Portuguese consensus document for the management of alpha-1-antitrypsin deficiency

www.journalpulmonology.org

CONSENSUS

Portuguese

consensus

document

for

the

management

of

alpha-1-antitrypsin

deficiency

A.P.

Lopes

_,

_M.A.

_Mineiro

_,

_F.

_Costa

_,

_J.

_Gomes

_,

_C.

_Santos

_,

_C.

_Antunes

_,

_D.

_Maia

_,

R.

Melo

_,

_M.

_Canotilho

_,

_E.

_Magalhães

_,

_I.

_Vicente

_,

_C.

_Valente

_,

_B.G.

_Gonc

_¸alves

_,

B.

Conde

_,

_C.

_Guimarães

_,

_C.

_Sousa

_,

_J.

_Amado

_,

_M.E.

_Brandão

_,

_M.

_Sucena

_,

M.J.

Oliveira

_,

_S.

_Seixas

_,

_V.

_Teixeira

_,

_L.

_Telo

a_{CentroHospitalareUniversitáriodeCoimbra(HUC)} b_{CentroHospitalLisboaCentral}

c_{CentroHospitalareUniversitáriodeCoimbra(HG)} d_{CentroHospitalardoPorto}

e_{CentroHospitalarLisboaNorte}

f_{HospitalProf.DoutorFernandodaFonseca} g_{CentroHospitalardeLeiria}

h_{CentroHospitalardaCovadaBeira} i_{CentroHospitalardoBaixoVouga}

j_{CentroHospitalardeTrásosMonteseAltoDouro} k_{HospitaldaSenhoradaOliveira---Guimarães} l_{CentroHospitalardeSãoJoão}

m_{UnidadeLocaldeSaúdedeMatosinhos} n_{CentroHospitaldeGaia-Espinho}

o_{InstitutodeInvestigac¸ãoeInovac¸ãoemSaúde,UniversidadedoPorto(I3S)}

p_{InstituteofMolecularPathologyandImmunologyoftheUniversityofPorto(IPATIMUP)} q_{Servic¸odeSaúdedaRegiãoAutónomadaMadeira(SESARAM)}

r_{Alpha-1-antitrypsindeficiencystudygroupcoordinator}

KEYWORDS Alpha1-Antitrypsin Deficiency;

Abstract Alpha-1-antitrypsindeficiency(AATD) isageneticautosomal codominantdisorder

caused by mutations inSERPINA1 gene. It is oneof the mostprevalent genetic disorders,

although itremainsunderdiagnosed.Whereasatinternationallevelthereareseveralareas

Abbreviations: AAT,Alpha-1-antitrypsin;AATD,Alpha-1-antitrypsindeficiency;ALT,Alanineaminotransferase;AST,Aspartate amino-transferase;ATS,AmericanThoracic Society;BODE,Body-massindex, airflowobstruction,dyspnea and exercise;c-ANCA,Cytoplasmic antineutrophilcytoplasmicantibody;COPD,Chronicobstructivepulmonary disease;CT,Computedtomography;DBS,Driedbloodspots; DLCO,Diffusingcapacityofthelungsforcarbonmonoxide;EDTA,Ethylenediaminetetraaceticacid;ER,Endoplasmicreticulum;ERS, Euro-peanRespiratorySociety;FDA,FoodandDrugsAdministration;FEV1,Forcedexpiratoryvolumeinonesecond;FRC,Functionalresidual

capacity;FVC,Forcedvitalcapacity;GGT,Gamma-glutamyltransferase;HIV,Humanimmunodeficiencyvirus;HRQoL,Health-relatedquality oflife;HU,Hounsfieldunits;IEF,Isoelectricfocusing;IgA,ImmunoglobulinA;IL8,Interleukin8;LTB4,LeukotrieneB4;MPO, Myeloperox-idase;NE,Neutrophilelastase;NHLBI,NationalHeart; ,LungandBloodInstituteRegistry;PCR,Polymerasechainreaction;PD10,10th percentiledensity;PR3,Proteinase3;RCL,Reactivecentreloop;RV,Residualvolume;SEPAR,SociedadEspa˜noladeNeumologíayCirugía Torácica;SERPINA1,SerpinpeptidaseinhibitorcladeAmember1;TLC,Totallungcapacity;URTI,Upperrespiratorytractinfections.

∗_{Correspondingauthor.}

E-mailaddress:plopeshuc@hotmail.com(A.P.Lopes). https://doi.org/10.1016/j.pulmoe.2018.09.004

2531-0437/©2018PublishedbyElsevierEspa˜na,S.L.U.onbehalfofSociedadePortuguesadePneumologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Consensus; Diagnosis; Therapeutics; Pulmonary Emphysema

of consensus on this disorder, in Portugal, inter-hospital heterogeneity in clinical

prac-tice and resources available have been adding difficulties in reaching a diagnosis and in

making therapeutic decisionsin this group ofpatients. This raised aneed to draft a

doc-ument expressing a national consensus for AATD. To this end, a group of experts in this

field was created within the Portuguese Pulmonology Society - Study group on AATD, in

order to elaborate the current manuscript. The authors reviewed the existing literature

and provide here general guidance and extensive recommendations for the diagnosis and

managementofAATDthat canbeadopted byPortugueseclinicians fromdifferent areasof

Medicine.

Thisarticleispartofasupplemententitled‘‘Portugueseconsensusdocumentforthe

mana-gement of alpha-1-antitrypsin deficiency’’ which is sponsored by SociedadePortuguesa de

Pneumologia.

©2018PublishedbyElsevierEspa˜na,S.L.U.onbehalfofSociedadePortuguesadePneumologia.

Thisisanopenaccess articleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

ALPHA-1-ANTITRYPSIN

AND

PATHOPHYSIOLOGY

OF

ITS

DEFICIENCY

Alpha-1-antitrypsin (AAT) --- the major serine protease inhibitor in the serum --- is primarily synthesized by hep-atocytes and also produced at lower concentrations by neutrophils, mononuclear phagocytes, and epithelial lung and intestine cells. Once secreted into the bloodstream, AAT weighs approximately 52 KDa, is composed of 394 aminoacids and threecarbohydrate side chainslinked to asparagineresidues.1,2_{Structurally,thisglobular} glycopro-teinpresentsthree␤-sheets(A-C),nine␣-helices(A-I)anda solventexposedstretchnamedreactivecentreloop(RCL). There,thepeptidebondMet358-Ser359 (P1-P1’)mimicsa protease substrate and, upon its cleavage, AAT initiates a major conformation rearrangement that culminates in the formation of a stable inhibitor-protease complex.3---5 Although AAT is well established asa potent inhibitor of neutrophilelastaseinthelung,itisalsocapableof inhibit-ingother proteinases, such asproteinase3, kallikreins7, matriptaseandcaspase-3.Morerecently,immunity-related andanti-inflammatoryfunctionshave alsobeenattributed toAAT.1,6

AAT is encoded by the SERPINA1 gene located in the chromosome 14q32.1 region. This gene encompasses approximately14kbandisorganizedintothreeuntranslated exons(Ia-Ic) containing differenttissue-specific transcrip-tionstartsitesandfourcodingexons(II-V).7

AAT deficiency (AATD) is inherited as an autosomal codominantcondition,whichiscausedbypathogenic muta-tionsinSERPINA1. Therearecurrentlyover 120identified allelesthatfollowaspecificcodingsystem,inwhich inher-itedAATvariantsareclassifiedbetween‘A’and‘Z’basedon theirmigrationinan isoelectricfocusing(IEF)pHgradient (‘A’denotes the mostanodal variantsand‘Z’ the slowest migratingalleles---seealsosection3.2.3.1).

AATvariantscanbecategorizedintofourgroups8_: - Normalvariants,characterizedbynormalAATserum

lev-els(20---53␮M,or80---220mg/dlbynephelometry). - Deficiency variants, characterized by AAT serum levels

below 20␮M, and, in the case of some alleles (i.e. Z),

concomitant decreased functional activity of the AAT molecule.

