - missense mutations Severity of disease ~ amount of function lost E.g. Congenital adrenal hyperplasia Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  5. Loss-of-function mutations - missense mutations Severity of disease ~ amount of function lost E.g. Congenital adrenal hyperplasia Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  6. Gain-of-functionmutations = mutationsthatenhanceoneor more of the normalfunctions of the protein • mutationsthatenhanceonenormalfunction of the protein • f.e.: the G380R mutation in • FGFR3causingachondroplasia • mutationsthatincrease the production of a normalprotein • in itsnormal environment • f.e.: trisomy 21 (Down syndrome) note: Alzheimer • duplication of PMP22 in Charcot-Marie-Toothdisease type 1A • chromosomalduplications in cancer Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  7. Novelpropertymutations = (missense) mutationsnovelproperty of the protein +/- normalfunction infrequent (most AA substitutionseitherneutralordetrimental) e.g. sicklecelldisease Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  8. Mutationsassociatedwith heterochronicorectopic gene expression • = mutationsthat alter the regulatoryregions of a gene • Examples: • oncogenemutations in cancer • hereditary persistence of HbF (continued expression of g-globin) • PITX1 • Liebenberg syndrome Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  9. Hemoglobinopathies • most commonsingle-gene disorders in humans • more than 5% of the world’spopulation is carrier of anabnormalglobin gene Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  10. Hemoglobin • 4 subunits: 2 a (like) and 2 b (like) - chains • each subunit is composed of : • - a polypeptidechain (globin) • - a prostheticgroup (heme): iron-containing pigment that combines with O2 • highlyconservedstructure • Hb A (adulthemoglobin): a2 b2 Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  11. Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  12. identical chromosome 16 differonly in 10/146 AA chromosome 11 In adultlife: >97% HbA 2% HbA2 <1% HbF commonancestral gene Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  13. Globinswitching Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  14. Globin switching Why? Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  15. ΒGlobin cluster 6 kb Chr. 11 LCR e Gg Ag yb d b • expression of b-globin gene controlledbynearbypromoterand LCR • locuscontrolregion (LCR): requiredfor the expression of all the genes • in the b-globin cluster • deletions of LCR results in egdb- thalassemia Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  16. Mutations affecting the globin chains Mutationsthat alter the structure of the globinprotein Reduced availability one or more globin chains (Thalassemias) mutationsthatimpair the globindevelopmentalswitching Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  17. 1. StructuralVariants • usuallydue to point mutationsin one of the globingenes • more than 400 abnormalhemoglobinvariants have been described • onlyabout 50% are clinically significant • three classes: • - mutantsthatcausehemolyticanemia • - mutantsthat alter oxygen transport • - mutantsthatreduce the abundance of the globinchain (thalassemias) Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  18. StructuralVariantsthatcauseHemolyticAnemia • the mutant makes the Hbtetramerunstable •  loss-of-function • e.g.: HbHammersmith (b-chain Phe42Ser mutation) • the mutant gives the globinchainanunusualrigidstructure • novelpropertymutations • f.e.: sicklecellglobin; HbC Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  19. SickleCellDisease • HbS: firstabnormalHbdetected (Glu6Val mutation in b-chain) • severeARcondition • common in equatorialAfrica; 1/600 AfricanAmericans is bornwith the disease • sicklecelltraitrefers to the heterozygous state • about 8% of AfricanAmericans are heterozygous • heterozygotes are protectedagainstmalaria Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  20. SickleCellDisease

