|Year : 2020 | Volume
| Issue : 8 | Page : 156-159
LRFN2 gene variant rs2494938 provides susceptibility to esophageal cancer in the population of Jammu and Kashmir
Ruchi Shah1, Varun Sharma1, Hemender Singh1, Indu Sharma1, Gulzar Ahmed Bhat2, Idrees Ayoub Shah2, Beenish Iqbal2, Rumisa Rafiq2, Najma Nissa2, Mansha Muzaffar2, Malik Tariq Rasool3, Ghulam Nabi Lone4, Sandeep Kaul5, Mohd Maqbool Lone3, Ekta Rai1, Nazir Ahmed Dar2, Swarkar Sharma1
1 Human Genetics Research Group, School of Biotechnology, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India
2 Department of Biochemistry, University of Kashmir, Srinagar, Jammu and Kashmir, India
3 Department of Radiation Oncology, SK Institute of Medical Sciences, Srinagar, Jammu and Kashmir, India
4 Department of CVTS, SK Institute of Medical Sciences, Srinagar, Jammu and Kashmir, India
5 Department of Surgical Oncology, SMVDNH Superspeciality Hospital, Katra, Jammu and Kashmir, India
|Date of Submission||26-Aug-2019|
|Date of Decision||24-Jan-2020|
|Date of Acceptance||26-Jan-2020|
|Date of Web Publication||22-Apr-2020|
Nazir Ahmed Dar
Department of Biochemistry, University of Kashmir, Srinagar - 180 001, Jammu and Kashmir
Human Genetics Research Group, School of Biotechnology, Shri Mata Vaishno Devi University, Katra - 182 320, Jammu and Kashmir
Source of Support: None, Conflict of Interest: None
Background: Leucine-rich repeat and fibronectin type 2 gene (LRFN2) variant rs2494938 has recently been found associated with esophageal cancer in a genome-wide association study in an Asian population. However, this association has not been replicated in any Indian population despite high incidence of the disease.
Materials and Methods: In the present case–control study, 166 cases and 459 controls were included. Taqman assay technique using real-time PCR was employed to investigate the association of the variant with esophageal cancer in the population of Jammu and Kashmir (J&K). The Hardy-Weinberg equilibrium for rs2494938 was assessed using the Chi-square test. The allele- and genotype-specific risk was estimated by odds ratio (OR) with 95% confidence interval (CI).
Results: Variant rs2494938 was observed to be significantly associated with esophageal cancer with an allelic OR of 1.59 (1.23–2.04 at 95% CI, P = 0.0003).
Conclusion: The study highlights LRFN2 as a candidate gene for esophageal cancer susceptibility in the population of J&K and calls for a detailed study with a large sample size and involving more ethnic groups of India.
Keywords: Candidate gene, case–control, esophageal cancer, Jammu and Kashmir, leucine-rich repeat and fibronectin type 2, odds ratio
|How to cite this article:|
Shah R, Sharma V, Singh H, Sharma I, Bhat GA, Shah IA, Iqbal B, Rafiq R, Nissa N, Muzaffar M, Rasool MT, Lone GN, Kaul S, Lone MM, Rai E, Dar NA, Sharma S. LRFN2 gene variant rs2494938 provides susceptibility to esophageal cancer in the population of Jammu and Kashmir. J Can Res Ther 2020;16, Suppl S1:156-9
|How to cite this URL:|
Shah R, Sharma V, Singh H, Sharma I, Bhat GA, Shah IA, Iqbal B, Rafiq R, Nissa N, Muzaffar M, Rasool MT, Lone GN, Kaul S, Lone MM, Rai E, Dar NA, Sharma S. LRFN2 gene variant rs2494938 provides susceptibility to esophageal cancer in the population of Jammu and Kashmir. J Can Res Ther [serial online] 2020 [cited 2021 Jan 25];16:156-9. Available from: https://www.cancerjournal.net/text.asp?2020/16/8/156/283076
| > Introduction|| |
Esophageal cancer is the carcinoma of the food pipe which connects the buccal cavity with the stomach. It is the eighth-most common type of cancer and the sixth-most common cause of cancer-related deaths worldwide. The highest incidence of esophageal carcinoma is reported in Asia and Africa. Esophageal carcinoma is categorized into two major subtypes esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). About 90% of esophageal carcinomas diagnosed worldwide are ESCC and only 5% are EAC., Esophageal cancer has a very complex and divergent etiology  and the number of risk factors such as smoking, alcohol consumption, and environmental factors are associated with esophageal carcinoma., Poor oral hygiene and tobacco products such as snuff (Nass) and Hukkah have been reported to be associated with ESCC in the population of Jammu and Kashmir (J&K)., In addition, recent literature has indicated the critical role of genetic factors in disease development., The genetic mutation  and genomic variations have a major role in disease development,, but this genetic landscape is heterogeneous and complex., A recent genome-wide association study (GWAS) in an Asian population indicated rs2494938 variant present in Leucine-rich repeat fibronectin type 3 domain containing 2 (LRFN2) gene to be associated with three types of cancers including ESCC. However, the association of this variant has never been replicated in any of the Indian population groups. J&K is located at the extreme north of India and primarily consists of hills and mountains. Esophageal and gastric cancer are among the most leading cancers in this population and account for almost 25%–34% of cancers. Because of the peculiar profile of this cancer, this region has been indicated as a cancer belt of India. With this background, we carried out thefirst replication study to find out the role of variant rs2494938 in association with ESCC in population of J&K in-India.
