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Structural, functional analysis and association of MSH6 rs1800932, rs1042821 polymorphisms with clinical outcome in North Indian lung cancer patients treated with platinum-based doublet chemotherapy

      Abstract

      Background

      In this study, we have done structural and functional analysis of rs1800932 rs1042821 polymorphisms and tried to estimate any association of these polymorphisms with clinical outcomes in north Indian lung cancer patients.

      Methods

      Genotyping of 500 lung cancer patients was completed utilizing PCR-RFLP (Polymerase chain reaction- Restriction fragment length polymorphism). MedCalc statistical software was used to calculate adjusted and unadjusted odds ratios. Various computational tools like SIFT PROVEAN are used for functional analysis. Structural analysis was completed via MODELLER and CHIMERA.

      Results

      In our study, patients suffering from small cell lung cancer (SCLC) and harboring heterozygous genotype (AG) for MSH6 (rs1800932) polymorphism have reported a significant increase in median survival time (MST) (20.6 vs. 7.6 months, p = 0.03). Furthermore, for MSH6 rs1042821 polymorphism, patients undergoing docetaxel and carbo/cisplatin combination chemotherapeutic regimen and carrying heterogeneous genotype (CT) reported a significant increase in MST (16.6 vs.8.36 months, p = 0.03) and a corresponding decrease in hazard ratio 0.42 (95% CI= 0.18–1.03). Structural and Functional analysis of rs1042821 polymorphism revealed that it is present in the non-coding region of MSH6 protein and is significantly associated with increased overall survival.

      Conclusions

      These results suggest that MSH6 rs1800932 rs1042821 polymorphisms are involved in increasing the overall survival of lung cancer patients, further confirmed by computational analysis.

      Key Indexing Terms

      Introduction

      Lung cancer is one of the most prevalent and foremost causes of malignancy-related deaths worldwide, especially in developing countries. In 2018, lung cancer alone was responsible for approximately 1.8 million deaths worldwide.
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      Cancer risk and low-penetrance susceptibility genes in gene–environment interactions.
      Several studies have shown that alteration in specific genes can increase an individual's susceptibility to cancer.
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      XPG polymorphisms and their association with lung cancer susceptibility, overall survival and response in North Indian patients treated with platinum-based doublet chemotherapy.
      There were reports where DNA repair pathways genes were evaluated to understand their role in preventing the occurrence of lung cancer.
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      DNA repair and cell cycle control genes and the risk of young-onset lung cancer.
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      Chromatin remodeling and mismatch repair: access and excision.
      It has been reported that defective MMR pathways may lead to various cancers such as colorectal and colon cancer.
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      BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair.
      MSH6 (MutS Homolog 6) gene is a post replicative DNA mismatch repair system (MMR). MSH6 protein (1360 amino acid) heterodimerizes with MSH2 to form MutSα, which binds DNA mismatches and initiates DNA repair.
      • Edelmann W.
      • Yang K.
      • Umar A.
      • et al.
      Mutation in the mismatch repair gene Msh6 causes cancer susceptibility.
      A polymorphism in the MSH6 gene will lead to changes in the structure of MSH6 protein, which may further lead to nonfunctional MSH6 protein losing its property to rectify DNA mismatches and thus leading to inefficient repair capacity, which might be further associated with an individual's susceptibility towards the development of lung cancer. At present, the SNP database of NCBI has more than 2121 single nucleotide polymorphisms, which are clinically significant for the MSH6 gene. Two polymorphic variants of MSH6 Pro92Pro and Gly39Glu have been identified, but the functional significance of these polymorphisms is still unknown. Pro92Pro polymorphism (rs1800932) is located on exon two and leads to A→G transition, whereas Gly39Glu polymorphism (rs1042821) is located on exon one leading to C→T transition. MSH6 polymorphism has been linked with the occurrence of various cancers such as thyroid, colorectal, colon, and breast cancer.
      • Santos L.S.
      • Silva S.N.
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      Mismatch repair single nucleotide polymorphisms and thyroid cancer susceptibility.
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      • Zheng B.
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      Genetic polymorphisms of DNA repair pathways in sporadic colorectal carcinogenesis.
      ,
      • Curtin K.
      • Samowitz W.S.
      • Wolff R.K.
      • et al.
      MSH6 G39E polymorphism and CpG island methylator phenotype in colon cancer.
      ,
      • Smith T.R.
      • Levine E.A.
      • Freimanis R.I.
      • et al.
      Polygenic model of DNA repair genetic polymorphisms in human breast cancer risk.
      No study so far has evaluated the role of the above two polymorphic variants in assessing its relationship as a prognostic marker and its role as a clinical predictor for platinum-based chemotherapy. Therefore, the present study was conducted on North-Indian lung cancer patients to understand the role of rs1800932 Pro92Pro and rs1042821 Gly39Glu polymorphism towards overall survival clinical response and clinic-pathological parameters of patients treated with platinum-based doublet chemotherapy. Further, these SNPs were analyzed by different bioinformatics approaches to understand the impact on protein structure and function.

