Increased Gastric Cancer Risk with PTEN IVS4 Polymorphism in a Turkish Population

Abstract

We aimed to investigate the association of the phosphatase and tensin homolog (PTEN) IVS4 polymorphism with a gastric cancer (GC) risk in the Turkish population. A hospital-based case-control study was conducted in 93 patients with GC, and 113 healthy controls. The PTEN IVS4 (rs no: 3830675) polymorphism was determined by using polymerase chain reaction-restriction fragment length polymorphism analysis. The PTEN IVS4 (−/−) genotype exhibited a significantly elevated risk for GC compared to controls (p<0.005; odds ratio: 1.6, 95% confidence interval: 1.19-2.14). Analyses on clinicopathological parameters showed that PTEN IVS4 genotypes were not associated with any of the variables of patients with GC (p>0.05). In conclusion, the PTEN IVS4 polymorphism might contribute to the development of GC in a Turkish population. Further studies, including comparison of the PTEN IVS4 polymorphism with plasma and tissue expressions of PTEN in larger study size groups will provide a further assessment of the PTEN IVS4 polymorphism in GC patients.

Introduction

Gastric cancer (GC) is an important cause of morbidity and mortality worldwide (Jemal et al., 2010). GC arises from the accumulation of genetic and epigenetic alterations (Vogiatzi et al., 2007). More recently, molecular epidemiological studies have described some relatively common genetic variants as biomarkers for genetic susceptibility to GC development, namely, single-nucleotide polymorphisms (SNPs) (Milne et al., 2009; Yin et al., 2009; Resende et al., 2010). Even though associations between some SNPs and a GC risk have been reported in previous studies, the definition of susceptibility genes for the GC risk remains to be determined.

The tumor suppressor phosphatase and tensin homolog (PTEN) located at 10q23.3 is a dual phosphatase, acting at both serine-threonine and tyrosine sites. PTEN regulates multiple mechanisms to control the cellular growth, the most important of which is by negatively controlling phosphoinositide 3-kinase activation (Wu et al., 1998). PTEN gene alterations were initially discovered in the glioma, prostate, kidney, and breast carcinomas (Li et al., 1997). Discoveries of PTEN alterations in gastrointestinal cancers (Okami et al., 1998; Jiang et al., 2003) suggest an association between PTEN and GC. Indeed, PTEN mutations in GC have been reported to be between 1.7% and 28.3% (Sato et al., 2002; Wang et al., 2003; Guo et al., 2008; Wen et al., 2010).

The PTEN IVS4 polymorphism occurring with an ACTAA insertion 109 bp downstream of exon 4 in intron 4 has been reported to be related with the onset of breast cancer at a younger age (Carroll et al., 1999), whereas it is not associated with a prostate cancer risk (George et al., 2001). Associations of the PTEN IVS4 polymorphism with increased esophageal and gastric cardia cancers risks have also been reported in a Chinese population-based study (Ge et al., 2008). Here, we aimed to determine that whether an association between the PTEN IVS4 (rs 3830675) polymorphism and a GC risk exists in a Turkish population in a hospital-based case-control study.

Patients and Methods

Study groups

The study was approved by the Committee for Ethics of Medical Experiment on Human Subjects, Faculty of Medicine, Istanbul University, Istanbul, Turkey. Submission of the individuals to the study was conditioned by an obtained written informed consent regarding the use of their blood samples for research studies. The study proceeded in agreement with the Helsinki Declaration approved by the World Medical Association meeting in Edinburgh. Blood samples were collected from 93 consecutive patients with GC at their initial staging. During the same time, 113 individuals who entered the hospital for health check-ups were enrolled as the control group. Peripheral blood samples were taken from patients, and controls were frozen at −20°C until analysis.

DNA extraction

A 5 mL sample of venous blood was collected from each subject into a test tube containing EDTA. Genomic DNA was extracted from peripheral whole blood according to a salting-out technique (Miller et al., 1988).

Polymerase chain reaction-restriction fragment length polymorphism analysis

Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis with an internal check on digestion was performed to determine the genotype of the polymorphism of the IVS4 region of the PTEN gene, as described previously (Ge et al., 2008). The 403-bp fragment encompassing the absence or presence of an ATCTT insertion downstream of exon 4 in intron 4 of the PTEN gene (PTEN IVS +4; rs 3830675) was amplified using specific primers 5′-GGGGGTGATAACAGTATCTA-3′ and 5′-CTTTATGCAATACTTTTTCCTA-3′ (Invitrogen; Carlsbad, CA). The amplification was performed with 100 ng of genomic DNA, 25 pmol of each primer, 200 μM total dNTP, 1.5 mM MgCl₂, 1x PCR buffer, and 2.5 U Taq DNA polymerase (Intron Bio, Sungnam, Kyungki-Do, Korea). The following cycling conditions were used: an initial melting step of 3 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 64°C, and 30 s at 72°C; a final elongation step 5 min at 72°C. After confirmation of successful PCR amplification by 1.5% agarose gel electrophoresis, each PCR product was digested overnight with 10 units of AfI II enzyme (New England Biolabs, Beverly, MA) at 37°C and electrophoresed on a 2% agarose gel containing 0.5 μg/mL ethidium bromide and visualized under UV illumination. AfI II recognizes of 5′-ATCTT-3′, so the transition from wild type to insertion in the variant is recognized by the enzyme and produces two bands (335 and 78 bp). To ensure quality control, genotyping was performed without knowledge of the subjects case/control status and a 15% random sample of cases and controls was genotyped twice by different persons; reproducibility was 100%.

