Illegitimate and Repeated Genomic Integration of Cell-Free Chromatin in the Aetiology of Somatic Mosaicism, Ageing, Chronic Diseases and Cancer

Round 1

Reviewer 1 Report

As the reviewer requested, authors corrected and added the definition of CfCh in the text.

However, I still wonder if CfCh truthfully integrate into genome as it is. The reviewer still wonders if Figure 5 and Figure 8 are explained well regarding methods. Especially in Figure 5, I wonder if almost all of these signals are outside nucleus. Moreover, the reviewer wonders if these signals are the true reflection of genomic integration of CfCh, but CfDNA.

Author Response

Comments and Suggestions for Authors (Reviewer 1 round 3)

As the reviewer requested, authors corrected and added the definition of CfCh in the text.

1) However, I still wonder if CfCh truthfully integrate into genome as it is. 2) The reviewer still wonders if Figure 5 and Figure 8 are explained well regarding methods. Especially in Figure 5, 3) I wonder if almost all of these signals are outside nucleus. 4) Moreover, the reviewer wonders if these signals are the true reflection of genomic integration of CfCh, but CfDNA

We thank the reviewer for raising these critical questions. Our response to them has helped to improve the paper greatly.

Reviewer’s query No. 1 and 4 are inter-related and have been addressed together on page 5, lines 177-231. Our response also includes a new figure (Figure No. 3).   

Query No. 2

The methodological details of Figures 6 (old figure 5) and 9 (old figure 8) have now been incorporated in their respective legends by referring to original source articles. The figure numbers have changed because of inclusion of a new figure (No. 3).

Query No. 3

With respect to the reviewer’s query as to whether the fluorescent cfCh signals seen in figure 5 are outside the nucleus, we have incorporated a clarification on page 7, lines 279-280.

 

Reviewer 2 Report

Authors have improved significantly the review in the light of recent and classical scientific literature dedicated to chromosomal mosaicism and heterogeneity. Still, I recommend an additional round of improving the manuscript’s text. Some phrases are still poorly comprehensible. Some phrases require to be shortened or to be separated into several phrases.

Author Response

Comments and Suggestions for Authors (Reviewer 2 round 3)

Authors have improved significantly the review in the light of recent and classical scientific literature dedicated to chromosomal mosaicism and heterogeneity. Still, I recommend an additional round of improving the manuscript’s text. Some phrases are still poorly comprehensible. Some phrases require to be shortened or to be separated into several phrases.

We appreciate the suggestion of the reviewer. We have gone through the manuscript closely and made several linguistic changes and other modifications to improve the paper. 

Round 2

Reviewer 1 Report

Now that paper is revised properly, the reviewer considers that this paper is worth publishing.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Golantra et al. discussed the recent literature describing the potential genomic integration of cell-free chromatin into cells. The potential impact of the outcome authors claim is the somatic mosaicism.

This reviewer sees the problem in the argument because there is no evidence that those events happen in cells with proliferating capacity, namely stem cells. Somatic mosaicism refers to the occurrence of two genetically distinct populations of cells within an individual, derived from a postzygotic mutation. Thus, proliferation after acquiring mutations are essential. It is an interesting idea, but this reviewer would say that the contribution of cell-free chromatin to somatic mosaicism is challenging to establish.

Second, authors proposed the model in which chromosomal integration of cell-free chromatin causes inflammation in host cells. The model is based on the article from the author's group. In this article, authors injected cell-free chromatin or cells from humans into mice. For that cell-free chromatin to get integrated into the genome of host cells, cell-free chromatin needs to get through the cytoplasmic part of cells before getting into the nucleus. There is growing evidence showing that cytoplasmic DNA causes inflammatory responses in cells (for instance, PMID 28759889 and 28738408). Thus, if any, the inflammation in cells is caused by the cytoplasmic DNA, rather than by the DNA damage during genomic integration.

These are the major criticisms from this reviewer who raises a concern that this manuscript relies heavily on the authors’ unique opinion, rather than a review article of the field.

Author Response

Reviewer 1

Golantra et al. discussed the recent literature describing the potential genomic integration of cell-free chromatin into cells. The potential impact of the outcome authors claim is the somatic mosaicism.

