Genetic variant rs2494938 of LRFN2 gene is associated with non-small cell lung cancer risk in North-Indian population

Abstract

Various Genome-wide association studies (GWAS) have reported the association of variant rs2494938 with lung cancer. However, genetic association of LRFN2 genetic variation with non-small cell lung cancer (NSCLC) in North Indian population remained unexplored. We conducted a case–control association study using TaqMan-based chemistry in which a total of 619 individuals, 189 NSCLC cases and 430 controls, were genotyped to explore the association of rs2494938 genetic variant of the LRFN2 gene with NSCLC patients from North India. The allele ‘G’ (risk allele) of the genetic variant rs2494938 was significantly associated with the NSCLC [OR = 1.51 (1.18–1.93 at 95% CI); p value = 0.0009]. Genetic association was also explored by applying different genetic models (Dominant, Additive). These results suggest that rs2494938 polymorphism of the LRFN2 gene is a risk factor in the North Indian populations to develop NSCLC. The LD (Linkage Disequilibrium) plot demonstrates the variant and its LD SNPs (r² > 0.8) and the variant has direct regulatory effect, which could affect the overall physiology of the gene. These findings could be used as diagnostic and prognostic markers in clinical studies of lung cancer patients in North Indian population groups. The present study also provides an important evidence on the genetic etiology of NSCLC in North Indian populations and further expounds GWAS findings on the role of LRFN2 in lung cancer risk. This study provides the holistic view about the non-small cell lung cancer in Jammu and Kashmir, North Indian population and it can be a hallmark of cancer if verified on a very large sample size (cohort).

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02403-1) contains supplementary material, which is available to authorized users.

Introduction

Lung cancer is a heterogeneous disease and a major global health concern. Histologically, 80% of lung cancers are non-small cell lung cancer (NSCLC) and 20% are small-cell lung cancer (SCLC). Despite making numerous efforts to increase the 5-year survival rate of lung cancer patients, yet it remains 15–20%. Since, lung cancer diagnosis and screening through conventional techniques have become less informative, nowadays, focus has shifted towards biomarkers for earlier diagnosis. Lung cancer accounts for 12.9% of all newly diagnosed cancer cases and 19.4% of all cancer-related deaths globally (Jemal et al. 2011; Torre et al. 2015). In India, it accounts for 63,000 newly diagnosed lung cancer cases each year with 52,000 deaths, thereby contributing 9.3% of all cancer-related deaths (Ganesh et al. 2011; Malik and Raina 2015). Among the North Indian States, Jammu and Kashmir (J&K) population, especially the Kashmir population is found to be at higher risk with high mortality rate associated with different cancers (Pandith and Siddiqi 2012). The population of J&K State is highly diverse, having different ethnic backgrounds, where cancer has been atypically reported as an endemic. In Kashmir valley, lung cancer was reported to be the second most common malignancy (Dhar et al. 1993). Whereas in Jammu region lung cancer and breast cancer are the most commonly occurring cancers followed by esophageal cancer (unpublished data from our research group).

While lung cancer is largely caused by tobacco smoking, both genetic and non-genetic factors contribute to its etiology, indicating that lung cancer is multifactorial in origin. Molecular/genomic alterations in the form of genetic polymorphisms have been recognized as chief factors responsible for lung cancer (Noronha et al. 2016). These molecular alterations have the transforming capability to drive the normal cell into cancer cell, thus characterize potentially an early event in carcinogenesis that fuels further sufficient genetic alterations.

The Genome-wide association studies (GWAS) have proven to be an important tool in the identification of novel loci associated with different complex diseases in diverse ethnic populations. Recent GWAS in ethnic population like Han Chinese, has found that genetic variant rs2494938 of the leucine-rich repeats and fibronectin type III domain containing 2 (LRFN2) gene is associated with the lung cancer risk (Jin et al. 2012). LRFN2 gene located at 6p21.1 position encodes LRFN2 protein, which is an integral member of synaptic adhesion-like molecules (SALMs) family that combines with the N-methyl D-aspartate (NDMA) receptor (The Human Gene Database 1996–2018:GeneCards® 1996–2018; Stelzer et al. 2016). NMDA receptor 2B (NR2B) gets methylated in ESCC and non-small cell lung cancer and exhibit a tumor-suppressive activity (Kim et al. 2006; Tamura et al. 2011). However, the precise mechanism is largely unknown though LRFN2 might function as a susceptibility gene for multiple cancers through LRFN2-NMDA receptor pathway.

