p53, also known as tumor protein P53 or cellular tumor antigen p53, is a critical cell-cycle check point protein that regulates the G1 phase of the cell cycle. It is also directly responsible for maintaining genomic DNA stability during the DNA damage repair stage, and it coordinates the repair of damaged DNA and the removal of DNA lesions before the cell enters S-phase. The p53 gene has been identified as one of the most frequently mutated genes in human cancers [1] [2] [3] [4] , but it remains unclear how far-reaching the effects of these mutations are on cell integrity. The p53 regulatory protein is crucial in the cell’s response to DNA damage as it plays a direct role in the DNA repair pathways, affecting the activities of a number of diverse regulatory proteins that collectively control the early stages of the cell cycle [5] - [19] . Mutation or the removal of p53 could lead to genomic instability, resulting in numerous chromosomal issues [20] - [26] . Apoptosis has also been reported to be affected by the presence of p53 mutants, as mutated cell lines show a decreased frequency of apoptotic death. This decrease could in turn lead to the accumulation of gene mutations and eventual progression of tumorigenesis [27] [28] [29] [30] .
In this study, the effects of ionizing radiation on two closely related WIL2-derived lymphoblastoid cell lines (TK6, or thymidine kinase 6, and WTK1) were analyzed. The WTK1 cell line is a TP53-knockout mutant; the p53 gene in the WTK1 cell line has a substitution at codon 237, which leads to a mutation from methionine to isoleucine in the p53 protein. This cell line will overexpress only mutant p53 protein. Therefore, no wild-type p53 protein is present in the WTK1 cells. The TK6 cell line is a thymidine kinase heterozygote cell line that is wild-type for p53 [31] [32] [33] [34] . Previous studies on the effects of ionizing radiation on these two cells lines indicate that the WTK1 cell line is more resistant to radiation-induced killing than the TK6 cell line. There is significantly less apoptosis in WTK1 when compared to TK6, and mutability was also shown to be drastically different, with TK6 exhibiting a 10-fold decrease when compared to WTK1 [35] [36] .
Our lab is interested in utilizing Multiplex Ligation-Dependent Probe Amplification (MLPA) to study the effects of ionizing radiation on specific regions of the chromosomes, focusing on the differences between the WTK1 and TK6 cell lines before and after irradiation at various levels of intensity. MLPA has become a preferred method for detecting deletions and amplifications in human genes [37] [38] [39] [40] . This study specifically focused on deletions or duplications in subtelomeric regions of genomic DNA. The probe set utilized—P070 (MRC Holland)—was selected as it can be utilized to analyze deletions and duplications in subtelomeric regions in genomic DNA. As per the manufacturer ( https://www.mrcholland.com/product/P070/1147 ), aberrant copy numbers of subtelomeric regions are a frequent cause of intellectual disabilities, developmental delays, and/or congenital abnormalities that lack distinct syndrome features.
2. Materials and Methods2.1. Cell Lines and Cell Culturing
Cells used were the thymidine kinase heterozygote cell line TK6 and the TP53 mutant cell line WTK1. Both cell lines were cultured and maintained as previously described [41] .
2.2. Irradiation of Cell Lines and Preparation for MLPA Analysis
The two lymphoblastoid cell lines (TK6 and WTK1) were treated with ionizing radiation prepared for MLP analysis as described previously [41] .
2.3. Preparation of Genomic DNA for MLPA Analysis
Total Genomic DNA was prepared as described previously [41] .
2.4. PCR Amplification
Amplification of the ligated probes chosen to study a region known to be associated with congenital abnormalities was carried out by polymerase chain reaction (PCR) using the SALSA MLPA Probemix P070 Subtelomeres Mix 2B. This mix is designed to probe for 41 subtelomeric regions of interest plus an extra probe for the subtelomeric regions on the short q-arm of the 5 acrocentric chromosomes. PCR amplification and fragment analysis was performed as previously described [41] .
2.5. Statistical Analysis
Statistical analysis was carried out as described previously [41] .
