Major forms of hemoglobin and its derivatives/hemoglobinopathies

Major forms of hemoglobin and its derivatives/hemoglobinopathies

1. Major forms of hemoglobin and its
derivatives/Hemoglobinopathies
Dr. Roshan Kumar Mahat
(PhD Medical Biochemistry)
Assistant Professor
Department of Biochemistry

2. Forms of Hemoglobin
Hemoglobinconsistsoftwopairsof
globinpolypeptidechainsassociated
withfourmoleculesofheme.
Structuraldiversityofpolypeptidechains:
Eachtypeofhemoglobinisatetrameroftwo
pairsofpolypeptidechains.
Thereare6differenttypesofpolypeptide
chains:α(alpha),β(beta),γ(gamma),δ
(delta),ε(epsilon)andζ(zeta).
ThevariousformsofHbdifferintheirprimary
structureofglobinchains.Thisiscalled
structuraldiversityofhemoglobin.

3. Different forms of hemoglobin include:
1.Adult hemoglobin
2.Fetal hemoglobin
3.Embryonic hemoglobin

4. Adult Hemoglobin
Adult hemoglobins include:
1.Hemoglobin A
2.Hemoglobin A
2
3.Glycated hemoglobin (HbA1C)
1.Hemoglobin A:
Itisthemajorhemoglobininadults(90-
95%),composedoffourpolypeptidechains
(twoαchainsandtwoβchains),held
togetherbynoncovalentinteractions.
TheaffinityofHbAforoxygenislowerthan
thatoffetalhemoglobinbecauseofhigh
affinityofadulthemoglobinfor2,3-BPG.

5. 2.Hemoglobin A
2:
It is composed of two α-globinchains and
two δ-globinchains (α
2δ
2).
Is a minor component of adult hemoglobin,
first appearing shortly before birth, and
ultimately constituting about 2% of total
hemoglobin.
3. Glycated hemoglobin (HbA1C):
The best index of long term control of blood
glucose level is measurement of glycated
hemoglobin or glycohemoglobin.

6. Enzymaticadditionofanysugartoproteinis
calledglycosylationwhilenonenzymatic
processistermedglycation.
Themostabundantformofglycated
hemoglobinisHbA1C.Ithasglucose
residuesattachedpredominantlytothe
aminogroupsofN-terminalvalinesoftheβ-
globinchains.
itaccountsfor4-6%oftotalHbinblood.
TheconcentrationofglycatedHbis
increasedindiabetesmellitusandits
estimationisusefulformonitoringlongterm
controlofhyperglycemiaindiabetesmellitus
patients.

7. Sincethehalflifeofanerythrocyteis
typically60days,thelevelofglycated
hemoglobinreflectsthemeanbloodglucose
concentrationoverthepreceding6-8weeks
Figure: Nonenzymicaddition
of glucose to hemoglobin.

8. Fetal Hemoglobin (HbF):
HbFisatetramerconsistingoftwoα
chainsidenticaltothosefoundinHbA,
plustwoγchains(α
2γ
2).
HbFisthemajorhemoglobinfoundinthe
fetusandnewborn,accountingforabout
60%ofthetotalhemoglobininthe
erythrocytesduringthelastmonthsof
fetallife.
HbFrepresentslessthan1%oftheHbin
mostadults,andisconcentratedin
RBCsknownasF-cells.

9. Embryonic Hemoglobin
They are produced from 3
rd
week to 8
th
week of gestation.
The hemoglobins that occur normally
during the embryonic period are:
1.Gower 1 (ζ
2ε
2)-minor form
2.Gower 2 (α
2ε
2)-major form
3.Portland 1 (ζ
2γ
2)
Note:However,portland-2(ζ
2β
2),another
embryonicHb,isfoundinfrequentlyandmailny
inanextremetypeofα-thaassemiawhereζ-
chainsubstituteforα-chainswhensynthesisof
laterisseverelyimpaired.

