Reviewer ’s report  
 
Title: Clinical and Molecular Characterization of a Patient with Mitochondrial 
Neurogastrointestinal  Encephalomyopathy  
 
Version: 0 Date: 20 Jan 2020  
 
Reviewer: Ramon Martí  
 
Reviewer's report:  
 
The manuscript is a MNGIE case report describing a patient with typical MNGIE clinical 
presentation and a novel nonsense mutation in TYMP. The main interest of the report is the 
publication of a novel mutation. The study would have bee n more complete if TP activity had 
been determined in order to fully demonstrate that the mutations causes TP dysfunction, but 
the authors did not have the chance to determine TP activity in this patient because of lack of 
laboratory resources. Considering  this limitation, since this is a nonsense mutation, the 
assumption that it will result in virtual loss of enzyme activity is reasonable.  
As indicated above, the interest of the report is the publication of a novel MNGIE mutation to 
be added to the list. H owever, the manuscript presents some inaccuracies and potential 
improvements that need to be considered before the manuscript is accepted:  
 
-The term used in this manuscript for MNGIE is "Mitochondrial neurogastrointestinal 
encephalopathy". This reviewer p refers the term "encephalomyopathy", because the term 
"encephalopathy" hides the muscle involvement, which is a cardinal feature of MNGIE. 
Although the term "encephalopathy" is used in some of the reference sites, such as OMIM, 
the term "encephalomyopathy"  is also used in other reference sites, such as Orphanet, and is 
the preferred term in the scientific literature published by the reference experts in MNGIE  
(Garone et al, Brain 2011; 134:3326 -32 ; Hirano et al, Neurologist 2004; 10:8 -17). 
 
-At the backgr ound section (line 21 and following), the statement "Recent evidence has 
suggested that the accumulation of these nucleosides is the main culprit for the development 
of the molecular and phenotypic aberrations reported in this disorder [4]", is misleading as it 
is not exact. The reference number (4) is indeed recent but it is not a primary original 
research work, but a review. The experimental evidence that nucleoside accumulation is the 
cause of the molecular phenotype (i.e., alterations in mtDNA) is not r ecent, but raised more 
than 15 years ago (Song et al,  J Biol Chem 2003; 278:43893 -6 ; Ferraro et al, J Biol Chem 
2005;280:24472 -80 ; Pontarin et al, J Biol Chem 2006;281:22720 -8 ; Gonzalez -Vioque et al, 
PLoS Genet 2011; 7:e1002035). Therefore, this senten ce should be reformulated.  
 
-First page of Discussion and Conclusion, line 44: TP does not catalyses the phosphorylation 
of mitochondrial dThd and dUrd. It catalyses the phosphorolysis of mitochondrial dThd and 
dUrd. Phosphorylation and phosphorolysis are different concepts.  
 
- First page of Discussion and conclusion, line 46. There is another statement here that is not 
correct and may mislead the readers. The authors say: "TP plays an important role in the 
metabolic pathways of various cells including thos e in the brain, muscle, RBCs, WBCs, and 
bone marrow…" This statement is inaccurate and incomplete because it hides the correct 
biochemical pathomechanism in MNGIE. Some of the affected organs in MNGIE, such as 
skeletal muscle and brain, do not express (or express very little) thymidine phosphorylase 
(Valentino et al, FEBS Letters 2007, 581:3410 -4). Therefore, it is not accurate to state that 
TP plays an important role in cells from these tissues. Instead, the absence of TP in other 
cells that express much T P (eg, white blood cells, but not RBC) produces systemic dThd and 
dUrd accumulation, and the toxic effect of them on other tissues (even though they do not 
express TP in healthy people) causes mtDNA mutations and the phenotype.  
 
-Figure 4 contains the ful l enzymatic network of pyrimidine metabolism taken from KEGG 
database, which seems to be too much context if the authors want just to depict what is the 
reaction catalysed by TP. The full and detailed anabolic and catabolic context it is perhaps 
not needed  here. Instead, the specific reaction catalysed by TP could be depicted in more 
molecular detail.  
 
-Legend of figure 4: the abbreviation TdR for thymidine is used by some authors, and it is 
correct, but the abbreviation of thymidine commonly used in this m anuscript is dThd. For 
formal consistency it should by always used the same abbreviation.  
 
