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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 3  |  Page : 780-785

Serum level of surfactant protein-D and its genetic polymorphisms in chronic obstructive pulmonary disease patients


Department of Biochemistry, Faculty of Medicine, Menoufia University, Shebin Elkom, Egypt

Date of Submission31-May-2017
Date of Acceptance13-Aug-2017
Date of Web Publication31-Dec-2018

Correspondence Address:
Manal A Ellaithy
Department of Biochemistry, Faculty of Medicine, Menoufia University, Shebin Elkom
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_406_17

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  Abstract 


Objective
The aim of this study was to study the serum level of surfactant protein-D (SP-D) and its genetic polymorphisms in chronic obstructive pulmonary disease (COPD) patients and its relation with severity.
Background
SP-D plays a role in COPD progression. It contributed toward the inflammatory response in the lungs. Single nucleotide polymorphisms of the surfactant protein-D gene (SFPTD) are believed to be associated with the risk of COPD.
Patients and methods
A case–control study was carried out on 76 individuals: group I included 50 patients with COPD and group II included 26 age-matched and sex-matched apparently healthy individuals. All participants were subjected to an assessment of history, clinical examination, BMI estimation, spirometry, serum SP-D by enzyme-linked immunosorbent assay, and genotyping of single nucleotide polymorphisms of SFPTD gene (rs721917, rs3088308) using the TaqMan allelic discrimination assay by real time PCR.
Results
Serum SP-D was significantly higher in patients than controls. The CT genotype of rs721719 was significantly more prevalent in patients. The T allele of both rs3088308 and rs721917 and smoking index were associated with increased SP-D levels. The T allele of rs721917 was associated with the risk of COPD. There was a significant difference between different genotypes of rs721917 in forced expiratory volume in 1 s (FEV1), forced volume capacity (FVC), FEV1/FVC, and serum SP-D.
Conclusion
SP-D can be used as a biomarker of the severity of COPD. The T allele of rs721917 might be a susceptible gene for COPD. The T allele of rs3088308 was associated with elevated serum SP-D, but not with COPD.

Keywords: chronic obstructive pulmonary disease, polymorphism, surfactant protein-D


How to cite this article:
Ghaniam NM, Badr IA, Dawood AA, Elshazly RM, Elwahsh RA, Ellaithy MA. Serum level of surfactant protein-D and its genetic polymorphisms in chronic obstructive pulmonary disease patients. Menoufia Med J 2018;31:780-5

How to cite this URL:
Ghaniam NM, Badr IA, Dawood AA, Elshazly RM, Elwahsh RA, Ellaithy MA. Serum level of surfactant protein-D and its genetic polymorphisms in chronic obstructive pulmonary disease patients. Menoufia Med J [serial online] 2018 [cited 2019 Jun 16];31:780-5. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/780/248745




  Introduction Top


Chronic obstructive pulmonary disease (COPD) is a common inflammatory disease characterized by chronic obstruction of airflow that is not fully reversible[1]. Pulmonary surfactant is a complex mixture of lipids (90%) and proteins (10%) that constitutes the mobile liquid phase covering the large surface area of the alveolar epithelium. It maintains minimal surface tension within the lungs to avoid lung collapse during respiration. Four surfactant proteins (SPs): SP-A, SP-B, SP-C, and SP-D are strongly associated with surfactant lipids in the lung[2].

SP-D plays an important protective role in the immune system against inhaled microorganisms and allergens. It plays a role in protection against viral, bacterial, and fungal infections, as well as apoptotic cells[3]. SP-D, a lung-derived innate immune protein, may be an important effecter molecule in the pathogenesis of COPD[4].

Within the SFTPD gene, the three single nucleotide polymorphisms (SNPs) Met11Thr, Ala160Thr, and Ser270Thr are of potential functional relevance[5]. Rs721917 is a missensesubstitution that leads to the replacement in position 11 of an ancestral methionine (Met) by a threonine (Thr) (Met11Thr). Patients carrying the TT genotype (Met/Met) have increased concentrations of SP-D in the plasma and show multimers, dodecamers, and monomers of subunits, whereas CC-carriers (Thr/Thr) produce almost exclusively monomers[6].

