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The determination of Lewis and secretor–nonsecretor functional alleles for clinical application of urinary CA19-9
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Forensic Science International: Genetics Supplement Series 1 (2008) 565–568 www.elsevier.com/locate/FSIGSS
Research article
The determination of Lewis and secretor–nonsecretor functional alleles for clinical application of urinary CA19-9 Yukio Itoh a,*, Chiyomi Nishida a,b, Naoko Tokiwa a,b, Koichi Satoh a,b, Tomoko Tokura c, Keiko Maeda c a
Department of Forensic Medicine, Juntendo University School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo 113-8421, Japan b Medico-Legal Section, Criminal Investigation Laboratory, Metropolitan Police Department, Tokyo, Japan c Atopy Research Center, Juntendo University School of Medicine, Tokyo, Japan Received 17 August 2007; accepted 10 October 2007
Abstract This paper describes the simultaneous detection of Lewis (Le) and secretor–nonsecretor (Se) blood groups by fragment analyses using the ABI Prism1 3130 genetic analyzer. We developed a method of simultaneously genotyping Le and Se by fragment analyses for the clinical application of urinary CA19-9. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Tumor marker; CA19-9; Lewis genotype; Secretor–nonsecretor genotype
1. Introduction The antigenic epitope of CA19-9, namely, sialyl Lewis A antigen, has been clinically applied as a tumor marker of pancreatic and colorectal cancer, as well as of other malignancies. The synthesis of CA19-9 is complex and involves the Lewis (Le) and secretor–nonsecretor (Se) genes, as well as the gene encoding sialyltransferase. The activities of the Le and Se enzymes are genetically controlled by genotypes of the Le and Se genes, respectively. Se gene dosage has been reported to negatively affect both CA19-9 and DU-PAN-2 values, whereas Le gene dosage positively affects the CA19-9 value and negatively affects DU-PAN-2 [1]. We examined the effects of different combinations of the Le and Se blood group genotypes on urinary levels of CA19-9 and DU-PAN-2 and found that urinary CA19-9 can function as a novel diagnostic tool in patients with bladder cancer who carry the functional alleles of both Le and Se [2]. However, the genotypes of both of these genes must be understood before the urinary CA19-9 can be clinically applied.
* Corresponding author. Tel.: +81 3 5802 1051; fax: +81 3 5802 1050. E-mail address: [email protected] (Y. Itoh). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.194
The present study aimed to simultaneously genotype Le and Se using PCR-based methods. Both Le and Se were genotyped using fragment analysis of multiplex PCR products. 2. Materials and methods We extracted DNA samples from blood or urine using QIAamp1 DNA Mini Kits (QIAGEN). We examined the nucleotide at position 59 on cDNA of the Le gene to determine the Le and le alleles [3] and the nucleotide at position 385 on cDNA of the Se gene and a Se pseudo gene to determine the Se, se2 and se5 alleles [4]. Samples of DNA were amplified in 25-ml reaction volumes. Template DNA (30 ng) was added to the reaction mixture containing PCR buffer (Invitrogen Life Technologies, Japan), 1.5 mM MgCl2 (Invitrogen Life Technologies), 0.2 mM dNTPs (Promega Corp., Japan), and 0.625 U platinum Taq DNA polymerase (Invitrogen Life Technologies), along with amplification primers shown in Table 1. The genes were amplified as follows: initial denaturation for 2 min at 94 8C, followed by 30 cycles of denaturation for 1 min at 94 8C, annealing for 1 min at 65 8C and extension for 1 min at 72 8C. Final extension proceeded for 30 min at 68 8C. The PCR products were analyzed using an ABI Prism1 3130 genetic analyzer and Gene Mapper Software (Applied Biosystems).
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Table 1 Primers for simultaneous genotyping Le and Se blood groups Primer
Sequence (50 ! 30 )
FCa (pmol)
b
6FAM-ACCCATGGATCCCCTcGGTGC 6FAM-GCCGCTGTCTGGCCGCAtT VIC-GCCGCTGTCTGGCCGCAaG tail-GGCAGTGATGTGGCAGTCGGC tail-TCAGGGGGATGTGGACGATCA tail-TGGGCATACTCAGCCCGTGT NED-ATGGACCCCTACAAAGGTGCC NED-CGGACGTACTCCCCCGGGAT tail-GGAGGAGGAATACCGCCACT
2 2 2 2 2 2 2 2 2
6FAM-Le1F(N6) 6FAM-Le21mF(f2) b VIC-Le-23mF Le4R Fut2-Z1 Fus-T1F b NED-Fut2-Z4R b NED-Fut2-T2R Fut2-NA1F b
a b
Specificity Le specific le specific
Se specific se2 specific se5 specific
Final concentration for PCR. Dye-labelled primer, sequence of small capital: mismatch.
