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Association between novel PLCE1 variants identified in published esophageal cancer genome-wide association studies and risk of squamous cell carcinoma of the head and neck.

Ma H, Wang LE, Liu Z, Sturgis EM, Wei Q - BMC Cancer (2011)

Bottom Line: AA: adjusted OR=1.30, 95% CI=1.03-1.64), while rs11599672 was associated with a significantly decreased risk (GG vs.Our findings suggest that PLCE1 variants may have an effect on risk of SCCHN associated with tobacco and alcohol exposure, particularly for those tumors arising at non-oropharyngeal sites.These findings, although need to be validated by larger studies, are consistent with those in esophageal and gastric cancers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT

Background: Phospholipase C epsilon 1 (PLCE1) (an effector of Ras) belonging to the phospholipase family plays crucial roles in carcinogenesis and progression of several cancers, including squamous cell carcinoma of the head and neck (SCCHN). A single nucleotide polymorphism (SNP, rs2274223) in PLCE1 has been identified as a novel susceptibility locus in genome-wide association studies (GWAS) of esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA) that share similar risk factors with SCCHN. Therefore, we investigated the association between potentially functional SNPs in PLCE1 and susceptibility to SCCHN.

Methods: We genotyped three potentially functional SNPs (rs2274223A/G, rs3203713A/G and rs11599672T/G) of PLCE1 in 1,098 SCCHN patients and 1,090 controls matched by age and sex in a non-Hispanic white population.

Results: Although none of three SNPs was alone significantly associated with overall risk of SCCHN, their combined effects of risk alleles (rs2274223G, rs3203713G and rs11599672G) were found to be associated with risk of SCCHN in a locus-dose effect manner (Ptrend=0.046), particularly for non-oropharyngeal tumors (Ptrend=0.017); specifically, rs2274223 was associated with a significantly increased risk (AG vs. AA: adjusted OR=1.29, 95% CI=1.01-1.64; AG/GG vs. AA: adjusted OR=1.30, 95% CI=1.03-1.64), while rs11599672 was associated with a significantly decreased risk (GG vs. TT: adjusted OR=0.54, 95% CI=0.34-0.86; TG/GG vs. TT: adjusted OR=0.76, 95% CI=0.61-0.95).

Conclusions: Our findings suggest that PLCE1 variants may have an effect on risk of SCCHN associated with tobacco and alcohol exposure, particularly for those tumors arising at non-oropharyngeal sites. These findings, although need to be validated by larger studies, are consistent with those in esophageal and gastric cancers.

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PCR-based genotyping for rs11599672.
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Figure 1: PCR-based genotyping for rs11599672.

Mentions: We extracted genomic DNA from the buffy-coat fraction of the blood samples using the Qiagen Blood DNA Mini Kit (Qiagen Inc., Valencia, Calif) according to the manufacturer's instruction and genotyped SNPs rs2274223 and rs3203713 using the TaqMan allelic discrimination assay on an ABI 7900 system (Applied Biosystems). Genotyping was performed without knowing the subjects' case or control status, and four negative controls (no DNA) and duplicated commercial positive controls (TaqMan Control Genomic DNA; Applied Biosystems) included in each 384-well plate were used for quality control. The accordance achieved 100% for the duplicates of 5% of samples. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method was also used to identify genotypes of rs11599672, because the Taqman assay was not applicable to this SNP. The antisense primer was introduced a mismatched A to replace T at +2bp from the polymorphic site to create an SspI restriction site (sense: 5'- GGAGAGACATTCTGTTGGGTGA-3'; antisense: 5'- CCTTCAAACCACCGCTGTAAT- 3'). A 155-bp fragment containing this T/G site was amplified in the PCR mixture consisted with approximately 20 ng of genomic DNA, 2 pmol of each primer, 0.1 mM each dNTP, 1 × PCR buffer (50 mM KCl, 10 mM Tris HCl, and 0.1% Triton X-100), 1.5 mM MgCl2, and 0.5 unit of Taq polymerase. The PCR profile consisted of an initial melting step of 95°C for 5 min; 35 cycles of 95°C for 30 s, 59°C for 45 s, and 72°C for 1 min; and final extension step of 72°C for 10 min. The PCR product was then digested with the restriction enzyme SspI (New England BioLabs, Beverly, MA) overnight at 37°C and separated on 3% agarose gel. The T allele has the restriction site and produces two bands of 134- and 21-bp, whereas the G allele lacks the SspI restriction site, resulting in one band of 155-bp (Figure 1). Finally, 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant. Direct sequencing was also conducted to confirm the genotypes of rs11599672 (Figure 2).


