ORIGINAL ARTICLESC reactive protein and alpha1-antitrypsin: relationshipbetween levels and gene variants

STEFANIA OTTAVIANI, MARINA GORRINI, ROBERTA SCABINI, ZAMIR KADIJA,ELENA PARACCHINI, FRANCESCA MARIANI, ILARIA FERRAROTTI, and MAURIZIO LUISETTI

PAVIA, ITALY

From the Center for Diagnosis

Laboratory of Biochemistry and G

Disease, IRCCS Policlinico San M

Pavia, Pavia, Italy.

Supported by grants from the Fonda

Corrente), Fondazione CARIPLO

Germany.

Submitted for publication Octob

December 24, 2010; accepted for p

332

The first step in laboratory diagnosis of alpha1-antitrypsin deficiency (AATD) is the de-termination of alpha1-antitrypsin (AAT) serum levels; these levels in turn are influ-enced by the inflammatory status. C reactive protein (CRP) has been proposed asa marker of systemic inflammation. Single nucleotide polymorphisms (SNPs) in theCRP gene have been associatedwith differences in baseline CRP levels. The purposeof this study was to investigate the relationship between CRP and AAT in the AATD di-agnostic setting and to verify whether variations in the CRP gene could influenceCRP. We determined AAT and CRP levels in 362 consecutive dried blood spot (DBS)samples submitted for AATD diagnosis and genotyped 3 CRP gene SNPs (rs1205,rs3093077, and rs3091244) associated with variations in serum CRP concentrations.To this aim, we developed amethod tomeasure CRP in a DBSwith a good correlationwith CRP measurement in serum (r2 5 0.9927). We showed then that systemic inflam-matory status parallels increased levels of AAT (80% of subjects with intermediateAATD and a CRP . 0.8 mg/dL had an AAT level above the cut-off of 113 mg/dL)and that this increase might mask the presence of AATD variants. No associationwas detected betweenCRP levels and the 3CRP gene polymorphisms. Simultaneousdetermination of CRP and AAT is useful in the correct diagnosis of heterozygotescarrying intermediate AATD genotypes; their genetic influence on the CRP level isnegligible. (Translational Research 2011;157:332–338)

Abbreviations: AAT ¼ alpha1-antitrypsin; AATD ¼ alpha1-antitrypsin deficiency; APR ¼ acutephase reactant; COPD ¼ chronic obstructive pulmonary disease; CRP ¼ C reactive protein;DBS ¼ dried blood spots; EDTA ¼ ethylenediaminetetraacetic acid; PI*MZ ¼ patients heterozy-gous for Z deficient allele; SNP ¼ single nucleotide polymorphism; VC ¼ variability coefficient

C hronic obstructive pulmonary disease(COPD) has currently reached critical levelsin Western countries.1 An effective treatment,

besides smoking cessation, is not yet available; how-ever, a great deal of effort is being spent on the search

of Inherited AAT Deficiency,

enetics, Institute for Respiratory

atteo Foundation, University of

zione IRCCS SanMatteo (Ricerca

, and Talecris Biotherapeutics,

er 11, 2010; revision submitted

ublication December 29, 2010.

for biomarkers in an attempt to achieve useful indicatorsto monitor disease progression.2,3 Some inflammatorymarkers are elevated in the blood of COPD patients,suggesting a possible relationship with systemicrepercussions of the disorder. C reactive protein

Reprint requests: Maurizio Luisetti, MD, Institute for Respiratory Dis-

ease, IRCCS Policlinico San Matteo Foundation, University of Pavia,

Viale Golgi 16, 27100 Pavia, Italy; e-mail: m.luisetti@smatteo.pv.it.

1931-5244/$ - see front matter

� 2011 Mosby, Inc. All rights reserved.

doi:10.1016/j.trsl.2010.12.014

AT A GLANCE COMMENTARY

Ottaviani S, et al.

Background

The first step in laboratory diagnosis of alpha1-

antitrypsin deficiency is the determination of

alpha1-antitrypsin (AAT) serum levels that in

turn are influenced by the inflammatory status.

The purpose of this study was to investigate the re-

lationship between C reactive protein (CRP) and

AAT in the AAT deficiency (AATD) diagnostic

setting.

Translational Significance

We had evidence that systemic inflammatory sta-

tus parallels increased levels of AAT and that

this increase might mask the presence of AATD

variants. We conclude that simultaneous determi-

nation of CRP and AAT is useful in the correct di-

agnosis of heterozygotes carrying intermediate

AATD genotypes.