Statistical analysis

Statistical analyses were performed using the SPSS software package (revision 11.5; SPSS, Inc., Chicago, IL). Data are expressed as means±SD. Differences in the distribution of PTEN IVS4 genotypes or alleles between cases and controls were tested using the χ² statistic. The Fisher's exact test was used if the number in any cell of the 2×2 contingency table was <5. The relative risk at 95% confidence intervals (CI) was calculated as the odds ratio (OR). A multivariate logistic regression model was performed to investigate the possible effects of genotypes and alleles after adjustment for age. Values of p<0.05 were considered statistically significant.

Results

Characteristic of study population

Table 1 shows selected characteristics of cases and controls. There were a total of 93 patients with GC and 113 controls.

Table 1.

Characteristics of Patients with Gastric Cancer and Controls

Parameters	Patients n=93 (%)	Controls n=113 (%)	p-Value
Age (years)
<50	41 (44.1)	56 (49.6)	0.260
≥50	52 (55.9)	57 (50.4)
Sex
Male	49 (52.7)	67 (59.3)	0.344
Female	44 (47.3)	46 (40.7)
Smoking status
Yes	53 (57)	73 (64.6)	0.166
No	40 (43)	40 (35.4)
Alcohol consumption
Yes	73 (78.5)	99 (87.6)	0.105
No	20 (21.5)	14 (12.4)
Tumor differentiation
Well/moderately	55 (59.1)
Poor	38 (40.9)
Tumor status
T1	6 (6.5)
T2	19 (20.4)
T3	40 (43)
T4	28 (30.1)
Lymph node status
N0	3 (3.2)
N1	47 (50.5)
N2	31 (33.3)
N3	12 (12.9)
Distant metastasis
Yes	30 (67.7)
No	63 (32.3)
Lymphovascular invasion
Yes	66 (71)
No	27 (29)
Stages
I	7 (7.5)
II	39 (41.9)
III	18 (19.4)
IV	29 (31.2)

p<0.05 is statistically significant.

Genotype frequencies distribution

The frequency distributions of the different genotypes and alleles for the PTEN IVS4 (−/+) polymorphism are presented in Table 2. (−) Allelic frequencies of patients were 0.80 and (+) allelic frequencies were 0.2 for patients with GC; these were different from those of controls [(−) allele, 0.68; (+) allele, 0.32], p<0.005, OR: 1.86, 95% CI: 1.54-2.24. The genotypic frequencies of the patients (n=93, χ²=1.4, df=1, p=0.236) and the controls (n=113, χ²=2.92, df=1, p=0.08) were in the Hardy-Weinberg equilibrium. Genotypic frequencies of the (−/−) genotype of the PTEN IVS4 gene in GC patients (0.61) were different than those of control (0.42) (p<0.005).

Table 2.

Genotype and Allele Frequencies for PTEN IVS4 (−/+) Genes in Gastric Cancer Patients and Controls

PTEN IVS +4 genotype/allele	Patients (n=93)	Controls (n=113)	p-Value	Exp (B) [95% CI]
General genotype
−/−	57 (61.3%)	43 (38.1%)	0.007^a	0.532 [0.337-0.839]
−/+	29 (31.2%)	60 (53.1%)
+/+	7 (7.5%)	10 (8.8%)
Allele frequency
(−) allele	86 (60.5%)	103 (59.5%)	0.007^a	1.822 [1.17-2.82]
(+) allele	36 (39.5%)	70 (40.5%)
Dominant genotype
(−/−)	57 (61.2%)	43 (46.2%)	0.001^a	0.374 [0.212-0.658]
(−/+)/(+/+)	36 (38.8%)	70 (54.8%)
Recessive genotype
(−/−)/(−/+)	86 (92.4%)	103 (91.1%)	0.732	0.838 [0.306-2.29]
(+/+)	7 (7.6%)	10 (8.9%)

p-Value obtained by χ² test.

p<0.05 is statistically significant.

PTEN, phosphatase and tensin homolog; CI, confidence interval.

