Phospholipase C Epsilon 1 ( PLCE 1 rs 2274223 A > G , rs 3765524 C > T and rs 7922612 C > T ) Polymorphisms and Esophageal Cancer Risk in the Kashmir Valley Manzoor

Carcinomas of the esophagus have diverse incidence patterns and risk factors (Brown et al., 2002). The etiology of these cancers is incompletely understood, like many other malignancies. Overall, esophageal cancer incidence and mortality have fallen dramatically over the past 70 years (Parkin, 2008). Despite its recent decline, esophageal cancer (EC) is the eighth most commonly occurring cancers in the world (Parkin et al., 1993). Within the Indian subcontinent, the Valley of Kashmir presents a strikingly different picture where incidence of esophageal cancer have been reported to exceed 40% of all cancers and incidence is 3-6 times higher than various metropolis cancer registries in India (Khuroo et al., 1992; Siddiqi et al., 1992). Some of the genetic and environment factors have been reported to be associated with an increased risk of esophageal cancer in Kashmir valley (Siddiqi et al., 1988; Malik et al., 2009; 2012; Chikan et al., 2012). A number of epidemiological studies have been used as a tool to find out common susceptibility markers of EC, however the results of only few studies were concordant when replicated in other independent populations.


Introduction
Carcinomas of the esophagus have diverse incidence patterns and risk factors (Brown et al., 2002).The etiology of these cancers is incompletely understood, like many other malignancies.Overall, esophageal cancer incidence and mortality have fallen dramatically over the past 70 years (Parkin, 2008).Despite its recent decline, esophageal cancer (EC) is the eighth most commonly occurring cancers in the world (Parkin et al., 1993).Within the Indian subcontinent, the Valley of Kashmir presents a strikingly different picture where incidence of esophageal cancer have been reported to exceed 40% of all cancers and incidence is 3-6 times higher than various metropolis cancer registries in India (Khuroo et al., 1992;Siddiqi et al., 1992).Some of the genetic and environment factors have been reported to be associated with an increased risk of esophageal cancer in Kashmir valley (Siddiqi et al., 1988;Malik et al., 2009;2012;Chikan et al., 2012).
A number of epidemiological studies have been used as a tool to find out common susceptibility markers of EC, however the results of only few studies were concordant when replicated in other independent populations.

RESEARCH ARTICLE
Phospholipase C Epsilon 1 (PLCE1 rs2274223A>G, rs3765524C>T and rs7922612C>T) Polymorphisms and Esophageal Cancer Risk in the Kashmir Valley Manzoor Ahmad Malik 1 , Meenakshi Umar 1 , Usha Gupta 1 , Showkat Ali Zargar 2 , Balraj Mittal 1 * The development of genome-wide association study (GWAS) has a major impact on the discovery of susceptibility genes for complex disease and exploring the relationship between a large number of single nucleotide polymorphisms (SNPs) and disease predisposition.A GWAS for PLCE1 was published in 2010 and identified significant associations with rs2274223 (Arg1927His) and rs3765524 (Ile1777Thr) on chromosome 10q23 in Chinese population.These two SNPs result in missense mutations in the coding region, rs2274223 (Arg1927His) and rs3765524 (Ile1777Thr) (Abnet et al., 2010;Wang et al., 2010).
The PLCE1 belongs to the phospholipase family that catalyzes the hydrolysis of polyphosphoinositides to generate secondary messengers, such as inositol-1,4,5 trisphosphate and diacylglycerol.Therefore, PLCE1 is involved in cell growth and differentiation (Wing et al., 2003).Epidemiological study have shown that PLCE1 functions as an effector of Ras and is a major factor in progression of various cancers, intestine (Li et al., 2009), skin (Bai et al., 2004), bladder (Ou et al., 2010), colorectal (Wang et al., 2008) and head and neck (Bourguignon et al., 2006).These findings suggest that single nucleotide polymorphisms (SNPs) in PLCE1 that affect the gene expression or protein functions, may affect the risk of some cancers.However, this finding lacks independent replication in many populations (Bye et al., 2012;Palmer et al., 2012).Therefore, we hypothesized that GWAS discovered SNPs in the PLCE1 gene are may be associated with esophageal cancer risk in patients from Kashmir valley.To test this hypothesis, we replicate association of three polymorphisms (rs2274223A>G, rs3765524C>T and rs7922612C>T) PLCE1 polymorphisms with susceptibility to EC in Kashmir valley a high risk area.