10. Hemoglobin Composition Percentage of totalHb
HbA α
2β
2 90-95%
HbA
2 α
2δ
2 1-2%
HbF α
2γ
2 1-2%
HbA
1C Glycosylated α
2β
2 4-6%
Different forms of hemoglobin present in adults:

11. Pathophysiology
Figure: The globingene clusters
with the α-genes on chromosome 16
and the β-genes on chromosome 11.

12. Figure: The switching of globinchain synthesis during
development.

13. HEMOGLOBIN DERIVATIVES
Thesearecompoundsthatareformedby
thecombinationofgasesorchemicals
withhemoglobin
NormalDerivatives
1.Oxyhemoglobin
2.Deoxyhemoglobin
3.Carbaminohemoglo
bin
AbnormalDerivatives
1.Methemoglobin
2.Carboxyhemoglobin
3.Sulfhemoglobin
4.Acid hematin

14. Detection of hemoglobinderivative:
1)Spectroscopicexamination:when
coloredsolutionsofHemoglobin
derivativesareviewedthrougha
spectroscope,darklinesorbands
areseenindifferentspectrumoflight
duetoabsorptionoflightbythe
derivedhemoglobinmolecule.
2)Microscopicexamination:crystalsof
acidhematin

15. Figure: Spectroscopic identification of hemoglobin
derivatives

16. 1. OXYHEMOGLOBIN
Formation:
Oxygenation: Oxyhemoglobin is formed
by the combination of oxygen with
hemoglobin.

17. Detection:
Appearanace:
cherry red coloured.
Spectroscopy:
Oxyhemoglobinshows2bandswhen
examinedusingspectroscope(between
DandElines).
Firstbandisbroad,seeningreenregion
(541nm)andsecondbandisnarrow,
seenintheyellowregion(577nm).
Dilutedsolution:twobandsseen
Concentratedsolution:singlediffuse
bandisseen.

18. 2. DEOXYHEMOGLOBIN
Formation:
Reduction:Deoxyhemoglobinisformed
bythereductionofoxyhemoglobinwith
concomitantreleaseofoxygen.

19. Detection:
Appearanace:
purple coloured.
Spectroscopy:
Deoxygenatedhemoglobinshowsonly
oneband(542nm)inthegreenregion
(betweenDandElines)whenexamined
usingspectroscope.
Oxyhemoglobin isreduced to
deoxyhemoglobinbytheadditionof
reducingagentssuchassodium
dithionate.

20. Significance:
Oxyhemoglobinanddeoxyhemoglobin
playimportantrolesinthetransportof
oxygenandcarbondioxide.

21. 3. CARBAMINOHEMOGLOBIN
Formation:
Carbaminohemoglobinisformedbythe
combinationofcarbondioxiodewith
hemoglobin.
Carbondioxidecombineswithterminal
aminogroupofhemoglobin.
Significance:
Transportofcarbondioxide:itisresponsible
fortransportofsmallamountofcarbon
dioxideintheblood.Itaccountsforabout2-
10%oftotalcarbondioxidetransportinthe
blood.

22. 4. METHEMOGLOBIN
Formation:
Oxidationofhemoglobin:methemoglobin
isahemoglobinderivativeinwhichironis
presentinferricform(oxidizedform)instead
offerrousform.
Henceitisalsocalledoxidizedhemoglobin.

23. Formation of methemoglobin occurs in
four ways:
1.Methemoglobinformationdueto
inheriteddefect:characterizedmy
mutationinglobinchain.Mutation
resultsinthepreventionofreductionof
methemoglobin.Eg.HemoglobinM.
2.Methemoglobinaccumulationdueto
inheriteddeficiencyofmethemoglobin
reductase (NADH-methemoglobin
reductase).