3. Results
This study specifically focused on using MLPA to investigate chromosomal copy number imbalances associated with telomere instability.
3.1. MLPA Analysis—TK6 Results
(a)--(b)--Figure 1. The data after 24 (black bars) and 48 (white bars) h incubation for 0 Gr exposure to irradiation for the TK6 cell line as a function of probe signal intensity. This is the control for this cell line. Each set of bars represents different probes in the P070 probe set.
(a)--(b)--Figure 1. The data after 24 (black bars) and 48 (white bars) h incubation for 0 Gr exposure to irradiation for the TK6 cell line as a function of probe signal intensity. This is the control for this cell line. Each set of bars represents different probes in the P070 probe set.
(a)--(b)--Figure 2. The data after 24 (black and gray bars) and 48 (white and hashed bars) h incubation for 2 Gy (black and white bars) and 3 Gy (grey and hashed bars) exposure to irradiation for the TK6 cell line as a function of probe signal intensity. Each set of bars represents different probes in the P070 probe set.
(a)--(b)--Figure 2. The data after 24 (black and gray bars) and 48 (white and hashed bars) h incubation for 2 Gy (black and white bars) and 3 Gy (grey and hashed bars) exposure to irradiation for the TK6 cell line as a function of probe signal intensity. Each set of bars represents different probes in the P070 probe set.
this probe set for 3 Gy irradiation show an increase in percent peak ratio between 24 and 48 h incubation, ranging from 9.9% to 2882.8%. More probes for the 3 Gy irradiation show a decrease as compared to the 2 Gy, with a minimum decrease of 1.9% and a maximum decrease of 93.9%. Interestingly, some of the probes experiencing some of the largest decreases (or increases) in percent peak ratio for the 2 Gy radiation between 24 and 48 h experienced the largest increase (or decrease) for the 3 Gy radiation with the same timepoints. In other words, the 2 Gy change was, for some of the probe sets, significantly different than that observed for the 3 Gy change. For example, VAMP7-8, which was the probe that experienced the largest decrease in percent peak ratio for 2 Gy, experienced the second largest increase in percent peak ratio for 3 Gy. It is unclear what drives this opposing reaction to the 2 and 3 Gy irradiation for certain probes.
Finally, the post-irradiation data indicates copy number imbalances in the TK6 cell line for eleven probes (ATG4B-7, ZNF10-5, PSPC1-1, PARP2-16, NDN-1, GAS8-11, RPH3AL-2, CTDP1-9, CHMP2A-3, UCKL1-7, and IL17RA-4). These eleven probes were present only in the control group; they were not present in either post-irradiation conditions (2 or 3 Gy).
3.2. MLPA Analysis—WTK1 Results
(a)--(b)--Figure 3. The data after 24 (black bars) and 48 (white bars) h incubation for 0 Gy exposure to irradiation for the WTK1 cell line as a function of probe signal intensity. Each set of bars represents different probes in the P070 probe set.
(a)--(b)--Figure 3. The data after 24 (black bars) and 48 (white bars) h incubation for 0 Gy exposure to irradiation for the WTK1 cell line as a function of probe signal intensity. Each set of bars represents different probes in the P070 probe set.
wild-type TK6 cell line. This result has been observed before by our group when analyzing these same two cell lines for radiation-induced instability using alternative probe sets.
Two probe sets—SEKTM1-4 and UCKL1-7—are present in the control results for the TK6 cell line yet are not present in the control results for the WTK1 cell line. One of those (UCKL1-7) also shows a loss in probe enhancement for the TK6 cell line pre- and post-irradiation. The implications of these results are not yet known.
