Reviewer's report 
 Lack of significant association of an insertion/deletion polymorphism in the angiotensin converting Title: 
enzyme (ACE) gene with tropical calcific pancreatitis 
 1  20 September 2006 Version: Date: 
 Joost P Drenth Reviewer: 
Reviewer's report: 
General 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be 
reached) 
COMMENTS TO THE AUTHORS 
The authors indicate that a substantial number of patients with tropical calcific pancreatitis carry mutations 
in the pancreatic secretory trypsin inhibitor (SPINK1) gene. They also note that mutated SPINK1 does not 
account for the disease in all patients. This is their prime reason to focus their attention to other genes, and 
in this case they focus on the gene encoding for angiotensin converting enzyme. they compared the 
frequency of a ACE gene insertion/deletion (I/D) polymorphism in a sample 91 tropical calcific pancreatitis and 80 fibro-calculous pancreatic diabetes 
patients to 99 healthy controls. 
They found that the same proportion of patients and healthy controls were carriers of a insertion/deletion 
(I/D) polymorphism. The presence or absence of the N34S SPINK1 mutation did not affect the results. The 
authors conclude that the ACE gene insertion/deletion (I/D) polymorphism is probably not a modifier of 
tropical calcific pancreatitis. 
What I like about this study is its straightforward character: the authors have identified a candidate gene, 
test a common polymorphism in a large set of patients and compare the frequency of that polymorphism 
with a group of healthy controls. 
Major comments 
In order to judge the study under review I have selected some key items. 
Selection of Candidate 
Key questions here are: 
1. Is the candidate gene biologically reasonable? 2. Have all variants identified? 
Ad 1 I was surprised that the authors consider the ACE gene to be a candidate gene in tropical calcific 
pancreatitis. The authors assert that”ACE and RAS activity is known to be enhanced during acute 
pancreatitis and chronic hypoxia in the experimental animals” and that “ACE Inhibitors have recently been 
demonstrated to attenuate pancreatic inflammation and fibrosis in rats”. The authors of the last study used 
glycodeoxycholic acid to induce acute pancreatitis and found that this was associated with increased expression of the pancreatic RAS component proteins. The mere fact that there is an association of experimental pancreatitis with increased ACE does not imply a causative role for ACE in pancreatitis. On 
the other hand, if there was experimental evidence that animals with an ACE gene insertion/deletion (I/D) 
polymorphism had a different course after induction of experimental pancreatitis compared to wild type animals this would bolster the authors’ thesis. At present I find the case for the ACE gene as a candidate gene quite meager. 
The argument that a previous study investigated the ACE gene insertion/deletion (I/D) polymorphism in a 
set of patients with chronic pancreatitis and that this study follows it up does not relieve the authors of the 
obligation to bolster the case for biological plausibility. 
Ad 2. Testing a single nucleotide polymorphism (albeit functional) does not rule out the ACE gene as a 
cause of tropical calcific pancreatitis. I would favor a haplotype study using several SNP’s in the region. 
Even if you do not have families in your sample so you do not know the phase of the haplotypes multilocus 
methods outperformed single locus tests. Combinations of SNPs, whether linked or 
unlinked, maximize the power to detect epistatic effects. 
Statistical analysis 
The authors state that the study was adequately powered to detect a relative risk of 3.0 in the TCP patients. 
Indeed a power of >90% is highly desirable. However do we really expect a relative risk of 3? I think it is 
unreasonable to expect this large effect size. If we consider the latest paper by the last author just published 
in GUT (with tropical calcific pancreatitis Association of cathepsin B gene polymorphisms) he finds a OR of 
2.1-2.15 for 2 cathepsin B gene polymorphisms. For diseases such as TCP that have only modest familial aggregation it is likely that only a few common polymorphisms of large effect (genotypic risk ratios of 2 or more) can exist (for example SPINK1) , and that there may be other 
polymorphisms of smaller effect (genotypic risk ratios 1•5 or less). By decreasing the OR, the authors need 
a much larger sample in order to maintain the power of the study. In other words I think that the study sample is too small to really refute the null hypothesis. 
