Reviewer's report 
 A novel mutation of the calcium sensing receptor gene is associated with chronic pancreatitis Title: 
in a family with heterozygous SPINK1 mutations 
Authors: 
Dr Peter Felderbauer ( peter.felderbauer@rub.de )
Peter Hoffmann ( ced-ambulanz@elis-stiftung.de )
Kerem Bulut ( kbulut@web.de )
Nikolaus Ansorge ( n.ansorge@gmx.de )
Henrik Einwaechter ( henrik.einwaechter@rub.de )
Dr Frank Schmitz ( frank.schmitz@rub.de )
Wolfgang E Schmidt ( wolfgang.e.schmidt@rub.de )
 2  23 Jul 2003 Version: Date: 
 Joost P Drenth Reviewer: 
 A paper of considerable general medical or scientific interest Level of interest: 
 Accept after discretionary revisions Advice on publication: 
A novel mutation of the calcium sensing receptor gene is associated with chronic 
pancreatitis in a family with heterozygous SPINK1 mutations 
Peter Felderbauer, Peter Hoffmann, Kerem Bulut, Nikolaus Ansorge, Henrik Einwachter Frank Schmitz, Wolfgang E. Schmidt 
In the present study the authors describe a family with familial hypocalciuric hypercalcemia (FHH). In 
their dataset at least 2 family members have FHH confirmed at the phenotypical and genotypical 
level. One of these two patients has concomitant CP, and both have a heterozygous N34S SPINK1 
allele. A third member of the family has CP and probably FHH but the SPINK1 status is not known. 
After exclusion of hereditary pancreatitis, the authors make a case that (if I understand their 
message well) that the combination of the N34S SPINK1 and CASR gene mutation might induce 
CP. 
I think that this is an interesting observation, that merits widespread attention. However, the 
message that this paper conveys is buried under redundant material. I think that if the authors could bring back focus in the introduction and discussion they will have a great story. I suggest that they 
limit themselves to the findings in their patients and do not give an elaborate literature review in their 
introduction. 
As the authors mention, the relation between FHH and CP is controversial but, given the relative 
rarity of both disorders, it seems unlikely that the coexistence (reported in several families) occurs 
just by chance. I agree with the authors that the role of SPINK1 in CP is very interesting. Indeed it is 
more likely that it modifies the disease (earlier onset; ref Gastroenterology. 2000 Sep;119(3):615-23; 
Gut. 2002 May;50(5):687-92.) than that it follows a clear mendelian mutated gene=disease relation. 
However, I think that the authors take it too far down the road when they state that possession of 
SPINK1 allele explains the presence of CP in FHH. Their observation is interesting and sheds new light on the possible role of SPINK1 in CP Nevertheless, the coexistence of two mutant alleles in a single patient with FHH and CP does not prove that the N34S SPINK1 mutation is a prerequisite for 
the development of CP. Indeed as the authors argue (in their discussion part), despite the obvious 
importance of N34S mutation in CP, causality is not proven. 
What is the age of individual III-1? This is relevant as CP is a disease that has its onset later in life, 
so it is important to know to judge whether she is still at risk for the disease. It would help to include the age for all individuals in table 2. 
The index patient was tested for PRSS1 mutations, most probably R122H and/or N29I. Although it is 
not very likely that this family co-inherited FHH and autosomal dominant hereditary CP, it is not beyond reality. I think that testing for the 2 most frequent PRSS1 mutations does not completely 
exclude hereditary pancreatitis. The authors should at least sequence the complete PRSS1 gene in 
one of the affected family members (index patient) to be certain that autosomal dominant pancreatitis is not the cause of the disease in this family. 
