(&(AORTOPATHY(( ABNORMALITIES(IN(EHLERS(DANLOS( SYNDROME((EDS)(&(SPORADIC( THORACIC(AORTIC(ANEURYSM(&( DISSECTION((TAAD)( ( ( ( ( ( ( ( Ruwan(A.(Weerakkody( Imperial(College(London,(Institute(of(Clinical(Sciences( ( ( (

June(2017(

(

(

(

A(thesis(submitted(for(the(degree(of:(Doctor&of&Philosophy&

(

( ( Supervisors:( T.(Aitman(&(N.(Cheshire( (

( DECLARATION+OF+ORIGINALITY+

I( declare( that( the( work( described( herein( is( my( own( and( where( collaborations( have( taken(place,(this(is(clearly(acknowledged(and(referenced(in(the(text.

( (

COPYRIGHT+DECLARATION+

The( copyright( of( this( thesis( rests( with( the( author( and( is( made( available( under( a( Creative( Commons( Attribution( NonWCommercial( No( Derivatives( licence.( Researchers( are(free(to(copy,(distribute(or(transmit(the(thesis(on(the(condition(that(they(attribute(it,( that(they(do(not(use(it(for(commercial(purposes(and(that(they(do(not(alter,(transform(or( build(upon(it.(For(any(reuse(or(redistribution,(researchers(must(make(clear(to(others(the( licence(terms(of(this(work.( (

! 2! ACKNOWLEDGEMENTS+

This(project(started(following(an(impromptu(discussion(between(its(two(supervisors(–(Professors( Tim( Aitman( and( Nick( Cheshire.( On( my( arrival( as( a( new( Academic( Clinical( Fellow( in( vascular( surgery((with(no(prior(knowledge(or(experience(of(genetics)(we(commenced(a(pilot(study.(This( initial( stab( in( the( dark( succeeded( largely( because( of( their( enthusiasm,( unwavering( confidence( and(continual(encouragement,(which(remains( today.( For( that,( I( owe( them( both( a(great(debt(of( gratitude.(( My(arrival(in(this(post(started(as(a(journey,(for(which(I(ought(to(acknowledge(those(who(started(me( on( it:( my( teachers( and( mentors( at( Gonville( &( Caius( College,( who( first( instilled( the( fundamental( importance(of(basic(scientific(investigation(to(Medicine:(Profs.(Roger(Carpenter(&(Dino(Giussani( (PDN,(Cambridge),(Prof.(KJ(Patel((MRC(LMB).(I’d(also(like(to(thank(clinical(mentors(who(pushed( me( in( the( direction( of( academic( pursuits( and( vascular( surgery( from( the( very( beginning( of( my( clinical(training:(Mr.(Stewart(Walsh(&(Mr.(Kevin(Varty((Vascular(surgery,(Addenbrooke’s(hospital)(&( Dr.( Marek( Czosnyka( (Clinical( Neurosciences,( Cambridge)( and( finally( Mr( Paul( Ziprin( (General( Surgery,(St(Mary’s(Hospital)(for(starting(me(on(the(ACF(programme.(( My( dayWtoWday( supervisor( throughout( this( PhD,( even( after( she( moved( on,( was( Dr( Jana( Vandrovcova.(I(thank(her(for(showing,(by(example,(how(science(should(be(conducted(and(for(her( passion(and(dedication(to(proper(scientific(rigor.(During(the(PhD,(I(learnt(many(lessons(from(Prof.( Aitman,(often(illustrated(by(anecdotes(from(his(own(experience.( His( mantra( throughout( the( PhD( was( that( good( science( sells( (not( his( exact( words),( whilst( also( demonstrating( how( to( sell( it( with( considerable(success`(other(lessons(included(the(need(to(focus,(and(the(“overestimate,(double(it( and(add(more”(rule(of(how(long(conceived(plans(take(to(come(to(fruition.(( The(main(reason(this(project(expanded(the(way(it(did(is(due(to(generous(clinical(collaborators,(who( showed(trust(and(patience(in(our(work,(readily(sharing(their(patients,(clinical(material(and(expert( knowledge.(First(and(foremost(are(Professor(F.(Michael(Pope(&(Dr(Anthony(Vandersteen((National( EDS( Diagnostic( Service,( Northwick( Park( Hospital),( who( were( our( first( collaborators,( soon( becoming(unofficial(coWsupervisors(and(indeed(friends.(I(thank(Mr.(Colin(Bicknell(&(Ms.(Celia(Riga( (Department(of(Vascular(Surgery(St(Mary’s(hospital)(for(introducing(me(to(the(world(of(academic( vascular(surgery(and(their(support(and(mentorship(in(the(very(early(days(of(the(project(as(well(as( Mr(Richard(Gibbs(&(other(Vascular(Consultants(at(St(Mary’s.(I(also(thank(Prof(John(Pepper(&(Mr( Ulrich(Rosendahl(from(the(Aortic(surgery(service,(Royal(Brompton(&(Harefield(Hospitals`(I(thank(Mr( Mark( Field( and( the( rest( of( the( Aortic( surgery( team( from( the( Liverpool( Heart( &( Chest( Hospital,( whose(high(quality(clinical(service(has(provided(a(cohort(of(loyal(patients,(highly(motivated(to(be( involved( in( research( studies.( I( especially( thank( Dr.( John( Elefteriades( (Director)( and( Dr( Bulat( Ziganshin(from(the(Yale(Centre(for(Thoracic(Aortic(Disease(for(their(hospitality(and(trust(in(sharing( a(huge(collection(of(aortic(material(and(clinical(database.(( Most(importantly,(I(must(thank(the(rest(of(the(team(at(the(MRC(CSC(and(many(others(with(whom(I( worked,(for(putting(up(with(me(W(a(hapless(surgical(trainee(trying(to(do(proper(research(W(had(the( patience( to( teach( me( and( the( generosity( to( contribute( to( the( project:( enumerating( their( contributions(required(a(whole(page((see(Specific(Attributions(below).(( This( acknowledgment( wouldn’t( be( complete( without( thanking( my( now( wife,( Angela,( whom( I( first( met( coincidentally( while( working( in( the( Hammersmith( hospital( laboratory( (probably( the( best( outcome(of(the(PhD(for(me!)(W((for(showing(me(what(really(matters(in(life.( R.W.,(June(2017( ( (

! 3! SPECIFIC+ATTRIBUTIONS( The(following(lists,(in(general(terms,(the(contributions(of(people(to(various(aspects(of(the(project.( Designations(were(at(the(time(of(contribution(and(all(were(based(at(the(MRC(CSC,*(Hammersmith( hospital,(Imperial(College(London(unless(specified(otherwise.(( Jana+ Vandrovcova,+ PhD( (Senior( PostWDoctoral( Fellow)( –( spearheaded,( troubleshooted( and( supported( all( laboratory( and( analytical( aspects( including:( the( Fluidigm( assay( design( and( setup,( wholeWexome( sequencing( strategies( (all( cases),( laboratory( protocols( for( targeted( sequencing( (Fluidigm( &( Sureselect)( as( well( as( SNP( genotyping( and( NGS,( initial( analysis( of( targeted( sequencing( and( exome( sequencing( data,( pathogenicity( assignment( of( variant( calls( as( well( as( providing(intellectual(input(into(all(three(manuscripts((2(published(and(1(ongoing)`(also(day(to(day( training(and(supervision(of(lab(work(carried(out(by(myself(and(other(research(assistants.(( Jennifer+Biggs,+Penny+Norsworthy,+Abdulshakur+Abdullah,+David+Ross((Research(Assistants)( W(helped(with(running(the(targeted(sequencing(assays((Fluidigm(and(Sureselect)(on(thousands(of( samples( and( validating( variants( by( Sanger( sequencing.( Jennifer+ Biggs+ also( helped( with( the( laboratory( aspects( of( exome( sequencing( and( DNA( extraction( from( over( a( thousand( aortic( specimens( from( the( Yale( TAAD( cohort.( Julia+ Dumfarth+ (Clinical( Research( fellow,( Yale( Aortic( Centre)(helped(with(sample(cataloguing,(DNA(extraction(and(phenotyping(in(this(cohort.( Dave+ Parry,+ PhD+ (Post( Doctoral( Fellow,( IGMM,( University( of( Edinburgh)+ and( David+ Ross+ (Research(Assistant,(IGMM,(Edinburgh)(continued(some(of(the(TAAD(analysis(independent(of(my( own(and(were(critical(to(its(continuity(and(completion(following(the(move(of(the(Aitman(laboratory( to(Edinburgh.(( Laurence+ Game,+ PhD+ &+ team+ at( the+ MRC( CSC+ Genomics( Facility( provided( all( sequencing( services.( Michael+ Mueller,+ PhD+ (Bioinformatician( &( Head)+ &+ Dalia+ Kasperavaciute,+ PhD+ (Bioinformatician),( Imperial( BRC( Genomics( Facility,( carried( out( most( of( the( analysis( of( the( raw( NGS(data(up(to(and(including(variant(calling.( Christina+Kanonidou+(Clinical(Research(Fellow),(helped(with(the(majority(of(clinical(phenotyping( for(the(EDS(patients((ca.(400(cases)(and(developed(a(detailed(clinical(phenotype(database`(she( also(contributed(to(patient(recruitment(and(to(the(EDS(manuscript.( Yousef+Ibrahim((visiting(medical(student),(helped(with(acquiring(the(majority(of(the(clinical(data(for( the(UK(TAAD(cohort(and(creating(the(TAAD(phenotype(database((over(500(cases).(( Claire+Neuwirth((Senior(research(nurse)(&(Hillary+BellQJoseph+(administrator)(helped(coWordinate( and( run( all( aspects( of( patient( recruitment,( correspondence( and( related( documentation,( with( additional(help(from:(Vera+ Weerasinghe,+ Alvin+ Alinsod,+ Dimple+ Chudasama,+ Vanessa+ Pearl,+ Laura+ MonjeQGarcia+ &( the( Imperial( College( Healthcare( NHS( Trust( Cardiovascular( Research( (CARRES)(team(W(Joannna+Smee,+Barbara+Kobson+&(Emily+Ashworth.(Yvonne+Tan+(research( nurse)(helped(set(up(ethical(approval(and(the(study(protocol(documentation(at(the(outset.(( Julie+ Leary( (coordinator( at( the( National( EDS( Diagnostic( Centre,( Northwick( Park)( along( with( Genetic( counsellors,( Carole+ Cummings,( Marion( Bartlett( and( other( Consultant( Clinical( Geneticists,( F.Michael+ Pope,+ Anthony+ Vandersteen,( Neeti+ Ghali,+ Angela+ Brady,+ Virginia+ Clowes+ helped( with( the( identification( and( recruitment( of( EDS( patients( as( well( as( obtaining( accurate(clinical(data. Helen+ Figueira( (PA( to( Prof( Aitman( until( midW2014),( Shalini+ Jadeja,+ PhD+ (lab( manager( to( Prof( Aitman(2013W14),(Rajdeep+Mehon+(lab(manager(up(to(2013)(provided(essential(administrative(and( logistical(support.++ Ellen+Thomas,+PhD(&(Tom+Oates,+PhD((previous(clinical(research(fellows)(and(Holly+Black,+PhD( (postWdoctoral(fellow,(IGMM,(University(of(Edinburgh)(provided(general(mentorship(&(training(in( various(theoretical(aspects.

(((((((((((((((((((((((((((((((((((((((( ((((((((

*!MRC!CSC,!Medical!Research!Council!Clinical!Sciences!Centre,!also!known!as!Institute!of!Clinical!Sciences,!Imperial!College!London! ! 4! ABSTRACT+

Introduction:+Ehlers(Danlos(Syndrome(EDS)(and(Thoracic(Aortic(Aneurysm(&(Dissection(TAAD)( comprise( rare( Mendelian( vascular( phenotypes( associated( with( significant( premature( morbidity( and(mortality.(New(sequencing(technologies(may(allow(the(better(characterisation(of(their(genetic( basis,(thereby(informing(clinical(management(and(identifying(novel(molecular(pathways.(

Methods:+A(combined(strategy(of(targeted(highWthroughput(sequencing(to(identify(the(mutational( spectrum(in(known(collagen(and(aortopathyWsusceptibility((and(wholeWexome(sequencing(to( identify(novel(candidate(genes(was(used(in(large(cohorts(of(EDS((n=600+)(and(TAAD((n=1200+).((

Results:+Using(targeted(sequencing(in(177(EDS(index(cases,(32(pathogenic(or(likely(pathogenic(

(P/LP)( variants( (including(9(new(diagnoses)(and( 21( Variants( of( uncertain( significance( (VUS’s)( were(identified.(In(1,050(TAAD(index(cases,(61(5.8%)(P/LP(variants((30(in(FBN1)(and(209(20%)(

VUS’s(were(identified.(Only(47%(of(P/LP(variant(in(FBN1(had(a(prior(clinical(diagnosis(of(Marfan( syndrome( and( only( 51%( of( P/LP( variants( in( any( gene( (40%( in( FBN1)( had( a( positive( family( history.( Patients( with( any( P/LP( variant( had( a( greater( likelihood( of:( aortic( dissection( (OR( 2.36),( younger(age(<50(years((OR(5.3)(and(positive(family(history((OR(3.4).(Exome(sequencing(in(189( unrelated(EDS(index(cases(without(a(genetic(diagnosis,(revealed(a(novel(diseaseWcausing(gene,(

C1R,&in(Periodontal(EDS.(Provisional(candidate(genes(were(prioritised(in(patients(with(classical(

EDS( features( without( COL5A1/2( .& Exome( sequencing( with( linkage( analysis( in( a( dominant(TAAD(pedigree(revealed(a(potentially(pathogenic(deep(intronic(variant(in(FBN1(and(3( provisional(candidate(genes(in(a(recessive(Kyphoscoliosis(EDS(pedigree.(

Conclusion:+ This( work( has( shown( that( targeted( sequencing( with( new,( highWthroughput( sequencing( technology( can( be( useful( in( revealing( new( genotypeWphenotype( relationships( and( directing(genotypeWspecific(clinical(management(in(EDS(and(TAAD.(Whole(exome(sequencing(in( well( selected( cases( was( also( found( to( be( useful( in( identifying( novel( genes( underlying( these( phenotypes,( thereby( revealing( a( new( pathogenic( mechanism( underlying( connective( tissue( disease.( (

! 5! TABLE+OF+CONTENTS+

DECLARATION+OF+ORIGINALITY+ 2+

COPYRIGHT+DECLARATION+ 2+

ACKNOWLEDGEMENTS+ 3+

ABSTRACT+ 5+

TABLE+OF+CONTENTS+ 6+

1.+GENERAL+INTRODUCTION+ 11+

1.1+Genetic+and+genomic+approaches+to+disease+ 11( 1.1.1(The(Mendelian(versus(complex(disease(paradigm( 11( 1.1.2(Traditional(approaches(to(the(genetic(analysis(of(human(diseases( 14( 1.1.3(New(genome(technologies((highWthroughput(genotyping,(next(generation(sequencing)18( 1.1.4(Impact(of(new(genomic(strategies(&(identifying(variant(pathogenicity( 20(

1.2+Genetic+strategies+for+detecting+Mendelian+subtypes+of+common+diseases+ 23( 1.2.1(What(are(Mendelian(subtypes(of(common(disease?( 23( 1.2.2(Current(genomic(strategies(for(studying(Mendelian(subtypes( 23( 1.2.3(Mendelian(subtypes(in(cardiovascular(disease( 24(

1.3+EDS+&+TAAD+as+examples+where+genomic+investigation+may+be+usefully+applied+ 25(

1.4+Aims+of+this+thesis+ 26(

2.+COMMON+METHODS+ 27+

2.1+Overview+of+common+methodologies+&+techniques+ 27(

2.2+Patient+cohorts+ 27(

2.3+DNA+extraction+ 29(

2.4+Targeted+exon+sequencing+(Fluidigm)+ 29(

2.5+Whole+exome+sequencing+(Agilent+SureSelect)+ 31(

2.6+PCR+and+capillary+sequencing+for+variant+confirmation+ 31(

2.7+SNP+genotyping+&+linkage+analysis+ 31(

2.8+Phenotyping+ 32(

2.9+Data+analysis+ 35( 2.9.1(Analysis(of(NGS(data:(read(mapping,(variant(calling(and(annotation( 35( 2.9.2(Analysis(of(capillary((Sanger)(sequencing( 35( 2.9.3(Linkage(analysis( 36( 2.9.4(Statistical(analysis( 36( ( +

! 6! 3.+TARGETED+SEQUENCING+IN+EDS+ 37+

3.1+Introduction+ 37(

3.2+Methods+ 40( 3.2.1(Selection(of(cases(with(EhlersWDanlos(syndrome( 40( 3.2.2(Characterisation(of(clinical,(biochemical,(histological(and(ultrastructural(phenotype( 41( 3.2.3(DNA(extraction(and(targeted(exon(sequencing( 41( 3.2.4(Pathogenicity(assignment( 42(

3.3+Results+ 42( 3.3.1(Clinical(phenotype( 42( 3.3.2(Results(of(previous(genetic(testing(by(Sanger(sequencing( 43( 3.3.3(Results(of(collagen((analysis(&(microscopy( 44( 3.3.4(Targeted(NGS(sequencing( 46( 3.3.5(Pathogenic(variants(identified(by(NGS( 46( 3.3.6(Variants(of(uncertain(significance(identified(by(NGS( 48( 3.3.7(In(silico(predictions(of(missense(variants(detected(by(NGS( 61( 3.3.8(Clinical,(genetic(and(pathological(correlates(of(collagen(gene(variants( 64( 3.3.9(Comparison(of(NGS(variants(with(results(of(previous(genetic(testing(by(Sanger( sequencing( 64(

3.4+Discussion+ 67(

4.+TARGETED+SEQUENCING+IN+TAAD+ 71+

4.1+Introduction+ 71(

4.2+Methods+ 76( 4.2.1(Selection(of(cases(&(characterisation(of(phenotype( 76( 4.2.2(Targeted(exon(sequencing,(variant(calling(&(downstream(analysis( 76( 4.2.3(Pathogenicity(assignment(and(validation( 78(

4.3+Results+ 78( 4.3.1(Clinical(phenotype( 78( 4.3.2(Assay(performance( 80( 4.3.3(Identification(and(classification(of(rare(genetic(variants( 80( 4.3.4(Pathogenic(&(Likely(Pathogenic(variants( 90( 4.3.5(Variants(of(Uncertain(Significance((VUS’s)( 90( 4.3.6(Validation(of(variants(by(Sanger(sequencing( 92( 4.3.7(GenotypeWphenotype(correlation( 92(

4.4+Discussion+ 95(

5.+WHOLE+EXOME+SEQUENCING+FOR+NOVEL+GENE+DISCOVERY+IN+ UNRELATED+INDIVIDUALS+WITH+RARE+PHENOTYPES+OF+EDS+ 101+

5.1+Introduction+ 101( 5.1.1(Discovery(of(a(causal(gene(in(PeriodontitisWtype(EDS((type(VIII)( 101( 5.1.2(Discovery(of(novel(genes(in(other(EDSWrelated(phenotypes( 102(

5.2+Methods+ 103( 5.2.1(Selection(&(stratification(of(phenotypes( 103( 5.2.2(Exome(sequencing(&(analysis( 107(

5.3+Results+ 108( 5.3.1(Data(output(&(assay(performance(in(the(8(EDS(VIII(samples( 108( 5.3.2(Prioritisation(of(candidate(diseaseWcausing(variants(in(EDS(VIII( 108( 5.3.3(Identification(of(candidate(genes(in(EDS(VIII( 111( 5.3.4(Identification(of(candidate(genes(in(other(EDS(phenotypes((Col.VWnegative(Classical( EDS(phenotype)( 113( ! 7! 5.4+Discussion+ 115( 5.4.1(Periodontal(EDS((VIII)(–(discovery(of(novel(genes(C1R(&(C1S( 115( 5.4.2(Other(EDS(spectrum(phenotypes( 119( 5.4.3(Conclusion( 119(

6.+WHOLE+EXOME+SEQUENCING+&+LINKAGE+ANALYSIS+FOR+NOVEL+GENE+ DISCOVERY+IN+EDS+&+TAAD+PEDIGREES+ 121+

6.1+Introduction+ 121(

6.2+Methods+ 124( 6.2.1(Selection(of(pedigrees( 124( 6.2.2(Exome(sequencing,(variant(calling(and(downstream(analysis( 127(

6.3+Results+ 127( 6.3.1(Family(1:(TAAD((dominant(inheritance)( 127( 6.3.2(Family(2:(EDS(VI((recessive(/(compound(heterozygous(Inheritance)( 131(

6.4+Discussion+ 132(

7.+CONCLUDING+REMARKS+ 135+

PUBLICATIONS+ARISING+FROM+THIS+WORK+ 139+

GLOSSARY+ 141+

BIBLIOGRAPHY+&+URLS+ 147+ ( (

! 8! “Nature&! is& nowhere& accustomed& more& openly& to& display& her& secret& mysteries& than& in& cases&where&she&shows&traces&of&her&workings&apart&from&the&beaten&pathI&nor&is&there& any&better&way&to&advance&the&proper&practice&of&medicine&than&to&give&our&minds&to&the& discovery&of&the&usual&law&of&nature,&by&the&careful&investigation&of&cases&of&rarer&forms& of&disease.”&&&& & & & & & & &

William&Harvey&(1578Q1657)&

( &

! 9! &

( (

! 10! 1.+GENERAL+INTRODUCTION+

1.1+Genetic+and+genomic+approaches+to+disease+

Genetics(&(genomics(are(at(the(vanguard(of(scientific(approaches(to(understanding(the( biological(basis(of(health(and(disease.(With(huge(technological(advances(in(genomics( in( the( past( decade,( which( have( allowed( us( to( read( the( DNA( sequence( of( whole( genomes,( identifying( variants( in( this( DNA( sequence( are( starting( to( prove( useful( in( a( more( immediate( way( to( the( clinician( (in( terms( of( risk( stratification( and( so( called( “personalised”( or( “precision( medicine”).( Thus,( their( potential( in( science( and( in( healthcare(have(also(been(recognised(at(the(policy(level(1,2(

In( this( project,( I( aimed( to( use( the( latest( genomic( approaches( to( investigate( two( overlapping( syndromes( with( important( cardiovascular( morbidities( –( Ehlers( Danlos( Syndrome((EDS)(and(Thoracic(aortic(aneurysm(&(dissection((TAAD)(W(((not(only(as(an( insight(into(molecular(aetiology(but(also(to(demonstrate(how(identified(genetic(variants( may( be( of( relevance( to( clinical( practice.( In( the( 16th( Century,( the( eminent( physicianW scientist,(William(Harvey(made(the(observation(above,(which(summarises(the(premise( of(this(thesis(–( that( is,( to( obtain( a( useful( insight( into( the( pathogenesis( of( a( common( cardiovascular( condition( –( aortic( aneurysm( W( by( studying( rarer( forms( of( the( same( phenotype((TAAD(&(EDS).(In(this(project,(I(hoped(to(demonstrate(that(new(advances(in( genomics(may(lend(very(well(to(such(an(approach.((

1.1.1+The+Mendelian+versus+complex+disease+paradigm+

Our(current(understanding(of(the(genetic(aetiology(of(disease(was(traditionally(thought( of( as( a( dichotomy( (Figure( 1A).( On( the( one( hand,( it( was( thought,( were( classical( Mendelian( diseases( (named( after( the( hybridisation( experiments( of( the( Augustinian( monk,(Mendel),(which(show(a(clear(inheritance(pattern(owing(to(the(passage(of(highlyW penetrant(monogenic(DNA(sequence(changes((or(variants)(from(one(generation(to(the( next.(Good(examples(are(sickleWcell(disease,(cystic(fibrosis(and(Marfan(syndrome.(On( the( other( hand,( are( the( genetically( complex( (polygenic)( diseases,( which( are( more( common( and( come( about( by( the( interaction( of( numerous,( lowWpenetrant( genetic( variants( with( numerous( environmental( factors,( for( example( ischaemic( heart( disease,( most(cases(of(dyslipidaemia,(insulin(resistance(and(most(cancers.(Depending(on(the( ‘burden’(of(genetic(and(environmental(factors,(these(diseases(typically(have(a(variable( onset( (usually( later( in( life),( variable( severity( (often( moderate)( and( without( a( clear( familial(pattern.(In(more(recent(times,(it(has(become(clear(that(these(two(phenomena(

! 11! really(exist(on(a(continuum,( underlain( by( genetic( variants( of( variable( effect( size( and( frequency((Figure(1B).((

Some(genetic(variants(are(inherited(from(parents(and(are(found(throughout(the(body( (so(called(“germline”(mutations)(whilst(others(occur(de(novo(during(a(person’s(lifespan( in( individual( cells( (somatic( mutations).(This(thesis(is(mainly(concerned(with(germline( variants.(

Owing( to( their( clear( phenotype( and( inheritance( pattern,( Mendelian( diseases( have( proven( more( tractable( for( identifying( specific( genes( responsible( for( the( observed( phenotypes.(Identifying(genes(underlying(specific(diseases(is(important,(because(once( identified,(this(opens(the(way(for(further(detailed(investigation(of(the(disease(and(preW clinical( detection( in( family( members.( Further( scientific( investigation( can( occur( along( two( broad( paths:( ( 1)( genotypeWphenotype( correlation:( identification( of( variants( in( the( DNA( sequence( of( those( genes( in( large( numbers( of( individuals( to( predict( the( clinical( and( pathological( consequences( of( genotype,( 2)( functional( assays( to( investigate( the( specific( molecular,( biochemical( and( histological( consequences( of( disrupted( gene( function( and,( ultimately,( to( identify( potential( avenues( for( disease( modification( and( therapy.

! 12! A(

B(

(

Figure+1.1+Mendelian+vs+Complex+diseases+&+their+underlying+genetic+basis.+A.+illustrative(examples( of(the(two(extremes(of(Mendelian(and(complex(disease(characteristics,(from(Peltonen(&(McKusick,( Science,(20013(and(B.+showing(how(the(majority(of(genetic(variants’(underlying(this(continuum(also(tend(to( vary(in(a(continuous(fashion(in(terms(of(their(frequency(and(contribution(to(phenotypic(effect,(from( Antonarakis(et(al.,(Nat&Rev&Genetics&2010.4

! 13! 1.1.2+Traditional+approaches+to+the+genetic+analysis+of+human+diseases+ Traditional( approaches( to( the( identification( of( diseaseWcausing( genes( both( for( Mendelian(and(polygenic(disease(rely(on(the(phenomenon(of(genetic(recombination.( Recombination( is( a( process( that( happens( in( germ( cells( (the( precursors( of( eggs( /( sperm)(in(the(process(of(meiotic(division:(it(is(the(phenomenon(whereby(sections(of(the( maternal( and( paternal( copies( of( equivalent( ( are( swapped,( so( that( the( ensuing(sperm/eggs(have(a(mixture(of(alleles(derived(from(the(individual’s(mother(and( father( (Figure( 1.2).( This( occurs( in(an(uneven(fashion(throughout(the(whole(genome,( centred( around( so( called( “recombination( hotspots”5.( Hence,( variants( on( parts( of( a( ( that( are( in( close( proximity( to( one( another( tend( to( be( passed( down( the( generations( together( (or( coWsegregate)–( these( are( called( “haplotype( blocks”( and( are( now(relatively(well(defined(in(the(human(species.(

Figure+ 1.2+ Recombination+ –+ illustrative+ example+ in+ one+ set+ of+ homologous+ chromosomes.( This( process( occurs( in( multiple( places( throughout( the( genome.( Thus,(two(genetic(markers(that(are(close(together(have(a(lower(probability(of(being( separated( by( intervening( recombination( events( and( therefore( will( tend( to( coW segregate.( In( the( example( above,( the( four( chromatids( shown( will( go( into( four( separate( gametes.( An( adaptation( from( Pierce,( Benjamin.( Genetics:& A& Conceptual& Approach,(2nd(ed.,(from(Nature(Education.6( (

! 14! Linkage&analysis&and&positional&cloning&in&Mendelian&disease&

Following( the( discovery( of( the( first( Mendelian( disease( genes( (most( notably( in( Duschenne( Muscular( Dystrophy7( and( Cystic( Fibrosis8)( in( the( midWlate( 80’s,( the( great( majority(of(subsequent(gene(discoveries(followed(the(same(approach:(a(combination(of( two( processes:( linkage( analysis( &( ( positional( cloning( followed( by( reWsequencing( of( those(genes(in(further(affected(individuals.((

Linkage( analysis( is( used( first( to( identify( the( approximate( chromosomal( location( of( a( causative( gene.( Essentially( it( is( a( complex( statistical( calculation,( which( uses( the( principle(that(variants(proximate(to(each(other(tend(to(be(inherited(and(passed(down( together(within(a(family:(thus,(by(calculating(the(recombination(fraction(between(known( SNP’s(and(the(trait(in(affected(and(unaffected(individuals(in(the(same(family,(one(can( identify(the(approximate(location(of(the(putative(causative(variant((or(trait(locus).((This( is(expressed(as(a(probability((specifically,(as(a(log(odds(ratio(or(LOD(score)(that(the( presence( of( the( disease( is( linked( to( any( given( SNP( and( its( surrounding( region.9,10( Thus,( in( the( ideal( situation,( one( would( observe( a( narrow( and( significant( LODWscore( peak( at( a( given( location( in( the( genome,( which( specifies( the( region( in( which( the( causative( gene( is( to( be( found.( This( method( is( most( effective( when( applied( to( large( families( affected( by( a( wellWdefined( phenotype,( segregating( in( a( clear( Mendelian( manner.( Outside( the( Mendelian( disease( paradigm,( linkage( analysis( tends( to( be( less( successful(and(other(approaches(are(required((see(next).((

Once( the( candidate( region( has( been( identified( a( list( of( genes( within( that( region( is( sought(and(a(shortlist(of(“candidate(genes”((those(of(higher(likelihood(to(be(causative)( is(made(therefrom,(based(on(prior(knowledge(of(gene(function,(homologies/functional( * relationships( with( other( genes( or( with( the( equivalent( genes( in( other( organisms. (In( Mendelian(disease,(because(the(underlying(genetic(defect(is(most(frequently(a(major( proteinWaltering,( highly( penetrant( ,( the( exons( (coding( part)( of( the( candidate( gene(are(sequenced(in(the(affected(and(unaffected(individuals(of(the(pedigree,(looking( for(rare(variants,(which(segregate(with(the(diseaseWphenotype(of(interest.(This(helps(to( identify(the(responsible(gene(in(that(particular(family(or(set(of(families(showing(linkage( to(the(same(haplotype(or(chromosomal(region.(

In(order(to(understand(its(contribution(to(the(phenotype(as(a(whole,(the(gene(is(then(reW sequenced( in( multiple( unrelated( individuals( affected( by( the( same( phenotype( and( a(

(((((((((((((((((((((((((((((((((((((((( (((((((( * !Before! the! human! genome! was! sequenced,! this! was! a! hugely! laborious! process! as! often! the! genes! present! within! a! given! chromosomal!location!were!not!known!and!the!process!of!sequencing!an!unknown!candidate!region!was!the!main!challenge;!this! underlies!the!“cloning”!part!of!the!term!positional!cloning.!! ! 15! * large( number( of( unaffected( controls. (Presence( of( variants( in( a( high( proportion( of( controls(reduces(the(likelihood(of(a(gene’s(contribution(to(disease`(on(the(other(hand,( presence( of( variants( which( are( very( rare( or( not( present( in( control( populations(but( at( relatively( high( frequency( in( affected( individuals( makes( that( gene( a( likely( causative( gene.(Alternatively,(it(may(be(that(particular(classes(of(mutation(or(variants(in(particular( exons(of(a(gene(are(enriched(in(the(diseased(state(compared(with(the(unaffected(state( even( though( the( overall( frequency( of( variants( in( controls( is( generally( high.( A( recent( example(of(how(such(genotypeWphenotype(correlation(analysis(is(able(to(elucidate(the( role(of(a(gene(that( has(a(relatively(high(prevalence(of(apparent(mutations( in( healthy( individuals( is( that( of( truncating( mutations( in( titin( (TTN)( gene( in( dilated( cardiomyopathy.11(Figure(1.3(summarises(this(approach.(

(

1.#Define#candidate#region

2.#Obtain#clones#of#all#DNA#in#the# candidate#region

3.#Identify#all#genes#in#the#region#

4.#Prioritise candidate#genes#for# resequencing

5.#Test#candidate#genes#for# candidate#disease#causing#variants#

+Figure+ 1.3( Flowchart+ summarising+ the+ positional+ cloning+ approach.+ Once( a( candidate( genomic( region( is( identified( (e.g.( by( linkage( analysis),( identifying( genes( within( that( region( in( the( past( required( systematically(cloning(the(region(and(identifying(exonic(regions(and(therefore(genes(within(it([2](but(since( the( publication( of( the( human( genome( reference( sequence( this( is( done( quite( simply( by( referring( to( the( reference(sequence(via(a(genome(browser.(Mutational(screening([5](is(done(by(sequencing(the(highest( priority( candidate( genes( and( identifying( variants,( in( the( past,( a( laborious( task( but( significantly( easier( following( the( advent( of( Next( Generation( Sequencing( (see( later,( Ch.1.1.2)`( assigning( pathogenicity( to( variants(is(a(challenge((also(dealt(with(later(in(this(chapter,(1.1.3).+Adapted(from(Strachan(&(Read,(2011.12(

(((((((((((((((((((((((((((((((((((((((( (((((((( * !In!some!cases,!e.g.!Cystic!Fibrosis!only!one!gene!is!responsible!for!the!whole!disease!and!indeed!most!cases!are!caused!by!the! same! DNA! sequence! variant! in! the! same! gene;! more! often! though! there! is! significant! genetic! heterogeneity! and! the! gene! identified!in!one!family!by!positional!cloning!will!only!be!responsible!for!a!proportion!of!cases.! ! 16! (Once( a( likely( candidate( diseaseWcausing( gene( is( identified,( one( begins( a( rigorous( process(of(proving(and(elucidating(its(molecular(contribution(to(disease.(The(first(part(is( often(genotypeWphenotype(correlation:(that(is,(to(compare(variants(in(the(gene(with(the( observed( phenotype( in( a( larger( number( of( individuals.( Then,( more( sophisticated( functional( assays,( e.g.( to( test( the( effects( of( knocking( out( the( gene( in( vitro( (tissue( culture)(or(in(vivo((rat(or(mouse(model)(and(conversely(to(test(the(ability(to(rescue(a( knockWout( phenotype( either( by( reinserting( the( gene,( editing( the( aberrant( part( or( modifying( a( related( molecule( implicated( in( the( molecular( pathway.( (The( latter( efforts( are(not(part(of(positional(cloning(but(the(necessary(corollary(to(substantiate(the(gene’s( role(in(pathogenesis).(

Association&studies,&linkage&disequilbrium&&&GWAS&in&genetically&complex&disease&

Complex(diseases(have(a(number(of(further(confounding(factors.(The(disease(in(any( one(individual(is(thought(to(be(caused(by(multiple(genetic(factors,(each(of(small(effect`( along( with( the( additional( influence( of( multiple( environmental( factors,( the( phenotypes( themselves( are( less( well( defined.( Therefore,( traditional( linkage( analysis( is( often( not( successful(due(to(the(lack(of(power(to(define(a(narrow(candidate(region.(Thus,(finding( genes( predisposing( to(complex(diseases(in(humans,( until( the( past( decade,( met( with( limited(success(mostly.13(((

In(the(wake(of(the(human(genome(project,(Risch(&(Merikangas(in(1996,(by(way(of(a( single( statistical(model,( argued( that,(whilst(linkage(analysis(would(not(feasibly(detect( the( small( effect( sizes( of( common( diseaseWcausing( variants,( association( studies( employing( a( sufficiently( large( number( of( polymorphisms( across( all( genes( in( the( genome( would( be( sufficiently( powered( to( detect( previously( undetectable( common( variants.((Previous(association(studies(had(been(limited(in(terms( of( the( scope(of(the( genome( studied( and( the( number( of( samples( sequenced).14( Although( at( the( time( a( controversial( theory,( it( is( argued( that( their( demonstration( predicted( and( indeed( catalysed(the(subsequent(genome(sequencing(and(SNP(mapping(efforts:(the(“genomeW wide(association(study”(GWAS),(which(was(born(from(this(principle,(have(proven(very( successful(in(identifying(common(variants,(which(reproducibly(seem(to(be(associated( with(the(presence(of(various(diseases.15,16(((

The( success( of( association( studies( relies( on( the( same( essential( principle( of( recombination( described( above.( That( is,( that( some( narrow( haplotype( blocks( are( preserved(within(the(species,(or(at(least(within(a(given(population,(having(been(passed( onto(apparently(unrelated(individuals(from(a(common(distant(ancestor.(This(is(reflected( by( the( observation( that,( across( a( group( of( unrelated( individuals,( particular( combinations( of( alleles( or( singleWnucleotide( polymorphisms(SNPs)( tend( to( coWoccur( more( frequently( than( one( would( expect( by( chance.( This( phenomenon( of( allelic(

! 17! association( (more( commonly( known( as( “linkage( disequilibrium”)( is( what( can( be( exploited( to( identify( potential( diseaseWcausing( variants( or( genes.( Furthermore,( it( is( thought( that( a( variant( that( contributes( to( a( complex( phenotype( in( a( small( way( W( as( opposed(to(a(largeWeffectWvariant(causing( a( Mendelian(phenotype( W( will(be(preserved( through(evolution(rather(than(being(negatively(selected(as(a(Mendelian(disease(variant( might(be.((

Thus,( if( one( tests( for( the( simple( statistical( association( of( known( single( nucleotide( polymorphisms( (SNPs)( with( the( presence( of( a( phenotype( without( any( prior( assumptions,( one( could( identify( a( SNP( or( a( few( of( them( which( were( in( linkage( disequilibrium(with((ie(in(close(proximity(to)(the(diseaseWcausing(variant.(By(sequencing( SNPs( at( sufficient( density,( evenly( spaced( across( the( whole( genome( in( sufficient( numbers(of(affected(individuals(and(controls((typically(thousands),(GWAS(have(been( highly(successful(at(identifying(SNPs(closely(associated(with(the(probability(of(disease( occurrence( on( a( population( level( for( a( wide( range( of( diseases.( (A( caveat( is( that( GWAS’s( can( pick( up( associations( due( to( other( reasons( apart( from( linkage( disequilibrium( including:( false( positives( “Type( 1( error”,( epistatic( effects,( coWincidence( with(another(trait(due(to(“population(stratification”,(etc.)15((

Though( GWAS( signals( or( “hits”( may( be( useful( as( screening( tools( for( disease( susceptibility( their( effect( sizes( have( been( found( generally( to( be( small,( and,( cumulatively,( to( leave( a( large( proportion( of( the( disease( heritability( unaccounted( for,( called(“missing(heritability”.17(More(importantly,(using(a(GWAS(hit(to(understand(more( about(disease(biology(in(the(same(way(as(a(Mendelian(disease(gene(is(not(as(feasible( e.g.( sequencing( genes( in( the( region( of( the( associated( SNP( does( not( necessarily( or( readily( identify( obvious( diseaseWcausing( variants( in( that( region( or( indeed( identify( a( gene(with(significant(contribution(to(the(disease.(This(is(understandable,(given(the(fact( that( genetic( variants( preserved( over( evolutionary( time( and( exerting( a( small( effect( on( physiology(are(more(likely(to(be(subtle(in(nature(W(their(effect(may(be(regulatory(rather( than( patently( damaging,( existing( in( the( far( more( vast( and( variable( intronic( and( intergenic(regions(of(the(genome,(rather(than(in(the(more(wellWdefined(exons(of(a(gene,( exerting(an(indirect(regulatory(effect(on(a(distant(gene(rather(than(being(found(in(the( causative(gene(itself.(Thus(a(large(part(of(the(heritability(of(complex(diseases(as(well( as(the(true(biological(significance(of(the(multiple(GWAS(hits(discovered(to(date(remain( unknown.18(

1.1.3+ New+ genome+ technologies+ (highQthroughput+ genotyping,+ next+ generation+sequencing)+

From( the( turn( of( the( millennium,( a( number( of( developments( dramatically( altered( the( success(of(gene(discovery.(Until(then,(knowing(the(actual(content(of(large(parts(of(the( ! 18! genome(and(sequencing(the(genes(therein(were(the(limiting(factor.(The(sequencing(of( the(entire(human(genome((or(94%(of(it)(in(the(year(200019(and(the(mapping(of(SNPs( and( haplotypes( on( a( genomeWwide( scale( (SNP( consortium20( &( International( HapMap( project21)(allowed(a(comprehensive(and(robust(human(reference(sequence(or(genetic( ‘road( map’.( The( subsequent( development( of( very( high( throughput( sequencing( technologies((W(Next(Generation(Sequencing((NGS)(W(quickly(allowed(multiple(genes(or( even(the(whole(genome(to(be(sequenced(on(a(large(scale(Table(1.1).22,23(This(paved( the(way(for(the(discovery(of(multiple(new(Mendelian(disease(genes,(positional(cloning( of( a( candidate( region( now( became( a( routine( exercise( executable( by( a( single( lab( technician( as( opposed( to( the( Behemothian( challenge( it(had(previously(been( (Figure( 1.4).(

Table+1.1+Illustrative(example(of(sequencing(a(single(human(genome(preW(and(postWNGS+ Platform( Year+ RunQtime+(days)( Cost+(US$)(

Traditional(Sanger( 2005( 1,500( 13,800,000( sequencing(

Next(Generation( 2015( <1(( +1,250( Sequencing(

Figures(are(from(the(National(Human(Genome(Research(Institute,(National(Institute(of(Health,(Maryland,( USA24,(rounded(to(the(nearest(3(significant(figures.((

(

Figure+1.4.+Progress+in+Mendelian+trait+and+complex+trait+gene+discoveries.+Note(the(steady(increase( in(Human(Mendelian(trait(gene(discoveries(following(the(establishment(of(positional(cloning(techniques(in( the(early(90s(and(the(exponential(increase(in(both(Mendelian(and(complex(trait(discoveries(following(new( genome(technologies(at(the(turn(of(the(millennium.(The(discovery(of(more(common(complex(trait(genes( still(trails(behind(Mendelian(gene(discoveries(significantly.(Adapted(from(Glazier(&(Aitman,(Science&2002.13( (

! 19! 1.1.4+Impact+of+new+genomic+strategies+&+identifying+variant+pathogenicity+

The( obvious( effect( of( largeWscale( sequencing( is( the( generation( of( vast( quantities( of( data( W( the( average( single( exome( sequence( by( NGS( generates( tens( to( hundreds( of( millions( of( sequence( ‘reads’( (see( Chapter( 5( &( 6),( typically( taking( 4W6( Gigabytes( of( computer(memory`(a(single(genome(is(a(100(times(larger((David(Parry,(University(of( Edinburgh,(Personal(Communication).25(This(has(required(a(whole(new(generation(of( bioinformatics( expertise( to( map( or( align( the( reads( to( the( reference( sequence( and( identify( variants( in( that( sequence( (“variant( calling”).( Furthermore,( large( numbers( of( DNA( sequence( variants( are( generated:( when( compared( to( the( reference( human( sequence,( the( exome( sequence( of( the( average( European( will( reveal( around( 20,000( variants.25( Many( of( the( DNA( sequence( variants( identified( by( NGS( will( have( no( relevance( to( the( disease( in( question( and( today( the( real( challenge( lies( in( identifying( those( variants( which( are( pathogenic( (contributory( to( pathology),( bearing( in( mind( that( the(vast(majority(of(human(genetic(variation(is(not(pathological.(A(number(of(tools(have( been(identified(to(help(the(process(of(prioritising(and(filtering(these(variants((see(Figure( 1.5( and( Table( 1.2( below( for( a( summary( W( they( will( be( discussed( in( more( detail( in( subsequent( chapters).( As( whole( exome( and( whole( genome( sequencing( come( into( routine( clinical( use,( this( task( becomes( more( important,( specifically( with( regard( to( incidental( findings( (mutations( in( clinically( important( genes( relating( to( an( unforeseen( pathology):(in(this(case(more(specific(guidelines,(clinical(and(ethical,(are(required.26,27(

( (

! 20! DNA$sequence$ Variant$ Intronic' '(non'splice'site)'

Exonic'&'' splice'site'

Synonymous' “Loss'of'Func3on”:' Splice,'Nonsense,'FS' Missense,' (indel)' Previously'reported' pathogenic'

known'domains'+'' 'inHsilico'predic3ons' [equivocal'cases]'

Segrega&on)with)phenotype)) /)absence)in)controls)

In)vitro)/)In)vivo)) func&onal))corrobora&on)

Likelihood'of'pathogenicity' (

Figure+ 1.5.+ Determining+ the+ pathogenicity+ of+ DNA+ sequence+ variants+ –+ a+ schema.+ FS,( frameshift( mutation.( SpliceWsite,( variant( predicted( to( disrupt( an( essential( spliceWsite.( Showing( a( basic( schema( for( initial(filtering(of(variant(and(the(variant(filtering(pipeline(used(by(the(Aitman(lab,(Imperial(College(London.( Variants( are( first( filtered( for( frequency( in( control( populations,( based( on( the( estimated( minor( allele( frequency,(which(is(a(function(of(the(disease(prevalence:(in(a(rare(disease,(typically,(one(would(filter(out( variants( with( a( reported( frequency( of( >1:1000.( Intronic( variants( not( predicted( to( affect( splicing( and( Synonymous(variants((those(which(do(not(result(in(a(change(in(the(amino(acid(sequence(of(the(resultant( protein)(are(considered(to(be(of(low(risk(of(pathogenic(effect((but(it(is(possible(they(may(exert(a(regulatory( role).(“Loss(of(function”(mutations(refer(to(those(which(cause(a(major(disruption(to(the(primary(structure(of( the( protein( and( are( therefore( considered( highly( likely( to( be( pathogenic:( essentially( they( completely( interrupt( the( synthesis( of( normal( protein( in( one( way( or( another:( “Nonsense”( mutations( (introduce( a( premature(“stop”(codon,(thereby(causing(a(truncated(protein(product),(SpliceWjunction(disrupting(mutations( (those( which( cause( abnormal( splicing( of( exons,( thereby( also( leading( to( an( abnormal( protein)( and( “frameshift”(mutations(which(change(the(frame(of(reference(of(the(transcription(apparatus(necessary(for( making( mRNA( from( the( gene,( again( often( leading( to( a( grossly( abnormal( protein( product.( Missense( mutations(lead(to(a(change(in(the(resultant(amino(acid(sequence(include(substitutions(of(one(DNA(base(to( another,(as(well(as(“Indels”((insertions(or(deletions(of(bases).(Missense(variants(are(of(variable(effect,(in( most(cases(they(may(have(no(effect(at(all(and(tend(to(be(the(most(difficult(for(determining(pathogenicity.(A( process( of( gathering( of( evidence( to( support( or( refute( likely( pathogenicity( includes:( previously( reported( evidence,( in( silico( predictions,( segregation( analysis( and( functional( corroboration( (see( Table( 1.2( for( elaboration).( N.B.( “Likelihood( of( pathogenicity”( scale( and( the( relative( position( of( variantWtypes( on( the( horizontal(axis(are(not(to(scale.([based(on(Kircher(et(al.28,(Richards(et(al.29].+ (

( (

! 21! +Table+ 1.2+ Determining( the( pathogenicity( of( a( Missense( variant( W( hierarchy( of( evidence.( [based(on(Kircher(et(al.28(Richards(et(al.29(and(Strachan(&(Read.12]+

Evidence( Explanation( Example(

Presence!in!a! If!the!gene!is!known!to!be!associated!with!the! A!novel!variant!in!FBN1!in!a! candidate!gene! phenotype,!this!provides!preliminary!evidence!of!the! marfanoid!patient!(see!later)! relevance!of!the!variant!to!the!phenotype!of!interest!

In'silico!predictions!! Various!bioinformatics!software!have!been!designed!to! Functional!&!conservation_based! predict!the!functional!effect!of!a!given!amino!acid! predictions:!SIFT,!Polyphen,! change!on!the!protein’s!structure!and!function.!These! MutationTaster,! are!broadly!based!on!two!principles:!! MutationAssessor,!cadd,!etc! 1)!The!resulting!change!in!chemical!composition!in!the! Conservation_based!predictions:! amino!acid!side!chains!(as!determined!by!the! PhyloP,!GERP++,!etc! Grantham!matrix!–!see!classic!paper!by!Grantham!in! 30 [see!Methods!for!a!full!list]! Science!1974! )!! ! 2)!Conservation!across!species!–!amino!acid!residues! which!are!more!conserved!across!species!are!more! likely!to!be!functionally!important!and!therefore!their! disruption!more!likely!to!be!pathogenic!! !

Affecting!a!known! If!the!site!of!the!variant!coincides!with!a!known! e.g.!Helical!Glycine!substitutions! functional!domain! functional!domain!e.g.!active!site!or!interacting! in!collagen!type!III!are!known!to! element.! be!pathological!in!vascular!EDS! (see!later!–Ch.3)! ! !

3D!structural! 1.!In!general!terms!an!amino!acid!change!from! ! predictions! hydrophobic!to!polar!on!the!inside!(e.g.!Glycine!to! other!amino!acid!in!collagen)!are!highly!likely!to! disrupt!the!tertiary!structure!of!a!protein;!similarly,! changing!a!polar!to!hydrophobic!amino!acid!on!the! outside!of!a!protein!makes!the!protein!more!sticky,! leading!to!protein!aggregation!(e.g.!sickle!cell!disease! and!some!neurodegenerative!diseases).! 2.!If!the!3D!structure!of!a!protein!or!related!protein! subunit!and!their!key!functional!domains!are!well! characterized,!X_ray!crystallography!can!be!used!to! visually!predict!the!impact!of!a!given!amino!acid! change!on!tertiary!structure!

Correlation!&! Very!high!evidence!of!pathogenicity!comes!from! ! segregation!with! segregation!analysis:!that!is!segregation!of!the!variant! phenotype!! within!a!family!with!presence!of!the!phenotype!(or! indeed!in!a!number!of!apparently!unrelated!individuals! affected!by!the!same!phenotype!who!carry!the!same! variant),!whilst!being!very!rare!or!absent!in!healthy! controls.!!

Functional!assays! Definitive!evidence!of!the!pathogenicity!of!any!variant! ! comes!from!a!robust!set!of!functional!experiments!in' vivo!or!in'vitro,!which!incontrovertibly!link!the! presence!of!the!variant!to!the!presence!of!the! phenotype!and!via!a!number!of!intervening! biochemical!stages.!