The present study was aimed to explore the association of variant rs2494938 with the risk of non-small cell lung cancer in North Indian population. This will help in the identification of the genetic etiology of lung cancer in the studied population and will highlight the underlying mechanism and pathways associated with it.

Materials and methods

Ethical statement

The Institutional Ethics Review Board (IERB) of Shri Mata Vaishno Devi University (SMVDU) approved the study under notification number (SMVDU/IERB/16/47). All the details have been recorded in a predesigned Performa and a written informed consent was obtained from each participant before filling the questionnaire. All experimental procedures were conducted according to the guidelines and regulations set by IERB, SMVDU.

The study consisted of blood samples of non-small cell lung carcinoma patients (all cancer cases were histopathologically confirmed) and normal healthy control subjects. Those which have history of TB, COPD and small-cell lung cancer were excluded from the study.

Sample collection

A total of 619 blood samples (189 non-small cell lung cancer cases and 430 ethnicity and age matched controls) were collected from different hospitals of Jammu and Kashmir, Union territory in India. Non-ethnic cases and cases from mixed genetic background were excluded from the study. The genomic DNA was isolated from the blood samples using Qiagen DNA Isolation kit (Catalogue No. 51206). Agarose gel electrophoresis was used to analyze the quality of the genomic DNA, and quantification was performed using UV spectrophotometer.

Genotyping

Genotyping of variant rs2494938 was performed using allele discrimination assay on MX3005p Agilent Real time PCR platform. Taqman Probe labelled with VIC and FAM (Thermofisher Scientific) and UNG Master Mix (Applied Biosystems, USA) were used for genotyping. The total volume of PCR reaction mix was 10 µl, comprising 2.5 µl of Taqman UNG Master Mix, 0.25 µl of probe, 3 µl DNA (5 ng/µl) and 4.25 µl nuclease-free water to make up the final volume. The following thermocycler conditions were adopted: hold for 10 min at 95 °C, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All the samples were run in 96-well plate with three NTCs (no template Control). The post-PCR detection system was used to measure allele specific fluorescence. Ninety-three random samples were re-genotyped for cross-validation of genotyping calls, and the concordance rate was 100%.

Statistical analysis

Statistical analysis of the data was performed using SPSS software (v.23; Chicago, IL). Chi-square test (χ²) was performed and genotypic frequencies were tested for total Hardy–Weinberg equilibrium. Binary Logistic regression was used to estimate the Odds Ratio (OR) at 95% confidence interval (CI) and respective level of significance as p value of < 0.05. Moreover, the genetic interaction was analyzed using String Software platform (https://string-db.org/).

Results

The clinical characteristic distribution of the cases and controls are given in Table 1. The mean age of cases was 61.16 (± 9.7) years and that of controls were 59.76 (± 9.7) years cases with no significant difference (p = 0.22). However, the average BMI of the cases (22.2) was significantly different from the controls (25.5) (p = 1.0 × 10^–4).

Table 1.

Clinical characteristics of cases and controls

Characteristics	Cases	Control	p value
Agea (in years)	61.16 ± 9.71	59.76 ± 14.4	0.22
BMIb (kg/m2)	22.2 ± 3.80	25.5 ± 4.96	< 1.0 × 10–4
Histological Subtypes
Adeno carcinoma	0.61	–	–
Squamous cell carcinoma	0.34	–
Undifferentiated carcinoma	0.05
Smoking (%)
Yes	0.82	–	–
No	0.18