24. 3.DecreasedavailabilityofNADPHdueto
inheriteddeficiencyofglucose-6-
phosphatedehydrogenase.NADPHisa
cofactorforNADPHdiphoraseII.Thisis
minorenzymeforreducingmethHb.
4.Combinationwithoxidisingchemicalsor
drugs:nitrates,aniline,acetaminophen,
benzene,phenacetin,amylnitriteand
sodiumnitroprusside,etc

25. There are three types of methemoglobin
reductase activities:
i.NADH dependent (75%)
ii.NADPH dependent (20%)
iii.Glutathione dependent (5%)
Figure: Methemoglobin Reductase System

26. Significance:
Harmful effects: Cyanosis
Normalconcentrationofmethemoglobin
islessthan1%oftotalhemoglobin.
(increasedconc.iscalledmethemoglobinemia)
Methemoglobin has very low capacity to bind
oxygen because of presence of iron in oxidized
state.
Therefore, accumulation of methemoglobin results
in impaired oxygenation of hemoglobin.
Tissue hypoxia
Cyanosis

27. Detection:
Appearanace:
dark brown compound.
Spectroscopy:
Methemoglobinshows2bandswhen
examinedusingspectroscope.
Firstbandisseeningreenregion
(542nm)andsecondbandisseeninthe
redregion(633nm).

28. 5. CARBOXYHEMOGLOBIN
Formation:
Reactionwithcarbonmonoxide:itis
formedbythecombinationofcarbon
monoxidewithhemoglobin.
Important sources of carbon monoxide
include:
a)Smokingofcigarettesorcigar:heavy
smokersmayhavemorethan20%oftheir
hemoglobinascarboxyhemoglobin.
b)Exposuretocoalmining:workersworking
incoalminefrequentlyhavehigh
carboxyhemoglobin.

29. c.Environmental pollution: by automobile
exhausts and industrial accidents.
Significance:
Harmful effects: Cyanosis
Affinity of CO to Hbis 200 times more thanO
2
Bindingof CO to one monomer of Hb(not
reversible) increases the affinity of other
monomers tooxygen
O
2 bound to other monomers are not released
Tissuehypoxia
Cyanosis

30. Clinicalfeatures:
Clinicalfeaturesdependsonamountof
carbonmonoxidesaturation,
Lessthan0.1% Normal
5-10% Asymptomatic
>30% toxictoxicsymptoms-cyanosis,
breathlessness,weaknes,irritability,
dizziness,headache,nausea,vomiting,
paininchest&abdomen.
>50% lifethreatening-comaanddeath

31. Treatment:
Theconditioncanbetreatedby
administrationofoxygenandblood
transfusion.
1.Oxygen:whentheconcentrationof
carboxyhemoglobinisminimal,no
treatmentisrequiredsincecarbondioxide
isgraduallyreplacedbyoxygen.
Administrationofoxygenhastensthe
recovery.
2.Bloodtransfusion:isrequiredwhenthe
levelsofcarboxyhemoglobinareveryhigh.

32. Detection:
Appearance:
cherryredincolour.
Colourresemblesthatofnormal
hemoglobin,henceitisdifficultto
distinguishcarboxyhemoglobinfrom
oxyhemoglobinbytheappearance.
(spectroscopic examination helps in differentiation)
Spectroscopy:
itshows2bands:oneingreenregion
(535nm)andoneinyellowregion
(570nm)underspectroscope.

33. 6. SULFHEMOGLOBIN
Formation:
Reactionwithsulphurcontaining
compounds:sulfhemoglobinisformedby
thecombinationofsulphurcontaining
compoundswithhemoglobin.
Formationofsulfhemoglobiniscausedby
ingestionofdrugssuchassulfonamides,
phenacetin,acetanilideanddapsone.
Significance:
Harmful effects: Cyanosis
Also impairs oxygenation of hemoglobin.
Excessive accumulation results in cyanosis.

34. 7. ACID HEMATIN
Formation:
Hydrolysisofhemoglobinandoxidation
ofheme:Acidhematin(ferrihemechloride)
isformedbyboilingofhemoglobininthe
presenceofsodiumchlorideanddilute
glacialaceticacid.
Undertheseconditions,hemoglobinis
hydrolyzedtohemeandglobin.Heme
interactswithsodiumchlorideandoxygento
formferrihemechloride.

35. Significance:
Detection of blood staining:
Sincehematincanbeformedfromtraces
ofblood,testingforthepresenceof
hematinisusedintheforensicmedicine
forthedetectionofbloodstains.