      Methods

      Patient recruitment

      This is a hospital-based study, where 500 cases were recruited from the department of pulmonary medicine of Post Graduate Institute of Medical Education and Research, Chandigarh, India. The ethics committee board of PGIMER has reviewed and approved this study. All procedures performed in studies involving human participants were following the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All the recruited patients provided informed written consent. This study recruited patients regardless of age, sex, smoking, histology, and TNM Classification of Malignant Tumors (TNM) staging. Patients having a previous history of cancer were not recruited in this study. All the patients recruited for this study were histopathologically confirmed to have non-small cell lung carcinoma (NSCLC) or Small cell lung carcinoma (SCLC). A trained interviewer collected the data about demographics and smoking habits from all recruited cases. Patient TNM staging, tumor stage, histology, treatment regimen, and chemotherapy cycles were retrieved from the patients' medical records. The patients were followed up every two months by telephone until the end of the patient's treatment or death, whichever was earlier.

      Chemotherapeutic regimen

      All the patients recruited for this study were given platinum-based doublet chemotherapy. The first line of chemotherapeutic drugs involved administrating carboplatin/cisplatin. The second line of drugs administered were either taxol-based drugs like docetaxel and paclitaxel or paclitaxel topoisomerase I inhibiter drug-like, irinotecan, or antifolate agent like pemetrexed. The patients were given a combination of carbo/cisplatin along with anyone of the second-line drugs as mentioned above in the following concentration: 75 mg/m2 docetaxel, 500 mg/m2 pemetrexed, or 75 mg/m2 irinotecan as a 1hr infusion along with 70 mg/m2 cisplatin or carboplatin for three hours. The treatment was repeated every 3–4 weeks. After four cycles of chemotherapy, tumor response was assessed, and patients were advised if they had to continue the treatment further.
      Chemotherapy was stopped in case of severe/ unacceptable toxicity to the patients. Two additional cycles of chemotherapy were administered if the patient showed an objective response towards the treatment (a maximum of 6 cycles can be given). In the current study, 105 patients were administered cis/carboplatin and pemetrexed combination, 45 patients were given cis/carboplatin and irinotecan combination, cis/carboplatin and docetaxel combination was given to 37 patients. In contrast, the combination of Paclitaxel cis/carboplatin and gemcitabine cis/carboplatin was given to 82 and 29 lung cancer patients, respectively.

      Follow up and response determination

      All the recruited subjects were followed up by telephone to obtain survival data once every two months until the subject's death or end of the study, whichever is earlier. The information obtained from the subject's survival time was determined from the date when cancer was first diagnosed until the end of the study or the patient's death. The study's objective was to determine an association between response to treatment and genetic polymorphism. A Response Evaluation Criteria in Solid Tumors (RECIST) criteria were employed further to assess how well a patient responded to the treatment. Based on this, subjects were divided into responders and non-responders. The subjects showing complete response (CR) or partial response (PR) were categorized into “responders,” whereas subjects showing stable disease (SD) or progression disease (PD) were categorized into “non-responders.”

      Genotyping of MSH6 variants

      Genomic DNA was isolated from 4 ml of blood using phenol-chloroform extraction protocol as mentioned by Singh and coworkers.
      • Singh A.
      • Singh N.
      • Behera D.
      • et al.
      Role of polymorphic XRCC6 (Ku70)/XRCC7 (DNA-PKcs) genes towards susceptibility and prognosis of lung cancer patients undergoing platinum based doublet chemotherapy.
      Two MSH6 polymorphisms, Pro92Pro and Gly39Glu genotype determination were completed using polymerase chain reaction-restriction length fragment polymorphism (PCR-RFLP) technique.
      • Walia H.K.
      • Singh N.
      • Sharma S.
      Association of NQO1Pro187Ser polymorphism with clinical outcomes and survival of lung cancer patients treated with platinum chemotherapy.
      PCR was carried out by using following primers: For MSH6 Pro92Pro polymorphism FP: 5′- CCTGCCATCAGCATTATACCA-3′, RP: 5′-CTGTACATGAACACGGACTGA-3′, for Gly39Glu polymorphism FP: 5′-TTAGGAGCTCCGTCCGACAGAAC-3′, RP: 5′- CCTCCGTTGAGGTTCTTCGCCTT-3′. PCR reaction was carried out in 20 μl volume. The reaction mixture was composed of 100 ng of genomic DNA, 0.5 μM of forward and reverse primer, 1x PCR buffer, 200 μM dNTPs, 1.5 mM MgCl2, 50 μg/ml BSA, and 1 U High fidelity Taq polymerase. The details of PCR cycling reaction are as follows: Stage 1: initial denaturation step of 5 min at 95°C, Stage 2: 29 cycles consisting of denaturation for 30 s at 94°C, annealing of the primers for Pro92Pro and Gly39Glu polymorphism at 58°C and 60°C respectively for 45 s and extension at 72°C for 30 s, Stage 3: final extension at 72°C for 5 min. PCR product of MSH6 Pro92Pro and Gly39Glu polymorphism is 434 bp and 286 bp long, which is confirmed by running the PCR product with 100 bp ladder on 1.0% agarose gel. 5 μl of PCR product of both polymorphisms was further digested by two units of NciI (New England Bio Labs, Beverly, MA) overnight at 37°C and was visualized on 3% agarose gel. For Pro92Pro, the homozygous AA genotype gave digested fragments of 313 and 121 bp, homozygous GG genotype gave undigested fragments of 434 heterozygous AG genotype gave three digested fragments of 434, 313, and 121 bp. For Gly39Glu polymorphism, the homozygous CC genotype gave two digested fragments of 177 and 109 bp, while the homozygous TT genotype gave one undigested fragment 286 and heterozygous CT genotype gave three digested fragments of 286, 177, and 109 bp.

