3.1 Identification of a Protective Single Nucleoti...
In the GWAS study，the minor T allele of a common single nucleotide polymorphism（SNP），rs10273639:C/T，located 408 base-pairs upstream of the translational initiation codon of the PRSS1 gene，was found to be significantly overrepresented in the control group as compared with the patient group（Whitcomb et al.2012）.This finding has been supported by several follow-up studies（Avanthi et al.2015；Derikx et al.2015；Giri et al.2016；Masamune et al.2015；Paliwal et al.2016）.Consistent with conferring a protective role against chronic pancreatitis，the minor T allele was associated with lower expression of PRSS1 mRNA in human pancreatic tissue as compared with the major C allele（Whitcomb et al.2012）.However，as with many GWAS findings［for a review，see（Zhang et al.2014）］，whether the rs10273639 T allele is the causal variant or not remains unknown.
In general，proximal promoter regions of tissue-specific genes are enriched for regulatory elements（Forrest et al.2014）.We wondered whether rsl0273639 was in linkage disequilibrium with another polymorphism within the proximal promoter of PRSS1.We therefore resequenced the promoter region of PRSS1 in 287 French individuals of European ancestry and found that rs4726576:C/A，which is located 204 bp upstream of the translational initiation codon of PRSS1，is in perfect linkage disequilibrium（r2=1）with rs10273639:C/T.Employing promoter reporter gene assays in both rat pancreatic acinar AR42J cell and mouse parenchymal pancreatic tissue models，we provided evidence to support the view that it is the minor A allele of rs4726576 which underlies the aforementioned protective effect（Boulling et al.2015）.(https://www.daowen.com)
The identification of a polymorphism other than that identified in the original studies as being the causal variant may have diagnostic implications since the two variants of interest may not always be in perfect linkage disequilibrium.Indeed，when we calculated linkage disequilibrium between rs4726576 and rs10273639 from the 1000 Genomes Project Phase 3 data，we found that the degree of linkage disequilibrium varies significantly between five metapopulations:r2 values in South Asian，European，Asian，American，and African were 1.000，0.996，0.994，0.977，and 0.797，respectively（Boulling et al.2015）.The problem appeared to be most serious in the African meta-population:whereas the TC diplotype（i.e.，rsl0273639T in cis with rs4726576C）carriers，who accounted for 5.5%of subjects，should be regarded as carrying a risk allele，they would have been ascertained as carrying a protective allele if the minor T allele of rs10273639 were thought to be causal（Nemeth and Sahin-Toth 2014）.
More recently，we extended our resequencing of the PRSS1 promoter region（Boulling etal.2015）to 626 French Caucasians（242 ICP patients and 384 controls）.We discovered three additional variants（c.-184G＞A，c.-173C＞T and c.-147C＞T），each being found only once in either a patient or a control.We analysed these three variants，together with a known PRSS1 promoter variant（c.-30_-28de1TCC）long considered to be causative for chronic pancreatitis（Férec et al.1999），by luciferase promoter reporter assay in AR42J cells treated with dexamethasone.This analysis revealed that c.-30_-28de1TCC resulted in reduced rather than increased PRSS1 gene expression，suggesting that it is not a chronic pancreatitis risk factor as originally claimed.Moreover，we provided evidence that c.-147C＞T probably confers protection against chronic pancreatitis by reducing the affinity of an ATF4 transcription factor binding site（Boulling et al.2016）.That loss of function regulatory variants in the promoter region of the PRSS1 gene protect against chronic pancreatitis（Boulling et al.2015，2016；Whitcomb et al.2012）complement previous observations that gains of trypsinogen copy number cause or predispose to the disease（Le Maréchal et al.2006；Masson et al.2008a）.