| Abstrakt: |
Aim: Rs16851030, a single-nucleotide variant located in the 3′-untranslated region of the ADORA1 gene, has been proposed as a potential marker of caffeine sensitivity in apnea of prematurity. Besides, it is associated with aspirin-induced asthma and the development of acute chest syndrome. However, its functional significance is still unconfirmed. This study aimed to elucidate the functional impact of rs16851030 by using CRISPR/Cas9 approach to induce the DNA variant and attendant physiological changes. Methods: Rs16851030 was introduced into HEK293 cells via homology-directed repair (HDR). Edited cells were fluorescence-enriched, sorted, isolated, and expanded into single-cell-derived clones. The edit was confirmed by Sanger sequencing. RNA sequencing was used to analyze affected pathways. Results: Rs16851030-mutant cells showed increased susceptibility to hypoxia, a condition related to apnea of prematurity. After 24 h of hypoxia, the viability of mutant clones 1 and 2 was low compared with wild-type cells (75.45% and 74.47% vs. 96.34%). RNA sequencing revealed transcriptomic changes linked to this increased vulnerability. Conclusion: Rs16851030 impairs cellular resistance to hypoxia, suggesting its role in conditions like apnea of prematurity. Further research should investigate the molecular mechanisms and transcriptomic alterations caused by rs16851030 under hypoxic conditions. Article highlights Background rs16851030, located in the 3′-untranslated region of ADORA1, was found to alter caffeine responsiveness in preterm infants. However, the exact mechanisms that underlie the functional impact of rs16851030 have not been determined. Objective This study aimed to elucidate the functional impact of rs16851030 by using the CRISPR/Cas9 gene editing approach to induce physiological changes associated with the DNA variant. Methods The rs16851030 variant was introduced into HEK293 cells through homology-directed repair induced by a combination of a sgRNA, a plasmid-encoded CRISPR enzyme, and a single-stranded oligodeoxynucleotide as the donor template. Edited cells were then fluorescence-enriched, sorted, isolated, and grown into single-cell clones. Sanger sequencing was used to confirm the presence of the rs16851030 variant. RNA sequencing was performed to compare the gene expression profiles of rs16851030-mutant and wild-type cells. Results Rs16851030-mutant cells showed increased susceptibility to hypoxia, a condition linked to apnea of prematurity. After 24 h of hypoxia, the viability of mutant clones (75.45% and 74.47%) was lower than that of wild-type cells (96.34%). Conclusion The rs16851030 variant can be a genetic marker for hypoxia sensitivity. Understanding the role of rs16851030 can aid in identifying infants at risk for hypoxia-related conditions, potentially guiding caffeine treatment strategies. [ABSTRACT FROM AUTHOR] |