      Statistical analysis

      In the present study, the population residing in the northern part of India was selected to analyze the effect of MSH2 polymorphism on the overall survival (OS) of lung cancer patients undergoing platinum-based doublet chemotherapy. Goodness-of-fit Chi-square test was used to determine if the case population follows the Hardy-Weinberg equilibrium. The univariate Kaplan-Meir regression method determined median survival time (MST) and unadjusted hazard ratio (HR). The Multivariate Cox regression method was used to determine adjusted HR taking genotype, age, sex, smoking ECOG, stage as confounding factors. A p-value of less than 0.05 was considered to be standard to determine if the analysis was significant or not. MedCalc statistical software version 14.8.1 was used to perform all the statistical analyses.

      Computational analysis

      MSH6 rs1800932 rs1042821 polymorphisms were analyzed using different prediction tools (SIFT, PROVEAN, PANTHER, and Poly-Phen) to check if these polymorphisms are deleterious or not.
      • Hasnain M.J.U.
      • Shoaib M.
      • Qadri S.
      • et al.
      Computational analysis of functional single nucleotide polymorphisms associated with SLC26A4 gene.
      To find any association of Pro92Pro and Gly39Glu polymorphism with the occurrence of disease, PhD-SNP, and SNP&GO tools were used; further I-mutant and MUpro servers were used to check the stability of proteins.
      • Ibrahim A.M.
      • Albakry A.M.
      • Alla N.W.
      • et al.
      Computational Analysis of Single Nucleotide Polymorphism (SNPs) in Human MYOC Gene.
      Homology modeling (via MODELLER 9.22) was used to generate the structure of wild MSH6 protein, followed by its energy minimization via the CHIMERA tool. This energy minimized MSH6 wild structure was further used to create rs1042821 mutant structure, and its subsequent energy minimization was also completed. The Ramachandran plot also validated these protein models via Procheck. Root Mean Square Deviation (RMSD) was also calculated for rs1042821 polymorphism via Pymol to estimate the structural deviation of mutant protein structures from wild MSH6 protein. The CONSURF server also performed conservation analysis to identify whether these mutations are conserved evolutionary.

      Results

      Patient characteristics and clinical predictors

      Five hundred lung cancer patients were recruited for this study. The main features of the investigation, such as sex, age, smoking pattern, pack-years, TNM staging, histology, and other clinical data, are shown in Table 1. The mean age of lung cancer patients was 60.5 ± 9.86. Lung cancer patients were further stratified based on histology: 41% of cases had squamous-cell carcinoma (SQCC), 40.6% cases had adenocarcinoma (ADCC), and 16.8% had small cell lung carcinoma (SCLC). The staging analysis of recruited patients showed that 1.2%, 3.8%, 38.6%, and 51.4% of patients belonged to Stage I, II, III, and IV, respectively. Further TNM data revealed that several patients with T3 and T4 were more than T1 and T2 (77.4% vs. 13.4%), patients having lymph node N0, N1 and N2 were significantly more than N3 (61.8% vs. 32.6%), patients belonging to metastasis stage M0 were 42% as compared to 52.6% belonging to M1 and 5.2% of patients’ metastasis level was unknown. Further, KPS and ECOG as performance indicators were used to evaluate patients; in our study, approximately 18% of cases have KPS below 60, 54.4% and 27.5% cases reported a KPS in the range of 70–80 and 90–100, respectively. For 44.9% of lung cancer patients, ECOG value was between 0 and 1, 39.49% have ECOG value of 2, and 15.54% of patients have ECOG value between 3 and 4.
      TABLE 1Demographic characteristics among cases.
      VariableTotal (N)Cases, n (%)ap-value
      Age (years)500
       Mean ± SD60.5 ± 9.860.103
       Range30–86
      Sex500
       Male401 (80.2)
       Female99 (19.8)<0.0001
      Smoking status500
       Smokers398 (79.6)
       Non-smokers102 (20.4)<0.0001
      Pack years500
       Mean ± SD20.82 ± 21.03<0.0001
      Histological types500
       SQCC205 (41.0)
       ADCC203 (40.6)
       SCLC84 (16.8)
       Others6 (1.4)
       Unknown2 (1.0)
      Overall survival500
       Dead356 (71.2)
       Alive119 (23.8)
      Staging500
       I6 (1.2)
       II19 (3.8)
       III193 (38.6)
       IV257 (51.4)
       Unclassified25 (5.0)
      Tumor Size500
       Tx19 (3.8)
       T126 (5.2)
       T241 (8.2)
       T385 (17)
       T4303 (60.6)
       Unknown26 (5.2)
      Lymph Node500
       Nx3 (0.6)
       N065 (13)
       N134 (6.8)
       N2210 (42)
       N3163 (32.6)
       N40 (0)
       Unknown25 (5)
      Metastasis500
       Mx1 (0.2)
       M0210 (42)
       M1265 (53)
       Unknown24 (4.8)
      Performance status476
       KPS (below 60)86 (18.06)
       KPS (70–80)259 (54.4)<0.0001
       KPS (90–100)131 (27.5)<0.0001
       ECOG (0–1)476214 (44.9)
       ECOG (2)188 (39.49)<0.0001
       ECOG (3–4)74 (15.54)<0.0001
      Chemotherapy regimen
       Pemetrexed cis/carboplatin341105 (30.79)
       Irinotecan cis/carboplatin41 (12.02)
       Docetaxel cis/carboplatin36 (10.6)
       Paclitaxel cis/carboplatin81 (23.75)
       Gemcitabine cis/carboplatin29 (8.19)
       Ceretinib1 (0.28)
       Others48 (13.55)
      Abbreviations: SD = Standard Deviation, n = total number of lung cancer cases or control subjects, a) p-values were derived from Pearson Chi – square test except age and pack-years; Student t-test was used for age and pack-years. All p-values are two – sided. p< 0.05 was considered statistically significant.
      As depicted in Table 1, out of 500 cases, 341 patients have undergone platinum-based doublet chemotherapy, out of which 30.79% received pemetrexed + cisplatin/carboplatin, 12.02% cases received irinotecan+cisplatin/carboplatin, 10.6% cases received docetaxel+cisplatin/carboplatin, 23.75% received paclitaxel+cisplatin/carboplatin and 8.19% cases received gemcitabine in combination with cisplatin/carboplatin.

