1. UNIVERSITY OF KENT
BI520 Metabolism and Metabolic
Disease
Case study and review of a genetic mutation on
the LDLR gene
B.S.Aston
Ba260
Word count:1,990

2. 2 Bjork Astonba260
In childrenplasmacholesterolisusuallybelow250mg/dl,butinthis4 yearold patientitisseento
be four timesthis,at1000mg/dl(1). Thiselevatedlevel of cholesterolsuggeststhatthe childhasa
highproportionof LDLs in the bloodstream. LDLsare formedbydepletedVLDLs(IDLsare an
intermediate product) andare the maintransporterof cholesterol totissuesinthe body.
The initial treatmentinvolvedalow-fatandlow cholesterol dietalongsidemedication(atorvastatin
and ezetimibe)inanattemptto lowerthe bloodcholesterol butasshownbythe laterbloodtest
results(500-600mg/dl)thismethoddid verylittle tothe levels of cholesterolinthe boy’sblood.This
isstrong evidence thatthere isageneticmutationcausingthe livertobe defective inlipid
metabolism.Notablyboththe motherandfatherexhibitraisedcholesterollevels.
The cholesterol depositsinthe eyesof the boy’sfather(arcusseniliscornea),althoughtheycanbe
confusedwiththe limbussignwhichreflectscalciumin the bloodnotcholesterol,suggestthathe
suffersfroma lipidmetabolismdisorderalso(2).Considering the conditionof bothhisparentsand
the patientsyoungage itwouldbe fairto say that hissubstantiallyraisedcholesterol isdue toan
autosomal dominantgeneticdisorderin whichhe ishomozygousfor,bothhisparents experiencea
lateronsetand mildersymptoms due toheterozygosity(3).
The elevatedlevelsof triglyceridesinthe blood(170mg/dl) canbe an indicatorof diabetesmellitus,
alcohol excessandchronicrenal failure butconsideringthatthe patientisstill ayoungchildthe
lattertwocan be ruledout. The diabetescanbe disregardeddue tothe livertransplantbeinga
successful treatmentforthe boy;he wouldcontinue tobe diabeticevenafterthe livertransplant
withoutanexplanationforhiscutaneousxanthomas. The presence of tendonxanthomas(onthe
Achillestendons)isthe mostrevealingsymptomasitispathogonomicforfamilial
hypercholesterolemiapatients,otherwiseknownashyperlipidemia(4).
The main geneticmutationthatwouldexplainthe patient’ssymptomsishomozygous dominant
familial hypercholesterolemia(HFH).Thisgeneticdisease affectsthe LDLRgene foundon
chromosome-19.MutantLDLRs or reducednumbersof functional LDLRscause highcholesterol
levelsdue tothe lackof re-absorptionof LDLsintothe liver(5).
LDLRs attach tothe ApoB-100 proteinfoundinLDLs. Thisrecognitionallows particlessuchasVLDLs
and IDLS to entercellsandtissuestodeliverthe triglyceridesorcholesterol theyare carrying.They
thenbecome remnantsandare transportedback to the liverinthe bloodstream.If the patient’s
LDLRs are dysfunctional thenLDLscannotbe absorbedintoadipose tissue orthe liverforeither
reuse or excretioninthe bile.Thismeansthatthe LDLs continue tocirculate inthe bloodstream
carryingcholesterol (4).
A highproportionof LDLs inthe bloodstreamcanresultintheircollection withinthe tunicaintimaof
bloodvessels.Here the LDLsare oxidisedand bindwith monocytestoforma foamcells.These cells
cause the bloodvesselstonarrowand increase the riskof a rupture/complete closureof ablood
vessel.If thisoccursto the coronaryarteriesthenitwill resultinaheart attack.
There are several drugsthatare usedtotreat highcholesterol including Atorvastatin.Itisprescribed
because itisa statinusedtolowerthe concentrationof LDLs and triglycerides inthe blood but
increase proportionof HDL’s (6).It worksby inhibitingthe enzymeHMG-CoA reductase.
Thisreduces the amountof cholesterolinextrahepatictissuesbyincreasingthe transportationof it
back to the liverwhile attachedtoHDLs.If there are fewerLDLsthenthere will be lesscholesterol
beingtransportedtoextrahepatictissuesalso,hencereducingthe bloodcholesterollevels.