The authors discount the observation made by Pearce et al. (1996) and suggest that because three 
different CASR mutations were found in 3 separate kindred's with FHH and CP this excludes a 
common genetic etiology. I do not think that this is true. For example, in familial Mediterranean fever, 
MEFV mutations cause the disease. A subtype of patients have concurrent amyloidosis and this 
occurs in patients with different MEFV mutations. The three mutations discovered by Pearce 
(Asn178Asp, Arg220Gln and Pro221Ser) were all in the same extracellular domain of the protein, and in theory (because of their similar location) could lead to a similar phenotype (FHH and CP). 
In the discussion I would like to see a little bit more on CP in FHH and little bit less on speculation 
with respect to SPINK1. 
Minor remarks 
The introduction is too long and should be shortened to a single page. The focus of their paper is to 
discuss the relation between FHH and SPINK1 using their family as a format. I do not think that an elaborate review of the discovery of the PRSS1 gene is fitting here. I suggest that the authors focus on FHH in relation to CP and the role of SPINK1 herein. 
The paper would be more succinct if the authors would delete sentences such as "We performed a 
screening for the recently published SPINK1 mutation N34S, which showed for the first time the presence of an additional genetic mutation. It could be shown that a heterozygous transition A>G 
causing a N34S SPINK1 mutation was apparent." This could easily be abbreviated (without loss of 
information) to " Sequencing of the SPINK1 gene revealed a heterozygous N34S mutation." 
Along the same line, the line: "It is therefore another indicator to strengthen the hypothesis of the 
SPINK1 mutations to be not a disease causing, but modifying factor' is tortuous, please shorten to 
something like: "This strengthens the hypothesis that SPINK1 mutations rather modify the disease phenotype than actually cause CP." 
The fact that the "the calcium sensing receptor was recently characterized in human pancreatic 
acinar and duct cells [29]" does not immediately infer a role of this receptor in the pathophysiology of 
pancreatitis. 
Figure 3 (and table 2) should tell the reader whole story, but are needlessly complicated. It is the 
most compelling piece of data the paper carries, yet it is difficult to ascertain. Why are there two 
lines for CASR wildtype and CASR mutation? You could argue that all patients with a CASR 
mutations have wildtype on the other allele? I suggest to delete the reference to wildtype here. In my 
PDF copy I could not see the difference between the signs for family members without genetic 
testing and those with familiar hypocalciuric hypercalcemia (FHH) as they are similar. Please 
correct. My suggestion is further that they indicate the N34S SPINK1 mutation directly in the figure, similar to what they have done for the CASR mutation (indicated by a star). 
Legends Figure 3: I:1 deceased in 971; probably in 1971. 
Conclusion 
"although from the symptomatic brother, where a FHH was diagnosed by clinical parameters, a blood sample for N34S testing could not be obtained. Looking at the family tree and his medical history of recurrent episodes of pancreatitis, a N34S mutation must be at least suspected." is 
speculative and certainly not a conclusive remark. If the authors feel that this sentence is critical, 
move it to elsewhere in the paper. 
Reference 16. Edited instead of Editeded by Katunuma N, Umezawa H, Holzer H. Tokyo: Springer; 
I had difficulties to extract the meaning of the following sentence "It is currently questioned that a 
mutation in this inhibitory system which is only potent enough to inhibit 20 % of trypsinogen triggers CP ? " I would be grateful if the authors could reword this. 
Although it is probably true that, on a equimolar basis, SPINK1 can inactivate 10-20% of active 
trypsin, can the authors comment whether to their knowledge SPINK1 is present in equimolar 
concentrations to trypsin in the acinar cell? If, for example SPINK1 is more abundantly present than 
trypsin , the actual in-vivo ratio changes in favor of SPINK1. 
Likewise I failed to grasp the meaning of the sentence "Looking at our family concerning N34S 
mutations, also both healthy and symptomatic family members are present indicating the missing 
link to be the CASR gene mutation." What do the authors want to tell us? I know that N34S occurs in 
healthy and symptomatic family members, but I do not see what this has to do with "the missing link" 
JPH Drenth 
Rockville, 20 July, 2003 
Competing interests: 
None declared. 