By again comparing the control data with data gathered under similar conditions post-irradiation, changes in the mutant cell line that may have occurred due to ionizing radiation can be identified much as they were for the wild-type cell line. Figure 4 shows the MLPA data for 2 Gy and 3 Gy exposures to irradiation after 24 and 48 h incubation for the WTK1 cell line as a function of probe signal intensity. All but 2 probes (92.0%) in this probe set for 2 Gy irradiation
(a)--(b)--Figure 4. The data after 24 (black and gray bars) and 48 (white and hashed bars) h incubation for 2 Gy (black and white bars) and 3 Gy (grey and hashed bars) exposure to irradiation for the WTK1 cell line as a function of probe signal intensity. Each set of bars represents different probes in the P070 probe set.
(a)--(b)--Figure 4. The data after 24 (black and gray bars) and 48 (white and hashed bars) h incubation for 2 Gy (black and white bars) and 3 Gy (grey and hashed bars) exposure to irradiation for the WTK1 cell line as a function of probe signal intensity. Each set of bars represents different probes in the P070 probe set.
show a decrease in percent peak ratio between 24 and 48 h incubation, ranging from 18.6% to 99.0%. Only two probe sets show an increase (THOC1-21 (134.9%) and HSPA13-2 (40.1%)). No probes in this probe set for 3 Gy irradiation show an increase in percent peak ratio between 24 and 48 h incubation; the decreases range from 76.5% to 87.1%. Finally, the post-irradiation data does indicate copy number imbalances in the WTK1 cell line, which is evident in the loss of probe enhancement for thirteen probes (SH3BP5L-3, ATG4B-7, FBXO25-8, RECQL4-17, DOCK8-24, ZNF10-5, PARP2-16, RPH3AL-2, CTDP1-9, CHMP2A-3, S100B-2, IL17RA-4, and VAMP7-8).
3.3. MLPA Analysis—Comparison of TK6 and WTK1 Results
SECTM1-4 and UCKL1-7 are found in the control for the TK6 cell line but are not found in the WTK1 cell line control. Three probes (PSPC1-1, NDN-1, and
<xref ref-type="bibr" rid="scirp.133707-"></xref>Table 1. Probes described in Section 3.3. Information is taken from genecards.org (accessed 4 Mar. 2024).Table 1. Probes described in Section 3.3. Information is taken from genecards.org (accessed 4 Mar. 2024).
Gene
Gene name
Role or function
Pathology
PSPC1
Paraspeckle component 1
Controls gene expression via an RNA nuclear retention mechanism
Inborne genetic disease
GAS8
Growth arrest-specific protein8
Putative tumor suppressor protein
Cillary dyskinesia
UCKL1
Uridine-cytidine kinase 1-like 1
Catalyzes the phosphorylation of uridine to uridine monophosphate
Inborne genetic disorder
SH3BP5L
SH3 binding domain protein 5-like
Involved in intracellular signal transduction andnegative regulation of kinase activity
Inborne genetic disease
S100B
S100 calcium binding protein B
Regulation of cell cycle progression andcell differentiation
Growth suppressor that facilitates the entry of the cell into cell cycle arrest; also interacts with p53 to inhibit cell growth
Prader-Willi syndrome
FBXO25-8
F-Box protein 25
Substrate-recognition component of the SCF (SKP1-CUL1-F-box protein)-type E3 ubiquitin ligase complex
A chromosomal aberration involving FBXO25 is a cause of X-linked intellectual disability (XLID)
RECQL4-17
RecQ-like helicase
May play a role in the repair of DNA damaged by ultraviolet light or other mutagens; magnesium-dependent ATP-dependent DNA-helicase activity
Recon Progeroid syndrome; hereditary breast ovarian cancer syndrome
DOCK8-24
Dedicator of cytokinesis 8
Activates small GTPase CDC42 by exchanging bound GDP for free GTP;
required for interstitial dendritic cell (DC) migration;
required for CD4(+) T-cell migration; involved in NK cell cytotoxicity
Hyper-Ige syndrome 2, autosomal recessive; combined immuniodeficiency due to Dock 8 deficiency; severe combined immuniodeficiency; autosomal dominant non-syndromic intellectual disability
GAS8-11) that are found in both control and post-irradiated WTK1 cell lines are found in the TK6 cell line control but are not found in the post-irradiation samples. Six probes (SH3BP5L-3, FBXO25-8, RECQL4-17, DOCK8-24, S100B-2, and VAMP7-8) that are found in both control and post-irradiated TK6 cell lines are found in the WTK1 cell line control but are not found in the post-irradiation samples. No probes were located post-irradiation that were not present in the controls for both cell lines. It appears from these results that radiation-induced telomere instability was more prevalent in the mutant cell line than in the wild type, which was to be expected.