Fig. 1. (a) Fragment chart of PCR-products obtained form Le/le, se2/se5 genotype sample and (b) fragment chart of PCR-products obtained form le/le, Se/Se genotype sample.
Y. Itoh et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 565–568
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Fig. 1. (Continued ).
3. Results and discussion Fig. 1a and b shows that the Le and Se blood groups were simultaneously genotyped. These results were directly comparable with respective individual genotyping. These results also correspond with the serological determinations. The frequency of patients having both Le and Se functional alleles is about 76% in the Japanese population. We have developed a method for simultaneous genotyping of Le and Se by fragment analyses for the development of clinical application of urinary CA19-9. Although simultaneous genotyping of Le and Se was successful, the peak height ratios for each allele were not satisfactory and we are attempting to
improve the peak height ratio of each allele in fragment analysis. Conflict of Interest None stated. References [1] H. Narimatsu, H. Iwasaki, F. Nakayama, et al., Lewis and secretor gene dosages affect CA19-9 and DU-PAN-2 serum levels in normal individuals and colorectal cancer patients, Cancer Res. 58 (1998) 512–518.
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Y. Itoh et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 565–568
[2] K. Nagao, Y. Itoh, K. Fujita, M. Fujime, Evaluation of urinary CA19-9 levels in bladder cancer patients classified according to the combination of Lewis and secretor blood group genotypes, Int. J. Urol. 14 (2007) 795–799. [3] Y. Itoh, K. Satoh, K. Takahashi, K. Maeda, T. Tokura, R. Kobayashi, Evaluation of Lewis genotyping by four PCR-based methods, Prog. Forensic Genet. 11 (ICS 1288) (2006) 819–821.
[4] K. Takahashi, Y. Itoh, K. Nagao, M. Fujime, Se genotyping by PCR with confronting two-pair primers (PCR-CTPP), DNA Polymorphism 11 (2003) 139–142.
Forensic Science International: Genetics Supplement Series 1 (2008) 565–568 www.elsevier.com/locate/FSIGSS
Research article
The determination of Lewis and secretor–nonsecretor functional alleles for clinical application of urinary CA19-9 Yukio Itoh a,*, Chiyomi Nishida a,b, Naoko Tokiwa a,b, Koichi Satoh a,b, Tomoko Tokura c, Keiko Maeda c a
Department of Forensic Medicine, Juntendo University School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo 113-8421, Japan b Medico-Legal Section, Criminal Investigation Laboratory, Metropolitan Police Department, Tokyo, Japan c Atopy Research Center, Juntendo University School of Medicine, Tokyo, Japan Received 17 August 2007; accepted 10 October 2007
Abstract This paper describes the simultaneous detection of Lewis (Le) and secretor–nonsecretor (Se) blood groups by fragment analyses using the ABI Prism1 3130 genetic analyzer. We developed a method of simultaneously genotyping Le and Se by fragment analyses for the clinical application of urinary CA19-9. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Tumor marker; CA19-9; Lewis genotype; Secretor–nonsecretor genotype
1. Introduction The antigenic epitope of CA19-9, namely, sialyl Lewis A antigen, has been clinically applied as a tumor marker of pancreatic and colorectal cancer, as well as of other malignancies. The synthesis of CA19-9 is complex and involves the Lewis (Le) and secretor–nonsecretor (Se) genes, as well as the gene encoding sialyltransferase. The activities of the Le and Se enzymes are genetically controlled by genotypes of the Le and Se genes, respectively. Se gene dosage has been reported to negatively affect both CA19-9 and DU-PAN-2 values, whereas Le gene dosage positively affects the CA19-9 value and negatively affects DU-PAN-2 [1]. We examined the effects of different combinations of the Le and Se blood group genotypes on urinary levels of CA19-9 and DU-PAN-2 and found that urinary CA19-9 can function as a novel diagnostic tool in patients with bladder cancer who carry the functional alleles of both Le and Se [2]. However, the genotypes of both of these genes must be understood before the urinary CA19-9 can be clinically applied.