36. Detection:
Microscopicappearance:
Bloodorelutedbloodstainsheatedwith
Nippe’sfluid(1%KCL,KBr&KIin
glacialaceticacid)overaglassslide.
Darkbrownrhomboidshapedcrystals
observedundermicroscope.
verysensitivebutansweredbyheme
partofbloodofallspecies.
Spectroscopy:
itshowsanarrowbandintheredregion
(650nm) when examined using
spectroscope.

37. HEMOGLOBINOPATHIES
Hemoglobinopathieshavetraditionally
beendefinedasafamilyofgenetic
disorderscausedbyproductionofa
structurallyabnormalHbmolecule,
synthesisofinsufficientquantitiesof
normalhemoglobin,or,rarely,bothdue
tomutation.
Morethan400differentmutationshave
beendescribed,whichresultsinasmany
structuralvariantsofthehemoglobin
polypeptidechains.

38. Type of defects
1)Qualitativedefects:
Leadtoproductionofstructurallyabnormal
hemoglobinmolecules.
Thesedefectsinvolvestructuralalterationof
thepolypeptidechainoftheHb,leadingto
functionalimpairement.
Anumberofqualitativedefects(over300)
havebeenidentified,whichmayaffecteither
ofthetwotypesofchains.
Example:Sicklecellanemia,theβ-chainis
affected,whereasHbMBoston,theα-chain
undergoesstructuralalteration.

39. 2)Quantitativedefects:
Resultinsynthesisofthenormalhemoglobin
molecules,butininsufficientquantities.
Aprimeexampleofthistypeisthe
thalassaemias,inwhicheithertheα-orβ-
chainareunderproduced.
Note:
Byconvention,thehemoglobinopathies
areusuallynamedwithacapitalletter
(e.g.HemoglobinS),ageographic
location(e.g.hemoglobinSeattle),or
both(e.g.hemoglobinMBostonorD
Punjab).

40. 3.Others:include
Methemoglobinemiaand
Hereditarypersistance offetal
hemoglobin(HPFH),resultfromdefects
inregulationofglobinchainsynthesis,
γ-βchainswitch

41. Mutated HbAffected
chain
Residue Substitution Notes
S β 6 GluVal Decreased
solubilityof Hb.
Sickingof
RBCs.
C β 6 GluLys Decreased
solubility,
Sicking
E β 26 GluLys
Zurich β 63 His Arg Affinityof Hbfor
O2 increased.
Solubility
decreased .
Seattle β 70 Ala Asp
Some examples of qualitativedefects

42. Mutated HbAffected
chain
Residue Substitution Notes
Hiroshima β 146 His AspHigh O2 affinity.
Salt bridges
stabilizing T
form impossible
to make.
Kansas β 102 AsnThr Low oxygen
affinity, cynosis
common.
D Punjab β 121 GluGln Migrates similar
to HbSon
electrophoresis.
Severe
condition
MBoston α 58 His TryCyanosis
common.

43. Sickle Cell Disease
1.Sicklecellanemia[HbSS]
2.Sicklecelltrait[HbAS]
3.HbCtrait[HbAC]
4.HbSCdisease[HbSC]-compoundheterozygote
5.Sickleβ
0
thalassemia(HbSβ
0
)
6.Sickleβ
+
thalassemia(HbSβ
+
)

44. Sickle cell anemia
Mostcommonlyoccurringhaemoglobinopathy.
Causedbyinheritedstructuralabnormalityinthe
β-globinpolypeptide.
Originallydescribedin1910byJamesHerrick,
thediseasewasthefirstinheriteddisorder
showntoarisefromaspecificaminoacid
changeinaprotein.
ThediseaseiscommoninAfrica,India,Middle
EastandSouthernEurope.

45. Genetics:
Autosomalrecessiveblooddisease.
HemoglobinSiscausedbysubstitution
ofavalineforglutamicacidresidueas
the6
th
aminoacidintheβ-globinchain.
Thissubstitutionresultsfromapoint
mutationinDNAduetosubstitutionof
thyminebyadenine.

