Activation Ratio Correlates with IQ in Female Carriers of the FMR1 Premutation

Round 1

Reviewer 1 Report

Dear author

The article need revision as attached

Comments for author File: Comments.pdf

Dear author

The article need revision as attached

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Very interesting manuscript. I do not have major criticisms. I just suggest to improve the section concerning  the limitations of the study, that is very concise. Could be worthwhile - for instance -  to study the level of FMRP ( by WB) and/or the ratio FMRP/AR in blood and other tissues (fibroblasts, iPS derived neurons) of these patients?

Author Response

Dear Editors,

Please find our revised paper entitled “Activation ratio correlates with IQ in female carriers of the FMR1 premutation”

submitted in Special Issue "The Molecular Basis of Fragile X Syndrome and Other FMR1-Associated

Conditions" of Cells.

 

We hope to have addressed the reviewers’ comments satisfactorily as reported below.

 

Please feel free to contact me if you have any questions or concerns.

 

 

REVIEWER 2

Very interesting manuscript. I do not have major criticisms. I just suggest to improve the section concerning the limitations of the study, that is very concise. Could be worthwhile - for instance - to study the level of FMRP (by WB) and/or the ratio FMRP/AR in blood and other tissues (fibroblasts, iPS derived neurons) of these patients?

Thanks for your comments. Likely it would be relevant to assess the levels of FMRP and the ratio in different tissues. However, we do not have fibroblast cell lines or derived iPSCs from the participants of this study. This goes beyond the scope of the study, and it will be certainly something to include in future studies given the recent attention that has been given to clinical involvements in female carriers of a premutation. We have added a comment in the text to this regard.

Reviewer 3 Report

The goal of this study was to measure the activation ratios of premutation FMR1 alleles and assess correlations with clinical outcomes. There were 30 female participants with premutation of the FMR1 gene in the range of 55-200 repeats. The average age was 60.5 years (range 40-84). Twelve participants had FXTAS, 11/24 FXPOI, 22 anxiety and 20 depression. Activation ratios were measured by two methods: Southern blot and methylation PCR.

 

The authors find: (1) higher activation ratios associated with lower FMR1 transcript levels for any given repeat length; (2) higher activation ratios by Southern blot associated with performance, verbal and full-scale IQ scores, odds of depression, number of medical conditions, and (3) activation ratios by methylation PCR not significantly associated with these measures.

 

Comments:

(1)   Since the FMR1 gene in premutation carriers expresses higher levels of FMR1 mRNA, it would be interesting if the data were also analyzed to correlate FMR1 mRNA level with clinical outcomes.

(2)   The font in Figure 1 is grainy and unreadable for the type superimposed on the Southern blot.

(3)   Line 256 states, “This would result in an apparent reduction in the fraction of normal alleles on the inactive X and thus a higher apparent AR.” Should the word “reduction” be “increase”? Sentences 251-257 are confusing. In line 252, the assay is measuring the normal allele, but then in lines 253-255, data is discussed for the PM allele. Perhaps an equation would be helpful for how AR is calculated.

(4)   Line 477 states, “In addition, this research revealed that higher ARs were associated with lower FMR1 transcript levels for any given repeat length confirming previous findings [5, 49, 50].” AR appears to be associated with lower FMR1 transcript levels in subjects with lower transcript levels, but with mid to higher transcript levels there seems to be divergence from a linear regression line (using unadjusted data from Table S1). Can the authors comment?

 

 

Author Response

Dear Editors,

Please find our revised paper entitled “Activation ratio correlates with IQ in female carriers of the FMR1 premutation”

submitted in Special Issue "The Molecular Basis of Fragile X Syndrome and Other FMR1-Associated

Conditions" of Cells.

 

We hope to have addressed the reviewers’ comments satisfactorily as reported below.

 

Please feel free to contact me if you have any questions or concerns.

 

REVIEWER 3

(1) Since the FMR1 gene in premutation carriers expresses higher levels of FMR1 mRNA, it would be interesting if the data were also analyzed to correlate FMR1 mRNA level with clinical outcomes.

Thanks for your comments. We analyzed correlation between FMR1 mRNA level and clinical outcomes. However, these correlations are not significant. Accordingly, we added this finding at the beginning of the Results section.

 

(2) The font in Figure 1 is grainy and unreadable for the type superimposed on the Southern blot.

We have improved the quality of the image.

 

(3) Line 256 states, “This would result in an apparent reduction in the fraction of normal alleles on the inactive X and thus a higher apparent AR.” Should the word “reduction” be “increase”? Sentences 251-257 are confusing. In line 252, the assay is measuring the normal allele, but then in lines 253-255, data is discussed for the PM allele. Perhaps an equation would be helpful for how AR is calculated.

We have edited the sentence for more clarity and added the formula of AR measure. The activation ratio measured by Southern blot is calculated on the signal intensity quantified by phosphorimaging of the appropriate bands on Southern blots as:

Activation ratio= Unmeth normal/Unmeth normal + Meth normal
and added the reference
\

(4) Line 477 states, “In addition, this research revealed that higher ARs were associated with lower FMR1 transcript levels for any given repeat length confirming previous findings [5, 49, 50].” AR appears to be associated with lower FMR1 transcript levels in subjects with lower transcript levels, but with mid to higher transcript levels there seems to be divergence from a linear regression line (using unadjusted data from Table S1). Can the authors comment?

Higher AR, which means that the majority the cells carry the normal allele on the active X chromosome, are associated with lower FMR1 transcript levels for any given repeat length as the premutation alleles (whatever the size may be) is active in small percent of cells.

Round 2

Reviewer 1 Report

Dear Author

the article has been modified as suggested and may be considered for publication