Testing of SNP 
It would be informative to know what technique they used to identify SPINK1/N34S mutants, as it is not mentioned in the “methods” 
Selection of Cases & Controls 
Were the controls comparable with the cases with respect to ethnicity and other environmental factors? As 
India is a ethnically diverse country, it is whorthwhile to know whether controls are from the same ethnic and 
social background and are sex-matched. The paper under review does not mention how the authors 
exclude that the cases had tropical calcific pancreatitis. Elsewhere I found that their control group had neither any complaints nor any evidence of pancreatitis on imaging. (Gut 2006) The authors migh be more 
specific and give some additional information what kind of imaging did they used. Did this control group 
also completed a structured questionnaire detailing their medical history? In any case, if the authors have performed the same tests (questionnaire and imaging) in their control population the authors have increased the standard for this type of case control studies and they should be commended for this. 
Minor comment 
Table1 refers to the age of onset as NA, which is not mentioned in the legend of the table1. How it should 
be understood as ´´not applicable´´ or ´´not available´´? 
Conclusion 
The major caveat of this study is the selection of the SNP (ACE gene insertion/deletion (I/D) polymorphism) 
as I think that the thesis for biological plausibility (that this gene is actually involved in pancreatitis) is rather 
weak. 
 Unable to decide on acceptance or rejection until the authors have responded to the major What next?: 
compulsory revisions 
 An article of limited interest Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
'I declare that I have no competing interests' 
Reviewer's report 
 Lack of significant association of an insertion/deletion polymorphism in the angiotensin converting Title: 
enzyme (ACE) gene with tropical calcific pancreatitis 
 1  20 September 2006 Version: Date: 
 Joost P Drenth Reviewer: 
Reviewer's report: 
General 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be 
reached) 
COMMENTS TO THE AUTHORS 
The authors indicate that a substantial number of patients with tropical calcific pancreatitis carry mutations 
in the pancreatic secretory trypsin inhibitor (SPINK1) gene. They also note that mutated SPINK1 does not 
account for the disease in all patients. This is their prime reason to focus their attention to other genes, and 
in this case they focus on the gene encoding for angiotensin converting enzyme. they compared the 
frequency of a ACE gene insertion/deletion (I/D) polymorphism in a sample 91 tropical calcific pancreatitis and 80 fibro-calculous pancreatic diabetes 
patients to 99 healthy controls. 
They found that the same proportion of patients and healthy controls were carriers of a insertion/deletion 
(I/D) polymorphism. The presence or absence of the N34S SPINK1 mutation did not affect the results. The 
authors conclude that the ACE gene insertion/deletion (I/D) polymorphism is probably not a modifier of 
tropical calcific pancreatitis. 
What I like about this study is its straightforward character: the authors have identified a candidate gene, 
test a common polymorphism in a large set of patients and compare the frequency of that polymorphism 
with a group of healthy controls. 
Major comments 
In order to judge the study under review I have selected some key items. 
Selection of Candidate 
Key questions here are: 
1. Is the candidate gene biologically reasonable? 2. Have all variants identified? 
Ad 1 I was surprised that the authors consider the ACE gene to be a candidate gene in tropical calcific 
pancreatitis. The authors assert that”ACE and RAS activity is known to be enhanced during acute 
pancreatitis and chronic hypoxia in the experimental animals” and that “ACE Inhibitors have recently been 
demonstrated to attenuate pancreatic inflammation and fibrosis in rats”. The authors of the last study used 
glycodeoxycholic acid to induce acute pancreatitis and found that this was associated with increased expression of the pancreatic RAS component proteins. The mere fact that there is an association of experimental pancreatitis with increased ACE does not imply a causative role for ACE in pancreatitis. On 
the other hand, if there was experimental evidence that animals with an ACE gene insertion/deletion (I/D) 
polymorphism had a different course after induction of experimental pancreatitis compared to wild type animals this would bolster the authors’ thesis. At present I find the case for the ACE gene as a candidate gene quite meager. 
The argument that a previous study investigated the ACE gene insertion/deletion (I/D) polymorphism in a 
set of patients with chronic pancreatitis and that this study follows it up does not relieve the authors of the 
obligation to bolster the case for biological plausibility. 
