# Association of LAMA1 Single-Nucleotide Polymorphisms with Risk of Esophageal Squamous Cell Carcinoma among the Eastern Chinese Population

Authors: Shaoyuan Zhang; Yong Fang; Feng Su; Tian Jiang; Jinjie Yu; Siyun Lin; Lu Lv; Tao Long; Huiwen Pan; Junqing Qi; Qiang Zhou; Weifeng Tang; Guowen Ding; Liming Wang; Lijie Tan; Jun Yin
Journal: Journal of Oncology
 (2023)

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## Abstract

Introduction. LAMA1, also known as laminin subunit α1, is a member of the laminin family, which is widely reported to be a key basement membrane molecule that affects various biological activities and is associated with many kinds of diseases. We aimed to investigate the association between LAMA1single-nucleotide polymorphisms and the occurrence and progression of esophageal squamous cell carcinoma in the Chinese population. Method. 2,186 participants were collected retrospectively between October 2008 and January 2017, including 1,043 ESCC patients and 1,143 noncancer patients. A 2 mL blood sample was obtained intravenously for the LDR for SNP analysis. The 6 SNP loci of LAMA1 were selected and examined. We analyzed the association of several genetic models of 6 LAMA1 SNP loci, sex, age, smoking and drinking status, and the occurrence of esophageal squamous cell carcinoma. Results. In the rs62081531 G > A locus, genotype GA was a protective factor for ESCC compared with GG (OR: 0.830, P=0.046), especially among the younger and nondrinkers. At rs607230 T > C, genotype TC was linked with a lower risk of ESCC compared with TT. (OR: 0.613, P=0.034). Haplotype Frequencies revealed that Ars62081531Grs621993Ars539713Trs566655Ars73938538Crs607230 (OR: 0.803, P=0.028) and Grs62081531Grs621993Ars539713Trs566655Crs73938538Crs607230 (OR: 0.679, P=0.010) were strongly associated with lower susceptibility of ESCC. Conclusion. The LAMA1 rs62081531, rs539713, rs566655, and rs607230 polymorphisms were demonstrated to be related to susceptibility to ESCC in the Chinese population. LAMA1 SNPs may have a significant impact on the occurrence of esophageal cancer and may serve as potential diagnostic biomarkers.


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## Body

## 1. Introduction

Esophageal cancer is a prevalent malignant tumor that has a high rate of morbidity and mortality worldwide. According to the Global Cancer Statistics 2020, esophageal cancer is more prevalent in East Asia, particularly in China, as well as West Asia and Africa [1]. There was a clear correlation between the pathological type of esophageal cancer and its geographic distribution. Squamous cell carcinoma is the most common type of esophageal cancer in developing countries. China has a high prevalence of esophageal squamous cell carcinoma (ESCC) of up to 90%. A variety of pathogenic factors may lead to esophageal squamous cell carcinoma, including smoking, drinking, eating habits, and viral infections [2, 3]. However, not everyone exposed to these risk factors develops esophageal cancer, suggesting that genetic susceptibility, particularly single nucleotide polymorphisms (SNPs), plays a significant role in the development of esophageal squamous cell carcinoma.LAMA1 is also known as laminin subunitα1. Laminins are a family of glycoproteins found in the extracellular matrix that comprises the basement membrane [4, 5]. Laminins have a heterotrimeric structure composed of an α, β, and γ chain [6]. Numerous biological processes are known to be directed by them, including cell adhesion, mitogenesis, differentiation, and metastasis, all of which contribute to carcinogenesis [7–9]. Tissue distribution of LAMA1 occurred mainly in early epithelial development and some adult epithelia. Recent reports have shown that laminin-1 acts as an efficient attachment protein for a large variety of cultured cell types in vitro [10]. Mutations in the LAMA1 gene result in a deficiency of the laminin α1 chain, which may lead to tumorigenesis and progression [11]. Recent research indicates that LAMA1 mutations or overexpression are linked with the occurrence and development of various malignant tumors, including colon cancer, pancreatic cancer, and ovarian cancer [12–15].However, the relationship between theLAMA1single-nucleotide polymorphism and ESCC remains unclear. Through multicenter large-sample case-control research, we aim to thoroughly investigate the association between LAMA1 single-nucleotide polymorphisms and the incidence and progression of esophageal cancer.


