PredictAAT: Accounting for Inflammation in the Diagnosis of Alpha 1 Antitrypsin Deficiency

Abstract

Alpha 1 antitrypsin deficiency (AATD) is a rarely diagnosed hereditary condition characterized by low alpha 1 antitrypsin (AAT) levels, which can lead to early-onset emphysema due to accelerated degradation of lung tissue. Similar to C-reactive protein (CRP), AAT is an acute phase reactant, meaning that blood levels rise in response to inflammation, injury or infection. Testing AAT levels is essential in the diagnosis of AATD; however, the presence of inflammation at the time of AATD testing can provide a false ‘normal’ level reading of the patient’s baseline AAT levels. Researchers from a US-wide screening program for AATD found that a large number of individuals with AATD variants (particularly with the PI*MZ genotype) presented with elevated CRP levels (≥5 mg/L), suggesting the presence of inflammation. Using a series of calculations, the relationship between AAT and CRP levels was characterized and found to be genotype specific. We have developed a publicly available, easy-to-use online calculator (PredictAAT) that enables the instant calculation of predicted baseline AAT levels in patients exhibiting elevated CRP levels that accounts for specific AATD genotype. There is a need to raise awareness of the importance of simultaneous determination of AAT and CRP levels to aid the accurate diagnosis of patients with AATD. The PredictAAT calculator is therefore a valuable tool for physicians to enhance early disease diagnosis and individualize treatment.

Keywords:

• Alpha 1 Antitrypsin Deficiency
• C-reactive protein
• multivariate analysis
• genotype
• variants
• algorithm

Article type: Commentary/perspectives (Based on Sanders, Ponte and Kueppers. COPD 2018;15:10-16. doi: 10.1080/15412555.2017.1401600)

Introduction

Alpha-1 Antitrypsin Deficiency (AATD) is a rare hereditary condition characterized by low Alpha 1 Antitrypsin (AAT) levels which can lead to early-onset of chronic obstructive pulmonary disease (COPD)/emphysema through the degradation of lung tissue [1]. Interest in AATD has grown significantly since its original description in 1963 [2] and AAT has become one of the best characterized human proteins, which is reflected in approximately 11,000 publications that focus on AAT in the PubMed database [3].

The original description of AATD was based on quantitative measurements of AAT concentration in plasma and the reduction of functional trypsin inhibiting activity [2]. With the recognition of the structural basis of the major deficiency, research interest has shifted to the investigation of mutations and sequence variations that could explain decreased elastase inhibiting activity, and consequently, lung and other tissue pathology. To date, over 500 single-nucleotide variants (SNVs) have been reported and over 70 sequence variants have been identified as clinically relevant [4]. However, the association with long-term disease risk, in terms of pathogenicity, is unknown or subject to debate for most rare/novel variants [4].

While the variability of the trypsin inhibiting activity of AAT in peripheral blood has been known for some time, the role of AAT in immunological processes has only been identified more recently [5]. AAT is an acute phase reactant protein (APRP), meaning that blood levels of the protein can rise by 75–100% in response to inflammation, injury or infection [6]. Normal levels of AAT range between 88.0–136.9 mg/dL [7] and during an acute phase response, AAT can rise to over 160 mg/dL [6]. The concentration of numerous APRPs increases together in a predictable manner during an acute phase response [8]. One protein that is a commonly used as an indicator of an acute phase reaction is C-reactive protein (CRP), levels of which can increase from between 0.8–10 mg/L (usually below 3.0 mg/L) up to 1000-fold in response to tissue damage [9]. For example, CRP plasma levels have been seen to increase from approximately 1 mg/L to over 500 mg/L within 24–72 h of severe tissue damage such as trauma and progressive cancer [10]. CRP belongs to the class of APRPs that can rapidly increase, whereas AAT belongs to the class of APRPs that increase more slowly; hence CRP is a stronger indicator of an acute phase response than AAT [11]. In the clinical setting, CRP has long been used as an inflammatory indicator, and due to its wide use in clinical testing, it is ideally suited as a benchmark for levels of other APRPs to be evaluated against.