46. ValHisLeuthrProValGlu
ValHisLeuThrProGluGlu
GAG
GUG
Point mutation
in DNAdue to
substitution of A
forT
Normal RBC
Sickle shaped RBC
βchain HbA( normalhemoglobin)
β chain HbS( Abnormalhemoglobin)
1 2 3 4 5 6 7
1 2 3 4 5 6 7

47. Pathophysiology:
Replacement of charged glutamate with the non-polar
(hydrophobic) valine
Protrusion (hydrophobic sticky patch) on the surface of β-
subunit of both oxy and deoxy HbS
Sticky patch fits into a complementary site on the β-chain of
another hemoglobin molecule in the cell [Note: Both HbAand
HbScontain a complementary site on their surfaces that is
exposed only in the deoxygenated T state]

48. At low oxygen tension, deoxy HbSpolymerizes inside the
RBCs.
A gelatinous network of fibrous polymers called tactoids
Tactoidsbecome stiff and distort the cell, producing rigid
erythrocytes (Sickled RBC)
Sickled cells frequently block the flow of blood in the narrow
capillaries

49. Interruption in the supply of oxygen
Localized anoxia in the tissues
Causing pain and eventually death of the cells in the vicinity of
the blockage

50. Complementarysite
to stickypatch
Stickypatch
HbShas decreased solubility in deoxygenatedstate.
OxyHb
DeoxyHbSOxyHbS
DeoxyHb

51. Polymerization of deoxyHbS
Oxy HbA deoxy HbA Oxy HbS deoxy HbS
Sickling occursunder deoxygenatedstate

52. Pathophysiology Pathophysiology of sickle
cell disease
Source: Bykersma, 2018

53. Conditions associated with increased
sickling phenomenon:
Sickling phenomenon is precipitated by
i.Infections
ii.Hypoxia
iii.Dehydration
iv.Acidosis
v.Cold
vi.Increased concentration of 2,3-BPG
vii.Increased pCO
2

54. Sickle cell anemia Sickle cell trait
Individual has received 2
mutant genes, one from each
parent
Individual hasreceived1 mutant
gene from one parent and
normal gene from otherparent
Homozygous (HbSS) Heterozyous(HbAS)
Clinicalsymptoms •No clinical symptoms
•At high altitudes –symptoms
Sicklecellanemia:resultsfromhomozygous
inheritanceofhemoglobinSgene(HbSS).
Sicklecelltrait:referstoheterozygous
inheritanceofhemoglobinSgene(HbAS).

55. Clinical features:
Clinical features result from either hemolytic
anemia or vaso-occulisive crisis.
Chronic hemolytic anemia:
Features of chronic hemolytic anemia are
i.pallor of mucous membranes,
ii.fatigue,
iii.dyspnea,
iv.jaundice may also occur.
v.The patients are also prone for gall stone
formation.

56. vaso-occulisive crisis:
Clinical features resulting from vaso-
occulivecrisis are
i.Leg ulcers
ii.Priapism
iii.Repeated episodes of pulmonary
infarction
iv.Pulmonary hypertension
v.Hematuria
vi.Stroke
vii.Aseptic necrosis of bone

57. Diagnosis
1. Hematological findings:
Hemoglobinlevelsaredecreased(6-8gm/dl)
Microcytichypochromicanemia
Reticulocytecount-increased
2. Sickling test:
Sicklingofredcellscanbeseenunder
microscope.Itisbetterseenbyinductionof
sicklingusingreducingagentssuchas
sodiumdithionite,sodiummetabisulfite.

59. 3. Sickle cell solubility test:
Turbidappearanceofhemoglobinsolutionin
thepresenceofreducingagentssuchas
sodiumdithionatesuggeststhepresenceof
sicklecellhemoglobin.
Turbidityisduetotheprecipitationof
hemoglobininthepresenceofreducing
agent.

