The Precise Breakpoint Mapping in Paracentric Inversion 10q22.2q23.3 by Comprehensive Cytogenomic Analysis, Multicolor Banding, and Single-Copy Chromosome Sequencing

Round 1

Reviewer 1 Report

The manuscript “Significance of the precise breakpoint mapping in paracentric inversion 10q22.2q23.3 by comprehensive cytogenomic analysis, multicolor banding and single-copy chromosome sequencing” suggests a potential diagnostic method for a patient with male infertility. Unfortunately, the authors found that their method was not diagnostically effective. However, at present, several problems in their method need to be carefully considered for a potential diagnostic method in the future. The authors should summarize alternatives to overcome the problems of their method.

 

Some points have to be corrected.

 

Major points

1. In the 2.6 section (page 4, line 40), the authors described the origin of the Microdissected DNA library. What is inv(10)? This means inversion 10q22.2q23.3? If there is more explanation, the reader is easy to understand.

 

2. It is a shame that there is no control data. If there is any report, please consider adding the information in the discussion section and discuss with results of this study.

 

3. If this method is used for diagnosis, which step is the proper timing for genetic analysis during fertility treatment?

Also, what kind of advice can it give to patients based on the results of the genetic analysis?

 

3. In the discussion section, it is better to add information about alternatives to overcome the problems of their method. For example, please describe the authors’ options for an alternative to detect more inversions and do long-read in NGS, and so on.  

 

 

Minor points

1. Page 1, Line 43: Please amend “of” to “for”.

2. Page 2, Line 9: Please amend “with ” to “by”.

3. Page 2, Line 30: Please add “,” after “Usually”. Also, add “is” before “at”.

4. Page 2, Line 33: Please add “the” before “case”.

5. Page 3, Line 18: Please add “was” before “diagnosed”.

6. Page 3, Line 22: Please amend “ejaculate” to “ejaculation”.

7. Page 13, Line 14: Please add “it” before “impossible”.

8. Page 13, Line 37: Please amend “significant” to “significantly”.

9. Page 14, Line 12 and 33: Please amend “detail” to “detailed”.

10. Page 14, Line 40: Please amend “in” to “into”.

Author Response

Dear Reviewer,

First of all, we would like to thank you for the detailed review and a substantial work on our manuscript. We agree and accept the absolute majority of your questions and comments.

 

Review Report Form 1

 

The manuscript “Significance of the precise breakpoint mapping in paracentric inversion 10q22.2q23.3 by comprehensive cytogenomic analysis, multicolor banding and single-copy chromosome sequencing” suggests a potential diagnostic method for a patient with male infertility. Unfortunately, the authors found that their method was not diagnostically effective. However, at present, several problems in their method need to be carefully considered for a potential diagnostic method in the future. The authors should summarize alternatives to overcome the problems of their method.

 

Response: A Part of Discussion was rewritten according to reviewer’s remarks. At present, the text is: ‘We suppose that for efficient genetic counselling the following problems should be resolved.

The first problem is associated with a possible gene disruption by the inversion. For the estimation of the possible significance of the chromosome rearrangement, the integrity of genes in the regions of breakpoints should be tested. According to our analysis, no essential genes are located near the breakpoints, and consequently one can expect that no gene is damaged. However, in other cases, testing of gene integrity at the region of breakpoints should be performed.

The next possible important problem of genome disturbance could be associated with changes in gene transcription regulation in the breakpoint regions. However, the analysis of efficiency of gene expression regulation is more complicated. Regulatory elements can be replaced at a new position, or TAD structure may be altered. Estimation of the significance of such changes for phenotype formation in an inversion carrier seems to be a complex task. Furthermore, variation among human genomes, especially in the regions enriched with repeats, makes this task almost insurmountable. Probably, the best way to test the gene regulatory system is direct analysis of the transcriptional activity of genes adjacent to the breakpoint regions with real-time or digital polymerase chain reaction.

Attempts to find the answer by performing comparison of the DNA sequences in the breakpoint regions and in corresponding region of reference genome can provide only suggestions of different level of credibility’.

Besides, we added the following fragment to the Discussion section:

‘…we suppose that in diagnostics involving a paracentric inversion, it is necessary to pay attention to the following points: a) the location of the breakpoints at least at the subband level; b) testing of integrity and transcriptional activity of the genes located in regions of the breakpoints; and c) looking for deletions and duplications in the inverted regions and in the regions of the breakpoints. The knowledge of the precise location of breakpoints allows to determine the region of interest for the subsequent analysis. Examination of gene integrity and transcriptional activity of these genes enables one to check for the damage of the genes and their expression regulation. aCGH can reveal a genomic imbalance associated with an inversion, but a small deletion of a region, especially a region enriched with repeats, can evade detection. We should mention that uneven crossover events inside an inverted region of a paracentric inversion depend on the size of the inverted region and typically lead to the formation of a dicentric chromosome and an acentric fragment. Such chromosome rearrangements usually yield such a substantial abnormality that some embryos are lost even before implantation [37]’.