-Legend of Figure 2: The Sanger sequencing fragments are usually resolved by capillary 
electrophoresis. Therefore, it is not a "chromatogram" but an "electropherogram", unless the 
laboratory where the DNA was analysed used chromatography to resolve the fra gments, 
which I think is unlikely.  
 
-References: the format of the references is not homogeneous. The year of publication is 
missing in many of them. Also, reference 10 is lacking information: journal?, book chapter?  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in th e manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of wri tten English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received rei mbursements, fees, funding, or salary from 
an organisation that may in any way gain or lose financially from the publication of 
this manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or  lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of 
the manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary fr om an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no  to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
Point 5:  My center is negotiating a license contract to promote Gene Therapy for MNGIE, 
and I  might be entitled to receive some payments in the future, once the license agreement is 
signed. I  do not see how this license agreement may constitute a competing interes t with a 
publication of a  MNGIE case report , but I quote this information in this point number 5 for 
the sake of clarity and  full transparency.Besides this, I declare that I have no competing 
interests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be 
included  on my report to the authors and, if the manuscript is accepted for publication, my 
named report  including any attachments I upload will be posted on the websi te along with the 
authors'  responses. I agree for my report to be made available under an Open Access Creative 
Commons  CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that 
any comments  which I do not wish to be included in my name d report can be included as 
confidential comments  to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Clinical and Molecular Characterization of a Patient with Mitochondrial 
Neurogastrointestinal  Encephalomyopathy  
 
Version: 0 Date: 20 Jan 2020  
 
Reviewer: Ramon Martí  
 
Reviewer's report:  
 
The manuscript is a MNGIE case report describing a patient with typical MNGIE clinical 
presentation and a novel nonsense mutation in TYMP. The main interest of the report is the 
publication of a novel mutation. The study would have bee n more complete if TP activity had 
been determined in order to fully demonstrate that the mutations causes TP dysfunction, but 
the authors did not have the chance to determine TP activity in this patient because of lack of 
laboratory resources. Considering  this limitation, since this is a nonsense mutation, the 
assumption that it will result in virtual loss of enzyme activity is reasonable.  
As indicated above, the interest of the report is the publication of a novel MNGIE mutation to 
be added to the list. H owever, the manuscript presents some inaccuracies and potential 
improvements that need to be considered before the manuscript is accepted:  
 
-The term used in this manuscript for MNGIE is "Mitochondrial neurogastrointestinal 
encephalopathy". This reviewer p refers the term "encephalomyopathy", because the term 
"encephalopathy" hides the muscle involvement, which is a cardinal feature of MNGIE. 
Although the term "encephalopathy" is used in some of the reference sites, such as OMIM, 
the term "encephalomyopathy"  is also used in other reference sites, such as Orphanet, and is 
the preferred term in the scientific literature published by the reference experts in MNGIE  
(Garone et al, Brain 2011; 134:3326 -32 ; Hirano et al, Neurologist 2004; 10:8 -17). 
 
-At the backgr ound section (line 21 and following), the statement "Recent evidence has 
suggested that the accumulation of these nucleosides is the main culprit for the development 
of the molecular and phenotypic aberrations reported in this disorder [4]", is misleading as it 
is not exact. The reference number (4) is indeed recent but it is not a primary original 
research work, but a review. The experimental evidence that nucleoside accumulation is the 
cause of the molecular phenotype (i.e., alterations in mtDNA) is not r ecent, but raised more 
than 15 years ago (Song et al,  J Biol Chem 2003; 278:43893 -6 ; Ferraro et al, J Biol Chem 
2005;280:24472 -80 ; Pontarin et al, J Biol Chem 2006;281:22720 -8 ; Gonzalez -Vioque et al, 
PLoS Genet 2011; 7:e1002035). Therefore, this senten ce should be reformulated.  
 
-First page of Discussion and Conclusion, line 44: TP does not catalyses the phosphorylation 
of mitochondrial dThd and dUrd. It catalyses the phosphorolysis of mitochondrial dThd and 
dUrd. Phosphorylation and phosphorolysis are different concepts.  
 