(

! 22! 1.2+ Genetic+ strategies+ for+ detecting+ Mendelian+ subtypes+ of+ common+diseases+

1.2.1+What+are+Mendelian+subtypes+of+common+disease?+

In(a(number(of(diseases,(there(has(been(a(notable(subset(of(patients(presenting(at(a( younger(age,(with(a(more(aggressive(natural(history(and/or(with(a(strong(family(history:( in(other(words,(common(phenotypes(presenting(in(a(Mendelian(fashion.(By(finding(the( genes(and(molecular(pathways(responsible(in(such(Mendelian(subtypes,(it(is(thought( that(this(may(shed(light(on(the(more(common(forms(of(the(same(phenotype(and(may( even(account(for(part(of(the(missing(heritability.4,17(

Good( examples( include:( Alzheimer’s( disease( (betaWamyloid( precursor( protein( and( presenilinW( 1( and( W2),( breast( cancer( (BRCAW1( and( W2),( colon( cancer( (familial( adenomatous( polyposis( and( hereditary( nonWpolyposis( colorectal( cancer),( diabetes( (maturity( onset( diabetes( of( youth),( primary( open( angle( glaucoma( and( cardiovascular( phenotypes,( such( as( inherited( cardiomyopathies( and( cardiac( conductive( system( disorders((see(later(–Ch.1.2.3).((

1.2.2+Current+genomic+strategies+for+studying+Mendelian+subtypes+

The( increased( sequencing( capacity( allowed( by( NGS( is( becoming( useful( in( two( respects.( Firstly,( in( allowing( the( mutational( spectrum( of( already( known( genes( to( be( determined((called(“targeted(sequencing”):(this(is(proving(relevant(not(only(in(classical( Mendelian( cases( with( a( known( familial( trait( but( also( in( identifying( DNA( sequence( variants( underlying( apparently( sporadic( cases( of( a( disease( (with( no( obvious( family( history( or( clinical( traits( suggestive( of( a( Mendelian( disease).( Secondly,( since( the( first( discovery( of( ( Mendelian( disease( gene( by( exome( sequencing( in( 2009,31( it( has( accelerated( the( discovery( of( new( genes( underlying( Mendelian( subtypes( by( exome( sequencing.25,32( The( advantage( of( exome( or( wholeWgenome( sequencing( for( gene( discovery(lies(in(the(fact(that,(as(well(as(providing(an(efficient(complementary(strategy( to(traditional(linkage(analysis(in(an(individual(family,(it(can(also(be(applied(successfully( to( unrelated( individuals( sharing( the( same( specific( phenotype.( An( excellent( review( of( the(strategies(employed(is(given(by(Gillisen(et(al((summarized(in(Figure(1.5(below).32(

! 23! +

Figure+1.5+A+summary+of+strategies+for+novel+gene+discovery+by+exome+sequencing.+From(Gillisen(et( al.32+

1.2.3+Mendelian+subtypes+in+cardiovascular+disease+

Many( cardiovascular( phenotypes( have( a( distinct( and( wellWdefined( Mendelian( subset,( including( hyperlipidaemias,( congenital( heart( defects,( cardiomyopathies,( arrhythmias,( and( aortic( aneurysm.33( These( Mendelian( (familial)( forms( have( been( variably( characterised( in( terms( of( genetic( aetiology,( of( which( the( least( well( characterised( are( the( aortic( pathologies.( Invariably,( the( Mendelian( forms( tend( to( present( with( more( aggressive( disease( (notably( sudden( cardiac( death)( and( earlier( onset:( subsequently( their( management( is( quite( different.( Clinically,( eliciting( their( genetic( aetiology( is( important(as(it(allows(preventative(screening(and(risk(stratification(and(even(treatment( based( on( genotypeWphenotype( correlations,( a( good( example( being( in( the( Long( QT( syndromes.33,34((

In(cardiovascular(diseases(as(well(as(in(other(phenotypes,(it(is(becoming(apparent(that( the( same( genes( responsible( for( some( Mendelian( forms( seem( to( underlie( a( larger( proportion( of( apparently( sporadic( cases.( A( good( example( are( truncating( mutation( in( TTN,(previously(thought(to(be(a(minor(cause(of(familial(DCM,(but(recently(have(been( identified(as(a(major(cause(of(DCM(overall.35(Finally,(and(perhaps(most(compellingly,( the( elucidation( of( genes( responsible( in( the( Mendelian( forms( uncover( molecular( pathways(that(may(account(for(disease(pathophysiology(and(therefore(provide(targets( for( therapeutic( intervention:( preliminary( forays( into( therapeutic( modification( based( on( such( discoveries( include( the( potential( for( the( use( of( fleicanide( in( Catecholaminergic( ! 24! polymorphic( ventricular( tachycardia( 11,36( and( Losartan( in( Marfan( aortic( aneurysm( (although( the( latter( failed( to( show( superiority( over( conventional( treatment( with( βW blockers).37((

1.3+EDS+&+TAAD+as+examples+where+genomic+investigation+may+ be+usefully+applied+

This( thesis( describes( the( application( of( some( of( the( genomic( strategies( described( above(to(two(overlapping(conditions(with(important(cardiovascular(consequences.((

•( Ehlers(Danlos(syndrome((EDS)(comprises(a(group(of(different(but(phenotypically( overlapping(rare(Mendelian(disorders(of(the(connective(tissue,(variably(affecting( the(skin,(bones/joints(and(viscera.(38,39(( ( •( Thoracic( aortic( aneurysm( and( dissection( (TAAD)( is( a( rare,( but( polygenic( condition,(which(has(a(distinct(Mendelian(subset.(The(latter(includes(syndromic( connective( tissue( disorders( such( as( Marfan( syndrome,( Loeys( Dietz( syndrome( and(EDS.(TAAD(is(considered(to(be(distinct(from(the(more(common(abdominal( aortic(aneurysm((AAA),(epidemiologically,(embryologically,(and(pathologically.40(

Both(of(these(conditions(are(clinically(important(because(of(their(implication(in(causing( significant(cardiovascular(mortality(and(morbidity,(often(occurring(at(a(young(age(and( with(clinically(insidious(course(until(vascular(rupture.(The(genetic(basis(of(both(of(these( conditions( is( limited( to( a( few( known( genes( and( the( mutational( spectrum( for( known( genes(in(the(overall(cohort(is(unknown.39((

In(EDS,(with(the(exception(of(patients(with(highly(specific(and(distinct(phenotypes((e.g.( Vascular( EDS,( Classical( EDS,( Kyphoscoliotic( EDS( –( see( later,( Chapter( 3( for( further( detail),( which( have( known( genetic( causes,( the( majority( of( EDS( patients( remain( undiagnosed(genetically.(Until(recently,(and(at(the(time(of(the(inception(of(this(project,( genetic( testing( was( phenotypeWdriven:( sequencing( individual( genes( by( the( traditional( Sanger( method( for( specific( diagnostic( criteria,( with( the( outcome( that( the( majority( of( patients(either(did(not(meet(these(criteria(or(had(negative(results(after(testing(the(most( likely( genes.( This( left( the( question( as( to( what( proportion( of( cases( would( have( abnormalities(in(the(known(genes(outside(specified(phenotypeWbased(criteria(and(how( would( the( mutational( spectrum( and( diagnostic( yield( change( if( known( genes( are( sequenced(in(all(patients,(irrespective(of(phenotype.(

In(TAAD,(genes(responsible(for(a(small(subset(of(syndromic(TAAD(and(some(familial( cases(have(been(identified,(but(their(relevance(to(the(wider(TAAD(cohort(remains(to(be( determined((although(preliminary(evidence(to(date(suggests(that(genes(associated(with( ! 25! the( Mendelian( forms( of( TAAD( may( also( be( important( in( the( sporadic( type).40W43( Furthermore,( TAAD( poses( a( special( challenge( to( surgeons,( as( optimal( management( and( riskWstratification( remain( controversial( and( surgical( repair( is( technically( complex( and( often( marred( with( morbidity.37,44W46( The( ability( to( risk( stratify( patients( based( on( genotype(would(provide(a(useful(tool(in(informing(prognosis(and(management.(

Their(genetic(epidemiology(and(genetic(approaches(used(to(date(will(be(discussed(in( detail(in(chapters(3(&(4.((

1.4+Aims+of+this+thesis+

This(thesis(aims(to(answer(the(following(specific(questions:(

1.(What(proportion(of(cases(of(a(mixed(EDS(cohort(sequenced(by(NGS,(have(a( pathogenic(mutation(in(one(of(the(known(genes(and(how(does(the(mutational(spectrum( underlying(EDS(correlate(with(previously(determined(genetic(abnormalities,(clinical(and( biochemical(phenotype(and(the(existing(classification(system?(( W(strategy:(To(undertake(targeted(sequencing(of(known(specific(genes(implicated(in(the( pathogenesis(of(EDS(in(a(cohort(of(clinically(diagnosed(EDS(patients,(using(next( generation(sequencing.+chapter+3(

2.(In(a(routine(clinical(cohort(of(TAAD(cases,(what(proportion(is(attributable(to(each(of( the(known(associated(genes(and(can(preliminary(genotypeWphenotype(relationships(be( identified?(( W&strategy:(To(undertake(targeted(sequencing(of(known(specific(genes(implicated(in(the( pathogenesis(of(TAAD(using(next(generation(sequencing.(chapter+4(

3.(In(the(cases(of(EDS(/(TAAD(without(mutations(in(the(known(genes(can(novel(genes( associated(with(these(diseases(be(identified:( a.(by(analysis(of(unrelated(individuals(sharing(rare(phenotypes?(( W(strategy:(use(whole(exome(sequencing(to(attempt(to(identify(novel(genes(underlying( these(phenotypes.(chapter+5( b.(by(analysis(of(families(with(a(Mendelian(phenotype?(( W&strategy:(undertake(linkage(analysis(and(whole(exome(sequencing(to(attempt(to( identify(novel(genetic(causes(of(EDS/TAAD.(chapter+6.&( (

! 26! 2.+COMMON+METHODS+

2.1+Overview+of+common+methodologies+&+techniques+

Figure(2.1(provides(a(summary(of(methodologies(used.((

EDS$/$TAAD$$ pa*ents$recruited$

DNA$extrac*on$

Phenotyping$

Targeted$sequencing$ (Fluidigm$assay)$ Whole$exome$ sequencing$

SNP$genotyping$(for$ Valida*on$(by$Sanger$sequencing)$ linkage$analysis)$

Analysis$

Figure+2.1+Overview(of(methods(used.(Dashed(arrows((W(W(W>)(indicate(that(only(selected(cases(from(the( previous(step(were(used.(

2.2+Patient+cohorts++

Patients( with( EDS( and( TAAD( were( selected( and( recruited( from( a( number( of( institutions,(summarised(in(Table(2.1(below.(All(patient(cohorts(were(recruited(during( this( project.( Ethical( approval( for( the( work( was( obtained( from( the( West( London( Research( Ethics( Committee( (REC( Ref( 11/LO/0883)( complemented( by( other( ethical( approval( from( collaborating( sites( (see( Table( 2.1).( In( addition,( an( incompletely( phenotyped( existing( archival( collection( of( DNA( from( approximately( 500( patients( and( fibroblasts(from(approximately(3,000(patients(was(used(selectively(for(assay(validation( and(for(sourcing(very(rare(EDS(phenotypes.(

(

( (

! 27! Table+2.1+Summary(of(cohorts(assembled(during(the(study+

Institution+ Disease+ Sample+Type++ Approx.+number++ Ethical+ category+ approval+

National(EhlersWDanlos( EDS( (&( some( VB(&(Saliva( 560((+30(TAAD)( 1,2(( syndrome(diagnostic(service( TAAD)( ( ( ( (London)( ( ( (

Hypermobility(Clinic,( EDS( VB(&(Saliva( 60( 1,2( Department(of(Rheumatology,( University(College(Hospital(

Vascular(Unit,(Imperial( TAAD( VB(&(Saliva( 170( 1( College(London(

Aortic(Centre,(Royal(Brompton( TAAD( Saliva( 100( 1( &(Harefield(hospitals(

Aortic(Centre,(Liverpool(Heart( TAAD( Saliva( 90( 1( &(Chest(Hospital(

Aortic(Institute,(Yale(School(of( TAAD( Aortic(tissue((fresh( >800( 3( Medicine,(Yale,(NH,(USA.(( frozen)(

EDS,( Ehlers( Danlos( syndrome.( TAAD,( Thoracic( aortic( aneurysm( and/or( dissection.( VB,( whole( venous( blood.( Ethical( approval:( 1.( West( London( Research( Ethics( Committee( 2.( NIHR( Bioresource( for( rare( diseases(3.(Yale(Institutional(Review(Board.(((

These(are(divisible(into(three(broad(groups:(

EDS(cohort:(index(cases(and(their(relatives(recruited(from(a(larger(group(of(patients( referred( to( the( National( EDS( Diagnostic( service( (London)( from( 2009W2014( and( later( from(selected(cases(diagnosed(with(EDS,(who(had(been(referred(to(the(Hypermobility( Clinic,( UCLH.( I( tried( to( select( a( mixed( cohort,( which( broadly( reflected( the( whole( spectrum(of(EDS(phenotypes.((

UK(TAAD(cohort:(I(tried(to(recruit(any(patient,(who(had(been(referred(to(one(of(three( UK(quaternary(referral(centres(for(TAAD((Table(2.1).(This(was(not(a(strictly(sequential( cohort((some(patients(had(passed(away(or(declined(to(take(part)(but(as(far(as(possible( reflects( a( mixed( (sporadic( and( familial)( TAAD( group( referred( to( three( major( TAAD( centres.(As(more(complex(cases(tend(to(be(referred(to(such(centres,(the(caveat(here(is( that(these(cases(reflect(the(referral(pattern(to(such(centres(rather(than(strictly(reflecting( the(entire(UK(TAAD(population.(

Yale(TAAD(cohort:(these(cases(comprised(a(sequential(series(of(mixed(TAAD(patients( operated( on( in( one( large( TAAD( centre( in( the( USA( over( an( approximately( 10Wyear( period.( They( had( previously( consented( to( research( studies( and( their( frozen( aortic( tissue(samples(were(available(for(DNA(extraction(and(analysis.(

! 28! 2.3+DNA+extraction*++

Saliva(samples(were(collected(using(the(Oragene(DNA(kit((Genotek,(Ontario,(Canada)( and( processed( according( to( the( manufacturer’s( protocol.( DNA( from( whole( blood( samples( was( extracted( using( the( QIAamp( DNA( Blood( Midi( kit( (Qiagen,( Venlo,( Netherlands).(DNA(from(fresh(frozen(aortic(specimens(was(extracted(using(the(DNeasy( Blood(&(Tissue(kit((Qiagen,(Venlo,(Netherlands).(

2.4+Targeted+exon+sequencing+(Fluidigm)†+

NGS( assays( were( designed( to( sequence( the( exons( and( exonWintron( boundaries( of( genes(commonly(associated(with(EDS/TAAD(and(overlapping(phenotypes.(For(targetW enrichment,(we(used(the(Fluidigm(Access(Array,(a(microfluidic(PCRWbased(system(for( amplifying( multiple( “amplicons”( in( multiple( cases( in( parallel( (Fluidigm,( South( San( Francisco,( USA).( This( technology( had( previously( been( shown( to( be( effective( in( targeted( sequencing( in( large( cohorts( of( individuals( and( we( therefore( selected( this( in( preference(to(the(hybridisation(based(capture(panels((e.g.(Sureselect)(as(it(allowed(a( much(greater(level(of(multiplexing,(with(a(more(efficient(workflow.47,48(For(sequencing( the( prepared( libraries,( we( used( the( Illumina( Miseq( or( Hiseq( systems( (Illumina,( San( Diego,( USA)( as( these( allowed( high( coverage( and( were( the( natural( companion( for( libraries(prepared(by(the(Fluidigm(system.((

The(following(gene(enrichment(panels(were(designed:(

(1)( Collagen( gene( panel:( 375( primerWpairs( for( COL1A1,( COL1A2,( COL3A1,( COL5A1( and( COL5A2& (Appendix( 1,( Table( S1).( These( are( the( genes( mainly( associated( with( EDS,( in( which( the( main( pathological( abnormality( is( thought( to( lie( with( abnormal( collagen( structure( or( availability39,49,50( (see( chapter( 3.1( for( further( details( on( their( specific(contribution(to(various(EDS(subtypes).(

(2)( Aortopathy( panel( 1( (“TAADWX”):( 363( primerWpairs( for& FBN1,& TGFBR1,& TGFBR2,& MYH11,&ACTA2,&SMAD3&and&MYLK&(Appendix(1,(Table(S2).(As(aortopathy(and(TAAD( are(observed(in(the(EDS( spectrum,(at(the(outset(I(also(selected(these(genes,(which( were(known(causes(of(syndromic(and(familial(TAAD,( (for(sequencing(in(a(mixed(EDS( cohort.52,53,54W57(

(3)(Aortopathy(panel(2((“TAADWZ”):(later(in(the(project,(I(designed(a(further(NGS(panel( to(incorporate(newly(discovered(TAAD(genes(as(well(as(the(collagen(genes(from(the(

(((((((((((((((((((((((((((((((((((((((( (((((((( *!Jennifer! Biggs,! Penny! Norsworthy,! Abdulshakur! Abdullah! David! Ross! and! Jana! Vandrovcova! helped! with! DNA! extraction!at!various!times,!totalling!over!2,000!samples.! †!Jana!Vandrovcova!helped!design,!set!up!and!troubleshoot!the!Fluidigm!assay!at!the!outset;!once!established,!Jennifer! Biggs,! Penny! Norsworthy,! Abdulshakur! Abdullah! and! David! Ross! helped! run! many! hundreds! of! samples! through! the! Fluidigm!pipeline.!!Aortopathy!panel!2!(TAAD_Z!assay)!was!designed!later,!with!help!from!Abdulshakur!Abdullah.! ! 29! first( panel( (with( the( knowledge( that( diseases( with( abnormalities( in( collagen( genes( predisposed(to(aortopathy),(to(be(applied(to(TAAD(patients(only:(495(primer(pairs(for( SKI,& TGFB2,& SLC2A10,& COL1A1,& COL1A2,& COL3A1,& COL5A1& and& COL5A2.& 58,59 (Appendix(1,(Table(S3). (

Thus,(the(following(NGS(panels(were(designed(for(each(group(as(follows:(

•( For(all(EDS(cases,(Collagen(gene(panel(&(Aortopathy(panel(1((TAADWX)(

•( For( all( TAAD( cases,( Aortopathy( panel( 1( (TAADWX)( and( Aortopathy( panel( 2(

(TAADWZ).((((

Transcript(ID’s(used(for(bioinformatics(analysis(are(shown(in(Table(S4.(Primers(were( designed(and(validated(using(Primer(3(software60(and(the(inWsilico(feature(of(the(UCSC( genome(browser,61(to(produce(individual(amplicons(of(300bp,(which(individually(or(in( combination(overlapped(single(exons(and(at(least(50bp(of(flanking(intronic(sequence(

(Figure(2.2).(The(following(settings(were(used:(size:(17W27bp((optimum:(20bp),(Tm:(60,( GC%:( 20W60,( Max( PolyWX:( 3,( maximum( target( size:( 200bp( (to( allow( for( primers( and( sequencing(adaptors(all(included(in(the(final(300bp(amplicon).((

Multiplex(PCR(was(performed(using(the(Access(Array((Fluidigm,(South(San(Francisco,( USA)( in( batches( of( 47( test( samples( and( one( negative( control( (water( blank)( as( previously( described.48( NinetyWsix( to( 288( purified( samples( (two( to( six( batches)( were( pooled(and(sequenced(on(a(single(lane(of(a(MiSeq(or(HiSeq2500(sequencer((Illumina,( San(Diego,(USA)(generating(150bp(pairedWend(reads.((

Amplicons*

Exons*of* targeted* gene*

+ Figure+2.2+Example+of+amplicon+design+to+cover+exons+and+flanking+intronic+sequence.(Viewed(in(UCSC(genome( browser,( this( example( shows( exons( 3( and( 4( of( the( COL3A1( gene.( In( some( cases( (e.g.( exon( 4( above)( two( or( more( overlapping(amplicons(were(designed(to(cover(a(target(region(either(due(to(large(target(or(limitations(in(primer(specificity( in(a(particular(location.(

( +

! 30! 2.5+Whole+exome+sequencing+(Agilent+SureSelect)*+

Genomic(DNA((3(µg)(from(each(sample(was(sheared(in(120uL(of(low(TrisWEDTA(buffer( (Covaris( S2,( 6( cycles( frequency( sweeping`( duty( cycle:( 10%,( intensity:( 5,( cycles( per( burst:( 200,( total( time:( 6( min,( peak( fragment( length:( 150bp).( After( SPRI( (solid( phase( reversible( immobilization)( magnetic( bead( purification( (AMPure( kit,( Agencourt( Biosciences,(Beckman(Coulter(Genomics,(USA)(and(analysis(on(the(Bioanalyser(2100( (Agilent),( these( were( used( for( the( construction( of( a( pairedWend( sequencing( library.( Enrichment(of(exonic(sequences(was(performed(for(each(library(using(the(SureSelect( Human( All( Exon( 50Mb( kit( (Agilent)( following( the( manufacturer’s( instructions.( ExonW enriched( DNA( was( precipitated( with( magnetic( beads( coated( with( streptavidin( (Invitrogen)(and(was(washed(and(eluted.(Exon(enrichment(was(validated(by(realWtime( PCR(in(a(7300(RealWTime(PCR(System((Applied(Biosystems).(Enriched(libraries(were( sequenced( in( one( lane( of( an( Illumina( HiSeq( 2000( sequencer,( using( the( standard( protocol.+

2.6+PCR+and+capillary+sequencing+for+variant+confirmation†+

Variants(detected(by(NGS(were(validated(by(conventional(capillary(sequencing(by(the( Sanger(method.(Primers(were(designed(using(Primer(3(software60(and(validated(using( the(in(silico(feature(of(the(UCSC(genome(browser,61(targeting(one(exon(and(50bp(of( flanking( sequence.( The( following( settings( were( used:( size:( 20W27,( opt:21,( Tm( 55W65,( opt:60,(GC%(40W60%(to(optimise(for(PCR(amplification(by(KOD(polymerase((Merck).( Approx(100ng(of(each(PCR(product(was(mixed(with(1(uL(of(3.2uM(primer(and(made(up( to( a( total( volume( of( 10uL( with( nuclease( free( water( and( sequenced( by( capillary( electrophoresis(on(the(3730XL(platform((Applied(Biosystems,(UK).(

2.7+SNP+genotyping+&+linkage+analysis‡++

DNA(sample(concentration(was(determined(using(the(Invitrogen(Picogreen(kit(following( the(protocol(described(in(the(Illumina(GoldenGate(Genotyping(assay(guide`(standard( lambda(DNA(was(used(to(make(a(standard(curve(and(the(standard(DNA(and(sample( DNA(dilutions(were(mixed(with(diluted(picogreen(reagent`(plates(were(read(separately( on(a(Mithras(LB(940(fluorometer((Berthold(Technologies,(Bad(Wildbad,(Germany)(and( analysed(using(MikroWin2000(software.(Five(microlitres(of(each(sample(at(50ng/ul(was(

(((((((((((((((((((((((((((((((((((((((( (((((((( *!Jana! Vandrovcova! supervised! and! Jennifer! Biggs! helped! with! library! preparation! for! whole! exome! sequencing!(2.5).! †!This! was! done! initially! by! myself;! later! by! Jennifer! Biggs,! Penny! Norsworthy,! Abdulshakur! Abdullah! and! David!Ross!at!various!points.! ‡!Linkage!analysis!was!done!by!Jana!Vandrovcova! ! 31! used( in( the( genotyping( assay.( Genotyping( was( carried( out( in( the( Genome( Centre,( Imperial(College(London(using(HumanCytoSNPW12(v2.1(DNA(Analysis(BeadChip(Kits( (* (Illumina)(according(to(the(Illumina(GoldenGate(Genotyping(assay(guide. (

2.8+Phenotyping†++

For(the(UK(cohorts(a(deep(phenotyping(strategy,(utilising(all(available(clinical(records( was(employed.(For(EDS(patients,(a(customised(database(was(created,(incorporating( all( 54( major( and( minor( criteria( for( EDS,( 24( Ghent( criteria( for( Marfan( syndrome,( 6( criteria( for( Osteogenesis( imperfecta( as( well( as( 596( standardised( human( phenotype( ontology( (HPO)( terms( divided( by( organ( system,( along( with( detailed( data( on( family( history( and( any( available( biochemical,( histological( and( genetic( diagnoses.(Table( 2.2( 62 38 and(Appendix(2).( ( ((

(((((((((((((((((((((((((((((((((((((((( ((((((((

*!Laurence!Game!&!team!provided!all!sequencing!services! †!Christina! Kanonidou! made! the! majority! contribution! to! phenotyping! in! EDS! and! led! the! design! and! implementation! of! the! EDS! phenotype! database;! Yousef! Ibrahim! led! the! phenotyping! for! the! UK! TAAD! cohort;!Phenotype!data!for!the!Yale!TAAD!cohort!was!provided!by!Bulat!Ziganshin! ! 32! (

Table+2.2+Phenotype(data(collected(for(EDS*(

Domain+ Phenotypes+recorded+ No.+of+criteria+ screened+for+

Demographics( Age(at(presentation((( ( &(General( Gender( ( criteria( Ethnicity( 17(ethnicities(( Family(history( ( Main(EDS(diagnosis( 23(diagnoses(

Cardiovascular(system( 38(HPO(terms( General( Abdomen(&(GI(tract( 24(HPO(terms( Phenotypic( Blood(and(bloodWforming(tissues( 10(HPO(terms( characteristics( Genitourinary(system( 16(HPO(terms( Head(&(Neck( 34(HPO(terms( Integument(&(connective(tissue( 35(HPO(terms( Respiratory(system( 6(HPO(terms( Skeletal(system(&(musculature( 84(HPO(terms( Nervous(system(&(Eye( 58(HPO(terms( Other( 20(HPO(terms(

Specific( Beighton(score((for(Hypermobility)63( ( syndromic( criteria( Villefranche(major(&(minor(criteria((for(EhlersWDanlos( 54(criteria( Syndrome)( ( Ghent(criteria((Marfan(syndrome)( 24(criteria( Osteogenesis(imperfecta((Sillence(criteria)( 6(criteria(

Investigations( Collagen(protein(analysis((electrophoresis)( ( Histology((light(and(electron(microscopy)( Urinary(crossWlink(assay((for(PLOD1)( Results(of(previous(genetic(testing(

HPO,(human(phenotype(ontology,64(see(http://humanWphenotypeWontology.github.io/((

(

( (

(((((((((((((((((((((((((((((((((((((((( (((((((( * Christina! Kanonidou! made! the! majority! contribution! to! phenotyping! in! EDS! and! led! the! design! and! implementation!of!the!EDS!phenotype!database!(see!Appendix!2!for!detailed!database!structure).! ! 33! For( TAAD,( a( detailed( phenotype( database( was( compiled( along( four( key( phenotypic( domains,( with( an( emphasis( on( phenotypes( known( to( be( associated( with( the( TAAD( spectrum( (summarised( in( Table( 2.3,( elaborated( in( Appendix( 2).( For( the( Yale( TAAD( cohort,( these( phenotypes,( as( far( as( available,( were( extracted( from( an( existing( phenotypic(database.(

Table+2.3+Phenotype(data(collected(for(TAAD*+

Domain+ Phenotypes+recorded+

Demographics( Age(at(presentation((( Gender( Ethnicity(

Medical(History( Cardiovascular(risk(factors((hypertension,(smoking(history,( hypercholesterolaemia,(diabetes(mellitus,(peripheral(vascular(disease)(

CoWexistence(of(valvular(heart(disease((aortic(regurgitation(and(mitral( valve(prolapse)(

Presence(of(specific(cardiovascular(developmental(anomalies((aberrant( subclavian(artery,(bicuspid(aortic(valve,(bovine(aortic(arch,(coarctation(of( the(aorta)(( Known(genetic(diagnoses(or(syndromic(features( Family(history(of(aortic(disease(or(sudden(cardiac(death((

Aortic(Anatomy(&( Aortic(pathology((aneurysm,(dissection,(intramural(haematoma,(aortic( Pathology( ulcer)((

Anatomical(extent(and(classification((based(on(Stanford(and(Crawford( classification(systems)(

Presence(of(multifocal(aneurysmal(disease((intraW(and(extraWaortic)(

Size(of(aneurysms(at(presentation((cm)(

Average(estimated(growth(rate(of(aneurysms((cm/year)(

Management(&( Mode(of(operative(intervention:(open(or(endovascular(surgery( Prognosis(

Rate(of(serious(and(commonly(associated(complications((endoleaks,( myocardial(infarction,(cerebrovascular(accident,(renal(failure((permanent( or(requiring(haemofiltration),(pneumonia,(deep(vein(thrombosis,(pulmonary( embolism,(paraplegia(and(death)(

( +

(((((((((((((((((((((((((((((((((((((((( (((((((( * !Yousef!Ibrahim!led!the!phenotyping!for!the!UK!TAAD!cohort;!Phenotype!data!for!the!Yale!TAAD!cohort!was! provided!by!Bulat!Ziganshin!(see!Appendix!2!for!detailed!database!structure)! ! 34! 2.9+Data+analysis+

* 2.9.1+Analysis+of+NGS+data:+read+mapping,+variant+calling+and+annotation + The( quality( of( NGS( reads( was( assessed( with( FastQC( v.0.11.2.65( Primer( sequences( were(removed(from(the(reads(using(cutadapt(v.1.7.1,(using(anchored(adapters(option.( Maximum(error(rate(allowed(per(primer(was(set(to(10%.(After(removal(of(primers,(reads( were(mapped(to(a(set(of(sequences(derived(from(the(human(GRCh37(assembly.(This( GRCh37Wderived(alignment(set(includes(chromosomal(plus(unlocalized(and(unplaced( contigs,( the( rCRS( mitochondrial( sequence( (AC:NC_012920),( Human( herpesvirus( 4( type( 1( (AC:NC_007605)( and( decoy( sequence( derived( from( HuRef,( Human( Bac( and( Fosmid( clones( and( NA12878.( (This( reference,( hs37d5( was( used( for( final( 1000( Genomes( alignments).( Reads( were( mapped( to( the( GRCh37/hg19( human( reference( sequence(using(BWAWMEM(v0.7.2.66(Picard(tools(v.1.85(and(GATK(v.3.3W0(were(used( to(collect(various(alignment,(coverage(and(quality(statistics.(

Sequence( reads( were( realigned( around( insertions/deletions( and( base( call( quality( scores( recalibrated( with( GATK( v2.6W5.67( SingleWnucleotide( variants( (SNVs)( and( small( insertions/deletions(were(called(with(GATK(UnifiedGenotyper.(Variant(annotation(was( carried( out( with( ANNOVAR( version( 2013aug2368( and( was( based( on( the( RefGene,( Genecode( and( Ensembl( transcripts.( The( annotations( included:( phastCons,69( GERP++,70(PhyloP,71(SiPhy72(conservation(scores,(minor(allele(frequencies(in(the(1000( genomes( (phase( 2( release)73( and( the( NHLBI( exome( sequencing( project74( data( sets,( Clinvar( references75( and( pathogenicity( prediction( from( SIFT,76( Polyphen2,77( MutationTaster,78( MutationAssessor,79( FATHMM,80( and( CADD.28( Polyphen2( scores( were( on( a( scale( from( 0W1.0( (1.0( indicating( highest( predicted( pathogenicity).( MutationTaster,(MutationAssessor(and(FATHMM(scores(were(converted(to(the(same( 0W1.0(scale.(Further(specific(filtering(of(variant(calls(are(elaborated(in(ensuing(chapters.(

2.9.2+Analysis+of+capillary+(Sanger)+sequencing†+

Sequence(data(generated(by(capillary(sequencing(was(analysed(using(the(Sequencher( software(version(4.8((GenCodes,(USA).(

(((((((((((((((((((((((((((((((((((((((( (((((((( *!All! NGS! data! were! generated! by! Laurence! Game! and! team.! For! most! cases! Michael! Mueller! and! Dalia! Kasperaviciute! carried!out!the!initial!analysis!of!raw!NGS!data!including! read!mapping,! variant!calling!and! annotation!(Ch.2.9.1);!for!the!first!few!batches,!I!carried!out!variant!annotation!helped!by!Jana!Vandrovcova.!! † !Sanger!sequencing!analysis!was!done!by!the!individual!who!performed!the!sequencing!(see!Ch.2.6).!!! ! 35! * 2.9.3+Linkage+analysis +

Initial( processing( of( linkage( data( was( carried( out( using( Illumina( GenomeStudio( software:(to(produce(output(W.map,(W.ped(and(W.dat(files((W.map(files(thinned(to(2.0(SNPs( per( centimorgan(cM)( using( MapThin( software).( Merlin( linkage( analysis( software( was( used(for(linkage(analysis.((

2.9.4+Statistical+analysis+

χ2(and(Fisher’s(exact(tests(were(used(to(examine(if(there(were(significant(differences(in( categorical( variables( between( individuals( in( different( genotype( or( phenotype( groups.& The( MannWWhitney( test( was( used( to( assess( statistical( differences( between( in( silico( prediction(scores((Chapter(3.3.6)(as(the(total(number(of(variables(tested(was(low(and( therefore(could(not(be(assumed(to(follow(a(normal(distribution.((

(((((((((((((((((((((((((((((((((((((((( ((((((((

* !This!was!done!by!Jana!Vandrovcova! ! 36! 3.+TARGETED+SEQUENCING+IN+EDS+

3.1+Introduction+

EhlersWDanlos( syndrome( (EDS)( comprises( a( number( of( overlapping( hereditary( disorders(of(connective(tissue((HDCT).38,39,81(The(predominant(traits(are(skin(fragility,( easy(bruising(and( joint( hypermobility39( and,( due(to( the( generalised(connective(tissue( weakness,( blood( vessels( and( internal( organs( can( also( be( affected.( Vascular( EDS( patients( can( present( at( any( age( W( sometimes( as( early( as( the( neonatal( period( W( with( vascular( aneurysm( or( rupture( or( sudden( spontaneous( rupture( of( the( bowel( or( other( viscera( and( 80%( of( these( patients( will( have( such( a( complication( by( the( age( of( 40( years.39,50,81( Effective( clinical( management( depends( upon( obtaining( an( accurate( diagnosis.( However,( EDS( represents( a( diagnostic( challenge( to( the( clinical( geneticist( owing(to(its(phenotypic(complexity(and(overlap(between(syndromic(subtypes.(

Traditionally,(the(classification(of(EDS(has(been(based(mainly(on(clinically(discernible( phenotypes.( At( the( time( of( writing,( EDS( was( classified( by( the( 1997( Villefranche( nosology38((Table(3.1),(which(is(the(nomenclature(I(shall(be(using(in(this(thesis`(very( recently( this( has( been( updated( to( the( 2017( International( Classification.82,( which( is( largely( based( on( the( former( but( takes( into( account( new( genotypeWphenotype( relationships(including(work(outlined(in(this(thesis.((The(three(major(EDS(groups(are( classical,(vascular(and(hypermobility(type(EDS,(whilst(others,(including(kyphoscoliosis,( dermatosparaxis,(arthrochalasia(are(extremely(rare.38,39((

The(prevalence(of(specific(vascular(complications,(including(aortopathy,(in(EDS(is(not( well( documented( in( the( literature.( Vascular( complications( are( not( restricted( to( the( vascular( type,( though( they( tend( to( predominate( and( be( more( aggressive( in( vascular( EDS,( where( they( are( major( contributor( to( the( reduced( lifeWexpectancy.50( In( a( phenotypic( study( of( 419( vascular( EDS( patients,( Pepin( et( al( identified( 272( arterial( complications(in(139((33%)(of(patients,(of(which(approximately(a(half((15%)(had(aortic( involvement.50(In(other,(much(smaller(studies(of(vascular(EDS(cases,(aortopathy(has( been( observed( in( 5( W( 13%( of( cases,( or( accounting( for( 10W50%( of( all( vascular( complications.83W85( In( other( types( of( EDS,( the( presence( of( aortic( dilatation( has( been( observed( in( upto( 33%( of( cases.85,86( There( is( likely( to( be( considerable( underW ascertainment(of(vascular(complications(in(EDS,(particularly(outside(the(vascular(EDS( type.((

The( central( pathological( abnormality( in( EDS( affects( collagen( packing( or( stability,( mainly( relating( to( collagen( types( I,( III,( and( V.( To( date( there( is( an( established(

! 37! understanding( of( genotypeWphenotype( correlation( in( a( proportion( of( cases,( based( on( Villefranche(criteria(with(most(classical(EDS(cases(caused(by(defects(in(collagen((Col.)( V((COL5A1,&COL5A2),(most(vascular(EDS(cases(caused(by(defective(Col.III((COL3A1)( and( Col.I( (COL1A1,& COL1A2)( defects( underlying( EDS( cases( that( overlap( with( osteogenesis(imperfecta(including(one(very(rare(EDS(type,(arthrochalasia(type.39,50(A( number( of( other( genes( encoding( extracellular( matrix( & (PLOD1,& CHST14,& FKBP14I& RIN2I& PRDM5,& ZNF469,& B4GALT7,& SLC39A13)( are( reported( as( causes( of( additional( very( rare( presentations.39( As( Table( 3.1( outlines,( however,( the( majority( of( cases( remain( genetically( unresolved.( Whilst( identifying( genetic( mutations( in( known( genes( in( individual( cases( provides( a( reasonable( indication( of( the( underlying( pathogenesis,( the( limitation( with( a( clinicalWphenotype( based( diagnostic( system( or( indeed( phenotypeWdriven( genetic( testing( is( the( phenotypic( heterogeneity( and( clinical( overlap(between(EDS(types.(Furthermore,(with(the(historical(use(of(phenotypeWdriven( Sanger(sequencing(for(genetic(testing,(the(majority(of(EDS(cases(remained(without(a( molecular( diagnosis38,39( and( the( mutational( spectrum( in( known( genes( and( their( correlation(with(clinical(phenotype(in(a(mixed(cohort(of(EDS(cases(was(unknown.(

The(aim(of(this(project(was(to(screen(a(mixed(cohort(of(EDS(patients(for(mutations(in(a( number( of( relevant( genes( in( parallel,( unbiased( by( preconceived( phenotype( or( by( previously(diagnosed(genetic(abnormalities.(For(this(I(developed(an(NGSWbased(panel( to(sequence(the(exons(of(key(collagen(genes(and(certain(other(genes(known(to(cause( EDS(or(related(connective(tissue(conditions:(COL3A1,(COL5A1&&COL5A2,(COL1A1(&& COL1A2,( FBN1,& TGBFR1,& TGFBR2,& ACTA2,& MYH11,& SMAD3,& MYLK.( The( aim( was( therefore(to(compare(the(mutational(yield(and(spectrum(in(these(genes(as(determined( by( NGS,( with( the( phenotype( spectrum( and( with( previously( diagnosed( genetic( abnormalities((carried(out(by(the(phenotypeWdriven(Sanger(method):(thus,(I(sought(to( ascertain( whether( this( approach( would( increase( the( proportion( of( genetic( diagnoses( and(help(improve(understanding(of(the(relationship(between(genotype(and(phenotype( in(EDS.(

( (

! 38! Table+3.1.+Classification(of(EDS(based(on(1997(Villefranche(classification38+

a Type+ ! Major+clinical+criteria+ Approx.+ Inherit Normally+associated+ prevalence+ ance! b+ + genes+

Classical((I/II)(49( Skin(hyperextensibility,(Widened( (1:20,000( AD( COL5A1,(COL5A2&+Q++ atrophic(scars,(Joint(hypermobility+((( (>50%)&

Hypermobility((III)( Generalised(Joint(hypermobility`(Skin( >1:10,000( AD( TNXB((W((<1%)+( 87,88(89( hyperextensibility(and/or(smooth( velvety(skin`(((

Vascular((IV)(50( Arterial/(Intestinal(/Uterine(rupture`(thin,( (1:250,000( AD( COL3A1&&&Q&(60%)& translucent(skin(with(extensive(bruising`( characteristic(facies.(((

Kyphoscoliosis((VI)( Severe(muscle(hypotonia(at(birth`( 60(described( AR( PLOD1((Lysyl(hydroxylase)( 90W92( Scoliosis(at(birth,(Scleral(fragility(and( worldwide( –((<50%),+FKBP14(,+CHST14+ rupture(of(the(ocular(globe,(Generalised( joint(laxity((((

Arthrochalasia((VIIa,( Congenital(bilateral(hip(dislocation,(( 30(described( AD( COL1A1,(COL1A2&( b)(51( Severe(Generalised(joint(hypermobility`((( worldwide(

Dermatosparaxsis( Severe(skin(fragility`(sagging,( 12(described( AR( ADAMTS2(( (VIIc)(51( redundant(skin(( worldwide( (Procollagen(NWpeptidase)( (

Miscellaneous+(extremely+rare+types+and+unspecified+forms)++

XWlinked(EDS((type( ( 1(pedigree( XL( no(specific(genetic(cause( V)(93( identified( ( (

Periodontitis(type( ( 7(pedigrees( AD( no(specific(genetic(cause( (type(VIII)(94( described(( identified,(but(linkage(to( 12p13(in(two(pedigrees(

FibronectinWdeficient( ( 1(pedigree( AR( ( EDS((type(X)(95( described( &

Progeroid(EDS((96( ( ( ?( B4GALT7& (

Unspecified(forms(( ( ( W( ( e.g.( SLC39A13( Spondylocheirodysp lastic((97(

Familial( Common(syndrome(overlapping(with( ( AD( ( hypermobility( EDS(III( syndrome((type(XI)(

AD,autosomal(dominant`(AR,(autosomal(recessive`(XL,XWlinked`(?,inheritance(pattern(unknown.(a.+EDS(type(is(according( to(Villefranche(classification,(Roman(numerals(in(paranthesis(indicate(previous(classifiation(according(to(the(1986(Berlin( nosology((EDS(types(I(W(IX).+b.+Overall,(the(majority(of(cases(comprising(Hypermobile(Type((III)(and(unspecified(foms(do( not(have(a(mutation(in(the(corresponding(usually(associated(genem+the+figure(for(PLOD1(in+Kyphoscoliosis(type((VI)(EDS(is( unestablished(in(the(literature(but(based(on(unpublished(data(from(approx.(15(cases(seen(in(the(National(EDS(Diagnostic( Service(UK.!(

( + ! 39! 3.2+Methods+

3.2.1+Selection+of+cases+with+EhlersQDanlos+syndrome+

177(unrelated(patients(with(suspected(EDS(referred(by(specialist((tertiary)(clinicians(to( the( National( EDS( Diagnostic( Service( (London,( UK)( were( selected( (Table( 3.2).( The( patients(were(predominantly(female((67%)(and(Caucasian((89%).(The(mean(age(of(the( cohort(was(33.6(years((range(2W78).(I(selected(cases(from(a(broad(range(of(presenting( clinical( phenotypes,( with( the( aim( of( reflecting( the( whole( spectrum( of( EDS( types( referred(to(the(national(referral(centre((see(Results,(Ch.3.3.1).(Patients(with(vascular( complications( were( preferentially( recruited( to( the( study,( because( of( their( prognostic( importance.(This(was(done(irrespective(of(previously(determined(genetic(abnormalities,( as(one(aim(of(the(study(was(to(compare(the(results(of(our(NGS(panels(with(previously( detected( genetic( mutations( by( the( traditional( method( of( phenotypeWdriven( Sanger( sequencing.(

Clinical(diagnoses(upon(first(interview,(based(only(on(clinical(features,(were(as(follows:( classical( EDS( (or( overlapping( syndrome)( 12%,( vascular( EDS( (or( overlapping( syndrome)( 12%,( EDS( hypermobility( type( or( benign( joint( hypermobility( syndrome( (BJHS)( 43%,( kyphoscoliotic( EDS( 2%.( In( addition,( 7%( of( cases( had( a( phenotype( overlapping(multiple(EDS(types,(not(falling(into(one(specific(EDS(type,(which(I(termed( Complex( EDS.( A( further( 24%( of( cases( had( features( predominantly( outside( the( EDS( spectrum(and(termed(either(“Other(hereditary(disorder(of(connective(tissue((HDCT)”(for( those(cases(with(nonWspecific(connective(tissue(signs(outside(the(EDS(spectrum(and(98( “Other( HDCT( (vascular)”( for( those( with( a( prior( history( of( an( arterial( complication( (defined(as(one(or(more(of:(aortic(or(peripheral(arterial(aneurysm,(dissection(or(rupture,( cerebral( aneurysm( /( subarachnoid( haemorrhage).( Clinical( categorisation( was( based( upon( 1997( Villefranche( criteria38( by( one( or( more+ specialist( EDS( clinicians( at( the( National( EDS( Diagnostic( Service,( London:( Professor( F.M.( Pope,( Dr( Anthony( Vandersteen,( Dr( Neeti( Ghali( ( (combined( experience( 51( years).( The( detailed( clinical( phenotypes(of(patients(in(each(group(and(their(relationship(to(the(existing(Villefranche( diagnostic( criteria( were( examined( and( deconstructed( (see( Results,( Ch.( 3.3.1).( 76( relatives( of( the( index( partients( were( also( phenotyped( and( recruited( to( the( study( for( segregation(analysis.(

! 40! 3.2.2+ Characterisation+ of+ clinical,+ biochemical,+ histological+ and+ ultrastructural+phenotype*+

Detailed(phenotypic(data(including(relevant(diagnostic(scores38,99(as(well(as(results(of( histological( and( collagen( protein( analysis( were( derived( along( with( results( of( clinical( genetic(testing(previously(carried(out(by(conventional(Sanger(sequencing(as(previously( described((chapter(2).(

Collagen(protein(analysis:(Fibroblast(cultures(were(prepared(from(skin(biopsies(taken( from( patients’( upper( inner( arms.( The( culture( of( fibroblasts,( collagen( labeling( and( sodium(dodecyl(sulfate(polyacrylamide(gel(electrophoresis((SDSWPAGE)(analysis(were( performed( as( described( previously.100( Light( Microscopy( (LM):( Dermal( tissue( blocks( stained( with( Haematoxylin( &( Eosin( /( Elastin( Van( Gieson,( were( assessed( by( light( microscopy( to( determine( changes( in( dermal( thickness( and( collagen:elastin( ratio.( Electron(microscopy((EM):(Skin(biopsies(from(the(upper(inner(arm(were(placed(directly( into(4%(glutaraldehyde(in(0.1M(phosphate(buffer(and(processed(for(routine(electron( microscopy(as(described(previously(with(the(following(modifications.101(Samples(were( orientated( to( allow( longitudinal( sections( through( the( depth( of( epidermis( and( dermis.( Thin( sections( (stained( with( uranyl( acetate( and( lead( citrate)( were( examined( for( ultrastructural( abnormalities( of( collagen( fibril( size( and( arrangement( and( the( appearances(of(the(endoplasmic(reticulum(within(the(fibroblasts(

3.2.3+DNA+extraction+and+targeted+exon+sequencing++

Two( Fluidigm( assays( were( designed( to( sequence( the( exons( and( exonWintron( boundaries(of(genes(commonly(associated(with(EDS(and(overlapping(phenotypes:((1)( Collagen(NGS(panel:(375(primerWpairs(for(COL1A1,(COL1A2,(COL3A1,(COL5A1(and( COL5A2`( and( (2)( Aortopathy( (“TAADWX”)( NGS( panel:( 363( primerWpairs( for& FBN1,& TGFBR1,& TGFBR2,& MYH11,& ACTA2,& SMAD3& and& MYLK.& (see( Chapter( 2.1( and( Appendix(1,(Tables(S1(&(S2).(

DNA(extraction(from(saliva(&(blood(samples,(normalisation(for(targeted(sequencing(by( the(Fluidigm(Access(Array(system(&(Miseq(next(generation(sequencer(and(mapping(of( NGS(reads,(followed(by(variant(calling(and(annotation(were(carried(out(as(previously( described((Chapters(2.4(&(2.9).(+

(((((((((((((((((((((((((((((((((((((((( (((((((( * !Fibroblast!cultures!and!protein!electrophoresis!in!all!the!cases!reported!in!this!chapter!was!carried!out!by! Dr!Philip!Sawle!at!the!National!EDS!Diagnostic!Service,!Northwick!Park!Hospital,!London;!Microscopy!was! carried!out!by!Prof.!David!Ferguson!at!the!Nuffield!Division!of!Clinical!and!Laboratory!Sciences,!University!of! Oxford.! ! 41! 3.2.4+Pathogenicity+assignment+

Synonymous( variants,( intronic( variants( located( outside( exon/intron( boundaries( and( SNVs(with(allele(frequency(>(0.1%(in(1000(Genomes(Project((phase(II(release)(or(the( NHLBI(exome(sequencing(project(data(sets((as(available(in(the(2013Aug23(version(of( Annovar)( were( excluded( from( further( analysis( as( the( aim( was( to( identify( rare,( functionally( significant( variants,( causing( significant( alteration( to( protein( primary( structure.(Splice(siteWdisrupting(variants,(nonsense(and(frameshift(coding(variants(with( allele(frequency(<0.1%(were(considered(pathogenic.(Other(variants(previously(reported( as( pathogenic( in( established( variant( databases( (LOVD102( and( HGMD103)( were( considered( pathogenic( unless( biochemical( (collagen( protein( analysis),( phenotype( and/or( segregation( data( suggested( otherwise.( Missense( variants( were( classified( according( to( American( College( of( Medical( Genetics( and( Genomics( (ACMG)( guidelines,29(with(the(special(additional(case(of(glycine(substitutions(in(collagen(helical( domains,( which( have( the( effect( of( disrupting( helix( formation( and( are( therefore( considered( to( be( pathogenic( because( of( loss( of( function.( All( other( variants( were( classified( as( variants( of( uncertain( significance( (VUS)( and( were( further( categorised( based( on( available( evidence( according( to( ACMG( criteria.29( Filtered( variants( were( validated( by( Sanger( sequencing( and( submitted( to( the( LOVD( database.102((A( single( falseWpositive(variant(was(found(in(exon(1(of(SMAD3(((ID(382)(and(was(removed(from( further(analysis.(

3.3+Results+

3.3.1+Clinical+phenotype++

Table(3.2(reflects(the(whole(range(of(phenotypes(as(initially(diagnosed(on(referral(to( the(National(EDS(diagnostic(service,(London((note,(the(numbers(of(each(EDS(subtype( do( not( reflect( their( relative( frequencies( in( the( general( population( as( this( is( not( a( sequential(or(representative(cohort:(cases(referred(to(the(clinic(tend(to(be(enriched(for( more( complex( and( prognostically( important( types).( For( each( EDS( subtype,( it( was( evident(that(a(large(proportion(of(cases(also(met(the(diagnostic(criteria(for(other(EDS( types,(reflecting(the(phenotypic(heterogeneity(and(overlap(between(these(groups.(As( per(previous(reports,(it(was(also(noteworthy(that(the(presence(of(vascular(complication( does( not( necessarily( coincide( with( the( presence( of( Vascular( EDS,( though( vascular( complications(were(enriched(in(the(Vascular(EDS(group((p=0.002).(((

Eighteen( patients,( distributed( across( diagnostic( groups( had( marfanoid( features( (of( which(two(had(Ghent(Marfan(systemic(scores(≥7).(Four(patients(diagnosed(as(Other( HDCT( had( signs( consistent( with( osteogenesis( imperfecta( (OI)( or( an( overlapping(

! 42! phenotype.(Fifty(per(cent(of(patients(with(an(initial(clinical(diagnosis(of(vascular(EDS( had(arterial(complications(at(presentation,(compared(with(17.5%(of(other(patients(with( EDS((p(=(0.001`(Table(3.2).(

(

Table+3.2+Clinical(spectrum(of(cohort(

c Initial+Clinical+ No.+of+ Mean+ Mean+ No.+(%)+meeting+Villefranche+EDS+criteria + Arterial+ a d Diagnosis+ + cases+ Age+ Beighton+ complication+ + (range)+ score+

Classical+ Vascular+ +++HM+ KPS+ + a Classical+ + 21( 34.8( 7.3( 21( 3((14%)( 15((71%)( 0( 2((10%)( (19W78)( (100%)(

30.7(( a 22( 4.8( 5((23%)( 21((95%)( 10((45%)( 0( e( Vascular+ + (6W48)( 11((50%)(

32.7(( Hypermobility++ 76( 5.8( 21((28%)( 14((18%)( 73((96%)( 8((11%)( 14((20%)( (2W67)(

Rare+&+ 22(( b 16( 6.2( 7((44%)( 4((25%)( 7((44%)( 6((38%)( 0( Complex+EDS! ++ (2W55)(

32.6( Other+HDCT+ 11( 6.0( 5((45%)( 1((9%)( 8((73%)( 3((27%)( 0( ((4W52)(

Other+HDCT+ 43.4( 31( 4.5( 11((35%)( 18((58%)( 22((71%)( 1((3%)( 31((100%)( (vascular)+ ((8W72)(

EDS,(EhlersWDanlos(syndrome`(HM,(EDS(Hypermobility(type,(KPS,(kyphoscoliosis(type(of(EDS`(HDCT,(hereditary(disorder( of(connective(tissue.( a.( Initial( Clinical( Diagnosis( refers( to( the( most(applicable(classification(at(first(specialist(EDS(consultation:( classical( cases( included( 9( cases( of( classical/hypermobility( type( overlap,( vascular( cases( included( 3( vascular/hypermobile( and( 2( vascular/classical( overlap.( b.( Rare( EDS( refers( to( EDS( types( outside( classical,( vascular( and( hypermobility( types.+ c.+ Villefranche(classification:(numbers((percentages)(indicate(the(number((percent)(of(patients(in(each(group(meeting(at(least( one( major( criterion( for( each( EDS( type.( d.( Arterial( complication:( history( of( one( or( more( of( aortic( or( peripheral( arterial( aneurysm,( dissection( or( rupture,( cerebral( aneurysm( /( subarachnoid( haemorrhage( at( first( EDS( consultation.( e.( p( =( 0.002( (vascular(vs.(other(EDS).(+

3.3.2+Results+of+previous+genetic+testing+by+Sanger+sequencing+

As(one(aim(of(the(study(was(to(compare(the(results(of(NGS(sequencing(with(previously( determined( genetic( abnormalities( by( the( Sanger( method,( we( then( determined( the( number( and( pattern( of( genetic( mutations( previously( identified( in( this( cohort.( Prior( genetic( testing( in( the( clinical( service( by( Sanger( sequencing( was( phenotypeWdriven,( usually(by(sequencing(one(or(two(of(the(most(likely(causative(genes,(based(on(clinical( and(laboratory(data,(followed(by(sequencing(of(additional(plausible(candidate(genes(in( some(cases.((

Reviewing( prior( genetic( test( results( by( the( traditional( method( in( this( cohort,( the( frequency(of(a(detected(mutation(in(the(anticipated(pathogenic(gene(was:(33%((7/21)( of( cases( clinically( diagnosed( with( classical( EDS( or( an( overlapping( phenotype,( 50%( ! 43! (11/22)( of( patients( initially( diagnosed( as( vascular( EDS( and( in( the( 4( patients( with( a( clinical(phenotype(of(EDS/OI(the(frequency(of(COL1A1/2(pathogenic(variants(was(75%( (Table(3.3).(Outside(of(the(anticipated(pathogenic(gene,(further(DNA(testing(revealed(a( specific( DNA( mutation( in( only( 7.8%( (12/155).( In( addition( to( these( short( variants( in( collagen(genes,(a(complex(rearrangement(was(identified(causing(complete(allelic(loss( of( COL3A1& && COL5A2( (Patient( ID( 444)( and( pathogenic( variants( were( identified( in( TNXB((Patient(ID(67),(FBN2((Patient(ID(1125)(and(FKBP14&(homozygous,&Patient(ID( 822)((Table(3.4).(Pathogenic(or(potentially(pathogenic(variants(were(also(identified(in( three(other(patients:(in(FBN1((Patient(ID(66),(TGFBR1&(Patient(ID(706),(and(SMAD3& (Patient(ID(382)((Table(3.4).(Several(incidental(genetic(abnormalities(not(considered(to( be(contributory(to(EDS(phenotype(were(also(observed,(including(a(heterozygote(VUS( in( TNXB( (Patient( ID( 79),( FLNA( (Patient( ID( 538)( mutation( and( 0.1Mb( polymorphic( deletion(identified(by(karyotyping((Patient(ID(801)((Table(3.4).((

3.3.3+Results+of+collagen+protein+analysis+&+microscopy+

Amongst(those(EDS(cases(undergoing(microscopy,(the(percentage(with(abnormalities( was( higher( for( classical( (73%)( and( vascular( EDS( (69%)( than( for( hypermobility( EDS( (27%)( (p=0.008( and( p=0.03( respectively,( Table( 3.3).( Amongst( those( undergoing( biochemical( analysis,( patients( with( an( initial( diagnosis( of( vascular( EDS( had( a( higher( proportion( of( abnormality( in( the( corresponding( collagen( protein( (64%)( than( those( diagnosed(as(classical(EDS((11%`(p(=(0.0281)((Table(3.3).((

+ +

! 44! Table+3.3+Previously(identified(genetic(and(pathological(abnormalities(

b Initial+Clinical+ No.+of+ +Gene+affected+ + Collagen+ LM/EM+ a c Diagnosis+ + cases+ abnormality+ + abnormality+ COL1A1& COL5A1& COL3A1& Other( COL1A2& COL5A2&

d Classical++ 21( 0/3( 1/7( 7/11( 1/2( 1/9((11%)( 11/15((73%)( (

Vascular++ 22( 0/1( 11/17( 0/1( 2/8( 7/11((64%)( 9/13((69%)(

Hypermobility++ 76( 0/6( 0/24( 0/11( 0/5( 0/21( 6/22((27%)(

Rare+&+ 16( 0/2( 0/6( 0/3( 1/1( 0/1( 3/7((43%)( Complex+EDS++

e Other+HDCT++ 11( 3/6( 0/1( 0/0( 0/2( 3/5((60%)( ( 5/6((83%)(

Other+HDCT+ 31( 0/4( 0/23( 0/5( (3/16( f 9/18((50%)(( (vascular)+ 3/9((33%)( ((

LM,(light(microscopy.(EM,(electron(microscopy.(HDCT,(hereditary(disorder(of(connective(tissue.(( a.(Initial(Clinical(Diagnosis(refers(to(most(applicable(classification(at(first(specialist(EDS(consultation((as(in(Table( 3.2).( b.( Pathogenic( variants( identified( by( Sanger( sequencing( (numerator,( number( of( pathogenic( variants`( denominator,( number( of( separate( genetic( tests( carried( out( in( each( category.( c.( Abnormality( of( the( corresponding( collagen( type( on( SDSWPAGE.( d.( Four( of( these( cases( had( collagen( rosettes( on( EM.( e.( These( three(patients(had(abnormalities(of(collagen(I(had(phenotypes(overlapping(with(osteogenesis(imperfecta`(two( had(pathogenic(variants(in(COL1A1/COL1A2.(f.+Two(of(these(three(cases(had(collagen(III(deficiency`(the(third( case(had(collagen(I(deficiency`(none(had(rare(variants(in(the(corresponding(genes.(( ( (

! 45! 3.3.4+Targeted+NGS+sequencing+

Amplicons(of(the(Collagen(panel(achieved(a(mean(coverage(of(602×(with(95.0%(bases( covered(above(100×,(whilst(those(of(the(Aortopathy(panel(achieved(a(mean(coverage( of( 926×( per( amplicon( with( 97.4%( targeted( bases( covered( by( >100( reads.( NGS( sequence(data(were(analysed(blind(to(previous(genetic(testing.(The(mean(number(of( variants( called( per( sample( was( 12.7( (range( 5W( 25)( and( 28.7( (range( 9W51)( with( the( Collagen(and(Aortopathy(NGS(panels,(respectively.(After(filtering(of(variants(by(allele( frequency( and( variant( type( as( described( in( the( Methods,( a( total( of( 28( pathogenic( variants,(4(likely(pathogenic(variants(and(22(VUS’s(were(identified.(These(are(listed(in( Tables( 3.4( (Pathogenic)( and( 3.5( (Likely( Pathogenic( &( VUS’s)( whilst( Table( 3.6( compares( the( NGS( findings( with( previously( identified( phenotype( and( genetic( background(in(detail.(

