Non-Invasive Detection of a De Novo Frameshift Variant of STAG2 in a Female Fetus: Escape Genes Influence the Manifestation of X-Linked Diseases in Females

Round 1

Reviewer 1 Report

REVIEW:  Non-invasive detection of a de novo frameshift variant of STAG2 in a female fetus: Escape genes influence the manifestation of X-linked diseases in females.  (Note new title!)

This interesting paper presents well documented evidence of the efficacy of prenatal DNA testing from blood samples of pregnant females and is therefore appropriate for this special issue of the journal.  It also discusses the influence of escape genes on female phenotype.

There are a few problems that need to be amended.

The first is the paper is not written in the best English.   I have made some suggestions for revisions, but perhaps your editors may have other suggestions. Cutting words will strengthen their presentation.

Second, I object to the use of dominant and recessive for X-linked traits.  There is a continuum

of expression mediated both by X inactivation, the type of mutation and degree of expression from the Y or inactive X.  These authors should avoid interpretating their data as dominant or recessive, or residual, which is used inappropriately, in the paper.

Third, the authors should use the term escape genes, (the proper term), and not escapees or escapers.  In addition, they should state somewhere that escape genes have 10-30-% the activity of the gene on the active female X and male X.

Figure 2 is difficult to understand as presented.  I still do not see the missing nucleotides.

Defining what is being highlighted would help.

My specific suggestions are as follows:

TITLE: Non-invasive detection of a de novo frameshift variant of STAG2 in a female fetus: Escape genes influence the manifestation of X-linked diseases in females.

Line 35.   Omit: on the contrary

Line 36.   Missense

Line 39.   Omit in determining the X-linked disorders as recessive or dominant.

Line 59.   The word are was omitted between which and compatible.

Line 62.   Omit semi dominant condition with

Line 221.  Use in instead of since

Line 233.  Use associate with instead of associate to

Line 236.  Omit in particular

Line 245.  Omit germinal

Line 247.  Use from instead of by

Line 254. Omit sentence starting with Therefore, every possible

Line 257. Omit act as semidominant; Substitute: is another escape gene

Line 258.  Omit as and use to be

Line 262.  Omit is not an escapee one.  Use does not escape X inactivation

Line 264.  Omit vary and use varies. 

Line 264.  Omit A paradigmatic. Use An example

Line 268.  Omit no phenomenon of inactivation occurs. Use where X inactivation does not occur.

Line 269.  Omit in males compared to females when analyzing expression in the Y homologues. Line 270-271.  Omit These data indicate that escapee genes do not totally escape X inactivation

Line 273.   Omit residual

Line 275.   Omit the senseless sentence. Indeed it is well known that LoF variants can result in partial effect with residual transcriptional activity at least for some transcripts.

Line 277.   Omit In agreement and would and semidominant inheritance.  Use We expect that expression is related to escape

Line 278.   Omit as indicated.  Use as shown  for DDX3X and intellectual developmental disorders

Author Response

We thank you for providing us the comments on our paper jcm-1814958

REVIEW:  Non-invasive detection of a de novo frameshift variant of STAG2 in a female fetus: Escape genes influence the manifestation of X-linked diseases in females.  (Note new title!)

This interesting paper presents well documented evidence of the efficacy of prenatal DNA testing from blood samples of pregnant females and is therefore appropriate for this special issue of the journal.  It also discusses the influence of escape genes on female phenotype.

There are a few problems that need to be amended.

The first is the paper is not written in the best English.   I have made some suggestions for revisions, but perhaps your editors may have other suggestions. Cutting words will strengthen their presentation.

Second, I object to the use of dominant and recessive for X-linked traits.  There is a continuum of expression mediated both by X inactivation, the type of mutation and degree of expression from the Y or inactive X.  These authors should avoid interpretating their data as dominant or recessive, or residual, which is used inappropriately, in the paper.

Third, the authors should use the term escape genes, (the proper term), and not escapees or escapers.  In addition, they should state somewhere that escape genes have 10-30-% the activity of the gene on the active female X and male X.

We added this sentence

These data indicate that escapee genes do not totally escape chromosome inactivation [28] maintaining only a proportion of the activity of the active female X and male X, final expression being mediated by X inactivation, the type of mutation, and the degree of expression in that specific tissue from either the Y or the active X.

