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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 – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  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 – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  3. Loss-of-functionmutations • deletion of the entire gene (and eventuallyalsocontiguousgenes) • examples: microdeletionsyndromes, monosomies (Turner), a-thalassemias • chromosomalrearrangements • nonsensemutationsor frameshift mutationsresulting in a premature stop codon • missensemutationsmayalsoabolishproteinfunction Severity of disease ~ amount of function lost PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  4. Gain-of-functionmutations = mutationsthatenhanceoneor more of the normalfunctionsof 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) • duplication of PMP22 in Charcot-Marie-Toothdisease type 1A • chromosomalduplications in cancer PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  5. Novelpropertymutations = mutationsthatgive a novelproperty to the protein without necessarilyalteringits normalfunction these mutations are rather infrequent since most amino acid substitutions are either neutralordetrimental to the functionorstability of the protein a classic example is sicklecelldisease PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  6. Mutationsassociatedwith heterochronicorectopic gene expression • = mutationsthat alter the regulatoryregions of a gene to causeinappropriate • expression, at anabnormal time or place • Examples: • oncogenemutations in cancer • hereditarypersistence of fetalhemoglobin (continuedexpression of the g-globin gene) PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  7. Genetic Disorders of the Central Nervous System – International Module on Pediatric Rehabilitation G Mortier – Department Medical Genetics – Ghent University Hospital