3.3.5+Pathogenic+variants+identified+by+NGS+

We(identified(pathogenic(variants(in(nine(cases((41%)(of(the(21(categorised(initially(as( classical( EDS( (including( nine( diagnoses( of( classical/hypermobility( overlap):( seven( in( the( COL5A1& or& COL5A2& genes( and( two( in( COL3A1( (Table( 3.4).( The( first( of( these( (Patient(ID(417)(had(overlapping(features(of(classical,(hypermobility(and(vascular(EDS`( the( second( (Patient( ID( 636)( had( convincing( clinical( features( entirely( consistent( with( classical(EDS((Table(3.7).(In(the(22(patients(initially(diagnosed(with(vascular(EDS,(11( had( pathogenic( variants( in( COL3A1,( and( 1( patient( with( overlapping( features( of( vascular( and( classical( EDS( had( a( pathogenic( variant( in( COL5A1( (functional( corroboration(was(not(possible(because(the(patient(declined(skin(biopsy—ID(1088,(see( Table(3.7).(Two(unrelated(patients((ID(405(and(765)(both(had(an(identical( variant( of( COL3A1( (c.1662+1G>A,( Table( 3.4).( One( of( the( patients( with( an( initial( diagnosis( of( hypermobilityWtype(EDS((ID(824)(carried(a(pathogenic(COL5A1(variant((Table(3.4(and( 3.7).104( Of( the( patients( with( an( initial( clinical( diagnosis( of( other( HDCT,( three( had( pathogenic(variants(in(COL1A1&(all(three(diagnosed(clinically(with(EDS/OI(overlap)&and( a(further(three(had(pathogenic(variants(in(FBN1((Tables(3.4(&(3.8).( + +

! 46! Table+3.4+Pathogenic(variants(identified(by(the(Collagen(and(Aortopathy(NGS(panels(

Initial+ ID+ Gene+ Variant+ Functional+ Novel/reported:+ Sanger+ b Clinical+ affected+ category+ phenotype+ detected+ + a Diagnosis+ +

Classical( 636( COL3A1& c.2329G>C:(p.Gly777Arg( Missense( Novel( N( c Classical( 417( COL3A1&& c.1922_1923+2delAAGT( Splice(site( Reported:(vascular(EDS( ( Y( Classical( 582( COL5A1& c.(2034+1G>T( Splice(site( Novel( Y( Classical( 31( COL5A1&& c.2903delC:(p.Pro968LeufsX106( Frameshift( Novel( Y( Classical( 429( COL5A1&& c.757C>T:(p.Gln253X(( Stop(gain( Novel( Y( d Classical( 581( COL5A1&& c.4552C>T:(p.Gln1518X( Stop(gain( Reported:(Classical(EDS( (( Y( Classical( 627( COL5A1&& c.831C>A:(p.Tyr277X( Stop(gain( Novel( Y( Classical( 1129( COL5A1&& c.1670dupT:(p.Leu557fsX?( Frameshift( Novel( Y( Classical( 62( COL5A2&& c.3445G>T:(p.Gly1149Cys(( Missense(GlyXY( Novel( Y( e Vascular( 448( COL3A1& c.4319C>T:(p.Pro1440Leu(( Missense( Reported:(Vascular(EDS( ( Y( d Vascular( 765( COL3A1& c.1662+1G>A( Splice(site( Reported:(Vascular(EDS( ( Y( d Vascular( 37( COL3A1&& c.2564G>A:(p.Gly855Asp( Missense(GlyXY( Reported:(Vascular(EDS( (( Y( Vascular( 42( COL3A1&& c.2417C>T:(p.Pro806Leu(( Missense( Novel( Y( Vascular( 46( COL3A1&& c.1771G>C:(p.Gly591Arg( Missense(GlyXY( Novel( Y( d Vascular( 76( COL3A1&& c.2771G>A:(p.Gly924Asp( Missense(GlyXY( Reported:(Vascular(EDS(( (( Y( d Vascular( 405( COL3A1&& c.1662+1G>A( Splice(site( Reported:(Vascular(EDS(( ( Y( d Vascular( 483( COL3A1&& c.2553+1G>A(( Splice(site( Reported:(Vascular(EDS( ( Y( d Vascular( 733( COL3A1&& c.2816G>A:(p.Gly939Asp(( Missense(GlyXY( Reported:(Vascular(EDS( ( Y( Vascular( 420( COL3A1&&& c.1150W2A>T( Splice(site( Novel( Y( Vascular( 443( COL3A1&&& c.3525+1G>A( Splice(site( Novel( Y( Vascular( 1088( COL5A1& c.3164T>A:(p.Leu1055X( Stop(gain( Novel( N( Hypermobility( 824( COL5A1& c.4564G>T:(p.Gly1522Cys(( Missense( Novel( N(

Other( HDCT( 527( COL1A1&& c.1265delG:(p.Gly422AlafsX119( Frameshift( Novel( Y( (OI/EDS)(

g Other( HDCT( 36( COL1A1&& c.643G>A:(p.Gly215Ser( Missense(GlyXY( Reported:(OI/OIWEDS( ( Y( (OI/EDS)(

Other( HDCT( 559( COL1A1&& c.662G>C:(p.Gly221Ala( Missense(GlyXY( Novel( Y( (OI/EDS)( f Other( HDCT( 66( FBN1& c.3781T>A:(p.Tyr1261Asn( Missense( Reported:(Marfan( ( Y( (vascular)( h Other( HDCT( 378( FBN1& c.1775G>A:(p.Gly592Asp( Missense( Reported:(Marfan( ( N( (vascular)( i Other( HDCT( 766( FBN1& c.3373C>T:(p.Arg1125X( Stop(gain( Reported:(Marfan( ( N( (vascular)( EDS,( EhlersWDanlos( syndrome`( HDCT,( hereditary( disorder( of( connective( tissue( (phenotypes( further( elaborated( in( Table( 3.6)`( OI,( osteogenesis(imperfecta.(Missense(GlyXY,(substitution(of(a(Gly(residue(in(the(helical(domain(of(the(corresponding(collagen(subtype.( a.+Initial(Clinical(Diagnosis(refers(to(most(applicable(EDS(type(at(first(specialist(EDS(consultation((though(some(cases(overlap(multiple( EDS( types)`( b.( variants( detected( (Y)( or( not( detected( (N)( by( previous( clinical( diagnostic( testing( (Sanger( method)`( c.( c.1923+2_+5delTAAG( reported( pathogenic( in( LOVD`( d.( LOVD`( e.( Pro1440Ser( reported( as( pathogenic( in( LOVD( (Morissette( et( al.,( 2014)(and(Pro1440Leu(pathogenicity(supported(by(structural(prediction((Vandersteen,(unpublished)`+ f.(Y1261C((HGMD(CM990591),( Y1261D((HGMD(CM547000)`(g.(Vandersteen(et(al.,(2013((h.(HGMD(CM013919((i.(HGMD(CM055245(

! 47! 3.3.6+Variants+of+uncertain+significance+identified+by+NGS+

Of( the( 26( VUS’s( identified,( 22( were( dispersed( amongst( either( hypermobile( or( other( HDCT(patients((Table(3.5).(Four(of(these(variants(in(COL1A1,(TGFBR1,&TGFBR2&and& SMAD3,&were(categorised(as(likely(pathogenic,(and(are(further(detailed(in(Tables(3.7(&( 3.8.( One( of( these( patients( (ID( 893)( carried( a( helical( domain( Arg>Cys( variant( in( COL1A1:( similar( Arg>Cys( variants( have( been( shown( to( be( pathogenic( in( previous( reports.104

Although(two(particular(variants(in(COL1A2(and(COL3A1(had(previously(been(reported( as( pathogenic,( I( classified( these( as( VUS’s( (Table( 3.6).( Patient( ID( 629( carried( a( p.Arg708Glu(variant(of(COL1A2,(previously(reported(in(an(EDS(patient(in(LOVD.(This( was(classified(as(a(VUS(because(of(nonWsegregation(in(the(index(case’s(firstWdegree( relatives( (Table( 3.7).105( Patient( ID( 655( carried( the( Lys1313Arg( variant( in( the( CW propeptide( domain( of( COL3A1,( previously( reported( in( two( unrelated( patients( with( vascular(EDS(in(LOVD.(I(classified(this(as(a(VUS(because(there(was(no(biochemical( abnormality(of(collagen(III(or(clinical(evidence(of(vascular(EDS(in(the(index(case(or(her( sister( who( carried( the( variant.( Further,( structural( prediction( by( XWray( crystallography( deemed(it(unlikely(pathogenic.106(Two(hypermobility(EDS(patients(were(each(found(to( have( two( separate( VUS’s:( Patient( ID( 38( in( COL3A1& (p.Ile66Met)( and( COL5A2& (p.Lys743Thr)`( Patient( ID( 39( in( COL1A2& (p.Ile954Thr)( and( COL3A1( (p.Glu682Lys)( (Tables(3.5(&(3.6).(One(patient(initially(categorised(clinically(as(classical(EDS((ID(636)( carried(an(intronic(VUS(in(COL5A2,&but(also(carried(a(pathogenic(COL3A1( mutation( (Tables(3.4(&(3.5).(

( (

! 48! Table+3.5+Variants(of(uncertain(clinical(significance(identified(in(the(Collagen(and(Aortopathy(NGS(panels+

Initial+Clinical+ ID+ Gene+ Variant++ Functional+ Novel/reported:+ Sanger+ Diagnosis+ affected+ category++ phenotype+ detected+ Classical( 49( COL3A1& c.3511G>A:(p.Glu1171Lys( Missense( Novel( N(

a Classical( 636( COL5A2& c.1402W10T>G(( Intronic( (( Novel( Y(

Vascular( 444( COL1A1& c.3755G>A:(p.Arg1252His( Missense( Novel( N(

Vascular( 384( COL1A1&& c.3466A>G:(p.Asn1156Asp( Missense( Novel( Y(

b ( Hypermobility( 893( COL1A1& c.2980C>T:(p.Arg994Cys( ((Missense( ( Novel( (N( Hypermobility( 478( COL1A1& c.4315A>G:(p.Ile1439Val(( Missense( Novel( N(

Hypermobility( 828( COL1A1& c.3301G>A:(p.Glu1101Lys(( Missense( Novel( N(

Hypermobility( 39( COL1A2& c.2861T>C:(p.Ile954Thr( Missense( Novel( N(

Hypermobility( 558( COL1A2& c.1159G>C:(p.Ala387Pro(( Missense( Novel( N(

Hypermobility( 1151( COL1A2& c.4012C>T:(p.Arg1338Cys( Missense( Novel( N(

Hypermobility( 38( COL3A1& c.198A>G:(p.Ile66Met( Missense( Novel( N(

Hypermobility( 39( COL3A1& c.2044G>A:(p.Glu682Lys( Missense( Novel( N(

Hypermobility( 655( COL3A1&&& c.3938A>G:(p.Lys1313Arg( Missense( Reported:( Y( c Vascular(EDS( ( Hypermobility( 34( COL5A1& c.4068G>A:(p.Ala1356Ala(( Predicted( Novel( N( splice(site( disruption( Hypermobility( 66( COL5A1& c.805G>A:(p.Glu269Lys((( Missense( Novel( N(

Hypermobility( 734( COL5A1& c.3257C>T:(p.Ala1086Val(( Missense( Novel( N(

Hypermobility( 799( COL5A1& c.2497C>T:(p.Pro833Ser(( Missense( Novel( N(

Hypermobility( 38( COL5A2& c.2228A>C:(p.Lys743Thr(( Missense( Novel( N(

Hypermobility( 671( COL5A2& c.470C>T:(p.Pro157Leu( Missense( Novel( N(

+b Hypermobility( 814( TGFBR2& c.1538T>C:(p.Val513Ala(( Missense ( Novel( N(

Other(HDCT( 475( TGFBR1& c.214A>G:(p.Ile72Leu(( Missense( Novel( N(

Other(HDCT( 804( COL1A1& c.584C>T:(p.Ala195Val( Missense( Novel( N(

Other(HDCT( 629( COL1A2& c.2123G>A:(p.Arg708Gl( Missense( Reported:( Y( d Marfanoid( ( Other(HDCT( 708( COL3A1& c.2002C>A:(p.Pro668Thr( Missense( Novel( N(

b Other(HDCT( 382( SMAD3& c.1218G>C:(p.Trp406Cys( Missense( ( Novel(( Y( (vascular)( b Other(HDCT( 706( TGFBR1& c.827T>C:p.Leu276Pro( Missense( ( Novel( Y( (vascular)( Column( headings( and( abbreviations( as( for( Table( 3.4.( a.+ possible( splice( site( disruption,( but( this( patient( also( carries( a( pathogenic( COL3A1( variant( and( his( definitive( diagnosis( was( altered( to( vascular( EDS.( b.+ these( four( variants( are( Likely( Pathogenic( (see( Tables( 3.7( &( 3.8)( c.( LOVD`( ExAC( database:( likely( benign( variant( (freq.( 1:500)( d.( HGMD( CM900074`( DBSNP(rs72658163.(c.+ &+ d.:(both(these(variants(have(been(previously(reported(as(pathogenic(but(are(classified(here(as( VUS((see(text).(

! 49! ! ! Table&3.6&(continued)&Detailed!phenotypic!and!genetic!background!of!patients!with!pathogenic!variants!or!variants!of!uncertain!significance! Table&3.6&Detailed!phenotypic!and!genetic!background!of!patients!with!pathogenic!variants!or!variants!of!uncertain!significance&

Patient&ID& Variants&identified&by&NGS&&& Genetic&testing&by& Age&& Sex&& Initial& Phenotype&details& No.& Affected& Beight VillefrancheI& Ghent&& OI& Bchem& LM&&&&&&&EM&&&&&% previous&Sanger& (yrs)& clinical& relatives& mut.pos.& on& &&No.&of&major&(minor)&criteria& [d]& [e]& [f]& [g]& [h]& sequencing&[a]&& diagnosis& affected&% /mut.neg.&& score&&&&& [b]& [c]& [c]& (0I9)& & & & & & % % && Gene& Variant&type& % && && && && && && && && && && && && & Other& Genes COL1A1% COL3A1 COL5A1% Vascular& /COL1A2 /COL5A2 Classical& Hypermob Other&EDS& Kyphoscol 31! COL5A1' Pathogenic! !! !! 1! !! 27! M! Classical!! !! 1! !! 7! 3(3)! !! 1(0)! !! !! !! !! !! +! ++! 49! COL3A1' VUS! !! !! !! !! 19! M! Classical!! !! 1! 1! 9! 3(3)! !! 2(0)! !! !! !! !! !! !! !! 62! COL5A2' Pathogenic! !! !! 1! !! 33! F! Classical!! !! 1! 1! 8! 3(3)! !! !! !! !! 4! !! !! K! ++! 429! COL5A1' Pathogenic! !! !! 1! !! 39! M! Classical!! Thin! skin! over! chest! &! spine,! 1! !! 4! 2(4)! 3(2)! !! !! !! 1! !! Normal! +! ++! hypermobile,! iliac! artery! aneurysm! and!rupture! 581! COL5A1' Pathogenic! !! !! 1! !! 42! M! Classical!! !! 1! 1! 7! 2(3)! 0(1)! !! !! !! !! !! !! +! ++! 582! COL5A1' Pathogenic! !! !! 1! !! 56! M! Classical!! !! !! !! 9! 1(1)! 0(1)! 2(0)! !! !! !! !! Col!V! +! ++! 627! COL5A1' Pathogenic! !! !! 1! !! 78! M! Classical!! Loose! fragile! skin,! atypical! for! 0! !! !! 3(1)! !! !! !! !! !! !! Normal! !! ++! Classical!EDS! 1129! COL5A1' Pathogenic! !! !! 1! !! 33! F! Classical!! Joint! hypermobillity,! fragile! skin,! 4! 1! !! 2(4)! 0(1)! !! !! !! !! !! !! !! +! Varicose!veins! 636*! COL3A1'' Pathogenic!!!! !! !! 1! TNXB:!0! 21! M! Classical!! Hyperextensible! skin,! generalised! !! 0! 8! 2(1)! 0(1)! 2(0)! !! !! 3! !! Normal! +! +! COL5A2' VUS! hypermobility! including! marked! distal! hypermobility,! facies! suggestive! of! Classical! EDS,! no! signs! of! Vascular! EDS! 67! '' !! !! !! !! TNXB:!1! 33! M! Classical! /!Hyperextensible! skin,! hypermobility! 3! 1! !! 2(4)! !! 2(1)! !! !! 1! !! !! K! +! Hypermob.!!! and!anal!prolapse!

417! COL3A1' Pathogenic! !! 1! !! !! 31! M! Classical! /!Widened! atrophic! scars,! bruisability! 1! !! 6! 2(0)! 1(2)! 1(0)! !! !! 1! !! Col!III! +! +! Hypermob./! and! friability! of! skin,! marked! Vascular!! generalised! hypermobility,! normal! facies,! history! of! recurrent! colonic! perforations!! 671! COL5A2' VUS! !! !! !! PLOD1:! 45! F! Classical! /!!! 3! 1! 7! 2(4)! 1(0)! 2(1)! 1(1)! !! 2! !! !! K! !! 0! Hypermob.!

37! COL3A1' Pathogenic! !! 1! !! !! 15! M! Vascular!! Popliteal!artery!rupture! 6! !! 6! 0(1)! 3(0)! 2(1)! !! !! !! !! Col!III! +! +! 46! COL3A1' Pathogenic! !! 1! !! !! 42! F! Vascular!! Vascular! EDS! facies! and! aortic! 0! !! 0! !! 3(1)! !! !! !! !! !! Col!III! !! +! rupture! 76! COL3A1' Pathogenic! !! 1! !! !! 26! F! Vascular!! Spontaneous!colon!perforation! 0! !! 9! !! 3(2)! 2(2)! !! !! !! !! Col!III! +! +! 384! COL1A1' VUS! 1! 0! !! FBN1:!0! 42! F! Vascular!! Acrogeria! suggestive! of! vascular! 2! !! 0! 1(1)! 3(3)! !! !! !! 0! !! Col!III! K! K! TGFBR1 EDS]!Ascending!aortic!aneurysm! :!0! TGFBR2 :!0! 405! COL3A1' Pathogenic! !! 1! !! !! 7! M! Vascular!! Acrogeria!&!easy!bruising! 3! !! !! !! 3(2)! !! !! !! !! !! !! !! !! ! ! ! ! !

! 51! Table&3.6&(continued)&Detailed!phenotypic!and!genetic!background!of!patients!with!pathogenic!variants!or!variants!of!uncertain!significance!

! Patient&ID& Variants&identified&by&NGS&&& Genetic&testing&by& Age&& Sex&& Initial& Phenotype&details& No.& Affected& Beight VillefrancheI& Ghent&& OI& Bchem& LM&&&&&&&EM&&&&&% previous&Sanger& (yrs)& clinical& relatives& mut.pos.& on& &&No.&of&major&(minor)&criteria& [d]& [e]& [f]& [g]& [h]& sequencing&[a]&& diagnosis& affected&% /mut.neg.&& score&&&&& [b]& [c]& [c]& (0I9)& & & & & & % % %

&& Gene& Variant&type& & && && && && && && && && && && && && ther& O Genes COL1A1% COL3A1 COL5A1% Vascular& /COL1A2 /COL5A2 Classical& Hypermob Other&EDS& Kyphoscol 443! COL3A1' Pathogenic! !! 1! !! !! 44! F! Vascular!! !! 2! 1! !! 1(0)! 3(3)! 1(0)! !! !! !! !! !! !! !! 448! COL3A1' Pathogenic! !! 1! !! !! 26! F! Vascular!! Typical!acrogeric!Vascular!EDS! 1! !! 5! !! 2(3)! !! !! !! !! !! Col!III! +! K! 483! COL3A1' Pathogenic! !! 1! !! !! 39! M! Vascular!! Spontaneous! colon! perforation! and! 1! 1! !! !! 3(4)! !! !! !! 5! !! !! +! +! positive! family! history! of! vascular! EDS! 733! COL3A1' Pathogenic! !! 1! !! !! 33! M! Vascular!! Spontaneous! intraKabdominal! 2! 2! !! 0(1)! 3(3)! !! !! !! !! !! !! !! !! haemorrhage! 1125! 4' !! !! !! !! FBN2:!1! 38! M! Vascular!! !! 1! 1! !! !! !! !! !! !! !! !! Normal! !! !! 444*! COL1A1' VUS! !! !! !! CNV!¶! 29! M! Vascular!! varicose! veins,! small! joint! 0! 0/0! 0! 0(0)! 0(2)! 0(1)! !! !! 2! !! Col!III! K! K! hypermobility,! mildly! thin! and! hyperextensible! skin,! learning! difficulties! 42! COL3A1' Pathogenic! !! 1! !! !! 38! F! Vascular! /! !! 6! 1/3!Δ! 4! !0(2)! 1(1)! !0(1)! !! !! 1! 1Ka!! Col!III! (+)! +! Hypermob.! (mild)!

765! COL3A1' Pathogenic! !! 1! !! !! 6! F! Vascular/! Subependymal! intracerebral! 1! !! 9! !! 3(0)! 1(1)! !! !! !! 1Ka! !! !! !! Hypermob.! haemorrhage! at! birth,! Hypermobility,! easy!bruising!

1088! COL5A1' Pathogenic! !! !! !! !! 40! M! Vascular/! !! 0! !! 3! 2(0)! 2(2)! !! !! !! 2! !! !! !! !! Classical!!! 66*! FBN15' Pathogenic]! !! 0! !! FBN1:!1! 55! M! Other! HDCT! Hypermobility! /! nonKMarfan! 3! !! !! 1(0)! !! 1(0)! !! !! 1! !! !! K! K! COL5A1' VUS! with!vascular!fibrinillopathy! (Ghent! =1),! short! phenotype! stature,!!ectopia!lentis,!Aortic!&!Mitral! valve! surgery! (aortic! dilatation,! MV! prolapse).!!! 378! FBN1' Pathogenic! !! 0! !! SMAD3: 42! F! Other! HDCT! Hypermobility,! Carotid! artery! 1! !! !! 0(1)! 1(0)! 1(2)! !! !! 0! !! !! !! !! 0' with!vascular!dissection,!early!onset!osteoarthritis! TGFBR1 phenotype! :0' TGFBR2 :0' 420! COL3A1' Pathogenic! !! 1! !! !! 27! F! Vascular/! Vascular!EDS!facies!(prominent!eyes,! 1! !! 9! 3(2)! 3(1)! 2(0)! !!!!!! !! !! !! !! !! +! Classical!! small!ear!lobes)!with!hyperKextensible! skin! and! extensive! hypermobilility.! Haemothorax.! 34! COL5A1' VUS! !! 0! !! !! 36! F! Hypermob./! Benign! connective! tissue! phenotype! 4!(both! !! 3! 2K 1(0)! !! !! !! !! !! !! K! K! BJHS! with!carotid(cervical)!artery!dissection! sides!of! mild( family)! 2)! 475! TGFBR1' Pathogenic! 0! 0! !! !! 33! F! Other!HDCT! Hypermobility,! soft! skin,! multiple! 3! !! 7! 0(3)! !! 2(1)! !! !! !! 2Ka.! !! K! K! fractures,! mild! blue! sclerae,! systolic! b! murmur!

! 52! Table&3.6&(continued)&Detailed!phenotypic!and!genetic!background!of!patients!with!pathogenic!variants!or!variants!of!uncertain!significance!

478! COL1A1' VUS! !! 0! 0! !! 41! F! Hypermob./! !! 3! !! 6! !! !! 1(3)! !! !! 0! !! Col!III! K! +! BJHS! ! ! Patient&ID& Variants&identified&by&NGS&&& Genetic&testing&by& Age&& Sex&& Initial& Phenotype&details& No.& Affected& Beight VillefrancheI& Ghent&& OI& Bchem& LM&&&&&&&EM&&&&&% previous&Sanger& (yrs)& clinical& relatives& mut.pos.& on& &&No.&of&major&(minor)&criteria& [c]& [d]& [e]& [f]& [g]& sequencing&[a]&& diagnosis& affected&% /mut.neg.&& score&&&&& [b]& [b]& (0I9)& & & & & & % % %

&& Gene& Variant&type& & && && && && && && && && && && && && Other& Genes COL1A1% COL3A1 COL5A1% Vascular& /COL1A2 /COL5A2 Classical& Hypermob Other&EDS& Kyphoscol 538! '4' !! !! 0! !! FLNA:!1!! 23! F! Hypermob./! Hypermobility,! periventricular! nodular! 3! 0(FLNA)! 7! 0(1)! 1(0)! 1(1)! !! !! 1! !! !! K! K! BJHS! heterotopia! (FLNA! mutation! not! segregating!with!EDS!signs)! 558! COL1A2' VUS! !! !! !! !! 22! M! Hypermob./! !! 0! !! 6! !! !! 1(0)! !! !! 0! !! !! K! +! BJHS! 655! COL3A1' VUS! !! 1! !! !! 28! F! Hypermob./! Marfanoid!hypermobility!syndrome! 1! 1! 7! 2(2)! !! 2(2)! !! !! 2! !! Normal! K! K! BJHS! 734! COL5A1' VUS! !! 0! 0! !! 35! F! Hypermob./! !! 0! !! 6! 3K 0(0)! 2(0)! !! !! !! 1Ka! !! K! +! BJHS! mild( 0)! 799! COL5A1' VUS! 0! 0! !! !! 46! F! Hypermob./! !! 1! !! 5! 1(3)! !(3)! 2(0)! !! !! !! !! !! !! +! BJHS! 801! '4' !! !! !! !! CNV⌘!!!! 9! F! Hypermob./! Hypermobility!with!significant!learning! 7! !! 8! 2(5)! !! 1(0)! 2(2)! !! !! !! !! !! !! NSD1:0! BJHS! difficulties!&!epilepsy! DMPK:!0!

814! TGFBR2' Pathogenic! !! 0! !! !! 34! F! Hypermob./! Marfanoid! hypermoblity,thin! skin,! 2! !! 8! !! 1(1)! 1(3)! 0(2)! !! 2! !! !! !! +! BJHS! varicose!veins,mild!blue!sclerae! 824! COL5A1' VUS! !! !! !! !! !! F! Hypermob./! !! 1! !! 9! 1(2)! !! 2(3)! !! !! !! !! !! !! !! BJHS! 828! COL1A1' VUS! !! 0! !! !! 30! F! Hypermob./! Hypermobility,! ! soft! skin,! ! easy! 3! !! '' 1(4)! !! 1(0)! !! !! 0! !! Normal! (+)! (+)! BJHS! bruising,! tall! stature,! ! arterial! ectasia,!! mild! varicose! veins,! ! early! onset! of! uterine!prolapse! 893! COL1A1' Pathogenic! !! 0! !! !! 51! F! Hypermob./! Hypermobility! with! pelvic! floor! 2! !! '' 2(3)! 0(1)! 2(3)! !! !! 0! !! !! K! K! BJHS! weakness,!fractured!tibia!and!fibula! 1151! COL1A2' VUS! !! 0! !! !! 65! F! Hypermob./! !! 2! !! 4! 1(2)! 0(1)! 2(0)! !! !! 0! !! !! +! K! BJHS! 38*! COL3A15' VUS]!!!!!!!!VUS! !! 0! !! !! 57! F! Hypermob./! Benign! connective! tissue! phenotype! 1! !! 7! 3(2)! 1(1)! 2(1)! !! !! !! !! Normal! +! +! COL5A2' BJHS! with!coronary!artery!dissection! 39*! COL1A25' VUS]!!!!!!!!VUS! !! 0! 0! CNV!§! 31! M! Hypermob./! Marke! hypermobility! with! bowel! 3(partial)! 3(TNXBK !! !! 1(0)! 1(1)! !! !! !! !! !! +! +! COL3A1' BJHS! fragility! hets)!

57! '4' !! !! !! !! PLOD1:! 30! F! Kyphoscolio !! 1! 1! 9! 3(3)! !! 2(2)! 3(4)! !! 3! 1Ka! !! !! !! 1!!! sis!! 732! '4' !! !! !! !! PLOD1:! 22! F! Kyphoscolio !! 1! !! 4! !! !! 2(0)! 3(3)! !! 3! !! !! !! !! 1!!! sis!!

! 53! Table&3.6&(continued)&Detailed!phenotypic!and!genetic!background!of!patients!with!pathogenic!variants!or!variants!of!uncertain!significance!

! ! ! Patient&ID& Variants&identified&by&NGS&&& Genetic&testing&by& Age&& Sex&& Initial& Phenotype&details& No.& Affected& Beight VillefrancheI& Ghent&& OI& Bchem& LM&&&&&&&EM&&&&&% previous&Sanger& (yrs)& clinical& relatives& mut.pos.& on& &&No.&of&major&(minor)&criteria& [d]& [e]& [f]& [g]& [h]& sequencing&[a]&& diagnosis& affected&% /mut.neg.&& score&&&&& [b]& [c]& [c]& (0I9)& & & & & & % % %

&& Gene& Variant&type& & && && && && && && && && && && && && Other& Genes COL1A1% COL3A1 COL5A1% Vascular& /COL1A2 /COL5A2 Classical& Hypermob Other&EDS& Kyphoscol 802! '4' !! !! !! !! PLOD1:! 10! F! Kyphoscolio !! 2!(partial)! 2(het)! !! 2(1)! 1(0)! 1(1)! !0(4)! !! !! 1Ka! !! !! !! 1!!! sis!! 822! '4' !! !! !! !! FKBP14:! 3! M! Kyphoscolio !! 2! 2(het)! !! !! !! !! 2(1)! !! 3! !! !! K! K! 1!! sis!! PLOD1:! 0! 36! COL1A1' Pathogenic! 1! !! !! !! 7! F! Other!HDCT! OI/EDS! overlap! (pelvic! fracture,! blue! 4! !! 6! 0(0)! !0(1)! 2(2)! !! !! 1! 2Ka,b! !! !! !! sclerae,! hyperextensible! skin,! hypermobility)! 527! COL1A1' Pathogenic! 1! !! !! !! 28! F! Other!HDCT! Silence! type! I! Osteogenesis! 2! 0/0! 5! 0!(1)! 0(0)! 1(0)! !! 1(0 3! 3K Col!I! +! +! Imperfecta/! EDS! VIIA! or! VIIB! :!! )! a,b,c! repeated! fractures,! deafness,! blue! VII sclerae,! kyphosis,! early! postural! hip! a! problems! 559! COL1A1' Pathogenic! 1! !! !! !! 48! M! Other!HDCT! OI/EDS! overlap! (blue! sclera,! 5! !! 4! 1(3)! !0(3)! !! !! !! 0! 2Ka,b! !! !! !! fractures,! slight! presenile! conductive! hearing! loss,! aortic! valve! disease,! easy! bruising,! hypermobility,! significant! family! history! of! vasculopathy)! 629! COL1A2' VUS! 1! !! !! !! 46! F! Other!HDCT! OI/EDS! overlap! (mild! blue! sclerae,! 7! 7/2! '' 0(2)! !! 2(0)! !! !! 2! 2Ka,! Col!I! +! K! fractures,! osteopenia,! hypermobility,! b! mild!skin!hyperextensibility),!history!of! recurrent!embolism! 804! COL1A1' VUS! !! !! !! PLOD1:! 5! F! Other!HDCT! Congenital! hip! dysplasia,! postural! 2(partial)! 0/0! '' 1(3)! 0(0)! 1(2)! 2(2)! !! 2! !! !! !! !! 0!TNXB:! kyphosis,!joint!hypermobility! 0! 79! '4' !! !! 0! !! TNXB:1' 47! M! Other! HDCT! Non! specific! connective! tissue! 2(mildly)! !! !! 2(2)! 0(1)! 2(1)! !! !! 4! !! Col!III! +! +! (VUS),!!'' with!vascular!phenotype! with! vascular! phenotype! FBN1:!0! phenotype! (venous! aneurysm),! generalised! hypermobility,!!skin!hyperextensibility,! some! features! of! Marfan! syndrome,! cutis! laxa! (of! the! face)! and! dysmorphic!features!! 706! TGFBR1' VUS! !! 0! !! TGFBR1 45! M! Other! HDCT! Descending!thoracic!aortic!dissection,! 2! !! 3! 1(1)! 1(1)! 1(1)! !! !! 1! !! !! K! K! :1!(VUS)' with!vascular!subclavian! artery! aneurysm,! pectus! phenotype! excavatum,!soft!skin! 708! COL3A1' VUS! !! !! !! !! 11! F! Other! HDCT! Marfanoid! Hypermobility,! joint! pain,! 5! 2! 4! 0! (1)! 1(2)! !! !! !! !! !! !! !! with!vascular!soft!thin!skin,!aortic!dilatation,!colitis!! ! phenotype! ! ! ! !

! 54! Table&3.6&(continued)&Detailed!phenotypic!and!genetic!background!of!patients!with!pathogenic!variants!or!variants!of!uncertain!significance!

! ! Patient&ID& Variants&identified&by&NGS&&& Genetic&testing&by& Age&& Sex&& Initial& Phenotype&details& No.& Affected& Beight VillefrancheI& Ghent&& OI& Bchem& LM&&&&&&&EM&&&&&% previous&Sanger& (yrs)& clinical& relatives& mut.pos.& on& &&No.&of&major&(minor)&criteria& [d]& [e]& [f]& [g]& [h]& sequencing&[a]&& diagnosis& affected&% /mut.neg.&& score&&&&& [b]& [c]& [c]& (0I9)& & & & & & . % % %

&& Gene& Variant&type& & && && && && && && && && && && && && Other& Genes COL1A1% COL3A1 COL5A1% Vascular& /COL1A2 /COL5A2 Classical& Other&EDS& Kyphoscol Hypermob 766! FBN1' Pathogenic! !! !! 0! !! 8! M! Other! HDCT! Generalized! joint! laxity! 0! !! 7! 0(0)! 0(0)! 2(0)! 1(1)! 1(1 6! 1Ka! !! !! !! with!vascular!(Hypermobility),! marfanoid! habitus,! )K phenotype! mild! aortic! dilatation,! narrow! palate,! VII precocious!puberty! a!

382! SMAD3' VUS! !! !! !! SMAD3: 49! F! Other! HDCT! Hypermobile! (9/9),! Coronary! artery! !! !! 9! 2(2)! 1(0)! 2(3)! !! !! 2! !! !! !! !! 1!(VUS)' with!vascular!dissection,! ASD,! Mildly! marfanoid! phenotype! features! EDS,! EhlersKDanlos! syndrome]! VUS,! variant! of! uncertain! clinical! significance]! Hypermob.,! EDS! Hypermobility! type]! BJHS,! Benign! Joint! Hypermobility! syndrome! (on! a! continuum! with! Hypermobility!EDS!but!not!meeting!criteria)]!Beighton!score,!describes!extent!of!joint!hypermobility!(/9)]!OI,!osteogenesis!imperfecta]!HDCT,!Hereditary!disorder!of!connective!tissue]!ASD,! atrial!septal!defect]!CNV,!copy!number!variant]!VIIa,!Arthrochalasia!type!EDS![under!Other!EDS,!cases!766!and!527].! a.!Genetic!testing!by!previous!Sanger!(performed!by!clinical!service,!independently!of!NGS):!1!=!alteration!identified,!0!=no!alteration!identified,![blank!cells]!=not!tested! b.!Initial!Clinical!Diagnosis!refers!to!most!applicable!classification!at!first!specialist!EDS!consultation,!‘/’!indicates!a!phenotype!overlapping!more!than!one!type!of!EDS!or!HDCT! c.!Segregation:!numbers!show!the!no.!affected!relatives!carrying!the!variant!/!no.!affected!not!carrying!variant!!("affected"!=any!relative!sharing!at!least!one!EDS!phenotype!with!index).!!! d.&Ghent!systemic!score!/20! e.!Signs!of!OI:!shows!the!number!and!type!of!signs!of!OI!according!to!Sillence!criteria107:!!a.!blue!sclera,!b.!hereditary!osteoporosis/fractures,!c.!presenile!conductive!hearing!loss! f.!Biochemical!abnormalities:!shows!collagen!type!harbouring!abnormality!by!SDSKPAGE! g.!LM,!Light!Microscopy:!'+'=abnormal]!!!'K'!=!normal]![blank!cells]=!not!tested!!! h.!EM,!Electron!Microscopy:!'+'=abnormal,!!'K'!=!normal,!![blank!cells]=!not!tested,!!'++'!=!collagen!rosettes!(only!in!Classical/COL5A1/2!cases)! *IDs!38,!39,!66,!444!had!more!than!one!rare!variant!coKexisting]!Karyotyping!revealed!0.1!megabase!deletion!to!chromosome!q23.1,!likely!to!be!a!polymorphic!CNV!(non!pathogenic)]!! §! TNXB! gene! duplication]!!¶! translocation! involving! COL3A1! &! COL5A2.! Not! shown:! 3! cases! of! Kyphoscoliotic! EDS! diagnosed! by! urinary! crossKlink! assay! (PLOD1)]!! Δ!Of!the!4!affected!relatives!tested!genetically,!1!carried!the!p.Pro806Leu!variant!and!the!3!who!did!not!carry!this!variant!had!joint!hypermobility!but!no!other!features!of!EDS&

&

! 55! ! & Table&3.7&Details!of!atypical!genotypeKphenotype!correlations!identified!by!the!Collagen!NGS!panel!

Patient& Variant& Family&history&/& Gene& Variant& Observed&phenotype& Functional&studies& Comments& ID& Classification& Segregation&

636! COL3A1' c.2329G>C:! Pathogenic! Classical!EDS.!Clear!phenotype!K! Collagen!Proteins:!normal! Not!known! Not!detected! a p.G777R! markedly!hyperextensible!skin,! proα1(III)!pattern ! in!clinical! generalised!hypermobility!(Beighton!8)! setting.!! including!marked!distal!hypermobility,! LM:!thickened!elastic!fibres.! facies!suggestive!of!Classical!EDS,!no! EM:!irregular!packing!of! criteria!met!for!Vascular!EDS! collagen!fibrils!(no!collagen! rosettes)!

417! COL3A1'' c.1922_1923 Pathogenic! Classical/Hypermobility/(vascular)!overlap.!!! Collagen!proteins:!absent! Mother[d.],!arterial!rupture]! K! +2delAAGT! Widened!atrophic!scars!(marked!forehead! proa1(III)!and!collagen!III]!!! segregation!unknown.!! scarring),!no!skin!thinning,!tissue!fragility,! marked!generalised!hypermobility! LM:!marked!collagen!depletion! (Beighton!6),!normal!facies,!history!of! &!increased!elastin!staining]! colonic!perforation.!! EM:!variable!collagen!fibril!size! &!shape,!dilated!rER!

1088! COL5A1' c.3164T>A:! Pathogenic! Vascular/classical!overlap.!Thin! [Not!done]! Not!known! Not!detected! b p.L1055X& ! translucent!skin!over!anterior!chest!wall,! in!clinical! bruising!of!the!shins,!with!moderate! setting.!! hyperextensibility!of!the!skin,!distal! hypermobility!!

824! COL5A1' c.4564G>T]! Pathogenic! Hypermobility!EDS.!Extensive!joint! [Not!done]! Mother!–hypermobility! Not!detected! p.G1522C! hypermobility!(Beighton!9/9)!with!recurrent! in!clinical! shoulder!dislocations,!easy!bruising]!soft! Maternal!grandfather!K! setting.!! nonKhyperextensible!skin.!! sudden!cardiac!death!!

! 56! !

893! COL1A1' c.2980C>T:! Likely! Hypermobility!EDS.!Predominant! Collagen!proteins:![not!done].!!! Incomplete!segregation!of! Not!detected! c p.R994C! ! Pathogenic!! hypermobility!trait!with!hyperextensible! LM,!EM:!nonKspecific! phenotype:!DaughterK! in!clinical! skin,!marked!pelvic!floor!weakness,! hypermobile,!variant! setting.!! history!of!fractured!tibia!and!fibula.!! negative]!Mother[d.]!K! hypermobile!with!pelvic!floor! weakness,!genotype! unknown!

629! COL1A2' c.2123G>A:! Uncertain! Other!HDCT.!Juvenile!hypermobility,!now! Collagen!proteins:!!broadening! Incomplete!segregation:!7! !K! d p.R708E! ! significance!! mild!hypermobility,!very!mildly!blue!sclera! of!Proα2(I)!band.!! offspring!affected!with!mild! 1/6,!osteopenia! connective!tissue!phenotype]! LM:!moderately!increased! 2/7!carry!variant!(one!had! elastin:collagen!ratio.!!! hypermobility!with!history!of! EM:!relatively!normal! fractures,!one!had! hypermobility!only)!

EDS,!EhlersKDanlos!syndrome,!VUS,!variant!of!uncertain!clinical!significance.!HGMD,!human!genetic!mutation!database.!(CMK!no's!are!specific!to!HGMD!entries).!LOVD,!Leiden!Open! Variation!Database.!Beighton,!score!for!extent!of!hypermobility!/9.!LM,!light!miscroscopy.!EM,!electron!microscopy.![d.],!deceased.!Variant!classification!is!as!described!in!the!Methods! and!for!those!variants!not!known!to!be!pathogenic,!using!ACMG!criteria29),!see!notes!c.!&!d.! a.& Pepsinised! medium! not! done! due! to! technical! reasons.! ! b.! pathogenic! COL5A1' variant! (p.Gly1537Val)! causing! vascular! EDS! phenotype! also! reported! in! LOVD! (Munroe! et! al.).&& c.&Helical!R/C!variants!are!known!to!be!pathogenic!in!COL1A1!(ACMG!criteria!supporting!this!variant:!2!Moderate!&!4!Supporting!criteria)]!R/C!variant!also!observed!in!COL1A2!in!Patient! ID!1151!K!hypermobility!EDS!with!mild!phenotype).!!d.&Previously!reported!in!a!marfanoid!hypermobile!patient!with!slow!migration!of!proa2(I)!on!SDS!PAGE!(HGMD!CM900074]!DBSNP! rs72658163),!this!variant!classified!as!“Uncertain!significance”!owing!to!contradictory!evidence!for!and!against!pathogenicity.! & &

! 57! ! Table&3.8&Rare!variants!identified!in!AortopathyKsusceptibility!genes&

Patient& Diagnosis& Clinical&details& Gene& Variant& Variant& Novel/reported:& Pathogenicity&details& Previously& ID& Classification& Phenotype& detected?&

66! Other!HDCT! Hypermobility!/!nonKMarfan! FBN1' c.3781T>A:! Pathogenic! Reported:! Known!interacting!domain!of! Y! a (vascular)! fibrinillopathy!(Ghent!=1),!short!stature,! p.Tyr1261Asn! Marfan! !! FBN1,!highly!conserved!residue.! ectopia!lentis,!Aortic!&!Mitral!valve! surgery!! ! In!silico!predictions:!deleterious! Family!history:!!sudden!death!(father)!&! mitral!valve!prolapse!(sister,!first!cousin)!

378! Other!HDCT! Hypermobility,!carotid!artery!dissection,! FBN1' c.1775G>A:! Pathogenic! Reported:! Known!pathogenic! N! b (vascular)! early!onset!osteoarthritis! p.Gly592Asp! Marfan! !! !

766! Other!HDCT! Generalized!joint!laxity!(hypermobility),! FBN1' c.C3373T:! Pathogenic! Reported:! Known!pathogenic! N! c (vascular)! marfanoid!habitus,!mild!aortic!dilatation!!! p.Arg1125X! Marfan! !! narrow!palate,!precocious!puberty,! parental!consanguinity! ! !

706! Other!HDCT! Descending!thoracic!aortic!dissection,! TGFBR1' c.T827C:! Likely! Novel! SerKThr!kinase!domain:!multiple! Y! d (vascular)! subclavian!artery!aneurysm,!pectus! p.Leu276Pro! Pathogenic! ! mutations!in!this!region!observed! excavatum,!soft!skin! in!LDS/Marfan!spectrum!K!p.267!is! closest.!Conserved!residue,! In!silico!predictions:!all!deleterious! !

Proximate!to!MH2!domain!(key! Hypermobile!(9/9),!coronary!artery! Other!HDCT! c.1218G>C:! Likely! interacting!domain!for!other! 382! dissection,!ASD,!Mildly!marfanoid! SMAD3' e Novel! Y! (vascular)! p.Trp406Cys! Pathogenic! !! SMADs)!! features!(Ghent!=!2)! In!silico!predictions:!all!deleterious!

! ! ! ' ! ! ! ! !

! 58! !

814! Hypermobile! Marfanoid!hypermobility,!thin!skin,! TGFBR2' c.T1538C:! Likely! Novel! SerKThr!kinase!domain:!multiple! N! f varicose!veins,!mild!blue!sclerae.! p.Val513Ala! Pathogenic! !! mutations!in!this!region!observed! ! in!LDS/Marfan!spectrum!K! Family!history!of!thoracic!aortic! ! p.C514R!is!closest.!!Conserved! rupture(sister),!marfanoid! residue,!! features(sister,!father)! In!silico!predictions:!mostly! deleterious! !

475! Other!HDCT! OI/EDS!overlap!(multiple!fractures,!mild! TGFBR1' 214A>G:! Uncertain! Reported:!BAV!! Extracellular!(ligand!binding)! N! g blue!sclerae,!soft!skin,!hypermobility,! p.Ile72Leu! significance! ! domain!–!variants!in!this!domain! systolic!murmur)! reported!in!LDS]!! ! In!silico!predictions:!equivocal! !

HDCT,!hereditary!disorder!of!connective!tissue]!Ghent,!Ghent!systemic!score!for!marfan!syndrome]!HGMD,!human!genetic!mutation!database]!LDS,!LoeysKDietz!syndrome]!SerKThr,! SerineKthreonine!kinase!(main!intracellular!signalling!mechanism!of!TGFBR)]!ASD,!atrial!septal!defect.!Variant!classification!is!as!described!in!the!Methods!and!for!those!variants!not! known!to!be!pathogenic,!using!ACMG!criteria!(Richards!et!al.!Genet'Med!2015![16]),!see!notes!dKg!below.! “Previously!detected”:!variants!detected!(Y)!or!not!detected!(N)!by!previous!clinical!diagnostic!testing!(Sanger!method).!!!a.!Y1261C!(HGMD!CM990591!ElKAleem!1999),!Y1261D!(HGMD! CM547000,!Arbustini!2005)!!!b.!HGMD!CM013919,!Loeys!2001!c.&HGMD!CM055245,!Rommel!2005!d.!ACMG!criteria!supporting!variant:!2!Moderate!+!3!Supporting!e.!2!Moderate!+!2! Supporting!!f.!2!Moderate!+!4!Supporting!!g.!this!variant!reported!by!Bonachea!et!al.,!BMC'Med'Genomics'2014!as!a!VUS!in!a!bicuspid!aortic!valve!(BAV)!series,!incomplete!evidence!of! pathogenicity.!! ! !

! 59! ! ! !

! 60! ! 3.3.7!In!silico!predictions!of!missense!variants!detected!by!NGS!

The! majority! of! missense! coding! variants! identified! by! NGS! that! were! classified! as! pathogenic!had!SIFT,!Polyphen2,!MutationTaster!and!MutationAssessor!scores!of,!or! close!to!1.0,!which!supported!this!classification!(Table!3.9).!By!comparison,!the!scores! for!VUS’s!versus!pathogenic!variants!were!more!variable!and!lower!(mean!(converted)! score! =! 0.77,! p=0.0009! for! SIFTP! 0.72,! p=0.02! for! Polyphen2P! 0.92,! p=0.13! for! MutationTasterP! 0.63,! p<0.0001! for! Mutation! Assessor)! (Table! 3.9).! Similarly,! predictions!of!evolutionary!conservation!of!residues!in!missense!variants!were!lower!for! VUS’s!compared!with!pathogenic!missense!variants!(Table!3.9).!The!two!variants!that! were! previously! reported! as! pathogenic! but! that! we! classified! as! VUS’s! (Patient! IDs! 629! and! 655,! see! above)! had! high! in! silico! prediction! scores!(Table! 3.9),! but! lacked! pathological! or! segregation! data! to! support! pathogenicity.! In! addition,! a! helical! Arg>Cys! mutation! in! COL1A2! (Patient! ID! 1151)! and! three! other! VUS’s! in! COL1A1,( COL3A1( and( COL5A1! (Patient!IDs!39,!824,!828)!also!had!high!in!silico!predictions! (Table!3.9).!In!the!Aortopathy!panel,!three!variants,!respectively!in!SMAD3!(Patient!ID! 382),!TGFBR2!(Patient!ID!814),!and!TGFBR1!(Patient!ID!706)!also!had!high!in!silico! predictive!scores:!these!three!variants!were!in!functionally!important!domains!and!the! clinical!phenotypes!of!these!patients!strongly!suggested!pathogenicity!of!the!variants! (Tables!3.8!&!3.9).!!

! 61! ! Table&3.9!!In$silico!predictions!of!missense!pathogenic!variants!and!variants!of!uncertain!significance& & Patie Classif Max.& Mutation& Mutation& GERP nt&ID& Gene& Variant& ication& Coordinates& Freq.& SIFT& Polyphen& &Taster& Assessor& LRT& FATHMM& CADD& ++& PhyloP& SiPhy& !!! !! !! !! !! !! score! pred! score! pred! score! pred! score! pred! score! pred& ! score! pred! !! !! !! !!

636! COL3A1' c.2329G>C:!p.G777R! Path.! 2:189866168! NA! 1.00! D! 1.00! D! 1.00! D! 0.87! H! 1.00! D! 0.61! D! 4.003,20.5! 5.38! 2.69! 19.49! 62! COL5A2'' c.3445G>T:!p.G1149C!! Path.! 2:189907903! NA! 1.00! D! 1.00! D! 1.00! D! 0.90! H! 1.00! D! 0.62! D! 4.220,21.9! 5.39! 2.68! 19.52! 36! COL1A1'' c.643G>A:p.G215S! Path.! 17:48275146! NA! 1.00! D! 1.00! D! 1.00! D! 0.84! H! 1.00! D! 0.63! D! 4.445,23.7! 4.80! 2.36! 17.02!

559! COL1A1'' c.662G>C:!p.G221A! Path.! 17:48275127! NA! 1.00! D! 1.00! D! 1.00! D! 0.87! H! 1.00! D! 0.63! D! 3.700,18.79! 4.80! 2.36! 17.02! 893! COL1A1' c.C2980T:!p.R994C! Path.! 17:48266329! NA! 1.00! D! 0.99! D! 1.00! D! 0.79! H! 1.00! D! 0.49! D! 3.479,17.80! 3.83! 2.00! 14.74!

37! COL3A1'' c.2564G>A:!p.G855D! Path.! 2:189868147! NA! 1.00! D! 1.00! D! 1.00! D! 0.80! H! 1.00! D! 0.59! D! 4.275,22.3! 5.61! 2.65! 19.65! 42! COL3A1'' c.2417C>T:!p.P806L!! Path.! 2:189867049! NA! 0.96! D! 0.06! B! 1.00! D! 0.62! L! 1.00! D! 0.54! D! 3.877,19.70! 5.77! 2.73! 19.99!

46! COL3A1'' c.G1771:!p.G591R! Path.! 2:189861900! NA! 1.00! D! 1.00! D! 1.00! D! 0.82! H! 1.00! D! 0.63! D! 4.290,22.4! 6.03! 2.87! 20.16! 76! COL3A1'' c.2771G>A:!p.G924D! Path.! 2:189868817! NA! 1.00! D! 1.00! D! 1.00! D! 0.84! H! 1.00! D! 0.63! D! 5.324,34! 5.50! 2.59! 19.41!

733! COL3A1'' c.2816G>A:!p.G939D!! Path.! 2:189868862! NA! 1.00! D! 0.98! D! 1.00! D! 0.87! H! 1.00! D! 0.63! D! 3.773,19.16! 5.63! 2.66! 19.69! 448! COL3A1' c.4319C>T:!p.P1440L!! Path.! 2:189876418! NA! 1.00! D! 1.00! D! 1.00! D! 0.78! M! 1.00! D! 0.49! D! 4.798,27.1! 5.70! 2.69! 19.83!

66! FBN1' c.3781T>A:!p.Y1261N! Path.! 15:48776072! NA! 1.00! D! 0.99! D! 1.00! D! 0.85! H! 1.00! D! 0.53! D! 5.006,29.4! 6.17! 2.37! 16.48! 378! FBN1' c.1775G>A:!p.G592D! Path.! 15:48800841! NA! 0.99! D! 1.00! D! 1.00! D! 0.74! M! 1.00! D! 0.43! T! 5.285,33.0! 5.71! 2.85! 18.78!

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 49! COL3A1' G3511A:!p.G1171K! VUS! 2:189872854! NA! 0.52! T! 0.81! P! 0.70! D! 0.61! L! 1.00! D! 0.52! D! 4.469,23.9! 5.51! 2.59! 19.42! 478! COL1A1' c.4315A>G:!p.I1439V! VUS! 17:48262943! NA! 0.92! T! 0.34! B! 1.00! D! 0.67! M! 1.00! D! 0.44! T! 1.873,12.22! 4.49! 1.89! 12.90!

828! COL1A1' c.3301G>A:!p.E1101K! VUS! 17:48265305! NA! 0.91! T! 1.00! D! 1.00! D! 0.61! L! 1.00! D! 0.52! D! 5.449,35.0! 5.00! 2.32! 17.08! 39! COL1A2' c.2861T>C:!p.I954T! VUS! 7:94055087! NA! 0.46! T! 0.00! B! 0.00! N! 0.44! N! 0.72! N! 0.52! D! 0.057,4.310! $0.42! $0.43! 5.62!

558! COL1A2' c.1159G>C:!p.A387P!! VUS! 7:94039801! NA! 0.023,4.135! 1151! COL1A2' c.C4012T:!p.R1338C! VUS! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 7:94059616! NA! 1.00! D! 1.00! D! 1.00! D! 0.74! M! 1.00! D! 0.44! T! 3.598,18.32! 5.35! 2.89! 19.95!

38! COL3A1' c.198A>G:!p.I66M! VUS! 2:189849604! 0.00010! 0.86! T! 0.05! B! 1.00! D! 0.59! L! 1.00! N! 0.42! T! 3.973,20.3! $4.78! $0.55! 14.85! 39! COL3A1' c.2044G>A:!p.E682K! VUS! 2:189864032! NA! 0.87! T! 1.00! D! 1.00! D! 0.66! M! 1.00! D! 0.52! D! 4.152,21.5! 4.96! 2.47! 18.61!

655! COL3A1''' c.3938A>G:!p.K1313R! VUS! 2:189875018! 0.00260! 0.95! D! 1.00! D! 0.86! D! 0.58! L! 1.00! D! 0.43! T! 3.731,18.95! 5.93! 2.26! 16.38! 66! COL5A1' VUS! c.G805A:!p.E269K!!! 9:137620534! NA! 0.08! T! 0.02! B! 0.95! D! 0.64! L! 0.99! U! 0.49! D! 1.159,9.720! 3.93! 1.72! 15.92!

734! COL5A1' c.3257C>T:!p.A1086V!! VUS! 9:137697059! NA! 0.39! T! 0.99! D! 1.00! D! 0.48! N! 1.00! U! 0.52! D! 2.645,14.80! 5.19! 2.41! 18.70! 799! COL5A1' c.2497C>T:!p.P833S!! VUS! 9:137676847! 0.00020! 0.43! T! 0.73! P! 0.74! D! 0.63! L! 1.00! U! 0.55! D! 1.614,11.35! 4.39! 2.00! 15.75!

! 62! !

824! COL5A1' c.4564G>T:!p.G1522C!! VUS! 9:137713952! NA! 1.00! D! 1.00! D! 1.00! D! 0.82! H! 1.00! U! 0.63! D! 3.577,18.22! 4.70! 2.14! 17.22!

38! COL5A2' c.2228A>C:!p.K743T!! VUS! 2:189923156! 0.00010! 0.99! D! 1.00! D! 0.60! L! 1.00! D! 0.52! D! 2.582,14.60! 5.56! 2.24! 16.02! 671! COL5A2' c.470C>T:!p.P157L! VUS! ! ! 2:189957133! 0.00010! 1.00! D! 1.00! D! 0.65! M! 1.00! D! 0.54! D! 4.745,26.5! 5.98! 2.84! 17.38! ! ! 804! COL1A1' c.584C>T:!p.A195V! VUS! 17:48275526! NA! 0.90! T! 0.08! B! 0.76! D! 0.59! L! 0.72! N! 0.53! D! 4.187,21.7! 4.10! 2.55! 14.50! 629! COL1A2' c.2123G>A:!p.R708Q! VUS! 7:94049588! 0.00080! 0.99! D! 0.99! D! 1.00! D! 0.57! L! 1.00! D! 0.54! D! 4.603,25.1! 5.84! 2.94! 20.53!