The effects of inflammation on AAT levels

To further evaluate the effect of inflammation on AAT protein levels, we assessed the quantitative relationship between AAT and CRP in a group of over 10,000 patients included in a national AATD screening program [6]. In this study, blood samples from patients who were suspected of having AATD were analyzed to determine AAT genotype/phenotype (PI*S, Z or F), as well as AAT and CRP levels. This study provided an opportunity to observe the relationship between AAT and CRP over a wide range of concentrations. Figure 1 shows this relationship for carriers of the common deficiency alleles (PI*MZ and PI*MS) as related to normal and elevated CRP levels [6]. Using a series of calculations and regression models, the relationship between levels of AAT and CRP was characterized and found to be genotype-specific. It was established that the presence of inflammation at the time of AATD testing can obscure the diagnosis because of an elevated level of AAT [6].

Figure 1. AAT level distribution according to genotype (PI*MM, PI*MS and PI*MZ) in those with (dashed lines) and without (solid lines) the presence of inflammation, defined as CRP ≥5 mg/L and <5 mg/dL, respectively.

Reproduced from Sanders, Ponte and Kueppers. COPD 2018;15:10-16 [6]

Several studies have identified that inflammation, indicated by elevated CRP levels, can affect observed AAT levels in genotypes associated with intermediate deficiency [7, 11, 12]. Other studies have employed multivariate analyses focusing on CRP adjustment to calculate a more accurate AAT level range that can be used as a reference marker for different genotypes [7, 13].

Although, individuals homozygous for the Z variant (PI*ZZ) only show a minimal quantitative acute phase response in terms of AAT level elevation, heterozygous carriers for a deficient AAT allele (PI*MZ) retain a substantial acute phase response [6, 14]. In the acute phase, AAT levels in these individuals rise to the quantitative range typical for AAT-normal individuals (PI*MM) (Figure 2) [14]. A study by Ottaviani et al. concluded that this may impact clinical diagnosis of the disease by potentially masking the presence of AATD variants [11], and that determining AAT and CRP levels simultaneously may be beneficial in the identification of intermediate-deficient AAT variants [11].

Figure 2. Changes in AAT levels of serum following the intravenous injection of 0.2 ml typhoid-paratyphoid vaccine (arrow) in the genetically different individuals. Homozygotes for common gene: solid line, heterozygotes for AATD gene: dashed line, homozygotes for deficiency gene: dotted line. At the right: standard error of the method.

Reproduced from Kueppers, Humangenetik 1968;6:207-14 [14]

Approximately a third of individuals with AATD variants (particularly with the PI*MZ genotype) presented with elevated CRP levels (≥5 mg/L), suggesting the presence of inflammation. Inflammation significantly masked the clinically relevant base AAT levels in some PI*MZ individuals by presenting higher results than would be observed in the absence of inflammation. Following the application of adjustment calculations to all patients with elevated CRP, some patients with AAT levels recorded as ‘normal’ were re-categorized to ‘low’ (<80 mg/dL), based on the thresholds specified by the American Thoracic Society/European Respiratory Society [15]. Overall, 40 patients (0.4% of patients in the study) with apparent normal AAT levels were adjusted to AAT <80 mg/dL, 77.5% of whom had the PI*MZ genotype. Patients with PI*ZZ genotypes displayed no correlation between CRP and AAT levels due to levels of the Z protein rising minimally in response to inflammation [6]. This could be attributed to the mechanism underpinning the disorder itself, i.e. polymerization of deficient AAT in hepatocytes preventing its secretion into the systemic circulation.

