Molecular mapping of aphidicolin-sensitive fragile chromosome sites in human iPSCs reveals instability of long clinically relevant genes

Background. Induced pluripotent stem cells (iPSCs) serve as an important model for studying the processes of embryogenesis and genetic diseases. At the same time, iPSCs are characterized by elevated levels of replication stress and genetic instability during mitosis. Under conditions of replication stress, breaks occur in the chromosomal material at constitutive fragile sites (CFS), which are observed on metaphase plates. Mapping CFS in iPSCs helps to better understand the functional consequences of replication stress for the derivatives obtained from iPSCs, as well as the mechanisms underlying somatic mutagenesis (including those occurring during the embryonic period). The current study continues the work on mapping constitutive fragile sites in human induced pluripotent stem cells in the context of investigating their genetic stability and safety.
Aim. Molecular mapping of constitutive fragile sites in iPSCs using FISH probes marking the boundaries of long genes.
Methods. Human iPSCs were cultured using a feeder-free method. Induction of chromatid breaks was performed by adding aphidicolin and caffeine to the medium. FISH probes specific to the boundaries of the studied genes were obtained using long range PCR and nick translation.
Results. The target genes were selected based on their size within the boundaries of the chromosomal bands where chromatid breaks were observed following treatment with replication inhibitors. Fluorescent signals from FISH probes specific to the boundaries of the genes ANKS1B, LRP1B, WWOX, NRXN3, LINGO2, and PRKN were detected on either side of the chromatid break, indicating the localization of the CFS within the target gene.
Conclusion. Replication instability has been revealed in iPSCs for six clinically significant genes that are functionally associated with oncogenesis and the development and functioning of nervous tissue. These results may clarify the role of replication stress in the formation of somatic mosaicism during embryogenesis. Additionally, mutations in these genes may affect the functional viability of differentiated derivatives of iPSCs.

1. Ahuja A.K., Jodkowska K., Teloni F., et al. A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells. Nature Communications 2016 7:1. 2016; 1(7): 1–11. DOI:10.1038/ncomms10660.

2. Milagre I., Pereira C., Oliveira R.A. Compromised Mitotic Fidelity in Human Pluripotent Stem Cells. International Journal of Molecular Sciences. 2023; 15(24): 11933. DOI:10.3390/IJMS241511933.

3. He Z., Feng W., Wang Y., et al. LRP1B mutation is associated with tumor immune microenvironment and progression-free survival in lung adenocarcinoma treated with immune checkpoint inhibitors. Translational Lung Cancer Research. 2023. 12(3): 510–529. DOI: 10.21037/TLCR-23-39/COIF.

4. Baryła I., Styczeń-Binkowska E., Bednarek A.K. Alteration of WWOX in human cancer, a clinical view. Experimental Biology and Medicine. 2015; 240(3): 305. DOI:10.1177/1535370214561953.

5. Husanie H., Abu-Remaileh M., Maroun K., et al. Loss of tumor suppressor WWOX accelerates pancreatic cancer development through promotion of TGFβ/BMP2 signaling. Cell Death and Disease. 2022; 13(12): 1–14. DOI:10.1038/s41419-022-05519-9.

6. Rouland L., Duplan E., dos Santos L.R., et al. Therapeutic potential of parkin as a tumor suppressor via transcriptional control of cyclins in glioblastoma cell and animal models. Theranostics. 2021; 20(11): 10047. DOI: 10.7150/THNO.57549.

7. Younis R.M., Taylor R.M., Beardsley P.M., McClay J.L. The ANKS1B gene and its associated phenotypes: focus on CNS drug response. Pharmacogenomics. 2019; 20(9):669. DOI:10.2217/PGS-2019-0015.

8. Shan D., Song Y., Zhang Y., et al. Neurexin dysfunction in neurodevelopmental and neuropsychiatric disorders: a PRIMSA-based systematic review through iPSC and animal models. Frontiers in behavioral neuroscience. 2024;18:1297374. doi: 10.3389/fnbeh.2024.1297374.

9. Yoshida F., Nagatomo R., Utsunomiya S., et al. Soluble form of Lingo2, an autism spectrum disorder-associated molecule, functions as an excitatory synapse organizer in neurons. Translational Psychiatry 2024 14(1): 1–12. DOI: 10.1038/s41398-024-03167-5.

10. Menon P.J., Sambin S., Criniere-Boizet B., et al. Genotype–phenotype correlation in PRKN-associated Parkinson’s disease. Npj Parkinson’s Disease 2024 10(1):1–12. DOI:10.1038/s41531-024-00677-3.

11. Al Baradie R., Mir A., Alsaif A., et al. Epilepsy in patients with WWOX-related epileptic encephalopathy (WOREE) syndrome. Epileptic disorders : international epilepsy journal with videotape. 2022. 24(4): 697–712. DOI: 10.1684/EPD.2022.1444.

12. Oliver K.L., Trivisano M., Mandelstam S.A., et al. WWOX developmental and epileptic encephalopathy: Understanding the epileptology and the mortality risk. Epilepsia. 2023; 64(5):1351–1367. DOI:10.1111/EPI.17542.

13. Fan Y., Goh E.L.K., Chan J.K.Y. Neural Cells for Neurodegenerative Diseases in Clinical Trials. Stem Cells Translational Medicine. 2023; 12(8):510–526. DOI: 10.1093/STCLTM/SZAD041.