| > Materials and Methods|| |
A total of 625 (166 ESCC and 459 age- and sex-matched controls) individuals participated in the present study [Supplementary Table 1]. The participants were enrolled at the University of Kashmir and Shri Mata Vaishno Devi University (SMVDU) Katra with informed consent. All the cancer cases were histopathologically confirmed and any other cancer type was excluded. A volume of 5 ml blood sample was collected from ethnically matched cases and controls without known family history of esophageal or any other cancer for two generations. The study was approved by the Institutional Ethical Review Board, SMVDU. Interaction of LRFN2 gene with other genes was evaluated using GeneMania Software of BioGRID version 3.4.
Genomic DNA was isolated from blood by phenol chloroform method and quantitated. DNA aliquots were made as per routine protocol in laboratories., The quality and quantity of the genomic DNA were analyzed by performing agarose gel electrophoresis and UV spectrophotometer, respectively, before genotyping.
The Taqman Allelic discrimination assay was adopted to perform the genotyping using real-time PCR (Agilent Mx3005p). Taqman Probe of rs2494938 and UNG Master Mix (Applied Biosystems, USA) were used. Samples were aliquoted in 96 well format and 3 nontemplate controls were used in each plate for quality control. The following thermal cycling conditions were adopted; 10 min at 95°C, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Genotyping call quality was checked by randomly genotyping 80 samples to check for 100% concordance.
Chi-square test was carried out to test genotype distribution of the SNP-single nucleotide polymorphism (SNP) for the Hardy–Weinberg equilibrium. Logistic regression analysis was performed to estimate odds ratio (OR) at 95% confidence interval (CI) and level of significance (P value). Age, gender, and body mass index [Supplementary Table 1] were used as the covariates in logistic regression analysis. The statistical analyses were performed using the Statistical Package for the Social Sciences SPSS software (version 23; Chicago, IL). Post hoc power of the study was also done by PS software version 3.1.2 (17).
| > Results and Discussion|| |
In this preliminary case–control study, 166 cases and 459 controls (n = 625) were analyzed. Genotypic and allelic frequency distribution was observed to be following HWE in the population. Risk allele frequency, OR, and other association details are summarized in [Table 1]. The allele G was observed to be associated with ESCC following recessive mode of inheritance (GG vs AG + AA) with OR 2.6 (1.5–4.3, at 95% CI, P = 0.0002). Other applied models and respective estimated OR are summarized in [Supplementary Table 2].
The present study replicated the association of variant rs2494938 in the LRFN2 gene with esophageal carcinoma in the population of J&K. In GWAS of multiple cancers in the Han Chinese population, the variant rs2494938 of LRFN2 was found to be significantly associated with ESCC. In GWAS, minor allele (A) frequency observed was 0.25 in cases and 0.23 in controls. In the present study, the allele frequency differs, the estimated risk/minor allele (G) frequency was 0.491 in cases and 0.378 in controls. In order to verify the frequency difference, we verified it with HapMap allele frequency of Gujrati Indians living in Houston, which shows “G” as the minor allele (0.465) and “A” as the major allele with frequency 0.534. There are multiple risk factors associated with esophageal carcinoma. Although there is a major reduction in mortality and incidence of esophageal carcinoma worldwide over the past 70 years, but in J&K region of India, the incidence of esophageal cancer is believed to be 3–6 times higher than other metropolis cancer registries in India. The present study replicated the association of variant rs2494938 of LRFN2 gene with esophageal carcinoma in the population of J&K.