Algorithm for the adjustment of baseline AAT levels in acute phase [6]

Based on these observations, an algorithm has been developed that correlates the levels of CRP, AAT and genotype using a complex regression model [6]. The algorithm has been condensed into a publicly available, easy-to-use online calculator that enables the instant calculation of predicted AAT levels. The PredictAAT calculator can be found at the webpage: https://predictaat.us. The user is asked to enter the levels of AAT (mg/dL) and CRP (mg/L) and the patient’s genotype in the appropriate fields and the corrected AAT level appears in a third box.

The validity of the algorithm has been verified by comparing the AAT levels in individuals with normal CRP levels to those calculated using the algorithm (Table 1) [6]. The adjusted levels of AAT calculated using the predictive algorithm showed significant agreement with observed AAT levels (p = 0.0001), thus demonstrating the accuracy of the algorithm.

Table 1. Comparison of observed and adjusted AAT levels calculated using the algorithm in the Sanders et al. cohort [6].

	Levels of Alpha -1 Antitrypsin mg/dL
Genotype	Initial Level	Level of AAT with normal CRP	Adjusted level using the predictor algorithm
PI*MM	162	142	140
PI*MS	136	117	115
PI*MZ	104	85	89
PI*MF	161	132	141
PI*SS	115	96	91
PI*SZ	66	54	50

Adapted from Sanders, Ponte and Kueppers. COPD 2018;15:10-16.

Significance and clinical context

Genotype-specific relationships between AAT and CRP were determined and a quantitative method for the adjustment of acute phase response levels of AAT based on elevated CRP was established.

AAT levels increase differently in the acute phase according to genotype, requiring genotype-specific adjustment of AAT levels. When AATD is suspected, genotyping is fundamental to achieve an accurate diagnosis and must be conducted prior to adjusting AAT level for inflammation. Physicians should be made aware of the potential for AAT levels to be elevated when inflammation occurs due to its nature of an acute phase reactant. It is recommended that if normal AAT levels are observed in an individual suspected of having AATD who exhibits signs of inflammation, i.e. elevated CRP levels, the individual is retested when CRP levels returns to normal. Use of the predictive algorithm can provide an instant indication of “true” AAT levels at baseline, and may, therefore, facilitate earlier intervention in patients with AATD or those with suspected AATD via recommendations such as smoking cessation and other lifestyle changes [6].

It’s application is most useful in carriers for a deficiency allele (such as MZ, SZ, SS) and other deficiency allele combinations with normal levels during an acute phase reaction, and can be helpful to decide whether augmentation therapy is indicated. We believe that the behavior of AAT during an acute phase is of interest, particularly if there is a discrepancy with the level of CRP and other APRPs. Online predictor tools have been shown to be successful and beneficial tools for physicians in other clinical settings. For example, in hematology, the WAPPS-Hemo calculator that estimates individualized pharmacokinetic values with different factor concentrates for patients with hemophilia A and B is currently used by 472 hemophilia treatment centers worldwide and has over 7000 registered patients [16, 17].

Conclusion

Testing AAT levels is important in the diagnosis of AATD; however, the presence of inflammation at the time of testing can cause bias. During an acute phase, levels of AAT can present higher than at baseline leading to the interpretation that AAT levels in the individual are above the threshold considered for AATD diagnosis. Thus, intervention with pharmacological and/or non-pharmacological therapies to slow disease progression may be delayed. Raising physician awareness of the need for simultaneous determination of AAT and CRP levels, in addition to genotypic/phenotypic analysis, is required. The PredictAAT calculator provides genotype-specific adjusted AAT levels in patients with elevated CRP levels. This publicly available online resource is a valuable tool for physicians to establish early disease diagnosis and individualized treatment.

Acknowledgments

Medical writing assistance was provided by Alice Walsh of Meridian HealthComms Ltd, Plumley, UK.

Declaration of interest

The author reports no conflicts of interest.

Additional information

Funding

CSL Behring funded medical writing assistance for preparation of this article. The sponsor had no role in the writing of the manuscript; and in the decision to submit the manuscript for publication.