^aAge in years

^bBMI in kg/m²

The allele frequency distribution in cases and controls is given in Table 2. The frequency of allele G was higher in cases (0.47) than in controls (0.37), showing a significant association of the rs2494938 allele G with NSCLC in the North Indian population (p = 0.0009). The association of rs2494938 variant with NSCLC was further evaluated by applying different genetic models as given in Table 3 to evaluate the maximum effect of risk allele G. Under dominant model scenario, the observed odds ratio (OR) was 2.2 (1.40–3.45 at 95% CI, p = 0.001) and under additive model scenario, the OR observed was 1.8 (1.32–2.47 at 95% CI, p = 0.0001) after correction with age and BMI. Thus, these findings suggest that genetic variant rs2494938 of LRFN2 has significant role in the etiology of NSCLC in the North Indian population. Furthermore, we have performed the sub-type analysis to evaluate the genetic association of rs2494938 with various subtypes of non-small cell lung cancer in the population of Jammu and Kashmir, North India in Table 4. It was observed that Adenocarcinoma (ADC) and Squamous cell carcinoma (SCC) subtype of non-small cell lung cancer showed the significant association with selected variant for the study as compared to undifferentiated carcinoma (UDC) subtype of non-small cell lung cancer.

Table 2.

Allelic frequency distribution among cases and controls in North Indian population

Gene/SNP	R.A	Cases (n = 189)	Controls (n = 430)	HWE	Allelic OR	p Value	Dominant OR	p Value
LRFN2/rs2494938	G	A = 52.8 G = 0.47	A = 62.9 G = 0.37	0.142	1.5 (1.18–1.93)	0.0009	1.84 [1.26–2.71]	0.001

Table 3.

Various genetic models and lung cancer risk

Genetic model	Genotype	Odds ratio	95% CI	p Value
Dominant	GG/AG vs AA	2.2a	1.40–3.45a	0.001a
Additive	–	1.81a	1.32–2.47a	0.01a

^aCorrected for age, BMI

Table 4.

Describes the genetic association of variant rs2494938 of LRFN2 with various subtypes of non-small cell lung cancer in the population of Jammu and Kashmir, North India

Genes	Chr. location	Location of the variant w.r.t gene	Genotype	Histological subtypes of non-small cell lung cancer			Controls
				ADC	SCC	UDC
LRFN2 rs2494938	6:40568389	Intronic variant	AA	32	12	03	163
			AG	64	35	07	215
			GG	20	16	00	52
			p value	0.03	0.0005	0.084	–

Bold values indicate that Adenocarcinoma (ADC) and Squamous cell carcinoma (SCC) subtype of non-small cell lung cancer showed the significant association with selected variant for the study

Discussion

In this case–control association study involving NSCLC cases and controls from North Indian population, we attempted to replicate the previously reported association of LRFN2 gene variant rs2494938 with NSCLC in Han Chinese (Jin et al. 2012) and East Asian (Wu et al. 2018) populations. In addition to NSCLC, the rs2494938 variant has been associated with multiple cancers, suggesting a critical role in cancer association (Jin et al. 2012). To the best of our knowledge, this variant has not been explored for its association with lung cancer in any North Indian population group. Consistent with earlier reports (Jin et al. 2012), our data support the association of rs2494938 in the North Indian population with “G” allele being the risk allele (OR = 2.2; 1.40–3.45 at 95% CI) and p = 0.001 corrected for age and BMI). Our similar studies in other cancer types of the same population suggest that the rs2494938 “G” allele is a risk factor in the esophageal cancer susceptibility with an OR = 1.59 (1.23–2.04 at 95% of CI) p = 0.0003; however, no significant association was found when explored in ovarian and breast cancer with p = 0.919 and 0.216, respectively (Verma et al. 2020). Although this genetic variant may confer different extents of increased risk in different ethnic populations for a variety of reasons, including differences in allele frequency, difference in genetic and environmental backgrounds that interact with the variant. LRFN2 gene is located at 6p21.1. LRFN2 gene encodes leucine-rich repeats and fibronectin type III domain containing 2 (LRFN2) protein. Synaptic adhesion molecules are very crucial in synapse formation and neuronal development (Ko et al. 2006; Nam et al. 2011).