The SNP rs3088308 causes a substitution of nucleotide adenine (A) in place of a thymidine (T) in the seventh exon of the SFTPD gene, leading to the replacement of serine (Ser) by threonine (Thr) at position number 270 in the carbohydrate recognition domain of the SP-D protein molecule. This residue corresponds to the carbohydrate recognition domain of the mature protein that binds various pathogens as well as apoptotic cells[7].

The aim of this study was to evaluate the serum level of SP-D and its genetic polymorphism in COPD patients and their relation to the severity of the disease.


  Patients and Methods Top


Patients

This case–control study was carried out at Medical Biochemistry and Chest Diseases and Tuberculosis Departments, Faculty of Medicine, Menoufia University. The study was carried out on 76 individuals. The patients were attendants of the inpatient and outpatient clinic in the Chest Diseases and Tuberculosis Department of Menoufia University Hospitals during the period from April 2014 to April 2015 (convenience sampling).

The participants studied were divided into two groups.

Group I included 50 patients with COPD. There were 43 men and seven women.

The diagnosis of COPD was made according to a clinical history suggestive of COPD and spirometry if the ratio of post bronchodilator (forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) greater than 0.7 and subclassified according to severity of airflow obstruction as follows. Mild: FEV1 less than 80% predicted; moderate: FEV150–79% predicted; severe: FEV130–49% predicted; and very severe: FEV1 less than 30% predicted[8].

Group II included 26 age-matched and sex-matched apparently healthy individuals. There were 18 men and eight women.

The exclusion criteria were as follows: acute exacerbation of COPD, hypertension, coronary artery disease, heart failure, a history of stroke, diabetes mellitus, chronic pulmonary disease (other than COPD), and chronic renal disease.

A written informed consent was obtained from all participants before participant characterization and sample collections. This study was approved by the Ethical Committee of Medical Research, Menoufia Faculty of Medicine.

Methods

Each participant was subjected to a full assessment of history including smoking history, general examination, and local chest examination, estimation of BMI by dividing body weight (kg) by the height in square meter (m2)[9], estimation of smoking index using pack-year index, number of packs in day multiplied by the number of years being smoking[10], spirometric measurements (pre and post bronchodilators) using a spirometer (Spiroanalyzer ST-90 supplied by Fukuda Sangyo, Tokyo, Japan), laboratory investigations including the serum SP-D level, which was determined by the enzyme-linked immunosorbent assay, and genotyping of two SNPs in the SFPTD gene (rs721917 and rs3088308) using real time PCR.

Sample collection

A volume of 5 ml of venous blood was collected from all participants included in this study by veni puncture from the cubital vein and collected as follows: 2.5 ml was collected into EDTA-containing tubes for genotyping of the SFPTD gene. A volume of 2.5 ml was collected in another plain tube, left for 10 min for coagulation, centrifuged at 3000 rpm for 10 min, and then the sera were separated into aliquots for the measurement of SP-D by enzyme-linked immunosorbent assay.

Assay

The quantikine kit for human SP-D immunoassay, catalog number DSFPD0 (R & D Systems Inc., Minneapolis, Minnesota, USA). GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Fisher Scientific Inc., V. A. Graiciuno, Vilnius, Lithuania)[11], was used.

Single nucleotide polymorphism genotyping of SFPTD (rs721917) and (rs3088308)

Genomic DNA was extracted from frozen EDTA-treated blood samples using the GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Fisher Scientific Inc.).

Detection of single nucleotide polymorphism

Single nucleotide polymorphism assay kit for rs721917

TaqMan genotyping assay kit contained both primers and probes as follows: the forward primer: 5'-CCCCATAGCAGAGGACAGAA-3', the reverse primer (5'-CCAGGGTGCAAGCACTGGAC-3'), and probe (VIC/FAM) (5'-CATGACCAGGGTG CAAGCACTGGGC[C/T]TTGTTCTGT GGGAGTAGGTCTTCAT-3').