Figure 2 is difficult to understand as presented.  I still do not see the missing nucleotides. Defining what is being highlighted would help.

Thanks for your suggestion, Figure 2 indicates the ultrasound of the fetus while the two nucleotides deletion is represented in Figure 4. We have improved the resolution and legend of Figure 4 

My specific suggestions are as follows:

In accordance with your suggestion, the title is now:

Non-invasive detection of a de novo frameshift variant of STAG2 in a female fetus: Escape genes influence the manifestation of X-linked diseases in females.

Line 35.   Omit: on the contrary. done

Line 36.   Missense done

Line 39.   Omit in determining the X-linked disorders as recessive or dominant. done

Line 59.   The word are was omitted between which and compatible. done

Line 62.   Omit semi dominant condition with done

Line 221.  Use in instead of since done

Line 233.  Use associate with instead of associate to done

Line 236.  Omit in particular done

Line 245.  Omit germinal done

Line 247.  Use from instead of by done

Line 254. Omit sentence starting with Therefore, every possible

We modified the sentence “Therefore, every possible relationship between X-inactivation and phenotype remains conflicting [6]. Interestingly, LoF variants in another X-linked gene involved in the co-hesion pathway, SMC1A, which is another escape gene, act as those in STAG2. Both STAG2 and SMC1A are reported as escapee genes [11, 28]. By contrast, missense and LoF variants in the other X-linked gene involved in Cornelia de Lange syndrome, HDAC8, result in severely affected males with either healthy or mildly affected carrier mothers.”

Line 257. Omit act as semidominant; Substitute: is another escape gene done

Line 258.  Omit as and use to be done

Line 262.  Omit is not an escapee one.  Use does not escape X inactivation done

Line 264.  Omit vary and use varies.  done

Line 264.  Omit A paradigmatic. Use An example done

Line 268.  Omit no phenomenon of inactivation occurs. Use where X inactivation does not occur. done

Line 269.  Omit in males compared to females when analyzing expression in the Y homologues. done

Line 270-271.  Omit These data indicate that escapee genes do not totally escape X inactivation

We modified the sentence: “These data indicate that escapee genes do not totally escape chromosome inactivation [28] maintaining only a proportion of the activity of the active female X and male X, final expression being mediated by X inactivation, the type of mutation, and the degree of expression in that specific tissue from either the Y or the active X.

Line 273.   Omit residual done

Line 275.   Omit the senseless sentence. Indeed it is well known that LoF variants can result in partial effect with residual transcriptional activity at least for some transcripts. done

Line 277.   Omit In agreement and would and semidominant inheritance.  Use We expect that expression is related to escape done

Line 278.   Omit as indicated.  Use as shown  for DDX3X and intellectual developmental disorders done

Reviewer 2 Report

Dear Authors,

thank you for preparing your paper on molecular diagnosis of a 20-week-old female fetus with a diaphragmatic hernia and other malformations revealed in ultrasound examination. You present a thorough analysis of fetal DNA from two sources – cell free fetal DNA from maternal blood and amniotic fluid, showing that non-invasive prenatal testing may be appropriate for the detection of de novo mutations. I appreciate that your approach takes into consideration the possibility of parental mosaicism and the phenomenon of X chromosome inactivation, making your analysis complete.

I only have a couple of comments you might wish to consider:

1.     Lines 93 and 95 – instead of rpm as centrifugal speed units use g force that is independent of rotor size.

2.     Figure 4 is very blurred and illegible, so you should either improve its resolution or maybe only describe it in a text.

Author Response

Dear Authors,

thank you for preparing your paper on molecular diagnosis of a 20-week-old female fetus with a diaphragmatic hernia and other malformations revealed in ultrasound examination. You present a thorough analysis of fetal DNA from two sources – cell free fetal DNA from maternal blood and amniotic fluid, showing that non-invasive prenatal testing may be appropriate for the detection of de novo mutations. I appreciate that your approach takes into consideration the possibility of parental mosaicism and the phenomenon of X chromosome inactivation, making your analysis complete.

I only have a couple of comments you might wish to consider:

1. Lines 93 and 95 – instead of rpm as centrifugal speed units use g force that is independent of rotor size.

      We modified the speed unit using g force

2. Figure 4 is very blurred and illegible, so you should either improve its resolution or maybe only describe it in a text.

Thank you for your suggestion, we improved the figure and the legend