The genetic sequences prone to copy number variation and single nucleotide polymorphism are linked to the repair of the poisoned DNA topoisomerase II

TOP2-poisoning bioflavonoids and pesticides are linked to the copy number variation-related autism and chromosome translocation-related leukemia. On the other hand, the poisoned DNA topoisomerase II (TOP2) can lead to chromosome aberration. However, except a limited number of genes such as the MLL fusion, other poisoned TOP2-targeted genes, as well as their relationships with any specific diseases, are not defined. We applied the γH2A.X antibodies to genome-widely immunoprecipitate the chromatins that were associated with the repair of the TOP2 poison etoposide-induced DNA double strand breaks. We identified many transcriptable protein- and nonprotein-coding DNA sequences that are the candidates of or associated with many gene copy number variation- and/or single nucleotide polymorphism-associated diseases, including but not limited to microdeletion and microduplication syndromes (which are phenotypically presented as developmental, autistic, neurological, psychiatric, diabetic, autoimmune, and neoplastic diseases among many others) as well as stature, obesity, metabolic syndrome, hypertension, coronary artery disease, ischemic stroke, aortic aneurysm and dissection, leukemia, cancer, osteoporosis, Alzheimer disease, Parkinson disease, and Huntington disease. Our data raise the possibility that the poisoned TOP2 might be linked to the specific genetic alterations contributing to these diseases, additional to the known copy number variation-related autism and chromosome translocation-related leukemia. According to our and others’ data, we propose a model that may interpret the features, such as mosaicism, polygenic traits and pleiotropy, of these diseases. Author Summary For the past several decades, the morbidity rate of many diseases, including autism, mental disorders, cancer, cardiovascular diseases, diabetes, and senile dementia, has world-widely been rising. Analysis of the genome of the patients and their family members has identified the genes, whose alterations, so called copy number variation (CNV) and single nucleotide polymorphism (SNP), contribute to the diseases. Moreover, the CNVs and SNPs are de novo, that is, they have occurred only in the recent generations. Epidemiologically, this indicates that for the past several decades, there have existed some unknown world-wide etiologies to which human beings are exposed. If the etiologies are identified, avoiding human’s exposure may reduce the morbidity of the diseases. We have found that the repair of the poisoned topoisomerase II involves many genes that contribute to the aforementioned diseases. As the topoisomerase II is known to be located at the genomic sites where the disease-associated CNVs occur, as the poisoned topoisomerase II is susceptible to chromosome aberration, and as the topoisomerase II poisons, such as dietary bioflavonoids, are widely distributed in the environment, our data raise the yet-to-be-confirmed possibility that the environmental topoisomerase II poisons might etiologically contribute to many CNV-associated diseases.

γH2A.X is a DNA repair-involved, post-translationally modified form of histone variant 7 110 capture Jurkat cell's genetic sequences that were associated with the repair of the 111 etoposide-induced DSBs. We determined to precipitate the chromatins associated with the 112 DNA repair rather than those only associated with the TOP2 cleavable complexes, for the 113 reason that the poisoned TOP2-induced chromosome aberration often results from the DNA 114 repair that involves DNA sequences far from the TOP2 cleavable complexes [8,29]. As 115 expected, we captured many genetic sequences that are known to contribute to leukemia and 116 autism. However, unexpectedly, a large number of genetic sequences that are related to many 117 other diseases, were also identified. The possible roles of the poisoned TOP2 in the 118 pathogenesis of these diseases are discussed and a hypothetical model is proposed to interpret 119 the complexed features of these diseases.

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Poisoned TOP2 has long been known to cause chromosome aberration. However, the vast 123 majority of the involved genes are undefined. Therefore, the only purpose of the current study 124 was to identify the genes that were associated with the repair of the poisoned TOP2 and to 125 find if these genes were related to any specific diseases. It was not our intention to study the 126 chemistry, biochemistry or biology of the poisoned TOP2 or its targeted genes, because a 127 large number of pioneering investigators have done many explicit studies. As a result, we 128 only present the data that are related to the genetic sequences and the associated diseases. We 129 do not show those sophisticated and exquisite figures or data that are generated by the 130 ChIP-seq computer software, the usual way that most ChIP-seq studies do. We would not like 8 131 to distract the readers from the main theme of the current study.