4. Discussion
This study was performed in order to understand the effects of ionizing radiation on the copy-number variations of the subtelomeric regions of chromosomes in two closely related cell lines: TK6 (the wild type) and WTK1 (the p53 mutant). The focus was to understand whether there was any connection between aberrant copy numbers and the presence or absence of p53.
WTK1 contains the mutant p53 (M237I) at the thymidine kinase (tk) locus [42] and has been shown to play a major role in maintaining genetic stability. Among other findings, p53 mutants have been shown to have loss of DNA binding function that prevents them from carrying out their regulatory role [43] . Mutant p53 appears able to engage in aberrant interactions with other cellular factors, which typically results in gain-of-function phenotypes [44] [45] [46] .
The application of Multiplex Ligation-Dependent Probe Amplification (MLPA) on TK6 and WTK1 has provided a wealth of information into the impact on chromosomal instability after being subjected to ionizing radiation. Our results show that there are unique alterations of probe enhancement for both TK6 and WTK1 cells. These results showed a number of notable gene deletions that are involved in general gene regulation, signal transduction, tumor suppression, regulation of the cell cycle and posttranslational modifications. The impact of abnormal gene expression, specifically loss of expression, could potentially lead to a unique set of pathologies that have been linked to developmental delay and congenital abnormalities (see for example Table 1 , which lists the 11 unique probes described in Section 3.3 above).
Finally, we have demonstrated that, while WTK1 and TK6 share genes that have been impacted in the same way by the irradiation, each cell line presents its own unique response profile. However, both cell types share changes in important cellular functions that can lead to cancer and disease. Each cell line seems equally impacted in the areas of regulation, signal transduction, tumor suppression, regulation of the cell cycle and posttranslational modifications. These results hold significant importance in the study of the effects of ionizing radiation in human cells, and how different cell lines can be affected in significantly different ways depending on the presence of wild type p53.
Acknowledgements
Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103449. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
References De Benedetti, V., Bennett, W.P., Greenblatt, M.S. and Harris, C.C. (1996) p53 Tumor Suppressor Gene: Implications for Iatrogenic Cancer and Cancer Therapy. Medical and Pediatric Oncology, 27, 2-11.>https://doi.org/10.1002/(SICI)1096-911X(1996)27:1+<2::AID-MPO2>3.0.CO;2-Z Greenblatt, M.S., Bennett, W.P., Hollstein, M. and Harris, C.C. (1994) Mutations in the p53 Tumor Suppressor Gene: Clues to Cancer Etiology and Molecular Pathogenesis. Cancer Research, 54, 4855-4878. Olivier, M., Eeles, R., Hollstein, M., Khan, M.A., Harris, C.C. and Hainaut, P. (2002) The IARC TP53 Database: New Online Mutation Analysis and Recommendations to Users. Human Mutation, 19, 607-614. >https://doi.org/10.1002/humu.10081 Duffy, M.J., Synnott, N.C., O’Grady, S. and Crown, J. (2022) Targeting p53 for the Treatment of Cancer. Seminars in Cancer Biology, 79, 58-67. >https://doi.org/10.1016/j.semcancer.2020.07.005 Williams, A.B. and Schumacher, B. (2016) p53 in the DNA-Damage-Repair Process. Cold Spring Harbor Perspectives in Medicine, 6, a026070.