- First page of Discussion and conclusion, line 46. There is another statement here that is not 
correct and may mislead the readers. The authors say: "TP plays an important role in the 
metabolic pathways of various cells including thos e in the brain, muscle, RBCs, WBCs, and 
bone marrow…" This statement is inaccurate and incomplete because it hides the correct 
biochemical pathomechanism in MNGIE. Some of the affected organs in MNGIE, such as 
skeletal muscle and brain, do not express (or express very little) thymidine phosphorylase 
(Valentino et al, FEBS Letters 2007, 581:3410 -4). Therefore, it is not accurate to state that 
TP plays an important role in cells from these tissues. Instead, the absence of TP in other 
cells that express much T P (eg, white blood cells, but not RBC) produces systemic dThd and 
dUrd accumulation, and the toxic effect of them on other tissues (even though they do not 
express TP in healthy people) causes mtDNA mutations and the phenotype.  
 
-Figure 4 contains the ful l enzymatic network of pyrimidine metabolism taken from KEGG 
database, which seems to be too much context if the authors want just to depict what is the 
reaction catalysed by TP. The full and detailed anabolic and catabolic context it is perhaps 
not needed  here. Instead, the specific reaction catalysed by TP could be depicted in more 
molecular detail.  
 
-Legend of figure 4: the abbreviation TdR for thymidine is used by some authors, and it is 
correct, but the abbreviation of thymidine commonly used in this m anuscript is dThd. For 
formal consistency it should by always used the same abbreviation.  
 
-Legend of Figure 2: The Sanger sequencing fragments are usually resolved by capillary 
electrophoresis. Therefore, it is not a "chromatogram" but an "electropherogram", unless the 
laboratory where the DNA was analysed used chromatography to resolve the fra gments, 
which I think is unlikely.  
 
-References: the format of the references is not homogeneous. The year of publication is 
missing in many of them. Also, reference 10 is lacking information: journal?, book chapter?  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in th e manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of wri tten English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received rei mbursements, fees, funding, or salary from 
an organisation that may in any way gain or lose financially from the publication of 
this manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or  lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of 
the manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary fr om an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no  to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
Point 5:  My center is negotiating a license contract to promote Gene Therapy for MNGIE, 
and I  might be entitled to receive some payments in the future, once the license agreement is 
signed. I  do not see how this license agreement may constitute a competing interes t with a 
publication of a  MNGIE case report , but I quote this information in this point number 5 for 
the sake of clarity and  full transparency.Besides this, I declare that I have no competing 
interests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be 
included  on my report to the authors and, if the manuscript is accepted for publication, my 
named report  including any attachments I upload will be posted on the websi te along with the 
authors'  responses. I agree for my report to be made available under an Open Access Creative 
Commons  CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that 
any comments  which I do not wish to be included in my name d report can be included as 
confidential comments  to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Clinical and Molecular Characterization of a Patient with Mitochondrial 
Neurogastrointestinal  Encephalomyopathy  
 
Version: 0 Date: 20 Jan 2020  
 
Reviewer: Ramon Martí  
 
Reviewer's report:  
 
The manuscript is a MNGIE case report describing a patient with typical MNGIE clinical 
presentation and a novel nonsense mutation in TYMP. The main interest of the report is the 
publication of a novel mutation. The study would have bee n more complete if TP activity had 
been determined in order to fully demonstrate that the mutations causes TP dysfunction, but 
the authors did not have the chance to determine TP activity in this patient because of lack of 
laboratory resources. Considering  this limitation, since this is a nonsense mutation, the 
assumption that it will result in virtual loss of enzyme activity is reasonable.  
As indicated above, the interest of the report is the publication of a novel MNGIE mutation to 
be added to the list. H owever, the manuscript presents some inaccuracies and potential 
improvements that need to be considered before the manuscript is accepted:  
 
-The term used in this manuscript for MNGIE is "Mitochondrial neurogastrointestinal 
encephalopathy". This reviewer p refers the term "encephalomyopathy", because the term 
"encephalopathy" hides the muscle involvement, which is a cardinal feature of MNGIE. 
Although the term "encephalopathy" is used in some of the reference sites, such as OMIM, 
the term "encephalomyopathy"  is also used in other reference sites, such as Orphanet, and is 
the preferred term in the scientific literature published by the reference experts in MNGIE  
(Garone et al, Brain 2011; 134:3326 -32 ; Hirano et al, Neurologist 2004; 10:8 -17). 
 