- Nullvariants(Q0),whicharecharacterizedbytheabsence ofcirculatingAATduetoeithertranscriptionalor transla-tionalerrorsthatpreventnormalproteinsynthesis. - Dysfunctional variants, which are characterized by an

abnormalfunction of AAT (i.e. reduced binding to neu-trophil elastase, which is the case of F and Z variants, or with alteredinhibitory activity, which is the case of anti-thrombinPittsburghvariant).9

Normalallelesarepresentin 85---90%ofindividualsand are designated asM; therefore, normal individuals reveal MMgenotypes.Ontheotherhand,mostprevalentdeficiency allelesaredesignatedasSandZ.10_{TheZalleleresultsfrom} aGlu342Lyssubstitutionthatcausesthelossofacrucialsalt bridge(Glu342-Lys290)andaffectsthestabilityoftheA ␤-sheet(breachandshutterdomains).3_{Thisstructuralchange} isthoughttofacilitatetheinsertionoftheRCLintothe shut-terofanotherAATmoleculepromptingtheaccumulationof Zpolymersintheendoplasmicreticulum(ER).2_TheSallele iscausedbyaGlu264Valreplacementthatalsoleadstothe disruptionofasaltbridge(Glu264-Lys387)linkingan␣-helix tothemoleculecore.3 _{TheSvariantisalsoproneto} poly-merizationintheER;however,itsrateisonlymarginalwhen comparedtotheZallele.1,11,12

EPIDEMIOLOGY

EpidemiologicalstudiesshowthatAATDisoneofthemost prevalentgeneticdisorders inhumansand themost com-monly diagnosed in adults. Over the past 10-15 years, diagnosis of AATD has markedly improved as a result of increasing awareness and the publicationof international diagnostic and management evidence-based recommen-dations. Nevertheless, this condition remains widely and persistentlyunder-diagnosedormisdiagnosedbyhealthcare providers.EveninSweden,whereAATDwasfirstrecognized and reported,Piitulainen andTanash statedrecently that only20%ofallexpectedcasesofsevereAATDinadultswere reportedtotheSwedishnationalregistry.13

Despitescarce epidemiologicalinformationona world-widebasis, severalstudies showedthat AATDisrelatively commonamongEuropeansand itsfrequency canbe com-paredtothatofcysticfibrosis, whichaffectsonein every 2000to4000people.14 _{Nonetheless,thestudy carriedout} bydeSerres15_{---whichincluded58countriesworldwideand} atotalpopulationofapproximately4.4billioninhabitants ---obtainedanestimateofatleast116millioncarryingAATDMS orMZandapproximately3.4milliondeficiencyallele com-binations(SS,SZ, andZZ).This study alsoconcludedthat AATDisaninheritedconditionaffectingallracialsubgroups, notjustEuropeansbutratherseveralAfricanpopulations, middleeasternJewsandArabs,andAsiansaswell.Blanco etal16 _{carriedout amulticentric studyinvolving 21} Euro-peancountriesinordertoestimatethenumberofcarriers forthetwomostcommondeficiencyalleles(SandZ).This study revealed thatItaly, Spain, Germany,France, United Kingdom, Latvia, Sweden, and Denmark have the highest number of ZZ carriers (5000-15000) followed by Belgium, Portugal,Serbia andMontenegro,Russia,Holland,Norway, andAustria,thelatterofwhichhavebetween1000and2000 carriers.

Over90%ofindividualswithclinicaldeficiencycarrythe ZZ genotype, while the remaining ones are explained by otherrarervariants.17_{TheZalleleismostcommonin} north-ernEurope,inthoseofScandinaviandescent,nearlyabsent amongAfricansandEasternpopulations,exceptwherethere is miscegenation with Europeans, while the S allele has increasedin the Iberian Peninsula. AATDis always associ-atedwithnon-ZallelesinAsianpopulations.Thenullallele occursinafrequencyof0.00017.14,17

Few epidemiological studies have been carried out in order to quantify the true prevalence of this genetic deficiency and information on the Portuguese population remains scarce. One study described the geographic dis-tribution of alleles across Portugal with S allele having a decreasing frequency from north to south and Z allele reaching maximum frequency in the north and minimum frequency in the centre.18 _{The most relevant data} pub-lished refers to a retrospective study that evaluated a sample of the Portuguese population tested for AATD over 10 years.19 _{Data from 1684 patients from almost} every region in Portugal --- whose blood/plasma samples were sent to an informal reference laboratory --- were analysed. It was possible to identify 427 MZ, 188 SZ, and 158 ZZ subjects. Different types of rare deficiency and null alleles were also detected: 59MMalton or MPalermo (p.Phe52del), 28 Q0Ourém (p.Leu353Phefs*24), 21 PLowell (p.Asp256Val), 17I (p.Arg39Cys), 6MWürzburg (p.Pro369Ser), 6MHeerlen (p.Pro369Leu), 4 T (p.Glu264Val), 2 Q0Madrid, 2 Q0Faro(c.-5+2dupT),2ZAugsburg(p.Glu342Lys),and1Q0Lisbon (p.Thr68Ile)variants.Fouralleleslinkedtonovelmutations were also identified: PGaia (p.Glu162Gly), Q0Oliveira do Douro (p.Arg281Lysfs*17),Q0VilaReal (p.Met374Leufs*19),andSGaia (p.Leu118Phe).20,21

DIAGNOSIS

OF

ALPHA-1

ANTITRYPSIN

DEFICIENCY

In1963,Carl-BertilLaurellandStenErikssonfirstdescribed AATD as a clinical entity.22 _{Since then, there have been}

impressive strides in understanding the pathobiology and clinical course of this inherited disorder.23 _{Nevertheless,} many studies continue to identify AATD as an under-recognized condition in symptomatic patients. A five to eight-yeardelay between the onset of the first symptom (usually dyspnea) and the recognition of AATD has been found in studies performed over a time span of 18 years uptill2013,andtherehasbeennooverallimprovementin earlydiseasedetection.24---27

CLINICALMANIFESTATIONS

AATDbyitselfisnotadisease,butratherapredisposition toa later developmentof several disorders.In fact, it is believedthatlowserumlevelsofAATtogetherwithother genetically determined characteristics and environmental influencesresultinthedevelopmentofadiseasestatethat mayresultinprematuredeath.Theavailabledataon pen-etration(the percentageof AATD individuals withclinical disease)iscurrentlyscantandthespectrumofAATD-related disordersandtheiragesofonsetarequitebroad.28

Inclinicalpractice,theriskofdiseaseismainlyrestricted tothedeficientZallele,accountingforapproximately95% ofAATDcases.Theremainingindividualsat riskcarryrare allelesincludingnullanddysfunctionalvariants.8

LowlevelsofAATrepresentasyndromeofclinical enti-ties, some more related to a deficiency condition while others resulting, in contrast, from a negative effect of proteinoverload. Itsmain clinical manifestations primar-ilyinvolvethelungs,liver,and---lessfrequently---theskin. SincepatientswithAATDaresusceptibletodevelopingall thesediseasesthroughouttheirlives,thedisorderis consid-eredasystemicdisease.29

Individuals may be identified after presenting clinical consequencesofthedeficiencyorthroughfamilyscreening ofanindexcase.13,30

Currently, the understanding of the natural history of AATDis stillincomplete.Withinthefirstthreedecadesof life,liver diseaseis the majorthreatand pulmonary dys-functionis not common.Besides that, thenatural history ofAATDpatients is less clear, andsurvival estimates vary widelyamongseries.

1.1. PULMONARYINVOLVEMENT

Inthelung,AATDpredisposesindividualstoprematureonset ofchronic obstructive pulmonary disease(COPD). Indeed, it is the major and best proven genetic risk factor for COPD,with1-2%ofallcasesestimatedtobeduetosevere AATD.28,31

1.1.1. Emphysema

Theprematureonsetofemphysemawasthefirstidentified clinicalmanifestationofAATD,describedin 1963,22 _andit is the most prevalent clinical correlate of AATD and the major cause of morbidity and mortality.32 _{Emphysema is} thoughttoprimarilyresultfroma‘lossoffunction’defect.8 It corresponds to a protease-antiprotease imbalance and proteolytic degradation of elastin andother extracellular matrix components of the respiratory tract by neutrophil elastase(NE)andotherproteases,theactivity ofwhichis

unopposedandenhancedbecauseofAATDinthepulmonary parenchyma and airways. Aside from a quantitative defi-ciencyintheserum, Z-typemolecules intheirmonomeric isoformsareapproximatelyfivefoldlessefficientinhibitors ofNEthannormalnativeM-typeAAT.33,34

Compoundingthelackofaproteolyticscreen,the tran-sition of monomeric Z-AAT to polymers inactivates its anti-proteinase function, thereby further depleting pro-tective levels of AAT, and also converts this natural broad-spectrum anti-inflammatory molecule into a proin-flammatory stimulus. Loop-sheet polymers of misfolded Z-typeshave been shown tocause chemotaxis and/or to activateinflammatorycells,hencesupportingthe hypothe-sisthatpolymersinthelung(eithercirculatingandtrapped in the lung or locally produced by alveolar macrophages and airway epithelial cells) may contribute to tissue destruction.35_{Moreover,theaccumulationofmisfoldedAAT} moleculeswithintheERlumenofmononuclearphagocytes andneutrophilsleads toER stressresponse thatmay also havearoleinthepathophysiologyoflungdisease.36_Indeed, the presence of polymers may explainthe progression of lung disease in Z homozygotes after smoking cessation despite appropriateintravenous replacement withplasma AAT.37

Individuals withgenotypes associated withplasma AAT levelsbelowtheputativeprotectivethresholdof11␮M (cor-responding to 57mg/dl by nephelometry or 80mg/dl by immunodiffusion),mostofallhomozygousforthedeficient Zallele,areconsideredtohavesevereAATD.8_{Consequently,} theyareatamarkedlyincreasedriskofearlydevelopment of panacinar emphysema,28 _{particularly when exposed to} cigarette smoke, environmental irritants, or infections.25 However,thepreciseriskofdevelopingemphysemain indi-vidualswithsevereAATDisnotcompletelyunderstood.