>https://doi.org/10.1101/cshperspect.a026070 Sancar, A., Lindsey-Boltz, L.A., Unsal-Kaçmaz, K. and Linn, S. (2004) Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints. Annual Review of Biochemistry, 73, 39-85. >https://doi.org/10.1146/annurev.biochem.73.011303.073723 Albrechtsen, N., Dornreiter, I., Grosse, F., Kim, E., Wiesmüller, L. and Deppert, W. (1999) Maintenance of Genomic Integrity by p53: Complementary Roles for Activated and Non-Activated p53. Oncogene, 18, 7706-7717. >https://doi.org/10.1038/sj.onc.1202952 Bertrand, P., Rouillard, D., Boulet, A., Levalois, C., Soussi, T. and Lopez, B.S. (1997) Increase of Spontaneous Intrachromosomal Homologous Recombination in Mammalian Cells Expressing a Mutant p53 Protein. Oncogene, 14, 1117-1122. >https://doi.org/10.1038/sj.onc.1200931 Bill, C.A., Yu, Y., Miselis, N.R., Little, J.B. and Nickoloff, J.A. (1997) A Role for p53 in DNA end Rejoining by Human Cell Extracts. Mutation Research/DNA Repair, 385, 21-29. >https://doi.org/10.1016/S0921-8777(97)00040-2 Blander, G., Kipnis, J., Leal, J.F., Yu, C.E., Schellenberg, G.D. and Oren, M. (1999) Physical and Functional Interaction between p53 and the Werner’s Syndrome Protein. Journal of Biological Chemistry, 274, 29463-29469. >https://doi.org/10.1074/jbc.274.41.29463 Dudenhöffer, C., Kurth, M., Janus, F., Deppert, W. and Wiesmüller, L. (1999) Dissociation of the Recombination Control and the Sequence-Specific Transactivation Function of p53. Oncogene, 18, 5773-5784. >https://doi.org/10.1038/sj.onc.1202964 Mathonnet, G., Leger, C., Desnoyers, J., Drouin, R., Therrien, J.-P. and Drobetsky, E.A. (2003) UV Wavelength-Dependent Regulation of Transcription-Coupled Nucleotide Excision Repair in p53-Deficient Human Cells. Proceedings of the National Academy of Sciences of the United States of America, 100, 7219-7224. >https://doi.org/10.1073/pnas.1232161100 Mekeel, K.L., Tang, W., Kachnic, L.A., Luo, C.M., DeFrank, J.S. and Powell, S.N. (1997) Inactivation of p53 Results in High Rates of Homologous Recombination. Oncogene, 14, 1847-1857. >https://doi.org/10.1038/sj.onc.1201143 Romanova, L.Y., Willers, H., Blagosklonny, M.V. and Powell, S.N. (2004) The Interaction of p53 with Replication Protein a Mediates Suppression of Homologous Recombination. Oncogene, 23, 9025-9033. >https://doi.org/10.1038/sj.onc.1207982 Saintigny, Y., Rouillard, D., Chaput, B., Soussi, T. and Lopez, B.S. (1999) Mutant p53 Proteins Stimulate Spontaneous and Radiation-Induced Intrachromosomal Homologous Recombination Independently of the Alteration of the Transactivation Activity and of the G1 Checkpoint. Oncogene, 18, 3553-3563. >https://doi.org/10.1038/sj.onc.1202941 Stürzbecher, H.W., Donzelmann, B., Henning, W., Knippschild, U. and Buchhop, S. (1996) p53 Is Linked Directly to Homologous Recombination Processes via RAD51/RecA Protein Interaction. The EMBO Journal, 15, 1992-2002. >https://doi.org/10.1002/j.1460-2075.1996.tb00550.x Tang, W., Willers, H. and Powell, S.N. (1999) p53 Directly Enhances Rejoining of DNA Double-Strand Breaks with Cohesive Ends in Gamma-Irradiated Mouse Fibroblasts. Cancer Research, 59, 2562-2565. Willers, H., et al. (2000) Dissociation of p53-Mediated Suppression of Homologous Recombination from G1/S Cell Cycle Checkpoint Control. Oncogene, 19, 632-639.