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ChIP captured a number of transcriptable protein-and nonprotein-coding 134 genetic sequences. 135 We treated the human leukemia Jurkat cells continuously with 100μM etoposide, waited for 136 5.5 hours and then immunoprecipitated the chromatins with the anti-γH2A.X antibodies. We 137 also immunoprecipitated the chromatins of the cells that were mock-treated with DMSO, 138 which was used to prepare the stock solution of etoposide, to control any endogenous DSBs 139 such as those described by Seo et al [5]. We designed our experiments in such ways for the 140 following reasons. 141 1) Unlike other TOP2 poisons such as the bioflavonoids that have multiple cellular effects, 142 including antioxidant, pro-oxidant, anti-mutagenic, mutagenic, carcinogenic, and 143 anti-carcinogenic activities, which have not been thoroughly characterized at the molecular 144 level [13], etoposide is well known to poison TOP2 and hence has been used by most 145 investigators to study the poisoned TOP2 [6-8]. We utilized etoposide just as a 146 TOP2-poisoning tool because our only purpose was to identify the genetic sequences 147 associated with the poisoned TOP2 and its repair. We did not intend to characterize any 148 specific TOP2 poisons. Therefore, the principle of the conclusion of the current study should 149 be applicable to other TOP2 poisons such the TOP2-poisoing bioflavonoids, although the 150 details are not likely to be exactly the same because of the disparate efficacy, potency and 151 DNA sequence selectivity among various TOP2 poisons. 2) Early studies have immunoprecipitated the TOP2 cleavable complex-or DNA 153 damage-associated chromatins [31,32]. We instead immunoprecipitated the γH2A.  We only captured the genes related to 66.4%, rather than all, of the microdeletion and 241 microduplication syndromes. Genes related to the remaining syndromes were not captured.

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Among many possible reasons that may be attributed to, one is that many deletion-and 243 duplication-susceptible DNA segments might be epigenetically silenced due to the Jurkat 244 cell-specific chromatin modification. These DNA segments are not accessible to TOP2. 245 However, investigating the true reason is beyond the purpose and scope of the current study.

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Much more work is required to find the true attributing reasons which could be related to the 247 ChIP-seq method, data analysis, statistics, cell biology, etc. 248 We did not either capture all the genes within the depleted and duplicated genomic DNA The poisoned TOP2-induced, γH2A.X-associated DNA was closely linked to many 256 genetic sequences related to autistic spectrum disorders. 257 To weigh how much the poisoned TOP2 would contribute to any specific types of the (26.9%) of them (Supporting Information S1 Table). Therefore, the poisoned TOP2-induced, 267 γH2A.X-associated DNA was closely linked to ASD.

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The poisoned TOP2-induced, γH2A.X-associated DNA was closely linked to 270 many genetic sequences related to leukemia and other types of cancers.  Table). 277 We next looked at whether the poisoned TOP2 might also contribute to other types of cancers.  However, except a limited number of genes such as the MLL gene, most involved genes are 316 not characterized, neither are their relationships with any specific diseases. Therefore, the one 317 and only purpose of the current study is to identify the genetic sequences associated with the 318 poisoned TOP2 and, particularly, its repair. To achieve our goal, we genome-widely 319 immunoprecipitated the genomic segments that were associated with the γH2A.X induced by 320 the TOP2 poison etoposide. As the signal of any γH2A.X-associated gene among a 321 heterogeneous cell population is likely to be low, to capture as much amount of the signal as Our repeated ChIP experiments reproducibly captured 3707 transcriptable protein-and 326 nonprotein-coding sequences. These sequences are likely to be TOP2-specific because they 327 include 75.0% of the known topoisomerase-regulated genes essential for neurodevelopment 328 (Supporting Information S3 Table) [46].

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The TOP2-poisoning anticancer drugs, bioflavonoids and pesticides have been linked to 330 leukemia and autism [7,13,116-18]. As expected, we did capture many genetic sequences 331 related to leukemia and ASD (Table 2). However, unexpectedly, we pulled down many 332 genetic sequences that belong with 66.4% of the CNV-related microdeletion and 333 microduplication syndromes (Table 1). Moreover, we also identified a large number, ranging 334 from 25% to 50%, of the genetic sequences whose CNVs and/or SNPs were associated with 335 the other 16 common diseases or conditions that we investigated ( Lastly, we hope that any researchers who are interested and are also funded can further the 534 investigation. Afterall, the development disorders, particularly autism, affect many children 535 across the world, particularly in the industrialized regions where the processed food is 536 routinely consumed.

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In conclusion, we have found that many genetic sequences associated with the repair of the 538 poisoned TOP2, are the candidates for not only leukemia and ASD but also a variety of other 539 CNV-and SNP-related diseases. Our results raise the possibility that the TOP2-poisoining 540 chemicals such as the dietary bioflavonoids might be involved in the genetic development of 541 many diseases. Additionally, according to our and, particularly, others' data, we propose a 542 yet-to-be-confirmed hypothetical model that seems able to interpret many features of the