708! COL3A1' c.C2002A:!p.P668T! VUS! 2:189863424! NA! 0.84! T! 0.85! P! 1.00! D! 0.65! M! 1.00! D! 0.55! D! 3.110,16.39! 5.93! 2.81! 19.95! 444! COL1A1' c.G3755A:!p.R1252H! VUS! 17:48264060! 0.00010! 1.00! D! 0.62! P! 0.99! D! 0.74! M! 1.00! D! 0.43! T! 3.060,16.21! 4.03! 2.06! 15.09!

384! COL1A1'' c.3466A>G:!p.N1156D! VUS! 17:48264441! NA! 0.67! T! 0.54! P! 1.00! D! 0.44! N! 1.00! D! 0.50! D! 2.782,15.27! 3.70! 1.67! 11.78!

475! TGFBR1' 214A>G:!p.I72L! VUS! 9:101891253! 0.00030! 0.16! T! 0.67! P! 1.00! D! 0.53! N! 1.00! D! 0.56! D! 2.997,16.00! 6.08! 2.33! 15.63!

814! TGFBR2' c.T1538C:!p.V513A! VUS! 3:30732925! NA! 0.99! D! 0.70! P! 1.00! D! 0.54! N! 1.00! N! 0.40! T! 4.401,23.3! 4.73! 1.03! 13.21!

382! SMAD3' c.1218G>C:!p.W406C! VUS! 15:67482814! NA! 0.97! D! 1.00! D! 1.00! D! 0.73! M! 1.00! D! 0.56! D! 4.218,21.9! 4.97! 2.32! 18.25!

706! TGFBR1' c.T827C:!p.L276P! VUS! 9:101904839! NA! 1.00! D! 1.00! D! 1.00! D! 0.73! M! 1.00! D! 0.41! T! 4.565,24.7! 5.87! 2.37! 15.56!

All!Pathogenic,!mean! ! 1.00! 0.92! 1.00! 0.81! 1.00! 0.57! 4.42+! 5.41! 2.58! 18.60! !!!!!!!s.d.! ! ! 0.01! ! 0.26! ! 0.00! ! 0.07! ! 0.00! ! 0.07! ! 0.57+! 0.62! 0.24! 1.70! ! ! ! ! ! ! ! All!VUS,!!!!!!!!!!!!mean!! ! ! 0.76! 0.71! 0.91! 0.62! 0.97! 0.50! 3.17+! 4.39! 2.04! 16.10! !!!!!!s.d.! ! ! ! 0.29! ! 0.36! ! 0.22! ! 0.10! ! 0.08! ! 0.06! ! 1.43+! 2.41! 0.91! 3.27! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 0.00 !0.004 p!(pathogenic!vs.!VUS)*!! !! !! !! 0.0009! !! 0.015! !! 0.13! !! <0.0001! !! 0.53! !! 0.0037! !! 0.0058! 0.024! 69! 2! Path.,!pathogenic!variant.!VUS,!variant!of!uncertain!significance.!Genomic!co$ordinates!are!based!on!GRCh37/hg19!human!reference!sequence.!Max.Freq.,!maximum!reported!minor!allele!frequency!in!1000!genomes! (phase!2!release)!and!the!NHLBI!exome!sequencing!project!data!sets.!NA,!novel!variant.!!s.d.,!standard!deviation.!*p!value!shows!statistical!significance!at!5%!level!(two$tailed!Mann$Whitney!test)!!In!silico!prediction!scores! are!based!on!ANNOVAR!version!2013aug23,!full!details!available!at:!http://www.openbioinformatics.org/annovar/!!!and!refer!to!the!following!specific!ANNOVAR!annotations:!Polyphen:!"LJB23_Polyphen2_HVAR_score",! Mutation! Taster:! "LJB23_MutationTaster_score_converted",! Mutation! Assessor:! "LJB23_MutationAssessor_score_converted",! LRT:! ! "LJB23_LRT_score_converted",! FATHMM:! ! "LJB23_FATHMM_score_converted"!!! Predictive&functional&scores:&SIFT,'Polyphen,'MutationTaster,'MutationAssessor,'LRT,'FATHMM:!all!scores!converted!to!0$1!scale,!higher!number!=!higher!probability!of!functional!significance!Cadd:!shown!as![raw!score,! scaled!score].!!Raw!score:!higher!number!=!higher!likelihood!of!deleterious!variant.!!Scaled!score:!phred$like!c$score!($10*log10(rank/total))!of!variant!pathogenicity!ranked!relative!to!all!possible!substitutions!of!the!human! genome!such!that,!a!scaled!score!>10!relates!to!the!top!10%!of!variants!ranked!for!pathogenicity,!scaled!score!of!>20!relates!to!the!top!1%!of!ranked!variants.!!Qualitative& predictions:!D,!deleterious.!T,!toleratedn!! [Mutation'Taster'only:!A,!disease!causing!automatic,!D,!disease!causingn!N,!polymorphismn!!P,!polymorphism!automatic]!![Mutation'Assessor'only:!!H(high),!!M(medium!and!L(low)!probability!of!functional!impact].! Conservation&scores:&(GERP++,!SiPhy,!PhyloP)!higher!number!!=!higher!conservation!across!species.

! 63! ! 3.3.8$ Clinical,$ genetic$ and$ pathological$ correlates$ of$ collagen$ gene$ variants$

Most%pathogenic%DNA%variants%in%collagen%genes%were%associated%with%a%corresponding% collagen%protein%abnormality%on%SDS:PAGE%but%protein%abnormalities%were%much%less% common%in%VUS’s%(81.8%%vs.%11.1%G%p=0.006).%A%similar%difference%in%light%and%electron% microscopy% abnormalities% was% observed% when% comparing% pathogenic% variants% with% VUS% (76.9%% vs.% 21.1%G% p=0.02).% There% was% no% significant% difference% (p>0.05)% in% the% frequency% of% presenting% arterial% complications% between% pathogenic% COL3A1% variants% (38%)% and% those% with% variants% of% other% genes% or% when% compared% with% mutation: negative% cases% (31%).% There% was% also% no% significant% difference% in% Beighton% score% based%on%gene%or%variant:type%(p>0.05).%

3.3.9$Comparison$of$NGS$variants$with$results$of$previous$genetic$testing$ by$Sanger$sequencing$

All% of% the% 22% short% pathogenic% variants% in% the% collagen% genes% and% the% pathogenic% variants%in%FBN1%and%SMAD3-that%were%identified%by%previous%Sanger%sequencing%were% also%identified%by%the%NGS%panels%(Figure%3.1A,%below).%Furthermore,%the%NGS%panels% newly%identified%seven%pathogenic%or%likely%pathogenic%variants% and%18%VUS%that%had% not% been% detected% by% phenotype:guided% Sanger% sequencing% (Tables% 3.4% and% 3.5G% Figure%3.1A).%The%Collagen%NGS%panel%identified%four%unexpected%pathogenic%or%likely% pathogenic%variants%in%genes%that%had%not%been%selected%for%Sanger%sequencing:%(i)%a% COL3A1%mutation%(Patient%ID%636,%p.Gly777Arg),%leading%to%the%diagnosis%of%vascular% EDS%in%this%patient.%This%patient%had%previously%been%diagnosed%as%classical%EDS%on% the% basis% of% clinical% phenotype% and% the% presence% of% an% intronic% variant% in% COL5A2,- which%had%possible%splice:site%disruptionG%(ii)%a%loss%of%function%mutation%was%identified% in%COL5A1%in%Patient%ID%1088,%for%whom%only%COL3A1%sequencing%had%been%selected,% leading% to% revision% of% the% diagnosis% to% classical% EDS% from% vascular/classical% overlap% (Table%3.7)G%(iii)%an%Arg>Cys%mutation%was%identified%in%the%helical%domain%of%COL1A1%in% a% patient% with% a% predominant% hypermobility% trait% with% fractures% (Patient% ID% 893,% Table% 3.7)%and%(iv)%a%helical%glycine%disrupting%COL5A1%variant%in%a%patient%with%a%predominant% hypermobility%trait%(Beighton%score%9/9)%but%minimal%skin%hyperextensibility%(ID%824)%(for% details%see%Table%3.6%and%3.7).%In%addition,%six%missense%variants%were%identified%with% maximal% or% near:maximal% in% silico% prediction% scores% for% pathogenicity% (Table% 3.9).% Because% these% variants% did% not% meet% ACMG% criteria% for% pathogenicity% or% likely% pathogenicity,%they%were%classified%as%VUS.%The%aortopathy%NGS%panel%identified%four% new% variants% not% previously% detected% by% clinical% or% genetic% investigation,% in% FBN1% ,% TGFBR1%and%TGFBR2%G%two%were%pathogenic%variants%of%FBN1%%(IDs%766%and%378,%both%

! 64! ! with%initial%diagnosis%of%other%HDCT)G%one%was%a%likely%pathogenic%variant%(TGFBR2,%ID% 814,%with%an%initial%clinical%phenotype%of%hypermobility:type%EDS)%and%the%fourth%variant% was% classified% as% uncertain% significance% (TGFBR1,% ID% 475,% with% an% initial% clinical% phenotype%of%other%HDCT)%(Table%3.8).%

The% three% previously% detected% variants% (in% TNXB,% FBN2% and% FKBP14)% outside% the% collagen% and% aortopathy% genes% covered% by% the% NGS% panels% and% the% complex% rearrangement% detected% by% array% CGH% were% not% detected% by% NGS.%

! 65! ! A Primary'/'secondary'care' Ter0ary'referral'' (General'Clinical'Gene0cist/' Hypermobility'clinic)'

Clinical'assessment'

Quaternary'referral'to'' Na0onal'EDS'Service''

Clinical'assessment' &'Laboratory'assessment' 'biopsy,'microscopy,'collagen'biochemistry'

NGS$Panel:$$ PhenotypeCdriven'single'gene' COL1A1,'COL1A2,'COL3A1,'COL5A1,'COL5A2,'FBN1,' sequencing'(Sanger'method)' TGFBR1,'TGFBR2,'ACTA2,'MYH11,'SMAD3,'MYLK'

No.'variants''idenBfied:'

Collagen( 22$ pathogenic' $ pathogenic' (genes( 26 ( 26$ 47$ 4$ VUS' $ VUS' ( 21 ( Aortopathy(( 1$ pathogenic' 3$ pathogenic' genes(((((( 3$ 7$ ( 2$ VUS' 4$ VUS' ( ( ( Other(( 4$ pathogenic' ( New(diagnoses((pathogenic(or(likely(pathogenic)( ( 5$ VUS' 1$ Collagen:'9'(5%)$ Aortopathy:''4''(2%)$

B( Primary(care( Secondary(care( Ter8ary(referral(( (General(Clinical(Gene8cist)(

Clinical(assessment(&(NGS(panel(

Collagen(variants(&(VUS’s((( Aortopathy(variants( No(Rare(Variant( (27%)& (4%)( (69%)&

Quaternary(referral(to(Na8onal( Specialist(Gene8c( Mul8disciplinary(team( EDS(Service(( counselling(&( review(&(quaternary( Cardiovascular(referral( referral(if(appropriate(

Clinical(assessment,(( Laboratory(tes8ng((microscopy,( collagen(biochemistry,(structural( predic8ons),(Segrega8on(analysis(

Defini8ve(diagnosis(

Figure$3.1%A.%Yield%of%rare%variants%using%traditional%Sanger%method%(left)%compared%with%the%NGS%panel% (right)%B.%Potential%alternative%clinical%pathway%to%genetic%diagnosis%in%EDS.%Boxed%numbers%show%the% number%of%rare%pathogenic%variants%and%VUS’s%identified%in%each%groupG%percentages%are%the%proportion%of% new%pathogenic%or%likely%pathogenic%rare%variants%in%our%cohort%of%177%EDS%referrals.%%“Other”%genetic% abnormalities%are%those%that%were%thought%to%underlie%the%EDS%phenotype%but%are%not%covered%by%the%NGS% panel%(3%pathogenic%variants%in%TNXB,%FBN2,%FKBP14%&%1%large%copy%number%variantG%1%VUS,%a%TNXB% gene%duplication).%Dashed%arrow%indicates%the%potential%for%NGS%panel%to%become%accessible%to%clinicians% in%secondary%care

! 66! ! 3.4$Discussion$

To%my%knowledge,%this%work%at%its%completion%represented%the%first%reported%use%of%NGS% to%sequence%a%panel%of%relevant%collagen%and%aortopathy%genes%in%a%large%mixed%cohort% of%patients%with%EDS%or%overlapping%HDCT.108%%

This% approach% used% NGS% to% identify% rare% functionally% significant% variants,% blind% to% previous% genetic% testing% and% in% an% unbiased% manner% (not% directed% by% phenotype),% to% seek% new% insights% into% genotype:phenotype% relationship% in% EDS% outside% the% known% paradigm.%%

Pathogenicity%was%assigned%according%to%variant%type%(frameshift,%nonsense%mutation% and%splice%site%disruption)%and%previous%reports%of%pathogenicity.%In%the%case%of%VUS’s,% in%silico%prediction%scores%and%clinical%and%functional%data%were%used%to%suggest%VUS’s% with%possible%or%likely%pathogenic%effects.%%

The%NGS%panel%was%able%to%detect%all%previously%identified%short%pathogenic%variants%in% collagen% and% aortopathy% genes% and% in% addition,% newly% identified% seven% pathogenic% variants% in% COL1A1,- COL3A1,% COL5A1(two- cases),- TGFBR2- and- FBN1- (2% cases),% leading% to% new% definitive% diagnoses% in% these% patients.% Of% 18% newly% detected% VUS,% a% significant%proportion%had%partial%evidence%of%pathogenicity,%based%on%previous%reports,% clinical%phenotype,%and%in%silico%prediction%score,%including%three%in%aortopathy%genes% (TGFBR1%and%TGFBR2)%and%four%in%COL3A1,%which,%if%shown%to%be%pathogenic,%alter% clinical% prognosis% and% management% because% of% the% associated% high% risk% of% arterial% aneurysm,%rupture,%and/or%bowel%perforation.%

Pathogenic%collagen%variants%did%not%always%correlate%with%the%expected%phenotype.%In% two%patients,%I%found%pathogenic%COL3A1-variants%associated%with%a%clinical%phenotype% of%classical%or%overlapping%classical%EDS,%one%of%whom%(Patient%ID%636)%also%had%an% intronic%COL5A2%variant,%possibly%splice%site%disrupting,%which%could%also%contribute%to% his%phenotype.%Classical%EDS%is%usually%associated%with%mutations%in%COL5A1/2,% but% has% rarely% has% been% associated% with% mutations% in% TNXB,% COL1A1% or% COL3A1.49,109% COL3A1-mutations%are%normally%considered%diagnostic%of%vascular%EDS,%with%reduced% life%expectancy%from%arterial%and%bowel%complications.50,110%

Of% 22% cases% classified% as% definite% or% likely% vascular% EDS,% 11(52%)% had% pathogenic% COL3A1-variants.%Since%previous%vascular%EDS%series%have%been%mostly%selected%by% clinical%criteria%(Villefranche)%along%with%collagen%type%III%protein%deficiency%or%COL3A1% mutations,%the%frequency%of%other%connective%tissue%gene%mutations%in%clinical%vascular% EDS% phenotypes% is% unknown.50% One% of% the% patients% clinically% classified% as% having% features% predominantly% of% vascular% EDS% (ID% 1088)% showed%a%Leu1055X%mutations%of% COL5A1.%One%pedigree%with%a%pathogenic%COL5A1%variant%segregating%with%a%vascular%

! 67! ! EDS%phenotype%including%arterial%ruptures%has%been%recently%reported%(Monroe%et%al.,% LOVD% ID% AN004203).111% Another% patient% (ID% 824)% for% whom% a% pathogenic% COL5A1% variant% was% found% had% an% initial% diagnosis% of% hypermobility:type% EDS% because% of% extensive%hypermobility,%soft%but%not%hyperextensible%skin,%and%family%history%of%sudden% cardiac%death%in%a%second:degree%relativeG%functional%corroboration%was%not%possible%for% this% patient% because% of% loss% to% follow:up.% As% far% as% one% is% aware,% such% a% phenotypic% pattern%has%not%been%observed%with%a%pathogenic%COL5A1%variant,%probably%because% COL5A1% sequencing% to% date% has% mostly% been% performed% in% phenotypically% classical% cases.39,81,112.%

Nine% VUS’s% with% high% in% silico% prediction% scores% or% other% evidence% for% pathogenicity% were% observed.% These% included% one% Arg>Cys% variant% within% the% helical% domain% of% COL1A2% (Patient% ID% 1151,% p.Arg1338Cys)% (Table% 3.9).% Arg>Cys% variants% at% helical% locations%in%COL1A1-have%been%reported%previously%as%predisposing%to%arterial%fragility% and% other% phenotypes.104% Although% the% COL1A2% variant% may% be% pathogenic,% little% evidence%exists%at%the%present%time%to%establish%its%pathogenicity.%

In% addition,% four% VUS’s% in% aortopathy% genes% TGFBR1,- TGFBR2,- SMAD3- were% observed,% three% of% which% were% considered% to% be% likely% pathogenic% and% the% other% possibly%pathogenic%on%the%basis%of%clinical%phenotype%and%functional%domain%affected% (Table%3.8).%Two%variants,%%one%in%TGFBR1-(Patient%ID%706)%and%the%second%in%TGFBR2% (Patient%ID%814),%were%termed%Likely%Pathogenic,%as%they%are%in%the%functionally%active% (Ser:Thr%kinase)%protein%domain%of%the%protein%which%so%far%have%harboured%the%majority% of%variants%in%Loeys:Dietz%syndrome%(LDS)%with%aortopathy.113%A%third%aortopathy%gene% VUS,% also% likely% pathogenic,% was% identified% in% SMAD3% (Patient% ID% 382).% This% was% adjacent% to% the% MH2% domain,% a% key% location% whereby% SMAD3% interacts% with% the% TGFBR1% receptor% and% regulates% TGF:beta% signalling.114% Coronary% artery% dissection% (seen% in% this% case)% and% other% vascular% complications,% especially% aortopathy,% have% previously%been%caused%by%SMAD3-variants.115%The%fourth%variant,%in%TGFBR1%(ID%475,% p.I72L)%was%in%a%patient%with%hypermobility,%bone%fragility%and%a%systolic%murmur%(and% normal% COL1A1/2).% This% variant% was% reported% previously% as% a% VUS% in% a% patient% with% bicuspid% aortic% valve.116% Bone% fragility% is% a% recent% addition% to% the% LDS% clinical% spectrum,117% so% this% variant% may% possibly% underlie% some% of% this% patient’s% features% but% the%evidence%is%unclear.%%

I% re:classified% two% variants% with% high% in% silico% prediction% scores% as% VUS’s,% though% previously%reported%as%pathogenic,%because%the%overall%evidence%for%pathogenicity%was% not%strong%enough%in%the%particular%patients%harbouring%them.%The%COL1A2%Arg708Glu% variant%detected%in%patient%ID%629%was%reported%previously%in%a%Marfanoid%patient,105%but% segregation% analysis% revealed% hypermobility% in% all% of% the% patient’s% seven% offspring,%

! 68! ! hypermobility%in%her%(unrelated)%husband,%multiple%fractures%in%one%of%the%two%offspring% who% carried% the% variant% and% in% none% of% the% remaining% five% offspring,% leading% it% to% be% classified%as%a%VUS.%The%COL3A1%p.Lys1313Arg%variant%detected%in%patient%ID%655%has% been% previously% reported% in% two% unrelated% patients% with% vascular% EDS.% However% collagen%microscopy%and%biochemistry%were%normal%in%the%patient,%neither%the%patient% nor% her% sister,% who% also% carried% the% variant,% had% features% of% vascular% EDS% and% subsequent% structural% studies% indicated% that% the% variant% was% unlikely% to% impair% C: propeptide:mediated%helical%winding.106%%

As% previously% reported,% I% observed% significant% phenotypic% overlap% between% our% EDS% diagnostic%categories%and%with%other%hereditary%disorders%of%connective%tissue%including% the%Marfan/LDS%spectrum%and%OI,%highlighting%the%difficulties%of%accurate%diagnosis%on% clinical% grounds% alone.% In% addition,% there% is% considerable% clinical% heterogeneity% and% overlap%amongst%patients%with%pathogenic%variants%in%individual%causative%genes.%These% factors% point% to% the% limitations% of% phenotype:driven% genetic% testing% of% individual% candidate%genes.%Although%there%was%a%low%frequency%of%genetic%diagnoses%amongst% patients%with%hypermobility%type%EDS%and%Other%HDCT,%the%NGS%approach%presented% here%permits%wider%genetic%testing%than%is%possible%with%traditional%Sanger%sequencing.% This% led% to% new% and% revised% diagnoses% of% patients% in% this% cohort% and% widened% the% phenotypic%spectrum%ascribed%to%individual%genes.%%

Based%on%the%previous%experience%of%the%clinical%collaborators%of%this%project%and%taking% the%results%from%this%study%as%a%whole,%some%general%clinical%recommendations%can%be% made.%For%cases%with%phenotypes%overlapping%classical%or%vascular%EDS,%the%yield%of% pathogenic%variants%is%high%in%the%normally%associated% gene% (COL3A1,% COL5A1% and% COL5A2),% and% occasionally% one% may% identify% pathogenic% variants% in% another% fibrillar% collagen%gene.%Cases%of%uncomplicated%hypermobility%type%EDS%with%no%family%history% of%vascular%complication%had%a%low%yield%of%pathogenic%variants%and%a%large%number%of% VUS%(most%of%which%will%not%be%fed%back%to%patients):%NGS%panel%testing%for%such%cases% is% unlikely% to% be% diagnostic.% Conversely,% potential% connective% tissue% disorder% patients% with% a% history% of% vascular% complication,% marfanoid% features,% or% a% significant% family% history%should%undergo%NGS%panel%testing%for%collagen%and%aortopathy%genes%because% these%results%indicate%a%reasonable%likelihood%of%identifying%pathogenic%variants%in%one% of% these% genes.% Similarly,% patients% with% EDS% or% other% HDCT% overlapping% with% osteogenesis% imperfecta% should% also% undergo% collagen% gene% testing% to% exclude% pathogenic%COL1A1%or%COL1A2%variants.%% The%interpretation%of%missense%variants%includes%correlation%with%the%complete%clinical% phenotype.%In%keeping%with%ACMG%guidelines,%I%classified%variants%supported%by%some% evidence%of%pathogenicity%(e.g.,%high%in%silico%scores%and%presence%in%functionally%active%

! 69! ! domains)%or%associated%with%clinical%features%such%as%aortopathy%as%“likely%pathogenic”:% these%patients%should%be%followed%clinically%with%vascular%imaging,%familial%segregation% studies,%and%available%structural%or%biochemical%studies,%including%electron%microscopy% or%protein%analysis.%Initial%genetic%counseling%for%such%patients%should%point%out%that%the% true% significance% of% the% variant% will% not% be% known% until% these% additional% tests% are% complete.%% Although%in%the%short:term,%the%increased%detection%of%VUS’s%bring%an%increased%burden% for% interpretation% and% may% add% uncertainty% to% the% diagnostic% process,% this% is% greatly% outweighed%by%the%prospect%of%making%specific%genetic%diagnoses%that%would%otherwise% go% undetected% and% form% the% basis% of% preventive% screening% in% relatives.% In% the% longer% term,%assignment%of%pathogenicity%is%likely%to%be%facilitated%by%data%from%ongoing%large: scale%genome%sequencing%projects%in%patient%and%control%cohorts.%

I% used% the% PCR:based% Fluidigm% Access% Array% followed% by% Illumina% MiSeq.% Mean% coverage% per% amplicon% was% greater% than% 140×% for% all% amplicons,% comparable% to% previously% reported% assays% using% this% methodology.47% Although% this% PCR:based% NGS% method%is%unable%to%detect%large%deletions%or%chromosomal%rearrangements,%these%are% rare% in% EDS.39% In% addition,% whilst% false% positive% variant% calls% have% been% previously% reported%with%this%approach,47%I%detected%only%a%single%variant%(in%exon%1%of%SMAD3)%that% could% not% be% confirmed% by% Sanger% sequencing.% Since% NGS% methods% have% higher% throughput,% achieve% greater% coverage% and% may% be% more% cost:efficient% than% conventional%Sanger%sequencing47,%they%may%be%applicable%to%wider%phenotypic%groups,% for%which%this%study%may%serve%as%a%guide%to%future%genetic%testing.%Further,%it%could%be% suggested% that% the% high% throughput,% lower% cost,% and% comparable% or% increased% diagnostic%yield%of%NGS%compared%to%conventional%Sanger%sequencing,%could%alter%the% optimal% diagnostic% pathway,% such% that% genetic% testing% with% NGS% panels% could% occur% earlier,% prior% to% quaternary% clinical% appraisal,% perhaps% even% giving% access% to% NGS: based%testing%to%physicians%in%secondary%or%tertiary%care%(Figure%3.1B).%%

Such%an%alternative%pathway%would%require%careful%consideration%for%its%implications%on% patient%consent,%counselling%regarding%negative%results%and%the%different%prognosis%of% vascular%EDS%versus%Marfan%or%other%primary%aortopathies.%

In%summary,%the%NGS%panels%have%allowed%new%molecular%diagnoses%in%genes%outside% the% expected% genotype:phenotype% relationship,% allowing% the% phenotypic% spectrum% associated% with% these% genes% to% be% extended.% Cases% with% apparently% mild% connective% tissue%phenotypes%may%harbour%clinically%actionable%pathogenic%variants%in%aortopathy% genes.%%

! 70! !

4.$TARGETED$SEQUENCING$IN$TAAD$

4.1$Introduction$

Aortic%diseases%are%the%third%leading%cause%of%cardiovascular%mortality%in%the%UK%and% US,% annually% accounting% for% over% 11,000% deaths% in% the% UK118% and% contributing% to% 151,500%deaths%in%the%US.119%Thoracic%aortic%aneurysm%and%dissection%(TAAD)%affects% patients%at%any%age%from%their%early%twenties%and%have%a%clinical%course%altogether%more% devastating%and%unpredictable%than%any%other%aortic%condition.120:122%TAAD%seems%to%be% increasing% in% incidence,% with% current% estimates% (in% the% range% of% 6:16% new% cases% per% 100,000%per%annum)%likely%to%under:estimate%the%true%incidence.%123,124%%

The%major%mortality%and%morbidity%associated%with%TAAD%lies%with%acute%dissection%or% rupture.% Both% are% difficult% to% predict% and% associated% with% high% mortality% and% morbidity.37,44,45,125%Dissection%weakens%the%aortic%wall%predisposing%to%rapid%growth%and% ruptureG% furthermore,% in% the% ascending% aorta% (“Type% A% dissection”),% retrograde% dissection%can%cause%aortic%valvular%failure%or%cardiac%tamponade%causing%acute%fatal% cardiac%failure%or%occlusion%of%the%coronary%ostia%causing%myocardial%infarctionG%in%the% descending% aorta% (“Type% B% dissection)% branch% vessel% occlusion% leads% to% end:organ% ischaemia.37,44,45,126%%Complicated%Type%A%and%Type%B%dissection%are%associated%with%in: hospital%perioperative%mortalities%of%up%to%63%%and%28%%respectively%according%to%the% International% Registry% of% Acute% Aortic% Dissection%(IRAD).119% Aortic%rupture,%which%may% occur%following%an%acute%dissection%or%a%period%of%clinically%silent%aneurysmal%growth,%is% associated% with% a% mortality% of% over% 85%% overall.% Early% perioperative% mortality% of% a% ruptured% aortic% aneurysm% remains% high% in% spite% of% all% recent% advances% in% surgical% technology% (28:46%)% compared% with% elective% treatment% of% unruptured% thoracic% aneurysms% where% surgical% results% have% improved% significantly% (<10%% mortality).127% TAAD%is%notoriously%poorly%detected%in%the%clinical%setting%due%to%the%non:specific%nature% of%its%clinical%presentation:%mimicking%the%more%common%myocardial%infarct%in%the%acute% phase%and%during%indolent%aneurysmal%growth%usually%remaining%silent.%%

The%current%management%paradigm%for%TAAD%uses%aortic%diameter%as%a%threshold%for% intervention,% along% with% an% arbitrary% pattern% of% surveillance% pre:% and% post:operatively.% This,% in% its% own% right,% is% considered% to% be% a% suboptimal% approach.% Furthermore,% the% choice% of% treatment% modality% :% endovascular% treatment% or% open% surgical% aortic% replacement%–%depends%quite%importantly%upon%the%presence%of%known%syndromes%such% as% Marfan% syndrome% or% indeed% any% underlying% monogenic% cause,% where% the% disease% course% is% more% aggressive.% In% this% context,% preliminary% evidence% suggests% that% these%

! 71! ! three% aspects% of% management:% aortic% surveillance,% size:thresholds% for% aortic% intervention%and%the%treatment%modality%may%be%informed%by%a%patient’s%genotype.128%In% the%context%that%there%may%be%a%significant%hidden%proportion%of%disease%caused%by%a% Mendelian%disease%gene,%this%is%of%special%significance%

Thus,% the% current% challenges% facing% clinicians% hinge% around% early% detection% of% TAAD% and,% once% detected,% prediction% of% aneurysmal% growth:rate% and% risk% of% rupture.% These% factors%are%critical%in%determining%the%optimum%timing%for%pre:emptive%elective%surgery% as% well% as% the% best% modality% for% surgery% (open% surgical% replacement% versus% endovascular%methods).46,122,127%%%

For% a% long% time% it% has% been% known%that%the%aetiology%of%TAAD%has%a%strong%familial% component,%which%may%or%may%not%take%the%form%of%a%clinically%apparent%syndrome%(e.g.% Marfan% (FBN1),% Loeys:Dietz% (TGFBR1,% TGFBR2),% Ehlers:Danlos% syndromes).40,113,129,130% Recent% advances% in% genome% technologies% have% increased% the% number%of%specific%genes%known%to%be%causative%in%familial%TAAD,%to%a%list%of%over%10% genes,% amongst% which% FBN1- (encoding% the% Fibrillin:1% protein)% seems% to% remain% predominant% (Figure% 4.1,% Table% 4.1).40:43,131,132% % What% is% not% known% is% how% prevalent% mutations% in% such% genes% :%%previously% considered% to% be% rare% causes% :% are% in% an% unselected% clinical% TAAD% cohort,% including% those% patients% with% no% apparent% family% history% or% syndromic% features.% Furthermore,% it% is% unclear% how% specific% genetic% abnormalities%would%contribute%to%the%phenotypic%behavior%of%TAAD%in%such%a%cohort.%

In%a%large%mixed%TAAD%cohort%from%two%countries,%the%UK%and%USA,%I%sought%to%outline% the%genetic%architecture%of%TAAD%in%genes%known%to%be%causative%of%TAAD%and%thereby% identify%any%clinically%relevant%genotype:phenotype%relationships.%

%

Figure$4.1%The%domain%structure%of%FBN1-(encoding%Fibrillin:1),%which%underlies%Marfan%syndrome%and%thought%to%be% a%predominant%cause%of%familial%TAAD.%Fibrillin%1%is%a%very%large%protein%(ca.%3,000%amino%acids),%comprising%46%EGFC like%repeats,%43%of%which%have%a%consensus%sequence%for%Ca:binding,%called%cbEGFClikeG%amongst%these%are%TGFClike% (TGF%binding%protein)%domains%and%a%number%of%cysteine:rich%Hybrid%motifs.%From%Tjeldhorn%et%al.%2015.133%

! 72! !

! 73! ! Table&4.1&Genes%found%to%be%causative%of%familial%TAAD% &Gene& Protein& Genome&co3 No.&Exons/&&&&&&&&&&&&&&&&&&&&&&&Protein&locus&&&function&& Other&Associated& No.&Pathogenic& No.&variants& Significant& Key&references&& ordinatesa& Transcript& &&Key&functional&domains& phenotypes&(in&addition&to& or&Likely& in&EXAC& Genotype3 length(bp)/& & familial&TAAD)& Pathogenic& database& Phenotype& Translated& mutations&in& correlations&in&& length(aa)&& ClinVar&(CNV)& TAAD&or&related& syndromes&

FBN1% Fibrillin%1% 15:48,700,503%@% 66/%11,756/% Extracellular.%Major%component%of%extracellular% Marfan%syndromeM%Acromicric% 672%(10)% 2,200% Nil%in%TAAD.% Pereira%et%al%1996%52M% 48,937,985% 2,871% microfibrils%@%structural%role%and%functional%in% dysplasiaM%familial%ectopia% Mutations%in%exons% Milewicz%et%al.%1997134M% TGFB%signallingM%% lentisM%Geleophysic%dysplasia% 24@32%associated% Le%Maire%et%al.%2011%42%% Domains:%46%EGF0like%repeats%(including%43% 2M%MASS%syndromeM%Stiff%skin% with%severe%Marfan% cb0EGF%like%domains)M%TGF0likeM%Hybrid%(see% syndromeM%Weill@Marchesani% syndrome%with% Figure%4.1)% syndrome%2%% presentation%in%early% childhood%%

TGFBR1% TGFbeta% 9:101,867,412%@% 9/%6,516/%503% Transmembrane.%Ser@Thr%kinase%receptor%@% Loeys@Dietz%syndromeM%Large% 52%(22)% 328% % Loeys%et%al.%Nat.% receptor,%type%1% 101,916,473% TGF@beta%pathway%signal%transduction% and%medium@sized%arterial% Genet.%2005%53% aneurysmM%susceptibility%to% multiple%self@healing% epitheliomas% TGFBR2% TGFbeta% 3:30,647,994%@% 7/%4,621/%567% Transmembrane.%Ser@Thr%kinase%receptor%@% Loeys@Dietz%syndromeM%Large% 60%%%(5)% 416% % Loeys%et%al.%Nat.% receptor,%type%2% 30,735,633% TGF@beta%pathway%signal%transduction% and%medium@sized%arterial% Genet.%2005%53% aneurysm%

SMAD3% mothers%against% 15:67358195%@% 9/%6,247/%425% Cytoplasm.%Transcriptional%modulator%(TGF@ Loeys@Dietz%syndromeM% 29%(6)% 369% % Van%de%Laar%et%al.% decaplentaplegic% 67487533% beta%pathway),%binds%TRE%element%in%various% Aneurysm@Osteoarthritis% Nat.Genet.%2011%56% homolog%3% gene%promoter%regions%in%complex%with% syndromeM%other%Aneurysm%&% SMAD2%&%SMAD4% Arterial%tortuosityM%Early%onset% Domains:&MH1%(aa%27@131)%and%MH2%(aa%226@ Osteoarthritis% 403)%are%key%domains%for%interacting%with%other% TBF@beta%pathway%intermediates% SMAD4% mothers%against% 18:48,494,410%@% % Cytoplasm.%(as%above)% Juvenile%PolyposisM%HHT% 161%(31)% 549% note%SMAD4%has% Teekakirikul%et%al.% decaplentaplegic% 48,611,415% syndromeM%Myhre%syndromeM% not%been%associated% AJMG%2013%135% homolog%4% Pancreatic%Ca.% with%TAAD% independently%of% JPS%

ACTA2% Alpha%Actin%@ 10:90,694,831%@% 9/%1,349/%377% Cytoplasm%(Smooth%muscle).%Major%role%in% Familial%TAAD%with%iris%floculli% 23%(5)% 387% Greater%association% Guo%et%al.,%Nat.%Genet.% smooth%muscle% 90,751,147% contractile%apparatus%of%smooth%muscle%% and%livedo%reticularis.%% with%ascending% 2007%55% aortic%aneurysm%

MYH11% Myosin%heavy% 16:15,797,029%@% 42/%6,921/% Cytoplasm%(Smooth%muscle).%Contractile% %Patent%Ductus%ArteriosusM% 86%(70)% 2,357% Duplications%more% Zhu%et%al.,%Nat%Genet,% chain%@%smooth% 15,950,890% 1,938% function% (rarely)Bicuspid%Aortic%Valve% common%in%cases%v.% 2006%54% muscle% controls%(OR%12.1),%%

! 74! ! MYLK% Myosin%Light% 3:123,328,896%–% % Cytoplasm%(Smooth%muscle).%Ca@Calmodulin@ % 12%(10)% 1,576% Mouse%KO:%cystic% Wang%et%al%AJHG% Chain%Kinase% 123,603,178% dependent%kinase%–%phosphorylates%Myosin% medial%degenerationM%% 2010%57% light%chain,%facilitating%Actin@Myosin%interaction% immunoblot%assay:% for%smooth@muscle%contractionM%from%a% defective%binding%with% separate%intronic%promoter%encodes%an% Ca%CaM%complex% independently%expressed%protein,%Tylokin,% which%stabilises%unphosphorylated%myosin%

TGFB2% TGF%beta%2% 1:218,518,676%–% 8/%5,053/%442%%% Extracellular.%Ligand%for%TGF@beta%receptors% Loeys@Dietz%syndrome%4% 35%(16)% 339% % Boileau%et%al.,%Nat.% 218,617,961% Gen.%2012%58% SKI% SKI%proto@ 1:2,160,134%–% 7/%5,613/%728% Cytoplasm.%Suppresses%TGFBeta%signalling% Shprintzen@Goldberg% 97%(82)% 483% % Doyle,%Nat.%Gen.% oncogene%(V@Ski% 2,241,652% by%interactions%with%pSmad’s%2,3%&%4%@% syndrome%% 2012%59% Avian%Sarcoma% repression%of%transcription%of%TGFbeta% % Viral%Oncogene% responsive%genes% Homolog)%

MAT2A% Homo%sapiens% 2:85,766,101%@% 9/%2,819/%395% Cytoplasm.%Catalyses%the%production%of%S@ % 3%(3)% 350% % Guo%et%al.%AJHG% methionine% 85,772,403% adenosylmethionine%(AdoMet)%from% 2015%136% adenosyltransfera methionine%+%ATP% se%II,%alpha%

PRKG1% Homo%sapiens% 10:52,834,234%@% 18/%6,928/%686% Cytoplasm%(Smooth%muscle,%also%platelets%and% %% %7%(5)% 956% %% %Guo%et%al.%AJHG% protein%kinase,% 54,058,110% CNS).%Encodes%the%soluble%isoforms%(1a%&%1b)% 2013%137% cGMP@ of%cGMP@dependent%kinase,%a%key%mediator%of% dependent,%type%I%% NO@mediated%smooth%muscle%relaxation,% inhibitor%of%platelet@aggregation%and%modulator% of%cellular%growth.%

Bp,%no.base%pairsM%aa,%amino%acid(s)M%fTAAD,%familial%thoracic%aortic%aneurysm%or%dissectionM%EGF,%epidermal%growth%factorM%Ca,%calciumM%HHT,%hereditary%haemorrhagic%telangiectasiaM%JPS,%juvenile% polyposis%syndromeM%CNS,%central%nervous%systemM%NO,%nitric%oxideM%KO,%knockout.%a.%Genome%co@ordinates%are%based%on%human%reference%sequence%GRCh37/hg19.%The%contribution%of%each%gene%to% familial%TAAD%is%unknown,%but%from%one%large%TAAD%series%from%Yale,%Marfan%syndrome%(FBN1)%accounted%for%approximately%10%%of%TAAD%cases.138%%%%

! 75! !

4.2$Methods$

4.2.1$Selection$of$cases$&$characterisation$of$phenotype$$

927$consecutive$cases$referred$to$the$Yale$Aortic$Centre,$Yale,$New$Haven,$USA$over$ a$ 10?year$ period$ and$ 423$ cases$ treated$ and$ followed$ up$ at$ three$ UK$ centres$ from$ 2000?2013$(St$Mary’s$hospital,$LondonJ$Royal$Brompton$&$Harefield$hospitals,$LondonJ$ Liverpool$Heart$&$Chest$Hospital,$Liverpool)$were$selected$for$recruitment.$From$these,$ after$exclusion$of$incorrect$phenotype,$duplicate$samples$and$relatives$of$index$cases,$ adequate$samples$from$796$patients$from$the$Yale$Aortic$centre$and$277$from$the$UK$ cohort$were$obtained$for$DNA$extraction.$Index$cases$were$defined$as$the$first$patient$ from$ a$ family$ to$ present$ to$ the$ service.$ Specific$ phenotypic$ data$ were$ obtained$ from$ existing$research$databases,$case?note$analysis$and$review$of$radiological$reports$and$ images$ (see$ Chapter$ 2.8).$ Whilst$ any$ patient$ diagnosed$ with$ a$ thoracic$ aortic$ pathology$and$referred$to$any$of$the$four$centres$above$was$included,$exclusion$criteria$ were:$traumatic$pathologies$(including$aortic$transection)$and$pathology$limited$to$the$ abdominal$aorta$without$the$thoracic$aorta.$Detailed$phenotype$data$including$patient$ demographics,$ aortic$ anatomy$ &$ pathology$ and$ relevant$ genetic$ and$ other$ clinical$ factors$ were$ obtained,$ as$ detailed$ in$ section$ 2.8$ and$ Appendix$ 2$ and$ summary$ statistics$were$compiled$for$these$variables$(see$Results,$Tables$4.2$and$4.3).$$

* 4.2.2$Targeted$exon$sequencing,$variant$calling$&$downstream$analysis $

Following$ DNA$ extraction,$ targeted$ exon$ sequencing$ was$ carried$ out$ using$ two$ Fluidigm$ assays,$ named$ in$ this$ thesis$ as:$ Aortopathy$ panel$ 1$ (TAAD?X),$ comprising$ 363$ primer?pairs$ for! FBN1,! TGFBR1,! TGFBR2,! MYH11,! ACTA2,! SMAD3! and! MYLK! (Appendix$1,$Table$S2)$and$Aortopathy$panel$2$(TAAD?Z)$comprising$495$primer$pairs$ for$ SKI,! TGFB2,! SLC2A10,! COL1A1,! COL1A2,! COL3A1,! COL5A1! and! COL5A2! (Appendix$1,$Table$S3),$as$previously$detailed$(Chapter$2.4).$

DNA$ extraction$ from$ saliva$ /$venous$blood$samples$(UK$cohort)$ &$ from$ fresh$ frozen$ aortic$samples$(Yale$cohort)$and$normalisation$for$targeted$sequencing$by$the$Fluidigm$ Access$ Array$ system$ &$ Illumina$ MiSeq$ or$ HiSeq$ next$ generation$ sequencer$ was$ carried$ out$ as$ detailed$ in$ Chapter$ 2.4.$ Samples$ were$ sequenced$ in$ batches$ of$ 48$ samples$ with$ 1$ negative$ control$ (water$ blank)$ per$ batch:$ where$ some$ samples$ had$ $$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ $$$$$$$$ *!Jana!Vandrovcova,!Jennifer!Biggs,!Penny!Norsworthy,!Abdulshakur!Abdullah,!David!Ross!at!various!times,! helped!with!the!laboratory!aspects!(library!preparation)!of!the!Fluidigm!targeted!sequencing;!Sequencing!on! the!Illumina!Miseq/Hiseq!of!prepared!libraries!was!carried!out!by!the!Imperial!Genomics,!led!by!Laurence! Game;!Initial!analysis!of!NGS!reads!including!read!mapping,!and!variant!calling!was!carried!out!by!Michael! Mueller!&!Dalia!Kasperavicuite!! ! 76! ! more$ than$ one$ DNA$ extraction,$ termed$ “replicates”$ these$ were$ run$ as$ independent$ samples,$to$optimise$coverage$and$validation.*$

Mapping$of$NGS$reads,$followed$by$variant$calling$and$annotation$were$carried$out$as$ described$ (Chapter$ 2.9).$ Median$ coverage$ of$ negative$ control$ samples$ was$ used$ to$ determine$sample$inclusion$thresholds.$Samples$that$had$a$median$coverage$<10x$of$ the$ negative$ control$ with$ the$ highest$ median$ coverage$ were$ identified$ and$ excluded$ from$downstream$analysis.$This$was$done$to$exclude$samples$which$could$potentially$ have$ >10%$ reads$ from$ contamination.$ Taking$ into$ account$ sample$ replicates,$ 29$ samples$ were$ excluded$ from$ the$ TAAD?X$ assay$ and$ 23$ from$ the$ TAAD?Z$ assayJ$ 1$ sample$ was$ excluded$ from$ both$ and$ therefore$ completely$ excluded$ from$ cohort$ analysis.$After$sample$exclusion,$the$median$coverage$of$amplicons$was$recalculated.$ The$ amplicons$ with$ median$ coverage$ <5$ were$ excluded$ from$ variant$ calling.$ 5$ amplicons$ from$ the$ TAADX$ assay$ and$ 28$ from$ the$ TAADZ$ assay$ were$ excluded.$ Details$of$excluded$amplicons$are$provided$in$Appendix$3.*$

From$ initial$ analysis$ of$ the$ transition$ transversion$ (Ti/Tv)$ ratio’s$ it$ was$ evident$ that,$ without$stringent$filtering$criteria,$there$may$be$a$significant$proportion$of$false$positive$ NGS$calls$resulting$from$amplified$PCR$errors$ or$other$PCR$artefacts.$Therefore,$ an$ initial$analysis$of$variant$calls$was$carried$out$to$determine$the$optimum$thresholds$for$ variant$filtering$based$on$allele$balance$(ABHet)$and$read$depth$(DP).$The$distributions$ of$ ABHet$ values$ and$ DP$ for$ variants$ in$ these$ two$ subsets$ were$ plotted.$ The$ plots$ showed$that$ABHet$would$be$a$useful$measure$to$discriminate$between$true$and$false$ positives,$ whilst$ DP$ does$ not$ discriminate$ between$ true$ and$ false$ positives$ well.$ Furthermore,$ to$ determine$ the$ filtering$ threshold,$ we$ fitted$ mixture$ distribution$ on$ ABHet$ values$ of$ all$ unfiltered$ dataset.$ For$ that,$ first$ we$ transformed$ the$ data$ to$ 1? ABHet.$Then$we$fitted$a$mixture$of$gamma$and$Gaussian$distributions$using$maximum$ likelihood$ estimation$ (Appendix$ 4).$ Then$ we$ chose$ filtering$ threshold$ to$ allow$ false$ discovery$ rate$ 5%.$ Based$ on$ this$ analysis,$ the$ filtering$ was$ done$ using$ ABHet$ thresholds$ 0.7083$ for$ taadX$ dataset$ and$ 0.6974$ for$ taadZ$ dataset.$ Detailed$ metrics$ and$ graphical$ analyses$ of$ ABHet$ filtering$ thresholds$ are$ provided$ in$ Appendix$ 4.$ In$ order$ to$ further$ refine$ the$ filtering$ process,$ the$ bam?file$ traces$ of$ variants$ with$ borderline?low$ABHet$values$(0.2$?$0.3)$or$DP$(<50$reads)$were$examined$using$IGV$ † software$and$manually$filtered. $

$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ $$$$$$$$ *![attributions!as!above]! †!Dalia!Kasperavicuite!calculated!the!optimum!filtering!thresholds!based!on!ABHet!and!DP!and!carried!out! initial! filtering! of! variants! based! on! these! thresholds.! Subsequent! (manual)! filtering! and! downstream! analysis!was!carried!out!by!myself.!! ! 77! ! 4.2.3$Pathogenicity$assignment$and$validation$

Non?rare$variants$(MAF>1:1000$in$any$database)$were$excluded$from$further$analysis.$ Of$the$rare$variants,$synonymous$and$intronic$variants$as$well$as$non?frameshift$indels$ were$ excluded.$ The$ remainder$ were$ named$“functionally$ significant”$ and$ divided$ into$ three$ groups$ based$ on$ available$ databases$ (ClinVar,$ HGMD),$ published$ data$ and$ ACMG$ guidelines:$ Pathogenic,$ Likely$ Pathogenic$ &$ VUS’s.$ Non?sense$ mutations,$ canonical$splice?site$disrupting$variants,$frameshift$indels,$along$with$well?established$ pathogenic$ missense$ variants$ from$ previous$ reports$ were$ counted$ as$ Pathogenic.$ In$ addition,$ helical$ Glycine$ substitution$ in$ collagen$ genes$ were$ considered$ Pathogenic.$ Other$missense$mutations$with$strong$evidence$of$pathogenicity$and$meeting$ACMG$ criteria$were$categorised$as$“Likely$Pathogenic”.$All$other$variants,$including$those$with$ conflicting$evidence$of$pathogenicity$were$categorized$as$VUS’s.$Unlike$in$the$previous$ chapter,$ where$ pathogenicity$ in$ relation$ to$ the$ well?characterised$ collagen$ genes$ is$ well?established$ and$ the$ relationship$ between$ an$ aortopathy$ susceptibility?gene$ and$ EDS$ is$ not$ clear,$ in$ this$ chapter,$ Pathogenic$ and$ Likely$ Pathogenic$ (P/LP)$ variants$ were$ grouped$ together,$ because$ the$ distinction$ between$ pathogenic$ and$ likely$ pathogenic$variants$is$more$blurred$(less$is$known$about$these$genes$in$general)$whilst$ the$ relationship$ between$ these$ genes$ and$ the$ aortopathy$ phenotype$ is$ established.$ Pathogenic$ and$ Likely$ Pathogenic$ variants$ were$ prioritised$ for$ validation$ by$ Sanger$ sequencing.$

4.3$Results$

4.3.1$Clinical$phenotype$

Tables$4.2$and$4.3$show$the$demographic$and$clinico?anatomical$characteristics$of$the$ cohort.$

Table$4.2$Demographic$characteristics$of$the$UK$and$Yale$TAAD$cohorts.$

$$ Yale$cohort$ UK$cohort$ Whole$cohort$ Number$ 773$ 277$ 1,050$ Mean$Age$(range)$ 61$(18?87)$ 57$(11?84)$ 60$(11?87)$ %$Male$ 69$ 67$ 68$ a Prop.$Caucasian(%)$ $$ ?$ 247/262*$(94)$ $ a.$no$ethnicity$data$were$available$for$the$Yale$cohort$nor$for$15$patients$in$the$UK$cohort$ $

! 78! ! Table$4.3$Clinical$&$Anatomical$characteristics$of$aortic$pathology.$

$$ Yale$cohort$$ UK$cohort$$$$$ Whole$cohort$$$$$$$$$$$$$$$$ a Primary$Aortic$pathology$ $ $ $ $ $$$$$$Aneurysm$only$ 689$(89.1)$ 185$(66.8)$ 874$(83.2)$ b $$$$$$Dissection$(+/?$aneurysm)$ $ 72$(9.3)$ 88$(31.8)$ 160$(15.2)$ $$$$$$Rupture$ 3$(0.4)$ 2$(0.7)$ 5$(0.5)$ $$$$$$Other$(PAU,$IMH)$ 12$(1.6)$ 4$(1.4)$ 16$(1.5)$ Anatomic$involvement$(primary$presentation)$ $ $ $ $$$$Ascending/Arch$only$ 696$(90.0)$ 143$(51.6)$ 839$(79.8)$ $$$$Descending/Thoracoabdominal$ 76$(9.8)$ 137$(49.5)$ 213$(20.2)$ $ $ $ $ Secondary$Aortic$event$ 108$(14.0)$ 35(12.6)$ 143$(13.6)$ Tertiary$Aortic$event$ 6$(0.8)$ 3(1.1)$ 9$(0.9)$ $ $ $ $ c Anatomic$involvement$(all$presentations)$ $ $$$$Ascending/Arch$ 726$$ 153$$ 879$$ $$$$Descending/Thoracoabdominal$ 160$ 165$$ 325$$ $ $ $ $ Maximal$aortic$diameter$Mean(range),$cm$ 5.2$(3.4$–$11.0)$ 5.6$(2.7?13.0)$ 5.3$(2.7?13.0)$ $ $ $ $ Extra?aortic$aneurysm$ 7$(0.9)$ 4$(1.4)$ 11$(1.0)$ $ $ $ $ Probable/proven$family$history$$$ 222$(28.7)$ 41$(14.8)$ 263$(25.0)$ d Known$syndrome$ $ $ $ $ $$$$$$$$$Marfan$/$LDS$ 4$(0.5)$ 26$(9.4)$ 30$(2.9)$ $$$$$$$$$EDS$ 0$$ 0$ 0$ $$$$$$$$Other$ 1$(0.1)$ 3$(1.1)$ 4$(0.4)$ $ $ $ $ e Management$ $ $ $ $ $$$$$$?$$conservative$/$awaiting$treatment$ $ 36$(13.0)$ $ $$$$$$?$$operative$intervention$ $ 223$(80.5)$ $ $$$$$$?$mean$no.$open$operations$per$patient$ 1.1$ 0.8$ 1.04$ $$$$$$?$ mean$ no.$ endovascular$ procedures$ per$ $ 0.3$ $ patient$ $$$$$?$unknown$(insufficient$data)$ $ 11$(4.0)$ $$ f Other$cardiovascular$interventions$ $ $ 49$(17.7)$ ?$ $ $ $ $

PAU,$penetrating$aortic$ulcerJ$IMH,$intramural$haematomaJ$LDS,$Loeys?Dietz$syndromeJ$EDS,$Ehlers?Danlos$ syndrome.$$a.$Primary$aortic$pathology$refers$to$presenting$aortic$pathologyJ$Secondary/Tertiary$events$refer$ to$ subsequently$ identified$ aortic$ involvement$ of$ a$ different$ part$ of$ the$ aorta$ distinct$ from$ the$ primary$ (presenting)$ pathology.$ $ b.$ 11$ dissections$ in$ the$ Yale$ cohort$ and$ 7$ in$ the$ UK$ cohort$ occurred$ as$ Secondary/Tertiary$pathologies$subsequent$to$other$aortic$presentations.$c.$Anatomic$involvement$is$defined$ relative$to$the$left$subclavian$artery$origin$(ascending$and$arch$disease$being$proximal$to$the$latter).$d.$Clinical$ data$on$known$syndromes$was$very$limited$in$the$Yale$cohort,$therefore$this$is$likely$to$underestimate$the$true$ incidenceJ$ "Other"$ refers$ to$ patients$ with$ suspected$ hereditary$ connective$ tissue$ disorder$ without$ a$ formal$ diagnosisJ$4$out$of$27$UK$Marfan/LDS$patients$had$a$known$diagnosis$of$LDS$e.$Complete$data$on$clinical$ management$ was$ not$ available$ for$ the$ Yale$ cohortJ$ operative$ intervention$ includes$ both$ open$ and$ endovascular$ procedures:$ in$ the$ UK$ cohort$ 226$ patients$ underwent$ 258$ open$ aortic$ operations$ and$ 89$ endovascular$procedures$(including$25$Hybrid$aortic$operations)$f.$49$patients$in$the$UK$cohort$had$55$other$ cardiovascular$ surgical$ interventions$ which$ comprised:$ 4$ iliac$ stents,$ 6$ femoro?femoral$ crossover$ grafts,$ 2$ lower$limb$bypasses,$1$visceral$artery$embolisation,$17$coronary$artery$bypass$graft$procedures,$6$coronary$ angioplasties,$ 6$ ablations/pacemaker$ insertion$ for$ arrhythmia,$ 1$ closure$ of$ VSD,$ 12$ mitral/tricuspid$ valvular$ procedures.$

! 79! ! 4.3.2$Assay$performance$

Amplicons$of$the$Aortopathy$panel$1$(“TAAD?X”)$achieved$a$mean$coverage$of$293×$ (range$ 71×?556×)$ with$ 91.9%$ and$ 86.5%$ bases$ covered$ by$ ≥50$ and$ ≥100$ reads,$ respectively.$ Those$ of$ Aortopathy$ panel$ 2$ (“TAAD?Z”)$ achieved$ a$ mean$ coverage$ of$ 214×$(range$88×?501×)$per$amplicon$with$89.7%$and$80.3%$of$targeted$bases$covered$ by$≥50$and$≥100$reads$respectively.$$

4.3.3$Identification$and$classification$of$rare$genetic$variants$$

Following$initial$filtering,$1,742$rare$variants$(MAF<1:1000)$were$identified$(Table$4.4).$ Following$exclusion$of$synonymous,$intronic$and$non?frameshift$variants,$the$remaining$ 270$functionally$significant$variants$were$then$classified$according$to$pathogenicity$as$ previously$ described.$ They$ comprised$ 61$ Pathogenic$ /$ Likely$ Pathogenic$ (P/LP)$ variants$and$209$VUS’s$(Table$4.4).$

Table$4.4$Summary$of$rare$variants$identified,$by$gene$

b Rare$functionally$significant$variants $ %$ of$ whole$ cohort$ with$ All$Rare$ P/LP$ variant$ a (any$ variants $ $$ functionally$ significant$ $$ Likely$ variant)$ Pathogenic$ Pathogenic$ VUS$ 2.9$(5.0)$ FBN1! 134$ 23$ 7$ 22$ 0.3$(3.9)$ MYH11! 150$ 1$ 2$ 38$ 0.7$(2.2)$ COL1A1! 144$ 0$ 7$ 16$ 0.3$(1.3)$ COL3A1! 182$ 3$ 0$ 11$ 0.1$(1.5)$ SKI! 296$ 1$ 0$ 15$ 0.5$(1.2)$ TGFBR2! 27$ 3$ 2$ 8$ 0.2$(3.2)$ MYLK! 147$ 2$ 0$ 32$ 0.4$(0.5)$ TGFBR1! 20$ 4$ 0$ 1$ 0.1$(1.9)$ COL5A1! 246$ 1$ 0$ 19$ 0.1$(1.1)$ COL5A2! 106$ 1$ 0$ 11$ 0$(1.3)$ COL1A2! 83$ 0$ 0$ 14$ 0.2$(0.9)$ SMAD3! 48$ 0$ 2$ 7$ 0.2$(0.9)$ SLC2A10! 38$ 2$ 0$ 7$ 0$(0.1)$ TGFB2! 37$ 0$ 0$ 1$ 0$(0.5)$ ACTA2! 14$ 0$ 0$ 5$ 0$(0.2)$ SMAD4! 70$ 0$ 0$ 2$ 5.8$(25.7)$ All$genes$ 1742$ 41$ 20$ 209$ VUS,$variant$of$uncertain$significance.$P/LP,$pathogenic$or$likely$pathogenic.$a,$any$variant$with$ maximum$ reported$ MAF<1:1000,$ including$ synonymous$ and$ intronic$ variants.$ b.$ functionally$ significant$ variants$ exclude$ synonymous,$ intronic$ or$ non?frameshift$ variants$ and$ are$ further$ classified$ according$ to$ predicted$ pathogenicity:$ Pathogenic,$ Likely$ Pathogenic$ and$ VUS.