## 2. Method

### 2.1. Patients and Study Design

Between October 2008 and January 2017, 2,186 participants were collected from the Affiliated People’s Hospital and the Affiliated Hospital of Jiangsu University (Zhenjiang, China). Totally, 1,043 cases of esophageal cancer were diagnosed and histologically confirmed as squamous cell carcinoma by two pathologists independently. Patients with a history of any other types of cancer or with metastasis or those who had received neoadjuvant therapy were excluded. Around the same time, 1,143 noncancer patients from both hospitals were enrolled, with a frequency matching by age (±5 years) and gender, and the majority of them were admitted for trauma.For 1,043 patients and 1,143 negative controls, baseline information such as age, gender, and other ESCC-related risk factors, such as smoking and drinking, were gathered through a questionnaire. A total of 1,143 control individuals and all case subjects provided feedback. Each participant had a 2 mL blood sample obtained intravenously for analysis.The protocol adhered to theDeclaration of Helsinki on the ethical conduct of research involving human/animal subjects and was approved by the Ethics Committee of Jiangsu University (Zhenjiang, China). All participants signed an informed consent form before recruitment.


### 2.2. Genomic DNA Extraction and Single-Nucleotide Polymorphism Analysis

The QIAamp DNA Blood Mini Kit was applied to amplify genomic DNA isolated from peripheral blood using PCR (Qiagen, Berlin, Germany). Samples were genotyped further using the ligation detection reaction (LDR) approach (supported by Genesky Biotechnology Inc., Shanghai, China). Six LAMA1 SNP loci (rs62081531, rs621993, rs539713, rs566655, rs73938538, and rs607230) were selected and analyzed. As a methodology of quality control, the analysis is repeated on 10% of randomly selected samples. In a preliminary study, we performed a linkage disequilibrium analysis on the 1000Genomes database, identified SNP loci with correlations, and further explored tag SNPs.


### 2.3. Surgical and Histological Evaluation

All patients underwent esophagectomy by qualified surgeons. Following the procedure, surgical specimens will be fixed to a cork and immersed in 10% formalin. All these patients’ specimens were systematically reevaluated by experienced pathologists specialized in thoracic oncology and restaged under the 8th edition AJCC/UICC staging of cancers of the esophagus and esophagogastric junction. The histopathological examination includes tumor size, grade of differentiation, the margin of resection, and lymph node status.


### 2.4. Statistical Analysis

Statistical analyses were performed using SPSS version 26 (IBM, Chicago, IL) andR software (Version 4.0.3, R Foundation for Statistical Computing, Vienna, Austria). The baseline characteristics were summarized using the R software package “tableone”. Clinical characteristics were compared using Fisher’s exact test or the χ2 test for categorical variables and the Student’s t-test for comparing continuous variables. For the genetic model (A as a major allele and B as a minor allele), (a) dominant model: allele B increases risk; (b) Recessive model: two copies of minor allele B are required for increased risk; (c) additive model: r-fold increased risk for AB and 2r increased risk for BB; (d) multiplicative model: r-fold increased risk for AB and r2 increased risk for BB [16]. Two-tailed p value < 0.05 is considered as statistical significance, whereas p values between 0.05 and 0.10 are considered borderline statistically significant. The crude odd ratio (OR) and its corresponding 95% confidence interval (CI) are calculated according to genotypes between the two groups. In most cases, the risk is compared using the parametric test. When the sample size in the group is small, we use the nonparametric test. For stratified analyses, we included age, gender, smoking and drinking status, and the SNP model in the analysis, resulting in an adjusted OR for the SNP model. The adjusted OR and its corresponding CI are calculated by logistic regression analysis, and the hierarchical analysis is carried out. Demographic data, including age, sex, alcohol, and smoking, were covariates. The genotype was a dummy variable, and the group was a dependent variable. The SHEsis online platform [17] was utilized to conduct linkage disequilibrium studies and visualize the results by using R 4.0.3 and the R software packages “LDheatmap” and “genetics,” and SHEsis was also used to conduct haplotype frequency analyses [18].


