Identification of ATP2B4 Regulatory Element Containing Functional Genetic Variants Associated with Severe Malaria
Abstract
:1. Introduction
2. Results
2.1. Prioritization and Annotation of Putative Regulatory SNPs in Linkage Disequilibrium with tagSNPs Associated with Severe Malaria
2.2. Association between the ATP2B4 tagSNP rs10900585 and Severe Malaria in a Senegalese Cohort
2.3. Association of rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255 with Severe Malaria in Senegalese Population
2.4. Association of rs10751450, rs10751451, and rs10751452 with Severe Malaria in Senegalese Population
2.5. Haplotype Analysis of the ATP2B4 SNPs
2.6. Lower Promoter Activity and Higher Enhancer Activity Are Associated with the Risk Haplotype of Severe Malaria
2.7. Genome Editing Confirmed the Regulatory Activity of the Region Containing the SNPs
2.8. K562 Deleted Clones Exhibited Higher Intracellular Calcium Concentration
2.9. PMCA4 Protein Is Not Expressed in K562 Deleted Clones
3. Discussion
4. Materials and Methods
4.1. Study Subjects, Blood Samples, and Phenotypes
4.2. Bioinformatic Prioritization and Functional Annotation of Genetic Variants
4.3. DNA Extraction, DNA Amplification, and Genotyping
4.4. Luciferase Gene Reporter Assay and Site-Directed Mutagenesis:
4.4.1. Promoter Activity
4.4.2. Enhancer Activity
4.5. CRISPR-Cas9 Genome Editing in K562 Cells
4.6. Reverse Transcription-Quantitative PCR
4.7. Calcium Measurement by Flow Cytometry in K562 Clones and Wild-Type Cells
4.8. Detection of PMCA4 by Flow Cytometry in K562 Clones and Wild-Type Cells
4.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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SNP rsID | Genomic Coordinate (hg38) | IW Scoring Rank | Number of ChIP-seq Peaks | eQTL Hits |
---|---|---|---|---|
rs11240734 | 203682695 | 1 | 54 | 3 hits |
rs1541252 | 203682798 | 2 | 90 | 3 hits |
rs10751450 | 203681816 | 3 | 46 | |
rs2228445 | 203698280 | 4 | 18 | 3 hits |
rs202111522 | 203682602 | 5 | 43 | |
rs1541254 | 203683011 | 6 | 104 | |
rs1541255 | 203683012 | 7 | 103 | |
rs10594838 | 203685540 | 8 | 2 | |
rs10625220 | 203686622 | 9 | 15 | |
rs1541253 | 203682911 | 10 | 149 | |
rs8176719 | 133257721 | 11 | 7 | 14 hits |
rs10751451 | 203681849 | 12 | 49 | 3 hits |
rs10751452 | 203681901 | 13 | 50 |
SNP | Position a | Minor Allele | MAF b | Risk Genotype | Controls% | Severe Malaria % | OR | 95% CI | p |
---|---|---|---|---|---|---|---|---|---|
rs10900585 (T > G) | 203684896 | G | 0.41 | TT | 32.9 | 48.7 | 1.94 | 1.07–3.50 | 0.029 |
rs11240734 (T > C) | 203682696 | C | 0.40 | TT | 31.6 | 53.8 | 2.52 | 1.39–4.58 | 0.002 |
rs1541252 (C > T) | 203682799 | T | 0.40 | CC | 31.6 | 53.8 | 2.52 | 1.39–4.58 | 0.002 |
rs1541253 (C > T) | 203682912 | T | 0.40 | CC | 31.6 | 53.8 | 2.52 | 1.39–4.58 | 0.002 |
rs1541254 (G > C) | 203683012 | C | 0.43 | GG | 31.6 | 53.8 | 2.52 | 1.39–4.58 | 0.002 |
rs1541255 (A > G) | 203683013 | G | 0.40 | AA | 31.6 | 53.8 | 2.52 | 1.39–4.58 | 0.002 |
rs10751450 (C > T) | 203681817 | T | 0.41 | CC | 32.9 | 50.4 | 2.07 | 1.15–3.75 | 0.016 |
rs10751451 (C > T) | 203681850 | T | 0.40 | CC | 34.2 | 56.4 | 2.49 | 1.38–4.50 | 0.002 |
rs10751452 (T > C) | 203681902 | C | 0.39 | TT | 35.4 | 55.6 | 2.27 | 1.26–4.09 | 0.006 |
SM vs. Ctrl | |||||
---|---|---|---|---|---|
Major Haplotype | Minor Haplotype | Risk Haplotype | p-Value | OR | 95% CI |
TCCGA | CTTCG | TCCGA/TCCGA | 0.002 a | 2.52 a | 1.39–4.58 a |
0.006 b | 2.38 b | 1.29–4.40 b | |||
CCTTCCGA | TTCCTTCG | CCTTCCGA/CCTTCCGA | 0.003 a | 2.67 a | 1.41–5.04 a |
0.005 b | 2.53 b | 1.32–4.86 b |
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Nisar, S.; Torres, M.; Thiam, A.; Pouvelle, B.; Rosier, F.; Gallardo, F.; Ka, O.; Mbengue, B.; Diallo, R.N.; Brosseau, L.; et al. Identification of ATP2B4 Regulatory Element Containing Functional Genetic Variants Associated with Severe Malaria. Int. J. Mol. Sci. 2022, 23, 4849. https://doi.org/10.3390/ijms23094849
Nisar S, Torres M, Thiam A, Pouvelle B, Rosier F, Gallardo F, Ka O, Mbengue B, Diallo RN, Brosseau L, et al. Identification of ATP2B4 Regulatory Element Containing Functional Genetic Variants Associated with Severe Malaria. International Journal of Molecular Sciences. 2022; 23(9):4849. https://doi.org/10.3390/ijms23094849
Chicago/Turabian StyleNisar, Samia, Magali Torres, Alassane Thiam, Bruno Pouvelle, Florian Rosier, Frederic Gallardo, Oumar Ka, Babacar Mbengue, Rokhaya Ndiaye Diallo, Laura Brosseau, and et al. 2022. "Identification of ATP2B4 Regulatory Element Containing Functional Genetic Variants Associated with Severe Malaria" International Journal of Molecular Sciences 23, no. 9: 4849. https://doi.org/10.3390/ijms23094849