! 80! ! Table&4.5!All!pathogenic!and!likely!pathogenic!variants!identified!

Genomic& & Family& Functional& Class. Age/& Known& co0 Gene& Variant& b c Sample&ID& Aortic&phenotype& history a effect && & Gender& d syndromes& ordinates & & 17:48266534! COL1A1& ex.40:c.C2932T:p.P978S! NSNV! LP! YJTF_50_38! 67M! Aneurysm!J!Asc! Y! !

17:48266534! COL1A1& ex.40:c.C2932T:p.P978S! NSNV! LP! YJDH_12_61! 65M! Aneurysm!J!Asc! ! !

17:48271380! COL1A1& ex.25:c.G1691A:p.R564H! NSNV! LP! YJDS_38_1! 43F! Aneurysm!J!Asc! Y! !

17:48273298! COL1A1& ex.16:c.G1042A:p.A348T! NSNV! LP! YJMH_17_1! 40F! Dissection!J!TA! ! !

17:48273298! COL1A1& ex.16:c.G1042A:p.A348T! NSNV! LP! YEM_109_21! 69F! Aneurysm!J!Asc! ! !

17:48278798! COL1A1& ex.1:c.G77A:p.G26D! NSNV! LP! YAL_48_91! 76F! Dissection!J!TA! ! !

17:48278798! COL1A1& ex.1:c.G77A:p.G26D! NSNV! LP! YWM_36_95! 24M! Aneurysm!J!Asc! ! !

2:189852809! COL3A1& ex.6:c.531delC:p.G177fs! fs!del! P! YJVW_130_31! 71F! Aneurysm!J!Asc! Y! !

2:189858792! COL3A1& ex.17:c.G1178A:p.G393D! NSNV! P! YJBC_5_23! 55F! Aneurysm!J!Asc! Y! Other!!

2:189858969! COL3A1& ex.18:c.G1204A:p.G402S! NSNV! P! YJRM_112_51! 52M! Aneurysm!J!Asc! ! !

9:137676854! COL5A1& ex.30:c.G2504C:p.G835A! NSNV! P! YJSD_68_20! 52M! Aneurysm!J!Asc/Arch! !

2:189948754! COL5A2& ex.12:c.G808A:p.G270S! NSNV! P! YJMC_1_1! 47M! Aneurysm!J!Asc! Y! !

15:48703298! FBN1& ex.66:c.8504dupC:p.P2835fs! fs!ins! P! YJAI_56_99! 31M! Aneurysm!J!Asc! Y! !

15:48707763! FBN1& ex.64:c.G8021A:p.C2674Y! NSNV! LP! UKJ896! 22M! Aneurysm!J!Asc! Y! Marfan!

15:48707778! FBN1& ex.64:c.G8006A:p.G2669D! NSNV! P! 24SB839\!840(son)!&! 67F! Aneurysm!J!Asc! Y! Marfan! 841(daughter)! 15:48707828! FBN1& ex.64:c.T7956A:p.C2652X! stopgain! P! YJDV_47_31! 51M! Aneurysm!J!Asc! Y! !

15:48712915! FBN1& ex.63:c.C7788A:p.Y2596X! stopgain! P! YJSB_94_61! 48M! Aneurysm!J!Asc! Y! !

15:48713883! FBN1& ex.62:c.A7571G:p.D2524G! NSNV! LP! YJDC_64_45! 31M! Aneurysm!J!Asc! ! !

15:48719928! & ex.58:c.7039_7040del:p.2347_2347del! ! ! UKJNH936! 28M! Dissection! J! Y! Marfan! FBN1& fs!del! P! Asc/Arch/Desc! 15:48719928! FBN1& ex.58:c.7039_7040del:p.M2347fs! fs!del! P! YJJD_26_51! 57M! Aneurysm!J!Asc! ! !

15:48729244! FBN1& ex.53:c.G6410A:p.C2137Y! NSNV! LP! YJPC_22_41! 31M! Dissection!J!Desc! ! ! 15:48737633! FBN1& ex.48:c.T5857C:p.S1953P! ! ! UKJ843\! shared! by! 43F! Aneurysm!J!Asc! ! Marfan! NSNV! LP! 844!(likely!daughter)! Y! ! 81! ! 15:48752502! FBN1& ex.43:c.5236_5237insTA:p.T1746fs! fs!ins! P! UKJ21JS0242! 16F! aneurysm!J!Asc! Y! Marfan!

15:48762884! FBN1& ex.36:c.G4406C:p.R1469P! NSNV! LP! YJAD_133_86! 18F! Aneurysm!J!Asc! Y! Marfan!

15:48764753! FBN1& ex.35:c.G4331A:p.C1444Y! NSNV! P! UKJSG931! 23M! Aneurysm! J! Y! Marfan! Asc/Arch! 15:48764814! FBN1& ex.35:c.C4270G:p.P1424A! NSNV! P! YJPK_86_81! 75F! Aneurysm!J!Asc! ! !

15:48776056! FBN1& ex.31:c.A3797T:p.Y1266F! NSNV! LP! YJMD_23_71! 51M! Aneurysm!J!Asc! ! !

15:48780423! FBN1& ex.27:c.G3224A:p.R1075H! NSNV! LP! UKJhg791! 78F! Dissection!J!Asc/Arch! !

15:48780580! FBN1& ex.26:c.3193delG:p.E1065fs! fs!del! P! YJEO_21_18! 30M! Aneurysm!J!Asc! Y! !

15:48782118! FBN1& ex.25:c.C3012G:p.Y1004X! stopgain! P! UKJ21JG0083! 66F! Dissection!J!TA! ! !

15:48782234! FBN1& ex.25:c.G2896T:p.E966X! stopgain! P! UKJ21ND0281! 38F! dissection!J!Asc/Arch/TA! !

15:48782270! FBN1& ex.25:c.C2860T:p.R954C! NSNV! P! UKJvg1113! 47F! Dissection! J! Y! Marfan! Asc/Arch/TA! 15:48786416! FBN1& ex.23:c.2710_2713del:p.904_905del! fs!del! P! UKJzs915! 11M! Aneurysm!J!Asc! Y! Marfan!

15:48787352! FBN1& ex.22:c.C2645T:p.A882V! NSNV! P! UKJ21MZ0003! 54f! Aneurysm!J!AA! ! !

15:48787441! FBN1& ex.22:c.2555_2556insAC:p.T852fs! fs!ins! P! UKJRP250! 39F! Dissection!J!Asc! Y! Marfan!

15:48788410! FBN1& ex.20:c.G2306A:p.C769Y! NSNV! P! UKJjb904! 41F! Aneurysm!J!Asc! Y! Marfan!

15:48789589! FBN1& ex.20:c.2168J1G>T:0! splicing! P! YJDI_82_41! 36M! Aneurysm!J!Asc! ! !

15:48812913! FBN1& ex.10:c.C1090T:p.R364X! stopgain! P! UKJ907! 16M! Aneurysm!J!Asc! Y! Marfan!

15:48812913! FBN1& ex.10:c.C1090T:p.R364X! stopgain! P! UKJ24UAM0916! (&! 18M! Aneurysm!J!Asc! Y! Marfan! 24PM917(sister)!! 15:48829873! FBN1& ex.7:c.G671A:p.C224Y! NSNV! P! UKJ21AY0270! 35M! dissection! J! Y! Marfan! Asc/Arch/TA! 15:48829918! FBN1& ex.7:c.G626A:p.C209Y! NSNV! P! YJAV_95_7! 24F! Dissection!J!Asc/Arch! !

15:48892413! FBN1& ex.5:c.G365A:p.R122H! NSNV! P! UKJ21RS0022! 27F! Dissection!J!TA! ! !

16:15812194! MYH11& ex.37:c.G5273A:p.R1758Q! NSNV! LP! YJGS_35_28! 40M! Aneurysm!J!Asc! ! Marfan!

16:15814100! MYH11& ex.34:c.A4861C:p.K1621Q! NSNV! LP! YJHE_20_10! 34M! Aneurysm!J!Asc! ! !

16:15844048! MYH11& ex.16:c.C2005T:p.R669C! NSNV! P! UKJ21VB1018! 74F! Dissection!J!Desc! ! !

3:123339147! MYLK& ex.30:c.T4915C:p.S1639P! NSNV! P! YJLL_84_18! 54F! Dissection!J!TA! Y! !

3:123426599! MYLK& ex.16:c.2183+2T>C:0! splicing! P! YJFC_19_18! 63M! Aneurysm!J!Asc! Y! ! ! 82! ! 1:2235346! SKI& ex.4:c.C1279T:p.Q427X! stopgain! P! YJGJ_139_21! 72M! Aneurysm!J!Asc! ! !

20:45354069! SLC2A10& ex.2:c.C394T:p.R132W! NSNV! P! YJKB_51_21! 33F! Aneurysm! J! Y! ! Asc/Arch! 20:45354323! SLC2A10& ex.2:c.C648G:p.Y216X! stopgain! P! YJRW_91_1! 84M! Aneurysm!J!Asc/Arch! !

15:67457304! SMAD3& ex.2:c.G146A:p.R49Q,SMAD3! NSNV! LP! UKJ26BW1201! 47M! Dissection!J!Asc/Arch/TA! !

15:67482843! SMAD3& ex.7:c.C662T:p.S221F,SMAD3! NSNV! LP! UKJRD726! 35M! Aneurysm!J!Asc! ! LDS!

9:101900268! TGFBR1& ex.3:c.471_473del:p.157_158del! nonfs!del! P! UKJ26EA1114! 39F! Dissection!J!Asc/Arch/TA! LDS!

9:101907012! TGFBR1& ex.6:c.974J2A>G:! splicing! P! YJHM_105_61! 50M! Dissection!J!Desc! ! !

9:101908876! TGFBR1& ex.6:c.C1009T:p.R337X! stopgain! P! YJJD_123_61! 56M! Aneurysm!J!Asc! Y! !

9:101908892! TGFBR1& ex.6:c.1024+1G>A:0! splicing! P! UKJ21GC1025! 45F! Dissection!J!Asc/Arch/Desc! !

3:30729968! TGFBR2& ex.6:c.C1489T:p.R497X! stopgain! P! YJJH_18_71! 54M! Aneurysm!J!Asc! Y! !

3:30730004! TGFBR2& ex.6:c.1524+1G>T:! splicing! P! UKJ24MU795! 29M! Aneurysm!J!Asc! ! LDS!

3:30732996! TGFBR2& ex.7:c.C1609T:p.R537C! NSNV! P! YJRV_112_30! 27M! Aneurysm!J!Desc! Y! !

3:30732997! TGFBR2& ex.7:c.G1610A:p.R537H! NSNV! LP! YJJH_19_37! 53M! Aneurysm!J!Asc! Y! !

3:30732997! TGFBR2& ex.7:c.G1610A:p.R537H! NSNV! LP! YJSM_61_74! 50F! Aneurysm!J!Asc! Y! ! ex.,!exon!no.\!NSNV,!non!synonymous!single!nucleotide!variant\!fs,!frameshift\!del,!deletion\!ins,!insertion\!P,!pathogenic\!LP,!likely!pathogenic\!M,!male\!F,!female\!Asc,!ascending! aorta\!Arch,!aortic!arch\!Desc,!descending!thoracic!aorta\!TA,!thoracoabdominal!aorta\!LDS,!LoeysJDietz!syndrome.!! a.!Genomic!coJordinates!are!based!on!GRCh37/hg19!human!reference!sequence.!b.!Functional!effect:!splicing!refers!to!predicted!spliceJsite!disruption,!stopgain!denotes!a!nonJ sense!(truncating)!mutation.!c.!pathogenicity!based!on!ACMG!guidelines!(Richards!et!al.)29!d.&Y,!denotes!a!known!or!probable!family!history!of!aortopathy,!ascertained!from! clinical!records.

! 83! !

Table&4.6!All!variants!of!uncertain!significance!identified! Genomic& Gene& Variant& Functi Sample&ID& Age/& Aortic&phenotype& Family& co0 onal& Gender& historyc& ordinates& effecta&& /& Known& syndro me& 10:90697957! ACTA2% ex.8:c.T851A:p.I284N! NSNV! YEAY_13_47! 36M! Dissection!E!TA! !

10:90699264! ACTA2% ex.7:c.G808A:p.G270R! NSNV! UKE21JM0336! 51F! Dissection!E!Asc/Arch!

10:90699264! ACTA2% ex.7:c.G808A:p.G270R! NSNV! YEMV_18_33! 58M! Aneurysm!E!Asc! !

10:90699264! ACTA2% ex.7:c.G808A:p.G270R! NSNV! YEDS_38_1! 43F! Aneurysm!E!Asc! Y!

10:90699305! ACTA2% ex.7:c.G767A:p.R256H! NSNV! YERH_40_81! 59M! Dissection! E! Y! Asc/Arch/Desc! 17:48263000! COL1A1% ex.51:c.G4258A:p.G1420R! NSNV! YEAZ_46_81! 38M! Aneurysm!E!Asc! !

17:48263689! COL1A1% ex.49:c.G3994A:p.D1332N! NSNV! YEJT_46_58! 36M! Aneurysm!E!Asc/Arch!

17:48265328! COL1A1% ex.45:c.G3278A:p.R1093H! NSNV! YERR_14_21! 69M! Aneurysm!E!Asc/Arch!

17:48265968! COL1A1% ex.43:c.C3130A:p.P1044T! NSNV! YEMP_72_51! 51F! Aneurysm!E!Asc! !

17:48266365! COL1A1% ex.41:c.C2944A:p.P982T! NSNV! YEJK_7_22! 49M! Aneurysm!E!Asc! Y!

17:48267399! COL1A1% ex.36:c.C2522A:p.P841H! NSNV! YEPW_28_1! 63M! Aneurysm!E!Asc/Arch!

17:48267399! COL1A1% ex.36:c.C2522A:p.P841H! NSNV! YEKK_23_2! 59M! Aneurysm!E!Asc/Arch!

17:48267725! COL1A1% ex.35:c.C2414T:p.P805L! NSNV! YEGW_76_1! 74M! Aneurysm!E!Asc/Arch!

17:48268252! COL1A1% ex.33:c.C2269G:p.P757A! NSNV! YEGR_79_1! 67M! Aneurysm!E!Asc/Arch!

17:48272183! COL1A1% ex.21:c.G1360A:p.V454I! NSNV! YE_48_96! 48F! Aneurysm!E!Asc! !

17:48277131! COL1A1% ex.2:c.T281C:p.V94A! NSNV! YEMK_80_61! 64F! Aneurysm!E!Asc/Arch!

17:48277144! COL1A1% ex.2:c.G268A:p.E90K! NSNV! YEJR_61_31! 32M! Aneurysm!E!Asc/Arch!

17:48277171! COL1A1% ex.2:c.T241C:p.C81R! NSNV! YEBM_62_1! 66M! Aneurysm!E!Asc/Arch! Y!

17:48277179! COL1A1% ex.2:c.C233T:p.T78I! NSNV! YEJG_89_31! 56M! Aneurysm!E!Asc! Y!

17:48278844! COL1A1% ex.1:c.C31T:p.L11F! NSNV! YERR_127_1! 70M! Aneurysm!E!Asc/Arch!

17:48278865! COL1A1% ex.1:c.T10C:p.F4L! NSNV! YEJA_98_30! 70M! Aneurysm!E!Asc! !

7:94029538! COL1A2% ex.5:c.G163A:p.E55K! NSNV! YELM_95_10! 73F! Dissection!E!TA! !

7:94033874! COL1A2% ex.7:c.A286G:p.M96V! NSNV! YEDW_44_51! 48F! Aneurysm!E!Asc! !

7:94034008! COL1A2% ex.8:c.C328T:p.P110S! NSNV! YEVF_116_91! 47M! Aneurysm!E!Asc! Y!

7:94039798! COL1A2% ex.21:c.G1156A:p.E386K! NSNV! YEAD_13_16! 68M! Aneurysm!E!Asc/Arch!

7:94040447! COL1A2% ex.23:c.C1331T:p.P444L! NSNV! YEVW_130_31! 71F! Aneurysm!E!Asc! Y!

7:94041965! COL1A2% ex.25:c.A1474G:p.I492V! NSNV! YEEB_41_40! 33M! Dissection!E!Arch/Desc!

7:94049584! COL1A2% ex.35:c.C2119T:p.P707S! NSNV! YEDB_71_1! 59F! Aneurysm!E!Asc/Arch!

7:94049588! COL1A2% ex.35:c.C2119T:p.P707S! NSNV! YEWW_90_1! 63M! Aneurysm!E!Asc/Arch! Y!

7:94049588! COL1A2% ex.35:c.G2123A:p.R708Q! NSNV! YECL_24_22! 82F! Aneurysm!E!Asc/Arch!

7:94051217! COL1A2% ex.39:c.A2356C:p.T786P! NSNV! YEDI_82_41! 36M! Aneurysm!E!Asc! !

7:94053724! COL1A2% ex.41:c.A2642C:p.E881A! NSNV! YEJZ_44_26! 30M! Aneurysm!E!Asc! !

! 84! !

7:94054937! COL1A2% ex.43:c.G2797A:p.D933N! NSNV! YELL_55_41! 39M! Aneurysm!E!Asc! !

7:94055158! COL1A2% ex.44:c.C2932T:p.R978C! NSNV! YEJA_66_1! 79M! Aneurysm!E!Asc! !

7:94055784! COL1A2% ex.46:c.C3047A:p.P1016H! NSNV! YERM_20_83! 69M! Dissection! E! Y! Arch/Desc! 2:189849623! COL3A1% ex.2:c.G217C:p.D73H! NSNV! YESW_11_18! 49F! Aneurysm!E!Asc! Y!

2:189851842! COL3A1% ex.5:c.C505T:p.L169F! NSNV! YEBW_42_25! 51M! Aneurysm!E!Asc! !

2:189851842! COL3A1% ex.5:c.C505T:p.L169F! NSNV! YERK_80_69! 60M! Dissection!E!Arch/Desc!

2:189854134! COL3A1% ex.8:c.C649T:p.P217S! NSNV! YEHM_135_71! 43M! Aneurysm!E!Asc! !

2:189858145! COL3A1% ex.16:c.A1109G:p.E370G! NSNV! YEBF_54_97! 35M! Aneurysm!E!Asc! !

2:189859026! COL3A1% ex.18:c.G1261A:p.A421T! NSNV! YEKH_50_25! 55F! Aneurysm!E!Desc! Y!

2:189869022! COL3A1% ex.40:c.G2863T:p.A955S! NSNV! YEBN_104_41! 77M! Aneurysm!E!Asc! !

2:189869086! COL3A1% ex.40:c.T2927C:p.V976A! NSNV! YEKS_5_82! 48M! Aneurysm!E!Asc! Y!

2:189872296! COL3A1% ex.45:c.G3326A:p.R1109Q! NSNV! YEAS_5_26! 75F! Aneurysm!E!Asc! Y!

2:189874915! COL3A1% ex.49:c.G3835A:p.V1279I! NSNV! YERM_20_83! 69M! Dissection!E!Arch/Desc!

2:189875435! COL3A1% ex.50:c.G4073A:p.R1358Q! NSNV! YEPB_95_81! 56M! Aneurysm!E!Asc! !

9:137591844! COL5A1% ex.3:c.C367G:p.Q123E! NSNV! YEJC_124_1! EM! Aneurysm!E!Asc/Arch!

9:137591847! COL5A1% ex.3:c.G370A:p.G124S! NSNV! YEDD_17_56! 72F! Aneurysm!E!Asc/Arch! Y!

9:137622272! COL5A1% ex.7:c.C1115A:p.A372D! NSNV! YEJT_140_1! 56M! Aneurysm!E!Asc! !

9:137623445! COL5A1% ex.8:c.C1268T:p.P423L! NSNV! YENN_30_48! 50F! Aneurysm!E!Asc! !

9:137623459! COL5A1% ex.8:c.T1282C:p.S428P! NSNV! YEFP_83_97! 36M! Dissection!E!TA! Y!

9:137650096! COL5A1% ex.18:c.G1889A:p.R630Q! NSNV! YECP_69_61! 75F! Aneurysm!E!Asc/Arch!

9:137676905! COL5A1% ex.30:c.A2555T:p.N852I! NSNV! YEVK_71_81! 53F! Aneurysm!E!Asc/Arch! Y!

9:137676905! COL5A1% ex.30:c.A2555T:p.N852I! NSNV! YEKV_99_51! 25F! Aneurysm!E!Asc/Arch! Y!

9:137694837! COL5A1% ex.39:c.C3110T:p.T1037M! NSNV! YEES_14_9! 72F! Aneurysm!E!Asc! !

9:137696891! COL5A1% ex.40:c.G3185A:p.R1062Q! NSNV! YERM_112_51! 52M! Aneurysm!E!Asc! !

9:137698068! COL5A1% ex.42:c.G3292T:p.A1098S! NSNV! YEPR_86_61! 64M! Aneurysm!E!Asc! !

9:137701090! COL5A1% ex.43:c.C3428T:p.P1143L! NSNV! YEMM_125_31! 48M! Aneurysm!E!Asc! Y!

9:137704518! COL5A1% ex.48:c.C3812T:p.P1271L! NSNV! YEMS_18_1! 79F! Aneurysm!E!Asc/Arch!

9:137705863! COL5A1% ex.49:c.C3887T:p.P1296L! NSNV! YEBH_24_54! 59M! Aneurysm!E!Asc/Arch!

9:137705881! COL5A1% ex.49:c.C3905T:p.P1302L! NSNV! YEJW_32_84! 49M! Aneurysm!E!Asc/Arch!

9:137707823! COL5A1% ex.52:c.C4111G:p.P1371A! NSNV! YERF_4_49! 61M! Aneurysm!E!Asc! !

9:137710526! COL5A1% ex.55:c.C4255T:p.P1419S! NSNV! YEJP_56_1! EM! Aneurysm!E!Asc/Arch!

9:137726832! COL5A1% ex.65:c.G5152A:p.A1718T! NSNV! YEJP_144_1! 73F! Aneurysm!E!Asc! !

9:137727018! COL5A1% ex.65:c.C5338T:p.P1780S! NSNV! YEMR_9_22! 65M! Aneurysm!E!Asc! !

2:189898934! COL5A2% ex.54:c.T4362A:p.N1454K! NSNV! YETP_105_1! 65M! Aneurysm!E!Asc/Arch!

2:189899700! COL5A2% ex.53:c.A4295T:p.D1432V! NSNV! YEBW_42_25! 51M! Aneurysm!E!Asc! !

2:189904214! COL5A2% ex.51:c.A3709G:p.M1237V! NSNV! YEJM_111_61! 76F! Dissection!E!Asc! !

! 85! !

2:189904265! COL5A2% ex.51:c.C3658T:p.P1220S! NSNV! YEKG_130_1! 44F! Aneurysm!E!Asc! !

2:189906386! COL5A2% ex.50:c.G3559A:p.E1187K! NSNV! YEBW_110_91! 55F! Aneurysm!E!Asc! Y!

2:189909945! COL5A2% ex.47:c.C3323T:p.P1108L! NSNV! YEFP_83_97! 36M! Dissection!E!TA! Y!

2:189910589! COL5A2% ex.46:c.G3246C:p.Q1082H! NSNV! YEVR_17_71! 73M! Aneurysm!E!Asc/Arch!

2:189915317! COL5A2% ex.43:c.T3017A:p.M1006K! NSNV! YESS_76_95! 37M! Aneurysm!E!Asc/Arch!

2:189916926! COL5A2% ex.41:c.C2741T:p.A914V! NSNV! YEKS_22_21! 52M! Aneurysm!E!Asc! !

2:189927911! COL5A2% ex.27:c.C1856T:p.P619L! NSNV! YEBV_110_31! 58M! Aneurysm!E!Asc/Arch!

2:189957049! COL5A2% ex.7:c.A554G:p.D185G! NSNV! YELH_12_34! 75F! Aneurysm!E!Asc! !

15:48703515! FBN1% ex.66:c.T8288G:p.I2763S!! NSNV! YERK_133_41! 73M! Aneurysm!E!Asc/Arch!

15:48707790! FBN1% ex.64:c.A7994G:p.N2665S! NSNV! YEJD_8_42! 71F! IMH!E!Asc! !

15:48720623! FBN1% ex.57:c.G6917A:p.R2306H! NSNV! YERW_91_1! 84M! Aneurysm!E!Asc/Arch!

15:48725102! FBN1% ex.55:c.G6700A:p.V2234M! NSNV! UKE794! 74M! Aneurysm!E!Asc! !

15:48725102! FBN1% ex.55:c.G6700A:p.V2234M! NSNV! UKE1114! 39F! Dissection! E! LDS! Asc/Arch/TA! 15:48729579! FBN1% ex.52:c.T6319C:p.F2107L! NSNV! YEAL_48_91! 76F! Dissection!E!TA! !

15:48730093! FBN1% ex.51:c.A6185C:p.Y2062S! NSNV! YERF_65_81! EM! Dissection!E!TA! !

15:48762930! FBN1% ex.36:c.A4360G:p.N1454D! NSNV! YEJR_50_61! 79M! Aneurysm!E!Asc/Arch! Y!

15:48764873! FBN1% ex.35:c.A4211G:p.D1404G! NSNV! YELL_55_41! 39M! Aneurysm!E!Asc! !

15:48779518! FBN1% ex.28:c.G3454A:p.A1152T! NSNV! YECO_10_16! 70F! PAU!E!Arch! !

15:48787669! FBN1% ex.21:c.A2536G:p.I846V! NSNV! YETP_105_1! 65M! Aneurysm!E!Asc/Arch!

15:48797302! FBN1% ex.16:c.G1880A:p.R627H! NSNV! YECA_30_91! 43F! Aneurysm!E!Asc! Y!

15:48797302! FBN1% ex.16:c.G1880A:p.R627H! NSNV! YETG_110_1! 72M! Aneurysm!E!Asc/Arch!

15:48800775! FBN1% ex.16:c.1837+4C>T:0! splicing! YERT_30_1! 63M! Aneurysm!E!Asc! Y!

15:48805742! FBN1% ex.14:c.1588+4T>A:! splicing! YEYZ_60_91! 47M! Aneurysm!E!Asc! !

15:48805763! FBN1% ex.13:c.C1571T:p.T524M! NSNV! YESB_94_61! 48M! Aneurysm!E!Asc! Y!

15:48808496! FBN1% ex.11:c.C1211G:p.P404R! NSNV! YESS_67_81! 27M! Aneurysm!E!Asc! !

15:48808539! FBN1% ex.11:c.T1168C:p.S390P! NSNV! YEEG_40_91! 74F! IMH!E!TA! !

15:48826363! FBN1% ex.8:c.G776A:p.G259E! NSNV! UKEbh1161! 67F! Aneurysm!E!Asc/Arch/Desc!

15:48829847! FBN1% ex.7:c.C697A:p.R233S! NSNV! UKEst908! 55F! Aneurysm!E!Asc! !

15:48888476! FBN1% ex.7:c.538+4A>G:0! splicing! YEMM_24_1! 54M! Dissection!E!Asc/Arch!

15:48936891! FBN1% ex.2:c.G76C:p.D26H! NSNV! YEMW_29_1! 46M! Aneurysm!E!Asc! !

16:15808820! MYH11% ex.40:c.C5732T:p.T1911M! NSNV! YEJD_26_51! 57M! Aneurysm!E!Asc! !

16:15808886! MYH11% ex.40:c.C5666T:p.A1889V! NSNV! YEJS_78_41! 66M! Aneurysm!E!Asc! Y!

16:15809049! MYH11% ex.39:c.G5585A:p.R1862H! NSNV! YEMN_35_30! 74M! Aneurysm!E!Asc! !

16:15812192! MYH11% ex.37:c.G5275A:p.V1759I! NSNV! UKE282! 74M! aneurysm!E!TA! !

16:15812192! MYH11% ex.37:c.G5275A:p.V1759I! NSNV! YEWA_110_61! 54M! Aneurysm!E!Asc/Arch!

16:15812194! MYH11% ex.37:c.G5275A:p.V1759I! NSNV! YENA_21_41! 62F! Aneurysm!E!Asc/Arch!

! 86! !

16:15812237! MYH11% ex.37:c.G5230A:p.E1744K! NSNV! UKE292! 68M! Aneurysm!E!TA! !

16:15813156! MYH11% ex.36:c.G5092A:p.A1698T! NSNV! UKE281! 38F! dissection!E!Asc/Arch/TA!

16:15814061! MYH11% ex.34:c.G4900A:p.D1634N! NSNV! YEGC_42_51! 63M! Aneurysm!E!Asc! Y!

16:15814153! MYH11% ex.34:c.C4808T:p.T1603M! NSNV! YEBD_98_31! 51M! Aneurysm!E!Asc! !

16:15814703! MYH11% ex.33:c.A4784G:p.Q1595R! NSNV! YEJR_50_61! 79M! Aneurysm!E!Asc/Arch! Y!

16:15814740! MYH11% ex.33:c.C4747T:p.R1583W! NSNV! YEJH_49_51! 66F! Aneurysm!E!Asc/Arch!

16:15814884! MYH11% ex.33:c.C4603T:p.R1535W! NSNV! YEGK_53_55! 64M! Dissection!E!Asc/Arch!

16:15815430! MYH11% ex.32:c.C4427G:p.A1476G! NSNV! YEPZ_47_71a!! 51F! Aneurysm!E!Asc/Arch!

16:15815454! MYH11% ex.32:c.C4403T:p.A1468V! NSNV! YEGS_128_61! 79M! Aneurysm!E!Asc! !

16:15815477! MYH11% ex.32:c.G4380T:p.E1460D! NSNV! YEEG_40_91! 74F! IMH!E!TA! !

16:15818206! MYH11% ex.31:c.G4177C:p.G1393R! NSNV! UKE338! 80F! PAU!E!Desc! !

16:15818647! MYH11% ex.30:c.C3973G:p.Q1325E! NSNV! UKEBW782! 75M! Aneurysm!E!Asc! !

16:15818780! MYH11% ex.29:c.G3928T:p.V1310L! NSNV! YEDB_71_1! 59F! Aneurysm!E!Asc/Arch!

16:15820736! MYH11% ex.28:c.C3827T:p.A1276V! NSNV! YEMC_65_41! 65F! Aneurysm!E!Asc! !

16:15820782! MYH11% ex.28:c.G3781A:p.V1261M! NSNV! YEVL_104_61! 76F! Aneurysm!E!Asc! !

16:15820797! MYH11% ex.28:c.A3766C:p.K1256Q! NSNV! YECG_22_77! EM! Aneurysm!E!Asc! !

16:15820797! MYH11% ex.28:c.A3766C:p.K1256Q! NSNV! YEJW_117_81! 74M! Aneurysm!E!Asc! Y!

16:15826416! MYH11% ex.29:c.3672+5T>G:0! splicing! YE_48_96! 48F! Aneurysm!E!Asc! !

16:15826416! MYH11% ex.29:c.3672+5T>G:0! splicing! YEBH_89_61! 59M! Dissection!E!Asc! Y!

16:15826416! MYH11% ex.29:c.3672+5T>G:0! splicing! YEEB_41_40! 33M! Dissection!E!Arch/Desc!

16:15826416! MYH11% ex.29:c.3672+5T>G:0! splicing! YEDW_28_75! 60M! Dissection!E!Asc/Arch/Desc!

16:15829354! MYH11% ex.26:c.G3375C:p.E1125D! NSNV! YERE_55_1! 70F! Aneurysm!E!Asc/Arch!

16:15829403! MYH11% ex.26:c.C3326A:p.A1109D! NSNV! YEAP_102_31! 62F! Aneurysm!E!Asc! !

16:15832533! MYH11% ex.24:c.C3010G:p.L1004V! NSNV! YECB_25_29! 36M! Aneurysm!E!Asc! !

16:15833981! MYH11% ex.23:c.C2924T:p.T975M! NSNV! UKE24SB839! 67F! Aneurysm!E!Asc! YEMFS!

16:15842009! MYH11% ex.17:c.C2075T:p.A692V! NSNV! UKE21AH0146! 43M! Dissection!E!Asc/Arch/TA!

16:15844149! MYH11% ex.16:c.C1904T:p.T635M! NSNV! YEAV_95_7! 24F! Dissection!E!Asc/Arch!

16:15850280! MYH11% ex.14:c.A1667G:p.E556G! NSNV! YEJR_50_61! 79M! Aneurysm!E!Asc/Arch! Y!

16:15853518! MYH11% ex.12:c.G1316A:p.R439H! NSNV! YERL_11_64! 68M! Aneurysm!E!Asc/Arch!

16:15917142! MYH11% ex.3:c.G472A:p.A158T! NSNV! YEAD_109_61! 78M! Aneurysm!E!Asc/Arch!

16:15931893! MYH11% ex.2:c.A217C:p.K73Q! NSNV! YEAP_102_31! 62F! Aneurysm!E!Asc! !

16:15932109! MYH11% ex.2:c.A1G:p.M1V! NSNV! YERW_91_1! 84M! Aneurysm!E!Asc/Arch!

16:15818073! MYH11% ex.9:c.T973C:p.S325P! NSNV! YEAA_77_31! 67M! Aneurysm!E!Asc! !

3:123333047! MYLK% ex.3:c.G367A:p.A123T! NSNV! YEAR_17_36! 55M! Aneurysm!E!Asc! !

3:123337509! MYLK% ex.2:c.C194T:p.A65V! NSNV! YEJH_129_21! 81F! Aneurysm!E!Asc/Arch!

3:123337509! MYLK% ex.2:c.C194T:p.A65V! NSNV! YEAZ_46_81! 38M! Aneurysm!E!Asc! !

! 87! !

3:123339177! MYLK% ex.30:c.G4885C:p.G1629R! NSNV! YEAY_13_47! 36M! Dissection!E!TA! !

3:123376245! MYLK% ex.23:c.G3809T:p.C1270F! NSNV! YEFL_25_40! 39M! Aneurysm!E!Asc! !

3:123383036! MYLK% ex.22:c.C3694T:p.R1232C! NSNV! YEML_107_81! 47M! Aneurysm!E!Asc/Arch!

3:123385095! MYLK% ex.21:c.A3595G:p.T1199A! NSNV! YEDV_47_31! 51M! Aneurysm!E!Asc! Y!

3:123385148! MYLK% ex.21:c.G3542A:p.R1181H! NSNV! YEPW_133_93! 63F! Aneurysm!E!Asc! !

3:123385148! MYLK% ex.21:c.G3542A:p.R1181H! NSNV! YECB_25_29! 36M! Aneurysm!E!Asc! !

3:123401086! MYLK% ex.19:c.G3430A:p.V1144M! NSNV! UKE26JW1055! 77M! Aneurysm!E!Asc/Arch!

3:123401086! MYLK% ex.19:c.G3430A:p.V1144M! NSNV! YEEG_47_91! 47F! Aneurysm!E!Asc! Y!

3:123401145! MYLK% ex.19:c.G3371T:p.S1124I! NSNV! YEJG_57_41! 61M! Aneurysm!E!Asc! !

3:123419143! MYLK% ex.17:c.A2965T:p.N989Y! NSNV! YEDS_103_41! 69F! Aneurysm!E!Asc! !

3:123419194! MYLK% ex.17:c.G2914A:p.A972T! NSNV! YELB_13_45! 61M! Aneurysm!E!TA! !

3:123419524! MYLK% ex.17:c.C2584T:p.R862W! NSNV! UKEGG247! 78M! Aneurysm!E!TA! !

3:123419614! MYLK% ex.17:c.C2494G:p.L832V! NSNV! YERT_90_81! 63M! Aneurysm!E!Asc! !

3:123420343! MYLK% ex.16:c.G2197A:p.E733K! NSNV! YECK_74_1! 73F! Aneurysm!E!Asc/Arch!

3:123427589! MYLK% ex.14:c.A1889G:p.N630S! NSNV! YENB_60_71! 59F! Aneurysm!E!Asc/Arch!

3:123427599! MYLK% ex.14:c.G1879A:p.E627K! NSNV! YENP_134_95! 71F! Aneurysm!E!Asc! !

3:123440977! MYLK% ex.12:c.A1595G:p.H532R! NSNV! YEKC_43_1! 50M! Aneurysm!E!Asc! !

3:123441071! MYLK% ex.12:c.A1501G:p.I501V! NSNV! YESD_109_48! 64F! Aneurysm!E!TA! !

3:123444833! MYLK% ex.11:c.G1402A:p.V468I! NSNV! UKEIH924! 65M! dissection!E!Asc/Arch/TA!

3:123451779! MYLK% ex.11:c.G1480A:p.G494S! NSNV! UKE323! 52M! Aneurysm!E!TA! !

3:123452670! MYLK% ex.10:c.C1173G:p.S391R! NSNV! UKE265! 65M! Aneurysm!E!TA! !

3:123452710! MYLK% ex.10:c.G1133A:p.R378H! NSNV! YENM_134_41! 76M! Aneurysm!E!Asc/Arch!

3:123453001! MYLK% ex.10:c.A842G:p.K281R! NSNV! YEHH_66_61! 71F! Dissection!E!TA! !

3:123456177! MYLK% ex.6:c.G802A:p.A268T! NSNV! YETT_39_76! 58M! Aneurysm!E!Asc! !

3:123471222! MYLK% ex.5:c.A329G:p.N110S! NSNV! YEJB_25_79! 58F! Aneurysm!E!Desc! !

3:123471291! MYLK% ex.5:c.G260A:p.G87E! NSNV! UKEQA1105! 78M! PAU!E!Desc! !

3:123471294! MYLK% ex.5:c.G257A:p.R86Q,! NSNV! UKE487! 42F! aneurysm!E!Desc! !

3:123512525! MYLK% ex.4:c.G164A:p.R55Q! NSNV! YEJN_10_75! 33M! Aneurysm!E!Asc! Y!

3:123512664! MYLK% ex.4:c.T25C:p.S9P! NSNV! YEEG_40_91! 74F! IMH!E!TA! !

1:2234432! SKI% ex.2:c.C985T:p.P329S! NSNV! YEJT_58_1! 72M! Aneurysm!E!Asc/Arch!

1:2234516! SKI% ex.2:c.C1069T:p.R357W! NSNV! YEJB_111_31! 81M! Aneurysm!E!Asc/Arch!

1:2235355! SKI% ex.4:c.G1288A:p.V430M! NSNV! YEKG_132_1! 58F! Aneurysm!E!Asc! YEMFS!

1:2235458! SKI% ex.4:c.C1391A:p.A464D! NSNV! YEGJ_139_21! 72M! Aneurysm!E!Asc! !

1:2235482! SKI% ex.4:c.C1415T:p.A472V! NSNV! YEJA_98_30! 70M! Aneurysm!E!Asc! !

1:2235503! SKI% ex.4:c.A1436G:p.D479G! NSNV! YEDP_136_1! 68M! Aneurysm!E!Asc/Arch!

1:2235531! SKI% ex.4:c.C1464A:p.S488R! NSNV! YERR_127_1! 70M! Aneurysm!E!Asc/Arch!

! 88! !

1:2235750! SKI% ex.5:c.C1493T:p.S498L! NSNV! YEJM_105_91! 62M! Aneurysm!E!Asc! !

1:2235756! SKI% ex.5:c.C1499T:p.S500F! NSNV! YEDR_136_81! 61M! Aneurysm!E!Asc! !

1:2237468! SKI% ex.6:c.T1777C:p.F593L! NSNV! YEKG_132_1! 58F! Aneurysm!E!Asc! YEMFS!

1:2237546! SKI% ex.6:c.C1855A:p.L619I! NSNV! YEPT_11_49! 64M! Dissection!E!Desc! !

1:2237597! SKI% ex.6:c.C1906A:p.R636S! NSNV! YESG_31_61! 50M! Aneurysm!E!Asc! !

1:2237648! SKI% ex.6:c.G1957A:p.G653S! NSNV! YEGG_119_91! EF! Aneurysm!E!Asc/Arch! Y!

1:2237652! SKI% ex.6:c.G1961A:p.C654Y! NSNV! YEAG_7_61! 64F! Aneurysm!E!Asc! Y!

1:2238024! SKI% ex.7:c.C2007G:p.D669E! NSNV! YEMS_18_1! 79F! Aneurysm!E!Asc/Arch!

20:45354042! SLC2A10% ex.2:c.G367A:p.V123M! NSNV! YESJ_48_61! 70F! Dissection!E!Asc! !

20:45354091! SLC2A10% ex.2:c.A416G:p.Y139C! NSNV! YEPB_33_81! EF! Aneurysm!E!Asc/Arch!

20:45354307! SLC2A10% ex.2:c.C632T:p.P211L! NSNV! YEEP_5_44! 66M! Aneurysm!E!Asc! !

20:45354822! SLC2A10% ex.2:c.C1147T:p.P383S! NSNV! YEJP_56_1! EM! Aneurysm!E!Asc/Arch!

20:45358006! SLC2A10% ex.4:c.T1426C:p.S476P! NSNV! YEPC_77_51! EM! Aneurysm!E!Asc! !

20:45358033! SLC2A10% ex.4:c.A1453C:p.T485P! NSNV! YEJZ_131_1! 48M! Aneurysm!E!Asc! !

20:45362418! SLC2A10% ex.5:c.G1571A:p.R524K! NSNV! YEKT_80_20! 61M! Aneurysm!E!Asc/Arch!

15:67358595! SMAD3% ex.1:c.G103A:p.V35I! NSNV! YETF_50_38! 67M! Aneurysm!E!Asc! Y!

15:67457280! SMAD3% ex.2:c.A122G:p.H41R! NSNV! YEAK_101_31! 66M! Dissection!E!Asc! !

15:67473647! SMAD3% ex.4:c.C142A:p.R48S! NSNV! YEPW_80_86! 49M! Dissection!E!TA! !

15:67479751! SMAD3% ex.6:c.C473A:p.A158D! NSNV! YERW_72_71! 73M! IMH!E!Desc! !

15:67479809! SMAD3% ex.6:c.C531G:p.I177M! ! UKEGW2! 720! Dissection!E!TA! Y! NSNV! 15:67479814! SMAD3% ex.6:c.T536A:p.M179K! NSNV! YESB_24_11! 35M! Aneurysm!E!Asc! Y!

15:67482814! SMAD3% ex.7:c.G633C:p.W211C! NSNV! YEML_107_81! 47M! Aneurysm!E!Asc/Arch!

18:48584818! SMAD4% ex.7:c.G896A:p.G299E! NSNV! YEBN_104_41! 77M! Aneurysm!E!Asc! !

18:48603029! SMAD4% ex.11:c.C1330T:p.H444Y! NSNV! YEAL_48_91! 76F! Dissection!E!TA! !

1:218578520! TGFB2% ex.2:c.C356T:p.P119L! NSNV! YEJK_7_22! 49M! Aneurysm!E!Asc! Y!

9:101907032! TGFBR1% ex.5:c.A761G:p.H254R! NSNV! YEAG_128_31! 41M! Aneurysm!E!Asc! !

3:30648428! TGFBR2% ex.1:c.C53T:p.T18M! NSNV! YEJT_41_61! 59F! Aneurysm!E!Asc/Arch!

3:30686248! TGFBR2% ex.2:c.A104G:p.D35G! NSNV! YERM_20_83! 69M! Dissection!E!Arch/Desc!

3:30686395! TGFBR2% ex.2:c.G251A:p.C84Y! NSNV! YEPZ_47_71! 51F! Aneurysm!E!Asc/Arch!

3:30691950! TGFBR2% ex.3:c.A452T:p.E151V! NSNV! YEBD_13_65! 39M! Aneurysm!E!Asc! !

3:30713213! TGFBR2% ex.4:c.A538G:p.I180V! NSNV! YEAC_12_42! 74F! Aneurysm!E!Asc/Arch!

3:30713250! TGFBR2% ex.4:c.A575G:p.N192S! NSNV! YEJR_50_61! 79M! Aneurysm!E!Asc/Arch! Y!

3:30713495! TGFBR2% ex.4:c.G820T:p.V274L! NSNV! YEEG_40_91! 74F! IMH!E!TA! !

3:30733045! TGFBR2% ex.7:c.C1658T:p.S553L! NSNV! YEZB_73_61! 73F! Aneurysm!E!Asc/Arch! Y!

![legend!as!for!Table!4.4]!

! 89! !

!

4.3.4&Pathogenic&&&Likely&Pathogenic&variants&&

The!majority!of!P/LP!variants!(30/61)!were!in!FBN1,!followed!by!a!significant!proportion!in! the! collagen! genes,! COL1A1! &! COL3A1! and! the! TGFEbeta! receptor! genes,! TGFBR1% &% TGFBR2.! Table! 4.5! shows! these! variants.! The! majority! of! FBN1% pathogenic/likely! pathogenic! variants! (19/30)! were! in! recognised! functionally! significant! domains! (15! in! calciumEbinding! epidermal! growth! factor! like! (cbEGF)! domains,! 4! in! the! Hybrid! #1! or! #2! domainsa!4!disrupted!Cysteine!residues!within!a!cbEGF!domain).!2!of!the!P/LP!variants!in! SMAD3!affected!the!MH1!or!MH2!domain!(required!for!SMAD!signalling).!

4.3.5&Variants&of&Uncertain&Significance&(VUS’s)&&

209!VUS’s!were!identified!across!all!genes,!with!the!majority!in!MYH11,!MYLK,!FBN1,%SKI! and! the! collagen! genes.(Table! 4.6)! Of! these,! approximately! 63! had! some! evidence! of! pathogenicity!(predicted!deleterious!by!at!least!4!inEsilico!tools!and/or!with!Cadd!score!>20)! or! were! within! a! functionally! important! domain,! though! overall,! they! lacked! sufficient! evidence!to!meet!criteria!for!pathogenicity!according!to!ACMG!guidelines.!15/23!VUS’s!in! FBN1!were!in!functionally!important!domains!(12!in!cbEGF,!2!in!TGFBP!and!1!in!Hybrid#1)a! 1!VUS!in!SMAD3!was!in!a!MH1!domain,!one!VUS!in!TGFBR1!was!in!its!SerineEThreonine! kinase!domain!and!a!further!VUS!in!SKI!was!in!the!SKIL(SKIEligand)Ebinding!domain.!Some! VUS’s! had! previously! been! reported! as! pathogenic! or! likely! pathogenic,! but! we! have! labelled! them! VUS’s! owing! to! conflicting! evidence! of! pathogenicity! (Table! 4.7).!

! 90! !

Table&4.7.&Variants(of(uncertain(significance(previously(categorised(as(pathogenic(or(likely(pathogenic&

Genomic& Sample&ID&& Phenotype&& Gene& Variant& &Domain& Predictive& Cadd" &Conservation( &Previous&reports& Co1ordinate& Scoresa& a&& Scores( a((((((((((((( SIFT,Polyphen GERP++,PhyloP, ,LRT,MT,MA,( SiPhy& FATHMM& & 7:94054937( YLLL_55_41( 39M(–(Asc.An( COL1A2' ex.43:(c.G2797A:( ( D,D,D,D,L,D( 4.56, 5.32,(9.657,(19.58( LOVD:(OI(I),(LP( p.D933N( 24.7( 15:48720623( YLRW_91_1( 84M(–(Asc.( FBN1' ex.57:(c.G6917A:( cbEGFLlike( T,P,D,D,L,D( 3.98, 5.76,7.818,19.57( ClinVar:(likely(pathogenic( +hemi.(An( p.R2306H( #36( 20.3( 15:48725102( UKL794a(( 74M(–(Asc.(An(( FBN1' ex.55:(c.G6700A:( cbEGFL T,B,N,N,L,D( 1.94, 1.24,0.876,12.2( HGMD:(MFSa(ClinVar:( ( ( p.V2234M( like#34( 12.44( MFS(probably( UKL1114( 39F(–(Asc.LArchL pathogenic)a(Comeglio(et( TA(Dissection( al.(Hum.(Mut.(2007(L( incomplete(MFS( ( 15:48764873( YLLL_55_41( 39M(Asc.(An( FBN1' ex.35:(c.A4211G:( cbEGFLlike( D,D,D,D,M,D( 1.93, 5.62,8.040,15.5( ClinVar:(likely(pathogenic( p.D1404G( #20( 12.42( 16:15932109( YLRW_91_1( 84M(–(Asc.+hemi.( MYH11' ex.2:(c.A1G:( ( D,B,D,D,.,D( 2.99, 5.82,6.181,15.36( ( p.M1V( 15.95( 9:101907032( YLAG_128_31( 41M(–(Asc.(An( TGFBR1' ex.5:(c.A761G:( SerLThr( D,D,D,D,H,D( 4.08, 5.73,9.271,15.301( ClinVar:(2(LP(variants(in(( p.H254R( kinase( 21.0( p.253((both(TAAD)( domain( ( 3:30713495( YLEG_40_91( 74F(–(TA(IMH( TGFBR2' ex.4:(c.G820T:( ( D,D,D,D,M,T( 4.84, 5.26,9.864,18.87( ClinVar:(K277QL p.V274L( 27.5( pathogenicLTAAD( M,(malea(F,(femalea(Asc.,(Ascending(aortaa(hemi,(hemiLarcha(TA,(thoracoabdominala(An,,(aneurysma(IMH,(intramural(haematomaa(ex.,(exon(numbera(cbEGF,(CalciumLbinding(Epidermal( Growth(Factor(like(domain(of(fibrillinL1a(MFS,(Marfan(syndromea(LOVD,(Leiden(Open(Variant(databasea(HGMD,(Human(Genetic(Mutation(Database.(( a.((In(silico(predictive(functional((scores(and(conservation(scores(are(based(on(ANNOVAR(version(2013aug23,(full(details(available(at:(http://www.openbioinformatics.org/annovar/(and( refer(to(the(following(specific(ANNOVAR(annotations:(Polyphen:("LJB23_Polyphen2_HVAR_score",(MT:("LJB23_MutationTaster_score_converted",(MA:( "LJB23_MutationAssessor_score_converted",(LRT:(("LJB23_LRT_score_converted",(FATHMM:(("LJB23_FATHMM_score_converted"aonly(qualitative(predictions(are(shown(here,(as( follows:(D,(deleterious.(T,(tolerateda(([Mutation(Taster(only:(A,(disease(causing(automatic,(D,(disease(causinga(N,(polymorphisma((P,(polymorphism(automatic](([Mutation(Assessor(only:(( H(high),((M(medium(and(L(low)(probability(of(functional(impact].(Cadd:(shown(as([raw(score,(scaled(score].((Raw(score:(higher(number(=(higher(likelihood(of(deleterious(variant.((Scaled( score:(phredLlike(cLscore((L10*log10(rank/total))(of(variant(pathogenicity(ranked(relative(to(all(possible(substitutions(of(the(human(genome(such(that,(a(scaled(score(>10(relates(to(the(top( 10%(of(variants(ranked(for(pathogenicity,(scaled(score(of(>20(relates(to(the(top(1%(of(ranked(variants.(Conservation(scores:((GERP++,(PhyloP,(SiPhy)(higher(number((=(higher( conservation(across(species.

