Serum concentrations of A disintegrin and metalloproteinase 9 (ADAM9) mRNA as a promising novel marker for the detection of pulmonary sarcoidosis

Abstract

Aims

This prospective study investigated the potential role of serum A disintegrin and metalloproteinase 9 (ADAM9) mRNA in the differential diagnosis of pulmonary sarcoidosis from other diseases with similar clinical presentations, such as tuberculosis (TB).

Methods

Serum samples were collected from patients with sarcoidosis, and compared with those from patients with TB and healthy control subjects. Real-time quantitative reverse transcription–polymerase chain reaction was performed to measure the concentration of ADAM9 mRNA relative to the internal control, glyceraldehyde-3-phosphate dehydrogenase.

Results

A total of 100 patients with sarcoidosis were compared with 50 patients with TB and 50 healthy control subjects. The serum concentration of ADAM9 mRNA in patients with sarcoidosis was significantly lower than that in healthy control subjects and patients with TB. The serum concentration of ADAM9 mRNA in patients with TB was significantly higher than that in healthy control subjects.

Conclusion

These data suggest that serum ADAM9 mRNA might be a potential biomarker for the differential diagnosis of pulmonary sarcoidosis.

Introduction

Sarcoidosis, a systemic inflammatory and noncaseating granulomatous disease of unknown origin affecting multiple organs and invariably the lung, typically undergoes spontaneous resolution.^1,2 The clinical presentation of sarcoidosis is highly variable. ³ A chronic course occurs in 10–30% of patients, at times resulting in significant impairment of lung function.^4,5 In addition, sarcoidosis is a diagnosis of exclusion because it shares similarities with the clinical presentations of other lung diseases (such as tuberculosis [TB], fungal infections, lung cancer and cryptogenic organizing pneumonia) which results in a substantial challenge in differentiating sarcoidosis from other granulomatous lung diseases, especially in cases of smear-negative TB.^6–8 Sarcoidosis biomarkers are urgently needed, to deliver targeted therapies in individuals with complicated sarcoidosis and to identify patients at risk for other lung diseases.

One of the first A disintegrin and metalloproteinase (ADAM) proteins to be identified and characterized, ADAM9, is a membrane-anchored metalloproteinase with an N-terminal prodomain followed by a metalloprotease domain, a disintegrin domain and cysteine-rich region, an epidermal growth factor repeat, a transmembrane domain and a cytoplasmic tail with potential Src homology 3 ligand domains. ⁹ Upregulated levels of ADAM9 mRNA and/or protein have been described in various diseases and have often been associated with adverse prognostic parameters or shorter patient survival times.^10–12 Levels of ADAM9 mRNA were shown to be significantly downregulated in bronchoalveolar lavage specimens from patients with pulmonary sarcoidosis compared with healthy control subjects, which suggested that this protein might act as a biomarker of pulmonary sarcoidosis. ¹³ The aim of the current study was to investigate the potential use of the serum ADAM9 mRNA concentration as a novel biomarker for the diagnosis of pulmonary sarcoidosis, using real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR).

Patients and methods

Study population and serum collection

This prospective study enrolled consecutive patients with pulmonary sarcoidosis and patients with TB who were being treated at the Department of Respiratory Diseases, Third Affiliated Hospital, Liaoning Medical University, Jinzhou, Liaoning Province, China between April 2009 and December 2012. There were no exclusion criteria for patients with pulmonary sarcoidosis or TB. Control subjects were recruited consecutively from among individuals who sought a routine health check-up at the Physical Health Examination Centre, Third Affiliated Hospital, Liaoning Medical University. Healthy control subjects were all nonsmokers with no history of lung disease, and were confirmed to be healthy on the basis of the results of chest radiography and bronchoscopy. The study was approved by the Ethics Committee of the Third Affiliated Hospital, Liaoning Medical University (no. 2012-LY-118) and written informed consent was obtained from all study participants.

A 5-ml sample of peripheral venous blood was drawn from all study participants after an overnight fast and placed at room temperature for 60 min. After centrifugation at 700 g for 15 min at 4℃, 0.5 ml aliquots were stored at −80℃ until they were used for real-time quantitative RT–PCR analysis.

Real-time quantitative RT–PCR

Total RNA was isolated from 200 μl of serum using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The quality and quantity of the RNA were verified using a NanoDrop 2000 instrument (Thermo Fisher Scientific, Wilmington, DE, USA).

Using 1 µg of RNA as a template, single-stranded cDNAs were generated by SuperScript® II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Real-time quantitative PCR experiments were conducted with an ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and SYBR® Green PCR Master Mix (Invitrogen) according to the manufacturers’ protocol (ADAM9 forward, 5′-TGCCCCCAAAGATTGTTTCAT-3′; reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′ [TaKaRa, Dalian, China]). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. The GAPDH primer sequences were as follows: forward, 5′-ACGGATTTGGTCGTATTGGG3′; reverse, 5′-ATCACCATCTTCCAGCG-3′ (TaKaRa). Each sample was normalized on the basis of its GAPDH mRNA content. The cycling programme involved preliminary denaturation at 95℃ for 2 min, followed by 40 cycles of denaturation at 95℃ for 15 s and annealing at 59℃ for 1 min. Analysis of the concentration of ADAM9 mRNA relative to the internal control GAPDH mRNA was calculated using the ΔΔC_T method, following the manufacturer’s guidelines. Data analysis was performed using ABI PRISM® 7900HT Sequence Detection System software (Applied Biosystems).