Translational ResearchVolume 157, Number 6 Ottaviani et al 333

(CRP), in particular, has been proposed as a biomarkerfor monitoring the systemic consequences of theinflammatory status in COPD.4,5

CRP is a member of the short-pentraxin subfamilyof pentraxins, which are radial pentameric structuredproteins that act as soluble pattern recognition receptorswith a wide variety of functions.6 In relationship withthis function, CRP is an acute phase reactant, producedby hepatocytes in response to inflammatory mediators,primarily interleukin-6.7 This property is shared byanother glycoprotein produced in the liver, namelyalpha1-antitrypsin (AAT).8 It is well known that thesevere, inherited AAT deficiency (AATD) representsa risk factor for developing pulmonary emphysema inthe 3rd to 5th decade of life in affected individuals,especially if they smoke.9 AATD is a rare disorderbecause it is largely underdiagnosed; more than 90%of possible AATD individuals remain unrecognized.10

To address this problem, the American ThoracicSociety/European Respiratory Society consensus docu-ment9 suggests submitting all individuals affected withCOPD and asthma without reversible airflow obstruc-tion to diagnostic testing for AATD.Laboratory diagnosis of AATD involves a complex

methodology, including different biochemical and mo-lecular methods to provide a complete and effective as-sessment of the AAT status.11 One problem in AATDlaboratory diagnosis is the lack of a definitive AAT se-rum level that represents the cut-off to decide whether

to proceed with other, expensive analyses. In fact, thepresence of an inflammatory status may increase theserum level of AAT, resulting in the masking of somedeficiency variant carriers.11,12 We previously haveestablished that the confidence limits for serum AATlevels in different AATD genotypes vary according tothe CRP serum level.13,14 Thus, it has beenhypothesized that CRP could be introduced usefullyas a reference to check whether the inflammatorystatus is present in a sample submitted for diagnostictesting. Interestingly, several single nucleotidepolymorphisms (SNPs) in the CRP gene already havebeen associated with differences in baseline CRPlevels in human populations.15-17 In particular, Zachoand colleagues found that some polymorphisms in theCRP gene are associated with marked increases inCRP levels.The present investigation therefore was designed to 1)

confirm the usefulness of CRPmeasurement as amarkerof systemic inflammatory status in AATD diagnosticsetting. Because the matrix currently used in diagnosticcenters for AATD is filter paper, also referred to as driedblood spots (DBS), in which drops of fresh blood are ab-sorbed and dried,11 we decided to develop a method tomeasure CRP from DBS; 2) verify whether variationsin the CRP gene influenced the physiological CRP con-centration in our study population to exclude factorsother than inflammatory status that could modify theCRP level. To do that, we genotyped a set of polymor-phisms representative of common variation patternsidentified in previous reports.15,17-20

METHODS

Development of an assay to measure CRP in DBSfluid. Blood supplemented with ethylenediaminetetra-acetic acid (EDTA) from 74 healthy donors or patientsadmitted to hospital was used. For each blood sample,DBS and plasma aliquots were obtained. A methodfor CRP determination in DBS was developed by mod-ifying the enzyme immunoassay described by McDadeet al.21 Briefly, a DBS standard curve was made bydiluting CRP (Liquicheck Elevated CRP Control level3; BIORAD Laboratories, Irvine, Calif) with EDTA-supplemented blood from donors or patients screenedfor CRP levels ,0.5 mg/dL. The final concentrationsof our curve ranged from 0.5 mg/dL to 2 mg/dL(8 points). One 3.2-mm paper filter disk (Schleicher &Schuell, Dassel, Germany), containing 6.77 mL ofserum or plasma (45% average hematocrit) from eachDBS (standard curve and samples) was eluted/diluted216 times in wash/elution buffer overnight at roomtemperature. Concentrations were calculated from thebest-fit linear regression curve (GOSA - pit software,http://www.bio-log.it) for the best matrix comparison

Fig 1. Relationship between DBS and serum/plasma CRP concentra-

tions in 74 paired samples.

Translational Research334 Ottaviani et al June 2011

between standards and unknown samples. Samplereadings .2 mg/dL were eluted as described; dilutedin wash buffer 5, 10, 20, 30, 40, and 60 times;assayed, and the results were multiplied by thedilution factor. CRP measurements in serum/plasmawere performed by a nephelometric assay (Array 360System; Beckman Coulter S.P.A., Milan, Italy)according to the manufacturer’s instructions.