! 91! ! 4.3.6%Validation%of%variants%by%Sanger%sequencing%

Not$ all$ variants$ have$ been$ validated$ by$ the$ time$ of$ writing,$ however,$most$ of$ the$ 61$ P/LP$variants$and$a$number$of$VUS’s$were$validated$by$Sanger$sequencing.$Of$those$ variants$which$were$tested$by$Sanger$sequencing,$variant$calls$with$allele$balance$<0.3$ or$ with$ coverage$ <50$ reads$ rarely$ validated,$ consistent$ with$ our$ earlier$ analysis$ of$ filtering$thresholds.$Excluding$failed$amplicons,$some$variants$with$allele$balance$and$ coverage$ above$ these$ thresholds$ did$ not$ validate.$ These$ included$ all$ 20$ variants$ in$ exon$ 1$ of$ SKI$ (an$area$of$high$GC$content$and$in$close$proximity$to$the$two$failed$ amplicons$ in$ this$ region,$ which$ also$ had$ high$ GC$ content)$ and$ seven$ others$ in$ five$ separate$samples$as$enumerated$below$(Table$4.8).$

$

Table%4.8.%Details%of%variants%which%failed%to%validate%

Genomic% coC Functional% DP/ABHet% Sample%ID% a Gene% Variant% b ordinates% % effect%% on%bam%file %

c YTJO_129_1$ $ 15:48780580$ FBN1( ex.26:c.3193delG:p.E1065fs$ fs$del$ 1039/0.44$

YTJR_50_61$ 7:94033896$ COL1A2( ex.7:c.G308A:p.G103D$ NSNV$ 508/0.56$

YTJR_50_61$ 9:137707423$ COL5A1( ex.51:c.G4016A:p.G1339D$ NSNV$ 310/0.31$

YTRR_101_81$ 2:189914134$ COL5A2( ex.44:c.G3086A:p.G1029D$ NSNV$ 117/0.41$

YTRR_101_81$ 7:94049748$ COL1A2( ex.36:c.G2179A:p.G727S$ NSNV$ 166/0.31$

YTJS_140_81$ 1:2235858$ SKI( ex.5:c.1601delC:p.A534fs$ fs$del$ 57/0.34$

YTRP_33_1$ 16:15832546$ MYH11( ex.26:c.3019T1G>A$ splicing$ 101/0.37$ ex.,$ exon$ no.$ NSNV,$ non$ synonymous$ single$ nucleotide$ variant.$ fs,$ frameshift.$ del,$ deletion.$$ a.$Genomic$coTordinates$are$based$on$GRCh37/hg19$human$reference$sequence.$b.%D/ABHet,$shows$number$ of$reads$T$read$depth(D)$and$allele$balance$(calculated$as$no.reads$for$Alt$allele$/$total$reads(D))$at$that$locus$for$ the$named$sample.$c.$YTJO_129_1$also$had$a$variant$in$SKI$exon$1$which$failed$to$validate.$$

4.3.7%GenotypeCphenotype%correlation%

Variants*detected*in*patients*with*clinically*apparent*syndromes*

There$were$9$cases$of$known$Marfan$syndrome,$who$had$confirmed$clinical$or$genetic$ diagnoses$of$Marfan$syndrome:$all$9$were$identified$by$the$assay$(in$four$of$these$nine$ cases$ where$ the$ specific$ DNA$ sequence$ variant$ was$ available$ in$ the$ clinical$ record,$ they$matched$our$findings).$In$16$other$patients$(12$from$the$UK$cohort$and$four$from$ the$Yale$cohort)$Marfan$syndrome$was$suspected$or$under$investigation:$in$7$of$these$

! 92! ! pathogenic$ or$ likely$ pathogenic$ FBN1( mutations$ were$ identified.$ In$ addition,$ in$ three$ cases$ of$ known$ or$ suspected$ LoeysTDietz$ syndrome$ –$ Patient$ ID’s$ 26EA1114,$ 24MU0795,$24RDA0726$T$we$identified$P/LP$variants$in$TGFBR1,$TGFBR2$and$in$the$ MH2$ (TGFTbeta$ signaling)$ domain$ of$ SMAD3( respectively.$ The$ patient$ with$ a$ P/LP$ variant$ in$ TGFBR1$ also$ had$ a$ VUS$ in$ FBN1.$ $ Four$ patients$ had$ other$ suspected$ hereditary$ disorders$ of$ connective$ tissue$ (HDCT):$ in$ one$ of$ these$ four$ patients,$ a$ pathogenic$ variant$ in$ COL3A1$ (indicative$ of$ Vascular$ EhlersTDanlos$ syndrome)$ was$ identifiedg$ the$ other$ three$ patients$ remained$ unresolved$ and$ were$ as$ follows:$ one$ patient$ with$ suspected$ EhlersTDanlos$ syndrome$ with$ kyphoscoliosis$ (under$ investigation)g$ one$ patient$ with$ severe$ kyphoscoliosis$ and$ suspected$ HDCT$ and$ one$ patient$with$other$suspected$HDCT.$$

Gene*Variants*detected*in*patients*without*clinically*apparent*syndromes*

In$addition$to$those$patients$with$known$or$suspected$syndromes,$we$newly$identified$ pathogenic$or$llikely$pathogenic$variants$in$the$following$syndromic$genes:$FBN1:(14$ patients,$ TGFBR1$ &$ TGFBR2:$ 4$ patients,$ COL3A1:$ 2$ patients,$ SMAD3:$ 1$ patient,$ representing$potentially$new$genetic$diagnoses.$Overall,$only$14/30$(47%)$patients$with$ pathogenic$ or$ likely$ pathogenic$ (P/LP)$ variants$ in$ FBN1$ had$ clinically$ suspected$ Marfan$syndrome$and$18/30$(60%)$had$a$probable$or$likely$family$history$from$clinical$ recordsg$for$all$P/LP$variants,$only$31/61$(51%)$had$a$probable$or$likely$family$history.$

Key* phenotypic* characteristics* in* patients* harbouring* a* pathogenic* or* likely* pathogenic*variant*

The$ whole$ cohort$ was$ divided$ into$ two$ groups:$ those$ in$ whom$ a$ pathogenic/likely$ pathogenic$variant$in$any$gene$was$identified$(“P/LP”$group,$n=62)$versus$the$rest$of$ the$cohort$(“No$P/LP”$group,$n=988).$Of$six$phenotypic$features$studied,$three$features$ significantly$correlated$with$the$presence$of$a$pathogenic$or$likely$pathogenic$variant:$ Age$ at$ presentation$ <50$ years$ (odds$ ratio$ (OR)$ 5.34),$ presence$ of$ a$ known$ or$ suspected$family$history$of$aortopathy$(OR$3.37)$and$presence$of$Dissection$(OR$2.36)$ (Table$4.9).$The$disparity$in$age$between$the$P/LP$group$and$the$No$P/LP$group$was$ also$evident$when$studying$the$overall$age$distribution$by$boxTplot$(Figure$4.2)$and$was$ more$ pronounced$ when$ we$ isolated$ those$ with$ just$ a$ P/LP$ variant$ in$ FBN1.$ There$ seemed$to$be$more$genderTparity$in$P/LP$group$(57%$males)$compared$with$the$No$ P/LP$group$(69%$male)$but$this$was$not$statistically$significant$(p=0.06).$Maximal$Aortic$ diameter$<5.5cm$seemed$to$be$somewhat$lower$in$the$P/LP$group$(54%$v.$63%),$but$ this$ did$ not$ reach$ statistical$ significance$ (p=0.1)g$ both$ groups$ had$ a$ roughly$ equal$ proportion$(5%)$of$large$aortic$aneurysms$>7cm.$Analysis$of$the$definitely$pathogenic$ group$(n=42)$in$isolation$revealed$no$significant$difference$from$the$pooled$P/LP$group$ in$terms$of$the$above$phenotypes$(Table$4.9).$ ! 93! ! The$extent$of$aortic$involvement$taking$into$account$all$presentations$and$pathologies$ did$ not$ differ$ significantly$ between$ the$ two$ groups$ except$ that$ the$ P/LP$ group$ had$ fewer$patients$with$involvement$of$the$arch$(20%$versus$41%,$p=0.0017,$Figure$4.3).$

%

Table%4.9%Key$phenotypic$characteristics$of$cohorts$with$and$without$a$pathogenic/likely$pathogenic$(P/LP)$ variant.% n(%)$of$Patients$ n(%)$of$ with$a$definitely$ n(%)$of$ Patients$ pathogenic$variant$$ Patients$with$ without$P/LP$ $$ P/LP$variant$$ variant$ OR$(95%$CI)$ pTvalue*$ n$ 42$ 62$ 988$ $ % Young$Age,$<50y$ 24$(56%)$ 35$(57%)$ 199$(20%)$ 5.34$(3.14T9.09)$ <0.0001% $ Known$or$probable$family$history$ 24$(56%)$ 31$(51%)$ 232$(23%)$ 3.37$(1.99T5.69)$ <0.0001% Presence$of$dissection$ 15$(35%)$ 19$(31%)$ 159$(16%)$ 2.36$(1.33T4.17)$ 0.003% Male$gender$ 24$(56%)$ 35$(57%)$ 681$(69%)$ 0.61$(0.36T1.03)$ 0.06$ Presence$of$recurrent/$multifocal$ $ disease$ 8$(19%)$ 11$(18%)$ 141$(14%)$ 1.32$(0.67T2.60)$ 0.42$ $ Max.$diameter$<5.5cm$ 23$(53%)$ 33$(54%)$ 620$(63%)$ 0.70$(0.42T1.18)$ 0.10$ Max.$diameter$>7cm$ 3$(7%)$ 3$(5%)$ 51$(5%)$ 0.95$(0.28T3.14)$ 0.90$ P/LP,$pathogenic$or$likely$pathogenic.$*P/LP$group$versus$patients$without$a$P/LP$variant$

$

%

Figure% 4.2% Age% distribution% boxCplots% for% FBN1,% P/LP% and% No% P/LP% groups.$ BoxTplots$ comparing$ the$ age$ distribution$ of$ those$ patients$ without$ a$ pathogenic$ or$ likely$ pathogenic$ variant$(No$P$/$LP$group,$top)$with$those$harbouring$any$P/LP$variant$(middle)$and$a$ FBN1$ pathogenic$or$likely$pathogenic$variant$(bottom)$

! 94! !

0% 20% 40% 60% 80% 100%

Asc

Arc h *

DT A

TAA No3P/LP3 (n=989)

IR P/LP3 (n=61)

$

Figure% 4.3% Comparison% of% anatomical% extent% in% P/LP% versus% No% P/LP% groups.%% Comparative$barTchart$showing$the$proportion$of$each$group$with$disease$involving$different$ parts$of$the$aorta:$those$without$a$pathogenic$/$likely$pathogenic$variant$(No$P/LP$group,$white$ bars)$versus$those$with$a$pathogenic$or$likely$pathogenic$variant$(P/LP$group,$shaded$bars).$ Asc,$ascending$aorta.$Arch,$aortic$arch.$DTA,$descending$thoracic$aorta$(from$left$subclavian$ to$diaphragm).$TAA,$thoracoabdominal$aorta$(disease$of$any$extent$of$the$thoracic$aorta$with$ contiguous$extension$into$the$abdominal$aorta).$IR,$disease$in$infrarenal$abdominal$aorta$not$ continuous$with$thoracic$aortic$pathology.$$*p=0.0017$(P/LP$versus$No$P/LP$group).$

4.4%Discussion%

This$work$represents$the$largest$targeted$sequencing$study$of$thoracic$aortic$disease$ to$ date,$ expanding$ on$ a$ previous$ study$ of$ 103$ TAAD$ patients$ from$ the$ Yale$ Aortic$ Centre,$USA,$undertaken$by$our$collaborators.131$$$

In$ this$ study,$ I$ performed$ targeted$ sequencing$ of$ known$ TAAD$ genes$ on$ over$ a$ thousand$unrelated$patients$from$two$countries,$the$aim$being$to$determine$the$overall$ prevalence$ and$ pattern$ of$ mutations$ in$ these$ genes$ as$ well$ as$ to$ identify$ any$ discernible$ genotypeTphenotype$ relationships.$ Information$ on$ the$ genetic$ spectrum$ underlying$ routine$ TAAD$ patients$ (where$ patients$ with$ a$ genetic$ aetiology$ may$ otherwise$be$undetectable$clinically)$may$be$highly$valuable$for$risk$stratification$and$ clinical$management.$

From$this$study,$the$overall$yield$of$rare$variants$was$26%,$which$comprised$61$(5.8%)$ known$ or$ likely$ pathogenic$ variants$ (according$ to$ ACMG$ guidelines)$ and$ 209$ (20%)$ VUS’s.$This$is$consistent$with$the$previous$exomeTsequencing$study$of$103$Yale$TAAD$ patients$ and$ preliminary$ work$ carried$ out$ by$ the$ Imperial$ College$ group.131,139$ FBN1( accounted$ for$ almost$ a$ half$ of$ all$ Pathogenic$ or$ Likely$ Pathogenic$ variants$ with( the$

! 95! ! majority$ affecting$ functionally$ significant$ domains$ of$ fibrillin,$ consistent$ with$ previous$ studies.$ 3%$ of$ the$ overall$ cohort$ and$ 7%$ of$ those$ with$ a$ probable$ or$ known$ family$ history$had$a$pathogenic$or$likely$pathogenic$FBN1$variant,$which$is$somewhat$higher$ than$ previous$ reports.140$ FBN1$ is$ clearly$ an$ important,$ probably$ the$ most$ important,$ contributor$to$Mendelian$cases$of$TAADg$furthermore,$recent$GWAS$has$shown$it$to$be$ the$most$significant$contributor$of$common$variants$to$sporadic$TAAD.42$

A$ relatively$ high$ proportion$ of$ pathogenic$ and$ likely$ pathogenic$ variants$ in$ TGFBR1$ and$TGFBR2$are$in$keeping$with$what$is$known$to$date$to$be$an$important$pathway$in$ TAAD$ pathogenesis.40$ The$ secondThighest$ proportion$ of$ pathogenic$ or$ likely$ pathogenic$ variants$ in$ this$ study$ were$ in$ the$ collagen$ genes,$ COL3A1( (3$ cases),( COL1A1(7$cases),(COL5A1(&(COL5A2(3$cases).$Though$collagen$genes$and$EhlersT Danlos$ syndrome$ are$ known$ to$ be$ associated$ with$ TAAD,$ the$ $ of$ these$ geneTmutations$in$the$overall$genetic$spectrum$of$TAAD$is$unexpected$and$we$believe$ reflects$ an$ emerging$ picture$ that$ hereditary$ connective$ tissue$ disorders$ (HDCT),$ of$ which$ TAAD$ is$ part$ may$ be$ contributed$ to$ by$ abnormalities$ in$ common$ signalling$ pathways.40,98,108,141(

In$ACTA2$we$observed$a$smaller$number$of$variants$than$expected–no$P/LP$variants$ and$ 5$ VUS’s$ (0.5%).$ From$ the$ studies$ of$ Dr$ Milewicz$ et$ al.$ on$ familial$ TAAD,$ pathogenic$ACTA2$variants$were$identified$in$10T14%$of$their$cohort$of$familial$TAAD$ from$Houston,$Texas,$USA55$–$this$is$partly$explained$by$the$fact$that$our$cohort$is$a$ mixed$ one,$ comprising$ only$ 20%$ of$ patients$ with$ a$ probable$ or$ known$ family$ history$ compared$ with$ that$ from$ the$ Houston$ group$ which$ comprised$ strong$ demonstrable$ multigenerational$fTAAD$from$one$centreg$it$is$also$important$to$note$that$our$VUS’s$in$ ACTA2$were$so$classified$owing$to$the$limited$knowledge$and$published$data$on$this$ gene,$ therefore$ another$ explanation$ may$ be$ the$ conservatism$ of$ pathogenicity$ assignment$adopted$in$our$study$(see$later).$$

The$ 49$ (5%)$ newly$ identified$ pathogenic/likely$ pathogenic$ variants$ in$ all$ genes$ and$ 29(3%)$ newly$ identified$ pathogenic$ or$ likely$ pathogenic$ variants$ in$ known$ syndromic$ genes,$ FBN1( (Marfan),( TGFBR1( &( TGFBR2( (Loeys$ Dietz$ syndrome),( COL1A1,( COL3A1( &( COL5A1( (EhlersTDanlos$ syndrome),( represent$ new$ genetic$ diagnoses,$ which$ to$ our$ knowledge$ had$ not$ been$ previously$ identified$ in$ the$ clinical$ setting.$ Of$ patients$ with$ clinically$ apparent$ or$ suspected$ syndromes$ all$ 9$ clinically$ confirmed$ cases$of$Marfan$syndrome$were$validated$and$a$further$7$out$of$16$cases$of$clinically$ suspected$but$unconfirmed$Marfan$syndrome$had$a$FBN1$ mutationg$the$remaining$9$ cases$were$unresolved$genetically,$which$might$be$expected,$especially$in$the$case$of$ incomplete$ Marfan$ (formal$ Ghent$ criteria$ were$ not$ assessed$ as$ part$ of$ the$ clinical$ record$in$these$patients):$from$Dr$Child’s$report$of$over$500$suspected$Marfan$referrals$

! 96! ! one$would$expect$a$proportion$of$27%$of$incomplete$MFS$and$82T91%$of$classic$MFS$ to$have$a$FBN1$mutation.142$$

Conversely,$ we$ also$ found$ that$ only$ 47%$ of$ those$ with$ a$ pathogenic$ or$ likely$ pathogenic$ (P/LP)$ variant$ in$ FBN1$ had$ a$ known$ or$ suspected$ clinical$ diagnosis$ of$ Marfan$syndromeg$similarly,$only$51%$of$those$with$a$pathogenic$or$likely$pathogenic$ variant$in$any$gene$(60%$in$FBN1)$had$a$known$or$probable$family$history.$Though$this$ phenomenon$may$be$partly$owing$to$an$incomplete$clinical$record,$it$is$also$consistent$ with$ the$ observation$ made$ over$ 20$ years$ ago$ by$ Dr$ Milewicz$ that$ FBN1$ mutations$ caused$TAAD$in$patients$who$did$not$have$clinical$Marfan$syndromeg$in$a$more$recent$ study$of$1,009$probands$with$FBN1$mutations,$Faivre$et$al.$showed$that$only$56T79%$ met$formal$clinical$criteria$for$Marfan$syndrome$by$Ghent$systemic$scores.143,144$Thus,$ it$would$appear$that$family$history$and$clinical$features$alone$are$insufficient$to$predict$ a$detectable$genetic$aetiology$in$TAAD.$

Taking$all$61$pathogenic$or$likely$pathogenic$variants$as$a$group$compared$with$the$ rest$ of$ the$ cohort,$ I$ identified$ three$ statistically$ significant$ correlations:$ a$ greater$ likelihood$ of$ younger$Age$at$presentation$<$50$years$(OR$5.3),$a$probable$or$known$ family$ history$ of$ aortic$ aneurysm$ (OR$ 3.4)$ and$ an$ over$ 2Tfold$ increased$ likelihood$ of$ Aortic$ Dissection. * $The$ first$ two$ of$ these$ factors$ are$ reasonable$ indicators$ of$ any$ Mendelian$ disease$ and$ may$ be$ suitable$ criteria$ for$ prioritizing$ patients$ for$ genetic$ testing.$ The$ third$ factor$–$ presence$of$dissection$T$ would$be$an$important$prognostic$ marker$ for$ clinical$ surveillance,$ as$ aortic$ dissection$ occurs$ suddenly,$ without$ a$ clinicallyTdetectable$ prodrome$ and$ is$ associated$ with$ high$ morbidity$ and$ mortality.$ These$analyses$will$need$to$be$reTevaluated$following$completion$of$variant$validation.$$

Assignment$of$pathogenicity$in$general$in$this$study$was$based$quite$strictly$on$ACMG$ criteria$ and$ therefore$ was$ relatively$ conservative$ as$ knowledge$ of$ the$ genotypeT phenotype$correlation$for$each$gene$is$currently$limited.$The$latter$point$is$reflected$in$ the$similar$phenotypic$characteristics$of$those$patients$carrying$a$definitely$pathogenic$ variant,$when$compared$to$the$pooled$pathogenic$&$likely$pathogenic$(P/LP)$group.$It$is$ probable$that$some$VUS’s$in$this$study$are$in$fact$pathogenic:$a$number$of$VUS’s$had$ some$ evidence$ of$ pathogenicity$ without$ meeting$ formal$ ACMG$ Pathogenic/Likely$ Pathogenic$ criteria$ and$ a$ significant$ proportion$ of$ FBN1( VUS’s$ were$ in$ functionally$ important$domains$and$represent$variants$which$would$have$clinical$priority$attached$to$

$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ $$$$$$$$ * This!last!factor,!Probability!of!Dissection,!on!subsequent!re>analysis!after!completion!of!variant!validation! by! David! Ross! and! David! Parry,! did! not! reach! statistical! significance;! instead! the! presence! of! Ascending! Aortic!versus!Descending!involvement!was!found!to!be!increased!in!the!P/LP!group;!this!work!is!currently!in! submission,! see:! Weerakkody! et! al.! Targeted! Genetic! Analysis! in! a! Large! Cohort! of! Familial! and! Sporadic! TAAD!Patients.! ! 97! ! them,$ requiring$ clinical$ followTup.$ Equally,$ other$ variants,$ which$ had$ in$ previous$ publications$been$reported$as$Pathogenic$or$Likely$Pathogenic,$we$labelled$as$VUS’s$ owing$to$conflicting$evidence$from$in(silico(evidence(and$clinical$phenotype$(Table$4.7).$ The$ possibility$ that$ VUS’s$ in$ general,$ though$ without$ a$ direct$ pathological$ role$ in$ Mendelian$inheritance,$may$also$contribute$to$the$phenotype$in$a$multigenic/$polygenic$ manner$must$also$be$considered:$their$relative$significance$will$not$however$become$ evident$until$larger$datasets$and$new$analytical$tools$emerge.$

The$ demographic$ characteristics$ of$ our$ cohort$ in$ terms$ of$ age$ and$ genderT predominance$broadly$reflects$previous$large$studies$of$TAAD$and$data$from$the$IRAD$ database$however,$it$should$be$born$in$mind$that$these$cases$are$recruited$from$large$ tertiary/quaternary$referral$centres$for$TAAD,$thereby$incurring$a$possible$bias$towards$ more$complex$phenotypes.125,130,145$In$terms$of$family$history,$a$larger$proportion$of$the$ Yale$ cohort$ had$ a$ proven$ or$ probable$ family$ history$ (28.7%)$ compared$ with$ the$ UK$ cohort(14.8%):$ this$ probably$ reflects$ the$ bias$ of$ clinical$ records,$ where$ family$ history$ data$ is$ incompletely$ recorded$ as$ part$ of$ the$ routine$ clinical$ record$ in$ many$ centres$ whereas$family$history$data$have$been$routinely$and$more$assiduously$recorded$in$the$ Yale$centre.$Data$regarding$family$history$from$IRAD$or$other$multicenter$studies$are$ sparse,$ but$ from$ available$ reports$ to$ date,$ one$ would$ predict$ a$ familial$ proportion$ of$ approximately$20%.130$146$$26$(9.4%)$of$the$UK$cohort$had$a$known$diagnosis$of$Marfan$ syndrome$ (MFS)g$ for$ the$ Yale$ cohort$ this$ was$ only$ 0.5%,$ although$ a$ previous$ phenotypic$ study$ of$ 520$ cases$ from$ the$ Yale$ aortic$ centre$ revealed$ MFS$ in$ 50/520$ (9.6%)138:$the$reason$for$this$discrepancy$is$unclear$but$most$likely$reflects$differences$ in$referral$practice$between$the$UK$and$USA$and$between$the$Yale$ specialist$ aortic$ centre$and$the$UK$NHS$vascular$surgery$centres.$We$did$not$have$available$detailed$ phenotypic$ characteristics$ (e.g.$ Ghent$ or$ Villefranche$ criteria)$ to$ confirm.$ There$ was$ one$ patient$ in$ this$ cohort$ recorded$ to$ have$ EhlersTDanlos$ syndrome$ and$ a$ small$ proportion$of$patients$(0.4%)$had$other$uncharacterized$syndromic$features.$

The$proportion$of$cases$with$Dissection$in$the$Yale$cohort$was$lower$than$in$the$UK$ cohort$ (9.3%$ versus$ 32%)g$ a$ previous$ phenotypeTbased$ study$ of$ 520$ Yale$ patients$ revealed$a$Dissection$rate$of$25%.$The$reason$for$the$apparently$lower$proportion$in$ this$cohort$from$the$Yale$centre$is$again$unclearg$the$higher$proportion$seen$in$the$UK$ cohort$ may$ reflect$ the$ referral$ pattern$ where$ somewhat$ more$ complex$ cases$ are$ referred$to$tertiary$aortic$centres.$The$number$of$ruptures$in$both$cohorts$is$very$low$ (0.5%$overall):$this$reflects$a$bias$of$ascertainment$as$those$with$ruptures$would$be$ less$likely$to$be$recruited$owing$to$their$high$associated$mortality$and$morbidity.$$$

The$relative$proportions$of$Ascending$versus$Descending$Thoracic$/$Thoracoabdominal$ aortic$ pathology$ reflect$ the$ referral$ pattern$ to$ the$ centres$ involved:$ the$ Yale$ aortic$

! 98! ! centre$taking$mainly$ascending/arch$aneurysms,$whilst$the$UK$centres$taking$all$aortic$ pathologies.$There$was$a$significant$rate$of$recurrence$of$aortic$disease$(secondary$or$ tertiary$ aortic$ events)$ of$ 14%$ in$ the$ whole$ cohort,$ which$ reflects$ that$ the$ primary$ pathology$ can$ be$ regarded$ not$ as$ a$ single$ phenomenon$ but$ as$ a$ manifestation$ of$ a$ generalized$aortopathy.$With$the$caveat$that$there$is$inevitable$bias$associated$in$any$ cohort$ that$ is$ formally$ recruited$ for$ research,$ we$ feel$ it$ otherwise$ as$ far$ as$ possible$ reflects$a$mixed$otherwise$unselected$cohort$of$TAAD$as$would$be$seen$in$the$tertiary$ healthcare$setting.$$

In$ addition$ to$ the$ limitations$ of$ pathogenicity$ assignment$ which$ is$ largely$ limited$ by$ current$ knowledge,$ discovery$ of$ genetic$ variants$ in$ this$ study$ is$ also$ limited$ by$ the$ study$design$whose$remit$was$to$identify$rare$and$functionally$significant$variants$in$the$ coding$region$of$specific$genesg$we$know$from$family$studies$that$a$large$proportion$of$ familial$ TAAD$ remains$ unaccounted$ from$ the$ known$ genes$ –this$ assay$ would$ not$ detect$ variants$ in$ as$ yet$ undiscovered$ genes,$ common$ variants$ nor$ intronic$ or$ copy$ number$variants.$The$assay$in$itself$has$its$own$limitations,$particularly$with$respect$to$ false$ positive$ calls,$ which$ is$ a$ known$ feature$ of$ PCRTbased$ targeted$ sequencing$ methods:$ we$ tried$ to$ circumvent$ these$ limitations$ bioinformatically$ by$ our$ filtering$ strategy$ and$ by$ independently$ validating$ variants$ by$ a$ different$ method$ (Sanger$ sequencing)47.$

In$ terms$ of$ clinical$ translation$ of$ these$ findings,$ based$ on$ the$ sizeable$ proportion$ of$ rare$functionally$significant$variants$in$known$aortopathy$genes$we$would$recommend$ routine$ genetic$ testing$ of$ any$ TAAD$ patient,$ prioritising$ those$ presenting$ below$ 50$ years$of$age$or$with$any$family$history$(and$taking$into$account$a$probably$higher$prior$ probability$ of$ genetic$ disease$ observed$ in$ cases$ referred$ to$ specialist$ quaternary$ referral$ centres$ for$ TAAD,$ from$ which$ our$ cases$ were$ selected).$ Preferably,$ future$ genetic$testing$would$be$by$whole$genome$sequencing$to$account$for$the$incomplete$ coverage$ of$ targeted$ sequencing$ methods$ and$ for$ as$ yet$ undiscovered$ genetic$ aetiologies.$One$potential$outcome$of$such$genetic$screening$would$be$to$identify$the$ firstTdegree$relatives$of$patients$who$carry$a$mutation$–$a$large$atTrisk$population$who$ would$otherwise$have$gone$undetected$T$thereby$reducing$the$frequency$of$morbidity$ and$ mortality$ due$ to$ late$ presentation$ and$ rupture).$ Consistent$ with$ this,$ we$ would$ advocate$ routine$ recording$ of$ detailed$ family$ history$ and$ preferably$ any$ syndromic$ features$specifically$according$to$Ghent$criteria,$owing$to$the$high$proportion$of$FBN1$ variants.$ A$ global$ registry$ of$ aortopathy$ genetic$ variants$ combined$ with$ detailed$ phenotypes$ is$ needed$ to$ draw$ further$ correlations$ between$ genotype$ and$ specific$ clinical$indices$and$outcomes,$with$the$eventual$aim$of$a$genotypeTbased$prediction$of$ critical$aortic$events$and$longTterm$prognosis.$

! 99! !

$ %

! 100! ! 5.% WHOLE% EXOME% SEQUENCING% FOR% NOVEL% GENE% DISCOVERY%IN%UNRELATED%INDIVIDUALS%WITH%RARE% PHENOTYPES%OF%EDS%%

5.1%Introduction%

Published$work$to$date$and$our$own$observations$(Chapter$3)$have$shown$that$EDS$is$ a$ genetically$ and$ phenotypically$ heterogeneous$ condition.39,108$ A$ subset$ of$ patients$ with$ rare$ Mendelian$ EDS$ phenotypes$ (e.g.$ classical$ EDS,$ vascular$ EDS,$ kyphoscoliosis$type$EDS)$do$not$appear$to$be$caused$by$mutations$in$the$genes$that$ have$ been$ previously$ reported$ to$ underlie$ EDS$ phenotypes.$ Given$ their$ Mendelian$ nature,$ the$ most$ likely$ hypothesis$ is$ that$ these$ represent$ locus$ heterogeneity$ T$ the$ same$ phenotype$ caused$ by$ pathogenic$ mutations$ in$ other$ genes.$ Other$ very$ rare$ phenotypes$within$the$EDS$spectrum$e.g.$periodontitisTtype$EDS$(VIII)$and$some$rare$ subtypes$of$hypermobilityTtype$EDS$(III)$do$not$have$an$identified$genetic$cause$at$all,$ despite$appearing$to$be$classical$Mendelian$diseases.$

WholeTexome$ sequencing$ (WES)$ has$ been$ particularly$ useful$ for$ identifying$ novel$ genes$underlying$Mendelian$diseases,$even$in$groups$of$unrelated$individuals$sharing$ the$ same$ rare$ phenotype.25$ The$ hypothesis$ here$ is$ that$ at$ least$ a$ proportion$ of$ unrelated$ cases$ presenting$ with$ the$ same$ rare$ phenotype$ will$ harbor$ pathogenic$ mutations$in$the$same$gene.25$The$aim$of$this$part$of$the$project$was$therefore$to$use$ whole$exome$sequencing$(WES)$in$an$attempt$to$identify$new$genes$underlying$certain$ rare$EDS$phenotypes,$all$of$which$were$initially$screened$for$pathogenic$mutations$by$ targeted$sequencing$of$collagen$genes$(Chapter$3).$

5.1.1%Discovery%of%a%causal%gene%in%PeriodontitisCtype%EDS%(type%VIII)%

This$very$rare$type$of$EDS$presenting$with$aggressive$periodontitis$was$first$identified$ by$Stewart$et$al.$in$1977$and$has$only$ever$been$reported$in$29$case$reports$and$seven$ pedigree$analyses$worldwide$(OMIM$#130080).94,147T149,150$$Until$the$work$outlined$here$ took$place,$a$causative$gene$had$not$been$identified.$In$this$project,$we$were$able$to$ identify$a$novel$causative$gene$accounting$for$at$least$some$of$the$cases$of$EDS$VIII,$ and$ in$ collaboration$ with$ partners$ in$ Austria$ (Prof.J.$ Zschocke)$ and$ the$ USA$ (Dr.$ Byers),$we$demonstrated$a$novel$mechanism$underlying$EDS$pathogenesis.$

Clinically,$EDS$VIII$is$an$extremely$rare$autosomal$dominant$connective$tissue$disorder$ whose$ main$distinguishing$feature$is$a$ rapidly$progressive$periodontitis$characterized$ by$severe$destruction$of$the$periodontal$attachment$apparatus,$leading$to$early$tooth$

! 101! ! loss.$Other$clinical$manifestations,$which$are$not$exclusive$to$this$type$but$form$part$of$ its$classification,$include$pretibial$hyperpigmentation,$acrogeria,$skin$and$gum$fragility,$ scarring,$generalised$and/or$distal$joint$hypermobility,$and$bruising$out$of$proportion$to$ trauma.$There$are$single$case$reports$of$lifeTthreatening$complications$like$arterial$or$ gastrointestinal$ruptures.151$

In$ 2003,$ Rahman$ et$ al.$ mapped$ a$ locus$ for$ periodontal$ EDS$ in$ a$ fourTgeneration$ Swedish$ family$ to$ a$ 7$ centiMorgan(cM)$ interval$ on$ chromosome$ 12p13,$ with$ LOD$ score$of$5.17.94$Analysis$of$further$pedigrees$with$EDS$VIII$revealed$linkage$intervals$ consistent$with$this$in$in$two$families$but$not$in$two$others,$leading$to$the$conclusion$ that$ EDS$ VIII$ must$be$genetically$heterogeneous,$with$one$causal$gene$being$in$the$ above$linked$region.94$Analysis$of$the$linked$region$did$not$reveal$any$known$collagen$ or$ collagenTprocessing$ genes.$ One$ candidate$ gene$ –$ MFAP2,( encoding$ microfibrilT associated$ glycoproteinT2$ T$ within$ the$ minimal$ interval$ was$ sequenced$ but$ did$ not$ reveal$any$likely$pathogenic$variants$segregating$with$the$phenotype.$

The$ work$ above,$previously$carried$out$ by$ our$collaborator,$ Prof.$F.M.Pope,$National$ Ehlers$Danlos$Syndrome$Diagnostic$Service,$Northwick$Park$Hospital,$Harrow,$UK,$laid$ the$groundwork$for$a$focused$reTanalysis$that$included$exome$sequencing$of$the$linked$ region$ in$ the$ index$ case$ from$ the$ original$ Swedish$ pedigree$ (DNA$ from$ other$ family$ members$ was$ not$ available)$ alongside$ a$ number$ of$ other$ EDS$ VIII$ patients$ subsequently$recruited$to$this$study.$

5.1.2%Discovery%of%novel%genes%in%other%EDSCrelated%phenotypes%

A$ significant$ proportion$ of$ cases$ with$ EDS$ have$ phenotypic$ features$ in$ keeping$ with$ specific$EDS$types$but$without$causative$mutations$in$the$gene$usually$associated$with$ those$phenotypes.$Still$others$have$less$specific$phenotypes$within$the$EDS$spectrum,$ not$ fitting$ into$ the$ existing$ classification$ system.$ In$ this$ project,$ I$ attempted$ to$ first$ phenotypically$prioritise$such$cases$and$then$identify$potential$novel$causative$genes$ underlying$them.$By$whole$exome$sequencing$the$aim$was$to$attempt$to$identify$rare$ functionally$significant$variants$in$the$same$gene,$in$two$or$more$unrelated$individuals$ sharing$the$same$phenotype.$$

$ $

! 102! ! 5.2%Methods%

5.2.1%Selection%&%stratification%of%phenotypes%%

EDS*VIII*(Periodontitis*type)*

I$ selected$ five$ unrelated$ individuals$ with$ a$ clinical$ phenotype$ known$ to$ be$ strongly$ indicative$of$or$overlapping$with$EDS$VIII.$They$included$the$proband$of$the$previously$ analysed$Swedish$pedigree$(20FB8035),$who$had$strong$features$of$EDS$VIII$and$two$ others$with$features$highly$suggestive$of$EDS$VIII$(Table$5.1,$below).$From$an$existing$ archival$ EDS$ DNA$ collection,$ I$ also$ identified$ three$ other$ cases$ with$ a$ diagnosis$ of$ possible$EDS$VIII,$but$without$any$detailed$phenotype$data$for$corroboration.$All$these$ cases$ were$ ranked$ according$ to$ the$ strength$ of$ the$ available$ phenotype$ information$ (Table$5.1).$

Table%5.1%Ranked$phenotypes$for$cases$with$known$or$suspected$PeriodontitisTtype$EDS$(VIII).$

Sample%ID% Clinical%diagnosis% Phenotype%summary% 20FB8035$ EDS$VIII$ Caucasian$ male.$ Index$ case$ of$ fiveTgeneration$ Swedish$ EDS$ VIII$ pedigree.$ Premature$ (childhood/adolescent$ onset)$ periodontal$ inflammation$and$gingival$recession$with$rapid$progression$through$ adolescence$ and$ complete$ loss$ of$ dentition$ by$ the$ third$ decade.$ Dental$ XR$ confirmed$ progressive$ periodontal$ loss.$ Generalised$ joint$laxity,$thin$atrophic$skin$(prominent$over$dorsum$of$hands$and$ feet),$ circumscribed$ hemosiderotic$ pretibial$ plaques.$ Tall$ stature$ with$armspan>height.$

22TP0427$ EDS$VIII$ Caucasian$ female.$ Sporadic$ /$ autosomal$ recessive$ EDS$ VIII$ T$ severe$periodontitis,$severe$bruising$of$shins,$premature$aging.$

20GT845$ EDS$VIII$ Caucasian$ female.$ Early$ onset$ periodontitis$ diagnosed$ aged$ 10.$ $ Complete$tooth$loss$at$age$43.$Generalised$gum$thinning.$No$joint$ hypermobility.$ Hyperextensible$ skin.$ Marked$ pretibial$ discolorations,$ prominent$ vasculature$ and$ thickened$ scars.$ Recurrent$ wound$ infections$ with$ delayed$ wound$ healing,$ frequent$ UTI’s/pyelonephritis.$ 22MMC0357$ Vascular$ /$ EDS$ South$ Asian$ (Pakistan)$ male.$ With$ acrogeria,$ easy$ bruising,$ VIII$overlap$ marked$generalised$periodontal$recession.$$ $ LM:$ slightly$ thin$ dermisg$ EM:$ relatively$ normal$ but$ with$ increase$ numbers$of$fibroblasts$some$with$dilated$ER.$$ 22EM0088$ Vascular$ /$ EDS$ Caucasian$ male.$ NonTspecific$ connective$ tissue$ phenotype$ with$ VIII$overlap$ features$ of$ vascular$ and$ EDS$ VIII.$ Generalised$ hypermobility$ (Beighton$ 7).$ Mild$ scoliosis.$ Normal$ Collagen$ (III)$ electrophoresis$ pattern$and$sequencing.$$ 20RP866$ ?EDS$VIII$ [no$phenotype$information]$ $ 20AJ1332$ ?EDS$VIII$ [no$phenotype$information]$ $ 20AC1004$ ?EDS$VIII$ [no$phenotype$information]$ EDS,$Ehlers$Danlos$syndrome.$UTI,$urinary$tract$infection.$LM,$light$microscopy.$EM,$electron$microscopy.$ER,$ endoplasmic$reticulum$T$dilated$ER$suggests$probable$dysfunction$with$postTtranslational$processing$(transport)$ of$ collagen$ molecules.$ “?”,$ used$ to$ denote$ suspected$ diagnosis$ based$ on$ available$ records$ but$ without$ any$ corroborating$phenotype$details.$

! 103! ! Other*EDS*

181$other$patients$with$EDS$phenotypes$overlapping$established$EDS$types$selected$ for$ exome$ sequencing.$ These$ were$ sequenced$ in$ collaboration$ with$ the$ NIHR$ Bioresource$ for$ Rare$ Diseases$ (BRIDGE)$ consortium.$ All$ cases$ were$ deeply$ phenotyped$ as$ described$ previously.$ They$ were$ divided$ into$ one$ of$ the$ following$ phenotype$groups$(some$groups$overlapped$in$terms$of$their$constituent$patients):$$

1.$ Col.(V)Tnegative$Classical$EDS$T$patients$with$features$of$classical$EDS$who$ had$screened$negative$for$mutations$in$the$known$genes,$COL5A1$or$COL5A2$

2.$ Col.(III)Tnegative$Vascular$EDS$–$patients$with$features$of$vascular$EDS$without$ mutations$in$the$known$gene,$COL3A1$$

3.$ EDS$with$extensive$hypermobility$trait$(defined$as$Beighton$score$>/=7)$$

4.$ EDS$with$Marfanoid$habitus$$

5.$ EDS$with$aortopathy$$

6.$ EDS$with$carotid$dissection$

7.$ EDS$with$cerebral$aneurysm$

8.$ EDS$with$severe$kyphoscoliosis$

As$ the$ features$ of$ these$ groups$ can$ be$ heterogeneous,$ in$ order$ to$ aid$ downstream$ variant$prioritisation,$patients$within$each$group$were$ranked$based$on$the$strength$of$ the$phenotype.$For$this,$I$developed$a$phenotypeTscoring$systems$to$prioritise$cases$ based$ on$ the$ number$ of$ relevant$ diagnostic$ criteria$ met$ using$ relevant$ Villefranche$ nosology$ major$ and$ minor$ criteria$ for$ the$ first$ two$ groups$ (classical$ and$ vascular$ groups),38$Beighton$score$for$group$3$(extensive$hypermobility)107$and$Ghent$criteria$for$ group$4$(Marfanoid$EDS).99$The$scoring$system$for$each$group$along$with$a$list$of$the$ patients$ comprising$ each$ group$ are$ detailed$ in$ Tables$ 5.2$ &$ 5.3$ and$ Appendix$ 5.$ Patients$within$each$group$were$ranked$based$on$their$phenotype$score.$In$addition,$ for$ classical$ EDS,$ where$ the$ overall$ clinical$ diagnosis$ by$ an$ EDS$ specialist$ was$ considered$ to$ be$ partially$ or$ strongly$ suggestive$ of$ classical$ EDS,$ this$ was$ also$ recorded$separately$(Table$5.3).$ $

! 104! ! $

Table%5.2%Summary$of$phenotype$groups% Phenotype%group% Approx.% No.% No.% Inclusion% criteria/% scoring% Reference% prevalence% Selec Sequ system% Table% ted% enced% Col(V)Tnegative$classical$EDS$$ $<1:20,000$ 71$ 37$ Phenotype$score$≥$6$and/or$ Table$5.7$ multiple$collagen$rosettesa$on$EM$ and/or$clinically$clear$diagnosis$of$ classical$EDS$

Col(III)Tnegative$vascular$EDS$$ $<1:50,000$ 18$ 9$ Phenotype$score$≥$6$or$clinically$ Appendix$5$ clear$diagnosis$of$vEDS$(cases$ with$a$vascular$event$only$not$ counted$unless$other$criteria$for$ vEDS$apparent)$

Extensive$hypermobility$ <1:1,000$ 87$ 28$ Beighton$score$≥$7,$ranked$by$raw$ Appendix$5$ Beighton$score$

Marfanoid$EDS$$$ <1:5,000$ 57$ 14$ Ghent$score$≥$5$ Appendix$5$

EDS$with$Aortopathy$ <1:10,000$ 34$ 11$ Any$aortic$aneurysm$or$dissection$ Appendix$5$ coTexisting$with$EDS$

EDS$with$Carotid$dissection$ $$<<1:1,000$ 10$ 8$ $ Appendix$5$

EDS$with$Cerebral$aneurysm$ <1:1,000$ 6$ 6$ Any$proven$cerebral$aneurysm$ Appendix$5$

EDS$with$Kyphoscoliosis$ <1:1000T 16$ 14$$ $6$EDSVIb,$5$EDSVIb/VIIa$ Appendix$5$ 1:10,000$ overlap,$3$EDS$VI/Marfan$ overlap)b$

EDS,$Ehlers$Danlos$syndrome.$Col.(V),$collagen$type$5,$the$usual$abnormality$in$classical$EDSg$Col.(III),$collagen$type$ 3,$ the$ usual$ abnormality$ in$ vascular$ EDSg$ EM,$ electron$ microscopy.$ a.$ multiple$ collagen$ rosettes$ on$ EM$ are$ pathognomonic$of$classical$EDS.$b.$these$refer$to$various$overlapping$types$of$kyphoscoliotic$(VI)$and$arthrochalasiaT type(VII)$EDS$(see$Chapter$3.1$for$detailed$EDS$classification)$

! 105! ! Table%5.3%Col.(V)Tnegative$classical$EDS$cases$selected$for$exome$sequencing,$ranked$by$phenotype$score$ No.% of% No.% of% Overall% Mendelian% BRIDGE% major% minor% Phenotype% Beighton% clinical% a% a b c ID% exome%ID% Age% Gender% Ethnicity% criteria criteria % score % score% diagnosis %

22EB0583$ B250043_A$ 19$ F$ White$T$British$ 3$ 4$ 11.5$ 8$ Moderate$ 22DP1528$ B250084_A$ 36$ M$ White$T$British$ 3$ 3$ 10.5$ ─$ Strong$ 22LA0534$ B250033_A$ 34$ F$ White$T$British$ 3$ 3$ 10.5$ 9$ Moderate$ 22JM0670$ B250032_A$ 36$ F$ White$T$British$ 2$ 5$ 8.5$ 8$ $ 22AC0404$ B250011_A$ 43$ M$ White$T$British$ 2$ 4$ 8$ 9$ $ 22AP0653$ B250073_A$ 21$ F$ White$T$British$ 2$ 3$ 7.5$ 9$ Moderate$ 22TF0595$ B250053_A$ 31$ M$ Not$Stated$ 2$ 3$ 7.5$ 6$ Moderate$ 22GS0564$ B250025_A$ 24$ M$ White$T$British$ 2$ 3$ 7.5$ 8$ Moderate$ 22DO0479$ B250021_A$ 24$ F$ White$T$British$ 2$ 3$ 7.5$ 6$ $ 22DF0428$ B250015_A$ 67$ F$ White$T$British$ 2$ 3$ 7.5$ ─$ $ 22MS0446$ B250017_A$ 49$ M$ White$T$British$ 2$ 2$ 7$ 4$ $ 22UM0100$ B250071_A$ 54$ F$ Asian$T$Indian$ 2$ 1$ 7$ 7$ $ 22WBJ0079$ B250048_A$ 47$ M$ White$T$British$ 2$ 2$ 7$ ─$ $ 22LH0778$ B250079_A$ 22$ F$ White$T$British$ 2$ 2$ 7$ 7$ $ 22SB0755$ B250076_A$ 42$ F$ White$T$British$ 2$ 2$ 7$ 4$ $ 22DW0682$ B250056_A$ 45$ F$ White$T$British$ 2$ 2$ 7$ 6$ $ 22MS1002$ B250081_A$ 53$ F$ White$T$British$ 2$ 2$ 7$ 7$ Moderate$ 22WK0810$ B250047_A$ 15$ M$ White$T$British$ 2$ 2$ 7$ 8$ Moderate$ 22PG0806$ B250064_A$ 16$ M$ White$T$British$ 2$ 2$ 7$ ─$ Moderate$ 22RT0681$ B250060_A$ 53$ F$ White$T$British$ 2$ 2$ 7$ ─$ $ 22SA0402$ B250050_A$ 31$ M$ Mixed$T$Other$ 2$ 2$ 7$ 6$ $ 22GC0560$ B250026_A$ 26$ F$ White$T$British$ 2$ 2$ 7$ 5$ $ 22BC0397$ B250009_A$ 21$ F$ White$T$British$ 2$ 2$ 7$ ─$ $ 22RC0395$ B250049_A$ 50$ M$ White$T$British$ 2$ 2$ 7$ ─$ $ 22LC0537$ B250037_A$ 30$ F$ White$T$British$ 2$ 2$ 7$ 8$ Moderate$ 22NW0383$ B250007_A$ 25$ F$ White$T$British$ 2$ 2$ 7$ 7$ Strong$ 22NW0075$ B250003_A$ 32$ F$ White$T$British$ 2$ 2$ 7$ 8$ Moderate$ 22AC0409$ B250012_A$ 46$ F$ White$T$British$ 2$ 2$ 7$ ─$ Moderate$ 22DB0536$ B250034_A$ 43$ M$ White$T$British$ 2$ 2$ 7$ 1$ $ 22JG0034$ B250001_A$ 37$ F$ White$T$British$ 2$ 2$ 7$ 3$ $ 22CB0136$ B250005_A$ 64$ F$ White$T$British$ 2$ 2$ 7$ ─$ Moderate$ 22NA0611$ B250063_A$ 39$ M$ White$T$British$ 2$ 1$ 6.5$ 7$ Moderate$ 22SP1482$ B250083_A$ 54$ F$ White$T$British$ 2$ 1$ 6.5$ 5$ Moderate$ 22HS0803$ B250046_A$ 28$ F$ White$T$British$ 2$ 1$ 6.5$ 8$ Moderate$ 22SC0396$ B250040_A$ 50$ F$ White$T$Other$ 2$ 1$ 6.5$ ─$ Moderate$ 22TH0566$ B250028_A$ 68$ M$ White$T$British$ 2$ 0$ 6$ 4$ $ 22AS0072$ B250002_A$ 53$ M$ White$T$British$ 2$ 0$ 6$ ─$ $$ M,$maleg$F,$female.$a.$Major$and$minor$criteria$are$those$for$classical$EDS,$according$to$the$1997$Villefranche$classification$b.$Phenotype$ score$calculation:$3$points$per$major$criterion,$0.5$points$per$minor$criterion.$In$addition,$0.5$points$added$for$nonTspecific$EM$changes$(e.g.$ occasional$collagen$rosettes)$which$applied$to$two$cases:$22EB0583$and$22UM0100$c.$Overall$clinical$diagnosis$was$based$on$the$initial$ clinical$assessment$by$an$EDS$specialist$irrespective$of$the$phenotype$score:$“Moderate”$for$an$overall$phenotype$suggestive$of$classical$ EDS$overlapping$with$another$EDS$type$or$“Strong”$in$cases$considered$strongly$suggestive$of$classical$EDS.

! 106! ! 5.2.2%Exome%sequencing%&%analysis% DNA$extraction$was$carried$out$for$all$cases$as$described$above$(Chapter$2.3).$The$8$ EDS$VIII$samples$were$processed$inThouse$and$for$these,$the$creation$of$libraries$for$ all$exon$target$enrichment$by$Sureselect$was$carried$out$as$detailed$in$Chapter$2.4T2.5.$ Enriched$exomeTlibraries$ from$ each$ sample$ were$ sequenced$ in$ a$ total$ cohort$ of$ 113$ exome$sequences,$run$on$the$Illumina$MiSeq.$Initial$analysis$in$terms$of$read$mapping$ and$variant$calling$were$carried$out$inThouse$as$described$above$(Chapter$2.9).$$

DNA$ samples$ extracted$ from$ the$ other$ 181$ patients$ with$ features$ of$ cEDS,$ vEDS,$ Hypermobility,$ aortopathy,$ Marfanoid$ EDS,$ cerebral$ aneurysm$ and$ carotid$ dissection$ for$ whom$ sequencing$ of$ the$ usual$ causative$ genes$ had$ not$ revealed$ abnormalities,$ were$ submitted$ for$ exome$ sequencing$ by$ the$ NIHR$ Bioresource$ for$ Rare$ Diseases$ (BRIDGE).152$Exome$sequencing$and$initial$analysis$including$variant$calling$for$these$ cases$was$carried$out$by$the$NIHR$BRIDGE$consortium.$$$

For$ all$ cases,$ after$ variant$ calling,$ synonymous$ and$ intronic$ variants$ and$ those$ with$ MAF>0.01$ were$ excluded$ from$ further$ downstream$ analysis.$ To$ prioritise$ candidate$ disease$ causing$ variants,$ rare$ (MAF<0.01),$ functionally$ significant$ variants$ were$ prioritised$ based$ on$ functional$ effect:$ loss$of$function$(LoF)$variants$(premature$stop$ codons,$ mutations$ in$ canonical$ spliceTsites$ and$ frameshift$ variants)$ were$ considered$ first,$ followed$ by$ other$ rare$ nonTsynonymous$ variants$ (NSNV’s).$ To$ identify$ potential$ diseaseTcausing$genes$in$each$phenotype$group,$we$first$attempted$to$identify$genes$ with$at$least$one$functionally$significant$variant$affecting$at$least$two$different$unrelated$ samples$ within$ the$ same$ phenotype$ group,$ prioritising$ individuals$ with$ the$ highest$ ranked$phenotypes.$The$Variant$Annotation$and$Analysis$Tool$(VAAST)153$was$used$to$ rank$ all$genes$according$to$the$overall$burden$of$pathogenic$variants$ for$ a$ particular$ phenotype$ group.$ The$ topTranked$ genes$ with$ p<0.05$ were$ selected$ and$ rare,$ functionally$significant$variants$in$those$genes$were$examined$as$above.*$

*

$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ $$$$$$$$ *!Bioinformatics$analysis$up$to$and$including$variant$calling$was$carried$out$by$Dalia$Kasperavaciute$of$the$ Imperial$ College$ Clinical$ Genomics$ Facilityg$ downstream$ bioinformatics$ analysis$ including$ variant$ prioritisation$based$on$phenotype$and$VAAST$burden$analysis$was$carried$out$with$the$help$of$Dr.$Jana$ Vandrovcova.! ! 107! ! 5.3%Results%

5.3.1%Data%output%&%assay%performance%in%the%8%EDS%VIII%samples%

177,404$ amplicons$ covering$ all$ exons$ and$ intron/exon$ boundaries$ were$ sequenced,$ with$each$amplicon$achieving$a$mean$coverage$of$42.9$reads$(range$0T2,148$reads)$in$ the$8$EDS$VIII$samples$(Table$5.4).$

Table%5.4%Sequencing$summary$for$the$8$EDS$VIII$probands% Sample$ID$ Sequencing$ID$ Total$reads$ %$reads$ Mean$ %bp$ covered$ on$target$ coverage$ by$>30$reads$ per$amplicon$

20FB8035$ IGFP000162$ 109,829,864$ 99.44$ 43.8$ 61$

22TP0427$ IGFP000688$ 45,517,652$ 98.82$ 19.7$ 27$ $ $ 20GT845$ IGFP000173$ 112,431,340$ 99.32$ 49.7$ 66$ $ $ $ 22MMC0357$ IGFP000635$ 104,550,376$ 98.97$ 46.8$ 63$ $ 22EM0088$ IGFP000534$ 158,143,650$ 98.68$ 42.0$ 59$ $ 20RP866$ IGFP000235$ 126,373,470$ 99.30$ 55.5$ 69$ $ $ $ 20AJ1332$ IGFP000129$ 113,377,410$ 99.39$ 55.9$ 69$ $ $ $ $ 20AC1004$ IGFP000121$ 118,132,508$ 99.44$ 47.7$ 61$

$ Bp,$base$pairs$ $

5.3.2%Prioritisation%of%candidate%diseaseCcausing%variants%in%EDS%VIII%

In$ the$ cohort$ of$ 8$ EDS$ VIII$ cases,$ 142,154$ total$ heterozygote$ variants,$ comprising$ 69,539$coding$variants$and$1,381$rare$variants$with$MAF<0.01$were$called.$The$mean$ number$of$variants$for$each$of$the$8$cases$of$EDS$VIII$was$15,795,$of$which$a$mean$of$ 7,727$ exonic$ (coding$ variants)$ and$ 153$ rare$ coding$ variants$ with$ MAF<0.01$ were$ detected$per$sample$(Table$5.5)$$

Rare(variants(in(the(previously(identified(linked(region(

One$of$the$cases$in$our$cohort–$20FB8035,$which$had$one$of$the$strongest$phenotypes$ consistent$ with$ EDS$ VIII$ T$ $ was$ the$ index$ case$ of$ the$ original$ Swedish$ pedigree$ to$ which$ a$ linked$ interval$ (genomic$ coTordinates$ 12:1T20,000,000,$ LOD$ score$ 5.1)$ had$ previously$been$mapped.$Therefore,$I$first$analysed$variants$called$within$this$region.$ Of$all$variants$called$in$the$overall$cohort$of$121$sequenced$exomes,$1,416$were$within$ this$region,$of$which$424$were$rare$coding$variants$and$38$were$rare$coding$variants$ unique$to$the$EDS$VIII$cohort.$These$are$elaborated$in$Table$5.5.$

! 108! ! Rare(candidate(diseaseNcausing(variants(outside(the(linked(region(

I$ then$ analysed$ rare$ variants$ outside$ the$ above$ interval,$ assuming$ genetic$ heterogeneity.$ Variants$ were$ divided$ into$ two$ groups:$ loss$ of$ function$ (LoF)$ variants$ and$nonTsynonymous$variants$(NSNV’s).$I$identified$33$rare$LoF$variants$unique$to$the$ EDS$ VIII$ cohort$ (25$ of$ these$ were$ exonic$ and$ the$ remainder$ in$ the$ 3’$ or$ 5’UTR$ or$ intronic$ regions).$ I$ also$ observed$ 504$ NSNV’s$ outside$ the$ linked$ region$ that$ were$ unique$to$the$EDS$VIII$cohort$(Table$5.3).$$

Table%5.5%EDS$VIII$cohort$–$summary$of$variants$called%

b Total$variants$ Rare$Coding$ In$linkage$ Outside$the$linkage$region $ a$ Mendelian$ID$ called$ variants$ region LoF$$$$$$$$$$$$$NSNV’s$ 20FB8035$ 15,309$ 140$ 3$ 8$ 14$ 20AJ1332$ 16,162$ 153$ 7$ 4$ 70$ 20GT845$ 15,671$ 174$ 7$ 0$ 15$ 20RP866$ 15,075$ 145$ 1$ 2$ 20$ 22EM0088$ 15,912$ 163$ 0$ 5$ 30$ 22MMC0357$ 16,169$ 151$ 8$ 13$ 63$ 20AC1004$ 15,500$ 144$ 2$ 0$ 1$ 20GC1005$ 16,950$ 147$ 3$ 0$ 0$ 22TP0427$ 15,406$ 164$ 4$ 0$ 0$ LoF,$ rare$ loss$ of$ function$ variantsg$ NSNV,$ rare$ nonTsynonymous$ single$ nucleotide$ variants.$ a.$This$column$shows$all$rare$exonic$variants$identified$in$the$previously$identified$linkage$region$(12:1T 20,000,000,$ LOD$ score$ 5.1)$ for$ the$ family$ of$ the$ first$ case,$ 20FB8035.$ b.$ This$ column$ shows$ rare$ functionally$significant$variants$unique$to$the$EDS$VIII$cohort,$outside$the$above$linkage$region.$

Combining$ the$ strength$ of$ phenotype$ of$ the$ first$ case,$ 20FB8035$ and$ the$ linkage$ region$ from$ that$ patient’s$ family,$ the$ highest$ priority$ variants$ were$ the$ three$ variants$ identified$in$the$linkage$region$for$that$patient.$Table$5.6$details$these$three$variants.$All$ three$ variants$ were$ nonTsynonymous$ (missense)$ variants,$ unique$ to$ the$ EDS$ VIII$ cohort$ (not$ present$ in$ 113$ other$ exomes$ sequenced$ inThouse).$ The$ first,$ in$ the$ C1R$ gene,$was$a$novel$variant,$not$previously$reported$in$any$populationTvariant$database.$ The$ other$ two,$ in$ TAS2R30( and$ PLEKHA5,( had$ previously$ reported$ minor$ allele$ frequencies$ of$ 2$ in$ 10,000$ and$ 2$ in$ 1,000$ respectively$ (Table$ 5.6).$ The$ TAS2R30( variant$(c.G920C)$was$also$observed$in$another$unrelated$EDS$VIII$patient,$22TP0427,$ who$also$had$a$highTranked$phenotype$for$EDS$VIII.$The$three$variants$had$variable$inT silico$ predictive$ scores$ and$ conservation$ scores$ and$ were$in$genes$not$known$to$be$ associated$ with$ collagen$ or$ connective$ tissue$ pathology$ and$ were$ therefore$ initially$ prioritised$ based$ on$ observed$ MAF$ alone$ (Table$ 5.6).$

! 109! !