Ad 2. Testing a single nucleotide polymorphism (albeit functional) does not rule out the ACE gene as a 
cause of tropical calcific pancreatitis. I would favor a haplotype study using several SNP’s in the region. 
Even if you do not have families in your sample so you do not know the phase of the haplotypes multilocus 
methods outperformed single locus tests. Combinations of SNPs, whether linked or 
unlinked, maximize the power to detect epistatic effects. 
Statistical analysis 
The authors state that the study was adequately powered to detect a relative risk of 3.0 in the TCP patients. 
Indeed a power of >90% is highly desirable. However do we really expect a relative risk of 3? I think it is 
unreasonable to expect this large effect size. If we consider the latest paper by the last author just published 
in GUT (with tropical calcific pancreatitis Association of cathepsin B gene polymorphisms) he finds a OR of 
2.1-2.15 for 2 cathepsin B gene polymorphisms. For diseases such as TCP that have only modest familial aggregation it is likely that only a few common polymorphisms of large effect (genotypic risk ratios of 2 or more) can exist (for example SPINK1) , and that there may be other 
polymorphisms of smaller effect (genotypic risk ratios 1•5 or less). By decreasing the OR, the authors need 
a much larger sample in order to maintain the power of the study. In other words I think that the study sample is too small to really refute the null hypothesis. 
Testing of SNP 
It would be informative to know what technique they used to identify SPINK1/N34S mutants, as it is not mentioned in the “methods” 
Selection of Cases & Controls 
Were the controls comparable with the cases with respect to ethnicity and other environmental factors? As 
India is a ethnically diverse country, it is whorthwhile to know whether controls are from the same ethnic and 
social background and are sex-matched. The paper under review does not mention how the authors 
exclude that the cases had tropical calcific pancreatitis. Elsewhere I found that their control group had neither any complaints nor any evidence of pancreatitis on imaging. (Gut 2006) The authors migh be more 
specific and give some additional information what kind of imaging did they used. Did this control group 
also completed a structured questionnaire detailing their medical history? In any case, if the authors have performed the same tests (questionnaire and imaging) in their control population the authors have increased the standard for this type of case control studies and they should be commended for this. 
Minor comment 
Table1 refers to the age of onset as NA, which is not mentioned in the legend of the table1. How it should 
be understood as ´´not applicable´´ or ´´not available´´? 
Conclusion 
The major caveat of this study is the selection of the SNP (ACE gene insertion/deletion (I/D) polymorphism) 
as I think that the thesis for biological plausibility (that this gene is actually involved in pancreatitis) is rather 
weak. 
 Unable to decide on acceptance or rejection until the authors have responded to the major What next?: 
compulsory revisions 
 An article of limited interest Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
'I declare that I have no competing interests' 
Please review this paper.
user
Reviewer's report 
 Lack of significant association of an insertion/deletion polymorphism in the angiotensin converting Title: 
enzyme (ACE) gene with tropical calcific pancreatitis 
 1  20 September 2006 Version: Date: 
 Joost P Drenth Reviewer: 
Reviewer's report: 
General 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be 
reached) 
COMMENTS TO THE AUTHORS 
The authors indicate that a substantial number of patients with tropical calcific pancreatitis carry mutations 
in the pancreatic secretory trypsin inhibitor (SPINK1) gene. They also note that mutated SPINK1 does not 
account for the disease in all patients. This is their prime reason to focus their attention to other genes, and 
in this case they focus on the gene encoding for angiotensin converting enzyme. they compared the 
frequency of a ACE gene insertion/deletion (I/D) polymorphism in a sample 91 tropical calcific pancreatitis and 80 fibro-calculous pancreatic diabetes 
patients to 99 healthy controls. 
They found that the same proportion of patients and healthy controls were carriers of a insertion/deletion 
(I/D) polymorphism. The presence or absence of the N34S SPINK1 mutation did not affect the results. The 
authors conclude that the ACE gene insertion/deletion (I/D) polymorphism is probably not a modifier of 
tropical calcific pancreatitis. 
What I like about this study is its straightforward character: the authors have identified a candidate gene, 
test a common polymorphism in a large set of patients and compare the frequency of that polymorphism 
with a group of healthy controls. 