Materials and Methods
This case control study comprised histopathologically confirmed cases with esophageal (EC) cancer ( 135) and healthy controls (195) from the population of Kashmir valley.The sample size of the present study was adequate to provide 80% power.All subjects were unrelated ethnic Kashmiri residents, referred from the Departments of Gastroenterology, Sher-i-Kashmir Institute of Medical Sciences, Srinagar from May 2006 to July 2008.Patients were excluded if they had non-malignant conditions like corrosive esophageal injury, Achalasia injury, Barrett's esophagus, gastro-esophageal reflux disease (GERD) and non-ulcer dyspepsia.The controls were also recruited from Sher-i-Kashmir Institute of Medical Sciences, Srinagar, who come for their routine checkup and were diagnosed as non-severe ailment and no malignancy.All the individuals were personally interviewed for their age, occupation, demographic features, dietary habits (Haak=brassica oleraceae; Wur=red chilies), usage of hot noon chai (salted tea) and smoking habits.Tobacco usage included smoking of cigarette, or hukka (water pipe).Written informed consent was obtained from all participants in the study.The research protocol was approved by the ethical committee of Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow (28 th Ethics Committee Meeting on May 14, 2004).Sample collection, storage and transport were complied with guidelines of the committee.Blood samples were collected in EDTA and the genomic DNA was extracted from peripheral blood leukocytes pellet using the standard salting-out method (Miller et al., 1998).The quality and quantity of DNA was checked by gel electrophoresis and spectrophotometry using Nanodrop Analyser (ND-1000) spectrophotometer (Nano Drop Technologies, Inc., Wilmington, DE, USA).The ratio of absorbance at 260 and 280 nm of DNA was around 1.7-1.9.The isolated DNA was stored at -70°C.

Genotyping
The genotyping of all three PLCE1 gene polymorphisms were carried through PCR RFLP method and detailed methodology is shown in Table 1.The genotypes of PLCE1 genetic variants were assigned on the basis of band sizes.In case of PLCE1 rs2274223 A>G polymorphism, A allele showed a band of 242 bp and G allele yielded two fragments of 155 bp and 87 bp.PLCE1 rs3765524 C>T C allele resulted in 326 bp fragment and the variant allele, designated T allele resulted in 200 bp and 126 bp fragments.The wild type C allele of PLCE1 rs7922612 C>T polymorphism on restriction digestion showed two fragments of 197bp and 143 bp, however the variant T allele was resistant to digestion and yielded a single fragment of 340 bp.More than 15% of the samples were randomly selected for confirmation, and the results were 100% concordant.

Statistical analysis
Demographic characteristics of patients and controls were described as frequencies and percentages, whereas descriptive statistics of patients and controls were presented as mean and standard deviations for continuous measures.Statistical significance of frequency differences between patients and control groups was evaluated using the χ 2 test.Deviation from the Hardy-Weinberg equilibrium in controls was assessed using the χ 2 test; p value was considered significant at <0.05 level.Effective sample sizes were calculated by Quanto software version 1.2.The same controls were used for analyzing two sets of cancer cases.Association was expressed as odds ratios (OR) for risk estimation with 95% confidence intervals (95% CI).All analyses were performed using the SPSS statistical analysis software, version 15.0 (SPSS, Chicago, IL, USA).

Population characteristics
The mean age of healthy subjects (controls) and patients with EC was 57.98yrs±12.67and 60.38yrs±8.41respectively (t-test p value=ns).EC was highly prevalent in males (68.1%) than in females.In patients with EC, squamous cell carcinoma (SCC) histopathology was common (76.3%).Smoking habit (Hukka) showed significantly higher risk in EC (OR=21.45;95%CI=11.63-39.55;p=0.0001) patients.Individuals consumed saltedtea in a range of 2-8 cups per day; and median consumption of tea was 4 cups per day.So, we grouped individuals in to ≤4 cups or >4 cups per day and individuals consumed salted tea >4 cups per day were regarded as high salted tea consumers.Higher consumption of salted tea was also found to be associated with increased risk of EC (OR=14.86;95%CI=8.42-26.25;p value=0.0001) (Table Table 1    2).None of patients or controls reported consumption of alcohol, so interaction of alcohol intake with genetic variations could not be analyzed.No association was observed between intakes of Brassica oleraceae or red chillies with EC (data not shown).

Association of genetic variants of PLCE1 (rs2274223A>G, rs3765524C>T and rs7922612C>T) polymorphism with susceptibility to EC
The genotype and allele distributions of three SNPs (rs2274223A>G, rs3765524C>T and rs7922612C>T) in cases and controls are shown in Table 3.The observed genotype frequencies for all these three polymorphisms were in Hardy-Weinberg equilibrium in the controls.The single locus analyses revealed that genotype distributions of these three polymorphisms were not significantly different between overall cases and controls (p=0.77for rs2274223, p=0.43 for rs7922612 and p=0.94 for rs3765524, respectively).Also at allele level we did not find any significant association (Table 3).

PLCE1 (rs2274223A>G, rs3765524C>T and rs7922612C>T) haplotype analysis and risk of EC
A total of eight haplotypes were observed in the study subjects.The frequency of haplotype G 2274223 T 3765524 T 7922612 was higher in patients as compared to controls and presented statistically significant increased risk of EC (OR=2.92;95% CI=1.30-6.54;P=0.009) even after Bonferroni's correction (0.027) (Table 4).