>https://doi.org/10.1038/sj.onc.1203142 Zurer, I., et al. (2004) The Role of p53 in Base Excision Repair Following Genotoxic Stress. Carcinogenesis, 25, 11-19. >https://doi.org/10.1093/carcin/bgg186 Lengauer, C., Kinzler, K.W. and Vogelstein, B. (1997) Genetic Instability in Colorectal Cancers. Nature, 386, 623-627. >https://doi.org/10.1038/386623a0 Lengauer, C., Kinzler, K.W. and Vogelstein, B. (1998) Genetic Instabilities in Human Cancers. Nature, 396, 643-649. >https://doi.org/10.1038/25292 Negrini, S., Gorgoulis, V.G. and Halazonetis, T.D. (2010) Genomic Instability—An Evolving Hallmark of Cancer. Nature Reviews Molecular Cell Biology, 11, 220-228.>https://doi.org/10.1038/nrm2858 Matsuno, Y., et al. (2019) Replication Stress Triggers Microsatellite Destabilization and Hypermutation Leading to Clonal Expansion in vitro. Nature Communications, 10, Article No. 3925. >https://doi.org/10.1038/s41467-019-11760-2 Matsuno, Y., et al. (2021) Replication-Stress-Associated DSBs Induced by Ionizing Radiation Risk Genomic Destabilization and Associated Clonal Evolution. iScience, 24, Article 102313. >https://doi.org/10.1016/j.isci.2021.102313 Rajagopalan, H. and Lengauer, C. (2004) Aneuploidy and Cancer. Nature, 432, 338-341. >https://doi.org/10.1038/nature03099 Korbel, J.O. and Campbell, P.J. (2013) Criteria for Inference of Chromothripsis in Cancer Genomes. Cell, 152, 1226-1236. >https://doi.org/10.1016/j.cell.2013.02.023 Bishop, A.J.R. and Schiestl, R.H. (2002) Homologous Recombination and Its Role in Carcinogenesis. Journal of Biomedicine&Biotechnology, 2, 75-85. >https://doi.org/10.1155/S1110724302204052 Hoeijmakers, J.H.J. (2009) DNA Damage, Aging, and Cancer. New England Journal of Medicine, 361, 1475-1485. >https://doi.org/10.1056/NEJMra0804615 Ciccia, A. and Elledge, S.J. (2010) The DNA Damage Response: Making It Safe to Play with Knives. Molecular Cell, 40, 179-204. >https://doi.org/10.1016/j.molcel.2010.09.019 Sebastian, R. and Raghavan, S.C. (2016) Induction of DNA Damage and Erroneous Repair Can Explain Genomic Instability Caused by Endosulfan. Carcinogenesis, 37, 929-940. >https://doi.org/10.1093/carcin/bgw081 Levy, J.A., Virolainen, M. and Defendi, V. (1968) Human Lymphoblastoid Lines from Lymph Node and Spleen. Cancer, 22, 517-524. >https://doi.org/10.1002/1097-0142(196809)22:3<517::AID-CNCR2820220305>3.0.CO;2-A Chuang, Y.Y., Chen, Q., Brown, J.P., Sedivy, J.M. and Liber, H.L. (1999) Radiation-Induced Mutations at the Autosomal Thymidine Kinase Locus Are Not Elevated in p53-Null Cells. Cancer Research, 59, 3073-3076. Little, J.B., Nagasawa, H., Keng, P.C., Yu, Y. and Li, C.Y. (1995) Absence of Radiation-Induced G 1 Arrest in Two Closely Related Human Lymphoblast Cell Lines that Differ in p53 Status. Journal of Biological Chemistry, 270, 11033-11036.