Althoughseveralstudiessuggestahighriskofemphysema inZZ subjects,some never develop lung disease. Accord-ingly, a post-mortem series from Sweden38 _{that included} computedtomography (CT)imagingstudies39 _showedthat 14to20%ofZhomozygotescanbefreeofemphysema.

Individualswithnullallelesareatriskofthemostsevere formofassociatedlungdisease,includingemphysema.

Although S homozygotes arenot associatedwith a sig-nificant risk of emphysema, SZ heterozygotes have an increased risk, particularly if associated with cigarette smoking.40---43 _{Moreover, several studies performed so far,} withdifferencesin terms of sample sizing,have provided contradictory results about an increased risk of COPD in MZindividuals.31,44---47 _{However,morerecent studiesbased} in large populations of patients and controls support an increased risk of COPD mainly influenced by exposure to cigarettesmoke.48

FindingsfromtheNational Heart,LungandBlood Insti-tuteRegistry(NHLBI)showthatindividualswithsevereAATD display the same symptoms as patients with ordinary, or non-AATD,COPD. Thesesymptoms (inorderof decreasing frequency) include dyspnea on exertion (84%), wheezing withupperrespiratorytractinfections(URTI)(76%), wheez-ingwithout URTI(65%),increasedcough andphlegm(50%) andchroniccough(42%).30

Holm analyzed cross-sectional data collected via self-report questionnaires completed by 480 individuals with non-AATD COPD and 578 individuals with AATD-associated

COPD.IndividualswithAATD-associatedCOPDdidnotreport more symptoms of depression (25% reported depressive symptoms in the clinical range) or anxiety (36% reported clinically relevant symptoms of anxiety) despite their youngerage;however, theydidreportmoreclinically sig-nificantdyspneaandgreaterimpairmenttohealth-related qualityoflife(HRQoL)thanotherindividualswithCOPD.49 In addition, a study which assessed the quality of life of PortuguesepatientswithAATDshowedthatfemalepatients hadworseHRQoL.Hospitalizationsandfunctional markers ofrespiratorydiseaseprogressionnegativelyinfluencedthe HRQoL.50

Considerablevariabilityinthetimeofsymptomonsethas beendescribed,butsymptomsrarelyappearbefore25years ofage.Severesymptomsaremostoftenseenincurrentor former smokersbut some smokersand manynon-smokers developnosymptomsatall.27_{Cigarettesmokingcan} accel-eratetheonsetofdyspneabyasmuchas22years.Inthree studies theageat onsetofdyspneawas48 to54 yearsin non-smokersand32to40insmokers.32,51,52

Distinctive features of emphysema associated with severe AATDareearly onset(i.e.age≤45),beingpresent inanon-smokerorminimalsmokerwhohasnooccupational riskfactorsandbasilar-predominantradiographicchanges.28 Individuals with severe AATD experience a widevariation inlungfunction,usuallywithanaccelerated decline. Esti-mates for the annualrate of declineof forced expiratory volumeinonesecond(FEV1)inZhomozygotesvarybetween 23and316mlandtherateofdeclineisstronglyaffectedby cigarettesmoking.8

The onset of airflow limitation typically occurs at a youngeragethan inusualfor COPD.Inamulticenter nat-uralhistorystudyofAlpha1-AntitrypsinDeficiencyRegistry Study Group, themeanFEV1 of the1129 participantswas 43±30%ofthatpredictedandtheiraverageagewas46±11 years.27_{Innon-AATDCOPDpatients,airflowlimitation} usu-allyappearsinthesixthandsevendecadesoflife.

Severalstudies have shown FEV1 values to bea major determinantof survival insevere AATD.Two year survival ispractically100%untilFEV1fallsto33%ofthatpredicted and,fromthispointon,itdecreasesexponentially,falling to50%whenFEV1is15%ofthatpredicted.53

WithCOPD, airflow obstruction usuallycoexists with a reduced capacity of carbon monoxide diffusion; however, discordanceisnotinfrequent.Evenpatientswithsevere air-ways obstructionand prominentpanacinar emphysemaon theCTscanmayhavenormalgastransfer.54

Variationsinthedistributionofemphysemamaybe asso-ciated with functional differences and therefore account fordiscordantphysiology.Basaldistributionofemphysema has been associated withgreater impairmentof FEV1 but lowerimpairmentofgasexchangethantheapical distribu-tion.Itispossiblethatrealphysiologicaldifferencesdoexist betweentheupperandlowerlungregions,andthesemay beresponsibleforthefunctionaldifferencesidentified.55

Thewiderangeofclinicalphenotypesthatareassociated withAATDcouldbecausedbyinteractionsbetweengenetic modifiers and environmental factors other than SERPINA1 geneclusterandjustsmoking.36

Thedetrimentalroleofexposuretocigarettesmokeon the clinical phenotype of AATD has been widely demon-strated, with a significant gene-by-smoking interaction.

Smokeexposureisthesinglegreatestdeterminantfor the progressionofemphysemainAATDandakeyelementinthe natural historyof thosepatients.56 _{There appearstobe a} dose-responserelationshipbetweencigaretteconsumption andchange inFEV1 overtime.57 Passivesmokingmayalso beariskfactorforrespiratorysymptoms,butno relation-shipwasfoundbetweensecond-handsmokinganddecreased lungfunction.58

Active smoking---often but notalways associatedwith lower FEV1 --- does not fully explain the variability, and therearestillvariations in theclinical course ofpatients whohave never smoked.59 _{The natural historyof AATDin} never-smokersisalteredatsomepointinsymptomatic indi-viduals,by an unknown trigger, worseningtheir condition and bringingthem tomedical attention.56 _{Other risk} fac-torsforairflowobstructionmaybemalegender,aparental historyofCOPD,andapersonalhistoryofasthma,chronic bronchitis, or pneumonia, among others.60,61 _{The precise} roleofoccupationalinhalationexposureinacceleratingthe loss of lung function in AATD patients is not completely understood.62,63 _{There are few data available to indicate} thatlong-termoutdoorairpollution(ozoneandprimary par-ticles) can worsenthe respiratorystatus andpredict lung functiondeclineinindividualswiththeZZgenotype.64,65

Inadditiontosmokingandenvironmentalexposure, fac-torsassociatedwithfasterdeclineoflungfunctioninAATD may include male gender, age 30 to 44 years, low body mass index, lower serum AAT level, exacerbation rate, bronchodilatorreversibility,andbaselineseverityfunctional capacity.66---68

1.1.2. COPDexacerbations

AATDexacerbationsareassociatedwithgreater inflamma-tionand elastase activity than is usual withCOPD,which may explain why exacerbations can be more frequent, prolonged,andsevere.69_{Theseepisodesmayrelateto} phys-iologicaldeclineeveninpatientsundergoingaugmentation therapy.70 _{In a large one year cohort study from United} Kingdom,neitherthepresencenorthefrequency of exac-erbationsshowedarelationshiptothedeclineinFEV1,but thenumberofexacerbationswasweaklyassociatedwitha declineinlunggastransferforcarbonmonoxide.71

1.1.3. Spontaneoussecondarypneumothorax

Thisacutediseasemaybethepresentingmanifestationof AATD72 _{or a complication of emphysema.}73 _{To date, the} mechanismsofspontaneouspneumothoraxarestillunclear. Preliminarydataofacasecontrolstudyof39patientswith thediagnosisofspontaneouspneumothoraxconfirmthe clin-icalimportanceofAATDphenotypesinthosepatients and theneedforAATDscreening.74

1.1.4. Asthma

ThereisanunclearrelationshipbetweenAATDandasthma. DiagnosticbiasandtheoverlapofCOPD withasthma may beaconfoundingfactorintheevaluationofasthma preva-lenceamongAATDsubjects.Althoughseveralstudiesfailed toproveanincreasedprevalenceofAATDamongasthmatic patients,75 _{asthmahasbeenfoundtobethemostcommon} respiratorycomplaintinpatientswithAATDpriortothe dis-easediagnosis.27

Areview published in 2010summarizes information on therelationshipbetweensevereAATD,asthma,andCOPD.75 Current literature indicates that asthma signs and symp-tomsarecommonandmayoccurearlyinthecourseofthe developmentofairflowobstructioninAAT-deficientpatients withorwithoutCOPD.Bronchodilatorresponseisarisk fac-torfor FEV1 declineandAATDitselfpredisposestoairway hyperresponsivenessthatisassociatedwithreversible air-flow obstruction. The difficulty in differentiating asthma andCOPDinthosepatientsisparticularlychallengingsince wheezing and airflow obstruction are common manifes-tations of both conditions. An increased awareness by cliniciansof the overlap between these twoconditions is fundamentaltoensureappropriatetherapeuticstrategiesin ordertopreventanaccelerateddeclineoflungfunction.75