## 2.1. Patients and Study Design

Between October 2008 and January 2017, 2,186 participants were collected from the Affiliated People’s Hospital and the Affiliated Hospital of Jiangsu University (Zhenjiang, China). Totally, 1,043 cases of esophageal cancer were diagnosed and histologically confirmed as squamous cell carcinoma by two pathologists independently. Patients with a history of any other types of cancer or with metastasis or those who had received neoadjuvant therapy were excluded. Around the same time, 1,143 noncancer patients from both hospitals were enrolled, with a frequency matching by age (±5 years) and gender, and the majority of them were admitted for trauma.For 1,043 patients and 1,143 negative controls, baseline information such as age, gender, and other ESCC-related risk factors, such as smoking and drinking, were gathered through a questionnaire. A total of 1,143 control individuals and all case subjects provided feedback. Each participant had a 2 mL blood sample obtained intravenously for analysis.The protocol adhered to theDeclaration of Helsinki on the ethical conduct of research involving human/animal subjects and was approved by the Ethics Committee of Jiangsu University (Zhenjiang, China). All participants signed an informed consent form before recruitment.


## 2.2. Genomic DNA Extraction and Single-Nucleotide Polymorphism Analysis

The QIAamp DNA Blood Mini Kit was applied to amplify genomic DNA isolated from peripheral blood using PCR (Qiagen, Berlin, Germany). Samples were genotyped further using the ligation detection reaction (LDR) approach (supported by Genesky Biotechnology Inc., Shanghai, China). Six LAMA1 SNP loci (rs62081531, rs621993, rs539713, rs566655, rs73938538, and rs607230) were selected and analyzed. As a methodology of quality control, the analysis is repeated on 10% of randomly selected samples. In a preliminary study, we performed a linkage disequilibrium analysis on the 1000Genomes database, identified SNP loci with correlations, and further explored tag SNPs.


## 2.3. Surgical and Histological Evaluation

All patients underwent esophagectomy by qualified surgeons. Following the procedure, surgical specimens will be fixed to a cork and immersed in 10% formalin. All these patients’ specimens were systematically reevaluated by experienced pathologists specialized in thoracic oncology and restaged under the 8th edition AJCC/UICC staging of cancers of the esophagus and esophagogastric junction. The histopathological examination includes tumor size, grade of differentiation, the margin of resection, and lymph node status.


## 2.4. Statistical Analysis

Statistical analyses were performed using SPSS version 26 (IBM, Chicago, IL) andR software (Version 4.0.3, R Foundation for Statistical Computing, Vienna, Austria). The baseline characteristics were summarized using the R software package “tableone”. Clinical characteristics were compared using Fisher’s exact test or the χ2 test for categorical variables and the Student’s t-test for comparing continuous variables. For the genetic model (A as a major allele and B as a minor allele), (a) dominant model: allele B increases risk; (b) Recessive model: two copies of minor allele B are required for increased risk; (c) additive model: r-fold increased risk for AB and 2r increased risk for BB; (d) multiplicative model: r-fold increased risk for AB and r2 increased risk for BB [16]. Two-tailed p value < 0.05 is considered as statistical significance, whereas p values between 0.05 and 0.10 are considered borderline statistically significant. The crude odd ratio (OR) and its corresponding 95% confidence interval (CI) are calculated according to genotypes between the two groups. In most cases, the risk is compared using the parametric test. When the sample size in the group is small, we use the nonparametric test. For stratified analyses, we included age, gender, smoking and drinking status, and the SNP model in the analysis, resulting in an adjusted OR for the SNP model. The adjusted OR and its corresponding CI are calculated by logistic regression analysis, and the hierarchical analysis is carried out. Demographic data, including age, sex, alcohol, and smoking, were covariates. The genotype was a dummy variable, and the group was a dependent variable. The SHEsis online platform [17] was utilized to conduct linkage disequilibrium studies and visualize the results by using R 4.0.3 and the R software packages “LDheatmap” and “genetics,” and SHEsis was also used to conduct haplotype frequency analyses [18].