      Association of the MSH6 Pro92Pro and Gly39Glu polymorphisms & clinic-pathological parameter

      To evaluate the effect of MSH6 Pro92Pro and Gly39Glu polymorphisms on various clinic-pathological features, the subjects were stratified based on the clinical stage (III vs. IV), tumor extension (T3 vs. T4), lymph node invasion (Nx+N0+N1 vs. N2+N3+N4), and metastasis (M0 vs. M1). As shown in Supplementary Table 1, no significant relationship between MSH6 Pro92Pro, Gly39Glu polymorphisms, and any clinic-pathological features was observed, suggesting that the above two polymorphic variants were not found to play a role in either correlating or modulating lung cancer.

      Association of MSH6 (rs1800932) Pro92Pro and Gly39Glu (rs1042821) polymorphism with chemotherapy response

      In order to determine any association between MSH6 rs1800932 and rs1042821 polymorphisms and patients' response towards chemotherapy, the odds ratio was evaluated after applying univariate logistic regression analysis. All the patients recruited in this study were given platinum-based doublet chemotherapy, based on how well a patient is responding to chemotherapy, and then they were the stratified basis of response into two groups: responders and non-responders. Patients showing complete remission or partial remission (CR+PR) were called responders, and patients who are not responding to chemotherapeutic drugs and continue to show stable or progressive disease (SD+PD) were called non-responders. As shown in Supplementary Table 2, two univariate regression analyses predicted no association of MSH6 polymorphism towards response to chemotherapy. Hence, none of the MSH6 polymorphisms can predict chemotherapy response rate.