Association between novel PLCE1 variants identified in published esophageal cancer genome-wide association studies and risk of squamous cell carcinoma of the head and neck.

Ma H, Wang LE, Liu Z, Sturgis EM, Wei Q - BMC Cancer (2011)

PCR-based genotyping for rs11599672.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142535&req=5

Figure 1: PCR-based genotyping for rs11599672.
Mentions: We extracted genomic DNA from the buffy-coat fraction of the blood samples using the Qiagen Blood DNA Mini Kit (Qiagen Inc., Valencia, Calif) according to the manufacturer's instruction and genotyped SNPs rs2274223 and rs3203713 using the TaqMan allelic discrimination assay on an ABI 7900 system (Applied Biosystems). Genotyping was performed without knowing the subjects' case or control status, and four negative controls (no DNA) and duplicated commercial positive controls (TaqMan Control Genomic DNA; Applied Biosystems) included in each 384-well plate were used for quality control. The accordance achieved 100% for the duplicates of 5% of samples. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method was also used to identify genotypes of rs11599672, because the Taqman assay was not applicable to this SNP. The antisense primer was introduced a mismatched A to replace T at +2bp from the polymorphic site to create an SspI restriction site (sense: 5'- GGAGAGACATTCTGTTGGGTGA-3'; antisense: 5'- CCTTCAAACCACCGCTGTAAT- 3'). A 155-bp fragment containing this T/G site was amplified in the PCR mixture consisted with approximately 20 ng of genomic DNA, 2 pmol of each primer, 0.1 mM each dNTP, 1 × PCR buffer (50 mM KCl, 10 mM Tris HCl, and 0.1% Triton X-100), 1.5 mM MgCl2, and 0.5 unit of Taq polymerase. The PCR profile consisted of an initial melting step of 95°C for 5 min; 35 cycles of 95°C for 30 s, 59°C for 45 s, and 72°C for 1 min; and final extension step of 72°C for 10 min. The PCR product was then digested with the restriction enzyme SspI (New England BioLabs, Beverly, MA) overnight at 37°C and separated on 3% agarose gel. The T allele has the restriction site and produces two bands of 134- and 21-bp, whereas the G allele lacks the SspI restriction site, resulting in one band of 155-bp (Figure 1). Finally, 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant. Direct sequencing was also conducted to confirm the genotypes of rs11599672 (Figure 2).

Bottom Line: AA: adjusted OR=1.30, 95% CI=1.03-1.64), while rs11599672 was associated with a significantly decreased risk (GG vs.Our findings suggest that PLCE1 variants may have an effect on risk of SCCHN associated with tobacco and alcohol exposure, particularly for those tumors arising at non-oropharyngeal sites.These findings, although need to be validated by larger studies, are consistent with those in esophageal and gastric cancers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT

Background: Phospholipase C epsilon 1 (PLCE1) (an effector of Ras) belonging to the phospholipase family plays crucial roles in carcinogenesis and progression of several cancers, including squamous cell carcinoma of the head and neck (SCCHN). A single nucleotide polymorphism (SNP, rs2274223) in PLCE1 has been identified as a novel susceptibility locus in genome-wide association studies (GWAS) of esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA) that share similar risk factors with SCCHN. Therefore, we investigated the association between potentially functional SNPs in PLCE1 and susceptibility to SCCHN.

Methods: We genotyped three potentially functional SNPs (rs2274223A/G, rs3203713A/G and rs11599672T/G) of PLCE1 in 1,098 SCCHN patients and 1,090 controls matched by age and sex in a non-Hispanic white population.

Results: Although none of three SNPs was alone significantly associated with overall risk of SCCHN, their combined effects of risk alleles (rs2274223G, rs3203713G and rs11599672G) were found to be associated with risk of SCCHN in a locus-dose effect manner (Ptrend=0.046), particularly for non-oropharyngeal tumors (Ptrend=0.017); specifically, rs2274223 was associated with a significantly increased risk (AG vs. AA: adjusted OR=1.29, 95% CI=1.01-1.64; AG/GG vs. AA: adjusted OR=1.30, 95% CI=1.03-1.64), while rs11599672 was associated with a significantly decreased risk (GG vs. TT: adjusted OR=0.54, 95% CI=0.34-0.86; TG/GG vs. TT: adjusted OR=0.76, 95% CI=0.61-0.95).

Conclusions: Our findings suggest that PLCE1 variants may have an effect on risk of SCCHN associated with tobacco and alcohol exposure, particularly for those tumors arising at non-oropharyngeal sites. These findings, although need to be validated by larger studies, are consistent with those in esophageal and gastric cancers.

Show MeSH
Related in: MedlinePlus