## 3. Results

Between October 2008 and January 2017, we included 1,043 patients and 1,143 negative controls. There was no statistically significant difference in median age between the case and control groups [63.00 (59.00, 68.00) versus 63.00 (54.00, 70.00),p=0.257]. Women accounted for 27.3% of the case group and 27.6% of the control group (P=0.941). The proportion of smoking and drinking in ESCC patients was higher than that in the control group (43.5% vs. 29.7%, P<0.001, 31.5% vs. 16.3%, P<0.001). Detailed clinicopathological information was described in Table 1.Figure 1
D′ (normalized D) or r2 (correlation coefficient) of linkage disequilibrium on 6 SNP loci ofLAMA1.Table 1
Distribution of clinicopathological characteristics in the ESCC case and control groups.
CaseControlp value(n = 1043)(n = 1143)Age (median (IQR))63.00 (59.00, 68.00)63.00 (54.00, 70.00)0.257Gender (%)Female285 (27.3)315 (27.6)0.941Male758 (72.7)828 (72.4)Smoke status (%)No589 (56.5)803 (70.3)<0.001Former/current454 (43.5)340 (29.7)Alcohol consumption (%)No714 (68.5)957 (83.7)<0.001Former/current329 (31.5)186 (16.3)BMI (median (IQR))22.27 (20.20, 24.35)23.88 (21.89, 25.88)<0.001Chronic disease (%)No797 (76.4)604 (52.8)<0.001Yes246 (23.6)539 (47.2)Hypertension (%)234 (22.4)416 (36.4)Diabetes (%)31 (3.0)216 (18.9)Cardiovascular disease (%)0 (0.0)59 (5.2)pT stage (%)Tis1 (0.1)T1a33 (3.2)T1b112 (10.7)T2382 (36.6)T3513 (49.2)T42 (0.2)pN stage (%)N0775 (74.3)N1201 (19.3)N245 (4.3)N322 (2.1)Differentiation (%)High368 (35.3)Moderate537 (51.5)Low138 (13.2)Data are no. (%) or mean (SD) or median (IQR); BMI: body mass index; pT stage: pathological T stage; pN stage: pathological N stage.The brief information on the six genotyped SNPs ofLAMA1 is shown in Table 2. All SNP genotyping experiments were successful at a rate greater than 95%. The control group’s minor allele frequencies (MAF) were similar to those found in East Asian groups in the 1000 Genomes and gnomAD-Genomes databases. The Hardy–Weinberg equilibrium (HWE) test revealed that all six SNPs in the control group had p values greater than 0.05, indicating that the control group was genetically equilibrium. Linkage disequilibrium of 6 SNP loci of LAMA1 is shown in Figure 1 and the coefficient of linkage disequilibrium and correlation coefficient test are described in Tables S7a and S7b.Table 2
Primary information of LAMA1.