3. 3 Bjork Astonba260
CholesterolsynthesisistoanextentinhibitedbyLDLs.LDLs inhibitthe synthesisof HMG-CoA
Reductase,an enzyme usedtoformmevalonate bybindingtoLDLRs.In HFH patientsthisbindingis
lackingdue to a mutationinthe gene thatcodesfor the LDLRs (7).Thismeansthat highlevelsof LDL
do notact as feedbacktopreventcholesterol synthesis;hence cholesterol isoverproducedaswell
as beingunable toenterthe liverwhile attachedtocholesterol.
The livercan formcholesterol viaaseriesof reactionsbeginningwiththe deliveryof glucose from
the small intestine.Viaglycolysisglucosecanbe convertedintopyruvate hence producingAcetyl
CoA.Cholesterol isformedfromAcetyl CoA infourdistinctstages;the firststage involves3Acetate
unitscondensingtoformHMG-CoA.ThisreactioniscatalysedbyHMG-CoA synthase.The second
stepinvolvedthe reductionof HMG-CoA usingthe enzyme HMG-CoA reductase toformmevalonate.
Thisstepis importantinregulatinghowmuchcholesterol ismade.Atorvastatinworksbyinhibiting
thisconversiontomevalonate asthe HMG-CoA cannotbe reducedwithoutthe enzyme.Preventing
the conversionof ketogroupstoalcohol groupsmeansthat mevalonate cannotthengoonto form
isoprene units.
Ezetimibe isusedtocontrol the exogenouslipidcycle by reducingplasmacholesterol levelsand
hence resultsin lesscholesterolinthe blood.Itworksbyreducingabsorptionof cholesterol bythe
small intestine(8).Cholesterol absorbedbythe small intestinealongside fattyacids,mono-glycerol
and apoproteinsform chylomicronswhichare comprisedof 85% triacylglycerols. Inahealthy
individualchylomicronsare producedinthe epithelial cellsof the small intestine.The apolipoprotein
ApoC-IIwhichisassociatedwithchylomicronsactivateslipoproteinlipaseinthe capillariesof the
heart,allowingthe release of free fattyacidstothe tissue.Once the chylomicronshave givenup
these fattyacidsto the tissue theybecome remnantsandare transportedinthe bloodbackto the
liver.
An excessin fattyacidsfromthe diet,resultsintheirpackaging intotriacylglycerolsinthe liver.
These bindtoVLDLs which release fattyacidsintothe extrahepatictissues,gradually changinginto
LDLs. Ezetimibe worksbyreducingthe absorptionof cholesterol bythe small intestine andhence
reducingthe amountof chylomicronsproduced. If the liverisstrugglingtostore the fattyacidsas
triacyglycerolsandtheyare leavingthe liverasVLDL’sthentheywill be releasingfattyacids into
bodytissuesandformingfattydeposits.If the uptake of cholesterolislimitedthenthe amountof
fattyacids releasedintothe extrahepatictissue will alsobe reduced.
Ezetimibe canbe usedalongside Atorvastatinandis prescribedforthisvery reasonasthe childis
youngand there isa highriskof a myocardial infarctionshouldhisconditioncontinue.
TreatmentsforhisconditionincludeLDL-apherisis;thisinvolvesthe removal of bloodand the
removal of LDLs, before returningittothe body.However,due tothe nature of thisprocedure
(similartodialysistreatment)anditsregularadministration,whichtakesapproximately3hours,it
wouldnotbe ideal fora growingboyto spendthat muchtime out of school and outof action(5).
A livertransplantwassuitableforthispatientisdue tohisage andthe earlydiagnosisof hisgenetic
condition.The damage due to, highlevelsof cholesterol inhisblood,resultedinmildsupra-valvular
aortic narrowing,distal transverseandproximal descendingaortanarrowingandluminal irregularity
inthe aorta.His cardiovascularassessmentwouldleadtothe conclusion alivertransplantisthe best
way to preventrisingcholesterol andfurtherdamage. Excesscholesterol woulddestroythe
chambersand bloodvesselssupplyinghisheartcausingthemtobe ischemic. The livertransplant
wouldresultin normal LDLRs andhence the cholesterol andtriglyceridelevelswoulddrop.

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Post-operationitisimportantthatthe childremains onimmunosuppressant’stoensure thathis
bodydoesnot rejectthe healthyliver.Unfortunatelythismeansthathe is vulnerable todiseaseand
illness.Inthiscircumstance itis the bestoptionandthe livertransplantwassuccessfulreflectedin
the patient’scholesterol levels returningtonormal.

5. 5 Bjork Astonba260
Usingthe webpage Ensembl,itiseasytocompare the givensequence tothe wildtype foundin
humansfor LDLR gene.
Thisfirstlyidentifysthatthe gene thatthe DNA sequence comesfromisthe LDLR gene.The redbox
on chromosome 19 belowoutlinesexactlywherethe sequence comesfrominthe code-
betweenp13.3and p13.2. This isinline withthe initial diagnosiscausingthe patienttohave
abnormal LDR receptorsinthe liver,resultinginthe increase of circulatingcholesterol.

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The screenshotbelowgivesamore indepthanalysisof LDLR.
The single base mutationcanbe foundhere,withthe codonhighlightedinyellow andthisexplains
whythe percentage identityis99.77% and not 100%:
TGTGATGGTGGCCCCGGCTGCAAGGACAAATCTGACGAGGAAAACTGCGGTATGGGCGGG
TGTGATGGTGGCCCCGACTGCAAGGACAAATCTGACGAGGAAAACTGCGGTATGGGCGGG
The E-value is4.0e-252 which showsthat
we can be confidentinthe datathe search
returnedasit istoo small a value toreflect
chance and therefore mustbe homology.
In additionthe 99.77% similaritybetween
the sequences we enteredand thatthat is
healthyshowsthatthere isa single
nucleotide mutation.