1.1.5. Bronchiectasis

Bronchiectasishasalsobeen associatedwithsevere AATD, but the relationship between these conditions remains unclearandtheestimatedprevalencevarieswidelyacross differentreports.41,76,77

ThePortugueserecommendationsarefortheaetiological diagnosisofbronchiectasis;giventhehighfrequencyof defi-cientallelesinthePortuguesepopulationandthebenefitof earlyAATDdetection,testingforAATDshouldbeundertaken forallsubjectswithbronchiectasis.78

EXTRAPULMONARY

DISEASE

In additionto extrapulmonary disease,liver and skin dis-orders may also occur in AATD. Clinical evidence of an association with other diseases such as bowel disease, glomerulonephritis,rheumatoidarthritis,fibromyalgia, vas-cularabnormalities(fibromusculardysplasiaofthearteries, abdominal and brain aneurysms, and arterial dissection), psoriasis, chronic urticaria, pancreatitis, neoplasia, and multiplesclerosis,israreandfurtherresearchstudies are requiredpriortoreachinganyconclusions.79

LiverDisease

Liverdiseasedoesnotoccurfrequently.AATDisonly diag-nosedthroughfindingofliverdiseasein3%ofAATDcases.80 Unlike pulmonary disease, AATD-related hepatopathy is not caused by functional loss or low protein serum lev-els but rather by the accumulation of AAT polymers in hepatocytes.81_{Inthiscontext,liverdiseaseistheresultof} adisruptedbalancebetweentheaccumulationofAAT poly-mers and the capacity of cellular mechanism todegrade thoseprotein aggregates.81,82 _{Hence, those AATDvariants} linkedtopolymerizationintheliver,suchasSIiyama,MDuarte, MMalton,andparticularlyZallele,mayallshowsignsof hep-atopathy. Liver disease has never been reported in Null (Q0Q0) patients. Unlike pulmonary disease, which is typ-ically a disorder of adulthood, liver disease may occur throughout a patient’s life; it is a frequent cause of dis-easeinnon-smokers.Theprevalenceofpost-mortemliver diseaseinnon-smokersis28%.Approximately10-15%of new-bornswithsevereAATDareatriskofdevelopingsomeform ofliverdisease.Similarly, about 10to15% ofadultsshow someevidenceof liverdisease,andalthoughitmayoccur

atanygivenage,itoccursmorefrequentlylaterinlife.80 Cholestaticjaundiceandhepatitis(neonatalhepatitis syn-drome)canbeobservedinsomenew-bornswithAATD,while later during childhood AATD patients may experience an unexplainedincrease inaminotransferases,hepatomegaly, or on rare occasions, cirrhosis and liver failure. In some cases,theonlysignsofliverdiseaseinyoungchildrenmay beanorexiaandagrowthdelay.83_{Ontheotherhand,adults} may show asymptomatic changes in transaminases, clini-calsignsofcirrhosis,andhepatocellularcarcinoma.Clinical manifestationsinpatientswithchronicliverdiseasedueto AATD may not be differentiated from those of any other etiology.

The development of liver disease and its evolution towards cirrhosis is the most common clinical sign after pulmonarydisease.Itisestimatedtooccurin2-43%ofZZ patients.Itmaybeafrequentcauseofdeathinnon-smokers withtheZZphenotype.84_{AccordingtoaSwedishstudy,out} ofapopulationof200000newborns,22/120 Zindividuals (18%)showedsignsofsomeliverdysfunction,whichincluded obstructivejaundice(12%)anddiscretelabchanges(7%).85 Theriskofcirrhosisinthesecaseswasapproximately50%, approximately 25% died during the first decade, and 2% developedcirrhosislateronintheirchildhood.86_The follow-upof127ZZnewbornsuntiltheir30sshowedthat3-7%had hightransaminaselevels,butnoneofthemshowedany clin-icalsymptomsofliverdisease.87_{Inasecondstudywhere246} ZZpatientswerefollowedoveran11-yearperiod,Larsson observedliverdiseasein12%ofthecases(cirrhosisin11.8%, neonatalhepatitisin0.4%,andhepatomain3.3%).32_ZZ indi-viduals who develop cirrhosis are at an increased risk of developing hepatocellular carcinoma,88 _especially middle-aged or older men, although therehave been some rare casesdescribed in children.89 _{However,thisrisk has been} difficulttoquantifyandmayvaryacrossstudies.88

Theriskofheterozygouspatientsdevelopingliverdisease isnotwellestablished.Manystudieshaveshownthatthere isagreaterprevalenceoftheMZgenotypeamongpatients withcirrhosisthanincontrolpopulationswithnon-cirrhosis liverdiseaseorcryptogeniccirrhosis.90---94 _{However,amore} recentstudyhasnotshownanyassociationbetweentheMZ genotypeandliverdisease.95_{Theriskofindividualswithone} Zalleledevelopinghepatocellularcarcinomaisalsonotas welldefinedasforhomozygouspatients.Heterozygosityfor theZallelemayincrease theriskor changeit inpatients withotherliverdiseases,suchaschronichepatitisC.96

Panniculitis

PanniculitiswasdescribedfortheveryfirsttimebyWarterin 1972andithasbeenassociatedwithAATDinapproximately 50reportedcases.Panniculitisisveryrarewithanestimated prevalenceof 1per 1000AATDcases,30 _{and withan} aver-age age of appearance of 40 years. As withemphysema, panniculitis is thoughttooccur due toa lack of protease inhibitory activity in the skin. It is characterized by pain andoneor more nodules showingexudingliquid and red-ness,andpainfulandwarmcutaneousplaques,whichmay suffernecrosisand berecurrent. Although itoccursmore oftenonthethighsandbuttocks,inonethirdofcasesitwill occurinplacesoftrauma.Thereareraresituationswhereit

maybeaccompaniedbypolyserositis.Itmayoccurinseveral diseasegenotypes,includingZZ,SZ,SS,andMS.97 Diagno-sis requires deep excisional biopsy, which shows areas of fat necrosisinterspersedamongnormal areas.A differen-tial diagnosis withother causes of panniculitis is difficult andthatiswhyAATlevelsshouldbemeasuredineverycase ofnecrotisingpanniculitis.

Vasculitis

Anassociationbetweenantiproteinase3(PR3)antibody vas-culitis (c-ANCA positive) and AATD was first reported in 1993.98,99_{Sincethen,severalserieshaveestablisheda} cor-relationbetweenAATDphenotypesandanti-PR3vasculitis. However,thereisalowincidenceofantiproteinase3 anti-bodiesin patientswithAATDanditspresence maynotbe relatedtothedevelopmentofsystemicvasculitis.100_Taking intoaccountthisassociation,AATDtestingmustbecarried outinadultswithc-ANCApositivevasculitis.28

LABORATORY

TESTS

DiagnosticteststoconfirmthepresenceofAATDaredivided intoquantitativeandqualitativetests.Therearefourtypes oftests:

--- Serum AAT Test, quantitative test for determining the amountofproteinintheblood;

--- AATPhenotyping, qualitativetestthatassessesthe pro-teinvariants inthe blood.This techniqueis capable of detectingnormalalleles(M)anddeficiencyalleles(S,I,Z, andothers)butitwillnotdetectnullalleles(i.e.Q0Ourém); --- AATGenotyping,

- Searching for S and Z mutations, qualitative test that assesses the presence or absence of the most common mutationsindeficiency-SandZalleles.

- SERPINA1 genesequencing, qualitative test that is con-sidered the reference method for identifying specific mutationsassociatedwithAATrarealleles,whetherthey aredeficientornull(I,MMalton,PLowell,Q0Ourém,etc.). SerumAATTEST

The concentrations ofAAT serumlevelsmaybemeasured using several different methods: immunoelectrophoresis, radialimmunodiffusionandnephelometry,andnormal val-ues will varydepending onthe method used101 _{(Table 1).} Nowadays, themost commonlyusedmethodis nephelom-etry. Radial immunodiffusion and nephelometry tend to overestimate theconcentrationofAAT whencomparedto thepurifiedstandardtestdevelopedbytheUSNational Insti-tuteofHealth.Inordertodifferentiatethevaluesobtained using the non-purified standard test from those obtained usingthepurifiedstandardtest,theformerareexpressedin mg/dlwhilethelatterarein␮M.Bothunitsarefrequently usedandareinterchangeableinmanyEuropeancountries, whatever thetest used.In ordertoconvert mg/dl (neph-elometry) to ␮M, one must multiply the concentration in mg/dlby0.1923.79

Table1 NormalandprotectiveserumlevelsofAATintheseveraltestingmethods.

Method Normalvalues Protectivethreshold

Purifiedstandardtest 20-53␮M 11␮M

Nephelometry 83-120a200-220mg/dl 57mg/dl

Radialimmunodiffusion 150-200a350-400mg/dl 80mg/dl

Table2 MainAATgenotypesandrelatedserumlevels.