* Corresponding author. Tel.: +81 3 5802 1051; fax: +81 3 5802 1050. E-mail address: [email protected] (Y. Itoh). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.194
The present study aimed to simultaneously genotype Le and Se using PCR-based methods. Both Le and Se were genotyped using fragment analysis of multiplex PCR products. 2. Materials and methods We extracted DNA samples from blood or urine using QIAamp1 DNA Mini Kits (QIAGEN). We examined the nucleotide at position 59 on cDNA of the Le gene to determine the Le and le alleles [3] and the nucleotide at position 385 on cDNA of the Se gene and a Se pseudo gene to determine the Se, se2 and se5 alleles [4]. Samples of DNA were amplified in 25-ml reaction volumes. Template DNA (30 ng) was added to the reaction mixture containing PCR buffer (Invitrogen Life Technologies, Japan), 1.5 mM MgCl2 (Invitrogen Life Technologies), 0.2 mM dNTPs (Promega Corp., Japan), and 0.625 U platinum Taq DNA polymerase (Invitrogen Life Technologies), along with amplification primers shown in Table 1. The genes were amplified as follows: initial denaturation for 2 min at 94 8C, followed by 30 cycles of denaturation for 1 min at 94 8C, annealing for 1 min at 65 8C and extension for 1 min at 72 8C. Final extension proceeded for 30 min at 68 8C. The PCR products were analyzed using an ABI Prism1 3130 genetic analyzer and Gene Mapper Software (Applied Biosystems).
566
Y. Itoh et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 565–568
Table 1 Primers for simultaneous genotyping Le and Se blood groups Primer
Sequence (50 ! 30 )
FCa (pmol)
b
6FAM-ACCCATGGATCCCCTcGGTGC 6FAM-GCCGCTGTCTGGCCGCAtT VIC-GCCGCTGTCTGGCCGCAaG tail-GGCAGTGATGTGGCAGTCGGC tail-TCAGGGGGATGTGGACGATCA tail-TGGGCATACTCAGCCCGTGT NED-ATGGACCCCTACAAAGGTGCC NED-CGGACGTACTCCCCCGGGAT tail-GGAGGAGGAATACCGCCACT
2 2 2 2 2 2 2 2 2
6FAM-Le1F(N6) 6FAM-Le21mF(f2) b VIC-Le-23mF Le4R Fut2-Z1 Fus-T1F b NED-Fut2-Z4R b NED-Fut2-T2R Fut2-NA1F b
a b
Specificity Le specific le specific
Se specific se2 specific se5 specific
Final concentration for PCR. Dye-labelled primer, sequence of small capital: mismatch.
Fig. 1. (a) Fragment chart of PCR-products obtained form Le/le, se2/se5 genotype sample and (b) fragment chart of PCR-products obtained form le/le, Se/Se genotype sample.
Y. Itoh et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 565–568
567
Fig. 1. (Continued ).
3. Results and discussion Fig. 1a and b shows that the Le and Se blood groups were simultaneously genotyped. These results were directly comparable with respective individual genotyping. These results also correspond with the serological determinations. The frequency of patients having both Le and Se functional alleles is about 76% in the Japanese population. We have developed a method for simultaneous genotyping of Le and Se by fragment analyses for the development of clinical application of urinary CA19-9. Although simultaneous genotyping of Le and Se was successful, the peak height ratios for each allele were not satisfactory and we are attempting to
improve the peak height ratio of each allele in fragment analysis. Conflict of Interest None stated. References [1] H. Narimatsu, H. Iwasaki, F. Nakayama, et al., Lewis and secretor gene dosages affect CA19-9 and DU-PAN-2 serum levels in normal individuals and colorectal cancer patients, Cancer Res. 58 (1998) 512–518.
568
Y. Itoh et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 565–568
[2] K. Nagao, Y. Itoh, K. Fujita, M. Fujime, Evaluation of urinary CA19-9 levels in bladder cancer patients classified according to the combination of Lewis and secretor blood group genotypes, Int. J. Urol. 14 (2007) 795–799. [3] Y. Itoh, K. Satoh, K. Takahashi, K. Maeda, T. Tokura, R. Kobayashi, Evaluation of Lewis genotyping by four PCR-based methods, Prog. Forensic Genet. 11 (ICS 1288) (2006) 819–821.
[4] K. Takahashi, Y. Itoh, K. Nagao, M. Fujime, Se genotyping by PCR with confronting two-pair primers (PCR-CTPP), DNA Polymorphism 11 (2003) 139–142.