Statistical analyses

All statistical analyses were performed using the SPSS® software package, version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. The mean ± SD mRNA concentrations (copies/µg RNA) for the three groups were compared using the Mann–Whitney U-test. A P-value < 0.05 was considered statistically significant.

Results

A total of 100 patients with pulmonary sarcoidosis (57 male and 43 female; mean ± SD age 44.2 ± 3.6 years) and 50 patients with TB (30 male and 20 female; mean ± SD age 46.1 ± 2.7 years) were enrolled in the study. A total of 50 healthy control subjects (22 male and 28 female; mean ± SD age 39.8 ± 3.2 years) formed the healthy control group.

Real-time quantitative RT–PCR was undertaken to measure the concentration of ADAM9 mRNA in the serum from patients with sarcoidosis, patients with TB, and healthy control subjects. As shown in Table 1, the concentration of ADAM9 mRNA in the serum of patients with sarcoidosis was significantly lower than that in patients with TB and healthy control subjects (P < 0.05). In addition, the concentration of ADAM9 mRNA in the serum of patients with TB was significantly higher than that in the healthy control subjects (P < 0.05).

Table 1.

Relative concentrations of A disintegrin and metalloproteinase 9 (ADAM9) mRNA in the serum of patients with pulmonary sarcoidosis, patients with tuberculosis (TB) and healthy control subjects, as determined using real-time quantitative reverse transcription–polymerase chain reaction.

Group	n	mRNA copies/µg RNAa
Patients with pulmonary sarcoidosis	100	17718.97 ± 10354.21b
Healthy control subjects	50	30423.15 ± 143315.80
Patients with TB	50	51974.28 ± 267081.74c

Data presented as mean ± SD.

a

Glyceraldehyde-3-phosphate dehydrogenase served as the internal control. The concentration of ADAM9 mRNA relative to the internal control was calculated using the ΔΔC_T method.

b

P < 0.05 compared with patients with TB and healthy control subjects; Mann–Whitney U-test.

c

P < 0.05 compared with healthy control subjects; Mann–Whitney U-test.

Receiver operating characteristic (ROC) curve analyses revealed that the concentration of serum ADAM9 mRNA was a valuable biomarker for differentiating patients with sarcoidosis and healthy control subjects, with an area under the ROC curve (AUC) of 0.803 (95% confidence interval [CI] 0.668, 0.891; Figure 1A), which indicated that ADAM9 was a disease-associated mRNA for sarcoidosis. Moreover, ROC curve analyses also showed that the concentration of serum ADAM9 mRNA was a valuable biomarker for differentiating patients with TB and healthy control subjects, with an AUC of 0.802 (95% CI 0.691, 0.885; Figure 1B), which implied that the concentration of serum ADAM9 mRNA might act as a biomarker for differentiating patients with TB and healthy control subjects.

Figure 1.

Receiver operating characteristic curves showing the ability of serum A disintegrin and metalloproteinase 9 (ADAM9) mRNA to discriminate between patients with pulmonary sarcoidosis and healthy control subjects (a) and to discriminate between patients with tuberculosis (TB) and healthy control subjects (b). Serum ADAM9 mRNA yielded an area under the receiver operating characteristic curve (AUC) of 0.803 (95% confidence interval [CI] 0.668, 0.891) with 75% sensitivity and 75% specificity (a). Serum ADAM9 mRNA yielded an AUC of 0.802 (95% CI 0.691, 0.885) with 76% sensitivity and 79% specificity (b).

Discussion

The diagnosis of sarcoidosis remains a substantial problem due to the need to exclude other lung diseases, especially TB, because multiple lung diseases share similar clinical presentations. The present study demonstrated that the concentration of ADAM9 mRNA in the serum of patients with sarcoidosis was significantly lower than that in healthy control subjects and patients with TB, which was consistent with ourprevious finding that ADAM9 mRNA levels in bronchoalveolar lavage samples were significantly downregulated in patients with pulmonary sarcoidosis compared with healthy control subjects. ¹³ In addition, the present results showed that the concentration of ADAM9 mRNA in the serum from patients with TB was significantly higher than that in healthy control subjects. These findings suggest that the concentration of ADAM9 mRNA in serum could be a useful biomarker for the diagnosis of pulmonary sarcoidosis and TB and also for the differential diagnosis of TB and pulmonary sarcoidosis.

Upregulated levels of ADAM9 mRNA have been associated with adverse prognostic parameters or shorter patient survival times.^{10–12,14,15} The present results were in accordance with these findings and demonstrated a prognostic value for serum concentrations of ADAM9 mRNA for pulmonary sarcoidosis. However, upregulation of ADAM9 mRNA has been described in a range of solid tumours including lung cancer,^{10–12,14–16} which seems to be incompatible with these current results. This inconsistency might be due to ADAM9 playing different biological roles in different types of disease, due to its involvement in various biological processes ¹⁷ such as: the regulation of the production of laminin, vitronectin, collagen types I and IV; ¹⁸ the regulation of cell–cell and cell–matrix interactions; ¹⁸ and the maintenance of the normal morphology and functions of cells through interactions between its disintegrin domain and the α1β1, α3β1, α6β1 and αvβ1 proteins in the β1 integrin family. ¹⁹ However, the detailed functions of ADAM9 and its effects in pulmonary sarcoidosis remain largely unknown, therefore further research is required.

In conclusion, the current study demonstrated that the concentration of serum ADAM9 mRNA was significantly downregulated in patients with pulmonary sarcoidosis in comparison with healthy control subjects and patients with TB, suggesting that decreased ADAM9 mRNA levels might be a useful diagnostic marker for the differential diagnosis of pulmonary sarcoidosis.