Reviewer's report 
 A novel mutation of the calcium sensing receptor gene is associated with chronic pancreatitis Title: 
in a family with heterozygous SPINK1 mutations 
Authors: 
Dr Peter Felderbauer ( peter.felderbauer@rub.de )
Peter Hoffmann ( ced-ambulanz@elis-stiftung.de )
Kerem Bulut ( kbulut@web.de )
Nikolaus Ansorge ( n.ansorge@gmx.de )
Henrik Einwaechter ( henrik.einwaechter@rub.de )
Dr Frank Schmitz ( frank.schmitz@rub.de )
Wolfgang E Schmidt ( wolfgang.e.schmidt@rub.de )
 2  23 Jul 2003 Version: Date: 
 Joost P Drenth Reviewer: 
 A paper of considerable general medical or scientific interest Level of interest: 
 Accept after discretionary revisions Advice on publication: 
A novel mutation of the calcium sensing receptor gene is associated with chronic 
pancreatitis in a family with heterozygous SPINK1 mutations 
Peter Felderbauer, Peter Hoffmann, Kerem Bulut, Nikolaus Ansorge, Henrik Einwachter Frank Schmitz, Wolfgang E. Schmidt 
In the present study the authors describe a family with familial hypocalciuric hypercalcemia (FHH). In 
their dataset at least 2 family members have FHH confirmed at the phenotypical and genotypical 
level. One of these two patients has concomitant CP, and both have a heterozygous N34S SPINK1 
allele. A third member of the family has CP and probably FHH but the SPINK1 status is not known. 
After exclusion of hereditary pancreatitis, the authors make a case that (if I understand their 
message well) that the combination of the N34S SPINK1 and CASR gene mutation might induce 
CP. 
I think that this is an interesting observation, that merits widespread attention. However, the 
message that this paper conveys is buried under redundant material. I think that if the authors could bring back focus in the introduction and discussion they will have a great story. I suggest that they 
limit themselves to the findings in their patients and do not give an elaborate literature review in their 
introduction. 
As the authors mention, the relation between FHH and CP is controversial but, given the relative 
rarity of both disorders, it seems unlikely that the coexistence (reported in several families) occurs 
just by chance. I agree with the authors that the role of SPINK1 in CP is very interesting. Indeed it is 
more likely that it modifies the disease (earlier onset; ref Gastroenterology. 2000 Sep;119(3):615-23; 
Gut. 2002 May;50(5):687-92.) than that it follows a clear mendelian mutated gene=disease relation. 
However, I think that the authors take it too far down the road when they state that possession of 
SPINK1 allele explains the presence of CP in FHH. Their observation is interesting and sheds new light on the possible role of SPINK1 in CP Nevertheless, the coexistence of two mutant alleles in a single patient with FHH and CP does not prove that the N34S SPINK1 mutation is a prerequisite for 
the development of CP. Indeed as the authors argue (in their discussion part), despite the obvious 
importance of N34S mutation in CP, causality is not proven. 
What is the age of individual III-1? This is relevant as CP is a disease that has its onset later in life, 
so it is important to know to judge whether she is still at risk for the disease. It would help to include the age for all individuals in table 2. 
The index patient was tested for PRSS1 mutations, most probably R122H and/or N29I. Although it is 
not very likely that this family co-inherited FHH and autosomal dominant hereditary CP, it is not beyond reality. I think that testing for the 2 most frequent PRSS1 mutations does not completely 
exclude hereditary pancreatitis. The authors should at least sequence the complete PRSS1 gene in 
one of the affected family members (index patient) to be certain that autosomal dominant pancreatitis is not the cause of the disease in this family. 