Association between PTEN IVS4 genotype and GC risk

To determine if there was a significant increased risk for GC development according to the PTEN IVS4 genotypes, we conducted logistic regression analysis, which showed that individuals with the (−/−) genotype of PTEN IVS4 had a 1.6-fold (95% CI=1.19-2.14, p<0.05) increased GC risk.

Subgroup analyses revealed that the frequencies of PTEN IVS4 genotypes were not associated with any clinicopathological parameters of patients with GC (Table 3, p>0.05).

Table 3.

Association of PTEN IVS +4 Polymorphism with Clinicopathological Features of Gastric Cancer

	PTEN IVS4 genotypes
	(−/−)	(−/+)/(+/+)	p-Value
Age
<50	28	13	0.155
≥50	29	23
Sex
Male	25	19	0.266
Female	32	17
Smoking status
No	31	22	0.337
Yes	26	14
Alcohol consumption
No	43	30	0.263
Yes	14	6
Tumor size
T1	4	2	0.231
T2	10	9
T3	29	11
T4	14	14
Nodal status
N0	1	2	0.477
N1	32	15
N2	17	14
N3	7	5
Metastases
No	42	21	0.123
Yes	15	15
Stages
I	5	2	0.218
II	28	11
III	10	8
IV	14	15
Differentiation
Well/moderately	33	22	0.76
Poor	24	14
Lymphovascular invasion
No	13	12	0.274
Yes	38	30

p-Value was obtained by χ² test.

p<0.05 was statistically significant.

Discussion

It is known that GC arises as the result of the accumulation of acquired genetic and epigenetic changes that transform a normal epithelium into an invasive carcinoma and eventually metastatic cancer. Recent studies have showed that SNPs are recognized as important risk factors for the development of GC.

This molecular epidemiological study investigated whether the PTEN IVS4 polymorphism could have an impact on susceptibility to GC in the Turkish population. PTEN was selected as a candidate gene because reported mutations of PTEN have been observed in GC, suggesting that SNPs of PTEN might play a role in the development and progress of GC. To the best of our knowledge, the possible role of PTEN IVS4 as a risk factor for GC has not been studied previously and our results suggest that PTEN IVS4 (−/−) genotype variations do influence susceptibility to GC development in the Turkish population. The potential functional role of the 5-bp insertion in intron 4 of the PTEN gene in the development of GC is not clarified. The mutations and the presence of the phosphatase core motif in the adjoining exon 5 have led to exon 5 being described as a hot spot for Cowden disease (Lynch et al., 1993; Marsh et al., 1998). Whether 210+109ins5 (IVS4) disrupts splicing or expression is unknown. The variant position, next to an exon that is a hot spot for Cowden disease mutations suggests that a splicing error deleting exon 5 could have a potentially significant effect. Alternatively, this variant might cause linkage disequilibrium with a functional mutation in another gene. Therefore, it is likely that this SNP is not the functional variant, but might be a surrogate marker for the underlying genetic variation within that region on the genome. The functional role of the PTEN IVS4 variant remains to be investigated and clarified. Even though an association between the PTEN IVS4 polymorphism and GC development has been detected, the effect of this variant might also be tissue-specific. Therefore, the role of the PTEN IVS4 variant in the development of other cancers also remains to be investigated. Results of the studies investigating the association between the PTEN IVS4 (rs 3830675) polymorphism and susceptibility to cancers are limited. Before our study, associations between SNPs in the PTEN gene and GC risk have been investigated only in one study that was focused on cardia tumors in comparison with esophageal squamous cancer (Ge et al., 2008). They have shown that PTEN IVS4 (−/+) gene polymorphisms are associated with susceptibility to esophageal squamous cancer and gastric cardia cancers in patients who have a family history of upper gastrointestinal cancers. They have reported that (+/−) genotype frequencies of PTEN IVS4 were significantly different in patients with cardia tumors than those of controls. Furthermore, they also reported that individuals who are carriers of the PTEN IVS4 (+/+) genotype had a reduced risk of development of gastric cardia tumors suggesting that the PTEN IVS4 region has a role in the regulation of carcinogenesis of GC. Beside this, an association of the PTEN IVS4 (−/+) polymorphism and cancer has been investigated in breast and prostate cancers (Carroll et al., 1999; George et al., 2001). In these studies, the PTEN IVS4 (+/+) variant has been reported to be associated with a younger onset of breast cancer (Carroll et al., 1999), and this genotype was found to be correlated with the tumor stage and grade for prostate cancer among men less than 69 years old (George et al., 2001).