60. 4. Hemoglobin electrophoresis:
Theperformanceofelectrophoresisusing
citratebufferisalsodonetodifferentiateit
fromotherabnormalhemoglobinssuchas
HbE.
DecreasedorabsenceHbA:
HbAwillbeabsentinsicklecelldiseaseand
decreasedlevelsareseeninsicklecelltrait.
IncreasedHbF:
HbFlevelsareincreasedinorderto
compensatefordecreasedorabsentHbS.

61. Hbelectrophoresis:
-+

62. Sickle cells and malaria
Malariaiscausedbytheparasite
plasmodiumfalciparum,whichisthemajor
causeofdeathintropicalareas(black
Africans).
Sicklecelldiseasehasbeenfoundto
provideprotectionagainstmalaria,which
canbeexplainedbyfollowingfacts:
Increasedlysisofsickledcells-shorterRBC
lifespan.
ParasitescauseacidityofRBC–increased
sickling
K
+conc.islowinsickledcell–parasitecannot
survive

63. Management:
1.Initialmanagementaimsatavoidinghypoxia
anddehydrationandadministrationof
cyanate,whichincreasestheoxygenaffinity
bycovalentmodificationoftheaminotermini
oftheglobinpolypeptides.
2.Antisicklingdrug:hydroxyureaisgiven,which
increasesthesynthesisofHbF.ExcessHbF
interfereswithsickling.

64. 3.5-Azacytidine:increases the production of
HbF(not used due to toxicity).
4.Sodium butyrate: increases the production
of HbF.
5.Repeated blood transfusion: in severe
anemia.
6.Hydration, analgesics, antibiotics: acute
painful crisis.
Emergingtreatments:
Stem cell transplantation,
Inducing HbFexpression
Gene therapy (in future)

65. Thalassemias
Heterogenousgroupofhereditaryanemias
thatconstitutethemostcommongene
disorderintheworldwithacarrierrateof
almost7%.
Thediseasewasfirstdiscoveredinthe
countriesaroundthemediterraneansea
andwasnamedforthegreekword
thalassa,meaningsea.However,itisalso
presentinareasextendingintoIndiaand
Chinathatareneartheequator.

66. Inheritedautosomalrecessiveblood
disorder.
Insufficientproductionofoneofthe
globinchainsofHbresultsinthelackof
coordinationbetweenthesynthesisof
theαandβ-chains.
Normallythesubunitsynthesisis
coordinatedinsuchawaythateach
newlysynthesizedβ-chainreadilypairs
withα-chain;andconversely,eachnewly
synthesizedα-chainpairswithβ-chain.

67. Thelackofcoordinationresultsin:
a)Formationofinsolubleaggregatesbythe
polypeptidechains.Theaggregatesare
damagingtothecellandreduceitslife
span.
b)Impairmentofhemoglobinsynthesis,so
thattheerythrocytesaresmallandpoorly
filledwithhemoglobin.

68. Thethalassemiasyndromearecaused
bymutationsthatdecreaseorabolishthe
synthesisoftheαorβ-chainsintheadult
hemoglobinAtetramer.
Individualsyndromesarenamed
accordingtothechainwhosesynthesis
isaffectedandtheseverityofthe
deficiency.

69. Types of Thalassemias:
Dependingonwhichchainisaffected,
thethalassemiasaredividedintotwo
majorclasses:
Thalassemias
α-thalassemia β-thalassemia

70. α-thalassemias
Are disorders of hemoglobin synthesis
caused by defective synthesis of α-globin
chain.
Genes for α-chains are clustered on
chromosome 16.
Genome of an individual contains four
copies (2 copies from each parent) of the
globingenes, two copies are located on
each of the chromosome 16.