The authors discount the observation made by Pearce et al. (1996) and suggest that because three 
different CASR mutations were found in 3 separate kindred's with FHH and CP this excludes a 
common genetic etiology. I do not think that this is true. For example, in familial Mediterranean fever, 
MEFV mutations cause the disease. A subtype of patients have concurrent amyloidosis and this 
occurs in patients with different MEFV mutations. The three mutations discovered by Pearce 
(Asn178Asp, Arg220Gln and Pro221Ser) were all in the same extracellular domain of the protein, and in theory (because of their similar location) could lead to a similar phenotype (FHH and CP). 
In the discussion I would like to see a little bit more on CP in FHH and little bit less on speculation 
with respect to SPINK1. 
Minor remarks 
The introduction is too long and should be shortened to a single page. The focus of their paper is to 
discuss the relation between FHH and SPINK1 using their family as a format. I do not think that an elaborate review of the discovery of the PRSS1 gene is fitting here. I suggest that the authors focus on FHH in relation to CP and the role of SPINK1 herein. 
The paper would be more succinct if the authors would delete sentences such as "We performed a 
screening for the recently published SPINK1 mutation N34S, which showed for the first time the presence of an additional genetic mutation. It could be shown that a heterozygous transition A>G 
causing a N34S SPINK1 mutation was apparent." This could easily be abbreviated (without loss of 
information) to " Sequencing of the SPINK1 gene revealed a heterozygous N34S mutation." 
Along the same line, the line: "It is therefore another indicator to strengthen the hypothesis of the 
SPINK1 mutations to be not a disease causing, but modifying factor' is tortuous, please shorten to 
something like: "This strengthens the hypothesis that SPINK1 mutations rather modify the disease phenotype than actually cause CP." 
The fact that the "the calcium sensing receptor was recently characterized in human pancreatic 
acinar and duct cells [29]" does not immediately infer a role of this receptor in the pathophysiology of 
pancreatitis. 
Figure 3 (and table 2) should tell the reader whole story, but are needlessly complicated. It is the 
most compelling piece of data the paper carries, yet it is difficult to ascertain. Why are there two 
lines for CASR wildtype and CASR mutation? You could argue that all patients with a CASR 
mutations have wildtype on the other allele? I suggest to delete the reference to wildtype here. In my 
PDF copy I could not see the difference between the signs for family members without genetic 
testing and those with familiar hypocalciuric hypercalcemia (FHH) as they are similar. Please 
correct. My suggestion is further that they indicate the N34S SPINK1 mutation directly in the figure, similar to what they have done for the CASR mutation (indicated by a star). 
Legends Figure 3: I:1 deceased in 971; probably in 1971. 
Conclusion 
"although from the symptomatic brother, where a FHH was diagnosed by clinical parameters, a blood sample for N34S testing could not be obtained. Looking at the family tree and his medical history of recurrent episodes of pancreatitis, a N34S mutation must be at least suspected." is 
speculative and certainly not a conclusive remark. If the authors feel that this sentence is critical, 
move it to elsewhere in the paper. 
Reference 16. Edited instead of Editeded by Katunuma N, Umezawa H, Holzer H. Tokyo: Springer; 
I had difficulties to extract the meaning of the following sentence "It is currently questioned that a 
mutation in this inhibitory system which is only potent enough to inhibit 20 % of trypsinogen triggers CP ? " I would be grateful if the authors could reword this. 
Although it is probably true that, on a equimolar basis, SPINK1 can inactivate 10-20% of active 
trypsin, can the authors comment whether to their knowledge SPINK1 is present in equimolar 
concentrations to trypsin in the acinar cell? If, for example SPINK1 is more abundantly present than 
trypsin , the actual in-vivo ratio changes in favor of SPINK1. 
Likewise I failed to grasp the meaning of the sentence "Looking at our family concerning N34S 
mutations, also both healthy and symptomatic family members are present indicating the missing 
link to be the CASR gene mutation." What do the authors want to tell us? I know that N34S occurs in 
healthy and symptomatic family members, but I do not see what this has to do with "the missing link" 
JPH Drenth 
Rockville, 20 July, 2003 
Competing interests: 
None declared. 
