Association of genetic polymorphism rs2071676 in carbonic anhydrase gene (CA9) with the risk of squamous cell carcinoma of lungs and esophagus


Esophageal and lung Squamous Cell Carcinoma (SCC) pose a serious threat to health as they are amongst the most prevalent cancers in Pakistan. Increased metabolic and energy needs within the proliferating cancer cells results in hypoxia, affecting the homeostatic balance within the tumor microenvironment. Under these circumstances, key hypoxia-regulated genes like CA9 have major role in the survival and invasiveness of malignant tumors. This study targeted the single nucleotide polymorphism rs2071676 in CA9 to find its association with the risk of ESCC and LSCC. The non-synonymous function of this polymorphism cause the base pair to change from guanine to adenine that changes the amino acid associated with the signal peptide domain of CAIX protein from valine to methionine. Blood samples and data from 127 cases with ESCC, 73 patients with LSCC and from their age and sex-matched healthy controls were collected. Genomic DNA were extracted through standard phenol-chloroform method. Assessment of targeted polymorphism was performed through tetra-primer Amplified Refractory Mutation System Polymerase Chain Reaction (t-ARMS PCR). Pearson’s chi-square value gave statistically significant association of the SNP with LSCC (χ2 = 47.104) and ESCC (χ2 = 87.804), both with p < 0.001. Further augmentation of the results was done by calculating odds ratio, the values for LSCC and ESCC were found to be as 11.814 (Confidence Interval: 5.425–25.727) and 13.64 (Confidence Interval: 7.378–25.221), respectively. Outcomes of this study suggest that rs2071676 may be involved in carrying the risk for LSCC and ESCC, which may bring better insights in genetic diagnostics and prognostics of patients.

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Fig. 1
Fig. 2

Data availability

Patients’ blood was drawn and data was collected at the hospital with the permission of the head of oncology department, JPMC. Laboratory work was performed at KIBGE, material was provided by the institute as part of the research work.



Esophageal Squamous Cell Carcinoma


Lung Squamous Cell Carcinoma

CA9 :

Carbonic Anhydrase Gene


Carbonic Anhydrase Protein


tetra-primer Amplified Refractory Mutation System Polymerase Chain Reaction


Deoxyribonucleic Acid


World Health Organization

pHi :

Intracellular pH

pHe :

Extracellular pH


Hypoxia Induced Factor-1

H+ ion:

Hydrogen Ion


Glucose Transporter 1 Gene


Monocarboxylate Transporter 1 and 4


Tumor Microenvironment


Single Nucleotide Polymorphism


Acid citrate dextrose


Ethylenediamine Tetra Acetic Acid


Polymerase Chain Reaction






Minor Allelic Frequency


Oral Squamous Cell Carcinoma


Metastatic Renal Cell Carcinoma


Confidence Interval


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No external funding was received for this research work. It was supported by the research institute The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, Karachi, Pakistan.

Author information




Saima SALEEM: Study conception and design, providing laboratory work material, literature and manuscript review.

Ramsha KHAN: Sample collection, DNA extraction, primer designing, PCR optimization and research article writing.

Ghulam HAIDER: Hospital IRB approval, providing blood samples and patients’ data.

Sarah HASAN: Sample collection and DNA extraction.

Faria FATIMA: PCR primer optimization and manuscript review.

Sitwat ZEHRA: Result statistical analysis and material approval.

Abid AZHAR: Study approval, guidance and reviewing manuscript.

All authors read and approved the final manuscript.

Corresponding author

Correspondence to Saima Saleem.

Ethics declarations

Approval for the research work and the use of human subjects were obtained as a preliminary step from ethical review committees of the concerned institutes KIBGE and JPMC, respectively. The procedures used in this study adhere to the tenets of the Declaration of Helsinki.

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Written informed consent was taken from all participants included in the study (Cases and Controls) before blood sample collection.

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Supported and approved by the research institute KIBGE, University of Karachi, Karachi.

Declaration of ethical approval and consent to participate

The study was reviewed and approved by Ethical Committee for Scientific Research at The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi. Written consent for research work was taken from each study participant.

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Saleem, S., Khan, R., Haider, G. et al. Association of genetic polymorphism rs2071676 in carbonic anhydrase gene (CA9) with the risk of squamous cell carcinoma of lungs and esophagus. Biologia (2021).

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  • CA9
  • CAIX
  • Lung squamous cell carcinoma
  • Esophageal squamous cell carcinoma
  • Tetra-primers amplified refractory mutation system polymerase chain reaction
  • Polymorphism