  8. PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  9. The Hemoglobinopathies • most commonsingle-gene disorders in humans • more than 5% of the world’spopulation is carrier of anabnormalglobin gene PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  10. The Hemoglobins • hemoglobin (Hb) is the oxygen carrier in vertebrate red bloodcells • the Hb A molecule contains 4 subunits: 2 a-chains and 2 b-chains • each subunit is composed of : • - a polypeptidechain (globin) • - a prostheticgroup (heme) which is aniron-containing pigment that • combines withoxygen • globinstructurehighlyconserved PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  11. PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  12. The Hemoglobins • hemoglobin (Hb) is the oxygen carrier in vertebrate red bloodcells • the Hb A molecule contains 4 subunits: 2 a-chains and 2 b-chains • each subunit is composed of : • - a polypeptidechain (globin) • - a prostheticgroup (heme) which is aniron-containing pigment that • combines withoxygen • globinstructurehighlyconserved • Hb A (adulthemoglobin): a2 b2 • and fiveotherhemoglobins PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  13. identical chromosome 16 differonly in 10/146 AA chromosome 11 In adultlife: >97% HbA 2% HbA2 <1% HbF commonancestral gene PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  14. Globinswitching PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  15. 6 kb Chr 11 LCR e Gg Ag yb d b • expression of b-globin gene controlledbynearbypromoter and LCR • locuscontrolregion (LCR): requiredfor the expression of all the genes • in the b-globin cluster • deletions of LCR results in egdb- thalassemia PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  16. Threebroadgroups of mutationsthat affect hemoglobin mutationsthat alter the structure of the globinprotein mutationsthatdecrease the synthesis of oneor more globinchains mutationsthatimpair the globindevelopmentalswitching PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  17. 1. HemoglobinStructuralVariants • 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 – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  18. HemoglobinStructuralVariantsthatcauseHemolyticAnemia • Pathogenesis • the mutant makes the Hbtetramerunstable • loss-of-functionmutations • f.e.: HbHammersmith (b-chain Phe42Ser mutation) • the mutant gives the globinchainanunusualrigidstructure • novelpropertymutations • f.e.: sicklecellglobin; HbC PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  19. SickleCellDisease • sicklecellhemoglobin (HbS) was the firstabnormalHbdetected • HbS: singenucleotidesubstitutionresults in Glu6Val mutation in b-chain • sicklecelldisease is due to homozygosityfor the Glu6Val mutation (AR condition) • sicklecelltraitrefers to the heterozygous state • common in equatorialAfrica; 1/600 AfricanAmericans is bornwith the disease • about 8% of AfricanAmericans are heterozygous • heterozygotes are protectedagainst malaria PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  20. SickleCellDisease • HbS has a normalability to bind oxygen in normalcircumstances • in deoxygenatedbloodHbS is lesssoluble as normalHb • in conditions of low oxygentension, the HbS molecules aggregatewhichdistort • the RBC to a sickleshape. These misshapenerythrocytes are lessdeformablethan • normal and cannotsqueeze in single file throughcapillaries, therebyblockingblood • flow and causinglocalischemia PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  21. 1. HemoglobinStructuralVariants • 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 – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  22. HemoglobinStructuralVariantsthat alter oxygen transport • these mutations have littleorno effect onhemoglobinstability • mutations in the region of the heme pocket will affect the heme-globin bond • in a waythatmakes the ironresistant to the enzymemethemoglobinreductase. • The result is accumulation of methemoglobin in the bloodwithcyanosis as a • consequence. • f.e. HbHyde Park (b-chain His92Tyr mutation) • heterozygotes are cyanoticbutusuallyasymptomatic. The homozygous state • is presumably lethal. PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  23. 2. HemoglobinSynthesis Disorders (The Thalassemias) • collectively the most commonhumansingle-gene disorders! • carriers have anprotectiveadvantageagainst malaria • > qalassa(sea): firstdiscovered in persons of Mediterraneanorigin • imbalance in the normal ratio of a : b chains • - reduction in synthesis of globinchains • - instability of globinchains • excess of normalchainswilldamage the RBCs (hemolyticanemia) • defect in Hbsynthesiswillcausehypochromic, microcyticanemia PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  24. The a - Thalassemias • decreased to absent a-globinproduction • affect the formation of bothfetal and adultHb • in the absence of a-globins: • - HbBart’s: g4 • - Hb H: b4 • hydropsfetalis in severeforms(due to severehypoxia) • anemia in mild forms(precipitation of Hbinclusions in RBCsdamage in spleen destruction) HomotetramericHbthat are ineffectiveoxygen carriers PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  25. The a - Thalassemias • most commonlydue to deletion of the a-globingenes Misalignmentwith PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  26. 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 – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  27. The a - Thalassemias • most commonlydue to deletion of the a-globingenes • otherformsrather rare: • - formdue to the ZF deletion(namedafterindividual ZF) • - the ATR-X syndrome PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  28. a – Thalassemiadue to the 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 – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  29. The ATR-X syndrome • association of mental retardation and a-thalassemia • due to mutations in the X-linked ATRX gene • ATRX codes for a chromatin remodelingprotein • the ATRX proteinfunctions in trans to activate the expression of • the a-globingenes and othergenes important fornormalbrainfunction • partialloss-of-functionmutationsresult in modest reduction of a-globinsynthesis • somatic (more severe) mutations in ATRX cause the • a-thalassemiamyelodysplasiasyndrome(ifgermline: hydropsfetalis!) PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  30. The ATR-X syndrome • X-linkedform of syndromic mental retardation • prevalence: 1-9/1.000.000 ? • 95% severe to profound mental retardation • usuallyno speech • 30% seizures; 75% microcephaly (postnatalonset) • spasticparaparesismaybe present • facialdysmorphism • 80% genitalabnormalities (undescended testes to ambiguousgenitalia) • 65% short stature; 20% heart defects • Hb H inclusions in 90% present, usually mild anemia • (Hb H inclusionsonlyappearafter 30%-40% reduction in a-globinsynthesis) PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  31. The ATR-X syndrome Erythrocytesafterincubation in briljant cresyl blue. Hb H inclusions in threecellsgivethem a golf balllikeappearance From Gibbons R. Orphanet Journal of Rare Diseases 2006;1:15 PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  32. The b - Thalassemias • decreasedb-globinproduction • onsetnotapparentuntil a few monthsafterbirth • samepathophysiology as in a-thalassemias: precipitation of the excessa-chains • results in hypochromic, microcyticanemia • increasedlevels of HbA2 (a2d2) and HbF(a2g2) • usuallydue to single-base pair substitutions (ratherthandeletions) • personswithtwoabnormalalleles are frequentlycompoundheterozygous • twob-thalassemiaalleles: usuallythalassemia major (severeanemia) • oneb-thalassemiaallele: thalassemia minor (mild anemia, noclinic) • simple versus complex b-thalassemia PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  33. of the 3’ end of the gene >> PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  34. Point mutationsthatcauseb-thalassemia are distributedthroughout the gene. They affect virtuallyeveryprocessrequiredfor the production of normalb-globin. PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  35. Posttranscriptionalmodifications of mRNA PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  36. RNA splicingmutations in b– Thalassemias (1) PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  37. RNA splicingmutations in b– Thalassemias (2) PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  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 PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  39. Complex b – Thalassemias (egdb)° thalassemia (illustratesimportance of LCR) (db)° thalassemia (Agdb)° thalassemia Hereditarypersistence of fetalhemoglobin PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  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 PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

  41. Fig 12.1 PostgraduatecourseHumanGenetics – 06/12/08 Geert Mortier, MD, PhD– Center forMedicalGenetics – GhentUniversityHospital

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