 

Major points

1: In the 2.6 section (page 4, line 40), the authors described the origin of the Microdissected DNA library. What is inv(10)? This means inversion 10q22.2q23.3? If there is more explanation, the reader is easy to understand.

 

Response 1: We have changed inv(10) to inv(10)(q?22.3q?23.3) according to reviewer recommendation. Additionally, we added description to explain what that means.

‘The microdissected DNA library derived from the chromosome inv(10)(q?22.3q?23.3). On the base of GTG-banding analysis of rearranged chromosome, it was suggested that region 10q22.3→q23.3 was inverted. GTG- banding did not allow precisely identification of the breakpoints but allowed to identify rearranged chromosome for its isolation with microdissection technique. We prepared microdissected DNA library from a single copy of the rearranged chromosome by DNA amplification with DOP-PCR and subsequent sequencing’. We should note that description of rearranged chromosome was written according to the recommendations of ‘An International System for Human Cytogenomic Nomenclature (2020)’.

 

2: It is a shame that there is no control data. If there is any report, please consider adding the information in the discussion section and discuss with results of this study.

 

Response 2: MCB was performed on metaphase chromosomes of both the patient and a healthy donor. In the figures, we showed images of the patient only because the metaphase plates from the patient contained both normal and rearranged chromosomes. Regarding the microdissected DNA library, its sequencing and analysis, we would like to note that the main problem appears to be genomic diversity of personal genomes. We had to use the reference genome for the analysis but it is different from the genome of the patient involved in the study. We discussed this problem in the original version but we also added a little more discussion in the revised version of our manuscript.

We agree that the best control would be unaffected chromosomes of the patient’s parents. Unfortunately, they are not available.

 

3: If this method is used for diagnosis, which step is the proper timing for genetic analysis during fertility treatment?

Also, what kind of advice can it give to patients based on the results of the genetic analysis?

 

Response 3: The diagnostics was performed for the patient of 33 years of age, male, diagnosed with infertility due to oligoasthenoteratozoospermia and necrozoospermia. There was no strong time limitation in this case. We should note that the generation of microdissected DNA libraries and DNA probes and testing their quality required 3-4 days. It is cheaper and faster than long read sequencing. On the other site, the generation of microdissected DNA libraries and DNA probes can be performed in few labs.

We discussed the question what can be additionally done for diagnostics of balanced rearrangements and what kind of information could be given to patients. We wrote already ‘Taking into account all the problems discussed above, we suppose that in diagnostics involving a paracentric inversion, it is necessary to pay attention to the following points: a) the location of the breakpoints at the subband level at least; b) testing of integrity and transcriptional activity of the genes located in regions of the breakpoints; and c) looking for  deletions and duplications in the inverted regions and in the regions of the breakpoints. Knowledge about the precise location of breakpoints allows to determine the region of interest for this subsequent analysis. Examination of gene integrity and transcriptional activity of these genes enables one to check for damage to the genes and their expression regulation. aCGH can reveal a genomic imbalance associated with an inversion, but a small deletion of a region, especially a region enriched with repeats, can evade detection. We should mention that uneven crossover events inside an inverted region of a paracentric inversion depend on the size of the inverted region and typically lead to one dicentric chromosome and one acentric fragment. Such chromosome rearrangements usually yield such a substantial abnormality that some embryos are lost even before implantation [37].

 

3: In the discussion section, it is better to add information about alternatives to overcome the problems of their method. For example, please describe the authors’ options for an alternative to detect more inversions and do long-read in NGS, and so on.

 

Response 4: We wrote already ‘Besides the molecular cytogenomic techniques employed in this work and additionally discussed above, there is a method that may also provide relevant information. HiC analysis can give additional information on breakpoint “location” in the reference genome assembly and about TAD chromosome structure. Unfortunately, the power of HiC analysis is low in regions enriched with repeats. Furthermore, a detected TAD disturbance can usually be converted into a diagnostic conclusion only in the form of a hypothesis’.

However, we revised this part of discussion and added some new cosiderations on application of NGS with long-reads. ‘Probably the best approach in genome description in patients with balanced chromosome rearrangements is NGS with long-reads and de novo genome assembly. Unfortunately, even this approach cannot solve all problems. It requires also an assembly of the parent genome containing the chromosome of interest before the rearrangement. A conventional comparison of the parent genome with a reference genome does not allow distinguishing some additional changes after chromosome rearrangement from the new genomic variant’.

 

Minor points. We corrected minor points, according to the recommendations.

Author Response File: Author Response.pdf

Reviewer 2 Report

In the paper entitled: “Significance of the precise breakpoint mapping in paracentric inversion 10q22.2q23.3 by comprehensive cytogenomic analysis, multicolor banding and single-copy chromosome sequencing”  the authors present a comprehensive cytogenomic approach for the breakpoint mapping in a rare paracentric inversion on 10q in a patient with oligoasthenoteratozoospermia and necrozoospermia that does not affect other features of phenotype. This study is interesting enough to attract the readers’ attention but it could be published after major revision.