>https://doi.org/10.1074/jbc.270.19.11033 Xia, F., et al. (1995) Altered p53 Status Correlates with Differences in Sensitivity to Radiation-Induced Mutation and Apoptosis in Two Closely Related Human Lymphoblast Lines. Cancer Research, 55, 12-15. Peng, Y., Zhang, Q., Nagasawa, H., Okayasu, R., Liber, H.L. and Bedford, J.S. (2002) Silencing Expression of the Catalytic Subunit of DNA-Dependent Protein Kinase by Small Interfering RNA Sensitizes Human Cells for Radiation-Induced Chromosome Damage, Cell Killing, and Mutation. Cancer Research, 62, 6400-6404. Amundson, S.A., Xia, F., Wolfson, K. and Liber, H.L. (1993) Different Cytotoxic and Mutagenic Responses Induced by X-Rays in Two Human Lymphoblastoid Cell Lines Derived from a Single Donor. Mutation Research, 286, 233-241.>https://doi.org/10.1016/0027-5107(93)90188-L Schouten, J.P., McElgunn, C.J., Waaijer, R., Zwijnenburg, D., Diepvens, F. and Pals, G. (2002) Relative Quantification of 40 Nucleic Acid Sequences by Multiplex Ligation-Dependent Probe Amplification. Nucleic Acids Research, 30, e57. >https://doi.org/10.1093/nar/gnf056 Kjaergaard, S., et al. (2010) Diagnostic Yield by Supplementing Prenatal Metaphase Karyotyping with MLPA for Microdeletion Syndromes and Subtelomere Imbalances. Prenatal Diagnosis, 30, 995-999. >https://doi.org/10.1002/pd.2604 Kirchhoff, M., Bisgaard, A.-M., Bryndorf, T. and Gerdes, T. (2007) MLPA Analysis for a Panel of Syndromes with Mental Retardation Reveals Imbalances in 5.8% of Patients with Mental Retardation and Dysmorphic Features, Including Duplications of the Sotos Syndrome and Williams-Beuren Syndrome Regions. European Journal of Medical Genetics, 50, 33-42. >https://doi.org/10.1016/j.ejmg.2006.10.002 Jehee, F.S., et al. (2011) Using a Combination of MLPA Kits to Detect Chromosomal Imbalances in Patients with Multiple Congenital Anomalies and Mental Retardation Is a Valuable Choice for Developing Countries. European Journal of Medical Genetics, 54, e425-e432. >https://doi.org/10.1016/j.ejmg.2011.03.007 Chant, A., Chaudary, A. and Kraemer-Chant, C.M. (2023) A Study of Radiation-Induced Instability for the Gene Locus Associated with Intellectual Disorders or Developmental Delays. Advances in Biological Chemistry, 13, 128-142. >https://doi.org/10.4236/abc.2023.134010 Zhang, Q., Liu, Y., Zhou, J., Chen, W., Zhang, Y. and Liber, H.L. (2007) Wild-Type p53 Reduces Radiation Hypermutability in p53-Mutated Human Lymphoblast Cells. Mutagenesis, 22, 329-334. >https://doi.org/10.1093/mutage/gem021 Kato, S., Han, S.-Y., Liu, W., et al. (2003) Understanding the Function-Structure and Function-Mutation Relationships of p53 Tumor Suppressor Protein by High-Resolution Missense Mutation Analysis. Proceedings of the National Academy of Sciences of the United States of America, 100, 8424-8429. >https://doi.org/10.1073/pnas.1431692100 Haupt, Y., Maya, R., Kazaz, A. and Oren, M. (1997) Mdm2 Promotes the Rapid Degradation of p53. Nature, 387, 296-299. >https://doi.org/10.1038/387296a0 Midgley, C.A. and Lane, D.P. (1997) p53 Protein Stability in Tumour Cells Is Not Determined by Mutation but Is Dependent on Mdm2 Binding. Oncogene, 15, 1179-1189. >https://doi.org/10.1038/sj.onc.1201459 Wiech, M., Olszewski, M.B., Tracz-Gaszewska, Z., Wawrzynow, B., Zylicz, M. and Zylicz, A. (2012) Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2. PLOS ONE, 7, e51426. >https://doi.org/10.1371/journal.pone.0051426