Reviewer ’s report 
 
Title: CTRC gene polymorphism (p.G60=; c.180 C>T) in acute pancreatitis  
 
Version: 1 Date: 16 Jun 2016  
 
Reviewer: Jonas Rosendahl  
 
Reviewer's report:  
 
Summary: The authors present the analysis of the CTRC gene in acute pancreatitis and identify 
the synonymous c.180C>T variant as a potential genetic risk factor (borderline significance after 
correction for multiple testing). In cases in that additional SPINK1 variants were identified the 
disease course was more severe (i.e. moderate sever e or severe).  
 
 
 
 
Major points:  
 
Since the authors present only borderline significance for the association (please check how 
Bonferroni correction needs to be done) of the c.180C>T variant with acute pancreatitis a 
replication cohort is recommended to confir m the association in an independent group (this is 
also recommended if the association is not borderline).  
 
The conclusion that severity is influenced by the presence of compound heterozygosity for 
SPINK1 and CTRC variants is based on the number of n=6 pati ents. Therefore, it might be better 
to be very careful with this assumption (maybe these patients had for example further factors that 
influenced the disease course?).  
 
Apart from the replication cohort the manuscript should be carefully revised so that it comes up 
with an easy to understand story (Why CTRC in acute pancreatitis? Is it recurring AP that makes 
the association? Do AP and CP share the same pathophysiological mechanisms? Etc). At the 
moment the reader would be confused and would not easily get t he main result.  
 
 
 
Minor points:  
 
The authors state in the introduction: „...but recent reports indicate a significant role of genetic 
mutations or polymorphism in the AP pathogenesis (2 -4)." This sentence might be misleading, 
since a genetic explanation (w ith low odds ratios) is found only in a few patients with acute 
pancreatitis. Maybe it is worth to correct the sentence and cite the relevant literature here.  
 
 
I do not understand this sentence: „...RAP is a risk factor of CP, but is not a significant fact or in 
itself [3]. ..." Can the authors explain it?  
 
 
In which way does CFTR prevent activation of pancreatic enzymes within the acinar cells?  
 
 
From my point of view CASR seems not to be a pancreatitis gene, since it alters Ca2+ 
concentrations (primary action), which than can lead to the development of CP.  
 
 
There are some new data on how CTRC acts on degradation of trypsinogen and trypsin. It rath er 
seems to be more effective in trypsinogen degradation? This might be added.  
 
 
The introduction needs rewriting so that it is easier to follow the thoughts of the authors.  
 
 
Are there no data on the functional properties of the c.180C>T variant? What about  GTex data? 
What about variants in linkage? There might be more information for the introduction available?  
 
 
In the methods section the definition of alcoholic acute pancreatitis is not clear. I guess the word 
alcoholic is missing (page 5 line 25)  
 
 
It woul d be nice to see more data on the controls (are they members of the hospital, the 
laboratory etc.).  
 
 
How many samples have been sequenced (sanger sequencing)? Can you give numbers on 
accuracy of your melting method?  
 
 
It does not seem reasonable to draw an y conclusion in the group of patients with a pancreatic 
carcinoma as the number of patients is very low (I would suggest to delete the part in the result 
section). The same holds true for the rare TT carriers in the different patient groups.  
 
 
Why was the g enotypic model used (what is the rationale behind this model?)?  
 
 
The discussion should be revised. The first page for example is a summary of earlier results in 
CTRC screening, something that fits into an introduction.  
 
It should be noted and highlighted t hat former studies investigated the c.180C>T variant in 
chronic pancreatitis patients (and these data cannot easily be compared to acute pancreatitis 
patients).  
 
 
In the discussion page 14 line 2: please correct Masona to Masson.  
 
 
The SPINK1 p.P55S variant has no pathogenic effect. This should be changed in the discussion.  
 
 
I do not understand the last sentence of the discussion, please clarify. (Simple, classic….)  
 
 
In Table 3 the percentages given are for the different genotypes in regard of the severity/et iology 
(e.g. 5 patients have TT and represent 100% of all patients in the mild group). Maybe it makes 
more sense to give the percentages for distinct mild/etiology groups overall i.e. in the mild group 
5 of 208 patients carry TT (2.4%)? Thereby you can see  that in the severe group the CT genotype 
is very uncommon (only 14%; Discussion?).  
 
 
In Table 4 the numbers of AP are n=299 for all genotypes (the same for the controls; should 
there be the numbers from Table 1 in regard of the c.180C>T genotype?), is this correct? If not 
all the numbers and calculations need to be revised. Or maybe the da ta should be presented in a 
different way (eg. heterozygous carriers in patients and controls).  
 
 
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Unable to assess  
 
Does the work include the neces sary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Unable to assess  
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
 
Are you able to asse ss any statistics in the 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 written English  
Please indicate the quality of language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
 
 
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the pa st five years received reimbursements, 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 t hat 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, f ees, funding, or salary from 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 p aper?  
 
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.  
 