Determination of AAT and CRP levels in DBSsamples. We evaluated 362 DBS samples shipped toour Diagnostic Center for Inherited AATD Diagnosisby physicians collaborating in the AATD targeted detec-tion program, already described in detail.13 AATDdiagnosis, including AAT nephelometric levels, wasperformed according to the algorithm previouslydescribed.11 CRP levels were determined on DBSaccording to the method described.

CRP genotyping. DNAwas extracted from DBS usingNucleoSpin (Macherey-Nagel, D€uren, Germany). Wegenotyped two SNPs in the CRP gene (12302G>A[rs1205] and 14899T>G [rs3093077]) using the LightCycler 480 (Roche, Rotkrenz, Switzerland). In addition,we genotyped a triallelic SNP (2390C>T>A [rs3091244]), located in the promoter sequence, with LightCycler 480 to identify the homozygous CC and TT sam-ples; we sequenced all remaining heterozygous sampleswith the CEQTM 8800 Genetic Analysis System (Beck-man-Coulter, Fullerton, Calif). Finally, genotyping wasverified by DNA sequencing of 30 random samples foreach SNP. Primers and probes used for genotyping andsequence reactions are available on request.

Statistical analysis. Statistical analysis was performedwith the MedCalc version 9.4.2.0 for Windows (Med-Calc Software, Mariakerke, Belgium). Rank correlationbetween AAT and CRP levels was calculated with theSpearman’s coefficient. Comparisons between the me-dians were performed with the Mann–Whitney test forindependent samples. Receiver operating characteristiccurve analysis was applied to patients heterozygous forZ deficient allele (PI*MZ). Haplotypes were analyzedwith the Hplus 2.5 web software (http://qge.fhcrc.org/hplus/).

RESULTS

Development of the assay to measure CRP in DBSfluid. We developed a convenient method to assayCRP in DBS fluid. The detection limit was 0.0020mg/dL. Within-assay precision estimates (variabilitycoefficient, VC) at a CRP concentration of 0.75 mg/dL was 0.6% and at 1.75 mg/dL was 1.3%. Thebetween-assay VC at 0.75 mg/dL was 5.4% and at1.75 mg/dL was 6.3%. Seventy-four paired DBS(assayed with the enzyme-linked immunosorbentassay method), serum/plasma samples (assayed with

the nephelometric method) were compared, and theCRP levels showed a linear relationship with a strongcorrelation (r2 5 0.9927; Fig 1).

Determination of AAT andCRP levels in DBS samples. Asa next step, and the major aim of the study, AAT andCRP levels were determined in DBS from filter papersubmitted for AATD diagnosis. A total of 362 consecu-tive samples were processed, and results of our pheno/genotyping analysis were as follows: 233 subjectswere diagnosed as normal (PI*MM), 107 subjectswere diagnosed as intermediate deficient (mostlyPI*MZ), and 22 were diagnosed as severely deficient(mostly PI*ZZ). Details are reported in Table I. Therelationship between AAT and CRP levels in allsamples is described in Fig 2. The Spearman’scoefficient of rank correlation was 0.299 (95%confidence interval 0.204 to 0.388); the weakcorrelation was related primarily to the geneticallydetermined variability in AAT levels. Inspection ofthis figure showed that most samples lay below theupper limit for both CRP and AAT (0.8 mg/dL and189 mg/dL, respectively). Several samples showeda simultaneous increase in both CRP and AAT values.Next, we stratified the subjects according to theirAATD status. As shown in Fig 3, in 2 genotypicalclasses, irrespective of their mean AAT plasma level,we observed values of CRP .0.8 mg/dL, denoting thepresence of an inflammatory condition, accompaniedby increased AAT plasma levels (P 5 0.0001 fornormal patients, P , 0.0001 for intermediate deficientpatients).In addition, we observed that 80% of subjects with in-

termediate AATD and a CRP .0.8 mg/dL had an AATlevel above the cut-off of 113 mg/dL (Fig 3, B). Thiscut-off previously was identified by our group as thelevel above which, under stable conditions, it wouldbe unlikely to detect a PI*MZ individual.22

CRP gene analysis. Finally, we decided to analyze,in our subjects, some polymorphisms previously

Fig 2. Correlation between CRP and AAT levels in DBS of 362 sam-

ples submitted for AATD diagnosis. Dots and dashes represent sub-

jects with CRP levels .0.8 mg/dL and CRP levels #0.8 mg/dL,

respectively. Red symbols: PI*MM individuals; blue symbols: sub-

jects with intermediate AATD; green symbols: subjects with severe

AATD. (Color version of figure available online.)

