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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 09/12/11 Bert Callewaert, MD, PhD Center forMedicalGenetics GhentUniversity Hospital

  2. The effect of mutationsonproteinfunction • mutationsresulting in a loss of function of 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 (nonsenseorframeshift 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. 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 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  5. Novelpropertymutations = (missense) mutationsnovelproperty of the protein +/- normalfunction infrequent (most AA substitutionseitherneutralordetrimental) e.g. sicklecelldisease PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  6. Mutationsassociatedwith heterochronicorectopic gene expression • = mutationsthat alter the regulatoryregions of a gene • Examples: • oncogenemutations in cancer • hereditarypersistence of HbF (continuedexpression of the g-globin gene) PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  7. Hemoglobinopathies • most commonsingle-gene disorders in humans • more than 5% of the world’spopulation is carrier of anabnormalglobin gene PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  8. 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 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  9. PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

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

  11. Globinswitching PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  12. 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 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  13. Groups of mutations Mutationsthat alter the structure of the globinprotein Reducedavailabilityoneor more globinchains mutationsthatimpair the globindevelopmentalswitching PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  14. 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) PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  15. 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 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  16. 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 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  17. SickleCellDisease

  18. 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) PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  19. HemoglobinStructuralVariantsthat alter oxygentransport • HbHyde Park (β-chain His92Tyr) • ~ normalhemoglobinstability • ironresistant to the enzymemethemoglobin reductase. • accumulation of methemoglobin → cyanosis (usuallyasymptomatic) • homozygousstate presumably lethal. • HbHammersmith (βchainPhe 42 Ser) •  instable Hb, lower O2 affinity • mutationsin α:β interface (HbKempsey) • prevent oxigenrelatedmovement • locked in high O2 affinity state •  Polycythemia PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  20. 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) PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  21. 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 • ↑normalchains: damage the RBCs (hemolyticanemia) • ↓Hbsynthesis hypochromic, microcyticanemia • DdIrondeficiency PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  22. α - globin production The a - Thalassemias Normal anemia Hydropsfoetalis HomotetramericHb: ineffectiveoxygencarriers  Hydropsfetalis • affect the formation of bothfetal and adultHb • in the absence of a-globins: • - HbBart’s: g4 • - Hb H: b4 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  23. The a - Thalassemias • most commonlydue to deletion of the a-globingenes Misalignmentwith PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  24. 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 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  25. The a - Thalassemias • Rare forms: • - formdue to the ZF deletion(namedafterindividual ZF) • - the ATR-X syndrome PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  26. ZF deletion transcribedfrom the opposite strand a2-gene is silenceddue to the generation of antisenseRNAsfrom the truncated LUC7L gene wild-typeantisensetranscripts do alsoexist and play a role in regulation of gene expression (f.e. X inactivation)! PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  27. The ATR-X syndrome • MR and a-thalassemia • due to mutations in the X-linked ATRX gene • encodes a chromatin remodelingprotein • activatesexpression in trans • partialloss-of-functionmutationsresult in modest reduction of a-globinsynthesis • somatic (more severe) mutations in ATRX cause the • a-thalassemiamyelodysplasiasyndrome(ifgermline: hydropsfetalis!) PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  28. The ATR-Xsyndrome • Profound MR (X-L) • MC • Short stature • Genital Δ • (Mild) anemia Erythrocytesafterincubation in briljant cresyl blue. HbH inclusions :‘golf ball’ From Gibbons R. Orphanet Journal of Rare Diseases 2006;1:15 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  29. 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 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  30. of the 3’ end of the gene >> PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  31. Point mutationsthatcauseb-thalassemia are distributedthroughout the gene. They affect virtuallyeveryprocessrequiredfor the production of normalb-globin. PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  32. Posttranscriptionalmodifications of mRNA PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  33. RNA splicingmutations in b – Thalassemias (1) PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  34. RNA splicingmutations in b – Thalassemias (2) PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  35. RNA splicingmutations in b – Thalassemias (3) Very frequent in SoutheastAsia Hb E: example of a single nucleotidesubstitutionthataffects both RNA splicing and the coding sequence PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  36. Complex b – Thalassemias (egdb)° thalassemia (illustratesimportance of LCR) (db)° thalassemia (Agdb)° thalassemia Hereditarypersistence of fetalhemoglobin PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

  37. 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 PostgraduatecourseHumanGenetics – 09/12/11 Bert Callewaert, MD, PhD– Center forMedicalGenetics – Ghent University Hospital

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