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Screening for polymorphisms in exon 5 of the glutathione S-transferase Pl gene
  1. FRANCISCO RODRIGUEZ-FRIAS,
  2. CARLOS GONZALEZ,
  3. XOSE COSTA,
  4. FRANCISCO CAMPOS,
  5. MONTSE COTRINA
  1. Department of Biochemistry
  2. Department of Pneumology
  3. Hospital Universitario Vall d'Hebron
  4. Barcelona 08035, Spain
  1. Dr R Jardi email: riardi{at}hg.vhebron.es
  1. ROSENDO JARDI,
  2. MARC MIRAVITLLES,
  3. RAFAEL VIDAL
  1. Department of Biochemistry
  2. Department of Pneumology
  3. Hospital Universitario Vall d'Hebron
  4. Barcelona 08035, Spain
  1. Dr R Jardi email: riardi{at}hg.vhebron.es
  1. TAKEO ISHII,
  2. SHINJI TERAMOTO
  1. Department of Geriatric Medicine
  2. Tokyo University Hospital
  3. 7-3-1 Hongo Bunkyo-ku
  4. 113-8655 Tokyo, Japan
  5. Department of Respiratory Medicine
  6. Yokohama City University
  7. School of Medicine
  8. Urafune General Medical Center
  9. 3-46 Urafune-cho
  10. Minami-ku
  11. Yokohama City
  12. 232-0024 Yokohama, Japan
  1. Dr T Matsuse email:matsuse{at}vrahp.yokohama-cu.ac.jp
  1. TAKESHI MATSUSE
  1. Department of Geriatric Medicine
  2. Tokyo University Hospital
  3. 7-3-1 Hongo Bunkyo-ku
  4. 113-8655 Tokyo, Japan
  5. Department of Respiratory Medicine
  6. Yokohama City University
  7. School of Medicine
  8. Urafune General Medical Center
  9. 3-46 Urafune-cho
  10. Minami-ku
  11. Yokohama City
  12. 232-0024 Yokohama, Japan
  1. Dr T Matsuse email:matsuse{at}vrahp.yokohama-cu.ac.jp

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A recent paper by Ishii et al 1 suggested an association between glutathione S-transferase P1 (GSTP1) polymorphism in exon 5 (Ile105Val) and the development of chronic obstructive pulmonary disease (COPD) in a sample from a Japanese population. The authors reported that GSTP1 homozygous wild type Ile105 polymorphism was found more frequently in patients with COPD than in controls (79% vs 52%).

In an ongoing COPD genetic study (unpublished preliminary results) we determined the prevalence of GSTP1 polymorphisms in exon 5 in 200 healthy Caucasian volunteers from the Barcelona area. Genetic variants were detected using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The amplification products were digested with the restriction enzyme BsmaIat 55°C, subjected to electrophoresis, and visualised by ethidium bromide staining. Of the 200 healthy volunteers, 99 (49.5%) were homozygous wild type Ile105 (additionally, 88 (44%) were heterozygote Ile105Val and 13 (6.5%) were homozygous Val105). This percentage was similar to that observed by Ishii et al in Japan and Harries et al in a Caucasian population in the UK,2 but differed from that observed by Morita et al in Japan3 and Lieshoust et al in a Caucasian population in the Netherlands.4

We now report the successful application of PCR and single strand conformation polymorphism (SSCP) to the rapid detection of Ile105Val genotypes. DNA was isolated from EDTA blood samples using the QIAamp Kit (Qiagen Ltd, Crawley, UK). Gene specific primers to amplify a 190 bp fragment (5′-CTCTATGGGAAGGACCAGCA-3′ and 5′-AGCCACCTGAGGGGTAAG-3′) were designed from the GSTP1 sequence (Genebank U12472). The PCR reaction was performed as previously described.5 The SSCP procedure and silver staining were carried out according to the manufacturer's instructions (Pharmacia Biotech. Sweden) with the following modifications: samples were denatured at 95°C for 15 minutes and electrophoresis was done at 15°C for two hours.

Using serum samples from 100 healthy volunteers, we compared in a blind experiment the results obtained by this method with those obtained by PCR-RFLP analysis. In all cases identical genotypes were obtained. Moreover, GSTP1 polymorphisms were confirmed by direct sequencing analysis of individuals predicted to be Ile105, Ile105Val, and Val105 from the assay results using a previously reported PCR sequencing method.5 Figure 1 shows the results obtained with PCR-SSCP and PCR-RFLP.

Figure 1

(A) PCR-SSCP based genotyping for GSTP1 polymorphism: (1) homozygous wild type Ile105, (2) heterozygous Ile105Val, and (3) homozygous Val105. (B) PCR-RFLP based genotyping corresponding to a patient (1) wild type, (2) heterozygous, and (3) homozygous for the Ile105Val mutation. Lane 4: 100 bp ladder molecular weight marker.

Studies on large series are required to investigate further the pronounced interethnic differences in allelic frequencies in exon 5 of the GSP1 gene in normal populations. The simple PCR-SSCP method we propose, with comparable sensitivity and specificity to that obtained with the PCR-RFLP method, could be useful for GSTP1 genetic screening. PCR-SSCP is a single step method that does not require digestion of PCR products with restriction enzymes, thus avoiding the inaccuracy resulting from incomplete digestion that can occur with PCR-RFLP. Moreover, PCR-SSCP is less expensive and time consuming than PCR-RFLP.

References

authors' reply The data presented by Rodriguez-Frias et al confirm the polymorphism in exon 5 of the glutathione S-transferase P1 (GSTP1) gene in healthy volunteers. Because this polymorphism varies in Asians and Caucasians, the current study is helpful in determining the prevalence of GSTP1 polymorphism in healthy subjects. In addition, the results suggest that the prevalence of GSPT1 polymorphism in healthy volunteers does not differ significantly between Caucasians and non-Caucasians. This is an important observation for understanding the pathogenesis of chronic obstructive pulmonary disease (COPD) in relation to cigarette smoking, irrespective of race.

We agree that PCR-SSCP is a better method for determining GSTP1 polymorphism than PCR-RFLP, since an ideal screening test should be simple, inexpensive, time saving, and have a high sensitivity with reasonable specificity. This screening test should be used in patients with marginal airflow limitation who have not yet developed COPD, and would be particularly effective for detecting patients with a predisposition to COPD. Because only 20% of heavy smokers develop COPD, the genetic susceptibility of lungs to cigarette smoke may be determined. Our study and the work by Rodriguez-Frias and coworkers further contribute to an understanding of the genetic background of COPD in both Asians and Caucasians.

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