Development of a novel microarray methodology for the study of SNPs in the promoter region of the TNF-α gene—Their association with obstructive pulmonary disease in Greek patients

doi:10.1016/j.clinbiochem.2007.03.024

Clinical Biochemistry

Volume 40, Issue 12, August 2007, Pages 843-850

https://doi.org/10.1016/j.clinbiochem.2007.03.024 Get rights and content

Abstract

Objectives:

Polymorphisms in promoter region of TNF-α gene were shown to interfere with the transcriptional activity of the gene resulting in the production of different levels of TNF-α product suggesting their involvement in susceptibility or severity of many inflammatory diseases. We set up a case–control study consisting of 117 COPD (Chronic Obstructive Pulmonary Disease), 62 DB (bronchiectasis) patients and two control groups (109 smokers without COPD-healthy smokers control group and 212 general population subjects) to evaluate involvement of TNF-α gene polymorphisms in the abovementioned diseases in a homogeneous population.

Methods:

The novel methodology of the NanoChip™ Molecular Biology Workstation (MBW Nanogen http://www.nanogen.com) was employed to genotype the 5 promoter SNPs.

Results and conclusions:

Genotype frequencies of the 5 SNPs showed no significant difference between the COPD and DB patient groups and the healthy smokers group. Statistical difference (p = 0.043) was only revealed between the haplotype frequencies in COPD patients compared to the general population control group. The NanoChip™ MBW is an accurate method for SNP screening.

Introduction

Chronic Obstructive Pulmonary Disease (COPD) is a major cause of chronic morbidity and mortality. The World Health Organization (WHO) listed COPD as the fifth leading cause of death in the world and it is estimated that COPD will be the third most common cause of death globally by 2020 [1]. In Greece the prevalence of the disease in subjects aged > 50 years ranges between 10.7% and 15.8%. The high proportion of the patients with mild COPD who are unaware of their illness highlights the need to increase the public awareness of COPD.

COPD is a disease state characterized by airflow limitation that is not fully reversible and associated with an abnormal inflammatory response of the lungs to noxious particles or gases (Global Initiative for Chronic Obstructive Pulmonary Disease (GOLD), http://www.goldcopd.com). It is a complex disease influenced by genetic and environmental factors. Cigarette smoking is the major environmental determinant of COPD, however only a minority (10–20%) of chronic heavy smokers develop symptomatic disease. The variability in disease susceptibility between individuals could be attributed to genetic factors interacting with cigarette smoking in the disease pathogenesis. Family and twin studies support this theory [2], [3].

Bronchiectasis (DB) is characterized by irreversible focal bronchial dilation, usually accompanied by chronic infection and associated with diverse conditions, some congenital or hereditary. Bronchiectasis may be focal and limited to a single segment or lobe of the lung or it may be widespread and affect multiple lobes in one or both lungs [4], [5], [6].

Tumor necrosis factor-α (TNF-α) is a key modulator of inflammatory response and has been found to be elevated in bronchoalveolar lavage (BAL) fluid, bronchial biopsy specimens and induced sputum of patients with COPD leading to the hypothesis that those patients who have abnormally elevated production of TNF-α are more likely to develop COPD following exposure to cigarette smoke [7]. The TNF-α gene is located in the HLA region of human chromosome 6, the most polymorphic region of the human genome.

There is evidence that polymorphisms in the promoter region of the TNF-α gene affect the amount of TNF-α production. Findings concerning the role of TNF-α promoter − 308G/A (rs1800629) polymorphism in the pathogenesis of cigarette smoke-induced COPD are inconsistent. The majority of studies found no association between the − 308G/A alleles with susceptibility to smoking related COPD [8], [9], [10]. Additionally other variants in the promoter region of the TNF-α gene such as: − 1031T/C (rs1799964), − 863C/A (rs1800630), − 857C/T (rs1799724) and − 238G/A (rs361525) have not yet been studied in COPD or DB [11].