71. Basedonthenumberofα-globingenes
absent,α-thalassemiaisclassifiedinto4
groups:
1)Silent carrier state
2)α-thalassemia trait
3)Hemoglobin H disease [α-thalassemia
minor]
4)HbBart’s disease [α-thalassemia
major]

72. Fig: Genetic basis of α-thalassemia
Source: Pielet al., 2014

74. Typesofhemoglobinsinthalassemic
patients:
Thoughsynthesisofα-chainishampered
inα-thalassemia,theβ-chainandγ-chain
proceedsasusual.Asaresultthere
occursaccumulationofcertainproteins
andare:
1.HbH(β
4tetramer)
2.HbBart’s(γ
4tetramer)

75. 1.HbH:
Itisthepredominanthemoglobininpatients
withdeletionofthreeαgenes.
Thisaberranthemoglobinisunstable;it
graduallydenaturestoforminclusion
bodiesinthecells.
2.HbBart’s:
havetenfoldhigheroxygenaffinitythan
hemoglobinAandthereforecannotdeliver
thebondoxygentotissues.
Thus,tissuehypoxiaisthemajor
consequenceandoxygendeficiency
symptomsrapidlyappear.

76. β-Thalassemias:
β-thalassemiasaredisordersofdefective
synthesisofβ-globinchainofhemoglobin.
However,α-globinchainsynthesisis
normal.Excessα-globinchainscannot
formstabletetramersandsoprecipitate,
causingtheprematuredeathofcells
initiallydestinedtobecomematureRBC.
IncreaseinhaemoglobinA
2[α
2δ
2]andF
[α2γ2]alsooccurs.

77. Causes:
β-thalassemiasarecausedbymutations
ofβ-globinchainthatresultsin:
i.Defectivetranscription,
ii.Defectiveposttranscriptionalprocessingof
mRNA(defectivesplicing)and
iii.Defectivetranslationcausedbyframeshift
mutationsornonsensemutations.

78. 1.Defectivetranscription:occursdueto
mutationinthepromoterorenhancer
region.
2.DefectiveRNAprocessing:occursdue
todefectivesplicingcausedbymutation
intheintron-exonregion.
3.Defectivetranslation:occursdueto
baseexchangemutationresultingin
formationofnon-sensecodonswith
prematureterminationofpolypeptide
chainsynthesis.Frameshiftmutations
causedbydeletionorinsertionofbases
willalsoresultindefectivetranslation.

79. Coding
region
Figure: Mutations causing defective synthesis of
β-globinchain in β-thalassemia
translation

80. Genetics:
Normallytherearetwogenes
responsiblefortheformationofβ-globin
chains,onefromeachparent.
Eachcopyofchromosome11hasone
geneforβ-globinchain.
Themutationinβ-thalassemiamay
involveasinglechromosome(i.e.the
heterozygousstate,calledβ-thalassemia
minor)oritisahomozygousstate,
involvingboththechromosomes(called
β-thalassemiamajor).

81. Figure: β-Globingene mutations in the β-thalassemias

82. Source: Herbert L et al., 2009

83. β-Thalassemia minor
Itisaheterozygousstate.
Affectedindividualsharboronenormalβ-
globingeneandonethatcarriesa
mutationleadingtoproductionof
reducedornoβ-globin.
Thalassemiaminorpatientsaregenerally
asymptomaticandlifeexpectancyis
alsonormalsincesomeamountofβ-
chainsisnormallysynthesizedbythese
individuals.

85. β-Thalassemia major
AlsocalledCooley’sanemia.
Characterizedbydefectinbothβ-globin
genes.
Isthehomozygousstateandmost
severeamongallcongenitalhemolytic
anemia.
Personswithbetathalassemiamajorare
almostneversymptomaticatbirth
becauseofthepresenceofHbF,but
symptomsbegintodevelopbysix
monthsofage.

87. Chipmunk facies
Bossing due to
extramedullary
hematopoesis

88. Diagnosis:
1.Hematological findings:
a)Decreased hemoglobin (usually less than
7mg/dl)
b)Reticulocytosis
c)Hypochromicmicrocyticanemia
d)Fragmentation of erythrocytes
e)Presence of inclusion bodies
2.Serum iron and ferritin: increased
3.Hbelectrophoresis: shows absence of
HbA(α
2β
2), increased HbA
2(α
2δ
2), and
increased HbF(α
2γ
2).