Please review this paper.
user
Reviewer's report 
 A novel mutation of the calcium sensing receptor gene is associated with chronic pancreatitis Title: 
in a family with heterozygous SPINK1 mutations 
Authors: 
Dr Peter Felderbauer ( peter.felderbauer@rub.de )
Peter Hoffmann ( ced-ambulanz@elis-stiftung.de )
Kerem Bulut ( kbulut@web.de )
Nikolaus Ansorge ( n.ansorge@gmx.de )
Henrik Einwaechter ( henrik.einwaechter@rub.de )
Dr Frank Schmitz ( frank.schmitz@rub.de )
Wolfgang E Schmidt ( wolfgang.e.schmidt@rub.de )
 2  23 Jul 2003 Version: Date: 
 Joost P Drenth Reviewer: 
 A paper of considerable general medical or scientific interest Level of interest: 
 Accept after discretionary revisions Advice on publication: 
A novel mutation of the calcium sensing receptor gene is associated with chronic 
pancreatitis in a family with heterozygous SPINK1 mutations 
Peter Felderbauer, Peter Hoffmann, Kerem Bulut, Nikolaus Ansorge, Henrik Einwachter Frank Schmitz, Wolfgang E. Schmidt 
In the present study the authors describe a family with familial hypocalciuric hypercalcemia (FHH). In 
their dataset at least 2 family members have FHH confirmed at the phenotypical and genotypical 
level. One of these two patients has concomitant CP, and both have a heterozygous N34S SPINK1 
allele. A third member of the family has CP and probably FHH but the SPINK1 status is not known. 
After exclusion of hereditary pancreatitis, the authors make a case that (if I understand their 
message well) that the combination of the N34S SPINK1 and CASR gene mutation might induce 
CP. 
I think that this is an interesting observation, that merits widespread attention. However, the 
message that this paper conveys is buried under redundant material. I think that if the authors could bring back focus in the introduction and discussion they will have a great story. I suggest that they 
limit themselves to the findings in their patients and do not give an elaborate literature review in their 
introduction. 
As the authors mention, the relation between FHH and CP is controversial but, given the relative 
rarity of both disorders, it seems unlikely that the coexistence (reported in several families) occurs 
just by chance. I agree with the authors that the role of SPINK1 in CP is very interesting. Indeed it is 
more likely that it modifies the disease (earlier onset; ref Gastroenterology. 2000 Sep;119(3):615-23; 
Gut. 2002 May;50(5):687-92.) than that it follows a clear mendelian mutated gene=disease relation. 
However, I think that the authors take it too far down the road when they state that possession of 
SPINK1 allele explains the presence of CP in FHH. Their observation is interesting and sheds new light on the possible role of SPINK1 in CP Nevertheless, the coexistence of two mutant alleles in a single patient with FHH and CP does not prove that the N34S SPINK1 mutation is a prerequisite for 
the development of CP. Indeed as the authors argue (in their discussion part), despite the obvious 
importance of N34S mutation in CP, causality is not proven. 
What is the age of individual III-1? This is relevant as CP is a disease that has its onset later in life, 
so it is important to know to judge whether she is still at risk for the disease. It would help to include the age for all individuals in table 2. 
The index patient was tested for PRSS1 mutations, most probably R122H and/or N29I. Although it is 
not very likely that this family co-inherited FHH and autosomal dominant hereditary CP, it is not beyond reality. I think that testing for the 2 most frequent PRSS1 mutations does not completely 
exclude hereditary pancreatitis. The authors should at least sequence the complete PRSS1 gene in 
one of the affected family members (index patient) to be certain that autosomal dominant pancreatitis is not the cause of the disease in this family. 