-At the backgr ound section (line 21 and following), the statement "Recent evidence has 
suggested that the accumulation of these nucleosides is the main culprit for the development 
of the molecular and phenotypic aberrations reported in this disorder [4]", is misleading as it 
is not exact. The reference number (4) is indeed recent but it is not a primary original 
research work, but a review. The experimental evidence that nucleoside accumulation is the 
cause of the molecular phenotype (i.e., alterations in mtDNA) is not r ecent, but raised more 
than 15 years ago (Song et al,  J Biol Chem 2003; 278:43893 -6 ; Ferraro et al, J Biol Chem 
2005;280:24472 -80 ; Pontarin et al, J Biol Chem 2006;281:22720 -8 ; Gonzalez -Vioque et al, 
PLoS Genet 2011; 7:e1002035). Therefore, this senten ce should be reformulated.  
 
-First page of Discussion and Conclusion, line 44: TP does not catalyses the phosphorylation 
of mitochondrial dThd and dUrd. It catalyses the phosphorolysis of mitochondrial dThd and 
dUrd. Phosphorylation and phosphorolysis are different concepts.  
 
- First page of Discussion and conclusion, line 46. There is another statement here that is not 
correct and may mislead the readers. The authors say: "TP plays an important role in the 
metabolic pathways of various cells including thos e in the brain, muscle, RBCs, WBCs, and 
bone marrow…" This statement is inaccurate and incomplete because it hides the correct 
biochemical pathomechanism in MNGIE. Some of the affected organs in MNGIE, such as 
skeletal muscle and brain, do not express (or express very little) thymidine phosphorylase 
(Valentino et al, FEBS Letters 2007, 581:3410 -4). Therefore, it is not accurate to state that 
TP plays an important role in cells from these tissues. Instead, the absence of TP in other 
cells that express much T P (eg, white blood cells, but not RBC) produces systemic dThd and 
dUrd accumulation, and the toxic effect of them on other tissues (even though they do not 
express TP in healthy people) causes mtDNA mutations and the phenotype.  
 
-Figure 4 contains the ful l enzymatic network of pyrimidine metabolism taken from KEGG 
database, which seems to be too much context if the authors want just to depict what is the 
reaction catalysed by TP. The full and detailed anabolic and catabolic context it is perhaps 
not needed  here. Instead, the specific reaction catalysed by TP could be depicted in more 
molecular detail.  
 
-Legend of figure 4: the abbreviation TdR for thymidine is used by some authors, and it is 
correct, but the abbreviation of thymidine commonly used in this m anuscript is dThd. For 
formal consistency it should by always used the same abbreviation.  
 
-Legend of Figure 2: The Sanger sequencing fragments are usually resolved by capillary 
electrophoresis. Therefore, it is not a "chromatogram" but an "electropherogram", unless the 
laboratory where the DNA was analysed used chromatography to resolve the fra gments, 
which I think is unlikely.  
 
-References: the format of the references is not homogeneous. The year of publication is 
missing in many of them. Also, reference 10 is lacking information: journal?, book chapter?  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in th e manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of wri tten English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received rei mbursements, fees, funding, or salary from 
an organisation that may in any way gain or lose financially from the publication of 
this manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or  lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of 
the manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary fr om an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no  to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
Point 5:  My center is negotiating a license contract to promote Gene Therapy for MNGIE, 
and I  might be entitled to receive some payments in the future, once the license agreement is 
signed. I  do not see how this license agreement may constitute a competing interes t with a 
publication of a  MNGIE case report , but I quote this information in this point number 5 for 
the sake of clarity and  full transparency.Besides this, I declare that I have no competing 
interests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be 
included  on my report to the authors and, if the manuscript is accepted for publication, my 
named report  including any attachments I upload will be posted on the websi te along with the 
authors'  responses. I agree for my report to be made available under an Open Access Creative 
Commons  CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that 
any comments  which I do not wish to be included in my name d report can be included as 
confidential comments  to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant