| Abstract |
Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of morbidity and mortality among the adult population worldwide. The major risk factor of COPD is undoubtedly cigarette smoking. However, only less than 20% of smokers develop clinically significant COPD. Thus, in addition to exposure, host factors are important in determining whether smokers develop lung disease. It has not been established if these susceptible smokers who develop COPD are genetically predetermined, or environmental (type of tobacco), dietary or other factors affect the development of the disease. Although several clinical studies exist on the pathophysiology of COPD, little is known as regards the molecular basis of the disease. Microsatellite DNA, is very short tandem nucleotide repeats and are found scattered throughout the human genome. Instability of tandem repeat DNA sequences or Microsatellite Instability (MI) has been correlated with high mutational rates and has been reported in various malignancies. In the first protocol of this study, we tried to investigate if Microsatellite DNA Instability (MI) is a detectable phenomenon in sputum cells of COPD patients and to perform an assessment of the mutational rate in those patients, as reflected by the incidence of Microsatellite Instability. Thirty-one cytological specimens from patients with COPD were analysed for Microsatellite Instability (MI). Polymerase Chain Reaction (PCR) coupled with assessment of electophoretic mobility of the amplified DNA fragments was employed to detect any irregularities in the generated pattern for each of the subjects. Six polymorphic microsatellite markers were targeted by utilizing specific oligonucleotide primer pairs for each of them. Genomic DNA isolated from sputum and blood samples from the subjects was used as template. The electophoretic pattern of each specimen was compared with the corresponding pattern of the peripheral blood and any difference in the mobility of the microsatellite alleles was interpreted as MI. Among 31 cases tested, 7 (23%) were interpreted as MI positive. In the 5 of these cases instability affected only one marker while in the remaining 2 cases, 2 markers were affected. It was postulated that MI is either a marker of potential malignancy or a genetic defect requirement for development of COPD. However, the design of the first protocol did not allow us to come to conclusions since we did not follow up the patients long enough or we did not compare them with non-COPD smokers. The second protocol was designed to study MI in smokers who develop COPD and those who do not. The hypothesis was that, if MI is a potential marker of malignancy, this marker should be rather evenly distributed among non-COPD smokers and COPD patients whereas, if this is a genetic alteration leading to COPD it would be more common in COPD patients. In order to investigate this hypothesis we compared smokers who develop COPD with smokers who did not (non-COPD). Seven highly polymorphic microsatellite markers were targeted on the DNA of sputum cells and of white blood cells. Sixty non-COPD smokers and 59 severe COPD patients with similar smoking history (mean ± SD) of 48±25 vs. 54±33 pack-years, respectively (p = 0.77) were studied. Non-COPD smokers were tested once and COPD-smokers were tested twice, with an interval of 24 months between tests. MI was detected in 14 COPD patients (24%) but in none of the non-COPD smokers. In 10 COPD patients MI positive instability was exhibited by one MI marker and in the remaining 4 by two different alleles. The most commonly affected marker was THRA1 on chromosome 17 (43%). No significant differences were found in the clinical or laboratory parameters, survival and in the development of lung cancer between MI positive and negative COPD patients, the revealed instability cannot be linked to the severity of the disease or previous smoking history. No change in the microsatellite alleles was found between the two tests made at interval of 24 months. Conclusions: This study demonstrated that MI was found exclusively in sputum cells of smokers with COPD. The results support the hypothesis that MI could be part of the complex genetic basis of COPD, and could be a marker of genetic susceptibility to cigarette smoking in order to develop COPD. To the best of our knowledge this is the first report of such a genetic alteration in smokers with COPD.
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Μελέτη της αστάθειας του μικροδορυφορικού DNA (Microsatellite instability) σε κυτταρολογικά δείγματα ασθενών με χρόνια αποφρακτική πνευμονοπάθεια