The authors discount the observation made by Pearce et al. (1996) and suggest that because three 
different CASR mutations were found in 3 separate kindred's with FHH and CP this excludes a 
common genetic etiology. I do not think that this is true. For example, in familial Mediterranean fever, 
MEFV mutations cause the disease. A subtype of patients have concurrent amyloidosis and this 
occurs in patients with different MEFV mutations. The three mutations discovered by Pearce 
(Asn178Asp, Arg220Gln and Pro221Ser) were all in the same extracellular domain of the protein, and in theory (because of their similar location) could lead to a similar phenotype (FHH and CP). 
In the discussion I would like to see a little bit more on CP in FHH and little bit less on speculation 
with respect to SPINK1. 
Minor remarks 
The introduction is too long and should be shortened to a single page. The focus of their paper is to 
discuss the relation between FHH and SPINK1 using their family as a format. I do not think that an elaborate review of the discovery of the PRSS1 gene is fitting here. I suggest that the authors focus on FHH in relation to CP and the role of SPINK1 herein. 
The paper would be more succinct if the authors would delete sentences such as "We performed a 
screening for the recently published SPINK1 mutation N34S, which showed for the first time the presence of an additional genetic mutation. It could be shown that a heterozygous transition A>G 
causing a N34S SPINK1 mutation was apparent." This could easily be abbreviated (without loss of 
information) to " Sequencing of the SPINK1 gene revealed a heterozygous N34S mutation." 
Along the same line, the line: "It is therefore another indicator to strengthen the hypothesis of the 
SPINK1 mutations to be not a disease causing, but modifying factor' is tortuous, please shorten to 
something like: "This strengthens the hypothesis that SPINK1 mutations rather modify the disease phenotype than actually cause CP." 
The fact that the "the calcium sensing receptor was recently characterized in human pancreatic 
acinar and duct cells [29]" does not immediately infer a role of this receptor in the pathophysiology of 
pancreatitis. 
Figure 3 (and table 2) should tell the reader whole story, but are needlessly complicated. It is the 
most compelling piece of data the paper carries, yet it is difficult to ascertain. Why are there two 
lines for CASR wildtype and CASR mutation? You could argue that all patients with a CASR 
mutations have wildtype on the other allele? I suggest to delete the reference to wildtype here. In my 
PDF copy I could not see the difference between the signs for family members without genetic 
testing and those with familiar hypocalciuric hypercalcemia (FHH) as they are similar. Please 
correct. My suggestion is further that they indicate the N34S SPINK1 mutation directly in the figure, similar to what they have done for the CASR mutation (indicated by a star). 
Legends Figure 3: I:1 deceased in 971; probably in 1971. 
Conclusion 
"although from the symptomatic brother, where a FHH was diagnosed by clinical parameters, a blood sample for N34S testing could not be obtained. Looking at the family tree and his medical history of recurrent episodes of pancreatitis, a N34S mutation must be at least suspected." is 
speculative and certainly not a conclusive remark. If the authors feel that this sentence is critical, 
move it to elsewhere in the paper. 
Reference 16. Edited instead of Editeded by Katunuma N, Umezawa H, Holzer H. Tokyo: Springer; 
I had difficulties to extract the meaning of the following sentence "It is currently questioned that a 
mutation in this inhibitory system which is only potent enough to inhibit 20 % of trypsinogen triggers CP ? " I would be grateful if the authors could reword this. 
Although it is probably true that, on a equimolar basis, SPINK1 can inactivate 10-20% of active 
trypsin, can the authors comment whether to their knowledge SPINK1 is present in equimolar 
concentrations to trypsin in the acinar cell? If, for example SPINK1 is more abundantly present than 
trypsin , the actual in-vivo ratio changes in favor of SPINK1. 
Likewise I failed to grasp the meaning of the sentence "Looking at our family concerning N34S 
mutations, also both healthy and symptomatic family members are present indicating the missing 
link to be the CASR gene mutation." What do the authors want to tell us? I know that N34S occurs in 
healthy and symptomatic family members, but I do not see what this has to do with "the missing link" 
JPH Drenth 
Rockville, 20 July, 2003 
Competing interests: 
None declared. 
assistant