Table I. Genotype classes of 362 patients analysed for AATD and CRP

Genotype classes

Normal Intermediate deficiency Severe deficiency

No. 233 107 22genotype PI*MM (233) PI*MS (20) PI*ZZ (18)

PI*MZ (57) PI*ZMmalton (1)PI*MZausburg (1) PI*ZMprocida (1)PI*MI (1) PI*Q0claytonQ0clayton (1)PI*MMmalton (5) PI*Q0ouremQ0ourem(1)PI* MMprocida (3)PI* MMwurzburg (1)PI*MPlowell (1)PI*MQ0amersfoort (1)PI*MQ0clayton (1)PI*MQ0granite falls (2)PI*MQ0lampedusa (8)PI*MQ0pordenone (1)PI* MQ0isola di procida (5)

Translational ResearchVolume 157, Number 6 Ottaviani et al 335

reported to be associated with elevated CRP levels:12302G>A [rs 1205] in the 30 untranslated region;14899T>G [rs 3093077] in the downstream region;2390C>T>A [rs 3091244] in the promoter sequence,respectively, of the CRP gene. The 3 SNPs agreedwith the Hardy–Weinberg equilibrium. We did notobserve any evidence of an association betweenCRP genotype and CRP level in our series (Table II).This lack of association was confirmed by thehaplotype analysis (Table III). In fact, no associationwas found among any combination of the 3 CRPSNPs and the CRP level (CRP # 0.8 mg/dL orCRP . 0.8 mg/dL).

DISCUSSION

CRP has become an attractive biomarker for COPD.Despite several studies that replicate findings on its ele-vation and relationship with the outcome of the dis-ease,4,5 the practical impact of CRP measurement inthe clinical management of COPD is not clear. In thisstudy, we provide evidence that validates the value ofCRP measurement in the diagnostic algorithm forAATD in virtue of its relationship with systemicinflammation.We demonstrate here that it is now possible to imple-

ment CRP measurement in DBS, which is the matrixcurrently used by most centers in the laboratory diagno-sis of AATD.23 In fact, CRP may indicate the presenceof systemic inflammation, and therefore, the clinicianshould question the level of AAT measured as it mightnot strictly reflect the range based on the SERPINA1gene variant detected. Of particular interest is thefact that, if we strictly apply the AAT cut-off of113 mg/dL previously identified, then in the presenceof a systemic inflammatory state, approximately 80%of individuals carrying an intermediate AATD genotypewould be missed—mostly PI*MZ subjects. However,one can argue that, looking at Figs 2 and 3C, it wouldbe impossible to miss the diagnosis of severe AATDbecause, even in the presence of intense systemicinflammation, AAT levels cannot surpass thedecisional cut-off level. We obviously agree on thispoint, but in our opinion, one should bear in mind2 points. First, as demonstrated by our work, genotypesat risk of being missed by the diagnostic procedure arethose heterozygous for 1 normal (M) allele and 1 defi-cient allele (usually Z). An increasing body of evidencehas been forming that PI*MZ individuals are at slightly

Fig 3. Stratification of the individuals submitted to AATD diagnosis

according to plasmaCRP concentrations. Median, 75� and 25� percen-tiles, highest and lowest values of AAT for normal subjects (A), inter-

mediate deficient patients (B), and severe deficient patients (C),

stratified according to CRP#0.8mg/dL and.0.8mg/dL, respectively.

Black markers represent outliers. (§P 5 0.0001 for normal patients,§§P , 0.0001 for intermediate deficient patients).

Translational Research336 Ottaviani et al June 2011

higher risk of developing COPD, with respect to normalM carriers,24,25 and that they display a decline in lungfunction over time,26 especially if they smoke.27 Thus,if a patient is made aware that he carries a genetic riskfactor for developing COPD, then this informationcould provide the additional motivation to stop smok-ing.13 Interestingly, in samples without systemic inflam-mation, the missed diagnoses concerned mostlysubjects carrying the PI*MS genotype, who are not atrisk of developing COPD.28 Second, centers performinglaboratory diagnosis for AATD actually performgenetic testing; therefore, a missed diagnosis mighthave legal implications, especially in the context of ge-netic counselling.Two factors might influence the role of CRP as an

acute phase reactant (APR) indicator. It is well knownthat both CRP and AAT, although both considered acutephase reactants, actually behave differently. In fact,CRP belongs to the class of APRs that rapidly increase(up to 5-fold to 1000-fold), whereas AAT belongs to theclass of APRs that increase more slowly (2/3-fold).29,30