This reviewer sees the problem in the argument because there is no evidence that those events happen in cells with proliferating capacity, namely stem cells. Somatic mosaicism refers to the occurrence of two genetically distinct populations of cells within an individual, derived from a postzygotic mutation. Thus, proliferation after acquiring mutations are essential. It is an interesting idea, but this reviewer would say that the contribution of cell-free chromatin to somatic mosaicism is challenging to establish.

Much of the work on somatic mosaicism has been conducted on adult brain cells which do not divide (1-7). Therefore, cellular proliferation is not essential for development in mosaicism, although this is true for haematopoietic stem cells (8, 9). Nonetheless, the reviewer has raised a pertinent question which needs to be addressed. This has now been incorporated (page 2, lines 82-85)

Second, authors proposed the model in which chromosomal integration of cell-free chromatin causes inflammation in host cells. The model is based on the article from the author's group. In this article, authors injected cell-free chromatin or cells from humans into mice. For that cell-free chromatin to get integrated into the genome of host cells, cell-free chromatin needs to get through the cytoplasmic part of cells before getting into the nucleus. There is growing evidence showing that cytoplasmic DNA causes inflammatory responses in cells (for instance, PMID 28759889 and 28738408). Thus, if any, the inflammation in cells is caused by the cytoplasmic DNA, rather than by the DNA damage during genomic integration.

We thank the reviewer for raising this important issue. It is certainly possible that internalized cfCh while in the cytoplasm may activate the cGAS-STING pathway to induce inflammation. This possibility has now been discussed in the text (page 12, lines 442-451).

These are the major criticisms from this reviewer who raises a concern that this manuscript relies heavily on the authors’ unique opinion, rather than a review article of the field.

Our paper is not a routine “Review” article, but rather is one that proposes a new theory. To that extent, it should be considered as a “Perspective” article.  Perhaps the editor would consider publishing this article as such. After all, the aim of the Special Issue of Genes states; “The aim of this Special Issue is to bring together a set of reviews and research articles focusing on significant discoveries, proofs of concept of new theories or relevant observations in genome instability and its related human diseases”.

References

1.     Heidenreich, E.; Novotny, R.; Kneidinger, B.; Holzmann, V.; Wintersberger, U. Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells. EMBO J. 2003, 22, 2274-2283.

2.     Cai, X.; Evrony, G.D.; Lehmann, H.S.; Elhosary, P.C.; Mehta, B.K.; Poduri, A.; Walsh, C.A. Single-Cell, Genome-wide Sequencing Identifies Clonal Somatic Copy-Number Variation in the Human Brain. Cell Rep. 2014, 8, 1280-9.

3.     Lodato, M.A.; Woodworth, M.B.; Lee, S.; Evrony, G.D.; Mehta, B.K.; Karger, A.; Lee, S.; Chittenden, T.W.; D’Gama, A.M.; Cai, X.; et al. Somatic mutation in single human neurons tracks developmental and transcriptional history. Science. 2015, 350, 94-98.

4.     Evrony, G.D.; Lee, E.; Park, P.J.; Walsh, C.A. Resolving rates of mutation in the brain using single-neuron genomics. Elife. 2016, 5, e12966.

5.     Lee, M.-H.; Siddoway, B.; Kaeser, G.E.; Segota, I.; Rivera, R.; Romanow, W.J.; Liu, C.S.; Park, C.; Kennedy, G.; Long, T.; et al. Somatic APP gene recombination in Alzheimer’s disease and normal neurons. Nature 2018, 563, 639-645.

6.     Richardson, S.R.; Morell, S.; Faulkner, G.J. L1 Retrotransposons and Somatic Mosaicism in the Brain. Annu. Rev. Genet. 2014, 48, 1-27.

7.     Upton, K.R.; Gerhardt, D.J.; Jesuadian, J.S.; Richardson, S.R.; Sánchez-Luque, F.J.; Bodea, G.O.; Ewing, A.D.; Salvador-Palomeque, C.; Van Der Knaap, M.S.; Brennan, P.M.; et al. Ubiquitous L1 mosaicism in hippocampal neurons. Cell. 2015, 161, 228-239.