LRFN2 protein is a member of SALMs family (synaptic-adhesion like molecule) that shows interaction with N-methyl D-aspartate (NMDA) receptor. Methylation of 2B receptor of NMDA has been observed to be associated with both ESCC and nonsmall cell lung carcinoma and LRFN2 has been proposed to serve as a susceptibility gene for multiple cancers through LRFN2-NMDA receptor pathway. In J&K, people have very peculiar dietary habits and smoking hukkah and cigarettes is a common practice. In addition, changes in lifestyle, psychological stress due to various social disturbances might have implications too. Evidence in literature exists extensively that indicate such environmental factors have huge potential in causing epigenetic alterations (methylation, histone modification, etc.). With this background, we hypothesize that the strong association of the variant in the gene LRFN2 indicate an interactive mechanism, either of the variant or other functional variants in LD, which is influencing LRFN2-NMDA pathway.
To evaluate, we looked into the interaction of LRFN2 gene using GeneMania Software of BioGRID version 3.4. We found a strong protein interaction of LRFN2 with DLG3 (discs large MAGUK scaffold protein 3) and (DLG1 discs large MAGUK scaffold protein 1) [Supplementary Figure 1]. DLG1 encodes multi-domain scaffolding proteins that are required for normal cellular development. These proteins may have a role in junction formation, signal transduction, cell proliferation, synaptogenesis, and lymphocyte activation and are known to interact with NMDA receptors. Interaction of all the three genes LRFN2, DLG1 and DLG3 with NMDA is shown in [Supplementary Figure 2]. DLG1 can acquire oncogenic attributes in some specific contexts.DLG3 encodes synapse associated protein 102 (SAP 102), a member of membrane-associated guanylate kinase protein family. SAP 102 consists of three domains: PDZ domain, SRC homology domain, and carboxyl-terminal guanylate kinase domain. NR2 subunit of NMDAR directly interacts with PDZ domain and other proteins. These interactions are responsible for NMDA receptor localization, immobilization, and signaling. Impaired functionality of SAP 102 can lead to disruption of domain-dependent distribution and anchoring of NMDA receptors within the plasma membrane which can lead to dysfunctional NMDA receptor signaling.
Keeping in view the interaction of LRFN2 gene with DLG1 and DLG3, we evaluated further the expression of both the genes in human protein atlas RNA seq in normal tissues., Both the genes show tissue specificity in the colon and esophageal tissues. DLG3 shows mean reads per kilobase million (RPKM) of 4.776 ± 0.738 in esophageal tissues and 6.228 ± 1.013 in the colon. DLG1 shows mean RPKM of 10.278 ± 1.624 in esophageal tissues and RPKM of 8.068 ± 1.267 in the colon [Supplementary Figure 3] and [Supplementary Figure 4]. These observations substantiated further our hypothesis that requires experimental validation.
| > Conclusion|| |
LRFN2 gene emerges as a candidate gene for ESCC susceptibility in the population of J&K, India. Further, screening of LRFN2 using sequencing methodologies may help to find functional variants. Furthermore, experimental studies involving these LRFN2 variations are required, which may exhibit functional correlation with respect to interaction with the NMDA receptor pathway.
RS and SS acknowledge Women Scientist Scheme (WOS-A), Department of Science and Technology, grant no. (SR/WOS-A/LS1067/2015) Government of India for financial support. RS, ND and SS has planned the study, VS, IS, HS and ER edited the manuscript critically.GA, IA, BI, RR, NN and MM helped in sampling and compilation of tables and figures. MTR, GNL, SK and MML provided the samples.
Financial support and sponsorship
Women Scientist Scheme (WOS-A), Department of Science and Technology, grant no. (SR/WOS-A/LS1067/2015), Government of India for financial support.
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Singhi AD, Arnold CA, Crowder CD, Lam-Himlin DM, Voltaggio L, Montgomery EA. Esophageal leukoplakia or epidermoid metaplasia: A clinicopathological study of 18 patients. Mod Pathol 2014;27:38-43.
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al
. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359-86.
Lin DC, Hao JJ, Nagata Y, Xu L, Shang L, Meng X, et al
. Genomic and molecular characterization of esophageal squamous cell carcinoma. Nat Genet 2014;46:467-73.
Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108.
Vizcaino AP, Moreno V, Lambert R, Parkin DM. Time trends incidence of both major histologic types of esophageal carcinomas in selected countries, 1973-1995. Int J Cancer 2002;99:860-8.
Mir MM, Dar NA. Esophageal cancer in Kashmir (India): An enigma for researchers. Int J Health Sci (Qassim) 2009;3:71-85.
Yang CS. Research on esophageal cancer in China: A review. Cancer Res 1980;40:2633-44.
van Rensburg SJ. Epidemiologic and dietary evidence for a specific nutritional predisposition to esophageal cancer. J Natl Cancer Inst 1981;67:243-51.
Dar NA, Islami F, Bhat GA, Shah IA, Makhdoomi MA, Iqbal B, et al
. Poor oral hygiene and risk of esophageal squamous cell carcinoma in Kashmir. Br J Cancer 2013;109:1367-72.
Dar NA, Bhat GA, Shah IA, Iqbal B, Makhdoomi MA, Nisar I, et al
. Hookah smoking, nass chewing, and oesophageal squamous cell carcinoma in Kashmir, India. Br J Cancer 2012;107:1618-23.
Testa U, Castelli G, Pelosi E. Esophageal cancer: Genomic and molecular characterization, stem cell compartment and clonal evolution. Medicines (Basel) 2017;4. pii: E67.
Huang FL, Yu SJ. Esophageal cancer: Risk factors, genetic association, and treatment. Asian J Surg 2018;41:210-5.
Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, et al
. Signatures of mutational processes in human cancer. Nature 2013;500:415-21.
Dulak AM, Stojanov P, Peng S, Lawrence MS, Fox C, Stewart C, et al
. Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat Genet 2013;45:478-86.
Nones K, Waddell N, Wayte N, Patch AM, Bailey P, Newell F, et al
. Genomic catastrophes frequently arise in esophageal adenocarcinoma and drive tumorigenesis. Nat Commun 2014;5:5224.
Weaver JMJ, Ross-Innes CS, Shannon N, Lynch AG, Forshew T, Barbera M, et al
. Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis. Nat Genet 2014;46:837-43.
Jin G, Ma H, Wu C, Dai J, Zhang R, Shi Y, et al
. Genetic variants at 6p21.1 and 7p15.3 are associated with risk of multiple cancers in Han Chinese. Am J Hum Genet 2012;91:928-34.
Khuroo MS, Zargar SA, Mahajan R, Banday MA. High incidence of oesophageal and gastric cancer in Kashmir in a population with special personal and dietary habits. Gut 1992;33:11-5.
Sharma V, Sharma I, Sethi I, Mahajan A, Singh G, Angural A, et al
. Replication of newly identified type 2 diabetes susceptible loci in Northwest Indian population. Diabetes Res Clin Pract 2017;126:160-3.
Mir MM, Dar NA, Gochhait S, Zargar SA, Ahangar AG, Bamezai RN. p53 mutation profile of squamous cell carcinomas of the esophagus in Kashmir (India): A high-incidence area. Int J Cancer 2005;116:62-8.
Brown LM, Devesa SS. Epidemiologic trends in esophageal and gastric cancer in the United States. Surg Oncol Clin N
Parkin DM. The role of cancer registries in cancer control. Int J Clin Oncol 2008;13:102-11.
Wang CY, Chang K, Petralia RS, Wang YX, Seabold GK, Wenthold RJ. A novel family of adhesion-like molecules that interacts with the NMDA receptor. J Neurosci 2006;26:2174-83.
Alegría-Torres JA, Baccarelli A, Bollati V. Epigenetics and lifestyle. Epigenomics 2011;3:267-77.
Stark C, Breitkreutz BJ, Reguly T, Boucher L, Breitkreutz A, Tyers M. BioGRID: A general repository for interaction datasets. Nucleic Acids Res 2006;34:D535-9.
Tarpey P, Parnau J, Blow M, Woffendin H, Bignell G, Cox C, et al
. Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation. Am J Hum Genet 2004;75:318-24.
Roberts S, Delury C, Marsh E. The PDZ protein discs-large (DLG): the 'Jekyll and Hyde' of the epithelial polarity proteins. FEBS J 2012;279:3549-58.
Fagerberg L, Hallström BM, Oksvold P, Kampf C, Djureinovic D, Odeberg J, et al
. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics 2014;13:397-406.