When DBS samples are shipped for AATD diagnosis,the handler is unaware of the presence or absence ofan acute inflammatory state in the patient. However,even in the remote case that the patient was in anacute illness at the moment of sample collection, itcould be supposed that, although CRP levels displaya rapid decrease, they remain above the normal limitfor a while. Assuming that most samples are shippedfrom patients under stable conditions, we shouldconsider that elevated CRP values reflect chronicstimulation. In this scenario, as in the case of COPDsubjects, APR levels remain elevated, but noinformation from longitudinal studies is found, to ourknowledge, on the differential behavior of APRs fromdifferent classes. The nonsignificant difference inAAT serum level in severe AATD patients witha CRP .0.8 mg/dL versus deficient subjects witha CRP # 0.8 mg/dL is probably a result of the lowAAT serum concentrations and small sample size.Nevertheless, the most important information ourresults provide is that intermediate AATD patients andnormal subjects showed increased AAT serum levelsduring an inflammatory state. These results underscorethe importance of using a biomarker to not missintermediate AATD patients when diagnostic testing isperformed in the presence of systemic inflammation.The second variable tested in this article was whether

CRP levels were influenced by individual factors. Infact, some reports seem to suggest that, in part, CRP var-iability could be ascribed to CRP gene variants. It hasbeen described that combinations of 4 CRP gene poly-morphisms (rs1205, rs1130864, rs3091244, and

Table II. Genotype frequencies in subjects with CRP # 0.8 mg/dL (group A) or CRP . 0.8 mg/dL (group B)

rs 1205 rs 3093077 rs 3091244

Group A Group B Group A Group B Group A Group B

GG 0.47 0.42 TT 0.89 0.90 CC 0.36 0.44GA 0.42 0.46 TG 0.11 0.10 CT 0.38 0.31AA 0.11 0.12 GG — — TT 0.20 0.23

CA 0.06 0.02

Table III. CRP haplotypes and serum CRP level

(group A 5 CRP#0.8 mg/dL, group B 5 CRP>0.8

mg/dL)

Haplotypes 1,2,3* Frequencies group A Frequencies group B

GTT 0.35 0.35ATC 0.31 0.35GTC 0.28 0.26GGA 0.03 0.01GGT 0.03 0.04

*1 5 rs1205; 2 5 rs3093077; 3 5 rs3091244.

Translational ResearchVolume 157, Number 6 Ottaviani et al 337

rs3093077) were associated with an increase in CRPlevels up to 64%.17 Kathiresan et al investigated 13CRP gene polymorphisms in their community-basedsamples and found that 12 clinical covariants accountedfor 26% of the interindividual variation in CRP, whereasa common triallelic CRP polymorphism (2390C>T>A[rs 3091244]) contributed only modestly because it wasassociated with only 1.4% of the total serum CRP vari-ation.15 Rhodes and his group found strong evidence forthe triallelic SNP alone in African American individ-uals. However, they noted that the effect was modest,contributing to only 5.2% of CRP variance.16 Brunneret al. described a strong association between commonmajor haplotypes and serum CRP levels.18 In our sam-ples, however, we found no evidence for an associationbetween CRP genotypes and/or haplotypes and CRPlevel. We propose that the contribution of the 3 CRPgene polymorphisms analyzed here, which are themost frequent ones associated with interindividual var-iability in CRP level, is irrelevant, when we consider the2 broad categories of subjects with CRP .0.8 mg/dLand #0.8 mg/dL.Despite the interesting results, we are aware that this

article has some limitations. Because of the nature ofthe screening procedure, clinical data of individualsundergoing AATD diagnosis are lacking, especiallysmoking status and lung function, andwe could not inves-tigate how they correlatewith systemic inflammatory sta-tus. Again, as discussed, some discrepancies occurredamong our CRP genotyping data and those reported inthe literature, showing a genetic influence on CRP level.

Discordancemight be a result of the different populationsand sample size investigated; however, and more impor-tantly, even in these reports, the individual influence ismuch less relevant than the systemic inflammatory status.In conclusion, in this study, we demonstrated that a sys-

temic inflammatory state, as suggested by the presence ofserumCRP levels.0.8mg/dL, is paralleled by increasedlevels of AAT and that such an increase might mask thepresence of AATD variants. Thus, during the laboratorydiagnostic procedure for AATD, CRP determinationand in turn the level of systemic inflammation, wouldbe useful to improve the accuracy of genetic testing.

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