      Survival analysis of MSH6 Pro92Pro and Gly39Glu polymorphism

      We have recruited 500 lung cancer patients in this study, but survival data was only available for 475 patients. The subjects' survival rate was followed up by telephone until the end of the study or the death of the subject, whichever occurred earlier. Median survival time (MST), log-rank p-value, and the unadjusted hazard ratio was calculated by Kaplan Meir regression method (univariate analysis), whereas adjusted HR was calculated by cox regression analysis method after adjusting based on genotype, age, sex, stage, smoking, stage of the tumor, chemotherapy regimen and ECOG. As shown in Table 2, for MSH6 pro92pro polymorphism, the patients carrying a single copy of the variant allele (AG) had a higher MST as compared to subjects who were carrying the wild genotype (AA) (MST=12.2 vs. 8.06, Log Rank p = 0.10). However, our analysis revealed no association between overall survival (OS) and MSH6 Pro92Pro (rs1800932) and Gly39Glu (rs1042821) polymorphisms.
      TABLE 2Association of MSH6 A>G and C>T polymorphism on overall survival in lung cancer cases and on basis of histology.
      Overall Survival
      MSH6 rs1042821 C>TCases N(%), N = 475Dead N(%), N = 356Alive N(%), N = 119Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted hr
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted hr
      hazards ratio for age, sex, smoking, histology, stage, KPS, ECOG.
      (95% CI)
      P
      CC238 (50.1)175 (49.1)63 (52.9)7.8
      CT190 (40.1)144 (40.4)46 (38.7)8.30.861.01 (0.81–1.27)1.10 (0.87–1.39)0.39
      TT47 (9.8)37 (10.5)10 (8.4)8.830.981.00 (0.70–1.43)0.95 (0.66–1.37)0.79
      CT+TT237 (49.8)181 (50.8)56 (47)8.560.891.01 (0.82–1.24)1.08 (0.87–1.34)0.47
      MSH6 rs1800932 A>GCases N(%), N = 475Dead N(%), N = 356Alive N(%), N = 119Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted hr
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted hr
      hazards ratio for age, sex, smoking, histology, stage, KPS, ECOG.
      (95% CI)
      P
      GG429 (90.3)326 (91.6)103 (86.6)8.06
      AG46 (9.7)30 (8.4)16 (13.4)12.20.100.73 (0.53–1.02)0.76 (0.51–1.11)0.16
      ADCC
      MSH6 rs1042821 C>TCases N(%), N = 191Dead N(%), N = 131Alive N(%), N = 60Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted hr
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted hr
      hazards ratio for age, sex, smoking, histology, stage, KPS, ECOG.
      (95% CI)
      P
      CC93 (48.7)64 (48.9)29 (48.3)8.5
      CT76 (39.8)50 (38.2)26 (43.3)9.70.500.88 (0.61–1.27)1.07 (0.72–1.58)0.72
      TT22 (11.5)17 (12.9)5 (8.4)8.20.751.08 (0.62–1.88)0.99 (0.97–1.01)0.57
      CT+TT98 (51.3)67 (51.1)31 (51.6)9.130.650.92 (0.65–1.30)1.06 (0.74–1.53)0.72
      MSH6 rs1800932 A>GCases N(%), N = 191Dead N(%), N = 131Alive N(%), N = 60Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted hr
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted hr
      hazards ratio for age, sex, smoking, histology, stage, KPS, ECOG.
      (95% CI)
      P
      GG174 (91.1)124 (94.7)50 (83.3)8.73
      AG17 (8.9)7 (5.3)10 (16.7)0.030.45 (0.26–0.78)0.38 (0.16–0.86)0.02
      SQCC
      MSH6 rs1042821 C>TCases N(%), N = 202Dead N(%), N = 155Alive N(%), N = 47Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted hr
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted hr
      hazards ratio for age, sex, smoking, histology, stage, KPS, ECOG.
      (95% CI)
      P
      CC104 (51.4)77 (49.7)27 (57.4)8.2
      CT78 (38.7)62 (40.0)16 (34.0)6.060.261.2 (0.86–1.69)1.26 (0.89–1.79)0.79
      TT20 (9.9)16 (10.3)4 (8.6)8.830.791.07 (0.61–1.86)1.19 (0.68–2.06)0.53
      CT+TT98 (48.5)78 (50.3)20 (42.5)7.160.301.17 (0.85–1.61)1.25 (0.90–1.74)0.17
      MSH6 rs1800932 A>GCases N(%), N = 202Dead N(%), N = 155Alive N(%), N = 47Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted hr
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted hr
      hazards ratio for age, sex, smoking, histology, stage, KPS, ECOG.
      (95% CI)
      P
      GG179 (88.6)136 (87.7)43 (91.5)7.96
      AG23 (11.4)19 (12.3)4 (8.5)7.10.431.20 (0.72–2.02)1.5 (0.92–2.48)0.10
      SCLC
      MSH6 rs1042821 C>TCases N(%), N = 75Dead N(%), N = 64Alive N(%), N = 11Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted hr
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted hr
      hazards ratio for age, sex, smoking, histology, stage, KPS, ECOG.
      (95% CI)
      P
      CC37 (49.3)31 (48.4)6 (54.5)7.13
      CT33 (44)29 (45.3)4 (36.4)100.881.03 (0.61–1.76)0.87 (0.46–1.63)0.67
      TT5 (6.7)4 (6.3)1 (9.1)14.90.490.70 (0.28–1.75)0.59 (0.19–1.81)0.36
      CT+TT38 (50.7)33 (51.5)5 (45.5)100.880.93 (0.58–1.59)0.76 (0.42–1.35)0.35
      MSH6 rs1800932 A>GCases N(%), N = 75Dead N(%), N = 64Alive N(%), N = 11Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted hr
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted hr
      hazards ratio for age, sex, smoking, histology, stage, KPS, ECOG.
      (95% CI)
      P
      GG69 (92.0)60 (93.8)9 (81.8)7.6
      AG6 (8.0)4 (6.2)2 (18.2)20.60.030.4 (0.20–0.82)0.40 (0.12–1.30)0.09
      a Unadjusted Hazards ratio for Kaplan meier analysis.
      b hazards ratio for age, sex, smoking, histology, stage, KPS, ECOG.
      Further, when we stratified patients based on histology, our results showed polymorphism for Pro92Pro (rs1800932). SCLC patients who were carrying a single copy of the variant allele (AG) for the rs1800932 polymorphism reported a significant increase in MST (20.6 vs. 7.6, log-rank p = 0.03, Fig. 1a) and a favorable death ratio (HR=0.4) as compared to those patients who were carrying both the alleles for the wild genotype (AA), as shown in Table 2. None of the other histology of lung cancer showed an association between the overall survival time and polymorphism for both the SNPs.
      Fig. 1
      Fig. 1a: Overall survival of MSH6 rs1800932 polymorphism. b: Overall survival with respect to docetaxel and carbo/cisplatin combination chemotherapeutic regimen of MSH6 rs104221 polymorphism.

      Association of MSH6 Pro92Pro (rs1800932) and Gly39Glu (rs1042821) polymorphic variant and their association with overall survival in lung cancer patients treated with different chemotherapy regimens