Single nucleotide polymorphism assay kit for rs3088308

TaqMan genotyping assay kit contained both primers and probes as follow: the forward primer (5'-ACGGAGGCACAGCTGCTG-3'), the reverse primer (5'-GGAAAGCAGCCTCGTTCT-3'), and probe (VIC/FAM) (5'-GCATTCTCAGCGGCA GAGCGTGGAG[A/T]GGCCAACTGT CCACCAGCCTGTGTG-3').

The DNA extract was used for SNP assay in a total reaction volume of 20 μl with 10 μl of TaqMan Genotyping Master Mix purchased from (Thermo Fisher Scientific Inc.). It is a universal ready-to-use solution composed of Hot Start Taq DNA polymerase and dNTPs in an optimized PCR buffer, 1.25 μl of 20× TaqMan genotyping assay kits containing both primers and probes and nuclease-free water. Using the 7500 real time PCR system (Applied Biosystems, Foster City, California, USA), the reaction conditions were as follows: 50°C for 1 min pre-PCR read, and then 95°C for 10 min and 45 cycles of 95°C for 15 s, 60°C for 1 min (cycling), and 60°C for 1 min (post-PCR).

Statistical analysis

The data were analyzed using the statistical package for the social sciences (IBM SPSS, version 20; IBM Corp, Armonk, New York, USA). Quantitative data were expressed as mean ± SD and analyzed using a t- test for comparison between two groups of normally distributed variables, whereas for comparison between two groups of not normally distributed variables, the Mann–Whitney test was used. Qualitative data were expressed as number and percentage, and analyzed using the χ2-test. A P value of less than 0.05 was considered statistically significant. The odds ratio describes the probability that individuals who are exposed to a certain factor will have a disease comparable to individuals who are not exposed to the factor. The Kruskal–Wallis test was used for comparison between three or more groups with non-normally distributed quantitative variables. Binary logistic regression is a special kind of multiple linear regression that measures the relationship between the categorical-dependent variable and one or more independent variables.


  Results Top


The current study showed that group I had significantly lower mean values of (FEV1, FVC and FEV1/FVC) and serum SP-D levels than group II (P < 0.001). However, there was a nonsignificant statistical difference between the two groups in the smoking index [Table 1].
Table 1: Statistical comparison between the studied groups in spirometric data (FEV1, FVC, FEV1/FVC), serum surfactant protein-D level, and smoking index

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For rs721917, the CT genotype was significantly more prevalent in group I than group II, whereas for rs3088308, there was a nonsignificant statistical difference in genotypes and alleles [Table 2].
Table 2: Statistical comparison between the groups studied in rs721917 and rs3088308 genotypes and allele frequencies

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There was a significant difference between different genotypes of rs721917 in (FEV1, FVC, and FEV1/FVC) and serum SP-D [Table 3].
Table 3: Statistical comparisons between different rs721917 genotypes in the groups studied in FEV1%, FVC, FEV1/FVC predicted, serum SP-D, and smoking index

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There was a significant difference between different genotypes of rs3088308 for serum SP-D, whereas there was a nonsignificant difference in the other parameters [Table 4].
Table 4: Statistical comparisons between different rs3088308 genotypes in the studied groups in FEV1%, FVC, FEV1/FVC predicted, serum SP-D, and smoking index

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In the regression model, the increasing level of SP-D was associated with an increase of the likelihood of COPD. Also, the presence of TT in rs721917 was 20-fold more likely to lead to COPD [Table 5]. The C allele of rs721917 was associated significantly with decreased SP-D (dependant) level (β = −33.39, P < 0.001), whereas the T allele of both rs3088308 (β = 19.45, P = 0.003) and rs721917 (β = 12.92, P = 0.04) and the smoking index (β = 0.31, P = 0.04) were associated with increased SP-D levels (not shown).
Table 5: Binary logistic regression analysis to detect risk factors of chronic obstructive pulmonary disease

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  Discussion Top


COPD is characterized by progressive airflow obstruction that is only partly reversible; disease is further aggravated by exacerbations, particularly in patients with severe disease, up to 78% of which are because of bacterial infections, viral infections, or both[12].