GeneLAMA1Genotyped SNPrs62081531rs621993rs539713rs566655rs73938538rs607230AlleleG > AG > AA > GT > GA > CT > CConsequenceSynonymous variantSynonymous variantStop gainedMissense variantSynonymous variantStop gainedRegulomeDB rank†745554Chromosome18Chromosome position‡698626070330387017323703450970085846980524MAF in controlA = 0.1810A = 0.1731G = 0.2768G = 0.1432C = 0.1142T = 0.1913MAF in 1000 genomesGlobalA = 0.1378A = 0.4065G = 0.5475G = 0.1502C = 0.1717T = 0.3063East AsianA = 0.1915A = 0.1319G = 0.2262G = 0.1042C = 0.1091T = 0.1756MAF in gnomAD-genomesGlobalA = 0.178903A = 0.468719G = 0.612173G = 0.203225C = 0.157585T = 0.319456East AsianA = 0.1986A = 0.1629G = 0.2540G = 0.1313C = 0.1097T = 0.1621p value for the HWE test in the controls0.94320.97430.99960.72120.86330.6770Genotyping methodLDR% genotyping value98.90%98.95%98.95%98.95%98.86%98.03%†https://www.regulomedb.org/. ‡Based on Genome Reference Consortium Human Build 38(GRCh38).The association of 6 SNPs inLAMA1 with esophageal squamous cell carcinoma is shown in Table 3. In the rs62081531 locus, G/A was a protective factor for ESCC compared with G/G (OR: 0.820, 95% CI: 0.677–0.993, P=0.042). We can also find in the dominant model that the minor allele A is a protective factor for ESCC (OR: 0.830, 95% CI: 0.691–0.997, P=0.046). At rs607230, T/C was a protective factor for ESCC compared with T/T (OR: 0.613, 95% CI: 0.389–0.965, P=0.034).Table 3
Association between LAMA1 single-nucleotide polymorphism and esophageal squamous cell carcinoma while controlling age and gender
LocusGenotypeESCC caseControlCodominant modelDominant modelRecessive modelAdditive modelMultiplicative modelOr (95%CI)pvalueOr (95% CI)pvalueOr (95% CI)pvalueOr (95% CI)pvalueOr (95% CI)pvaluers62081531G/G729 (69.9)765 (66.9)Ref0.830 (0.691–0.997)0.0460.968 (0.606–1.545)0.8910.870 (0.743–1.017)0.0810.866 (0.739–1.015)0.076G/A261 (25.0)334 (29.2)0.820 (0.677–0.993)0.042A/A34 (3.3)39 (3.4)0.915 (0.571–1.465)0.711rs621993G/G704 (67.5)777 (68.0)Ref0.981 (0.818–1.177)0.8391.114 (0.682–1.818)0.6660.997 (0.852–1.167)0.9700.997 (0.851–1.167)0.970G/A288 (27.6)328 (28.7)0.969 (0.803–1.17)0.744A/A33 (3.2)33 (2.9)1.104 (0.674–1.807)0.695rs539713A/A525 (50.3)595 (52.1)Ref1.044 (0.881–1.236)0.6210.805 (0.576–1.124)0.2020.991 (0.866–1.135)0.9000.992 (0.868–1.133)0.902G/A436 (41.8)456 (39.9)1.084 (0.909–1.292)0.371G/G64 (6.1)87 (7.6)0.834 (0.591–1.175)0.299rs566655T/T738 (70.8)832 (72.8)Ref1.057 (0.875–1.277)0.5631.340 (0.736–2.441)0.3381.071 (0.904–1.268)0.4301.070 (0.904–1.266)0.432T/G263 (25.2)286 (25.0)1.037 (0.853–1.259)0.717G/G24 (2.3)20 (1.7)1.353 (0.741–2.469)0.325rs73938538A/A829 (79.5)891 (78.0)Ref0.844 (0.684–1.042)0.1150.854 (0.373–1.957)0.7100.857 (0.706–1.042)0.1220.859 (0.707–1.042)0.124C/A184 (17.6)234 (20.5)0.845 (0.682–1.048)0.125C/C10 (1.0)13 (1.1)0.827 (0.361–1.896)0.653rs607230T/T49 (4.7)37 (3.2)Ref0.657 (0.425–1.016)0.0591.046 (0.875–1.25)0.6240.981 (0.844–1.140)0.8030.981 (0.842–1.142)0.801T/C293 (28.1)361 (31.6)0.613 (0.389–0.965)0.034C/C664 (63.7)739 (64.7)0.678 (0.437–1.053)0.084OR: odds ratio; CI: confidence interval.Simultaneously, we performed stratified analyses on 6 loci ofLAMA1, and detailed information is present in Tables S1–S6. In rs62081531 G > A, in the younger population, the dominant model (OR: 0.680, 95% CI: 0.527–0.877, P=0.003), the recessive model (OR: 0.741, 95% CI: 0.594–0.923, P=0.008), or the multiplicative model (OR: 0.754, P=0.010) all imply a negative link with ESCC incidence. In those without alcohol consumption, we found that either the dominant model (OR: 0.777, 95% CI: 0.627–0.963, P=0.022), the recessive model (OR: 0.796, 95% CI: 0.660–0.961, P=0.018), or the multiplicative model (OR: 0.800, 95% CI: 0.664–0.964, P=0.019) reveals that it is a protective factor for esophageal cancer (Table S1). At the rs539713 A > G locus, in the former or current drinking population, we found that in the recessive or multiplicative model, it was associated with a reduced risk of ESCC (OR: 0.443, 95% CI: 0.224–0.876, P=0.019 and OR: 0.744, 95% CI: 0.556–0.996, P=0.047) (Table S3). At the rs566655 T > G locus, we discovered that both the additive model (OR: 1.270, 95% CI: 1.006–1.603, P=0.044) and the multiplicative model (OR: 1.274, 95% CI: 1.007–1.611, P=0.044) were linked with an elevated risk of ESCC in individuals younger than 65 years old (Table S4). It was found that the rs607230 T > C variant was associated with a decreased risk of esophageal cancer in adults aged 65 years (OR: 0.427, 95% CI: 0.196–0.930, P=0.032) (Table S6). After stratified analysis, rs621993 G > A and rs73938538 A > C did not seem to be linked with esophageal cancer susceptibility (Table S2 and S5). We also analyzed the association of LAMA1 6 SNPs with tumor differentiation and lymph node metastasis, and the detailed information is shown in Table S8. We found that neither the lymph node positivity rate nor the degree of differentiation was significantly associated with the mutation status of the 6 loci. Haplotype frequencies analysis revealed that Ars62081531Grs621993Ars539713Trs566655Ars73938538Crs607230 (OR: 0.803, 95% CI: 0.660∼0.977, P=0.028) and Grs62081531Grs621993Ars539713Trs566655Crs73938538Crs607230 (OR: 0.679, 95% CI: 0.504∼0.913, P=0.010) were associated with less susceptibility of ESCC (Table 4).Table 4