      Overall survival of patients treated with the different chemotherapeutic regimen and their association with MSH6 Pro92Pro (rs1800932) and Gly39Glu (rs1042821) polymorphisms is shown in Table 3. Homozygous wild genotype (CC) was considered as the reference for MSH6 Pro92Pro (rs1800932) polymorphic variant, whereas for Gly39Glu (rs1042821) polymorphic variant homozygous mutant type (GG) genotype was considered as reference. As shown in table 3 for MSH6 Pro92Pro polymorphism, it was observed that the patients who were harboring a single copy of the variant allele (CT) and administered carbo/cisplatin and docetaxel chemotherapy regimen reported a significant increase in MST (16.6 vs. 8.36, Log-rank p = 0.03, Fig. 1b) and a lower hazard ratio (HR= 0.42), in comparison to those patients who were carrying the wild genotype (CC) and underwent same chemotherapeutic treatment. On the contrary, our data also suggests no significant association between the overall survival of a patient-administered carboplatin/cisplatin along with irinotecan, paclitaxel, and pemetrexed in both MSH6 rs1800932 and rs1042821 polymorphic variants.
      TABLE 3Association of MSH6 A>G and C>T genotype on survival in lung cancer cases on basis of chemotherapeutic regimen.
      Docetaxel
      MSH6 rs1042821 C>TCASES n (%), N = 36DEAD n (%), N = 28ALIVE n (%), N = 8Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted HR
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted HR
      hazards ratio for age, sex, smoking, histology, stage, ECOG.
      (95% CI)
      P
      CC15 (41.7)13 (46.4)2 (25)8.36
      CT15 (41.7)9 (32.1)6 (75)16.660.030.42 (0.18–1.03)0.53 (0.18–1.58)0.26
      TT6 (16.6)6 (21.5)08.50.761.15 (0.42–3.11)1.02 (0.25–4.10)0.97
      CT+TT21 (58.3)15 (53.5)6 (75)110.100.55 (0.25–1.22)0.57 (0.23–1.38)0.22
      MSH6 rs1800932 A>GCASES n(%), N = 36DEAD n (%), N = 28ALIVE n (%), N = 8Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted HR
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted HR
      hazards ratio for age, sex, smoking, histology, stage, ECOG.
      (95% CI)
      P
      GG33 (91.7)25 (89.3)8 (100)8.73
      AG3 (8.3)3 (10.7)0 (0)13.30.741.21 (0.33–4.48)3.03 (0.66–13.7)0.15
      Paclitaxel
      MSH6 rs1042821 C>TCASES n (%), N = 81DEAD n (%), N = 62ALIVE n (%), N = 19Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted HR
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted HR
      hazards ratio for age, sex, smoking, histology, stage, ECOG.
      (95% CI)
      P
      CC48 (59.3)36 (58.1)12 (63.2)6.9
      CT28 (34.6)22 (35.4)6 (31.6)6.10.501.19 (0.68–2.08)1.21 (0.70–2.10)0.49
      TT5 (6.1)4 (6.5)1 (5.2)4.10.631.28 (0.40–4.02)1.23 (0.42–3.58)0.69
      CT+TT33 (40.7)26 (41.9)7 (36.8)5.80.461.20 (0.71–2.03)1.19 (0.71–2.00)0.50
      MSH6 rs1800932 A>GCASES n (%), N = 81DEAD n (%), N = 62ALIVE n (%), N = 19Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted HR
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted HR
      hazards ratio for age, sex, smoking, histology, stage, ECOG.
      (95% CI)
      P
      GG68 (83.9)52 (83.8)16 (84.2)7.5
      AG13 (16.1)10 (16.2)3 (15.8)2.90.391.33 (0.63–2.83)1.42 (0.70–2.88)0.32
      Irinotecan
      MSH6 rs1042821 C>TCASES n (%), N = 41DEAD n (%), N = 33ALIVE n(%), N = 8Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted HR
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted HR
      hazards ratio for age, sex, smoking, histology, stage, ECOG.
      (95% CI)
      P
      CC18 (43.9)15 (45.5)3 (37.5)7.13
      CT19 (46.4)17 (51.5)2 (25)100.681.15 (0.57–2.33)1.15 (0.48–2.71)0.74
      TT4 (9.7)1 (3.03)3 (37.5)0.050.18 (0.06–0.55)0.14 (0.01–1.36)0.09
      CT+TT23 (56.1)18 (54.5)5 (62.5)13.20.700.87 (0.43–1.76)0.76 (0.34–1.72)0.52
      MSH6 rs1800932 A>GCASES n (%), N = 41DEAD n (%), N = 33ALIVE n (%), N = 8Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted HR
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted HR
      hazards ratio for age, sex, smoking, histology, stage, ECOG.
      (95% CI)
      P
      GG39 (95.1)33 (100)6 (75.0)8.66
      AG2 (4.9)0 (0)2 (25.0)0.04000.96
      Pemetrexed
      MSH6 rs1042821 C>TCASES n(%), N = 105DEAD n(%), N = 77ALIVE n(%), N = 28Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted HR
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted HR
      hazards ratio for age, sex, smoking, histology, stage, ECOG.
      (95% CI)
      P
      CC53 (50.5)40 (51.9)13 (46.4)7.22
      CT40 (38.1)29 (37.7)11 (39.3)11.00.340.79 (0.49–1.27)0.77 (0.46–1.27)0.31
      TT12 (11.4)8 (10.4)4 (14.3)15.70.350.70 (0.35–1.38)0.76 (0.35–1.63)0.48
      CT+TT52 (49.5)37 (48.1)15 (53.5)12.20.240.77 (0.49–1.20)0.76 (0.48–1.21)0.26
      MSH6 rs1800932 A>GCASES n (%), N = 105DEAD n (%), N = 77ALIVE n (%), N = 28Univariate analysisMultivariate analysis
      MST (months)Log rank pUnadjusted HR
      Unadjusted Hazards ratio for Kaplan meier analysis.
      Adjusted HR
      hazards ratio for age, sex, smoking, histology, stage, ECOG.
      (95% CI)
      P
      GG97 (92.4)74 (96.1)23 (82.1)7.56
      AG8 (7.6)3 (3.9)5 (17.9)0.040.32 (0.15–0.65)0.33 (0.10–1.07)0.06
      a Unadjusted Hazards ratio for Kaplan meier analysis.
      b hazards ratio for age, sex, smoking, histology, stage, ECOG.