Surfactant is the first barrier that pathogens encounter within one of the largest exposed surfaces of the human body[13].

Pulmonary surfactant is a complex mixture of lipids and proteins, and lipids account for more than 90% of the surfactant. However, the protein composition is critical for normal surfactant function. SP-A and SP-D belong to the collectin protein family. They are directly related to the innate host defense of the lung and recognize, bind, and eliminate pathogens[14].

The aim of this study was to detect the value of serum level of SP-D and its genetic polymorphism in COPD patients and their relation with the severity of the disease.

This study showed that there was a statistically significant increase in serum SP-D in patients with COPD compared with controls. This is in agreement with the results of Shakoori et al.[4], El-Deek et al.[15], and Moreno et al.[16]. Shakoori et al.[4] reported that alveolar damage in COPD causes leakage of SP-D from the pulmonary compartment into the systemic circulation, resulting in lowered bronchoalveolar lavage fluid, but high serum SP-D levels.

For rs721917 genotypes, the CT genotype and the T allele were significantly more prevalent in patients than in controls. This is in agreement withShakoori et al.[4] andJohansson et al.[17], who reported that the T allele is a susceptible gene for COPD. In contrast, Foramen et al.[18] and Shakoori and Sin[7] reported lack of a significant association between rs721917 and lung function. Differences in the findings may be explained by differences in the genetic and ethnic backgrounds of the groups studied.

For rs3088308 genotypes, this study showed that there was no statistically significant difference between both groups. This is in agreement with Shakoori et al.[4] and Johansson et al.[17], who found that rs33088308 is associated with serum SP-D, but there was no association or significant correlation with lung function. Shakoori and Sin[7] reported in their study a similar association between rs3088308 with serum SP-D in healthy smokers. In contrast, they found in their study that most of the lung function variables were significantly higher in patients carrying the major allele. This difference between their results and those of the current study may be explained by different group sizes and backgrounds of the patients.

The current study showed that there was a significant difference between different genotypes of rs721917 in terms of spirometric measurements (FEV1, FVC, and FEV1/FVC), being the lowest in the CT genotype. This is in agreement withShakoori and Sin[7], who found that the T allele of rs721917 was linked to low FEV1, but the association did not reach statistical significance. For serum SP-D, this study showed that there was a statistically significant difference between different genotypes of rs721917, being the highest in the TT genotype. This is in agreement with Shakoori and Sin[7], who reported that the C allele of rs721917 showed lower serum SP-D levels than the T allele. Sun et al.[19] also reported that the expressions of SP-D of the genotypes CC, CT, and TT of rs721917 in the COPD group were significantly different, being higher in TT than CT and CC.

This study showed that there was a significant difference between different genotypes of rs3088308 in serum SP-D, being the highest in the TT genotype. This is in agreement withJohansson et al.[17] and Shakoori and Sin[7], who reported that SNP rs3088308 was found to modulate serum SP-D levels and may be a risk factor for the development of COPD.


  Conclusion Top


It can be concluded that the T allele of rs721917 might be a susceptible gene for COPD as it was associated with higher serum SP-D levels and lower spirometric data. For rs3088308, the T allele was associated with increased serum SP-D, but not with the development of COPD.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Johansson S, Tan Q, Holst R, Christiansen L, Hansen N, Hojland A, et al. Surfactant protein D is a candidate biomarker for subclinical tobacco smoke-induced lung damage. Am J Physiol Lung Cell Mol Physiol 2014; 306:L887–L895.  Back to cited text no. 17
    
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Foreman M, Kong X, De Meo D, Pillai S, Hersh C, Bakke P, et al. Polymorphisms in surfactant protein-D are associated with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2011; 44:316–322.  Back to cited text no. 18
    
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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