Genotype AATserumlevel

mg/dl* _␮M** MM 103-200 20-39 MS 100-180 19-35 SS 70-105 14-20 MZ 66-120 13-23 SZ 45-80 9-15 ZZ 10-40 2-8 Q0Q0 0 0 * _nephelometry ** _{purifiedstandardtest)}

The reference values for each method are shown in

Table1.Inordertomakeiteasiertointerpretresults,each laboratorymustpreviouslydeterminethenormalvaluesof AATintheserumsamplesofnormalindividuals.79

Bearinginmindthatdifferentallelesdeterminea differ-entpercentageof proteinproduction,thereareexpected serumlevelsforeachphenotype79_(Table2).

AAT behaveslikean acutephaseprotein,whichis why itsserumlevelsmaybefalselyaugmentedduring inflamma-toryandinfectiousprocesses.Therefore,AATtestsmustbe carriedout preferablyoutsidetheseepisodes.Higher AAT valuescanbealsofoundduringpregnancyandaftertaking oralcontraceptives.Inthesecases,normalserumlevelsmay be detected in patients withmoderate deficiency. Serum levelsarelowerinchildrenthaninadults.79

DETERMININGTHENEUTROPHILELASTASE INHIBITORYCAPACITY

Inrarecircumstances, anothertestmaybeusedtoassess if a patient with normal circulating levels has an AATD. TheinhibitionofNEbyAATisdirectlyrelatedtoitsserum concentrationinsuchawaythatagreateramountofAAT cir-culatingwillhavegreaterinhibitoryeffect.However,some patients maypresent normal or high AAT levelsand little anti-elastase activity due to the fact that not every pro-teinisactive(80%beoxidised,destroyed,ornotcapableof linkingtoneutrophils).

Measuring the capacity to inhibit elastase from neu-trophils is especially indicated in the following cases: 1) studying patients with COPD where AATD is suspected althoughwithanormalAATserumlevel;2)when determin-ingifduringanexacerbationofCOPDtheincreaseinAATis proportionaltoanti-elastaseactivity;3)whenassessingthe implicationofAAT deficiencyintheseverityofpulmonary

disease,sincequite oftenAAT serumconcentration isnot relatedtoclinicalprogress.79

DETERMININGPHENOTYPEANDGENOTYPE

Qualitative tests detect the different mutations of the SERPINA1 gene. While phenotyping is carried out using isoelectricfocusing(IEF)techniquesthatallowforthe iden-tification of several protein variants in the blood (S, Z, M, andothers), genotypingusually covers different varia-tionstothepolymerasechainreaction(PCR)method,which enablestheanalysisofdifferentmutationsinmoreorless extensiveregionsoftheSERPINA1gene.

1.1.6. Phenotyping

Severalproteinvariantspresentintheblood,corresponding todifferentallelesoftheSERPINA1gene,maybeidentified throughisoelectricfocusing.

Although phenotyping permits the characterisation of approximately30AATDvariants,thismethodalonewillnot besufficient for a complete diagnosis in the case of null variantswithverylow(MHeerlen)orvirtuallynon-detectable (Q0Ourém)proteinserumlevels.Phenotypingmayalsoleadto apartialdiagnosisinthecaseofM-like(MMaltonandMWurzburg) variantswhich,in additiontopresentingreduced concen-trationsofprotein,showaverysimilarmigrationpatternto Mprotein.

1.1.7. Genotyping

Differentmethods basedonthePCR techniquehave been used for genotyping the mutations that define the S (rs17580, p.Glu264Val or c.863A>T) and Z (rs28929474, p.Glu342Lysorc.1096G>A)alleles,whichuseeitherprimer oligonucleotidesor specificfluorescentprobes(inthecase of real time PCR detection), detecting exclusively the mutated(S andZ)or non-mutated(non-S andnon-Z) posi-tion in the DNA sequence.102 _{In spite of these methods} beingtechnicallysimpler andmorelikelytobeappliedto abroadergeneticscreeningofAATD,theydonotenableus todetect the presenceof other mutationswhich occurin Portugal.21 _{IfwhileusingthesetechniquesSand/orZ} alle-lesarenotdetected,patientswillbefrequentlycatalogued ascarrying the M allele(homozygosity or heterozygosity) sinceinthecaseofthePortuguesepopulationthemost fre-quentalleles areM(80-86%)followed byS (12-18%)andZ (1-2.5%).103,104_{Thisfieldisrapidlyadvancingandsoonthere} willbediagnostickitscapableofdetectingagreaternumber ofmutationsoftheSERPINA1gene.

Onlywhengenotypingincludestheamplificationof SER-PINA1codingregions(exonsIItoV)andDNAsequencingdoes itbecomepossibletoassessinasystematicmannertheAATD geneticvariation. Itmaybecomplementedby ananalysis

Clinical suspiction of AATD

Measure AAT serum levels

Phenotyping or genotyping (by allelle specific methods)

Intermidiate deficiency 57-110mg/dL

Others

Genotyping

(gene sequencing in reference laboratory) SZ,MZ, MS,SS MM ZZ

(SZ) Probably without AATD

(probable MM phenotype) >110mg/dL Others (null-null,znull, z-rare, null-rare) Severe deficiency < 57mg/dL <110mg/dL

Figure1 AATDdiagnosticalgorithm. oftheexon-intronregionsandthepromoter(exonsIatoIc)

inordertoruleouteventualmutationsthatmayaffectthe normalprocessingofmRNA(Q0OportoandQ0Faro).104Although thisformofgenotypingmayrepresentanexcellentmethod for diagnosing AATD,currently itscosts and technological requirements,particularlyin less specialisedlaboratories, stillprohibititsuseasascreeningtool.Hence,this excep-tionalmethodisonlyusedtoconfirmdiagnosisinthecaseof rareAATDallelesthathavebeenidentifiedthrough isoelec-tricfocusing(phenotyping), in particularsituations where serumlevels diverge fromphenotype and/or genotype (S andZ mutation search),when results areinconclusive or donotagreewiththeclinicalmanifestations,orwhenever otherrareornullvariantsaresuspected.79

ConsideringthecomplexityoftheAATDgenetic diagno-sis,partlyduetothelargenumberofrarevariants,104_and thespecificitiesandlimitationsofeachdifferenttechnique --- genotyping and phenotyping ---, the differentreference laboratories inEuropehave established different diagnos-ticalgorithms, which often combine the measurement of AATserumlevels,thesearchforSandZmutations, isoelec-tricfocusing,andtheSERPINA1genesequencinginorderto reducethenumberoffalsenegativesandpositives.105

Phenotypeand/orgenotypemaybedeterminedinatotal blood samplepreferably resortingtothe ethylenediamine tetraaceticacid(EDTA)anticoagulant,whichshouldbe pro-cessedintotwosegmentswithinanapproximatetimespanof 48h:plasmaorserum(forisoelectricfocusing)and mononu-clearbloodcellsortotalremainingblood(forgenomicDNA extraction).Inanycase,thedifferentblood fractionsmay bestored at -4◦F (-20◦C).79 _{The timely execution of this} procedureiscrucialinordertoidentifydeficientvariants, suchastheZallele,givenitslabilityandconsequentriskof deterioration.

Recently, as an alternative to collecting samples by venipuncture, rapid genetic screening methods (kits) have been provided,which enablea quick assessment of the presence of S and Z alleles in blood (Alphakit®_{) or} saliva samples. However, these tests still require result confirmationusingmoreconventionalmethods,particularly ifone of thesealleles is detected, or ifAATD is strongly suspected.Currently,therearealso AATDgenetic screen-ing programmes, which favour sample collections using

minimally invasive methods by means of finger puncture and collection of a small amount of blood on dry filter paper(DBS:driedbloodspots),whichmaybesent bypost without any extra care. Several studies have shown that testing andsearching for S andZ mutations by means of adried blooddropsample isfeasible anditisparticularly suitableforthepurposeofscreeninginalargerpopulation, since it is a simpler and cheaper sampling method.105---109 However, due to the lower quantity and quality of the obtained sample, in some cases --- particularly regarding theSERPINA1genesequencingandphenotyping---itmaybe necessaryorevendesirabletoconfirmresultsbymeansof atotalbloodsamplecollectedbyvenipuncture.28,105

1.1.8. AATDGenotypes

Genotypes thatincrease therisk of developingpulmonary diseaseareassociatedwithserumvalueslowerthan11␮M or 57mg/dl (protective threshold) which may resultfrom differentcombinationsofdeficient(Z,MMaltonorotherrare alleles)andnull(Q0Ourémorothers)allelesinhomozygosity or heterozygositysuchasZZ,SQ0,ZQ0,Q0Q0or with lev-elsofserumsimilartothese(Table2).IntermediateAATD is characterised byserumconcentrations between 11 and 20␮Mandassociated toseveral genotypecombinations in homozygosityorheterozygosity,suchasSS,SZ,MZ,MS,SQ0, andMQ0,amongothers79,101_(Table2).