There are several limitations that need to be addressed regarding the present study. We performed a hospital-based study, and the sample size of our study was small for a final conclusion of SNP analysis. Even so, the calculated sample size was sufficient (an alpha error of 5%, beta error of 50% was 25 samples) and the sample error of this study was calculated as 7.3% (95% CI in total population) with 91.8% statistical power (alpha error of 5%) (DSS Research Program, Fort Worth, TX); further studies with an enlarged sample size and a higher statistical power will be needed to verify our results. We also did not perform PTEN expressions in blood and tissue samples of patients with GC. Therefore, we did not assess the significance of the PTEN IVS4 polymorphism with PTEN expression in our patients with GC. A prospective clinical study, including a comparison of the PTEN IVS4 polymorphism with plasma and tissue expression of PTEN will provide a further assessment of the PTEN IVS4 polymorphism in GC patients. Other limitations are the size of the study groups and that it was hospital based. Studies need to be performed in larger study groups to confirm our preliminary results. Also, the exact role of the PTEN IVS4 polymorphism on PTEN expression and tumor development remain to be clarified with further studies.

In conclusion, our results demonstrate for the first time that the (−/−) genotype of PTEN IVS4 might have a role in susceptibility to GC development. Because this is the first report concerning the PTEN IVS4 polymorphism and the risk of GC in the literature, further independent studies are required to validate our findings in a larger series, as well as clarifying the exact role of the PTEN IVS (−/+) polymorphism in carcinogenesis of GC, determining PTEN expression of tumors and in patients of different ethnic origins and to better understand the underlying mechanism of PTEN polymorphisms and GC risk.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Carroll

, Couch

, Rebbeck

, Weber

. 1999. Polymorphisms in PTEN in breast cancer families. J Med Genet, 36:94-96.

, Cao

, Cgen

et al. 2008. Pten polymorphisms and the risk of esophageal carcinoma and gastric cardiac carcinoma in high incidence region of China. Dis Esophagus, 21:409-415.

George

, Shepard

, Ma

et al. 2001. PTEN polymorphism (IVS4) is not associated with risk of prostate cancer. Cancer Epidemiol Biomarkers Prev, 10:411-412.

Guo

, Xu

, Wu

, Liu

. 2008. PCR-SSCP-DNA sequencing method detecting PTEN gene mutation and its significance in human gastric cancer. Worl J Gastroenterol, 14:3804-3811.

Jemal

, Center

, DeSantis

, Ward

. 2010. Global patterns of cancer incidence and mortality rates and trends. Cancer Epidemiol Biomarkers Prev, 19:1893-1907.

Jiang

, Fan

, Jiang

et al. 2003. Expression and significance of PTEN, hypoxia-inducible factor-1 alpha in colorectal adenoma and adenocarcinoma. World J Gastroenterol, 9:491-494.

, Yen

, Liaw

et al. 1997. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science, 275:1943-1947.

Lynch

, Smyrk

, Watson

et al. 1993. Genetics, natural history, tumor spectrum, and pathology of hereditary nonpolyposis colorectal cancer: an updated review. Gastroenterology, 104:1535-1549.

Marsh

, Coulon

, Lunetta

et al. 1998. Mutation spectrum and genotype-fenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation. Hum Mol Genet, 7:507-515.

10.

Miller

, Dykes

, Polesky

. 1988. Simple salting-out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res, 16:1215.

11.

Milne

, Carneiro

, O'morain

, Offerhaus

. 2009. Nature meets nurture: molecular genetics of gastric cancer. Hum Genet, 126:615-28.

12.

Okami

, Wu

, Riggins

et al. 1998. Analysis of PTEN/MMAC1 alterations in aerodigestive tract tumors. Cancer Res, 58:509-511.

13.

Resende

, Ristimaki

, Machado

. 2010. Genetic and epigenetic alteration in gastric carcinogenesis. Helicobacter, 15:34-39.

14.

Sato

, Tamura

, Tsuchiya

et al. 2002. Analysis of genetic and epigenetic alterations of the PTEN gene in gastric cancer. Virchows Arch, 440:160-165.

15.

Vogiatzi

, Vindigni

, Roviello

et al. 2007. Deciphering the underlying genetic and epigenetic events leading to gastric carcinogenesis. J Cell Phsiol, 211:287-295.

16.

Wang

, Huan

, Chen

et al. 2003. Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in advanced gastric carcinomas. Virchows Arch, 442:437-443.

17.

Wen

, Wang

, Zhou

et al. 2010. Mutation analysis of tumor suppressor gene PTEN in patients with gastric carcinomas and its impact on PI3K/AKT pathway. Oncol Reports, 24:89-95.

18.

, Senechal

, Neshat

et al. 1998. The PTEN/MMAC1 tumor suppressor phosphotase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc Natl Acad Sci U S A, 95:1558-15591.

19.

Yin

, Hu

, Tan

et al. 2009. Molecular epidemiology of genetic susceptibility to gastric cancer: facus on single nucleotide polymorphisms in gastric carcinogenesis. Am J Transl Res, 1:44-54.