The synaptic adhesion molecule SALM (synaptic adhesion-like molecule) commonly known as LRFN belong to leucine-rich repeat (LRR)-containing adhesion molecule superfamily. Various integral members of SALM family have been attributed in the regulation of neurite outgrowth, branching, synapse formation and maturation. Despite sharing the similar domain architect, individual SALM members have distant function (Wang et al. 2006). SALM usually interacts with the N-methyl D-aspartate (NMDA) receptor (Wang et al. 2006).

Various studies have demonstrated that the LRFN2 can disrupt hematopoietic differentiation to enhance erythropoiesis and lead to erythroblastosis in cooperation with MYC (Castellanos et al. 2007). NMDA receptor is an example of glutamate receptors, which includes NR1 and NR2 (2A–2D) subunits (Fernandes et al. 2007; Skeberdis et al. 2006). NMDA receptor 2B (NR2B) gets methylated in ESCC and non-small cell lung cancer and exhibits a tumor-suppressive activity, although the aberrant methylation pattern can drive the cell toward oncogenesis especially non-small cell lung cancer (NSCLC) (Kim et al. 2006; Tamura et al. 2011). However, the precise mechanism is largely unknown though LRFN2 might function as a susceptibility gene for multiple cancers through LRFN2–NMDA receptor pathway. Besides genetics, lung cancer is mainly caused by smoking. In North India, Jammu and Kashmir, the practice of smoking is more. J&K is usually known as the lung cancer capital of India. There are ample confirmations in the literature which indicate that such environmental agents can lead to epigenetic modulations like methylation, histone changes, chromatin remodeling, etc. With this background, we hypothesize that strong association of the variant of the LRFN2 gene might affect the physiology of LRFN2–NMDA receptor pathway.

To correlate, we used String Software v.11.0 (Szklarczyk et al. 2015) Supplementary Fig. 1 and observed the strong association of LRFN2 gene with DLG1, DLG3 and DLG4. Discs Large MAGUK Scaffold Protein 1 (DLG1) is actually a scaffold protein that has multiple roles in signal transduction, cell proliferation, synaptogenesis and lymphocyte activation. DLG3 plays a critical role in clustering of NMDA receptors at excitatory synapses. However, cellular proliferation is negatively regulated by DLG3 by interacting with C-terminal region of APC-tumor-suppressor protein. DGL3 gene encodes a member of the membrane-associated guanylate kinase (MAGUK) family (Miko et al. 2011). DLG4 coordinates with another MAGUK protein, DLG2 and is recruited into NMDA receptor and potassium channel clusters (Miko et al. 2011). The interactions of these two genes are attributed to clustering of receptors, ion channels, and associated signaling proteins (Stelzer et al. 2016). Alteration in any of the genes along with LRFN2 can have critical effect in the physiology of LRFN2–NMDA receptor pathway as shown in Fig. 1.

Fig. 1.

demonstrating the interaction of LRFN1, DLG1, DLG3 and DLG3 with NMDA. Interaction of DLG1 and DLG4 is attributed to clustering of receptors and associated pathways. DLG3 helps in clustering of NMDA receptors at excitatory synapse. LRFN2 interacts with the NMDA and the LRFN2-NMDA receptor pathway is critical for the susceptibility of cell towards oncogenesis. Aberrant methylation patters in NMDA receptor pathways can lead to NSCLC

We also tried to evaluate the LD (Linkage Disequilibrium) of the genetic variant rs2494938 of LRFN2 using genetic variant-centered annotation browser SNIPA 3.0 (Arnold et al. 2014) as in Supplementary Fig. 2. The plot demonstrates the sentinel variant and its LD SNPs (r² > 0.8) and the variant has direct regulatory effect, which could affect the overall physiology of the gene. These findings could be used as diagnostic and prognostic markers in clinical studies of lung cancer patients in North Indian population groups.

In conclusion, we observed that the genetic variant rs2494938 of LRFN2 is associated with an increased risk of non-small cell lung cancer in North Indian population and is candidate gene for the NSCLC in North Indian population. These findings may also enhance our understanding of inter-population differences in lung cancer etiology and strengthens GWAS findings as well, although the biological functions of these LRFN2 SNPs need to be explored in different population groups with large sample size.

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Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.