Ontheotherhand,genotypesthathavebeendescribed as being associated with a greater risk of liver disease are those associated with homozygosity or heterozygos-ityfor alleles withproven susceptibilitytoformpolymers in hepatocytes (Z, S, MMalton, MPalermo, MNichinan, SIiyama and MWurzburg).2,21

DIAGNOSTIC

ALGORITHM

TheAATDdiagnosticalgorithm(Fig.1)beginswithaclinical suspicionofAATD(Table3).Inthiscase,thequantification ofAATserumlevelsisrecommendedbydoingaquantitative test,oftenbynephelometry.28

The consensus document from the American Tho-racicSocietyandEuropeanRespiratorySociety(ATS/ERS)28 suggests the combination of the quantitative test and

Table 3 Clinical cases where an AATD screening is recommended.

ClinicalSuspicionofAATD:CandidatesforAATvaluesto

bedetermined

AllCOPDpatients

Earlypulmonaryemphysema(patientsyoungerthan45)

Emphysemawhennotexposedtoknownriskfactors

(smokingandoccupationalfactors)

Panlobularemphysemathatispredominantlybasal

Asthmawithspirometrythatdoesnotnormalizeinspite

ofadequatetherapy

Adultswithbronchiectasis

Teenagerswithapersistentobstructioninlungfunction

tests

Aclinicalhistoryofdyspneaandchroniccoughingin

severalmembersofthefamily

Hepatopathyofunknowncause

Decreaseinthepeakofthealpha-1proteininthe

proteinogram

Panniculitisorvasculitisofunknowncause

RelativesofAATDpatients(brothersandsisters,

offspring,parents,others)

phenotypingasgoldstandardforthediagnosisofAAT defi-cient states. In a recent document updating the Spanish guidelines,110_{thisalgorithmcontinuestoberecommended} withhighqualityscientificevidence,sinceitenablesusto clarifymostAATDcases.However,someauthorsdefendthe association of a quantitative test withgenotyping as the bestmethodfordiagnosis.105,111,112_{Whateverthetestused,} thereshouldbeaconcordancebetweentheidentified phe-notype/genotypeandthemeasuredserumlevel(Table2).

According to ATS/ERS28 _{recommendations, individuals} withaAATserumlevelbelowthenormalvalue,orbetween 90-140mg/dl throughnephelometry,whichareconsidered as borderline values, should undertake a qualitative test that includes phenotyping and/or genotyping, sincethese values maycorrespond toa phenotype withintermediate deficiency(SZ, SS,MZ)and theremayberelatives witha severedeficiency.AccordingtoFerrarotti,113_thefollowing cut-offpointswereproposed:1)Ifthepurposeisto iden-tifygenotypeswithanincreasedriskofemphysema,thena 100mg/dLcut-offpointissufficient(95.8%sensitivity,94.8% specificity);2)Ifthepurposeistodetectallpatients carry-ingZandSalleles,thena110mg/dLcut-offpointshouldbe used(73.4%sensitivity, 88.5%specificity);3)Otherstudies haveuseda113mg/dLcut-offpoint, sincetherearenoZ allelesor SSindividualsabovethis value(100%sensitivity, 78%specificityforAATDand79%sensitivityand83% speci-ficityfordetectingZandSalleles).Thereisahighnumber offalsepositivesabovethesevalues.114

Basedonthis,werecommendaqualitativewheneverthe serumlevelisbelow110mg/dl.

The genetic diagnosis of AATD should be supported by reduced serum levels (<110mg/dl) and a compatible phenotypeor genotypethatisrecognisedasbeingrelated to the disease. Whenever the serum levels, phenotype, or genotypedonot agreewitheach other or withclinical manifestations,thenarare,deficient,ornullvariantshould

beconsidered,anditshouldbeinvestigatedbyareference laboratoryinordertoclarifythesituation.

SCREENING

In light of the misdiagnosis and delay in the diagnosis of AATD,inPortugalandintherestoftheworld,itismandatory toscreen riskgroups ifwewishtochangethis panorama. Early diagnosis and proper follow-up should lead to pre-ventive attitudes and lifestylechanges that will certainly changetheprogressofthisdisease---smoking,occupational exposure, andinfections ---, early treatment of symptoms andexacerbationsandadministrationofreplacement ther-apyinthe earlystages ofthediseasein ordertoimprove lifequalityandexpectancyofthesepatients.Therefore,it isimperativetoidentifythediseaseassoonaspossible.

However,identifyingindividualswithAATDmayalsocarry somerisks,suchasgeneticdiscrimination,ahigh psycholog-icalburden,andmayhaveemploymentimplications.Hence, beforedoingthescreening,doctorsshouldweigh thepros andconsofdiagnosisanddiscussthemwiththepatient.28

Screeningisrecommendedforpatientswhoareclinically suspectedforAATD(Table3).Inthecaseofscreening rela-tivesoftheindexcase,ithasnotyetbeenestablishedhow widethis screening shouldbe, although some advisethat firstdegreerelativesshouldbetested.115

Thescreeningmethodshouldbesimilartothatusedin thediagnosisandfollowthesamealgorithm.

The implementation of national screening programmes isleadingtoanincreaseinthenumberofdetectedcases. Countrieswherescreeningprogrammeshave alreadybeen implemented116---119_{showdifferentdetectionrates.Manyof} these programmes are carried out as an initiative of the AATDNational Registries.Whendesigning ascreening pro-grammefordetectingAATD,onemustconsiderthesample process protocol as well as criteria for including candi-dates, since both factors will have a decisive impact on the programme results and cost.120 _{We suggest screening} programmesusingaccurateandsimplemethodswithdried blood drop samples where AAT is tested and phenotyping and/orgenotypingiscarriedoutusingfastmethodswhich arecost-effective.

Key

Points:

Diagnosis

- AATDdiagnosis beginswithaclinicalsuspicionthat shouldbeconfirmedbyAATserumlevelsand pheno-typingand/orgenotyping.

- AATserumlevels<110mg/dl(nephelometry)should prompttophenotypingand/orgenotyping

- There should be agreement between serum levels andthephenotype/genotype,otherwisewecouldbe inthepresenceofarareallele.

- Ifthereissuspicionofarareallele,samplesshould besenttoareferencelaboratoryinordertomakea correctdiagnosis.

Table4 AdditionalexamsintheearlystagesofconfirmedAATD.

PulmonaryAssessment LiverAssessment

Spirometry Liverfunctiontests

Pulmonaryvolumes Abdominalultrasound(orFibroscanTM₎

DLCO

Arterialbloodgasanalysis

ChestX-ray

ChestCT(withemphysemaquantification,ifavailable)

Six-minutewalktest

INITIAL

ASSESSMENT

OF

A

PATIENT

SUFFERING

FROM

ALPHA-1

ANTITRYPSIN

DEFICIENCY

TheinitialassessmentofapatientwithAATDshouldbegin withamedicalhistoryandphysicalexamwithspecialfocus onpulmonarydiseaseaswellasintheotherclinical mani-festationsofthedisease,suchasliverandskindisease.

InadditiontotheAATtestandphenotyping/genotyping, whichwerecarriedoutduringscreeninganddiagnosis, addi-tionalexamsshouldbeundertakenintheearlystagesofa patientwithconfirmedAATD(Table4).

PULMONARY

ASSESSMENT

LungFunctionTests

TheinitialassessmentofapatientwithAATDshouldinclude: spirometry---beforeandafterabronchodilator---,pulmonary volumes---measuredeitherthroughplethysmographyorthe helium dilution technique or nitrogen-washout ---, carbon monoxidediffusioncapacity,arterialbloodgasanalysis,and 6-minutewalktest.28_{Otherrespiratoryfunctionexamsmay} becarriedoutinselectedcases.

Spirometryisthemostsuitableexamforthediagnosisof COPDandforassessingitsprogression.Thereisusuallyan obstructivepattern(FEV1/FVC<0,7)withadecreaseinFEV1 andnormal or decreasedforced vitalcapacity(FVC). The flow-volumecurveshowsanotchintheexpiratoryportion leadingtoahighdecreaseinflowwithprogressivelylower volumes.Mostpatientsdonothaveapositivebronchodilator response,28_{althoughtheremaybemoderatereversibilityin} upto49%cases(≥12%and≥200mlinFEV1).121,122Theremay alsobereversibilityin12.5%ofpatientswithnormalFEV1, suggestingthathyperreactivitymaybeanearly character-isticofthisdisease.121

Inthelaterstagesofthedisease,lossinthelung elas-ticrecoilwillresultinanincreasedpulmonary compliance with hyperinflation, which translates into a decrease in FVC and an increase in total lung capacity (TLC) and in residualvolume (RV).Hence, addingmeasurement of the pulmonaryvolumestospirometrywillallowforbetterand morecompletepatientassessment.Duetotheeffectofair entrapment,thepulmonary volumesmeasuredby plethys-mography are usually greater than those measured using helium-dilutionbasedtechniques28_{;therefore,theyshould} bemeasuredpreferablyusingthefirstmethod.

Measuring carbon monoxide diffusing capacity (DLCO) will assess gas exchange through the alveolar-arterial

membrane, which is usually reduced. Although thesetwo aspectsbelongtothesameprocess(emphysema),FEV1and DLCOdonotalwayscorrelate.28 _{Arterialbloodgasanalysis} alsoenablesustoassessgas exchange. Theremaynotbe any changes in the early stages, but as the disease pro-gresses,hypoxemiawillappearwithexerciseandthenwhile resting,andwilllateronbeassociatedwithhypercapnia.

Inthelaterstagesofthediseasetheeffectofemphysema onthe muscular activityof the chestand diaphragm may beassessedbymeasuringinspiratoryandexpiratorymuscle pressures.

Respiratory function in exercise may be assessed by meansofexercisetestssuchasthesix-minutewalktestor thecardio-pulmonaryexercisetest.Inlaterstagesofthe dis-ease,cardio-pulmonaryexercisetestmayshowadecrease in the PaO2 and an increase in the alveolar-arterial dif-ference.PatientssufferingfromAATDmayhaveincreased respiratoryrateswhilerestingandtheirminuteventilation mayexceed80%ofpredictedmaximumvoluntaryventilation duringmildexercise,whichisanindicationthatventilation maylimithigherlevelsofexercise.28_{The6-minutewalktest} assessesnotonlytheexercisefunctionalcapacity,butitis alsoimportanttoestimatediseaseprognosisbycalculating theBODEindex.

ImagingExams

ChestX-ray shouldbecarriedoutduringtheinitial assess-ment of all patients. However, high-resolution CT of the chestinordertoassessthebronchopulmonarymorphology orchestCTwiththickersectionsfor densitometryarethe bestexams for detectingand quantifyingthe presence of emphysema.

ChestX-ray

Chest X-rayisthenormintheearlystagesofthedisease. Asthediseaseprogressesthetypicalsignsofhyperinflation, suchasanincreaseintheanteroposteriorandlateral diam-eter of the chest, higher transparency of the pulmonary fields, adecrease inthe vascular markings, horizontaliza-tionoftheribs,anincreasein theintercostalspaces, low and rectified hemidiaphragm, and vertical heart, appear. Thesechangesareusuallymoreprominentinthelowerlung fields.28_{BullouschangesdonotoccurveryofteninAATDand} whentheydooccur(35%)theirmostlikelylocationisinthe inferiorlobes.123

ChestCT

Chest CT is the best exam in vivo for characterising the severityandmorphologyofpulmonaryemphysema,124 bet-terthanachestX-rayorrespiratoryfunctiontests.28 _Ithas apositivecorrelationwithpathologicalfindings,andthatis whyithasbeensuggestedasthebestmethodfor monitor-ingemphysemaprogression.101_{Thepossibilityofmeasuring} pulmonary density enablesquantificationof the extentof emphysemawhichcanbeusedtomonitordisease progres-sion.

In AATD, the emphysema is usually of the panlobular type28 _{andin2/3 patientsitparticularlyaffectsthe basal} regions, in contrastto the centrilobular emphysema pre-vailing in the upper lobes which is typical of emphysema causedbysmoking.However,inupto36%ofthecasesthe emphysemamaybediffuseoreventuallyapical.55

InCT,theemphysemaareasarecharacterisedbya par-ticularly low attenuation, in contrast to the surrounding parenchymaindensityvaluesinHounsfieldunits(HU).Chest CT provides for calculation of densitometry parameters, makingitpossibletoassesstheextentoftheemphysema. There are several methods for calculating these densito-metricparameters: the ‘‘density mask’’, the ‘‘percentile method’’, and the ‘‘average parenchyma density’’. The densitymaskmethodusesacut-offvalue(usually-910HU) and all pixels with density below this value will be clas-sified as emphysema and compared in percentage to the entire lung parenchyma volume. The percentile method quantifiestheemphysemathroughthecut-offpoint,which definesapercentiledatumfromthehistogram.For exam-ple, the 10th _{percentile density (PD10) is derived from a} histogramrecordingthedensitiesinHUofalllungvoxelsand isdefinedasthethresholdvalueforwhich 10%ofalllung voxelshavealowerdensity.Asatruedensitymeasurethis valuewilldecreaseasemphysemaworsens.Intheaverage density method,the average pulmonary parenchyma den-sity is calculated,which will belower asthe emphysema progresses.124

Lungattenuationvalues(inHU)canbeconvertedtolung tissue density values(g/l) by adding 1000 tothe HU. For example,a15th_{percentiledensityvalueof-950HUequalsa} lungdensityof50g/l,meaningthat15%ofthepixels/voxels haveadensityvaluebelow50g/l.125

InchestCT,other changesbesidestheemphysemamay be seen, such as the thickening of the bronchial walls andbronchiectasis (25%)withawidening ofthebronchial diameterat thesegmentalandsub-segmentallevel.When present,bronchiectasisarecylindricalorsaccularand pre-vailinthelobeswhereemphysemaismoreextensive.101

Liverassessment

AllpatientscarryingallelesleadingtoAATaccumulationin theliver (Z, MMalton, MDuarte,SIiyama,etc.) must undergoan initialassessment ofliver changesbymeans ofabdominal ultrasoundandserumtests.76

The serum liver assessment should include transami-nases(ASTandALT)aswell asalkalinephosphatase,GGT, bilirubin, albumin, coagulation tests, platelets, fat sol-uble enzymes, and alpha-fetoprotein.76,79 _{An abdominal}

ultrasound will evaluate liver changes --- steatosis, portal hypertension,andcirrhosis ---andthepresenceof hepato-cellularcarcinoma.76 _FibroscanTM_{,anoninvasivetechnique} usingaprobewithanultrasoundtransduceranda mechan-ical vibrating device, exerts dynamic stress on the body surfacetogenerateshear waves.The shearwave velocity canthenbeconvertedintoliverstiffness,whichisexpressed inkilopascals.Itmaybeusedasanalternativetoultrasound toassesssteatosisandliverfibrosisinpatientswithAATD.126 Liverbiopsyshouldnotbeusedfordiagnosis, sincethe pathologicalchangesvaryandarenotspecific.The diagnos-ticmethodshouldbephenotypingorgenotyping.However, liverbiopsymaybeindicatedincertaincases,particularly whenitbecomesnecessarytoassessthelevelofliver dam-age,itsprogression,ortoinvestigatethepresenceofother relateddiseases. Thereis usuallylobeinflammation, vari-ablehepato-cellularnecrosis, fibrosis, cirrhosis, steatosis, andPAS-positivediastase-resistantglobulesinsome,butnot all,hepatocytes.76

KeyPoints:

Initial

Patient

Evaluation

- Initial evaluation of patients with AATD should include: spirometry, lung volumes, DLCO, arterial blood gas,chestX-ray,chestCTscan(with emphy-semaindexanalysisifavailable),6-minutewalktest - In phenotypes with liver involvement, liver func-tionstestsandabdominalultrasound(orFibroscanTM₎ shouldbedone.

TREATMENT

[Researchstudies inthe1980srecognisedthat plasma ␣1-antitrypsinlevelscouldberestoredbyintravenousinfusions ofpurifiedhumanprotein.Initiallicensingofaugmentation therapyin1987wasbaseduponanassumptionthatits suc-cessinboostingcirculatingandlunglevelsof␣1-ATwould beof clinicallymeaningful benefitanda logical approach tolong-term medicaltreatment. Thisassumptionhas sub-sequentlybeenexplored,androbustdemonstrationofsuch outcomeshasprovenproblematicin trials(sensitive mea-suresoflungdestructionv.clinicalefficacy)].127,128

Several scientific societies recommend, based on the availableclinical evidence,theuseof augmentation ther-apy,although withsomedifferencesin theindications for treatmentasaresultoftheevaluationofclinicalstudiesand dateofpublication.Basedona studypublishedin2003129 onthecost-effectiveassessmentoftreatment,therevised ERSguidelines,127_{reportalargeincreaseincostsfor} rela-tivelylittleincreasein maintainingpatientqualityoflife. Moreover,ERS guidelinesalso highlight that fact that the cost-benefitimplicationsoftheapparentreductionin exac-erbationfrequencyandseverityremaintobequantified.127 In Portugal, health authorities recognize the impor-tanceofpatientstohaveaccesstoaugmentationtherapy; however,theappreciationof medicalevidencesupporting the efficacy of this therapy by different prescribers and

medicalcentresisstillunderdiscussionandmayvary, caus-ing inequalities in terms of clinical requirements for the approvalofthetherapy.