Haplotype frequencies in the case and control groups and risk of ESCC.
Haplotype (LAMA1)Case (freq)Control (freq)Chi2Or [95% CI]p valueArs62081531Ars621993Grs539713Grs566655Ars73938538Crs60723038.73 (1.9%)63.18 (2.8%)——Ars62081531Ars621993Grs539713Grs566655Crs73938538Crs6072304.99 (0.2%)2.60 (0.1%)——Ars62081531Ars621993Grs539713Trs566655Ars73938538Crs6072302.33 (0.1%)4.12 (0.2%)——Ars62081531Grs621993Ars539713Trs566655Ars73938538Crs607230200.31 (10.0%)269.91 (11.9%)4.8230.803 [0.660∼0.977]0.028Ars62081531Grs621993Ars539713Trs566655Crs73938538Crs60723013.40 (0.7%)15.93 (0.7%)——Ars62081531Grs621993Grs539713Trs566655Ars73938538Crs60723038.33 (1.9%)56.03 (2.5%)——Grs62081531Ars621993Ars539713Grs566655Ars73938538Crs6072307.10 (0.4%)4.11 (0.2%)——Grs62081531Ars621993Ars539713Trs566655Ars73938538Crs6072301.29 (0.1%)3.52 (0.2%)——Grs62081531Ars621993Grs539713Grs566655Ars73938538Crs607230168.31 (8.4%)171.67 (7.6%)0.7311.102 [0.881∼1.379]0.393Grs62081531Ars621993Grs539713Grs566655Ars73938538Trs60723018.19 (0.9%)30.69 (1.4%)——Grs62081531Ars621993Grs539713Grs566655Crs73938538Crs60723029.83 (1.5%)39.59 (1.7%)——Grs62081531Ars621993Grs539713Grs566655Crs73938538Trs6072303.90 (0.2%)7.69 (0.3%)——Grs62081531Ars621993Grs539713Trs566655Ars73938538Crs60723029.88 (1.5%)35.63 (1.6%)——Grs62081531Ars621993Grs539713Trs566655Ars73938538Trs60723012.55 (0.6%)18.16 (0.8%)——Grs62081531Ars621993Grs539713Trs566655Crs73938538Crs6072306.78 (0.3%)3.95 (0.2%)——Grs62081531Grs621993Ars539713Trs566655Ars73938538Crs607230845.42 (42.2%)909.38 (40.1%)1.0661.072 [0.939∼1.224]0.302Grs62081531Grs621993Ars539713Trs566655Ars73938538Trs607230259.49 (12.9%)275 (11.9%)0.7201.083 [0.900∼1.303]0.396Grs62081531Grs621993Ars539713Trs566655Crs73938538Crs60723074.63 (3.7%)119.97 (5.3%)6.6260.679 [0.504∼0.913]0.010Grs62081531Grs621993Ars539713Trs566655Crs73938538Trs60723040.47 (2.0%)43.28 (1.9%)——Grs62081531Grs621993Grs539713Trs566655Ars73938538Crs607230117.31 (5.9%)114.20 (5.0%)1.1271.155 [0.885∼1.508]0.288Grs62081531Grs621993Grs539713Trs566655Ars73938538Trs60723034.02 (1.7%)59.00 (2.6%)——Grs62081531Grs621993Grs539713Trs566655Crs73938538Crs6072308.56 (0.4%)15.83 (0.7%)——Freq: frequency; OR: odds ratio; CI: confidence interval; p < 0.05 is regarded as statistically significant value.


