Review for 10.1186/1471-230X-3-34 (2003)

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  17 Jul 2003 Version: Date: 
 Heiko Witt Reviewer: 
 A paper whose findings are important to those with closely related research Level of interest: 
interests 
 Other (see below) Advice on publication: 
Felderbauer and co-workers describes a family with familial hypocalciuric hypercalcemia, 
pancreatitis and mutations in CASR and SPINK1. Although the subject is of interest, the manuscript is poorly written and contain many errors. 
Introduction 
"Risk factors for CP were identified in the last decades, which can be summarized as 
toxic-metabolic, idiopathic, genetic, autoimmune, recurrent and obstructive (TIGAR-O risk factor classification) [3]." Recurrent is a clinical appearance of pancreatitis, not a risk factor. Idiopathic means the absence of 
any known risk factor, "non-A cannot be A" (Aristoteles). 
"Since 1996 the understanding of genetic risk factors for CP extended with the discovery of different 
gene defects causing hereditary pancreatitis (HP), setting, as Whitcomb et al. described, "the stage 
for new preventive and management strategies for hereditary and sporadic acute and chronic 
pancreatitis" [4]." 
Which new preventive and management strategies were set by the discovery of these gene defects ? Both sentences are citations only, I know, but are they well chosen ? 
"A French and an American group were the first to identify chromosome 7q35 as one putative locus 
for HP [5,6]." 
A French and two American groups (Pandya and co-workers, Genomics 1996). 
"The disease gene was mapped to be a point mutation A>T in the exon 3 of the protease serine 1 
(PRSS1) gene which encodes for cationic trypsinogen (R122H represents 70% of the cases with HP." 
A gene cannot be a point mutation. 
R122H represent maybe 70% of trypsin positive cases, but not 70% of cases with HP. 
"The originally described mutation R122H makes trypsin resistant to hydrolysis while the additional 
mutations that were supposed to facilitate early trypsin activation..." Old concept of R122H action. See the publications of Sahin-Toth and colleagues. 
Pfutzer et al. showed, that neither an autosomal dominant disorder with a low penetrance pattern ... 
nor an autosomal recessive trait can be used as a model for disease inheritance [18] ... and the thesis of a modifying, but not causative factor, for SPINK1 mutations was proposed by different groups [18,20]." 
In both references (18 and 20) Dave Whitcomb and John Neoptolemos as group leaders are 
authors. What is a "modifier gene" ? E.g. MBL2 might be a pulmonary modifier in CF-related lung disease, but will not cause the disease i.e. you have to bear two CFTR mutations to get CF, but the 
pulmonary phenotype is not related to the class or type of CFTR mutation. To suffer from CF you 
need (in the vast majority) 2 CFTR mutations. Without these mutations you will not get CF. As "proof 
of concept", the modifier gene mutation will not cause disease alone and will be found in a similar 
frequency in the general population. This is clearly not true for SPINK1. Approximately 10% of 
patients with ICP are homozygous for N34S, compared to an estimated homozygosity frequency of 
1:10,000 to 1:40,000 (assuming a heterozygote frequency of 1% to 2% in Caucasians). Which genetic defect in which gene should be modified by SPINK1 ? First you have to identify the gene, than you might talk about modifier genes... 
Gene symbols are labelled in italics. 
"The correlation was first observed in patients with hyperparathyroidism, while trypsin activation and 
stabilization are the suspected mechanism [4]." 
Secretion stimulus ? 
Case Presentation 
The index patient did not show clinical improvement. Were calcium levels normal or elevated after 
1996 ? 
Marseilles-Rome 
Methods 
"Coded DNA samples..." 
Looking at the family tree I assume that the samples were not made anonymous. 
I assume that the fluoresceine is 3-prime in both FRET probes and not 5-prime. 
Discussion 
"...is an elevated Ca 2+ concentration in the extracellular compartment of the pancreatic acinar cell." 
What is an extracellular compartment of a cell ? 
"Although these investigations underscore the importance of N34S mutations in ICP, they do not 
provide for a causative line. Hence, SPINK1 was suggested by Threadgold et al. to "rather act as a 
disease modifying factor but not causing the disease" [20]. Moreover, the role of SPINK1 mutations 
as a disease modifier is further substantiated as independent groups published data of SPINK1 
mutations (N34S) in patients with tropical pancreatitis [32,33]. " 
Why do these studies underscore ? 
Again, association studies can show an association between a genetic variation and a disease or 
even the lack of any association. If there is an association, this association may be strong or week. 
That's all. Functional studies or animal models may can give more answers. (By the way, SPINK 
knock-out mice do not possess any pancreas.) The very strong association of TCP and SPINK1 substantiates more are causative than modifying influence. 
And see above... (introduction) 
Conclusion 
"The presence of two putatively CP related mutations makes our case unique." 
Numerous studies have shown the presence of two CP related mutations. E.g. Noone et al. 
Gastroenterology 2001, Audrezet et al. Eur J Hum Genet 2002, Gomez-Lira Eur J Hum Genet 2003. 
"Coincidence seems unlikely, 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." 
Last week, I've written a record of genetic testing for a young boy with ICP. He was heterozygous for 
N34S and the mutation was inherited by an unaffected parent. The sister suffers also from ICP, but 
bears 2 wild-type alleles. 
"This is therefore the first report to show a "modifying role" of the SPINK1 mutation in one family and 
the first case to show that mutations in the CASR gene might play a role SPINK1 related chronic pancreatitis." 
I guess if you screen 100 SPINK1 mutated ICP patients, you will not find any CASR mutation. 
Nevertheless, you may find SPINK1 mutations in patients with hyperparathyroidism as described by Joost Drenth and co-workers (Gut 2002). 
References 
2: DiMagno 
12,24: Pfutzer, Kruger (we Germans love our umlauts) 
6,8,19: French people love their accents (Ferec, Raguenes etc) 
33 should be cited before 32 (earlier submission and acceptance). 
Moreover, the most important reference is missing (G Chandak, J Med Genet 2002). 
This was the first study published about TCP and SPINK1, and is by far the best study of all studies published dealing with TCP and SPINK1. 
Heiko Witt, MD 
Competing interests: 
None declared.