!

Table&5.6!The!three!rare,!functionally!significant!variants!identified!in!the!linked!region!for!sample!FB8035& Genomic&co0 Gene& Protein& Transcript&ID& Variant& Frequency& Observed&frequencies&of& In0silico&predictions&of& a& within&the& b ordinates variants&in&databases & pathogenicity,&functional& EDS&VIII& c cohort& score(qualitative&prediction) & & !! !! !! !! !! & Max& 10000 ESP6500& &SIFT&M&&&Polyphen&M&&LRT&M&&&MT&M&&MA& Genom& FATHMM&M&RadialSVM&M&LR&& GERP&M&PhyloP&M&SiPhy& 12:7241449! C1R$ complement! component! 1,! ENST00000542 exon6:! c.G899C:! 1! NA! NA! NA! 0.79(T)!R!0.709(P)!R!0.995(N)!R!0(N)!R!S(S)!! r!subcomponent! 285.1! p.R300P! 0.317(T)! R! 0.258(T)! R! 0.052(T)!! !S10.1R!S1.641R!14.643!

12:11285924! TAS2R30$ taste! receptor,! type! 2,! NM_001097643! exon1:! c.G920C:! 2! 2.00ES04! NA! 2.00ES04! 0.97(D)! R! 0(B)! R! S(S)! R! 0(N)! R! 0.481(N)!! member!30! p.R307P! 0.353(T)! R! 0.244(T)! R! 0.036(T)!!!! S3.76R!S0.389R!3.029!

12:19500060! PLEKHA5$ pleckstrin! homology! NM_001256787! exon18:! c.C2290T:! 1! 0.0022! NA! 0.0018! 0.4(T)!R!0.046(B)!R!0.998(N)!R!1(D)!R!S(S)!! domain! containing,!! p.P764S! 0.448(T)! R! 0.314(T)! R! 0.259(T)!! family!A!member!5! 4.77R!1.379R!10.866!

Max,!maximum!observed!minor!allele!frequency!in!all!databasesR!1000SGenom,!frequency!in!1000!Genomes!project!database!(phase!2!release)R!ESP6500,!frequency!in!ESP6500!database!total! and!European!subsetR!NA,!novel!!variant.!! a.&Genomic!coSordinates!are!based!on!GRCh37/hg19!human!reference!sequence.!b.!All!three!variants!were!not!present!in!ClinVar!or!HGMD.!c.&In!silico!prediction!scores!are!shown!as!a!predictive! functional! score! (with! qualitative! prediction! in! brackets)! based! on! ANNOVAR68! version! 2013aug23! and! refer! to! the! following! specific! ANNOVAR! annotations:! Polyphen:! "LJB23_Polyphen2_HVAR_score",! Mutation! Taster:! "LJB23_MutationTaster_score_converted",! Mutation! Assessor:! "LJB23_MutationAssessor_score_converted",! LRT:!! "LJB23_LRT_score_converted",!MT:!MutationTaster,!MA:!MutationAssessor,!FATHMM:!!"LJB23_FATHMM_score_converted"!!!Predictive!functional!scores!for!SIFT,!Polyphen,!MutationTaster,! MutationAssessor,!LRT!&!FATHMM!were!converted!to!a!0S1!scale!with!a!higher!number!denoting!a!higher!probability!of!functional!significance.!!Qualitative!predictions:!D,!deleterious.!T,!toleratedR!! for!Mutation!Taster!only:!A,!disease!causing!automatic,!D,!disease!causingR!N,!polymorphismR!!P,!polymorphism!automaticR!!for!Mutation!Assessor!only:!!H(high),!!M(medium!and!L(low)!probability! of! functional! impact.! Conservation! scores! (GERP++,! SiPhy,! PhyloP):! higher! number! ! =! higher! conservation! across! species

! 110! ! 5.3.3$Identification$of$candidate$genes$in$EDS$VIII$

For$the$three$putative$candidate$genes$identified$above,$I$then$sought$to$identify$other$ rare$ functionally$ significant$ exonic$ variants$ in$ these$ genes.$ (The$ observed$ MAF’s$ for$ the$ variants$ in$ TAS2R30$ and$ PLEKHA5$ effectively$ excluded$ them$ from$ being$ likely$ diseaseAcausing$ genes$ as$ these$ outweigh$ the$ disease$ frequency$ of$ EDS$ VIII,$ but$ variants$ in$ these$ genes$ were$ investigated$for$ completeness).$ $ For$C1R,$ 4$ other$rare$ exonic$variants$were$observed:$2$of$these$were$NSNV’s$(observed$in$2$other$EDS$VIII$ patients$apart$from$the$index$case$above)$and$2$were$synonymous$variants$observed$ in$ 2$ individuals$ without$ signs$ of$ EDS$ VIII.$ For$ TAS2R30,1 two$ other$ variants$ were$ observed:$one$was$a$rare$NSNV$in$a$patient$with$no$signs$of$EDS$VIII$and$the$other$ was$a$common$polymorphism$(maximum$MAF$0.53)$observed$in$53$of$113$ inAhouse$ control*$exomes.$ One$ further$ NSNV$ in$ a$ related$ gene,$ TAS2R10,$ was$ observed$ in$ a$ single$individual$with$EDS$VIII:$this$variant$had$a$maximum$reported$MAF$of$1A3%$(in$ the$ 1000$ Genomes$ database).$ Nine$ other$ variants$ in$ PLEKHA5$ (&$ 7$ variants$ in$ a$ related$ gene,$ PLEKHG6)$ were$ observed$ but$ none$ were$ exclusive$ to$ the$ EDS$ VIII$ cohort.$ The$ functionally$ significant$ exonic$ variants$ unique$ to$ the$ EDS$ VIII$ cohort$ are$ summarised$in$Table$5.7$below.$$

Table$5.7$All$functionally$significant$variants$identified$in$candidate$genes$from$the$linked$region$for$FB8035$$ b Candidate$ Genomic$co< Type$ Variant$ $ Max.$MAF$ Freq.$in$ Freq.$in$ Samples$ a gene$ ordinates$ $ EDS$VIII$ other$ containing$ cohort$ exomes$ this$variant$ #c (n=8)$ (n=113) $

d C1R1 12:7241449$ NSNV$ exon6:c.G899C:p.R300P$ NA$ 1$ 0$ 20FB8035 $ C1R1 12:7241461$ NSNV$ exon6:c.G887A:p.G296D$ NA$ 1$ 0$ 20GT845$ C1R1 12:7241482$ NSNV$ exon6:c.A866G:p.D289G$ NA$ 1$ 0$ 22TP427$ #d TAS2R301 12:11285924$ NSNV$ exon1:c.G920C:p.R307P$ 2.00EA04$ 2$ 0$ 20FB8035 $,$ 22TP427$ TAS2R101 12:10978607$ NSNV$ exon1:c.T262C:p.W88R$ 0.03$ 1$ 0$ 22MMC357$

Max.$ MAF,$ maximum$ observed$ minor$ allele$ frequency$ across$ all$ population$ databases_$ Freq.,$ frequency$$ a.$Genomic$coAordinates$are$based$on$GRCh37/hg19$human$reference$sequence.$b.$Transcript$ID’s:$C1R:$ENST00000542285.1,$ TAS2R30:1NM_001097643,$TAS2R10:1NM_023921.$c.$113$other$EDS/TAAD$cases$without$signs$of$EDS$VIII.$d.$This$sample$is$the$ index$case$from$the$Swedish$EDS$VIII$pedigree$to$which$the$linkage$region$pertains.$

Assuming$genetic$heterogeneity,$I$finally$considered$other$variants$apart$from$the$three$ putative$candidates$from$the$linked$region$of$20FB8035$mentioned$above.$The$25$LoF$ variants$that$were$unique$to$the$cohort$(Table$5.5)$were$identified$in$24$different$genes,$ all$of$which$were$outside$the$linkage$region$above.$These$genes$(listed$in$Table$5.8$ below)$were$then$compared$with$a$list$of$candidate$genes$known$to$be$associated$with$

$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ $$$$$$$$ *!The!“control”!exomes!in!this!case!refer!to!105!other!EDS/TAAD!cases!without!features!of!periodontal!EDS.! ! 111! !

EDS$and$connective$tissue$matrix$function$(see$Appendix$6).$No$matches$were$found$ with$ the$ candidate$ gene$ list.$ Following$ literature$ review,$ none$ of$ these$ 24$ potential$ candidates$ showed$ specific$ relevance$ to$ EDS$ pathogenesis,$ although$ four$ genes$ –$ LAD1,$ SNX8,$ EXOSC3$ and$ TBC1D13$ did$ show$ relevant$ tissueAlevel$ expression$ (in$ skin$and/or$soft$tissue).$I$also$searched$for$rare,$functionally$significant$variants$in$other$ components$ of$ the$ complement$ system$ and$ identified$ two$ variants$ in$ the$ same$ individual,$AC1004:$$p.E320D$in$C5$(complement$component$5)$and$p.V896L$in$c8A$ (complement$component$8,$alpha$subunit)$with$maximum$reported$MAF’s$of$1:10,000$ and$2:1,000$respectively_$both$genes$are$associated$with$high$levels$of$mutation$in$the$ general$population154$and$do$not$interact$directly$with$C1r$or$C1s.$$

Table$ 5.8$ Other$ candidate$ genes$ for$ EDS$ VIII$ outside$ the$ linkage$ region,$ in$ which$rare$LoF$variants$were$identified$$

Gene$ Protein$ CEP85$ centrosomal$protein$85kDa$ ZMYM1$ zinc$finger,$MYMAtype$1$ LAD1*$ ladinin$1$ FAM71A$ family$with$sequence$similarity$71,$member$A$ GGPS1$ geranylgeranyl$diphosphate$synthase$1$ SCN9A$ sodium$channel,$voltageAgated,$type$IX,$alpha$subunit$ MCTP1$ multiple$C2$domains,$transmembrane$1$ AQPEP$ Laeverin$ STMND1$ Stathmin$Domain$Containing$1$ RAB44$ RAB44,$member$RAS$oncogene$family$ MYB†$ vAmyb$myeloblastosis$viral$oncogene$homolog$(avian)$ SNX8*$ sorting$nexin$8$ TFR2$ transferrin$receptor$2$ PKHD1L1$ polycystic$kidney$and$hepatic$disease$1$(autosomal$recessive)Alike$1$ EXOSC3*$ exosome$component$3$ PPP6C†$ protein$phosphatase$6,$catalytic$subunit$ TBC1D13*$ TBC1$domain$family,$member$13$ MALRD1$ MAM$And$LDL$Receptor$Class$A$Domain$Containing$1,$aka$c10orf112$ SLC37A4$ solute$carrier$family$37$(glucoseA6Aphosphate$transporter),$member$4$ PDZRN4$ PDZ$domain$containing$ring$finger$4$

USP44$ ubiquitin$specific$peptidase$44$

CCDC168$ CoiledACoil$Domain$Containing$168,$aka$c13orf40($ PCID2$ PCI$domain$containing$2$ CRNKL1†$ crooked$neck$preAmRNA$splicing$factorAlike$1$(Drosophila)$

*these$ genes$ show$ high$ expression$ in$ skin/soft$ tissue$$$ †These$ genes$ are$ the$ least$ likely$ to$ be$ tolerant$ of$ loss$ of$ function$ variation,$ based$ on$ constraint$metrics$(pLI$scores:$MYB:$0.88,$PPP6C:$0.99,$CRNKL1:$0.88)_$the$next$highest$ pLI$scores$were$for$GGPS$(0.67)$and$TBC1D13$(0.61)_$all$other$genes$had$low$pLI$scores.$ Constraint$metrics$(from$Exac$database$154)$refer$to$the$degree$of$tolerance$of$a$gene$based$ on$the$observed$mutation$rate$of$the$gene$(pLI$score$closer$to$1$indicates$less$tolerance).$ ! 112! ! 5.3.4$ Identification$ of$ candidate$ genes$ in$ other$ EDS$ phenotypes$ (Col.V< negative$Classical$EDS$phenotype)$ Of$the$other$phenotypeAgroups$identified,$sequence$data$from$the$highest$priority$group$ –$the$classical$EDS$group$A$$were$analysed.$Due$to$time$constraints$and$as$all$samples$ had$ not$ undergone$ sequencing$ by$ the$ time$ of$ writing,$ the$ other$ groups$ were$ not$ analysed.$$

Though$ all$ cases$ had$ already$ been$ screened$ for$ mutations$ in$ the$ collagen$ genes$ normally$associated$with$EDS$A$$COL1A1/2,1COL3A1,1COL5A1/2$–$in$order$to$exclude$ the$possibility$of$a$missed$collagen$gene$mutation$I$first$searched$for$rare,$functionally$ significant$ variants$in$these$ genes.$ $ In$one$patient$A$ 22DP1528$–$ with$a$highAranked$ phenotype,$one$nonsense$mutation$in$COL5A11was$identified.$

Secondly,$ I$ searched$ for$ functionally$ significant$ variants$ in$ the$ topAranked$ genes$ for$ this$phenotype,$based$on$the$VAAST$burden$analysis$of$variant$pathogenicity$for$this$ cohort.$The$top$9$ranked$genes$met$statistical$significance$(p<0.05)$and$included$one$ gene$ –$ EXPH51 >$ with$ a$ known$ high$ level$ of$ expression$ in$ the$ skin$ and$ one$ gene$ –$ DMTF11>$with$high$constraint$metrics$according$to$the$ExAC$database154,$suggesting$a$ low$level$of$background$mutation$and$therefore$a$higher$probability$that$a$functionally$ significant$ variant$ is$ likely$ to$ be$ damaging$ (see$ Table$ 5.9).$ Variants$ in$ these$ genes$ were$examined,$specifically$looking$for$functionally$significant$exonic$variants$unique$to$ the$classical$EDS$cohort$but$none$were$rare$variants$(Table$5.9).$$

$ $

! 113! !

Table$5.9$Top$ranked$statistically$significant$genes$identified$by$VAAST$burden$analysis$ Gene$ Protein$ Expression$&$associated$ p$$ Constraint$ No.$of$variants$in$Col.(V)$ phenotypes$ metrics$from$ negative$classical$EDS$ ExAC$database$ cohort$ a (z31 Keratin$ Mainly$hair$follicle$(very$low$in$ 0.0001$ A1/08,$0.00$ $ 1$ 0$ associated$ skin)$ protein$ OR2W31 Olfactory$ Plasma$membrane$receptor,$ 0.0002$ A1.49,$0.08$ $ 1$ 0$ receptor$2W3/8$ widely$expressed$ DSPP1 Dentin$ Mainly$extracellular_$wide$ 0.0011$ >0.31,10.001 1 11 01 Sialophosphopr expression:$ECM$degradation$ otein$A$$ pathway,$collagen$binding,$(ph:$ dentinogenesis$imperfecta)$ BAG31 BAGproteins1A5$$ Intracellular:$widely$expressed$ 0.0153$ A1.01,$0.53$ 1$ $ 0$ including$skin$and$smooth$ muscle$/$inhibits$chaperone$ activity$of$HSP70$A$protein$ folding,$intracellular$trafficking$/$ ph:$cardiomyopathy$A$gwas$hit:$ PMID:$21459883$ DMTF11 $ Spleen,$testis,$thymus.$Cause$of$ 0.0167$ 1.73,$0.96$ $ 1$ 0$ 40%$of$nonAsmall$cell$lung$ cancer$ EXPH51 $ Skin,$brain,$placenta.$Exosome$ 0.0173$ A2.63,$0$ 1$ $ 0$ secretion$and$intracellular$ trafficking.$Associated$with$ epidermolysis$bullosa$ COL9A11 $ Minor$component$of$hyaline$ 0.0266$ A1.15,$0$ 3$ $ 0$ cartilage.$Associated$with$OA.$ OR6C741 $ Olfactory$receptor.$ 0.0313$ A0.26,$0.08$ 1$ $ 0$ p,$pAvalue$from$VAAST$burden$analysis$associating$each$gene$with$the$col.VAnegative$classical$EDS$phenotype$group_$MAF,$minor$allele$ frequency_$ECM,$extracellular$matrix_$ph,$specific$associated$phenotype_$OA,$osteoarthritis.$a.constraint$metrics$(from$Exac$database$154)$ refer$to$the$degree$of$tolerance$of$a$gene$based$on$the$observed$mutation$rate$of$the$gene,$to$missense$variation$(zAscore$–$a$positive$ score$indicates$less$tolerance)$and$Loss$of$function$variants$(pLI$–$a$score$closer$to$1$indicates$less$tolerance).$$ $

! 114! ! 5.4$Discussion$

The$aim$of$this$chapter$was$to$identify$novel$genes$underlying$specific$phenotypes$of$ EDS,$which$remain$unresolved$genetically:$

1)$A$wellAdefined$subtype$of$EDS,$Periodontal$EDS$(Type$VIII).$$

2)$ Specific$ phenotypes$ observed$ in$ the$ context$ of$ EDS$ –classical$ EDS$ features,$ vascular$ EDS$ features,$ extensive$ Hypermobility,$ vasculopathy,$ Marfanoid$ habitus,$ kyphoscoliosis,$without$mutations$in$known$genes.$$

The$likelihood$of$novel$gene$discovery$was$highest$in$the$first$group$owing$to$the$rarity$ and$specificity$of$the$phenotype$and,$as$this$led$to$the$identification$of$a$highApriority$ candidate$gene$in$that$group,$the$majority$of$this$chapter$is$devoted$to$that.$$$

5.4.1$Periodontal$EDS$(VIII)$–$discovery$of$novel$genes$C1R$&$C1S$

Periodontal$ EDS$ (type$ VIII),$ is$ a$ distinct$ entity$ and$ appears$ to$ segregate$ in$ an$ autosomal$dominant$manner.$The$cardinal$clinical$feature$in$EDS$VIII$is$severe$early$ onset$ periodontitis$ with$ marked$ gingival$ recessions,$ underlined$ by$ a$ chronic$ inflammatory$reaction$of$periodontal$tissues.94,155$There$is$no$evidence$of$an$immune$ deficiency$in$EDS$VIII,$although$some$individuals$report$recurrent$infections.$$

The$presence$of$data$from$previous$linkage$analysis$from$a$large$ Swedish$EDS$VIII$ pedigree$along$with$DNA$from$other$unrelated$individuals$sharing$the$same$very$rare$ and$ distinct$ phenotype,$ allowed$ me$ to$ focus$ on$ a$ relatively$ narrow$ genomic$ interval$ and$identify$one$clear$candidate$gene$in$the$index$case$of$the$Swedish$pedigree,$C1R$ (which$ encodes$ one$ of$ two$ subunits$ of$ the$ complement$ 1$ protein)_$ the$ presence$ of$ novel,$ functionally$ significant$ variants$ in$ 3$ separate$ unrelated$ EDS$ VIII$ individuals$ in$ C1R$made$this$the$highest$priority$candidate$gene,$likely$to$be$diseaseAcausing$in$at$ least$part$of$EDS$VIII.$$

This$finding$was$corroborated$by$the$work$of$Prof$Zschoke$&$colleagues$in$Innsbruck,$ Austria$who$independently$discovered$C1R$in$a$large$Austrian$EDS$VIII$pedigree$(15$ affected$individuals)$and$by$Prof$Byers$&$colleagues$at$the$University$of$Washington,$ USA$ who$ identified$ the$ related$ gene$ C1S$ in$ a$ large$ US$ pedigree$ (6$ affected).$ Subsequent$targeted$sequencing$of$these$two$genes$in$a$further$10$families$with$EDS$ VIII$(43$affected$individuals)$demonstrated$novel$point$mutations$in$C1R$or$C1S1(Table$ 5.10).$ These$ variants$ segregated$ with$ disease$ status$ and$ were$ not$ observed$ in$ unaffected$relatives$or$in$variant$databases.$We$did$not$identify$functionally$significant$ variants$in$these$other$complement$proteins,$which$interact$directly$with$C1r$and$C1s_$ the$ two$ variants$ identified$ in$ the$ same$ individual$ in$ two$ downstream$ complement$ components,$ C5$ and$ C8A,$ are$ of$ unknown$ significance,$ but$ owing$ to$ their$ reported$ ! 115! !

MAF$ and$ the$ high$ level$ of$ mutation$ in$ these$ latter$ genes,$ have$ a$ low$ probability$ of$ being$pathological.$$

Table$5.10$Summary$of$all$variants$discovered$in$the$candidate$genes,$C1R$and$its$target$C1S1$ Family$ $Details$ Variant$ Affec Gene$ DNA$(c.)$ Protein$(p.)$ Domain$ discovery$ ted$ (GRCh38)$ strategy$ (n)$ 1$ Austrian$ Linkage$(8$+$ 15$ C1R1 c.149_150T p.Val50Asp$ CUB1$(EGF)$ pedigree$ 4)$&$WES$ C>AT$

2$ UK$EDS,$ID$ WES$ 1$ C1R1 c.869A>G$ p.Asp290Gly$ C1q$binding$ 22TP427$$ site$ 3$ UK$EDS,$ID$ WES$ 1$ C1R1 c.890G>A$ p.Gly297Asp$ CUB2$ 20GT845$$ 4$ $$ Targeted$seq.$ 4$ C1R1 c.899T>C$ p.Leu300Pro$ CUB2$(near$ CCP1)$ 5$ UK$EDS$ID$ (LinkageA 17$ C1R1 c.902G>C$ p.Arg301Pro$ CUB2$(near$ 20FB8035,$ previously)$&$ CCP1)$ Swedish$ WES$ pedigree$ 6$ $$ Targeted$seq.$$ 7$ C1R1 c.905A>G$ p.Trp302Cys$ CUB2$(near$ CCP1)$ 7$ $$ Targeted$seq.$$ 1$ C1R1 c.927C>G$ p.Cys309Trp$ Sushi$CCP1$ (near$CCP2)$ 8$ $$ Targeted$seq.$$ 3$ C1R1 c.927C>G$ p.Cys309Trp$ Sushi$CCP1$ (near$CCP2)$ 9$ $$ Targeted$seq.$$ 4$ C1R1 c.1012T>C$ p.Cys338Arg$ Sushi$CCP1$ (near$CUB2)$ 10$ $$ Targeted$seq.$$ 3$ C1R1 c.1073G>T$ p.Cys358Phe$ Sushi$CCP1$ (near$CCP2)$ 11$ $$ Targeted$seq.$ 1$ C1R1 c.1092G>C$ p.Trp364Cys$ Sushi$CCP1$ (near$CUB2)$ 12$ $$ Targeted$seq.$ 1$ C1R1 c.1113C>G$ p.Cys371Trp$ Sushi$CCP1$ (near$CUB2)$ 13$ $$ Targeted$seq.$ 10$ C1R1 c.1200_1215 p.Arg401_Tyr4 Sushi$CCP2$ delinsTCAT 05delinsHisVal GTAATA$ Ile$ 14$ USA$pedigree$ WES$ 7$ C1S1 c.880T>C$ p.Cys294Arg$ Sushi$CCP1$ (near$CUB2)$ 15$ $$ Targeted$seq.$$ 9$ C1S1 c.945A947del$ p.Val316del$ Sushi$CCP1$ (near$CUB2)$

1

The$ central$ role$ of$ complement$ protein$ C1$ in$ the$ innate$ immune$ response$ makes$ it$ compatible$with$causality$in$a$disease$in$which$inflammation$is$a$major$feature.$C1q,$ C1r$and$C1s$together$constitute$the$first$of$the$complement$complex$in$serum.$Upon$ binding$ of$ C1q$ to$ appropriate$ targets$ such$ as$ antigen–antibody$ complexes,156$ C1r$is$ autoAactivated$and$cleaves$C1s,$which$in$turn$cleaves$C4$and$C2$to$form$the$classical$ pathway$C3$convertase$(C4b2a).157A159$The$interaction$and$subsequent$activity$of$C1r$ and$ C1s$ depends$ upon$ the$ formation$ of$ a$ tetramer,$ which$ is$ mediated$ by$ the$ interaction$domains$of$these$two$proteins.$$

There$is$significant$evidence$that$altered$complement$function$plays$an$important$role$ in$ the$ pathogenesis$ of$ periodontitis.160$ Induction$ of$ experimental$ gingivitis$ in$ humans$ causes$progressive$complement$activation$(demonstrated$by$C3$conversion$in$gingival$ ! 116! ! crevicular$ fluid),$ which$ is$ correlated$ with$ increased$ inflammation$ clinically.161$ Conversely,$ traditional$ periodontal$ treatment$ can$ lead$ to$ decreased$ complement$ activity162$ and$ C3$ downregulation.163$ Local$ inhibition$ of$ C3$ reduced$ experimental$ periodontitis$ in$ nonAhuman$ primates,$ and$ this$ strategy$ has$ been$ suggested$ as$ a$ treatment$in$humans.164$$

$ Figure$ 5.1$ Domain$ structure$ of$ C1r$ and$ C1s.$ From$KapfererASeebacher$et$ al.,$2016165$

Complete$deficiency$of$C1r$or$C1s$caused$by$homozygous$C1R$or$C1S$null$mutations$ causes$a$lupusAerythematosusAlike$syndrome$with$increased$susceptibility$to$infections$ and$increased$risk$of$developing$autoimmune$diseases.$However,$that$condition$is$not$ associated$with$periodontal$destruction,$and$individuals$heterozygous$for$C1R$or$C1S$ null$mutations$are$reported$asymptomatic.157,166,167$$

Furthermore,$when$considering$the$mutations$that$we$observed$in$the$various$EDS$VIII$ patients,$none$of$them$were$loss$of$function$mutations$and$none$were$associated$with$ obvious$inAsilico$predictions$suggestive$of$deleterious$effect.$This$prompted$the$theory$ that,$in$fact,$the$heterozygous$C1R$or$C1S$mutations$observed$in$these$patients$might$ have$ gainAofAfunction$ effects$ on$ other$ targets,$ thereby$ leading$ to$ the$ changes$ observed.$

To$assess$their$functional$effect,$the$positions$of$the$identified$missense$variants$were$ mapped$ onto$ XAray$ crystallographic$ structures$ of$ C1r$ and$ C1s.$ Thus,$ it$ became$ ! 117! ! apparent$that$these$clustered$around$three$key$functionally$important$domains:$CUB2$ * and$CCP1$in$C1r,$and$CCP1$in$C1s$(Figure$5.1).$ $$

Most$ of$ the$ EDS$ VIII$ mutations$ identified$ seemed$ to$ alter$ residues$ that$ cluster$ at$ the$ hinges$ between$ the$ interaction$ and$ catalytic$ domains$ of$ C1r$ or$ of$ C1s$ (Figure$ 5.1$ above).$ These$ hinges$ are$ the$ sites$ of$ a$ conformational$ change$ that$ allows$ interaction$ with$ C1q.$ Other$ mutations$ identified$ by$ us$ and$ our$ collaborators$ seemed$ to$ affect$ cysteine$residues$(positions$309/358$and$338/371)$of$C1r,$required$for$forming$stabilizing$ intraAchain$disulfide$bonds$close$to$the$C1r/C1s$interface$which$are$essential$for$tetramer$ assembly$.The$C1R1mutation$p.Asp290Gly$(Family$2)$involves$a$C1q$binding$site$and$ may$interfere$with$the$assembly$of$the$C1$complex,$as$previously$shown$for$a$previously$ reported$mutation$p.Asp290Ala168.$The$C1R$p.Val50Asp$substitution$is$close$to$another$ C1q$binding$site$but$may$also$affect$the$calciumAdependent$interaction$of$C1r$with$C1s.$ Finally,$ the$ indel$ (p.Arg401_Tyr405delinsHisValIle)$ at$ residues$ 401A405$ of$ C1r$ may$ affect$the$disulfide$bond$at$aa$407A471.$

To$further$evaluate$the$functional$effects$of$C1R$mutations$observed$in$the$EDS$VIII$ cohort,$ three$ of$ these$ mutations$ p.Val50Asp$ (Family$ 1,$ Austrian$ pedigree),$ p.Cys309Trp$ (Families$ 7$ &$ 8),$ and$ p.Cys371Trp$ (Family$ 12),$ were$ overexpressed$ in$ HEK293$cells.165$Western$blots$of$cells$and$supernatants$indicated$that$the$abnormal$ C1r$proteins$are$retained$in$the$cells$but$can$undergo$autoactivation,$which$may$lead$to$ interaction$ with$ offAtarget$ substrates$ intracellularly.$ This$ was$ evidenced$ by$ the$ mutationAtransfected$ cells$showing$an$increased$proportion$of$dilated$RER$cisternae,$ similar$to$that$seen$in$dermal$fibroblasts$of$EDS$VIII$patients.$Compellingly,$it$has$been$ identified$ that$ the$ C1rAC1s$ tetramer$ normally$ binds$ to$ a$ collagenous$ domain$ of$ C1q$ which$contains$a$phylogenetically$conserved$hexapeptide$motif$HypAGlyALysAXaaAGlyA (Pro/Tyr/Asn),169,170$similar$to$HypAGlyALysAAsnAGlyA(Asp/Glu/Ala)$sequences$present$in$ the$triple$helical$domains$of$type$I$collagen$proα1(I)$and$proα2(I)$chains,$as$well$as$the$ proα1(III)$ chains$ of$ type$ III$ collagen.$ Thus,$ these$ may$ represent$ alternative$ C1r/C1s$ binding$ sites,$ with$ which$ retained,$ autoAactivated$ C1r/C1s$ proteins$ may$ interfere.$ Furthermore,$it$became$apparent$that$gain$of$function$mutations$in$the$CUB$domains$of$ c1r$and$c1s$may$also$lead$to$abnormal$interaction$with$collagen$or$procollagen$(CUB$ domains$are$also$present$in$a$number$of$proteins,$including$procollagen$CAproteinase$ enhancers$ (PCPE1)$ and$ boneAmorphogenetic$ protein$ (BMP1),171$ known$ to$ bind$ the$ tripleAhelix$of$collagen$via$their$CUB$domains).172A176$It$is$conceivable$therefore$that$a$ major$pathogenetic$contributor$to$periodontal$EDS$(VIII)$are$gain$of$function$mutations$ in$ C1r/C1s,$ leading$ to$ inappropriate$ intracellular$ retention$ of$ these$ proteins$ and/or$

$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ $$$$$$$$ *!All!functional!work!was!carried!out!by!our!collaborator,!Johannes!Zschocke!and!colleagues!in!Innsbruck!! ! 118! ! abnormal$ binding$ to$ connective$ tissue$ precursors$ such$ as$ collagen$ proteins.$ Further$ work$is$needed$to$further$elucidate$these$mechanisms$and$indeed,$identify$any$other$ underlying$genes$or$mechanisms$underlying$EDS$VIII.$

5.4.2$Other$EDS$spectrum$phenotypes$

In$terms$of$identifying$novel$genes$for$the$other$EDSAsubtypes$and$related$phenotypes$ characterised$ in$ this$ chapter,$ following$ detailed$ phenotypeAcharacterisation,$ the$ sequencing$ and$ downstream$ analysis$ is$ ongoing,$ though$ some$ preliminary$ observations$have$been$made.$The$cases$of$classical$EDS$phenocopies$represent$a$ specific$ and$ rare$ phenotype,$ and$ therefore$ one$ might$ expect$ at$ least$ one$ other$ Mendelian$ gene$ to$ contribute$ to$ the$ phenotype.$ Though$ initial$ analysis$ was$ able$ to$ identify$some$genes$that$were$statistically$correlated$with$the$phenotype,$no$candidate$ disease$ causing$ variants$ were$ identified$ in$ these$ genes.$ The$ group$ of$ patients$ with$ features$of$vascular$EDS$without$a$COL3A1$variant$also$represent$a$rare$and$specific$ phenotype$ which$ would$ show$ potential$ for$ novel$ disease$ gene$ discovery.$ $ The$ remaining$ phenotypes$ –$ kyphoscoliosis,$ aortopathy,$ marfanoid$ habitus,$ carotid$ and$ cerebral$arterial$accidents$A$may$be$less$tractable$as$these$represent$less$rare$and$less$ specific$ phenotypes,$ which$ may$ all$ be$ different$ generic$ manifestations$ of$ a$ common$ generalized$HDCT$spectrum.98,108$The$evidence$for$such$phenotypes$being$Mendelian$ traits$ is$ mixed$ or$ controversial:$ $ evidence$ in$ favour$ includes$ the$ fact$ that$ they$ often$ occur$in$the$context$of$rare$Mendelian$conditions,$e.g.$EDS.$However,$the$fact$that$they$ are$less$rare$and$overlap$with$multiple$diseases$(some$Mendelian$and$some$sporadic)$ and$indeed$the$fact$that$the$traits$in$the$individual$pedigrees$concerned$have$variable$ penetrance$contravenes.$$

5.4.3$Conclusion$

In$this$chapter$I$have$identified$EDS$subtypes$and$distinct$phenotypes$within$the$EDS$ spectrum,$which$remain$unaccounted$for$genetically$in$terms$of$the$usually$associated$ genes.$For$one$of$these,$periodontal$EDS$(type$VIII),$I$have$identified$a$novel$genetic$ cause$ and,$ with$ our$ collaborators,$ shown$ that$ the$ phenotype$ results$ from$ specific$ classes$ of$ heterozygous$ mutations$ in$ C1R$ and$ C1S$ (novel$ genes,$ previously$ not$ known$to$be$involved$in$EDS$aetiology$but$which$are$likely$to$mediate$their$connective$ tissue$phenotypes$by$interaction$with$collagen).$The$exact$mechanism$of$pathogenesis$ remains$ to$ be$ elucidated.$ To$ evaluate$ potential$ novel$ genetic$ causes$ of$ other$ phenotypes$associated$with$EDS,$ completion$of$exome$sequencing$and$downstream$ analysis$is$required.$ $

! 119! !

$ $

! 120! ! 6.$ WHOLE$ EXOME$ SEQUENCING$ &$ LINKAGE$ ANALYSIS$ FOR$ NOVEL$ GENE$ DISCOVERY$ IN$ EDS$ &$ TAAD$PEDIGREES$

6.1$Introduction$

WholeAexome$sequencing$has$been$particularly$successful$in$identifying$novel$genes$ underlying$ rare$ recessive$ phenotypes,$ but$ has$ also$ been$ useful$ in$ large$ autosomal$ dominant$ pedigrees$ in$ conjunction$ with$ linkage$ analysis.25,132,177,178$ Various$strategies$ for$ filtering$ and$ prioritising$ the$ thousands$ of$ variants$ detected$ by$ exome$ sequencing$ based$on$mode$of$inheritance$and$pedigree$structure$has$been$described$in$detail$over$ recent$years.25,179$

The$aim$of$this$part$of$the$project$was$to$combine$some$of$the$approaches$described$in$ the$literature$along$with$those$mentioned$in$the$previous$chapter$to$attempt$to$identify$ novel$ causative$ genes$ in$ EDS/TAAD$ pedigrees$ in$ which$ the$ pattern$ of$ inheritance$ suggested$Mendelian$inheritance.$$

In$this$chapter$I$describe$two$such$pedigrees:$

Family$ 1:$ TAAD$ family$ –$ large$ pedigree$ with$ autosomal$ dominant$ inheritance$ of$ aortopathy.$

Family$ 2:$ EDS$ VI$ family$ –$ very$ rare$ phenotype$ inherited$ in$ an$ autosomal$ recessive$ manner.$$

The$ molecular$ basis$ of$ TAAD$ has$ already$ been$ described$ in$ detail$ in$ Chapter$ 4.$ Familial$ TAAD$ is$ the$ subset$ of$ TAAD$ patients$ who$ have$ one$ or$ more$ firstAdegree$ relatives$ affected$ by$ TAAD.$ It$ is$ a$ rare$ condition$ (approximately$ 1A3:100,000).$ Approximately$ 75%$ of$ familial$ TAAD$ cases$ (from$ the$ literature180)$ and$ 77%$ (61%$ including$ VUS’s)$ of$ our$ own$ cohort$ of$ TAAD$ (Chapter$ 4.3)$ who$ have$ a$ known$ or$ probable$family$history$do$not$have$a$mutation$in$a$known$TAAD$gene.$$

The$ EDS$ kyphoscoliotic$ type$ or$ EDS$ Type$ VI$ is$ an$ autosomal$ recessive$ disorder$ characterized$mainly$by$a$congenital$or$earlyAonset$progressive$kyphoscoliosis,$severe$ neonatal$muscular$hypotonia$with$delayed$gross$motor$development,$generalised$joint$ hypermobility,$ and$ various$ dermatological,$ ocular$ and/or$ arterial$ manifestations$ (detailed$in$Table$6.1$ below).$ This$ is$ considered$ one$ of$ the$ rarest$ subtypes$ of$ EDS,$ with$ a$ few$ reports$ worldwide$ A$ significantly$ rarer$ than,$ for$ instance$ classical$ EDS$ (prevalence$1:10,000).$$

! 121! ! Two$ subtypes$ have$ been$ recognized:$ EDS$ VIA$ (caused$ by$ PLOD1$ mutations)$ and$ EDS$VIB,$whose$genetic$basis$is$largely$undiscovered.$The$first$subtype,$referred$to$as$ EDS$VIA,$is$caused$by$deficient$activity$in$the$enzyme,$Lysyl$hydroxylase$(encoded$by$ the$PLOD1,$a.k.a.$LH11gene)$whose$function$is$to$create$stable$intermolecular$crossA links$between$collagen$fibrils.$PLOD1(LHA1)$hydroxylates$lysyl$residues$in$the$(GlyAXA Lys)$triplets$of$collagen$type$I,$which$are$required$for$the$formation$of$these$crosslinks,$ the$ function$ of$ which$ are$ to$ provide$ tensile$ strength$ and$ mechanical$ stability$ to$ the$ collagen$fibrils$and$to$serve$as$attachment$sites$for$carbohydrate$units,$which$modulate$ the$lateral$packing$of$collagen$molecules$into$fibrils.$

EDS$ Type$ VIA$ can$ be$ diagnosed$ by$ sequencing$ the$ PLOD1$ gene$ to$ show$ a$ homozygous$or$compound$heterozygous$mutation,$by$demonstrating$decreased$LHA1$ activity$ in$ cultured$ dermal$ fibroblasts$ or$ by$ detecting$ an$ increased$ ratio$ of$ lysylpyridinoline$to$hydroxylysylpyridinoline$crosslinks$in$the$urine$with$the$latter$being$ the$ main$ investigation$ in$ clinic.$ A$ homozygous$ multiAexon$ duplication$ accounts$ for$$ ≈20%$of$mutations$reported$so$far,$but$missense,$nonsense$and$small$indel$mutations$ leading$to$lossAofAfunction$of$PLOD1$have$also$been$identified.181$$

Phenotypically$ overlapping$ with$ EDS$ VI,$ with$ predominant$ ocular$ signs$ is$ the$ brittle$ cornea$ syndrome$ (BCS$ –$ Table$ 6.1),$ also$ a$ rare$ recessive$ condition$ caused$ by$ mutations$in$ZNF469$and$PRDM5,$which$are$both$part$of$the$C2H2$zinc$finger$family$ and$thought$to$modulate$Wnt$signalling$pathways,$based$on$zebrafish$experiments.182$

Those$ patients$ who$ fit$ into$ the$ EDS$ VI$ phenotypic$ spectrum,$ who$ do$ not$ have$ mutations$in$PLOD1$have$been$termed$EDS$VIB.$In$this$group,$many$patients$remain$ undiagnosed,$ though$ in$ some$ cases,$ mutations$ in$ the$ brittleAcornea$ syndrome$ gene,$ ZNF469,$ have$ been$ identified.$ Other$ cases$ of$ EDS$ VIB$ have$ been$ associated$ with$ CHST14,$ which$ encodes$ dermatanA4AsulfotransferaseA1$ (D4STA1)$ a$ known$ cause$ of$ adducted$ thumb$ clubfoot$ syndrome(ACTS).$ EDS$ VIB$ patients,$ though$ largely$ overlapping$ with$ EDSVIA$ phenotypically,$ also$ show$ some$ features$ distinct$ from$ the$ latter$ including$ characteristic$ craniofacial$ abnormalities,$ joint$ contractures,$ wrinkled$ palms,$ tapered$ fingers$ and$ gastrointestinal$ /$ genitourinary$ manifestations.183$ As$ one$ would$ expect,$ there$ is$ phenotypic$ overlap$ with$ ACTS.$ The$ implication$ of$ CHST14$ provides$important$evidence$for$the$role$of$proteoglycans$in$EDS,$recently$supported$ by$structural$evidence$showing$a$key$role$for$proteoglycans$in$maintaining$the$correct$ spatial$ distance$ between$ collagen$ fibrils.184$ Overall,$ the$ majority$ of$ patients$ with$ features$of$EDSVI$remain$undiagnosed$genetically.$

$ $

! 122! ! Table$6.1$Phenotype$spectrum$of$Kyphoscoliosis$type$EDS$(VIA$&$VIB)$and$overlapping$syndromes$

Phenotype$ VIa$ VIb$ BCS$ ATCS$ PLOD1$ CHST14,$$ ZNF469,1 CHST141 PRDM5#

Early$onset$progressive$kyphoscoliosis,$ 1*$ 1*$ 1$ 1$ Severe$neonatal$muscular$hypotonia$with$delayed$gross$motor$ development,$ 1*$ 1*$ $ 1$

Generalized$joint$hyperlaxity,+/A$talipes$equinovarus$(clubfoot)$ 1$ 1$ 1$ 1*$ Osteopenia$ 1$ $ $ $

Fragile,$hyperextensible$and$bruisable$skin,$ 1$ 1$ $ 1$

Ocular$features$including:$Microcornea,$scleral$fragility/rupture$ (also,$blue$sclera,$myopia,$retinal$detachment,$glaucoma)$ 1$ 1$ 1*$ 1$

Rupture$of$mediumAsized$arteries.$ 1$ $ $ $

Craniofacial$abnormalities$ $ 1$ $ 1$

Joint$contractures$ $ 1$ $ 1$

Wrinkled$palms$ $ 1$ $ $

Tapered$fingers$ $ 1$ $ $

GI$&$GU$symptoms$ $ 1$ $ 1$

Via,$EDS$Kyphoscoliosis$Type$(VI)$caused$by$deficient$lysyl$hydroxylase$(LHA1,$encoded$by$the$PLOD1$gene)$VIb,$EDS$ Type$VI$with$normal$LHA1(PLOD1)$activity.$BCS,$Brittle$cornea$syndrome.$ACTS,$adducted$thumb$clubfoot$syndrome.$(in$ italics),$genes$encoding$each$of$the$syndromes$listed$above$them.$“1”$indicates$the$presence$of$the$phenotype$in$the$ syndrome_$“1*”$indicates$the$predominant$phenotype$for$each$syndrome.$

$

$ $ $ Figure$ 6.1$ Structural$ relationship$ between$ collagen$ fibrils$ and$ proteoglycans.$ Collagen$ fibrils$ in$ blue$ and$ proteoglycansA chondroiton$ sulphate$ &$ dermatan$ sulphate$ in$ violet.$ The$ panels$ show$ longitudinal[left]$ and$ transverse[right]$ 3D$ reconstructions$ of$ keratanaseAtreated$Bovine$corneal$specimens.$ Adapted$from$Lewis$et$al.$2010184$

$

! 123! ! 6.2$Methods$

6.2.1$Selection$of$pedigrees$ Available$individuals$from$each$family$were$recruited$and$phenotyped$as$described$in$ chapter$2.8$(see$also$Appendix$2).$$$

Family$ 1$ is$ a$ fourAgeneration$ Caucasian$ pedigree$ from$ Southampton,$ England,$ affected$by$familial$TAAD$(Figure$6.2).$Phenotype$status$was$assigned$as$follows:$$“1”,$ individuals$known$to$be$affected$by$TAAD_$“0”,$individuals$unaffected,$on$the$basis$of$ not$being$directly$related$to$the$index$case$and$assuming$TAAD$is$a$rare$disease_$“U”,$ individuals$ whose$ phenotype$ status$ was$ unknown,$ assuming$ TAAD$ is$ a$ condition$ of$ variable$onset$(usually$in$the$7th$decade$onwards)$and$variable$penetrance$A$this$was$ the$ case$ for$ the$ majority$ of$ relatives$ of$ the$ index$ case$ who$ were$ not$ known$ to$ be$ affected$by$TAAD$ (Table$6.2).$ Family$2$is$ a$nuclear$family$ with$partial$consanguinity$ (the$ parents$ are$ second$ cousins$ from$ the$ same$ village$ in$ Pakistan).$ Similarly,$ phenotype$status$was$assigned$as$1$(affected)$or$0$(unaffected).$Figure$6.3$shows$the$ pedigree$structure$for$Family$2$and$their$phenotypic$details$are$elaborated$in$Table$6.3.$

$

$

Figure$6.2.$Family$1:$TAAD.$Squares$indicate$males,$circles$females.$Phenotype$status:$Black$=$affected,$white/unshaded$=$ unaffected,$grey$=$unknown.$$Arrow$indicates$the$index$case$(3.10,$JS0107).$

$ $

! 124! ! Table$6.2$Clinical$characteristics$of$individuals$recruited$from$Family$1(fTAAD)$

Patient$ ID$ from$ Relationship$ Phenotype$ pedigree$6.2$ to$index$ Age$ Gender$ status$$ Phenotype$detail*$

2.3$ 22VB0127$ aunt$ 73$ F$ U$

2.5$ 22MS0112$ mother$ 72$ F$ 0$ $

2.6$ 22DS0111$ father$ 77$ M$ U$ $

3.1$ 22SD0129$ first$cousin$ 41$ M$ 1$ $

spouse$ of$ $ 3.2$ 22CMK0119$ cousin$ 55$ M$ 0$

3.3$ 22KK0118$ first$cousin$ 48$ F$ U$ $

3.5$ 22LB0122$ first$cousin$ 51$ F$ U$ $

3.7$ 22CP0113$ sister$ 49$ F$ U$ $

3.9$ 22ST0125$ sister$ 45$ F$ U$ $

3.10$ 22JS0107$ index$ 50$ M$ 1$ $Type$B$dissection$

daughter$ of$ 4.1$ 22SK0120$ cousin$ 24$ F$ U$

4.2$ 22CK0121$ son$of$cousin$ 22$ M$ 1$ $

daughter$ of$ $ 4.3$ 22LB0123$ cousin$ 24$ F$ U$

daughter$ of$ $ 4.4$ 22EB0124$ cousin$ 25$ F$ U$

daughter$ of$ $ 4.5$ 22JB0128$ cousin$ 12$ F$ U$

4.6$ 22MP0115$ nephew$ 21$ M$ U$ $

4.7$ 22SP0117$ nephew$ 16$ M$ (1)$ $

4.8$ 22AP0116$ nephew$ 23$ M$ U$ $

4.9$ 22AP0114$ nephew$ 19$ M$ U$ $

4.10$ 22CT0126$ nephew$ 22$ M$ U$ $

4.11$ 22PS0108$ son$ 17$ M$ 1$ $

4.12$ 22SS0109$ daughter$ 15$ F$ U$ $

4.13$ 22CS0110$ son$ 19$ M$ U$ $$

1,$affected_$0,$unaffected_$(1),$possibly$affected$but$phenotype$information$not$available.$*Phenotypes:$Type$B$ dissection$ refers$ to$ aortic$ dissection$ starting$ in$ the$ descending$ thoracic$ aorta$ distal$ to$ the$ origin$ of$ the$ left$ subclavian$ artery$ as$ per$ the$ Stanford$ classification_$ phenotype$ details$ were$ not$ accessible$ at$ the$ time$ of$ writing$but$are$available$on$request$from$Prof.T.Aitman,$Institute$of$Genomic$&$Metabolic$Medicine,$University$ of$Edinburgh,$UK.$ $

! 125! ! $

$

Figure$ 6.3.$ Family$ 2:$ EDS$ VIb.$ Squares$ indicate$ males,$ circles$ females.$ Phenotype$ status:$ Black=affected,$ white/unshaded=unaffected.$ Arrow$ indicates$the$index$case$(2A2,$620).$

$ Table$6.3$Clinical$characteristics$of$Family$2$(EDSVIb)$

$ Age$ Gender$ Phenotype$ Phenotype$details$ status$

1A1$#617$ 54$ F$ 0$ Asymptomatic$

1A2$#616$ 60$ M$ 0$ Mild$ hypermobility,$ otherwise$ asymptomatic$

2A1$#618$ 29$ M$ 1$ Severe$kyphoscoliosis$

2A2$#620$ 26$ F$ 1*$ Severe$congenital$kyphoscoliosis$(multiple$ operations),$congenital$hypotonia,$delayed$ walking,$hypermobility,$marfanoid$habitus$$$

2A3$#619$ 27$ F$ 0$ Asymptomatic$

1*,$index$case$

$ $

! 126! ! 6.2.2$Exome$sequencing,$variant$calling$and$downstream$analysis$

DNA$extraction,$shearing,$purification$and$creation$of$pairedAend$libraries$for$all$exon$ target$ enrichment$ by$ Sureselect$ (as$ detailed$ in$ Chapter$ 2.3A2.5)$ was$ carried$ out$ for$ each$ “affected”$ case$ described$ above.$ Enriched$ exomeAlibraries$ from$ each$ sample$ were$sequenced$in$a$total$cohort$of$113$exome$sequences,$run$on$the$Illumina$MiSeq.*$ SNP$ genotyping$ and$ linkage$ analysis$ was$ carried$ out$ for$ Family$ 1$ (TAAD)$ to$ help$ define$a$linkage$interval$for$variant$prioritization$(Chapter$2.7).†$

In$all$cases,$after$variant$calling,$synonymous$and$intronic$variants$were$excluded$from$ further$ downstream$ analysis.$ Remaining$ variants$ were$ divided$ into$ two$ groups:$ nonA synonymous$ single$ nucleotide$ variants$ (NSNV’s)$ and$ loss$ of$ function$ (LoF)$ variants,$ which$ included$ premature$ stop$ codons,$ mutations$ in$ canonical$ spliceAsites$ and$ frameshift$ variants.$ Variants$ were$ filtered$ based$ on$ maximum$ reported$ population$ frequency,$ in$ two$ tiers:$“somewhat$rare”$(MAF<1%)$and$“rare”$(MAF<0.1%)$ to$ allow$ variant$ prioritization.$ LoF’s,$ NSNV’s,$ candidate$ homozygous$ and$ compound$ heterozygous$ variants$ relevant$ to$ affected$ individuals$ in$ each$ family$ were$ identified$ using$custom$Perl$scripts.185$‡$$

6.3$Results$

6.3.1$Family$1:$TAAD$(dominant$inheritance)$

Linkage#analysis#

SNP$ genotyping$ of$ 1,516$ genomeAwide$ markers$ in$ 23$ individuals$ from$ family$ SH$ (4$ affected,$2$unaffected,$17$unknown$–$Table$6.4)$was$achieved.$NonAparametric$linkage$ analysis$revealed$5$regions$with$Lod$scores$>1.0$(Table$6.4).$The$two$intervals$with$the$ highest$LOD$ scores$were$found$on$chromosome$10$(a$5Mbp$region$with$LOD$ score$ 1.4)$and$chromosome$15$(a$1.7Mbp$region$with$LOD$ score$ 1.2).$These$regions$and$ genes$ within$ them,$ of$ which$ one$ was$ a$ known$ candidate$ gene,$ FBN1$ are$ shown$ in$ Figure$6.4.$

$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ $$$$$$$$ *!Exome!sequencing!was!supervised!by!Jana!Vandrovcova!and!Jennifer!Biggs!assisted! †!SNP!genotyping!&!analysis!was!carried!out!by!Jana!Vandrovcova! ‡!Most! of! the! bioinformatics! analysis! up! to! and! including! variant! calling! was! carried! out! by! Dalia! Kasperavaciute! of! the! Imperial! College! Clinical! Genomics! Facility;! Jana! Vandrovcova! assisted! with! downstream!bioinformatic!analysis!including!variant!prioritisation.! ! 127! ! Table$6.4$Linked$regions$from$linkage$analysis$in$Family$1.$ Chr$ Position$/cM$ Genomic$ co< LOD$score$ p

10$ 92.198$ 10:$ 73335042$ A$ 1.42A1.44$ 0.005247$ rs1867998,$ rs2688610,$ rs12254441,$ 78299651$ rs1907308$ 15$ 38.836$A50.14$ 15:$ 39229103$ A$ 1.09A1.20$ 0.009A0.012$ rs1433887,$ rs2004175,$ rs2925345,$ 53006147$ rs1995939,$ rs16952667,$ rs1426932,$ rs281265,$ rs514327,$ rs1048975,$ rs479062,$rs2017176$ 15$ 46.262A48.815$ 15:$ 47675602$ A$ 1.18A1.20$ 0.0094A0.0099$ rs281265,$rs514327,$rs1048975$ 49417546$ 20$ 44.35$ 20:$17311577$ $ 1.13$ 0.01138$ rs926492,$rs728481$ cM,$ centimorgans_$ LOD,$ log$ odds$ ratio$ that$ the$ locus$ is$ linked$ to$ the$ phenotype_$ SNP$ ID,$ known$ single$ nucleotide$ polymorphisms$in$each$region,$from$DBSNP$database$

$

$

Figure$ 6.4.$ Linked$ regions$ for$ family$ SH$ from$ the$ UCSC$ genome$ browser$ on$ chromosome$ 10$ (5Mbp$region$with$LOD$score$1.4,$top$panel)$and$chromosome$15$(1.7Mbp$region$with$LOD$score$ 1.2,$ bottom$ panel).$ Only$ Refseq$ genes$ are$ shown$ for$ clarity.