Major comments 
In order to judge the study under review I have selected some key items. 
Selection of Candidate 
Key questions here are: 
1. Is the candidate gene biologically reasonable? 2. Have all variants identified? 
Ad 1 I was surprised that the authors consider the ACE gene to be a candidate gene in tropical calcific 
pancreatitis. The authors assert that”ACE and RAS activity is known to be enhanced during acute 
pancreatitis and chronic hypoxia in the experimental animals” and that “ACE Inhibitors have recently been 
demonstrated to attenuate pancreatic inflammation and fibrosis in rats”. The authors of the last study used 
glycodeoxycholic acid to induce acute pancreatitis and found that this was associated with increased expression of the pancreatic RAS component proteins. The mere fact that there is an association of experimental pancreatitis with increased ACE does not imply a causative role for ACE in pancreatitis. On 
the other hand, if there was experimental evidence that animals with an ACE gene insertion/deletion (I/D) 
polymorphism had a different course after induction of experimental pancreatitis compared to wild type animals this would bolster the authors’ thesis. At present I find the case for the ACE gene as a candidate gene quite meager. 
The argument that a previous study investigated the ACE gene insertion/deletion (I/D) polymorphism in a 
set of patients with chronic pancreatitis and that this study follows it up does not relieve the authors of the 
obligation to bolster the case for biological plausibility. 
Ad 2. Testing a single nucleotide polymorphism (albeit functional) does not rule out the ACE gene as a 
cause of tropical calcific pancreatitis. I would favor a haplotype study using several SNP’s in the region. 
Even if you do not have families in your sample so you do not know the phase of the haplotypes multilocus 
methods outperformed single locus tests. Combinations of SNPs, whether linked or 
unlinked, maximize the power to detect epistatic effects. 
Statistical analysis 
The authors state that the study was adequately powered to detect a relative risk of 3.0 in the TCP patients. 
Indeed a power of >90% is highly desirable. However do we really expect a relative risk of 3? I think it is 
unreasonable to expect this large effect size. If we consider the latest paper by the last author just published 
in GUT (with tropical calcific pancreatitis Association of cathepsin B gene polymorphisms) he finds a OR of 
2.1-2.15 for 2 cathepsin B gene polymorphisms. For diseases such as TCP that have only modest familial aggregation it is likely that only a few common polymorphisms of large effect (genotypic risk ratios of 2 or more) can exist (for example SPINK1) , and that there may be other 
polymorphisms of smaller effect (genotypic risk ratios 1•5 or less). By decreasing the OR, the authors need 
a much larger sample in order to maintain the power of the study. In other words I think that the study sample is too small to really refute the null hypothesis. 
Testing of SNP 
It would be informative to know what technique they used to identify SPINK1/N34S mutants, as it is not mentioned in the “methods” 
Selection of Cases & Controls 
Were the controls comparable with the cases with respect to ethnicity and other environmental factors? As 
India is a ethnically diverse country, it is whorthwhile to know whether controls are from the same ethnic and 
social background and are sex-matched. The paper under review does not mention how the authors 
exclude that the cases had tropical calcific pancreatitis. Elsewhere I found that their control group had neither any complaints nor any evidence of pancreatitis on imaging. (Gut 2006) The authors migh be more 
specific and give some additional information what kind of imaging did they used. Did this control group 
also completed a structured questionnaire detailing their medical history? In any case, if the authors have performed the same tests (questionnaire and imaging) in their control population the authors have increased the standard for this type of case control studies and they should be commended for this. 
Minor comment 
Table1 refers to the age of onset as NA, which is not mentioned in the legend of the table1. How it should 
be understood as ´´not applicable´´ or ´´not available´´? 
Conclusion 
The major caveat of this study is the selection of the SNP (ACE gene insertion/deletion (I/D) polymorphism) 
as I think that the thesis for biological plausibility (that this gene is actually involved in pancreatitis) is rather 
weak. 
 Unable to decide on acceptance or rejection until the authors have responded to the major What next?: 
compulsory revisions 
 An article of limited interest Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
'I declare that I have no competing interests' 
assistant