I declare that I have no competing inter ests 
 
I agree to the open peer review policy of t he 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 website along with the authors'  
responses. I agree for my report to be made available under a n 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 named 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: CTRC gene polymorphism (p.G60=; c.180 C>T) in acute pancreatitis  
 
Version: 1 Date: 16 Jun 2016  
 
Reviewer: Jonas Rosendahl  
 
Reviewer's report:  
 
Summary: The authors present the analysis of the CTRC gene in acute pancreatitis and identify 
the synonymous c.180C>T variant as a potential genetic risk factor (borderline significance after 
correction for multiple testing). In cases in that additional SPINK1 variants were identified the 
disease course was more severe (i.e. moderate sever e or severe).  
 
 
 
 
Major points:  
 
Since the authors present only borderline significance for the association (please check how 
Bonferroni correction needs to be done) of the c.180C>T variant with acute pancreatitis a 
replication cohort is recommended to confir m the association in an independent group (this is 
also recommended if the association is not borderline).  
 
The conclusion that severity is influenced by the presence of compound heterozygosity for 
SPINK1 and CTRC variants is based on the number of n=6 pati ents. Therefore, it might be better 
to be very careful with this assumption (maybe these patients had for example further factors that 
influenced the disease course?).  
 
Apart from the replication cohort the manuscript should be carefully revised so that it comes up 
with an easy to understand story (Why CTRC in acute pancreatitis? Is it recurring AP that makes 
the association? Do AP and CP share the same pathophysiological mechanisms? Etc). At the 
moment the reader would be confused and would not easily get t he main result.  
 
 
 
Minor points:  
 
The authors state in the introduction: „...but recent reports indicate a significant role of genetic 
mutations or polymorphism in the AP pathogenesis (2 -4)." This sentence might be misleading, 
since a genetic explanation (w ith low odds ratios) is found only in a few patients with acute 
pancreatitis. Maybe it is worth to correct the sentence and cite the relevant literature here.  
 
 
I do not understand this sentence: „...RAP is a risk factor of CP, but is not a significant fact or in 
itself [3]. ..." Can the authors explain it?  
 
 
In which way does CFTR prevent activation of pancreatic enzymes within the acinar cells?  
 
 
From my point of view CASR seems not to be a pancreatitis gene, since it alters Ca2+ 
concentrations (primary action), which than can lead to the development of CP.  
 
 
There are some new data on how CTRC acts on degradation of trypsinogen and trypsin. It rath er 
seems to be more effective in trypsinogen degradation? This might be added.  
 
 
The introduction needs rewriting so that it is easier to follow the thoughts of the authors.  
 
 
Are there no data on the functional properties of the c.180C>T variant? What about  GTex data? 
What about variants in linkage? There might be more information for the introduction available?  
 
 
In the methods section the definition of alcoholic acute pancreatitis is not clear. I guess the word 
alcoholic is missing (page 5 line 25)  
 
 
It woul d be nice to see more data on the controls (are they members of the hospital, the 
laboratory etc.).  
 
 
How many samples have been sequenced (sanger sequencing)? Can you give numbers on 
accuracy of your melting method?  
 
 
It does not seem reasonable to draw an y conclusion in the group of patients with a pancreatic 
carcinoma as the number of patients is very low (I would suggest to delete the part in the result 
section). The same holds true for the rare TT carriers in the different patient groups.  
 
 
Why was the g enotypic model used (what is the rationale behind this model?)?  
 
 
The discussion should be revised. The first page for example is a summary of earlier results in 
CTRC screening, something that fits into an introduction.  
 
It should be noted and highlighted t hat former studies investigated the c.180C>T variant in 
chronic pancreatitis patients (and these data cannot easily be compared to acute pancreatitis 
patients).  
 
 
In the discussion page 14 line 2: please correct Masona to Masson.  
 
 
The SPINK1 p.P55S variant has no pathogenic effect. This should be changed in the discussion.  
 
 
I do not understand the last sentence of the discussion, please clarify. (Simple, classic….)  
 
 
In Table 3 the percentages given are for the different genotypes in regard of the severity/et iology 
(e.g. 5 patients have TT and represent 100% of all patients in the mild group). Maybe it makes 
more sense to give the percentages for distinct mild/etiology groups overall i.e. in the mild group 
5 of 208 patients carry TT (2.4%)? Thereby you can see  that in the severe group the CT genotype 
is very uncommon (only 14%; Discussion?).  
 
 
In Table 4 the numbers of AP are n=299 for all genotypes (the same for the controls; should 
there be the numbers from Table 1 in regard of the c.180C>T genotype?), is this correct? If not 
all the numbers and calculations need to be revised. Or maybe the da ta should be presented in a 
different way (eg. heterozygous carriers in patients and controls).  
 
 
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Unable to assess  
 
Does the work include the neces sary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Unable to assess  
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
 
Are you able to asse ss any statistics in the 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 written English  
Please indicate the quality of language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
 
 
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the pa st five years received reimbursements, 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 t hat 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, f ees, funding, or salary from 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 p aper?  
 
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.  
 