7. 7 Bjork Astonba260
The top hitfrom the sequence foraprotein isan LDL receptor. Thissuggeststhatthe base mutation
will alterthe shape of the proteinandhence resultinthe formationof abnormal LDLRs.
As shown above the mutation is atthe 221 amino acid. Itis a basechange from A to G and hence the codon
which normally codes for Aspartic acid (GAC-D) now codes from Glycine(GGC-G). This is cohearentwith the
initial basechangedisplayed above.
Aspartic acid
C4H5NO3
Asp D 115.026943 115.0874
The aminoacid ischangedfrom D
(asparticacid)toG (glycine) inthe
sequence.
The end of the alignmentdoesnot
completelymatchup because we are
comparinggenomicDNA tosplicedDNA sothe intronsare presentinthe genomicDNA.
Glycine
C2H3NO
Gly G 57.021464 57.0513

8. 8 Bjork Astonba260
1AJJ isthe known3D structure whichisan extracellulardomainof the LDL receptor.Thismeansthat
the mutationisinthis sectionof the protein.
The same mutationisvisibleinthe tophitof the search.
Belowisthe initial paperstudying1AJJ,itshowsthatit waspublishedin1996 by Blacklow andKim.
The title of the studymatcheswiththe diagnosisof HF and illustratesthatthese mutationsonthe
LDLR gene affectthe proteinfoldingandcalciumbindingof the protein.

9. 9 Bjork Astonba260
The above pymol figure showsthe base structure of the protein,below ithasbeenconvertedinto
cartoons viewwhichallowsthe secondarystructure tobe more easilyviewedandaccessible.
Sheetstructuresare
showninyellow within
the proteinstructure.
Loop structuresinthe
proteinare shownin
green
Helix structureswithin the
proteinare showninred.
Oxygenatomsinwaters
boundto the protein.

10. 10 Bjork Astonba260
The Ca2+ ion isnowshownas a sphere in the residue.
The model of the
normal residue does
make contact withthe
CA2+
ion.The grey structure inthe picture isthe normal AsparticAcidresidue,howeverinthe
mutantform thisresidue iscodedasGlycine andtherefore the hydrogen ‘R’groupcannotform a
polarcontact withthe calciumion. The bindingof the calciumiontothe structure is veryimportant
as itsbondscreate the bindingsite forthe LDL particles.Inthe mutantLDLR these bondsare not
presentandhence the LDLs cannot bind,causingFHand the symptomsexhibited inthe patient.
The 3D structure and mappingof the DNA sequence outlineshow asingle base change causesthe
active site aminoacidto change and hence resultsinmutantLDLRs,causingall the symptomsinthe
FH patient.
The yellow dots
portrayedinthe picture
representthe non
covalentbondsformed
withinthe structure.
AsparticAcid
Residue

11. 11 Bjork Astonba260
References
1- HovinghGK,DavidsonMH, KasteleinJJ,O'ConnorAM.Diagnosisandtreatmentof familial
hypercholesterolaemia. Eur.HeartJ. 34(13), 962–971 (2013).
2- US national Libraryof Medicine,Medline Plus,Medical encyclopaedia familial
hypercholesterolemia, https://www.nlm.nih.gov/medlineplus/ency/article/000392.htm,
27/11/2015
3- NIH- National HumanGenome ResearchInstitute,Health,Specificgeneticdisorders-Familial
Hypercholesterolemia, https://www.genome.gov/25520184, 27/11/2015
4- Clinical biochemistrysecondedition,A.Gaw,R.Cowan,D,O’Reilly,M.Stewart,J.Shepherd,pg
120-123, Churchill livingstone publisher,1999
5- The Rogosininstitute,Familial hypercholesterolemia, http://www.rogosin.org/treatment-
areas/treatment-cholesterol-lipid-disorders/treatment-familial-hypercholesterolemia.php,
27/11/2015
6- Drugs.com,Drugs A to Z-Atorvastatin, http://www.drugs.com/atorvastatin.html,27/11/2015
7- BindingandDegradationof LowDensityLipoproteinsbyculturedhumanfibroblasts
(comparisonof cellsfromanormal subjectand froma patientwithhomozygousfamilial
hypercholesterolemia),JosephGoldsteinandMichael Brown,The Journal of Biological
Chemistry,receivedforpublicationJanuary14-1974, page 5153,
http://web.mit.edu/7.61/restricted/readings/Brown_Goldstein_1974.pdf,27/11/2015
8- Drugs.com,Drugs A to Z- Ezetimbe, http://www.drugs.com/ezetimibe.html,27/11/2015