## 4. Discussion

Through our multicenter large-samplecase-control study, we found that rs62081531 locus G > A and rs607230 locus T > C ofLAMA1 were independent protective factors for esophageal squamous cell carcinoma, especially in those younger than 65 and nondrinkers.Haplotype frequency analysis revealed that Ars62081531Grs621993Ars539713Trs566655Ars73938538Crs607230 and Grs62081531Grs621993Ars539713Trs566655Crs73938538Crs607230 were associated with less susceptibility to ESCC among the Chinese population. To our knowledge, this is the first report on LAMA1 single-nucleotide polymorphisms and susceptibility to esophageal squamous cell carcinoma based on large-scale multicenter clinical research.LAMA1 (laminin subunitα1) is a part of laminin, a glycoprotein found in the extracellular matrix that constitutes the basement membrane, and has been shown to be involved in the occurrence and development of various diseases [11]. Tissue distribution of laminin subunit α1 is mainly in early epithelial development and some adult epithelia [8]. In terms of carcinogenesis, laminin plays an essential role in cell adhesion, mitosis, differentiation, and even metastasis [19]. Laminin is a fundamental functional component of the basement membrane of several tissues, including the endothelium of the vessel wall, and different isoforms may contribute to vascular homeostasis [20]. The α1 subunit of laminin is typically confined to capillary walls and is expressed in the basal layer of capillaries in the central nervous system [21]. A recent study demonstrates that laminin-1 functions as a chemoattractant for both stromal and vascular cells, as well as in epithelial/stromal cell interactions for the structure of the basement membrane and segregation of integrins, hence signaling the proliferation of epithelial cells [13]. Similarly, in colorectal cancer, Wu et al. reported novel driver mutations occurring during adenoma and cancer evolution by single-cellwhole-exome sequencing (scWES), with LAMA1 (PI3K-Akt signaling pathway) being one of the most critical pathways for CRC evolution [22]. Likewise, Gudjonsson and coworkers revealed that laminin-1 plays a vital role in the replacement of myoepithelial cells in polarity reversal in breast cancer [23]. LAMA1 (laminin α1) mutations are highly related to retinal avascularity and neovascularization in nontumor fields, such as the Poretti–Boltshauser syndrome [24]. Regardless of tumor or other nontumor diseases, LAMA1 is essential for vascular homeostasis and the basal layer of blood vessels. Interestingly, Velling et al. found that none of the colon cancer cell lines synthesized the laminin α1 protein, and they suggested that mutations in the LAMA1 gene may underlie the lack of laminin α1 chains observed in some colon cancers [25]. In our study, LAMA1 mutation showed protective factors in both rs62081531 G > A and rs607230 T > C, and basic research also found that LAMA1 deficiency could inhibit the proliferation and invasion of esophageal cancer [26, 27]. Clearly, not all LAMA1 SNPS are protective factors. People younger than 65 years of age indicate that the mutation at rs566655 T > G increases the risk of esophageal cancer, which may be correlated to the function of a certain SNP.However, there are few reports onLAMA1 and esophageal cancer. Most of the research is limited to the genetic function of LAMA1. Meng and colleagues found that laminin α1 (LAMA1) is highly expressed in ESCC tissue and mediates the FAK-PI3K-Akt signaling pathway [27]. Zhou et al. revealed that LAMA1 was significantly upregulated in ESCC tissues and positively correlated with an aggressive oncogenic phenotype [26]. Nevertheless, the relationship between LAMA1 SNP and disease in malignancies has not been demonstrated. Previous research has focused chiefly on nontumor studies such as chronic disease or degenerative disease. Zhao et al. showed that rs2089760 T > G, which is located in the LAMA1 promoter region, may be associated with myopia in Chinese populations [28]. Similarly, the LAMA1 rs2089760 G > A mutation was reported to reduce transcription factor binding ability and transcription initiation activity and negatively control the gene transcription of LAMA1, playing a crucial role in pathological myopia [29]. In a study on degenerative diseases, D’Aoust and colleagues were the first to discover that LAMA1 rs73938538 A > C was positively related to Alzheimer’s disease in the Amish community [30]. Due to the single-nucleotide polymorphism of LAMA1, the mutated site seems unable to effectively translate LAMA1 into laminin subunit α1 so as to exert its specific biological function, hence preventing esophageal cancer susceptibility. We observed that the consequences of two SNP loci that were related to esophageal susceptibility were stop-gain mutations, while the rest were synonymous and missense variants. This is largely in accordance with our expectations, especially when terminal gain mutations and missense variants can dramatically alter protein function, even with single nucleotide changes. However, we also reveal that in the ESCC population, LAMA1 polymorphisms did not show a statistically significant association with the degree of differentiation, lymph node positivity, or T stage.The main limitation of our study is that we only included populations from a specific region in eastern China, which may result in a certain geographical specificity and may not be generalized to the entire ESCC population. In addition, our study lacks replication in independent cohorts. Furthermore, we only discovered SNP sites in peripheral blood in our investigation. How these SNPs ofLAMA1 translate into biological function in the evolution of esophageal carcinoma is definitely a primary subject of our future investigation, which is currently technically difficult due to the lack of biological tools in our lab.In conclusion, we found a strong association ofLAMA1 rs62081531, rs539713, rs566655, and rs607230 polymorphisms with esophageal cancer susceptibility in the Chinese population. LAMA1 SNPs may significantly impact the occurrence of esophageal cancer and may serve as effective diagnostic biomarkers.



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