I declare that I have no competing inter ests 
 
I agree to the open peer review policy of t he 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 website along with the authors'  
responses. I agree for my report to be made available under a n 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 named 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: CTRC gene polymorphism (p.G60=; c.180 C>T) in acute pancreatitis  
 
Version: 1 Date: 16 Jun 2016  
 
Reviewer: Jonas Rosendahl  
 
Reviewer's report:  
 
Summary: The authors present the analysis of the CTRC gene in acute pancreatitis and identify 
the synonymous c.180C>T variant as a potential genetic risk factor (borderline significance after 
correction for multiple testing). In cases in that additional SPINK1 variants were identified the 
disease course was more severe (i.e. moderate sever e or severe).  
 
 
 
 
Major points:  
 
Since the authors present only borderline significance for the association (please check how 
Bonferroni correction needs to be done) of the c.180C>T variant with acute pancreatitis a 
replication cohort is recommended to confir m the association in an independent group (this is 
also recommended if the association is not borderline).  
 
The conclusion that severity is influenced by the presence of compound heterozygosity for 
SPINK1 and CTRC variants is based on the number of n=6 pati ents. Therefore, it might be better 
to be very careful with this assumption (maybe these patients had for example further factors that 
influenced the disease course?).  
 
Apart from the replication cohort the manuscript should be carefully revised so that it comes up 
with an easy to understand story (Why CTRC in acute pancreatitis? Is it recurring AP that makes 
the association? Do AP and CP share the same pathophysiological mechanisms? Etc). At the 
moment the reader would be confused and would not easily get t he main result.  
 
 
 
Minor points:  
 
The authors state in the introduction: „...but recent reports indicate a significant role of genetic 
mutations or polymorphism in the AP pathogenesis (2 -4)." This sentence might be misleading, 
since a genetic explanation (w ith low odds ratios) is found only in a few patients with acute 
pancreatitis. Maybe it is worth to correct the sentence and cite the relevant literature here.  
 
 
I do not understand this sentence: „...RAP is a risk factor of CP, but is not a significant fact or in 
itself [3]. ..." Can the authors explain it?  
 
 
In which way does CFTR prevent activation of pancreatic enzymes within the acinar cells?  
 
 
From my point of view CASR seems not to be a pancreatitis gene, since it alters Ca2+ 
concentrations (primary action), which than can lead to the development of CP.  
 
 
There are some new data on how CTRC acts on degradation of trypsinogen and trypsin. It rath er 
seems to be more effective in trypsinogen degradation? This might be added.  
 
 
The introduction needs rewriting so that it is easier to follow the thoughts of the authors.  
 
 
Are there no data on the functional properties of the c.180C>T variant? What about  GTex data? 
What about variants in linkage? There might be more information for the introduction available?  
 
 
In the methods section the definition of alcoholic acute pancreatitis is not clear. I guess the word 
alcoholic is missing (page 5 line 25)  
 
 
It woul d be nice to see more data on the controls (are they members of the hospital, the 
laboratory etc.).  
 
 
How many samples have been sequenced (sanger sequencing)? Can you give numbers on 
accuracy of your melting method?  
 
 
It does not seem reasonable to draw an y conclusion in the group of patients with a pancreatic 
carcinoma as the number of patients is very low (I would suggest to delete the part in the result 
section). The same holds true for the rare TT carriers in the different patient groups.  
 
 
Why was the g enotypic model used (what is the rationale behind this model?)?  
 
 
The discussion should be revised. The first page for example is a summary of earlier results in 
CTRC screening, something that fits into an introduction.  
 
It should be noted and highlighted t hat former studies investigated the c.180C>T variant in 
chronic pancreatitis patients (and these data cannot easily be compared to acute pancreatitis 
patients).  
 
 
In the discussion page 14 line 2: please correct Masona to Masson.  
 
 
The SPINK1 p.P55S variant has no pathogenic effect. This should be changed in the discussion.  
 
 
I do not understand the last sentence of the discussion, please clarify. (Simple, classic….)  
 
 
In Table 3 the percentages given are for the different genotypes in regard of the severity/et iology 
(e.g. 5 patients have TT and represent 100% of all patients in the mild group). Maybe it makes 
more sense to give the percentages for distinct mild/etiology groups overall i.e. in the mild group 
5 of 208 patients carry TT (2.4%)? Thereby you can see  that in the severe group the CT genotype 
is very uncommon (only 14%; Discussion?).  
 
 
In Table 4 the numbers of AP are n=299 for all genotypes (the same for the controls; should 
there be the numbers from Table 1 in regard of the c.180C>T genotype?), is this correct? If not 
all the numbers and calculations need to be revised. Or maybe the da ta should be presented in a 
different way (eg. heterozygous carriers in patients and controls).  
 
 
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Unable to assess  
 
Does the work include the neces sary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Unable to assess  
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
 
Are you able to asse ss any statistics in the 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 written English  
Please indicate the quality of language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
 
 
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the pa st five years received reimbursements, 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 t hat 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, f ees, funding, or salary from 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 p aper?  
 
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.  
 
I declare that I have no competing inter ests 
 
I agree to the open peer review policy of t he 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 website along with the authors'  
responses. I agree for my report to be made available under a n 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 named 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