Association of PLCE1 rs2274223A>G, rs3765524C>T and rs7922612C>T gene polymorphisms with tumor histopathology and environmental factors like smoking and salted tea
While analyzing the genotypes with histology (squamous cell carcinoma and adenocarcinoma) none of the PLCE1 genetic variants were associated with risk in EC (data not shown).We examined the possible interactions of PLCE1 (rs2274223, rs7922612 and rs3765524) genotypes with smoking and salted tea consumption and risk of EC in study subjects.Although both smoking and salted tea were independent risk factors for EC in the Kashmir Valley, we did not find any enhanced risk of EC with PLCE1 genotypes in subjects with smoking and salted tea intake habits (Table 5 and Table 6).

Discussion
In present study, we investigated the associations between three novel, potentially functional SNPs of PLCE1 (rs2274223, rs3203713 and rs11599672) with risk of EC in Kashmir valley.In the present study, we observed that although PLCE1 polymorphism did not independently modulate risk of EC, however, one PLCE1 haplotypes was found to be associated with significant increased risk of EC in Kashmiri population.
Recently in Han Chinese populations, polymorphism in PLCE1 was identified as a risk factor for GC and EC by two contemporary independent GWAS (Abnet et al., 2010;Wang et al., 2010).Also two replicated independent studies have also validated the association of PLCE1 rs2274223A>G polymorphism with head and neck cancer and gastric cancer in Chinese population (Luo et al., 2011;Ma et al., 2011).Recently, a metaanalysis of PLCE1 rs2274223 A>G polymorphism also suggested its involvement in conferring increased risk of esophageal cancer risk (Umar et al., 2013).
PLCE1 rs2274223 A>G was found to increase the risk of esophageal and gastric cancers in genome wide association study (GWAS).This nonsynonymous SNP present in exon 26 results in histidine to arginine amino acid change in the Ca2 + -dependent lipid binding domain of the protein.Although the exact functional relevance of the polymorphism is not known, Hu et al. (2000) have shown significant reduced staining of PLCE1 protein in the ESCC tissues of the rs2274223 GG genotype compared to that in the AG genotype.Also, one study have shown a downregulation of the PLCE1 mRNA expression in tumor tissues of colorectal cancer, gastric adenocarcinoma, and esophageal squamous cell carcinoma compared to respective normal tissues (Wang et al., 2008).
In addition to possible exposure to well known risk factors (like smoking and salted tea) for esophageal and gastric cancers, the people of valley have many unique social, cultural and dietary features which are different from rest of world.Salted tea used by people is prepared by using baking soda (sodium bicarbonate) and common salt (sodium chloride) and boiled for few hours before consuming.It has been suspected that the salts might cause thermal injury to esophageal and gastric epithelium (Khuroo et al., 1992).Several previous studies have attributed high incidence of esophageal and gastric cancers in Kashmir to considerable amount of nitroso compounds in raw foodstuffs and use of hot salted tea (Khuroo et al., 1992;Siddiqi et al., 1992).Salted tea used in Kashmir valley has considerable amounts of N-nitrosoproline (NPRO) (360 micrograms/kg) and N nitrosopipecolic acid (NPIC) (5870 micrograms/kg) which may impart risk for EC in this area (Siddiqi et al., 1988).In the present study salted tea was significantly associated with increased high risk for EC (OR=18.179;p=0.001).Also our results show significant independent association of smoking (Hukka) with EC (OR=21.465;p=0.0001).But in gene environment interactions no such association was observed.Brassica oleraceae or red chillies also did not show any association with EC.It appears that genetic variants of PLCE1 modulate susceptibility to EC independent of environmental interactions.
In conclusion, the association of the PLCE1 polymorphisms seem important in carcinogenesis of EC in Kashmiri Valley.It is worthwhile conducting additional population-based studies in large sample size before its clinical application.

Table 2 . Demographic Characteristics of Esophageal Cancer Patients and Healthy Controls of Kashmir Valley
*Age and gender adjusted odds ratio; **Data missing

Table 3 . Frequency Distribution of PLCE1 rs2274223A>G, rs3765524C>T and rs7922612C>T and Risk Assessment in Esophageal Cancer Cases and Control's
*Age and gender adjusted odds ratio

Table 5 . Interaction of PLCE1 rs2274223A>G, rs3765524C>T and rs7922612C>T Genotypes and Smoking in Modulation of EC Risk
*Data missing in some subjects; **Age and gender adjusted odds ratio; OR was calculated for smokers only

Table 6 . Interaction of PLCE1 rs2274223A>G, rs3765524C>T and rs7922612C>T Genotypes and Salted Tea in Modulation of EC Risk
Data missing in some subjects; **Age and gender adjusted odds ratio; OR was calculated for >4 cup tea consumption in cases and controls *