 

My observations are the following:

 

 

·         English revision of the entire manuscript is necessary

 

·         The abstract should describe the results more than comment or make considerations. So the authors should rewrite much of the abstract otherwise the reader does not understand what results the authors have reached.

 

·         I suggest that the authors also put "reproductive failure" as a keyword.

 

 

·         All the “ml” should become “mL”

 

·         Honestly, I find it a bit confusing to talk about the male partner's problems and the female partner's problems. Perhaps the work should be set up differently and focused on one specific aspect, the male aspect

 

·         Apart from the sperm count/mL what are the other characteristics of this man's semen? Often alterations at the molecular level are not observed even in patients with a normal spermiogram.

 

·         The man considered for the study what lifestyles does he have? could this chromosomal alteration be due to his environment or lifestyle? In this regard I would expand the introduction by also putting other factors that may affect male infertility, such as environmental pollution. I suggest reading and possibly citing the following recent works:

doi: 10.3390/ijms21186710

doi: 10.3390/ijms21124198

 

·         The message the authors want to give in the conclusion needs to be clearer as well as some parts of the results and discussion. In short, the authors should be a little more didactic to capture the reader's attention and to make their results well understood.

 

 

 

 

 

Author Response

Dear Reviewer,

First of all, we would like to thank you for the detailed review and a substantial work on our manuscript. We agree and accept the absolute majority of your questions and comments.

Review Report Form 2

 

In the paper entitled: “Significance of the precise breakpoint mapping in paracentric inversion 10q22.2q23.3 by comprehensive cytogenomic analysis, multicolor banding and single-copy chromosome sequencing” the authors present a comprehensive cytogenomic approach for the breakpoint mapping in a rare paracentric inversion on 10q in a patient with oligoasthenoteratozoospermia and necrozoospermia that does not affect other features of phenotype. This study is interesting enough to attract the readers’ attention but it could be published after major revision.

My observations are the following:

English revision of the entire manuscript is necessary

 

Response: English was revised by a native speaker.

 

• The abstract should describe the results more than comment or make considerations. So the authors should rewrite much of the abstract otherwise the reader does not understand what results the authors have reached.

 

Response 1: The abstract has been significantly rewritten in accordance with the reviewer's recommendation.

 

• I suggest that the authors also put "reproductive failure" as a keyword.

 

Response 2: Done.

 

• All the “ml” should become “mL”

 

Response 3: Done.

 

• Honestly, I find it a bit confusing to talk about the male partner's problems and the female partner's problems. Perhaps the work should be set up differently and focused on one specific aspect, the male aspect

 

Response 4: May be it is true, but from our point of view, reproductive problems are common problems even in case when only one partner has medical problem. However, in our case both patient and his wife did have reproductive problems related to morphological, anatomical and biochemical defects. Therefore, we can’t ignore this fact.

 

• Apart from the sperm count/mL what are the other characteristics of this man's semen?

 

Response 5: The morphology parameters have already been presented in the manuscript. We have also added values for the teratozoospermia index (TZI) and the total motility index with link to the WHO reference values

 

• Often alterations at the molecular level are not observed even in patients with a normal spermiogram.

 

Response 6: It is true, alterations at the molecular level could be observed in patients with a normal spermiogram and on the contrary no alteration at the molecular level could be observed in patients with an abnormal spermiogram. Inversions are alterations at genome level. Karyotypes with reciprocal translocations or inversions usually considered as balanced ones, but the question on the significance such chromosome rearrangements remains open. In result of chromosome rearrangement, the genes located in the breakpoints could be damaged or regulation of their transcription could be changed. In this study we tried to estimate modern possibilities revealing genomic alterations hidden behind the balanced karyotype performing analysis on the genomic level. It is appeared that even precise localization of the breakpoints of chromosome rearrangements did not give the ultimate answer. We discussed this problem in more details.

 

• The man considered for the study what lifestyles does he have? could this chromosomal alteration be due to his environment or lifestyle? In this regard I would expand the introduction by also putting other factors that may affect male infertility, such as environmental pollution. I suggest reading and possibly citing the following recent works:

doi: 10.3390/ijms21186710

doi: 10.3390/ijms21124198

 

Response 7: It is true. Environment and lifestyle of the patient can lead to different type of mutations. However, this patient has been living in the ecologically safe region. In his lifestyle there is no indication to any factor that may affect male infertility. We suppose that discussion of this problem is beyond the frame of present paper.

 

• The message the authors want to give in the conclusion needs to be clearer as well as some parts of the results and discussion. In short, the authors should be a little more didactic to capture the reader's attention and to make their results well understood.

 

Response 8: Conclusions and discussion were significantly rewritten according to reviewer remarks.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Accept in present form