Lung adenocarcinoma frequently develops in non-smokers, particularly in Asian populations, pointing to an important role for germline genetic susceptibility beyond tobacco exposure. Despite advances in TNM staging and targeted therapies for drivers like EGFR and ALK, approximately 35 to 40 percent of patients experience unanticipated treatment failure, indicating unexplained clinical heterogeneity that demands new biomarkers.
Methylenetetrahydrofolate reductase is a pivotal enzyme in folate-mediated one-carbon metabolism that converts 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, directing the folate pool toward homocysteine remethylation and S-adenosylmethionine synthesis. Two functional polymorphisms, rs1801131 (A1298C) and rs1801133 (C677T), significantly reduce MTHFR enzymatic activity, impairing the methyl supply needed for normal DNA methylation.
DNA hypomethylation resulting from impaired MTHFR activity is a well-established precursor to chromosomal instability, oncogene activation, and tumor suppressor silencing. These mechanisms link MTHFR variants mechanistically to carcinogenesis, yet most existing studies have focused on European or Han Chinese populations while excluding the Zhuang ethnic group, the largest minority group in China with a distinct genetic background.
Lactate dehydrogenase is a well-established biomarker of tumor glycolysis and the Warburg effect, correlating with aggressive phenotypes, metastasis, and poor survival in non-small cell lung cancer. No prior study had integrated germline MTHFR genotyping with serum LDH and tumor protein expression to investigate their combined prognostic effect in LUAD.
This retrospective case-control study enrolled 306 histologically confirmed lung cancer patients from the First Affiliated Hospital of Guilin Medical University between July 2021 and March 2024, compared against 156 age- and sex-matched Zhuang healthy controls. The 306 patients comprised 160 LUAD, 77 LUSC, and 63 SCLC cases, all pathologically verified according to the WHO Classification of Thoracic Tumours 2021 and CSCO clinical guidelines.
MTHFR rs1801131 and rs1801133 genotyping was performed using the SNPscan multiplex fluorescence PCR platform with three locus-specific probes per SNP and more than 99 percent concordance across duplicate replicates. All genotype distributions conformed to Hardy-Weinberg equilibrium in controls, confirming assay validity.
Serum LDH was measured at an ISO 15189-accredited laboratory using Roche Cobas analyzers. An LDH level at or above 180 U/L was classified as elevated, consistent with established clinical thresholds. Tumor MTHFR protein expression was quantified in representative LUAD formalin-fixed paraffin-embedded sections using immunohistochemistry, with staining scored by two board-certified pathologists blinded to genotype and confirmed by digital image analysis using ImageJ.
Survival outcomes including overall survival and progression-free interval were assessed using multivariate Cox proportional hazards regression and Kaplan-Meier analysis. Only covariates with P below 0.10 in univariate screening were entered into multivariate models, with a prespecified strategy to limit overfitting. Haplotype analysis was performed using SHEsis software combining the two MTHFR polymorphisms.
Chi-square analysis revealed a significant difference in MTHFR rs1801131 GG genotype distribution between lung cancer patients and healthy controls (P = 0.021), with the recessive model showing a stronger association (P = 0.017). In the LUAD subgroup, GG genotype carriers showed a trend toward increased risk compared to TT genotype carriers, with an adjusted odds ratio of 2.14 (95% CI: 0.99 to 4.65, P = 0.054) after adjustment for age and sex.
In contrast to rs1801131, the rs1801133 polymorphism showed no significant association with lung cancer susceptibility under any genetic model tested, suggesting that the A1298C substitution rather than the C677T substitution is the more relevant susceptibility locus in this population.
Haplotype analysis combining both polymorphisms identified the GG haplotype (rs1801131-G combined with rs1801133-G) as significantly associated with a 1.53-fold increased LUAD risk (OR = 1.527, 95% CI: 1.062 to 2.195, P = 0.022). Conversely, the TG haplotype was associated with a protective effect on LUAD risk (OR = 0.708, P = 0.029). No significant haplotype associations were observed for overall LC, LUSC, or SCLC, confirming specificity to the adenocarcinoma subtype.