SNPs are the most common form of human sequence variation occurring, on average, every 1000–2000 bases. They are very useful for human haplotype analysis and are well suited to automated, high-throughput genotyping. Case–control studies using SNPs test whether single locus alleles or genotype/haplotype frequencies differ between cases and controls. This is a case–control study and the first to investigate the genetic contribution of all 5 TNF-α promoter gene SNPs alone or in haplotype combinations on the pathogenesis of COPD and DB in Greek patients in comparison to a clinically tested resistant control group of smokers and a general population control of the same ethnic origin.

Additionally we sought to develop an advanced methodology for SNP/mutation identification, which is sensitive, simple to perform, rapid, inexpensive and amenable to high throughout applications and automation. The most modern DNA diagnostic tool is the “DNA-chip”, a small device that permits the rapid analysis of genetic information by miniaturization. We used the NanoChip™ Molecular Biology Workstation (MBW) (Nanogen http://www.nanogen.com) to genotype the above 5 SNPs in the 5′ flanking promoter/enhancer region of the TNF-α gene. No similar Analyte-specific reagents (ASR) assay or home brew method has been published for the MBW concerning the 5 SNPs.

The MBW technology is based on the electronic manipulation of an electric field in a semiconductor device. Through the use of microelectronics, this technology enables the active movement and concentration of charged molecules to designated test sites. A biotinylated PCR primer is used to allow for attachment of an amplicon to streptavidin in the agarose permeation layer of the microarray.

Section snippets

Patients

This was a case–control study. The patient groups consisted of (a) 117 stable COPD patients (mean age 68.8 ± 8.84; range 45–90 years) and (b) 62 patients with DB (mean age 63.1 ± 14.87; range 45–87 years). Whole blood was obtained from the COPD/Bronchiectasis Outpatient Clinic of the Respiratory Department of the University of Athens at “Sotiria” Hospital. COPD was diagnosed on the basis of history, physical examination and spirometric data, according to the Global Initiative for Chronic

Results

The duplex PCR yielded a 501-bp amplicon for SNPs − 1031T/C, − 863C/A and − 857C/T, and a 317-bp amplicon for SNPs − 308G/A and − 238G/A (Fig. 1 in the Supplementary data).

Both amplicons were addressed to the same pad and genotyping for the 5 SNPs was performed sequentially under the conditions shown in Table 2. No cross-hybridization for any of the 5 sets of probes was obtained. An example of the red to green ratios and final genotyping results obtained for the detection of the five SNPs is shown

Discussion

Chronic pulmonary diseases such as COPD and bronchiectasis have become a major problem worldwide. COPD, in particular, is the sixth leading cause of death in the Western world, accounting for much morbidity and mortality and it is estimated that this will rise to the third-leading cause by the year 2020 [20]. Especially in Greece, an increase in the number of regular smokers is observed and since cigarette smoking is considered the major environmental risk factor for disease development the

Acknowledgments

This work was funded by an Athens University Research Grant (ELKE no. 70/4/1661) and a grant given to P. Latsi by Glaxo-Smith-Kline (GSK 2003, Grant #1013).

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^☆: Human genes: tumor necrosis factor alpha (MIM#191160), TNFa GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide accession number NM_000594). NCBI SNP Database: http://www.ncbi.nlm.nih.gov/.

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Development of a novel microarray methodology for the study of SNPs in the promoter region of the TNF-α gene—Their association with obstructive pulmonary disease in Greek patients☆

Abstract

Objectives:

Methods:

Results and conclusions:

Introduction

Section snippets

Patients

Results

Discussion

Acknowledgments

Paediatr. Respir. Rev.

Chest

J. Mol. Diagn.

Am. J. Hum. Genet.

Am. J. Hum. Genet.

Lancet

Respir. Med.

GOLD Scientific Committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI?WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary

Am. J. Respir. Crit. Care Med.

Chronic obstructive pulmonary disease. 1: Susceptibility factors for COPD the genotype–environment interaction

Thorax

Genetic risk factors of chronic obstructive pulmonary disease

Swiss Med. Wkly.

Genetics of chronic obstructive pulmonary disease and disseminated bronchiectasis

Monaldi Arch. Chest Dis.

Bronchiectasis

Internal. Med. J.

Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma

Am. J. Respir. Crit. Care Med.

Tumour necrosis factor-α gene promoter polymorphism in chronic obstructive pulmonary disease

Eur. Respir. J.