AUGMENTATIONTHERAPYWITHAAT

[International expert treatment guidelines indicate that optimal management of Alpha -1 should include strict lifestylestandards, includingsmokingcessation,exercise, dietandsymptomatic treatment withregularreview with augmentation therapy intended toslow down progression andincrease thechancesof preventingirreversible tissue damage].130

The augmentation therapy available for AATD consists oftheadministrationofanintravenousinfusionofpurified humanAATextractedfromapoolofdonorplasma.Themain rationaleforthistherapyistomodifythecourseofthe dis-easebypreventingprogressionoftheemphysema,reducing thenumberofexacerbations,andimprovingqualityoflife. In1990,theobservationthatnon-smokingindividualswith SZphenotypeshowed a low risk of developing pulmonary diseaseledtothegeneralacceptancethatAATserumlevels foundintheseindividualscouldrepresentaminimum pro-tectivethreshold.131_{Later,Campbell}132_{demonstratedthat} themeanareaofdamageexertedbyneutrophilswas unre-lated toAAT concentration, untilits level droppedbelow 10␮M. Since then, the target for circulating AAT to give properlungprotectionhasbeenwidelyacceptedtobe11␮M (57mg/dlasdeterminedbynephelometry).

ThegroundsforusingAATinthetreatmentofpulmonary diseasearebasedonitsbiochemicalroleandclinical effi-cacy.Itsintravenousadministrationresultedinanincrement ofAATserumlevelabovetheprotectivethreshold,increased concentrationin the alveolar lining fluid, and neutralised activityoftheserumandalveolarelastasefromneutrophils throughouttheintervalbetweendoses.133---136_Adecreasein the degradation of elastase has also been shown through thequantificationofserum,urinaryandalveolarliningfluid levelsofdesmosineandisodesmosinemarkers.137,138

Stockley demonstrated a decrease in leukotriene B4 (LTB4), myeloperoxidase (MPO) and interleukin 8 (IL8), albeitonlysignificantlyreducedinpatientsundergoingAAT augmentationtherapy.136

Clinicalefficacyshouldaddressthefollowingquestions: does AAT augmentation therapy have a positive effect in loweringthe decline of pulmonary function and in delay-ing the progression of emphysema? Does it decrease the numberandseverityofexacerbations?Doesitimprove func-tional capacity, patient symptoms and survival? Is it safe andcost-effective?8_{Hence, medicalgroundsarebasedon} theresultsofvariousstudieswithdifferentdesigns (obser-vationalorrandomizedandplacebo-controlled)comparing thedeclineinpulmonaryfunction,variationoflungdensity assessedbychestCT,mortalityrate,frequencyandseverity ofexacerbations,impactonthequalityoflifeandfunctional capacitybetween groupsofpatientssubmittedtotherapy andpatientswhohavenotbeentreated.Amongthese,the randomizedstudiescontrolledwithplacebostandout,since theyhavedemonstratedasmallerlossinlungdensity quan-tifiedby CT insubjects that have been submitted toAAT

therapy. Table 5 summarizes the main results for studies supportingtheuseoftheAATtherapy.

Selectingcandidates---treatmentcriteria

Age---theagerangeforstartingtreatmentisunknown; how-ever,mostpatientsareexpectedtobeinthefourthdecade oflifewhendiagnosed,sincetheemphysemausually devel-opsapproximatelytenyearsearlierthaninsmoking-related COPD.Theminimalageforinitiatingtherapyis18andany exceptionmustbejustified.Themaximumagelimitmaybe establisheddependingoneachcase.Table6

Genotype-pulmonarydiseaseinAATDisassociatedwith genotypessuchasZZ,ZQ0, Q0Q0,andothercombinations withraredeficientornullvariants.Theclinicalefficacyof theAATtherapyisbetterdocumentedinthetreatment of ZZpatients.

Importantly,knowingthatapproximately10%ofpatients withSZgenotypehave aserumlevelbelowtheprotective threshold,itseemsacceptabletoincludethesesubjects,as longasallothercriteriafortherapyareverified.However, forSZindividualsthereisnoscientificevidencesupporting clinicalefficacyofthetherapy.

AATLevel---Decisionalcut-off-Patientswithserumlevels ≤57mg/dl(nephelometry)arecandidatesforAAT replace-menttherapy.Table7

PulmonaryFunctionalassessmentandCTDensitometry Observational studies indicate a smaller decline in pulmonary function of patients undergoing therapy with airways obstruction withinFEV1 31-65%or FEV1 35-49% of predictedvalues.Patientswithmoresevereobstructionsdo not appear tohave benefitted from therapy.67,139 _{A study} carriedoutin2009andcomprising164patients,concluded that a beneficialeffect wasobserved in the group of ex-smokerswithFEV1 <50% predictedvalue.141 Furthermore, inameta-analysisthatincludedfiveclinicaltrialsand1509 patients undergoing therapy vs. controlled patients, the treatedgroupregisteredalowerfunctionaldeclinewiththis resultshowinggreatersignificanceinthegroupofpatients withFEV1between30-65%ofpredictedvalue.145

The analysisof thesestudies suggests thatthe decline in FEV1 is not linear and drops faster in the moderate obstruction interval.150 _{Nevertheless, Wencker}140 _carried out a multi-centre and retrospective study to assess the progressofemphysemabeforeandduringtheAATtherapy (assessmentofthe47monthspriorandthe50months dur-ingtherapy---inthe60mg/Kg/weekdose),whichincluded 96patientssplitintothreefunctionalgroups---FEV1<30%, between30-65%and>65%,andtheyverifiedtheexistence of patients with severe deficiency, preserved pulmonary function (FEV1> 65%) and fast declining pulmonary func-tion (> 120ml/year). Moreover, in this latter group, the lossofpulmonaryfunctionpriorandduringtreatmentwas 255±70.4 and 52.7±61.3ml, respectively and according totheseauthors,an earlyimplementationof AATtherapy significantlyreducedpatientdeclineofrespiratoryfunction. More recently, randomized studies controlled with placebo, in which the emphysema progression is quanti-fiedbyCTscans,haveproposedthistechnologyasthebest indicatoroftheefficiencyofthistherapy.Theauthorsfind thatCTdensitometryismoresensitivethanthevariationof

Table5 StudiescarriedoutovertheyearstoevaluateefficacyofAATaugmentationtherapy.

Authors Dose TypeofStudy PrimaryEndpoint Results

Seersholmetal.

(1997)139

60mg/Kg/week Observationalwith controlledgroup n=295

FEV1decline LowerFEV1declineinthe grouptreated.Significant effectonlyforthegroup withFEV131-65%of predictedvalue(p=0.04) AmericanAAT DeficiencyRegistry StudyGroup(1998)67 33%weekly43%every twoweeks 24%monthly Observationalwith controlledgroup n=1129 FEV1decline Mortality

LowerFEV1declineinthe treatedgroupwithFEV1 35-49%ofpredictedvalue (p=0.03) Reducedmortalityin36%at 60months(p=0.02) Wenckeretal. (2001)140 60mg/Kg/week Observational withoutcontrolled group(beforevs. aftertherapy) n=96(3groupsFEV1 <30%,FEV130-65%, andFEV1>65%)

FEV1decline LowerFEV1declineduring treatmentperiod;inthe wholegroup49.2vs.34.2 ml/year(p=0.019). Significanteffectinasmall group(n=7)ofrapid declinerswithFEV1>65% (256vs.53ml/yr.P=0.001) Tonellietal.

(2009)141

60mg/Kg/week Observationalwith controlledgroup (n=164)

FEV1decline Mortality

Significanteffectinthe grouptreated(+10.6±21.4 vs.-36.96_±12.1ml/year; p=0.05)

Beneficialeffectinthe groupwithinitialFEV1<50% ofpredictedvalueandin formersmokers

Mortality-NS Dirksenetal.

(1999)142

250mg/Kg/month (foratleastthree years) RCT n=56(ZZ) FEV130-80% predictedvalue FEV1decline Secondaryendpoint: Lungdensity VitalCapacity DLCO

FEV1declineoverlapsin bothgroups

(26.5_±15.1vs.

25.2±22.0ml/year)p=0.96 Tendencyforareducedloss oflungdensityinthe treatedgroup(p=0.07) VitalCapacityandDLCO -NS Dirksenetal. (2009)143 EXACTLEtrial 60mg/Kg/week Week RCT n=77(FEV125-80%) (2-2.5years) Lungdensity Secondaryendpoint: Pulmonaryfunction Exacerbations Qualityoflife

Reducedlossoflungdensity inthetreatedgroup (p=0.49)

FEV1andDLCO-NS Numberofexacerbations -NS(2.55v.2.19;p=0.265; lessseriousinthetreated group) SGRQ-NS(1.48vs.2.37) p=0.695 Stockleyetal. (2010)144 250mg/Kg/month (n=56)60 mg/Kg/week(n=77) Meta-analysis(of bothDirksenstudies)

Lungdensity FEV1decline

Reducedlossoflungdensity inthetreatedgroup (p=0.006)

(meanchangefrombaseline -4.082vs.-6.379g/Lwitha treatmentdifferenceof 2.297(95%CI0.669-3.926) FEV1decline-NS