LC patients as a group exhibited substantially elevated LDH compared to healthy controls (median 216.50 versus 160.50 U/L, P less than 0.001), alongside lower albumin, altered bilirubin fractions, and elevated alkaline phosphatase and globulin, a biochemical profile consistent with systemic metabolic effects of malignancy.
Multivariate Cox regression confirmed that the rs1801131 GG genotype was independently associated with shorter overall survival in the entire lung cancer cohort (HR = 1.926, 95% CI: 1.071 to 3.465, P = 0.029) after adjusting for stage, treatment type, primary therapy outcome, and other clinical variables. Within the LUAD subgroup specifically, GG genotype carriers showed significantly worse overall survival (HR = 2.210, 95% CI: 1.048 to 4.661, P = 0.037) and worse progression-free interval (HR = 1.979, 95% CI: 1.103 to 3.552, P = 0.022).
Kaplan-Meier analysis corroborated these multivariate findings. The rs1801131 GG genotype was associated with the worst overall survival in the full LC cohort (P = 0.033), and with both the worst overall survival (P = 0.043) and worst progression-free interval (P = 0.023) specifically within the LUAD subgroup.
Combined stratification by rs1801131 genotype and LDH level revealed a synergistic prognostic effect. Patients with both the GG genotype and elevated LDH had significantly worse overall survival than patients with TG or TT genotypes combined with elevated LDH, in both the overall LC cohort (P = 0.034) and the LUAD subgroup (P = 0.042).
Immunohistochemical analysis of LUAD tissues confirmed the mechanistic basis for these clinical findings: MTHFR rs1801131 GG genotype carriers showed significantly lower tumor MTHFR protein expression compared to TG and TT carriers (P less than 0.05). This reduction in protein expression was specific to LUAD and was not observed in LUSC or SCLC tissues.
The prognostic effect of rs1801131 in the Guangxi Zhuang population contrasts with null associations reported in European cohorts, reflecting ethnic-specific genetic architectures that shape how common polymorphisms interact with tumor biology. East Asian populations show stronger MTHFR effects in LUAD subgroup analyses than European cohorts, consistent with ancestry-specific molecular contexts that may amplify or suppress the functional consequences of folate pathway disruption.
The mechanistic link between the GG genotype and elevated LDH reflects a model of metabolic dysregulation. Impaired MTHFR activity from the rs1801131 variant disrupts folate cycling, which may alter NAD plus and NADH homeostasis, a key regulator of LDH activity. This may explain why the allele is associated with elevated serum LDH even in non-oncogenic contexts, and why its combination with LDH elevation represents a compounded metabolic stress state in LUAD.
The absence of significant rs1801133 associations in this population, despite rs1801133 being the more extensively studied variant, suggests that the specific enzymatic impairment introduced by the A1298C substitution has distinct biological consequences in the folate pathway that are relevant to LUAD pathogenesis in this ethnic context.
Limitations include single-center design limiting generalizability, small SCLC subgroup numbers making those findings preliminary, and the absence of a formal sample size calculation. No mechanistic studies were performed to directly confirm the proposed NAD-LDH interaction, and external validation in independent Zhuang or multi-ethnic Asian cohorts is needed before clinical application.
This study provides the first evidence that the MTHFR rs1801131 GG genotype may serve as a population-specific marker for LUAD susceptibility and poorer prognosis in the Guangxi Zhuang population, supported by mechanistic evidence of reduced tumor MTHFR protein expression in GG carriers.
The combination of rs1801131 genotype with serum LDH offers supplementary prognostic information beyond standard clinical staging variables, identifying a high-risk patient subgroup with both impaired folate metabolism and elevated tumor glycolysis who may warrant closer surveillance and more aggressive treatment planning.
Because LDH is a widely available routine clinical test and genotyping is increasingly accessible and affordable, this two-biomarker panel has practical clinical appeal if validated in larger multicenter cohorts. The testing approach is particularly relevant for lung adenocarcinoma patients from minority ethnic populations in southern China who may currently be underrepresented in precision oncology risk stratification tools.
Future research priorities include multi-ethnic validation studies across Zhuang and other southern Chinese minority populations, functional investigations to confirm the proposed MTHFR-NAD-LDH interaction at the cellular level, and integration of rs1801131 into larger polygenic risk score frameworks for LUAD susceptibility prediction.