      Computational results

      Structural analysis of SNPs

      A homology modeling approach was used to build MSH6 wild protein structure as a complete MSH6 protein structure was unavailable in the PDB database. BLAST results showed that structures (1EWQ, 1FW6, 1NNE, and 2O8B) have the maximum coverage and percentage similarity with the query. Hence these were selected as templates for creating MSH6 structure and named MSH6_WILD. Furthermore, this MSH6_WILD structure was submitted to energy minimization to find a set of coordinates representing the minimum energy conformation of the structure. The CHIMERA tool utilizes the steepest descent and conjugate gradient optimization algorithms for energy minimization. After energy minimization of MSH6_WILD protein, the energy got significantly reduced to 49,992.2857 KJ/mol from 1,626,512.431 KJ/mol (lower is better), which signifies that all the forces on the atoms in that protein molecule are balanced. The mutant structure of rs1042821 was also generated by MODELLER 9.22 using previously generated energy minimized MSH6_WILD structure with appropriate mutations as a template. Mutant structure for rs1800932 polymorphism was not generated because it is a synonymous mutation. These generated structures were also subjected to energy minimization, rs1042821 polymorphisms' initial energy was 233,954.65 KJ/mol, and energy minimization resulted in a more stabilized structure having final energy −62,352.722 KJ/mol. These structures were further subjected to Ramachandran plot analysis via PROCHECK. Ramachandran plot results for MSH6_WILD represent 96.4% residues in the allowed region compared to rs104282 having 96.8% amino acid residues in the allowed region. Root mean square deviation (RMSD) values of rs1042821 mutant structure were 0.179, which signifies there is not much deviation from the MSH6_WILD protein. Further, these two mutations, rs1042821 (Gly39Glu) and rs1800932 (Pro92Pro), were also mapped on the MSH6 protein. MSH6 protein has five domains, and rs1042821 rs1800932 SNPs are present in the disordered domain or N-terminal region, which appears not to have significant protein activity (Fig. 2a & b).
      Fig. 2
      Fig. 2(A) Diagrammatic representation of different domains in MSH6 protein. (B) Energy minimized MSH6 protein structure, five domains of MSH6 are colored differently, and the two polymorphisms rs1042821 Gly39Glu and rs1800932 Pro92Pro are shown with green sphere shape. N-terminal Region (red), Domain 1 (blue), domain 2 (Yellow), domain 2 (Cyan), domain 4 (green), domain 5 (Purple).

      Functional analysis of SNPs

      Computational analysis was carried out for rs1042821 (Gly39Glu) with different prediction tools to understand the functional impact. The synonymous mutation was for rs1800932 (Pro92Pro); hence it is not a functional analysis. SIFT, PROVEAN, PANTHER, and Polyphen tools predict the deleterious and damaging nature of nsSNP's where only PANTHER reported possibly damaging (Table 4).
      Table 4Functional analysis of rs1042821 with the employment of different algorithms.
      Prediction algorithmrs1042821 (Gly39Glu)
      SIFTTolerated
      PROVEANNeutral
      PANTHERPossibly damaging
      Polyphen-2Benign
      PhD SNPNeutral
      SNP&GONeutral
      I mutantIncrease
      MuProIncrease
      PhD SNP and SNP&GO servers were used to analyze whether single amino acid substitution is associated with the occurrence of a disease or not, and both servers predicted rs1042821 to have a neutral effect. For estimating the stability of mutant proteins, I-mutant and MUpro servers were used, which were predicted to have increased stability. Amino acid, which plays a crucial role in protein-protein interactions, is more highly conserved than other amino acids. Hence, the CONSURF server was utilized to determine the conservation status of these SNPs; the CONSURF server predicted that rs1042821 Gly39Glu to have a conservation score of 3 respectively, which signifies most minor conservation. If a mutation is present in the conserved region, it is much more damaging than polymorphism present in the variable region of the protein. These results show that rs1042821 Gly39Glu does not affect the protein's function.