  21. 1. StructuralVariants • usuallydue to point mutations in one of the globingenes • more than 400 abnormalhemoglobinvariants have been described • onlyabout 50% are clinically significant • three classes: • - mutantsthatcausehemolyticanemia • - mutantsthat alter oxygen transport • - mutantsthatreduce the abundance of the globinchain (thalassemias) Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  22. HemoglobinStructuralVariantsthat alter oxygen transport • HbHyde Park (β-chain His92Tyr) • ~ normalhemoglobinstability • ironresistant to the enzymemethemoglobin reductase. • accumulation of methemoglobin → cyanosis (usuallyasymptomatic) • homozygous state presumably lethal. • HbHammersmith (βchainPhe 42 Ser) •  instable Hb, lower O2 affinity • mutations in α:β interface (HbKempsey) • prevent oxigenrelatedmovement • locked in high O2 affinity state •  Polycythemia Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  23. 1. StructuralVariants • usuallydue to point mutations in one of the globingenes • more than 400 abnormalhemoglobinvariants have been described • onlyabout 50% are clinically significant • three classes: • - mutantsthatcausehemolyticanemia • - mutantsthat alter oxygen transport • - mutants that reduce the abundance of the globin chain (thalassemias) • mutations in the coding region • rate of syndthesis↓ • severe instability of the chains. Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  24. 2. HemoglobinSynthesis Disorders (Thalassemias) • collectively the most commonhumansingle-gene disorders! • carriers: protectiveadvantageagainst malaria • > qalassa(sea): firstdiscovered in Mediterraneanarea • imbalance in a : b chainratio • - ↓synthesis • - instability (cfr supra) • ↑normal chains: damage to the RBCs (hemolytic anemia) • ↓Hbsynthesis hypochromic, microcyticanemia • DdIrondeficiency Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  25. α - globin production The a - Thalassemias Normal anemia Hydropsfoetalis HomotetramericHb: ineffectiveoxygen carriers  Hydropsfetalis • affect the formation of bothfetal and adultHb • in the absence of a-globins: • - HbBart’s: g4 • - Hb H: b4 Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  26. The a - Thalassemias • most commonlydue to deletion of the a-globingenes Misalignmentwith Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  27. The a - Thalassemias clinicalconditionnumber of functionala-globin gene a-chain a-genesgenotypeproduction Normal 4 aa/aa 100% Silent carrier 3 aa/a- 75% a-thalassemiatrait 2 a-/a- or 50% (mild anemia, microcytosis)aa/- - * Hb H (b4) disease 1 a-/- - 25% (moderatelyseverehemolyticanemia) Hydropsfetalis (HbBart’sg4) 0 - -/- - 0% * Carriers frequent in SoutheastAsia Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  28. The a - Thalassemias • Rare forms: • - formdue to the ZF deletion(namedafterindividual ZF) • - the ATR-X syndrome Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  29. The a - Thalassemias ZF deletion transcribedfrom the opposite strand a2-gene is silenceddue to the generation of antisenseRNAsfrom the truncated LUC7L gene wild-type antisense transcripts do also exist and play a role in regulation of gene expression (f.e. X inactivation, siRNA, lnRNA)! Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  30. The a - Thalassemias The ATR-X syndrome • MR and a-thalassemia • due to mutations in the X-linked ATRX gene • encodes a chromatin remodeling protein (methylation) • activatesexpression in trans • partialloss-of-functionmutationsresult in modest reduction of a-globinsynthesis • somatic (more severe) mutations in ATRX cause the • a-thalassemiamyelodysplasiasyndrome(ifgermline: hydropsfetalis!) Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  31. The ATR-Xsyndrome • Profound MR (X-L) • MC • Short stature • Genital Δ • (Mild) anemia Erythrocytesafterincubation in briljant cresyl blue. Hb H inclusions :‘golf ball’ From Gibbons R. Orphanet Journal of Rare Diseases 2006;1:15 Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  32. The B- Thalassemias • ↓b-globinproduction • twob-thalassemiaalleles: usuallythalassemiamajor (severeanemia) • oneb-thalassemiaallele: thalassemiaminor (mild anemia, noclinic) • postnatal • precipitation of excessa-chains hemolysis • low β-chain production  hypochromic, microcyticanemia • ↑HbA2 (a2d2) and ↑ HbF (a2g2) • >> single-base pair substitutions (ratherthandeletions) • >> compoundheterozygous • simple versus complex b-thalassemia Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  33. of the 3’ end of the gene >> Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  34. Point mutationsthatcauseb-thalassemia are distributedthroughout the gene. They affect virtuallyeveryprocessrequiredfor the production of normalb-globin. Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  35. Posttranscriptionalmodifications of mRNA Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  36. RNA splicingmutations in b – Thalassemias (1) Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  37. RNA splicingmutations in b – Thalassemias (2) Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  38. RNA splicingmutations in b – Thalassemias (3) Very frequent in SoutheastAsia Hb E: example of a single nucleotidesubstitutionthataffects both RNA splicing and the coding sequence Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  39. Complex b–Thalassemias (egdb)° thalassemia (illustratesimportance of LCR) (db)° thalassemia (Agdb)° thalassemia Hereditarypersistence of fetalhemoglobin Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  40. 3. GlobinDevelopmentalSwitching Disorders • hereditarypersistence of fetalhemoglobin • group of clinicallybenignconditions • production of higherlevels of Hb F than is seen in (db)° thalassemia • theyimpair the perinatal switch fromg-globin to b-globinsynthesis Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

  41. Thanks for your attention ! Hemoglobinopathies– Bert Callewaert, MD, PhD – 09/12/2011 Ghent University Hospital – Center for Medical Genetics

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