8.     Jaiswal, S. et al. Age-related clonal hematopoiesis associated with adverse outcomes. N. Engl. J. Med. 2014,  371, 2488–2498.

9.     Jacobs, K. B. et al. Detectable clonal mosaicism and its relationship to aging and cancer. Nat. Genet. 2012, 44, 651–658.

Reviewer 2 Report

Authors discussed an interesting topic which was cell-free chromatin related. The topic is interesting and the authors introduced a lot of details on the origin, releasing, uptake, integration, and effects of cell-free chromatin. I have only one suggestion as follows:

I suggest authors to focus on the effect of cfCh and the relationship among cfCh, somatic mosaicism, ageing, cancer, and diseases since the title is so. I accordingly recommend authors to rewrite the section 2 to section 7 as a brief introduction because the related studies had been summarized by several reviews already.

Author Response

Reviewer 2

I suggest authors to focus on the effect of cfCh and the relationship among cfCh, somatic mosaicism, ageing, cancer, and diseases since the title is so. I accordingly recommend authors to rewrite the section 2 to section 7 as a brief introduction because the related studies had been summarized by several reviews already.

We accept the reviewer’s suggestion. We have now merged and much abridged sections 2-7 which has resulted in the deletion of nearly 800 words.

Reviewer 3 Report

Mittra et al. have presented a review that is dedicated to illegitimate and repeated genomic integration of cell free chromatin in the etiology of somatic mosaicism, ageing, chronic diseases and cancer. This is rather provocative and interesting review. However, there are serious omissions, which hinder the understanding and, thereby, diminish the scientific merit of this contribution to biology of chromosomal heterogeneity and human diseases. The main problem of the submission is that it completely falls apart from the main topic of the section — Chromosomal Heterogeneity and Human Diseases. Chromosomal heterogeneity and mosaicism are not properly addressed (i.e. chromosomal mosaicism is left aside). The problem persists through the whole text. Therefore, extended editing is required. For instance, abstract does not have even a word chromosome/chromosomal leading to the lack of correspondence to the topic. Numerous basic and timely references should be addressed to solve this problem. Here, some of points with references missed from authors’ scope are given.

The second paragraph lacks views on basic aspects of somatic mosaicism and chromosomal mosaicism:

Campbell, I.M.; Shaw, C.A.; Stankiewicz P.; Lupski, J.R. Somatic mosaicism, implications for disease and transmission genetics. Trends Genet. 2015, 31, 382-392.

Heng, H.H. Missing heritability and stochastic genome alterations. Nat Rev Genet. 2010, 11, 813.

Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Somatic genome variations in health and disease. Curr. Genomics. 2010, 11, 387-396.

Ye, C.J.; Regan, S.; Liu, G.; Alemara, S.; Heng, H.H. Understanding aneuploidy in cancer through the lens of system inheritance, fuzzy inheritance and emergence of new genome systems. Mol Cytogenet. 2018, 11, 31.

 

Brain-specific chromosomal changes were also left aside. Authors can extend their ideas towards a possibility of cell-free chromatin being a biomarker for brain-specific chromosomal mosaicism (since it cannot be uncovered in living individuals per se). References:

Arendt, T. Cell cycle activation and aneuploid neurons in Alzheimer's disease. Molecular neurobiology. 2012, 46, 125-135.

Arendt, T.; Brückner, M.K.; Mosch, B.; Lösche, A. Selective cell death of hyperploid neurons in Alzheimer's disease. Am J Pathol. 2010, 177, 15-20.

Arendt, T.; Brückner, M.K.; Lösche, A. Regional mosaic genomic heterogeneity in the elderly and in Alzheimer's disease as a correlate of neuronal vulnerability. Acta Neuropathol. 2015, 130, 501-510.

Arendt, T.; Stieler, J.; Ueberham, U. Is sporadic Alzheimer's disease a developmental disorder? J Neurochem. 2017, 143, 396-408.

Bushman, D.M.; Chun, J. The genomically mosaic brain: aneuploidy and more in neural diversity and disease. Semin Cell Dev Biol. 2013, 24, 357-369.

Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Chromosomal variation in mammalian neuronal cells, known facts and attractive hypotheses. Int Rev Cytol. 2006, 249, 143-191.

Iourov, I.Y.; Vorsanova, S.G.; Liehr, T.; Yurov, Y.B. Aneuploidy in the normal, Alzheimer's disease and ataxia-telangiectasia brain: differential expression and pathological meaning. Neurobiol Dis. 2009, 34, 212-220.

Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Somatic cell genomics of brain disorders: a new opportunity to clarify genetic-environmental interactions. Cytogenet Genome Res. 2013, 139, 181-188.

Potter, H.; Granic, A.; Caneus, J. Role of trisomy 21 mosaicism in sporadic and familial Alzheimer's disease. Curr Alzheimer Res. 2016, 13, 7-17.

Rohrback, S.; Siddoway, B.; Liu, C.S.; Chun, J. Genomic mosaicism in the developing and adult brain. Dev Neurobiol. 2018, 78, 1026-1048.

Yurov, Y.B.; Vorsanova, S.G.; Iourov, I.Y. The DNA replication stress hypothesis of Alzheimer's disease. ScientificWorldJournal. 2011, 11, 2602-2612.

 

Early ontogenetic chromosomal heterogeneity (i.e. changes of genomes between fetal/embryonic cells during prenatal development) is also needed to be highlighted:

Vanneste, E.; Voet, T.; Le Caignec, C.; Ampe, M.; Konings, P.; Melotte, C.; Debrock, S.; Amyere, M.; Vikkula, M.; Schuit, F.; Fryns, J.P.; Verbeke, G.; D'Hooghe, T.; Moreau, Y.; Vermeesch, J.R. Chromosome instability is common in human cleavage-stage embryos. Nat Med. 2009, 15, 577-583.

Yurov, Y.B.; Iourov, I.Y.; Vorsanova, S.G.; Liehr, T.; Kolotii, A.D.; Kutsev, S.I.; Pellestor, F.; Beresheva, A.K.; Demidova, I.A.; Kravets, V.S.; Monakhov, V.V.; Soloviev, I.V. Aneuploidy and confined chromosomal mosaicism in the developing human brain. PLoS One. 2007, 2, e558.

Robberecht, C.; Vanneste, E.; Pexsters, A.; D'Hooghe, T.; Voet, T.; Vermeesch, J.R. Somatic genomic variations in early human prenatal development. Curr Genomics. 2010, 11, 397-401.

Yurov, Y.B.; Vorsanova, S.G.; Iourov I.Y. Ontogenetic variation of the human genome. Curr Genomics. 2010, 11, 420-425.

English is required to be seriously checked. Additionally, terminology should be appropriate (e.g. triple pathologies in the abstract are erroneous term).

Reviewer 4 Report

This article reviews on cell-free DNA found in nucleosome as DNA short fragment, which could influence human diseases such as cancer, aging and cell inflamation via integration of them to genome as somatic mosaicism. This article is well written and cites many recent articles and well review current topics.

However, I might be misundersood, but the the definition of the terminology "cell-free chromatin, cfCh" is not explained enogh to discriminate well known terminology "cell-free DNA".

Unless the authors explain the difference between the well known "cell-free DNA" and "cell free chromatin", this article could be misleading to wide range of readers. Is the short fragmented DNA in nucleosomal fragment all cfChs, not cell-free DNA?

The authors showed "cell free Ch" by labelling histone protein and showed they influence DNA DSB repair. Is this only caused by cell-free Ch and not by cell-free DNA?

Round 2

Reviewer 1 Report

Much of the work on somatic mosaicism has been conducted on adult brain cells which do not divide (1-7). Therefore, cellular proliferation is not essential for development in mosaicism, although this is true for haematopoietic stem cells (8, 9). 

The somatic mosaicisms we see in adult brain cells are developed during development when cells are dividing.

I do not see the connection between the mechanisms authors are proposing and somatic mosaicism.

Reviewer 2 Report

I do not have further concerns except for a minor suggestion as follows.

References and permissions of reprint are missing for figure 5 and figure 8.