      Discussion

      The process of replication and recombination happens with incredible accuracy with the help of particular enzyme-like DNA polymerase, which ensures the bases are added to the growing strand and are correctly paired with the template strand. However, these enzymes sometimes make mistakes at a rate of 1 per every 100,000 nucleotides.
      • Pray L.
      DNA replication and causes of mutation.
      So, to repair these errors, cells are equipped with various repair mechanisms, e.g., mismatch repair (MMR) pathways that identify and repair the incorrect insertions, misincorporation, and deletion of bases. Many studies have associated DNA repair pathways' mutation with various cancers.
      • Aleskandarany M.
      • Caracappa D.
      • Nolan C.C.
      • et al.
      DNA damage response markers are differentially expressed in BRCA-mutated breast cancers.
      To date, several studies have addressed whether some genetic variation (SNPs) affects various clinical outcomes in lung cancer patients. Hence, in this study, we have analyzed the association of two MSH6 polymorphisms (rs1800932 and rs1042821) with various clinic-pathological parameters and overall survival of lung cancer patients treated with platinum-based doublet chemotherapy.
      Data from our research revealed that both MSH6 Pro92Pro (rs1800932) and Gly39Glu (rs1042821) polymorphisms did not show any significant association towards stage III and IV lung tumors and for both lymph node invasion and metastasis. As far we know that this is the first study conducted in north Indian lung cancer patients, which has estimated the association of MSH6 polymorphism towards overall survival of patients administered platinum-based doublet chemotherapy. Our study concludes that neither of the two polymorphisms showed any significant association with the overall survival of lung cancer patients. However, when classified based on histological subtypes, SCLC patients who were heterozygous for the Pro92Pro (rs1800932) polymorphism reported a significant increase in SCLC patients (p = 0.03). Our results were corroborated by a previous study, where Vymetalkova and coworkers reported no association of rs1800932 polymorphism in colorectal cancer patients with overall survival in the Czech Republic population.
      • Vymetalkova V.
      • Pardini B.
      • Rosa F.
      • et al.
      Variations in mismatch repair genes and colorectal cancer risk and clinical outcome.
      However, another study conducted by Dong and coworkers on pancreatic cancer patients contradicted our findings and reported a 2-fold increased risk and a corresponding decrease in survival time in American lung cancer patients for rs1042821 polymorphism
      • Dong X.
      • Jiao L.
      • Li Y.
      • et al.
      Significant associations of mismatch repair gene polymorphisms with clinical outcome of pancreatic cancer.
      This study analyzed survival data for MSH6 Pro92Pro (rs1800932) and Gly39Glu (rs1042821) polymorphisms based on the chemotherapeutic regimen. As far as our knowledge is concerned, this is the first-time lung cancer patients' overall survival data based on different chemotherapeutic regimens are being reported. All the patients were given either docetaxel, paclitaxel, irinotecan, or pemetrexed as a second line of chemotherapy drugs along with platinum-based drugs like carboplatin/ cisplatin. Our analysis revealed that heterozygous patients for the Gly39Glu polymorphism and administered docetaxel, and carbo/cisplatin had an increased median survival time and lower hazard ratio.
      A previous study by Mazaheri and coworkers demonstrated that in silico analysis serves as an essential tool for analyzing the effect of polymorphism on protein structure and functionality.
      • Mazaheri M.
      • Karimian M.
      • Behjati M.
      • et al.
      Association analysis of rs1049255 and rs4673 transitions in p22phox gene with coronary artery disease: a case-control study and a computational analysis.
      The rs1042821 is a non-synonymous mutation with Glycine to Glutamine substitution at position 39, whereas rs1800932 is synonymous with Proline-to-Proline substitution at position 92. Computational analysis was carried out for non-synonymous mutation (rs1042821), but most of these tools revealed that these particular SNPs do not affect the structure and function of the protein. Since both SNPs are present in the N-terminal region / disordered domain of MSH6 protein, they cannot alter MSH6 protein structure and function. Disorder domain of MSH6 protein ranges from 4 to 361 amino acids having PCNA binding motif and PWWP domain. PWWP domain binds to dsDNA without preference for mismatches/nicks with 20 times more affinity than ssDNA. Stec and coworkers have also demonstrated that the PWWP domain is involved in protein-protein interactions due to its close presence with specific amino acids and the pattern of other domain nearby domains.
      • Stec I.
      • Nagl S.B.
      • van Ommen G.J.B.
      • den Dunnen J.T.
      The PWWP domain: a potential protein–protein interaction domain in nuclear proteins influencing differentiation?.
      A study led by Santos demonstrated that rs1042821 polymorphism is associated with an increased risk of thyroid cancer in the Caucasian Portuguese population. The computational results follow the polymorphism study where we did not find any association of rs1042821 polymorphism with overall survival, stage III, IV, and TNM staging of lung cancer.
      A significant strength of our study lies in the number of patients recruited for this investigation and computational analysis of the selected SNPs; previous studies elucidating the role of MSH6 polymorphism reported to date have a minimal number of subjects enrolled makes our investigation much more dependable. The current study focuses on the following criteria: clinic-pathological features, overall survival (OS), OS based on the chemotherapeutic regimen, and computational analysis of SNPs selected for this study. Our investigation also has certain limitations: although the patients recruited for this study were huge, the patients falling under the subcategories of lung cancer are still small. Moreover, since the smoking patterns and pack years difference influence the investigation, our study can also be considered a limitation. Another limitation would be that the subjects recruited for this study belong to one particular area in northern India, which may cause biased results.

      Funding details

      Since we have no funds to research, Dr. Siddharth Sharma and Mr. Sidhartha Singh funded this work from their savings.

      Disclosure

      The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

      Author contribution statement

      SS1 performed experiments; SS2 conceived and designed the research; SS1, and NS analyzed data; NS provided the samples and clinical data for analysis. SS1 wrote the paper, and all authors revised and approved the paper.

      Conflict of interest

      All the authors of this manuscript report that they have no conflict of interest.

      Acknowledgements

      We would like to express gratitude to all the subjects who participated in this study. The authors thanks Department of Pulmonary Medicine, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India, for providing samples, and Thapar Institute of Engineering and Technology, Patiala, Punjab for providing the necessary infrastructure to carry out the research work.

      Appendix. SUPPLEMENTARY MATERIALS

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