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Gene & Protein in Disease Association of GST1 with lung cancer

and two SNVs from the list of 22: rs2207950 and rs743590, patients, were included. We collected 10 mL of peripheral
which potentially regulate the XMGs GSTA1 and SULT1A1, blood from each participant under the supervision of
respectively. GSTA1 encodes an enzyme involved in adding collaborating clinicians, after obtaining informed consent.
glutathione to target electrophilic compounds, including The study, involving human subjects, was conducted at
carcinogens, as an important step in their detoxification. the University of Calcutta, IPGME&R, Kolkata, and Saroj
GSTA1 is known to protect cells from reactive oxygen Gupta Cancer Centre and Research Institute, with approval
species and the products of lipid peroxidation. SULT1A1 from their respective Ethics Committees. The approvals
catalyzes the sulfate conjugation of various compounds, were as follows:
including xenobiotics. Sulfonation increases the water i. University of Calcutta Ethics Committee (Ref No:
solubility of most xenobiotic compounds, thereby 0024/16-117/1434; dated: October 24, 2016)
facilitating their renal excretion. Polymorphisms in both ii. IPGME&R Ethics Committee (Memo No. Inst/
genes can influence their detoxification abilities. IEC/2015/545; dated: December 10, 2015)
2. Materials and methods iii. Saroj Gupta Cancer Centre and Research Institute
Ethics Committee (IEC SGCCRI Ref No-2017/MS/1;
2.1. Selection of genes and SNVs dated: October 11, 2017)
Utilizing a plethora of omics datasets such as the Regulatory The study complied with the regulations of the Indian
Elements Database, GTEx, Roadmap Epigenomics, and Council of Medical Research (ICMR) and adhered to the
TCGA, along with predictive tools such as rSNPbase, principles of the Declaration of Helsinki, 1964. Genomic
RegulomeDB, and HaploReg, we analyzed a list of 2,984 DNA was isolated using the conventional phenol-
DNAse hypersensitive sites for 23 XMGs and 25 DRGs. chloroform method. Due to a lack of sufficient DNA
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These genes are known to be differentially expressed samples, the number of smoker controls had to be reduced
between healthy smokers and smokers with lung cancer. from the original study.
From this analysis, we identified 22 regulatory variants
potentially responsible for modulating 14 candidate genes 2.3. Genotyping of SNVs
expressed in the lungs. For the current study, we selected Genotyping was carried out utilizing polymerase chain
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two rSNVs from the XMGs – GSTA1 and SULT1A1 – to reaction (PCR) followed by restriction fragment length
investigate their genetic association with lung cancer in polymorphism (RFLP) analysis. Primers were designed
an eastern Indian cohort. The SNP rs2207950 (A/G) is using the freely available Primer3 software and procured
located in the insulator region of GSTA1, while rs743590 from IDT (United States). PCR was performed in a 20 μL
(A/G) is located in the active enhancer region of SULT1A1. reaction volume containing 30 – 50 ng of genomic DNA,
Figure 1 illustrates a violin plot representing a genotype- 20 pM of each primer, and 10 μL of 2× GoTaq PCR Master
specific expression of GSTA1 (left) and SULT1A1 (right) Mix (Promega, United States) and nuclease-free water
for varying genotypes of rs2207950 and rs743590 from (Milli-Q grade) in a Biorad T100 thermocycler (Biorad,
the GTEx portal. The plot illustrates the expression United States). Following PCR amplification, products were
distributions of homozygous reference, heterozygous, and digested with the appropriate restriction enzymes, after
homozygous alternative alleles in lung tissue. In this plot, quality assessment via polyacrylamide gel electrophoresis
major and minor alleles are represented by G and A, with (PAGE), following the manufacturer’s protocol (NEB,
subject counts for each genotype. Statistically significant United States). The digested PCR products were then
alterations in genotype-specific eQTLs are highlighted electrophoresed on 8% polyacrylamide gels alongside a
(P-value). 100 bp DNA marker. Only PCR products showing a single

2.2. Selection of study subjects, collection of blood amplification product, with no non-specific amplification,
samples, and isolation of DNA were used for subsequent RFLP analysis and genotype
calling.
This case–control analysis study included 101 lung cancer
patients and 252 smoker controls, selected from a larger For the GSTA1 rs2207950 variant (229 bp), PCR
pool of 401 subjects as specified in Sengupta et al. products were digested with HaeIII (NEB, USA) at 37°C
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Study participants were recruited from the Saroj Gupta overnight. After digestion, the AA genotype yielded a
Cancer Centre and Research Institute, while the controls single 229 bp band; the AG genotype yielded fragments
comprised smokers recruited from the Department of of 229 bp, 206 bp, and 23 bp, while the GG genotype
Chest Medicine at IPGME&R in Kolkata, India. Clinico- yielded fragments of 206 bp and 23 bp. For the SULT1A1
radiologically established healthy smokers over 55 years rs743590 variant (327 bp), PCR products were digested
of age, residing in the same geographical region as the with BseYI (NEB, USA) at 37°C. The AA genotype
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Volume 3 Issue 3 (2024) 3 doi: 10.36922/gpd.3928