! 128! ! Exome#sequencing#

Exome$ sequencing$ was$ carried$ out$ in$ 3$ affected$ individuals,$ including$ the$ proband$ (Table$6.5).$$

Table$6.5$Sequencing$data$for$Family$1$(TAAD)$

Subject$$ 3<10$ 3<1$ 4<2$ 22JS0107$ 22SD0129$ 22CK0121$ [index]$ [first$ [son$ of$ cousin]$ other$ first$ cousin]$ Total$reads$ 115,008,862$ 122,567,366$ 108,690,784$ $ $ $ $ %$Mapped$reads$ 99.9$ 99.6$ 99.8$ $ Mean$coverage$ 55.9$ 59.5$ 51.2$

%$bp$>30X$ 69$ 69$ 65$

Total$variants$called$ 25,315$ 25,650$ 25,487$

Rare$variants$(MAF$<$0.001)$$ 903$ 911$ 906$ $ Rare,$functionally$significant$ 188$ 174$ 182$ $ $$$$$Rare$LoF$ 32$ 32$ 25$ $ $$$$$Rare$NSNV$ 156$ 142$ 157$ MAF,$ minor$ allele$ frequency_$ LoF,$ loss$ of$ function$ variants_$ NSNV,$ nonA synonymous$single$nucleotide$variant$

Prioritisation#of#candidate#disease=causing#variants##

Of$ all$ variants$ called,$ I$ first$ considered$ functionally$ significant$ (amino$ acid$ altering)$ exonic$ and$ canonical$ spliceAsite$ variants.$ These$ were$ divided$ into$ nonAsynonymous$ variants$ (NSNV’s)$ and$ loss$ of$ function$ (LoF)$ variants$ and$ are$ further$ elaborated$ in$ Table$6.6.$

Table$ 6.6$ Summary$ of$ rare$ functionally$ significant$ exonic$ and$ spliceAsite$ variants$from$the$exome$sequences$of$3$affected$individuals$from$Family$1.$ NSNV$ LoF$ Total$ $ 288$ 31$ Heterozygous,$exonic$variants$ 207$ 21$ Shared$by$2$affected$ 31$ 3$ Shared$by$all$3$affected$ 5$ 0$ a b In$linked$regions$ 1$ $ 1$ $ In$candidate$gene$list$ 0$ 0$

NSNV,$nonAsynonymous$single$nucleotide$variants_$LoF,$loss$of$function$variants$(nonsense$ mutations$ and$ canonical$ splice$ site$ variants).$ a.$ This$ variant,$ in$ CDH23,$ is$ present$ in$ all$ three$affected$individuals$(see$Table$6.7).$b.$This$is$a$novel$nonsense$mutation$present$only$ in$ the$ index$ case,$ 22JS0107$ in$ FUT11 (transcript NM_173540:exon1:c.T639G:p.Y213X).$

! 129! ! First,$ I$ considered$ genes$ in$ which$ rare$ variants$ were$ identified$ in$ all$ three$ affected$ cases$and$not$present$in$110$other$unrelated$exomes$sequenced$inAhouse.$There$were$ no$ LoF$ variants$ meeting$ this$ criterion$ but$ four$ NSNV’s,$ one$ of$ which$ was$ within$ the$ previously$ identified$ linked$ regions$ and$ are$ detailed$ in$ Table$ 6.7$ below.$ All$ four$ are$ previously$ unreported$ variants$ of$ uncertain$ significance,$ with$ no$ clear$ indications$ of$ pathogenicity$from$in$silico$predictions,$although$for$the$p.M828V$variant$in$MEGF11,$ two$ of$ four$ in$ silico$ predictions$ were$ deleterious.$ One$ of$ these$ four$ variants$ –$ p.R1334Q$in$CDH231>1$was$in$the$previously$identified$linked$region$on$chromosome$ 10.$

Table$6.7$Details$of$nonAsynonymous$variants$present$in$all$three$affected$individuals$from$Family$1$ b #b Genomic$co< In$ Gene$ Protein$ Variant$ Predictive$scores$<$ $ Conservation$scores$< $ a ordinates$ $ linked$ SIFT,Polyphen.LRT,MT$ GERP++$,$PhyloP,$SiPhy$ region$ 8:10465940$ N$ RP1L11 retinitis$pigmentosa$ ex.4:$ 0.89(T),$0.33(NA),$ A0.30,$0.33,$A$$ 1Alike$1$ c.G5668A:$ 0.62(NA),$0.001(N)$ p.V1890I$ 8:17810675$ N$ PCM11 pericentriolar$ ex.9:$ A$ A$ material$1$ c.A1268G:$ p.Q423R$ 15:66206303$ N$ MEGF111 multiple$EGFAlikeA ex.20:$ 1.0(D),$0.246(P),$ 4.3,$0.25,$A$ domains$protein$11$ c.A2482G:$ 0.99995(U),$0.77(D)$ p.M828V$ c 10:73492029$ $ Y$ CDH231 cadherinArelated$23$ ex.31:$ A$ A$ c.G4001A:$ p.R1334Q$ Y,$yes_$N,$no_$Ex.,$exon$no.$ a.$Genomic$coAordinates$are$based$on$GRCh37/hg19$human$reference$sequence.$b.$In$silico$prediction$ scores$are$shown$as$a$predictive$functional$score$(with$qualitative$prediction$in$brackets)$based$on$ANNOVAR68$version$2013aug23$and$ refer$ to$ the$ following$ specific$ ANNOVAR$ annotations:$ Polyphen:$ "LJB23_Polyphen2_HVAR_score",$ MT:$ "LJB23_MutationTaster_score_converted",$ LRT:$ $ "LJB23_LRT_score_converted",$ Predictive$ functional$ scores$ for$ SIFT,$ Polyphen,$ MutationTaster,$ $ LRT$ were$ converted$ to$ a$ 0A1$ scale$ with$ a$ higher$ number$ denoting$ a$ higher$ probability$ of$ functional$ significance.$$ Qualitative$ predictions:$ D,$ deleterious.$ T,$ tolerated_$ $ for$ Mutation$ Taster$ only:$ A,$ disease$ causing$ automatic,$ D,$ disease$ causing_$ N,$ polymorphism_$ $ P,$ polymorphism$ automatic_$ Conservation$ scores$ (GERP++,$ SiPhy,$ PhyloP):$ higher$ number$ $ =$ higher$ conservation$ across$species.$c.$this$variant$was$within$the$linked$region$with$highest$LOD$score$and$shared$by$all$three$affected$individuals.$$

Of$all$the$heterozygous$variants$identified$above$none$were$present$in$genes$in$a$list$of$ 123$high$priority$candidate$genes$I$previously$identified$as$associated$with$connective$ tissue$disease$and$aortopathy$(Appendix$6).$$

As$one$of$the$candidate$genes,$FBN1,$was$in$the$linked$regions$above$and,$owing$to$ its$ significant$contribution$to$TAAD$pathogenesis$overall$(Chapter$4),$we$reAanalysed$ variants$ in$ FBN11 to$ include$ intronic$ variants.$ One$ deep$ intronic$ variant$ in$ intron$ 54,$ 147bp$upstream$of$exon$55$(chr15:48725332,$C>T)$was$observed$in$all$three$affected$ individuals$and$validated$by$Sanger$sequencing.$$This$variant$was$not$predicted$to$alter$ splicing$from$a$number$of$splice$prediction$programmes.$It’s$presence$in$the$unaffected$

! 130! ! members$ of$ the$ same$ family$ remains$ to$ be$ determined,$ as$ does$ the$ effect$ on$ gene$ expression$(if$any).*$

6.3.2$Family$2:$EDS$VI$(recessive$/$compound$heterozygous$Inheritance)$

Exome#sequencing#

Exome$sequencing$was$carried$out$for$all$five$members$of$family$2$(Table$6.8).$$

Table$6.8$Sequencing$data$for$Family$2$(EDSVIb)$

Subject$$ 1<1$ 1<2$ 2<1$ 2<2$ 2<3$ a #617$ #616$ #618$ $ #620$ #619$ Total$reads$ 126,522,072$ 137,063,456$ 19,488,462$ 133,234,748$ 141,433,158$ $ $ $ $ $ $ %$Mapped$reads$ 99.1$ 99.5$ 99.8$ 98.6$ 98.7$ $ Mean$coverage$ 57.7$ 64.0$ 8.1$ 77.1$ 64.8$ $ %bp$>$30X$ 63.1$ 71.6$ 1.8$ 71.0$ 72.1$ $ Total$variants$called$ 137,452$ 137,448$ 137,448$ 138,004$ 137,328$ $ $ $ $ $ Total$rare$variants$ 39,767$ 39,001$ 29,496$ 39,545$ 39,114$ $ $ $ $ $ Rare,$coding$variants$ 662$ 610$ 618$ 658$ 620$ $ $ $ $ $ $ _$ $$Arare$LoF$ 39$ 37$ 45$ 38$ 40$ _$ $$Arare$NSNV$ 134$ 99$ 175$ 121$ 116$ Lof,$ loss$ of$ function$ variants$ (nonsense$ mutations$ and$ canonical$ splice$ site$ variants)_$ NSNV,$ nonA synonymous$ single$ nucleotide$ variants_$ Rare$ variants$ were$ defined$ as$ those$ with$ MAF<0.001$ a.$Sample$#618$was$poorly$covered:$median$coverage$7$reads,$IQR$4A12,$%bp$covered$>4X:69.5,$>10X:$ 32%,$>20X:7.1$

Prioritisation#of#candidate#disease=causing#variants##

Firstly,$ assuming$ an$ autosomal$ recessive$ mode$ of$ inheritance,$ I$ looked$ for$ homozygous$ variants$ (Methods,$ Ch.6.2.2)$ observed$ in$ at$ least$ one$ of$ the$ affected$ siblings$ that$ was$ heterozygous$ in$ both$ parents$ and$ either$absent$or$heterozygous$in$ the$unaffected$sibling.†$Of$three$rare$homozygous$variants$in$the$affected$index$case,$ #620,$one$NSNV$in$the$SDSL1gene$met$these$criteria,$although$it$was$not$detected$in$ the$other$affected$sibling$#618$(Table$6.9).$None$of$the$homozygous$variants$for$the$ second$affected$sibling,$#618,$met$these$criteria.$

I$then$searched$for$compound$heterozygote$variants$for$each$of$the$affected$individuals$ (that$is,$more$than$one$heterozygote$variant$observed$in$the$same$gene$per$individual)$ where$each$was$inherited$from$an$alternative$parent$(see$Ch.6.2.2$for$methodology).$

$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ $$$$$$$$ *!Jana!Vandrovcova!helped!with!downstream!analysis,!including!variant!prioritisation!and!identification!of!the!deep! intronic!variant!in!FBN1;&David!Ross!validated!by!Sanger!sequencing!! † !Jana!Vandrovcova!helped!with!iidentifying!candidate!homozygous!and!compound!heterozygous!variants!! ! 131! ! For$patient$#620,$there$were$30$compound$heterozygotes$in$13$genes_$of$these,$two$ pairs$of$variants$in$c10orf901and1LPXN$respectively,$were$compatible$with$the$pedigree$ structure$ observed$ (see$ Table$ 6.3.$ Figure$ 6.3).$ For$ patient$ #618,$ there$ were$ 45$ compound$heterozygote$calls$in$21$genes_$of$these,$three$pairs$were$consistent$with$ inheritance$ of$ one$ variant$ from$ each$ parent,$ but$ only$ one$ pair$ of$ variants$ (also$ identified$ in$ the$ other$ affected$ sibling,$ #620),$ in$ c10orf90$ was$ completely$ compatible$ with$ the$ pedigree$ structure$ shown$ prevously(Table$ 6.3$ &$ Figure$ 6.3).$ Table$ 6.9$ elaborates$ these$ candidate$ homozygous$ and$ compound$ heterozygous$ variants$ for$ Family$2.$

Table$6.9$Candidate$homozygous$and$compound$heterozygote$variants$for$Family$2.$

Genomic$ co< Categ Max.$ a Candidate$ Variant$details$ Allele$balance $ Gene$ ordinates$ ory$ observe d$MAF$ AV0620$ TV0618$$ SV0619$$ SV0616$ HV0617$ $$ <$ index$$ <$ sibling$ <$ sibling$ <$ parent$ <$ parent$ $$ $$ $$ $$ (1)$ (1)$ (0)$ (0)$ (0)$ SDSL1 12:113873316$ NSNV$ exon7:c.C626T: 0.0013$ 1/1:$$ 0/0$ 1/0:$ 1/0:$ 1/0:$ p.A209V$ 39$ 5$ 21/36$ 26/20$ 14/14$ $ C10orf901 10:128192895$ Indel$ exon3:c.873_87 0.0012$ 1/0:$ 1/0:$ 0/0:$$ 0/0:$$ 1/0:$ 4insTCG:$ 49/73$ 9/7$ 117$ 157$ 40/57$ p.L292delinsSL$ C10orf901 10:128193125$ NSNV$ exon3:c.G644A: 0.0044$ 1/0:$ 1/0:$ 0/0:$$ 1/0:$ 0/0:$ p.G215E$ 74/59$ 7/8$ 112$ 51/62$ 109$ $ LPXN1 11:58331673$ NSNV$ exon3:c.C188T: NA$ 1/0:$ 0/0:$$ 0/0:$$ 0/0:$$ 1/0:$ p.A63V$ 23/28$ 6$ 49$ 44$ 28/33$ $ LPXN1 11:58338162$ NSNV$ exon2:c.G53T:$ NA$ 1/0:$ 1/0:$ 1/0:$ 1/0:$ 0/0:$$ p.R18L$ 109/84$ 5/5$ 58/65$ 72/137$ 117$ NSNV,$ non$ synonymous$ variant_$ Indel,$ insertion/deletion$ variant_$ MAF,$ minor$ allele$ frequency$ (1),$ affected_$ (0),$ unaffected.$ a.$ Alleles:$ 1/0,$ heterozygous$ for$ variant_$ 1/1,$ homozygous$ for$ variant_$ 0/0,$ homozygous$ for$ reference$ allele$ (numbers$ show$ the$ number$of$NGS$reads$for$each$allele,$e.g.$“1/0:$21/57”$shows$a$heterozygote$with$21$reads$for$the$variant$allele$and$36$reads$for$ the$reference.$Transcript$ID’s:$C10orf90:1NM_001004298$LPXN:1NM_001143995.$

6.4$Discussion$

This$chapter$focussed$on$two$pedigrees$–$one$large$multigenerational$pedigree$with$a$ dominantlyAinherited$trait$of$aortopathy$(Family$1,$TAAD)$and$one$nuclear$family$with$ autosomal$recessive$inheritance$of$a$rare$phenotype$suggestive$of$kyphoscolioticAtype$ EDS$(Family$2,$EDS$VIb).$

The$ index$ case$ of$ Family$ 1$ had$ already$ undergone$ sequencing$ to$ rule$ out$ exonic$ variants$ in$ the$ known$ aortopathy$ genes$ (listed$ in$ Chapter$ 4.1).$ Candidate$ genes$ include$those$already$linked$to$aortopathy$and$other$enzymes$of$the$TGFBR$pathway.$ The$majority$of$causative$variants$discovered$in$TAAD$are$exonic$variants$in$the$FBN1$ gene,$ affecting$ key$ functional$ domains$ such$ as$ the$ EGFAlike$ domains.$ $ On$ initial$ search,$which$was$focused$mainly$on$exonic$variants,$we$were$unable$to$find$a$likely$ causative$variant$in$any$of$the$known$candidate$genes.$A$NSNV,$with$some$predictions$ ! 132! ! of$deleterious$effect$by$in$in$silico$tools$and$present$in$all$three$affected$individuals$in$ one$gene,$MEGF,$was$observed.$MEGF11$encodes$a$large$protein$(largest$transcript,$ 1,044$ aa$ in$ Ensemble186),$ with$ a$ higher$ than$ expected$ level$ of$ natural$ mutation$ as$ indicated$ by$ a$ ZA(constraint)score$ of$ (A0.73)$ for$ missense$ variation$ according$ to$ the$ Exac$ database,154$ suggesting$ that$ this$ is$ not$ likely$ to$ be$ a$ pathological$ variant.$ Segregation$analysis$of$this$variant$in$the$pedigree$may$however$be$useful$to$further$ clarify$its$significance.$

Linkage$ analysis$ was$ not$ strong,$ owing$ to$ the$ fact$ that$ the$ affected$ status$ of$ the$ individuals$ without$ known$ aortopathy$ is$ unknown$ (aortic$ disease$ remains$ clinically$ silent$until$later$in$life)$and$because$the$pedigree$is$relatively$small.$However,$one$of$ the$ linked$ regions$ contained$ the$ FBN11 gene.1 Although$ there$ were$ no$ functionally$ significant$exonic$variants$in$this$gene$in$Family$2,$by$relaxing$our$filtering$criteria,$we$ were$able$to$identify$an$intronic$variant$in$(intron$54)$of$FBN1,$which$was$present$and$ subsequently$ validated$ in$ all$ three$ affected$ members$ of$ the$ family$ by$ Sanger$ sequencing.$ This$ VUS$ may$ be$ important$ in$ terms$ of$ causality$ either$ by$ distantly$ influencing$splicing$/$gene$expression$or$may$be$in$linkage$disequilibrium$with$another$ variant$ that$ is$ causal.$ Further$ work$ in$ terms$ of$ segregation$ analysis$ and$ reverse$ transcription$PCR$are$needed$to$further$clarify$its$significance.$It$is$noteworthy$that$a$ previously$ reported$ deep$ intronic$ variant$ in$ a$ neighbouring$ FBN1$ region$ (intron$ 56)$ creating$a$new$pseudoAexon$in$the$TGFAlike$domain,$has$been$described$as$causative$ in$a$single$case$of$Marfan$syndrome.187$It$is$possible$that$many$genetically$unresolved$ Mendelian$ cases$ of$ HDCT$ may$ indeed$ be$ caused$ by$ intronic$ variants$ in$ known$ candidate$ genes.$ $ Finally,$ CNV’s$ (such$ as$ multiexonic$ deletions$ or$ whole$ gene$ deletions)$ would$ not$ be$ identified$ by$ exome$ sequencing$ and$ would$ also$ need$ to$ be$ tested.$$

In$ EDS$ VI$ (Kyphoscoliotic$ type$ EDS),$ there$ exists$ a$ significant$ number$ of$ patients$ whose$phenotype$overlaps$with$the$EDSVI$spectrum$but$do$not$have$mutations$in$the$ known$genes$PLOD1(EDS$VIA)$or$CHST14(for$EDSVIB).$Indeed,$from$the$experience$ at$the$National$EDS$referral$centre,$London,$UK,$a$large$part$of$the$EDSVI$cases$with$ normal$ PLOD1$ activity$ remain$ genetically$ uncharacterised$ (F.$ M.$ Pope$ &$ A.$ Vandersteen,$unpublished$data).$This$is$the$group$into$which$our$“Family$2”$above$fits.$ The$ findings$ to$ date$ from$ overlapping$ syndromes$ and$ the$ modulatory$ functions$ from$ proteoglycans$provide$a$useful$list$of$candidate$genes.$In$ family$ 2,$ therefore$ we$ first$ looked$ for$ potential$ diseaseAcausing$ variants$ in$ candidate$ genes$ in$ the$ above$ categories$ as$ well$ as$ those$ known$ to$ play$ a$ role$ in$ collagen$ processing$ and$ matrix$ metabolism$(Appendix$6).$Of$the$three$genes$identified$in$this$family$whose$rare$exonic$ variants$fitted$into$the$homozygous/compound$heterozygous$model$–$LPXN,$c10orf90$

! 133! ! and$SDSL,$none$are$present$on$our$list$of$candidate$genes,$nor$do$they$appear$to$have$ high$expression$in$skin$or$connective$tissue.188$$$

LPXN$ has$ recently$ been$ shown$ to$ influence$ cytoskeletal$ dynamics$ by$ causing$ ERKA dependent$phosphorylation$of$the$actin$binding$protein,$Caldesmon$(CaD),$which$leads$ to$ dynamic$ actin$ structures$ shown$ to$ induce$ cell$ migration$ in$ an$ inAvitro$ model$ for$ prostate$ cancer.189$ The$ role$ in$ cytoskeletal$ dynamics$ and$ its$ interaction$ with$ caldesmon/actin$ complex$ does$ make$ it$ a$ viable$ candidate$ gene$ in$ EDS$ pathogenesis.189 Not$ a$ great$ deal$ is$ known$ about$ SDSL$ (serine$ dehydratase$ like),$ except$ that$ it$ seems$ to$ be$ an$ isoform$ of$ the$ hepatic$ enzyme,$ serine$ dehydratase$ (SDH),$ which$ is$ one$ of$ three$ hepatic$ enzymes$ known$ to$ metabolise$ Serine$ and$ Glycine.190$Whether$it$has$a$regulatory$role$outside$the$liver$particularly$with$regard$to$ Glycine$ residues$ in$ collagen$ (which$ are$ structurally$ critical$ to$ the$ collagen$ helix)$ or$ indeed$ to$ Serine$ residues$ of$ TGFABeta$ ligands$ is$ unknown$ but$ potentially$ of$ value.$ C10orf90,$ more$ recently$ known$ as$ FATS$ (fragileAsite$ associated$ tumor$ suppressor$ gene)$is$thought$to$modulate$p53$activity$and$has$been$implicated$in$breast$cancer.191$ Its$relation$to$HDCT$pathogenesis$is$unknown.$

The$ major$ limitation$ in$ the$ study$ of$ Family$ 2$ was$ the$ poor$ coverage$ of$ the$ second$ affected$ individual,$ TV618.$ Further$ work$ would$ entail$ reAsequencing$ this$ individual$ followed$by$reAanalysis$to$identify$the$homozygous$or$compound$heterozygous$variants$ shared$by$both$affected$offspring,$using$homozygosity$mapping$to$focus$on$regions$of$ autozygosity_$VAAST$analysis$to$further$refined$the$list$of$candidate$genes$may$also$be$ helpful.$Again,$large$structural$variants$(CNV’s)$cannot$be$reliably$identified$by$exomeA sequencing$and$would$be$a$focus$for$analysis.$It$can$be$argued$that$all$of$the$above$ would$reliably$be$served$by$the$routine$use$of$whole$genome$sequencing$as$a$‘frontA line’$test.192,193$$

$ $

! 134! ! 7.$CONCLUDING$REMARKS$

The$work$outlined$in$this$thesis$has$highlighted$a$number$of$important$points$regarding$ the$ genetic$ basis$ of$ the$ two$ conditions$ studied$ –$ EDS$ and$ TAAD$ –$ and$ HDCT$ in$ general.$$

EDS$ has$ traditionally$ been$ a$ condition$ defined,$ categorised$ and$ treated$ mainly$ on$ phenotypic$grounds.$However,$from$the$work$carried$out$here,$we$have$highlighted$the$ significant$ phenotypic$ and$ genetic$ heterogeneity$ that$ exists.$ The$ conclusion$ that$ can$ be$ drawn$ is$ that$ the$ very$ distinct$ phenotypes$ of$ EDS,$ which$ meet$ the$ expected$ genotypeAphenotype$relationship$and$can$be$defined$on$clinical$grounds,$are$relatively$ few_$ the$ majority$ of$ cases$ cross$ traditional$ phenotype$ boundaries$ and$ these$ cases$ either$ remain$ unaccounted$ for$ genetically$ or$ show$ mutations$ in$ genes$ outside$ the$ expected$ genotypeAphenotype$ relationship.$ There$ is$ clinical$ significance$ in$ this$ observation,$ as$ it$ potentially$ alters$ the$ diagnostic$ approach$ to$ the$ patient$ presenting$ with$ EDS$ or$ a$ related$ hereditary$ connective$ tissue$ disorder$ for$ whom$ alternative$ pathways$and$approaches$to$clinical$management$may$arise.$

In$a$mixed$cohort$of$over$a$thousand$TAAD$patients$(the$majority$sporadic$and$without$ any$known$syndromic$features$or$family$history),$we$identified$a$significant$proportion$ of$over$20%$with$rare$variants$in$known$aortopathyApredisposing$genes$(a$quarter$of$ these$ are$ pathogenic$ or$ likely$ pathogenic,$ by$ conservative$ criteria).$ We$ showed$ that$ those$who$do$harbor$a$known$pathogenic/likely$pathogenic$variant$were$predisposed$to$ a$higher$chance$of$younger$age$of$onset$of$the$disease$(<50$years),$to$have$familial$ * disease$ and$ to$ present$ with$ aortic$ dissection. $Importantly$ we$ have$ also$ shown$ that$ traditional$ indicators$ of$ genetic$ disease$ such$ as$ presence$ of$ syndromic$ features$ or$ family$history$do$not$reliably$predict$these$cases$(indeed$only$approximately$a$half$of$ patients$in$whom$a$pathogenic$/$likely$pathogenic$variant$was$identified$had$a$positive$ family$ history).$ These$ preliminary$ findings$ are$ of$ clinical$ significance$ as$ they$ would$ suggest$a$change$in$clinical$management$of$TAAD:$one$would$suggest$routine$genetic$ testing$ for$ all$ patients$ with$ TAAD$ (prioritising$ those$ with$ younger$ age,$ syndromic$ features$or$a$family$history),$as$well$as$closer$surveillance$and$family$cascade$testing$ for$those$found$to$have$clinically$significant$variants.$$

$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ $$$$$$$$ *!NB! on! re_analysis! after! completion! of! variant! validation! by! David! Ross! and! David! Parry,! this! last! factor,! presence!of!Dissection,!was!more!common!in!the!P/LP!group!but!did!not!reach!statistical!significance!(see! Weerakkody! et! al.! Targeted! Targeted! Genetic! Analysis! in! a! Large! Cohort! of! Familial! and! Sporadic! TAAD! Patients,!manuscript!currently!in!submission).! ! 135! ! A$ number$ of$ phenotypes$ within$ the$ EDS/TAAD$ spectrum$ were$ identified$ and$ exome$ sequencing$ carried$ out$ to$ try$ to$ ascertain$ novel$ genetic$ causes.$ One$ new$ gene$ discovery$and$a$number$of$useful$preliminary$observations$were$made.$

By$ wholeAexome$ sequencing$ of$ eight$ unrelated$ probands$ (supported$ by$ previous$ linkage$analysis$from$one$proband),$we$were$able$to$identify$a$novel$candidate$gene$ underlying$EDS$Periodontitis$Type$(VIII),$a$distinct$and$rare$EDS$subtype,$which$had$ remained$unsolved$prior$to$this.$With$further$sequencing$and$functional$corroboration$ from$our$collaborators,$it$was$demonstrated$that$Periodontitis$Type$EDS$results$from$ specific$classes$of$heterozygous$mutations$in$C1R$and$C1S,$which$are$likely$to$bind$ and$disrupt$collagen$proteins$and/or$other$ECM$proteins,$though$the$exact$mechanism$ remains$to$be$solved.$$

The$finding$that$a$class$of$molecules$involved$in$inflammation,$which$is$quite$distinct$ from$ the$ collagen$ and$ matrix$ genes$ identified$ in$ EDS$ pathogenesis$ to$ date,$ is$ an$ interesting$one:$along$with$the$recent$implication$of$the$key$role$of$TGFAbeta$pathway$ signaling$initially$identified$from$its$role$in$LoeysADietz$syndrome$$(and$later$in$TAAD$in$ general)$ and$ Wnt$ signaling$ (in$ EDS$ type$ VI$ and$ Brittle$ cornea$ syndrome,$ an$ overlapping$ phenotype)$ this$ further$ highlights$ the$ role$ for$ aberrations$ in$ common$ biochemical$ pathways$ and$ not$ only$ major$structural$defects$ underlying$ HDCT’s.$This$ raises$ an$ interesting$ hypothesis$ in$ relation$ to$ the$ paradigm$ of$ Mendelian$ subsets$ of$ complex$ disease$ mentioned$ in$ the$ introduction,$ if$ rare$ Mendelian$ disease$ such$ as$ familial$TAAD$or$EDS$are$caused$by$major$alterations$in$common$molecular$pathways,$ it$is$probable$that$milder$phenotypes$in$the$same$spectrum$might$be$caused$by$more$ subtle$aberrations$in$these$same$pathways.$

On$the$hypothesis$that$other$genetic$causes$for$TAAD$exist,$as$evidenced$by$the$60%$ of$cases$who$had$a$positive$family$history$in$whom$a$genetic$variant$was$not$identified$I$ carried$out$exome$sequencing$and$linkage$analysis$in$one$large$pedigree$with$familial$ TAAD.$ From$ this$ I$ made$ the$ preliminary$ identification$ of$ a$ possible$ spliceAsite$ disrupting$intronic$variant$in$FBN1$as$the$causal$factor$(though$further$work$is$required$ to$validate$this$finding).$

Further$ work$ from$ this$ thesis$ includes:$ from$ targeted$ sequencing_$ refining$ the$ genotypeAphenotype$ relationship$ in$ EDS$ &$ TAAD$ by$ ongoing$ sequencing$ of$ larger$ cohorts_$and$from$the$exome$sequencing$studies,$further$sequencing$and$reAanalysis$of$ larger$cohorts$of$the$specific$phenotypeAgroups$identified.$This$thesis$has$shown$that$ HDCT’s$such$as$EDS$&$TAAD$exist$on$an$overlapping$spectrum$and,$in$many$cases$ are$ caused$ by$ aberrations$ in$ common$ and$ unexpected$ signalling$ pathways.$ The$ genetic$ origin$ for$ the$ majority$ of$ these$ cases$ remains$ unresolved,$ but$ possibly$ lie$ in$ these$same$genes$or$pathways.$It$may$also$be$that$a$number$of$genetically$unresolved$ ! 136! ! cases$may$be$caused$by$nonArare,$intronic$or$other$regulatory$elements$or$indeed$by$ multiple$ pathogenic$ genes$ segregating$ within$ the$ same$ family.$ Novel$ bioinformatics$ and$experimental$methodologies$will$be$needed$to$identify$these$from$large$genomic$ datasets.$

! 137! !

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! 138! ! PUBLICATIONS$ARISING$FROM$THIS$WORK$$

Original$journal$articles$ Weerakkody$RA,$Vandrovcova$J,$Kanonidou$C,$Mueller$M,$Gampawar$P,$Ibrahim$Y,$ Norsworthy$ P,$ Biggs$ J,$ Abdullah$ A,$ Ross$ D,$ Black$ HA,$ Ferguson$ D,$ Cheshire$ NJ,$ Kazkaz$H,$Grahame$R,$Ghali$N,$Vandersteen$A,$Pope$FM,$Aitman$TJ.$Targeted$next$ generation$ sequencing$ makes$ new$ molecular$ diagnoses$ and$ expands$ genotypeA phenotype$ relationship$ in$ EhlersADanlos$ syndrome.$ Genetics1 in1 Medicine$ 2016,$ doi$ 10.1038/gim.2016.14.$[Epub$ahead$of$print].$[Impact$factor$7.5]$ $ Ines$ KapfererASeebacher$ et$ al.$ Periodontal$ EhlersADanlos$ syndrome$ is$ caused$ by$ mutations$ in$ C1R1 and$ C1S,$ which$ encode$ subcomponents$ C1r$ and$ C1s$ of$ complement.$American1Journal1of1Human1Genetics120161[Impact$factor$11.2]$ $ Weerakkody$RA,$Ross$D,$Parry$D,$Ziganshin$B,$Vandrovcova$Gampawar$P,$Abdullah$ A,$ Biggs$ J,$ Bicknell$ C,$ Field$ M,$ Pepper$ J,$ Elefteriades$ J,$ Cheshire$ NJ,$ Aitman$ TJ.$ Targeted$Genetic$Analysis$in$a$Large$Cohort$of$Familial$and$Sporadic$TAAD$Patients.$ [In$submission]$ $ $ Other$publications$ $ Published1abstract1 Weerakkody$RA,$Vandrovcova$J,$Bicknell$D,$Aitman$TJ,$Cheshire$NJ.$Genetic$analysis$ of$ aneurysm$ and$ dissection$ of$ the$ descending$ thoracic$ aorta$ using$ nextAgeneration$ sequencing.$British1Journal1of1Surgery.$2014_101(Suppl$2):8A9.$ $ Conference1Proceedings1(chapter)11 Weerakkody$ RA,$ Vandrovcova$ J,$ Aitman$ TJ,$ Cheshire$ NJ.$ What$ role$ will$ genomics$ play$ in$ thoracic$ dissection$ and$ thoracic$ aneurysm$ management?$ Proceedings$ of$ the$ 10th$International$Symposium$on$Endovascular$Therapeutics$(ISTE)$2013.$$ $ Book1chapter1 Weerakkody$RA$&$Cheshire$NJ.$Thoracoabdominal$aneurysm$repair$In:$Child$A,$editor.$ Diagnosis$&$Management$of$Marfan$syndrome.$London:$Springer$2016.$ $

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! 140! ! GLOSSARY$

Gene$ or$ protein$ domain$ names$ (e.g.$ FBN1),$ proper$ nouns$ relating$ to$ specific$ equipment$ or$ software$(e.g.$Fluidigm)$and$SI$units$are$not$included.$Explanations$are$specific$to$the$subject$of$ this$thesis$and$not$exhaustive.$Bold$text$indicates$a$term$listed$in$the$glossary,$to$allow$crossA referencing.$ aa$ (No.$of)$amino$acid(s),$in$this$thesis$generally$used$to$convey$the$length$of$a$ protein$ AAA$ Abdominal$aortic$aneurysm$

ABHet$ Allele$ balance$ A$ the$ proportion$ of$ NGS$ reads$ giving$ the$ alternate$ versus$ reference$allele$at$a$specific$locus,$in$this$thesis$calculated$as$no.$Alt.alleles/$ Total$reads$(it$is$an$experimental$annotation$that$attempts$to$estimate$whether$ the$ data$ supporting$ a$ variant$ call$ fits$ allelic$ ratio$ expectations$ (the$ perfect$ unbiased$ heterozygote$ variant$ would$ have$ ABHet$ of$ 0.5).$ See$ GATK$ documentation$ (at:$ https://software.broadinstitute.org/gatk/gatkdocs/3.5A0$ for$ more$detail)$ ACMG$ American$ College$ of$ Medical$ Genetics$ guidelines$ for$ interpreting$ the$ guidelines$ pathogenicity$of$a$variant$29$$$ Allele$balance$ see$ABHet$

Amplicon$ Length$ of$ DNA$ targeted$ for$ amplification$ (by$ PCR,$ thereby$ allowing$ it$ to$ be$ sequenced)$ Annotation$ The$ process$ of$ identifying$ the$ functional$ effect$ of$ a$ variant,$ its$ frequency$ in$ population$ databases$ and$ its$ predicted$ pathogenicity$ by$ inAsilico$ tools.$ This$ process$ is$ carried$ out$ bioinformatically$ using$ programmes$ such$ as$ ANNOVAR.68$ Aortic$extent$ The$involvement$or$extent$of$aorta$affected$by$pathology$is$clinically$described$ in$ terms$ of$ its$ surgical$ anatomy:$ Aortic$ root,$ Ascending$ aorta,$ Arch,$ Descending$thoracic$aorta$(from$origin$of$left$subclavian$artery$to$diaphragm),$ Abdominal$ aorta$ (described$ relative$ to$ the$ origin$ of$ the$ renal$ arteries$ into$ suprarenal,$ juxtarenal$ and$ infrarenal),$ Thoracoabdominal$ (contiguous$ involvement$of$the$thoracic$and$abdominal$aorta)$ ASD$ Atrial$septal$defect$

BAV$ Bicuspid$aortic$valve$

BCS$ Brittle$cornea$syndrome$(overlaps$with$Kyphoscoliotic$EDS$and$is$included$in$ the$latest$classification$for$EDS82).$$ Beighton$score$ Score$ (0A9)$ of$ the$ extent$ of$ hypermobility$ based$ on$ the$ number$ of$ affected$ joints63$ bp$ Base$pairs$ c.$ Denotes$the$cDNA$change$describing$a$variant$according$to$Human$Genome$ Variation$Society$(HGVS)$nomenclature$(e.g.$c.869A>G)$194$ Ca.$ Calcium$

Capillary$ see$Sanger$sequencing$ sequencing$ Ch.$ Chapter$(e.g.$Ch.2.1.2$refers$to$chapter$2,$section$1.2)$

Chr.$ Chromosome$number$(usually$defining$a$genomic$coAordinate)$

! 141! ! cM$ CentiMorgan$ A$$standardised$ measure$ of$ genetic$ distance,$ which$ reflects$ recombination$ frequency.$ One$ centimorgan$ is$ equal$ to$ a$ 1%$ chance$ that$ a$ marker$at$one$genetic$locus$will$be$separated$from$a$marker$at$a$second$locus$ due$ to$ crossing$ over$ in$ a$ single$ generation:$ in$ the$ human$ genome$ 1$ centimorgan$is$equivalent,$on$average,$to$1$million$base$pairs.$ CNV$ Copy$ number$ variant$ A$$where$ a$ section$ of$ DNA$ is$ repeated$ or$ deleted,$ essentially$ refers$ to$ large$ insertions$ or$ deletions$ (often$ multiexonic$ or$ multigenic)$$ Compound$ Two$ different$ mutations$ affecting$ the$ same$ gene$ (or$ locus)$ in$ the$ same$ heterozygous$ individual$ mutation$ Cutis$laxa$ Describes$ the$ phenotype$ of$ sagging,$ inelastic$ skin$ (a$ feature$ of$ connective$ tissue$disorder)$ DBSNP$ Database$ of$ single$ nucleotide$ polymorphisms$ (SNP's),$ available$ at:$ https://www.ncbi.nlm.nih.gov/SNP/snp_summary.cgi$$ DCM$ Dilated$cardiomyopathy$

Dissection$ Dissection$ of$ the$ aorta$ (aortic$ dissection)$ refers$ to$ the$ splitting$ of$ the$ aortic$ intima$ from$ the$ media,$ allowing$ blood$ to$ track$ between$ these$ layers$ and$ create$a$“false$lumen”.$DIssection$may$also$affect$other$arteries$e.g.$coronary$ artery,$ carotid$ artery,$ etc.$ It$ is$ a$ distinct$ pathological$ phenomenon$ from$ aneurysm,$though$may$coAexist$with,$or$lead$to$aneurysmal$dilatation.$Aortic$ dissection$is$conventionally$divided$by$the$Stanford$classification$into$Type$A$ (involving$ the$ ascending$ aorta$ and$ arch)$ and$ Type$ B$ (confined$ to$ the$ descending$thoracic$and/or$abdominal$aorta).$ DNA$sequence$ see$Variant$ variant$ Domain$ Different$parts$of$a$protein$have$functional$significance$or$homology$with$other$ proteins$and$these$are$generically$termed$domains.$

DP$ Read$depth.$The$number$of$NGS$reads$covering$a$specific$locus.$

EDS$ Ehlers$ Danlos$ Syndrome.$ Previously$ classified$ by$ the$ 1986$ Berlin$ nosology$ (EDS$ I$ –$ IX),$ then$ by$ the$ 1997$ Villefranche$ criteria,$ which$ have$ been$ most$ recently$updated$in$2017.82$ EGF$ Epidermal$ growth$ factor.$ EGFAlike$ domains$ are$ a$ key$ functional$ domain$ of$ fibrillinA1$(FBN1)$ EM$ Electron$microscopy$

ER$ Endoplasmic$ reticulum.$ Dilated$ ER$ on$ electron$ microscopy$ of$ fibroblasts$ suggests$ deficient$ transportation$ of$ collagen$ molecules$ (part$ of$ the$ postA translational$processing$of$collagen$molecules).$ ex.$ Exon$ number$ A$ $ for$ each$ gene,$ the$ exons$ are$ conventionally$ numbered$ sequentially$ from$ 1$ to$ n.$ The$ exon$ number$ for$ a$ given$ gene$ can$ give$ an$ indication$of$the$likely$importance$of$a$functionally$significant$DNA$sequence$ variant$in$that$exon.$ EXAC$ Exome$Aggregation$Consortium$database$of$variants$and$related$metrics$from$ database$ >60,000$exome$sequences.$Available$at:$http://exac.broadinstitute.org/$$ Exon,$exome$ The$ coding$ part$ of$ the$ DNA$ sequence$ or$ its$ mRNA$ transcript$ (that$ which$ is$ translated$ into$ amino$ acids)_$ each$ gene$ is$ comprised$ of$ multiple$ exons,$ separated$ by$ introns$ (nonAcoding$ DNA)_$ the$ whole$ exome$ refers$ to$ the$ entirety$of$the$exons$of$the$genome.$ FTAAD$ Familial$Thoracic$Aortic$Aneurysm$and$Dissection$(also$referred$to$as$HTAD,$ hereditary$Thoracic$Aortic$Disease),$in$this$thesis$used$to$mean$anyone$with$ at$ least$ one$ first$ degree$ relative$ affected$ by$ TAAD$ or$ other$ aortopathy$ including$AAA.$

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GC$ The$proportion$of$G&C$versus$A&T$bases$in$a$given$DNA$sequence$affects$ the$annealing$temperature$and$therefore$potentially$the$efficacy$of$PCR$ Genetic$ Single$phenotype$caused$by$mutations$at$any$one$of$multiple$genetic$loci_$the$ heterogeneity$ corollary$ of$ this$ being$ "pleiotropy"$ (same$ gene$ leading$ to$ multiple$ different$ phenotypic$effects)$$$ Genotype$ A$particular$combination$of$alleles$at$a$given$locus$

Ghent$criteria$ Formal$clinical$criteria$used$for$the$diagnosis$of$Marfan$syndrome.$

GI$ Gastrointestinal$

GMP$ Guanosyl$monophosphate$

GRCh37/hg19$ This$ refers$ to$ the$ specific$ version$ of$ the$ assemblyAsequence$ of$ the$ human$ genome$ used$ in$ this$ thesis:$ note$ the$ exact$ coAordinates$ of$ specific$ loci$ will$ vary$depending$on$the$version.$ GU$ Genitourinary$

GWAS$ GenomeAwide$association$study$

Haplotype$ A$ group$ of$ linked$ loci$ or$ genes$ that$ are$ inherited$ together$ from$ one$ parent$ (see$Ch.1.1.2)$ HDCT$ Hereditary$disorder$of$connective$tissue$(for$a$good$review$see$MurphyARyan$ et$al.$(47))$ HGMD$ Human$ Gene$ Mutation$ Database103,$ available$ at:$ http://www.hgmd.cf.ac.uk/ac/index.php$$ HGVS$ Human$ Genome$ Variation$ Society$ –$ conventional$ nomenclature$ for$ variant$ annotation$194$ HHT$ Hereditary$haemorrhagic$telangiectasia$

HPO$ Human$phenotype$ontology$64$–$standardised$nomenclature$system$for$human$ phenotypes$(see$http://humanAphenotypeAontology.github.io/)$ ID$ Identification$number$

IMH$ Intramural$haematoma$(see$TAAD)$

In$silico$tools$ Refers$ to$ software$ used$ to$ analyse$ variants,$ specifically$ to$ predict$ likely$ pathogenicity$e.g.$SIFT,$Polyphen,$etc.$(see$Ch.2.9.1$and$legend$to$Table$3.9)$ Indel$ Insertion$deletion$variant,$whereby$bases$are$inserted$and/or$deleted$from$the$ DNA$sequence$at$a$specific$position.$ Intron$ NonAcoding$segments$of$DNA$within$a$gene,$which$intervene$the$exons$of$the$ gene$and$are$spliced$out$during$translation.$Intergenic$sequences$are$the$nonA coding$ DNA$ that$ lie$ between$ genes.$ Introns,$ though$ not$ translated$ into$ proteins,$are$thought$to$have$a$regulatory$role$e.g.$in$transcription$or$splicing.$ IRAD$ International$registry$of$aortic$disease$145$

Karyotype,$ Chromosome$ constitution$ of$ a$ cell$ or$ organism$ (e.g.$ 46XY$ is$ the$ normal$ karyotyping$ karyotype$of$a$human$male)$ Kinase$ An$ enzyme$ which$ activates$ its$ substrates$ by$ phosphorylating$ specific$ residues.$ LDS$ Loeys$Dietz$syndrome$

Linkage$ $“Genetic$linkage”$refers$to$the$concept$that$alleles$physically$proximal$to$each$ other$on$a$given$chromosome$tend$to$be$inherited$together.$

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Linkage$ Process$ of$ locating$ the$ approximate$ chromosomal$ location$ of$ a$ causative$ analysis$ gene,$based$on$the$concept$of$genetic$linkage,$in$a$group$of$(usually$related)$ individuals$ sharing$ the$ same$ phenotype$ thought$ to$ be$ caused$ by$ the$ same$ gene$(see$Ch.1.1.2).$$ Linkage$ The$observation$that,$in$a$given$population$particular$combinations$of$alleles$ disequilibrium$ tend$ to$ coAoccur$ more$ frequently$ than$ one$ would$ expect$ by$ chance.$ (also$ known$as$“allelic$association”.)$ LM$ Light$microscopy$

Locus$ A$specific$location$of$a$gene$or$gene$sequence$along$a$chromosome,$can$be$ used$to$refer$to$the$gene$itself$ LOD$score$ Log$ odds$ ratio$ that$ the$ presence$ of$ the$ disease$ is$ linked$ to$ any$ given$ haplotype$or$genomic$region$(see$chapter$1.1.2)$ LoF$ Loss$of$function$variant(s)$–$in$this$thesis$includes$nonsense$mutations,$splice$ site$disrupting$variants$and$frameshift$mutations$as$per$standard$convention.$ (see$Figure$1.5)$ Long$QT$ Arrhythmia$characterised$by$prolonged$"QAT"$interval$on$electrocardiogram$ syndrome$ LOVD$ Leiden$Open$Variant$Database$102$

MAF$ Minor$ allele$ frequency.$ This$ is$ the$ frequency$ of$ the$ second$ most$ common$ allele$at$a$particular$locus.$It$is$conventionally$used$to$define$or$categorise$the$ population$ frequency$ of$ variants,$ with$ rare$ variants$ generally$ defined$ as$ having$MAF$1A3%,$and$MAF<1%$variants$described$as$very$rare$(though$this$ definition$is$somewhat$fluid).$A$rare$Mendelian$disease$is$likely$to$be$caused$ by$a$rare$or$novel$variant.$ Marfan$ A$ hereditary$ disorder$ of$ connective$ tissue$ (HDCT)$ characterised$ by$ tall$ syndrome$ stature,$ arachnodactyly$ and$ a$ number$ of$ other$ skeletal$ manifestations$ $ A$ outlined$ in$ the$ Ghent$ classification(85)$ $ A$ as$ well$ as$ aortic$ aneurysm.$ It$ is$ caused$ by$ mutations$ in$ FBN1$ gene$ (fibrillin$ 1),$ though$ a$ pathogenic$ FBN1$ mutation$alone$is$not$a$sufficient$condition$for$positive$diagnosis.$$ Marfanoid$ Describes$ skeletal$ manifestations$ overlapping$ Marfan$ syndrome,$ but$ not$ fulfilling$Ghent$criteria$for$a$diagnosis$of$Marfan$syndrome.$$ MASS$ Syndrome$ typically$ involving$ Mitral$ valve,$ Aorta,$ Skeleton$ &$ Skin,$ which$ syndrome$ overlaps$with$Marfan$syndrome$and$is$also$caused$by$FBN1$mutations$ Max$PolyAX$ PCR$condition:$maximum$number$of$consecutive$bases$of$the$same$type$in$a$ primer$ Mendelian$ Strictly$ refers$ to$ the$ laws$ of$ inheritance$ proposed$ by$ Mendel$ in$ 1865,$ but$ conventionally$refers$to$a$monogenic$disease$or$inheritance$pattern$(see$Ch.$ 1.1.1)$$ Missense$ A$DNA$sequence$variant$where$one$base$is$replaced$by$another.$ variant$ Mutation$ Used$to$mean$a$DNA$sequence$change$away$from$the$normal,$and$informally$ used$to$mean$any$pathogenic$variant_$can$also$mean$the$actual$event$leading$ to$a$DNA$sequence$change.$ NGS$ Next$generation$sequencing$refers$to$the$major$technological$advance$(since$ ca.2005)$from$traditional$Sanger$sequencing$that$has$significantly$increased$ sequencing$capacity.$ NHLBI$(ESP)$ National$ Heart$ Lung$ and$ Blood$ Institute$ Exome$ Sequencing$ Project$ database,$also$known$as$ESP6500$ is$a$variant$database$from$6500$exome$ sequences$from$a$variety$of$cohorts,$representing$mixed$phenotypes$(not$all$ healthy$controls).$

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Nonsense$ A$ DNA$ sequence$ variant$ leading$ to$ the$ introduction$ of$ a$ "stop"$ codon,$ one$ variant$ that$ terminates$ translation$ prematurely,$ thus$ leading$ to$ a$ truncated$ protein$ product.$ NSNV$ Non$synonymous$single$nucleotide$variant$A$a$missense$variant$leading$to$an$ alteration$in$the$amino$acid$sequence$ OI$ Osteogenesis$Imperfecta$

OMIM$ Online$Mendelian$Inheritance$in$Man$(available$at:$https://www.omim.org/$)$

OR$ Odds$ratio$ p.$ Denotes$the$amino$acid$change$resulting$from$a$specific$variant.$See$HGVS$ nomenclature$ P/LP$ Pathogenic$or$Likely$Pathogenic$variant$(ACMG$criteria$were$used)29$

PAU$ Penetrating$aortic$ulcer$(see$TAAD)$

PCR$ Polymerase$chain$reaction$ –$ process$used$to$amplify$(make$multiple$copies$ of)$ a$ specific$ portion$ of$ DNA_$ this$ is$ done$ using$ a$ DNA$ Polymerase$ which$ amplifies$the$DNA$that$intervenes$two$primers$designed$to$complement$and$ bind$either$side$of$the$part$of$DNA$one$wants$to$amplify.$ Phenotype$ The$observable$physical$characteristics$of$an$organism$(or$cell)$

Polymerase$ Enzyme$used$to$amplify$DNA$bases.$

Polymorphism$ see$SNP$

Positional$ The$process$of$sequencing$a$region$of$interest$(or$candidate$genes$within$that$ cloning$ region)$that$has$been$linked$to$a$specific$phenotype$in$order$to$identify$novel$ diseaseAcausing$genes$(see$Ch.$1.1.2)$ Primer$ PreAdesigned$ length$ of$ DNA,$ which$ complements$ and$ therefore$ binds$ to$ a$ specific$portion$of$DNA$flanking$a$target$which$one$wants$to$amplify$by$PCR.$ Read$mapping$ NGS$produces$multiple$"reads"$of$a$given$DNA$sequence,$which$are$aligned$ or$ ‘mapped’$ to$ the$ reference$ sequence,$ allowing$ sequence$ variants$ to$ be$ identified.$ REC$ Research$ethics$committee.$

Recombination$ During$ meiosis$ (gamete$ formation)$ in$ any$ individual,$ the$ process$ whereby$ parts$of$the$maternal$and$paternal$copies$of$each$chromosome$are$swapped$ (see$ Ch.1.1.2).$ This$ ensures$ that$ the$ DNA$ passed$ down$ to$ the$ next$ generation$in$any$given$gamete$will$not$be$purely$from$the$individual's$father$ or$mother,$but$a$mixture$of$the$two,$thereby$promoting$genetic$diversity$in$the$ species.$ RER$ Rough$endoplasmic$reticulum.$(see$ER)$

Sanger$ (Technically$known$as$Capillary$Sequencing),$the$traditional$method$of$DNA$ sequencing$ sequencing,$ based$ on$ Fred$ Sanger’s$ dideoxy$ chain$ termination$ method$ originally$described$in$1975.$ SDSAPAGE$ Sodium$ dodecyl$ sulphate$ polyacrylamide$ gel$ electrophoresis$ A$ method$ for$ separating$molecules$based$on$molecular$weight$ Segregation$ The$pattern$of$inheritance$of$a$specific$genetic$variant$in$relation$to$that$of$a$ particular$ phenotypic$ trait.$ If$ a$ particular$ functionally$ significant$ variant$ segregates$in$parallel$with$a$specific$phenotype,$that$provides$good$evidence$ of$the$variant$being$causative$of$the$phenotype.$ SerAThr$kinase$ SerineAThreonine$kinase$

SNP$ Single$ nucleotide$ polymorphism,$ usually$ refers$ to$ a$ common$ variant$ (frequency$>1%)$at$a$particular$position$in$the$genome,$usually$benign$

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SNV$ Single$nucleotide$variant$

Synonymous$ A$variant$that$does$not$result$in$a$change$in$the$amino$acid$sequence$of$the$ variant$ resultant$protein.$ TAAD$ Thoracic$aortic$aneurysm$&$dissection$A$refers$to$aortopathy$affecting$any$part$ of$the$thoracic$aorta,$forms$part$of$the$spectrum$of$Acute$Aortic$Syndromes,$ encompassing$ Aneurysm,$ Dissection,$ Penetrating$ Aortic$ Ulcers$ (PAU)$ and$ Intramural$ Haematoma$ (IMH).$ (Not$ to$ be$ confused$ with$ a$ similar$ acronym,$ TAD,$which$refers$to$Type$A$aortic$dissection.)$$ TGF$ Transforming$ growth$ factor$ (beta)A$ refers$ to$ superfamily$ of$ cytokines$ and$ signalling$ proteins$ that$ underlie$ a$ variety$ of$ intracellular$ processes$ and$ implicated$in$TAAD$pathogenesis$40$ Tm$ PCR$condition:$Melting$temperature$$

UTI$ Urinary$tract$infection$

Variant$ Refers$ to$ the$ phenomenon$ where$ a$ DNA$ base$ at$ a$ given$ locus$ is$ different$ from$the$reference$human$genome.$Any$given$individual$will$have$hundreds$of$ thousands$ of$ variants,$ most$ of$ which$ are$ of$ no$ pathological$ significance$ (Benign$ variants)_$ some$ variants$ significantly$ alter$ protein$ structure$ or$ function$ and$ are$ therefore$ pathological$ (Pathogenic$ variant)_$ others$ cause$ some$ change$ to$ the$ amino$ acid$ sequence$ but$ are$ of$ uncertain$ pathological$ significance$and$termed$Variant$of$Uncertain$Significance$(VUS).$ Variant$calling$ The$process$of$identifying$variants$within$a$given$DNA$sequence$(this$is$done$ bioinformatically$by$software$such$as$GATK)$ Villefranche$ Clinical$classification$system$for$Ehlers$Danlos$Syndrome$(EDS)$ criteria$ VSD$ Ventricular$septal$defect$

VUS$ Variant$of$uncertain$significance$(see$Variant)$

WES$ Whole$exome$sequencing$(see$also$Exon,$Exome)$ $ $

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! 156! ! APPENDICES$ The$Appendices$to$this$thesis$are$in$a$separate$file$and$their$details$are$as$follows.$

Appendix$1:$Primers$used$for$the$three$targeted$sequencing$assays$(Tables$S1A3)$and$ transcript$ID's$for$genes$covered$therein$(Table$S4).$ Appendix$2:$Phenotyping$details.$ Appendix$3:$List$of$excluded$amplicons$from$the$TAAD$assays$(TAADAX$and$TAADAZ).$ Appendix$4:$Details$of$the$calculation$of$filtering$thresholds$for$the$TAAD$cohort.$ Appendix$5:$Details$of$the$EDS$phenotype$groups$prioritised$for$exome$sequencing.$ Appendix$6:$List$of$highApriority$candidate$genes$in$EDS$&$TAAD.$ Appendix$7:$Permission$requests$for$published$extracts$from$other$publications.$

! 157!