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LRFN2 gene variant rs2494938 provides susceptibility to esophageal cancer in the population of Jammu and Kashmir


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 Submission26-Aug-2019
Date of Decision24-Jan-2020
Date of Acceptance26-Jan-2020
Date of Web Publication22-Apr-2020

Correspondence Address:
Nazir Ahmed Dar,
Department of Biochemistry, University of Kashmir, Srinagar - 180 001, Jammu and Kashmir
India
Swarkar Sharma,
Human Genetics Research Group, School of Biotechnology, Shri Mata Vaishno Devi University, Katra - 182 320, Jammu and Kashmir
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_613_19

PMID: 32880595

 > Abstract 


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 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 [Epub ahead of print] [cited 2020 Oct 24]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=283076




 > Introduction Top


Esophageal cancer is the carcinoma of the food pipe which connects the buccal cavity with the stomach.[1] 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.[2] Esophageal carcinoma is categorized into two major subtypes esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC).[3] About 90% of esophageal carcinomas diagnosed worldwide are ESCC and only 5% are EAC.[4],[5] Esophageal cancer has a very complex and divergent etiology [6] and the number of risk factors such as smoking, alcohol consumption, and environmental factors are associated with esophageal carcinoma.[7],[8] 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).[9],[10] In addition, recent literature has indicated the critical role of genetic factors in disease development.[11],[12] The genetic mutation [13] and genomic variations have a major role in disease development,[14],[15] but this genetic landscape is heterogeneous and complex.[14],[16] 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.[17] 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.[18] 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 Top


Sampling

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.



DNA isolation

Genomic DNA was isolated from blood by phenol chloroform method and quantitated. DNA aliquots were made as per routine protocol in laboratories.[19],[20] The quality and quantity of the genomic DNA were analyzed by performing agarose gel electrophoresis and UV spectrophotometer, respectively, before genotyping.

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.

Statistical analyses

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 Top


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].


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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.[17] 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.[21] Although there is a major reduction in mortality and incidence of esophageal carcinoma worldwide over the past 70 years,[22] 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.[18] 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.[23] 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.[17] 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.).[24] 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.[25] 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.[26] 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.[27]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.[26]



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.[26],[28] 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 Top


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.

Acknowledgment

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.



 
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