89. 4.X-ray: shows characteristic hair on end
(crew cut appearance) appearance in
the skull.
5.Prenataldiagnosis: can be made using
amniotic fluid or chorionic villous
sampling.

90. Treatment:
a)Blood transfusion
b)Folic acid supplementation
c)Iron chelation therapy
d)Hematopoietic stem cell transplantation
Note:Althoughregulartransfusionofbloodislife
saving,thecumulativeeffectoftransfusionsis
ironoverload(asyndrome called
hemosiderosis).Forthisreason,patientis
treatednotonlywithbloodtransfusionbutalso
withdesferrioxamine.

91. Desferrioxamine:
anironchelatorthatisbestadministered
throughasubcutaneousinfusionpump.
Itformsasolubleironcomplexthatcanbe
excretedbythekidneys.

92. Methemoglobinemia
Definition:
Methemoglobinemiaisablooddisorder
inwhichanabnormalamountof
methemoglobinisproduced.
Methemoglobins arehemoglobins
containingironatomsasferricions
insteadofferrousions.

93. Types:
1)Hereditary methemoglobinemia
2)Acquired methemoglobinemia
Hereditary methemoglobinemia:
Caused by deficiency of methemoglobin
reductase and M hemoglobins.
Deficiency of methemoglobin reductase is
caused by mutation of the gene coding this
enzyme.
M hemoglobins are caused by mutations in
the globingene. Example for M hemoglobin
is hemoglobin Boston.

94. HbBostoniscausedbysubstitutionof
tyrosineforhistidineinα-chain.Themutation
nearthehemepocketresultsinformationof
stablemethemoglobinwithironinferricstate
insteadofferrousstate.
Acquired methemoglobinemia:
Iscausedbyexposureofindividualsto
compoundssuchasnitrites,nitrates,
sulfonamides,anilinedyes,phenacetin,
etc.
Acquiredmethemoglobinproductionis
morecommon.

95. Clinical features:
Include cyanosis, headache, dyspnea and
mental retardation.
Cyanosis:
Methemoglobin has very low capacity to bind
oxygen because of presence of iron in oxidized
state.
Therefore, accumulation of methemoglobin results
in impaired oxygenation of hemoglobin.
Tissue hypoxia
Cyanosis
Normal concentration of methemoglobin is less
than 1% of total hemoglobin

96. Headache and dyspnea:
Areseen ifconcentrationof
methemoglobinincreasedbeyond30%
ofhemoglobin.Methemoglobinlevels
morethan50%maybefatal.
Mental retardation and early death:
Mentalretardationandearlydeathis
seeninmethemoglobinreductase
deficiency.

97. Diagnosis:
1.Colourofblood:bloodmayappear
chocolatebrownincolourincongenital
disorders.
2.Methemoglobinreductaseactivity:islowin
congenitaldeficiencybutnormalin
methemoglobinemiacausedbyglobinchain
mutationsortoxicdrugs.
3.Methemoglobinlevels:inbloodare
increased.Itismeasuredbyoptical
absorptionofmethemoglobin.
4.Oxygensaturation:isnormal(normal
arterialpO
2)

98. Treatment:
Methemoglobinemia istreatedby
reducingmethemoglobintohemoglobin
bytheadministrationofmethyleneblue
orascorbicacid.
a)Methyleneblue:isabletogenerateNADPH
linkedmethemoglobinreducingsystem.It
inducesNADPHdiaphoraseII.
b)AscorbicAcid:Methemoglobinreductase
deficiencyismoreefficientlytreatedbythe
administrationofascorbicacid.
Congenitalmethemoglobinemiaistreatedwithascorbic
acidormethyleneblue.
Acquiredmethemoglobinemiaistreatedwithmethylene
blue.

99. Assignments:
1.Define haemoglobinopathy. Describe the
biochemical basis of different types of
thalassemia along with their clinical
presentation.
Short Notes:
1.Biochemical basis of sickle cell anemia
2.Methemoglobinemia
3.List the derivatives of hemoglobin
4.Different forms of hemoglobin.
5.Sickle cell anemia gives protection against
malaria. Give reason.