2.3 Genetic Risk Factors
Familial clustering of TCP is well documented（Mohan et al.1989；Kambo et al.1989；Pitchumoni 1970）.One of the earliest studies reported familial aggregation in nearly 8%of TCP patients（Mohan et al.1989）.It provided evidence of vertical transmission or horizontal distribution of the disease，which hinted at the involvement of genetic factors in pathogenesis of TCP.Ours and others’attempts to fine map genetic risk factors for TCP have revealed that genes regulating both“Trypsin-Central”and“Trypsin-independent”pathways contribute to its pathogenesis.The major factors contributing to these pathways are discussed below.
2.3.1 Trypsin-Central Pathway
Observations made more than a century ago by Chiari，a German pathologist engendered the belief that pancreatitis is an autodigestive disease caused due to inappropriate activation of digestive enzymes within the pancreas itself（Chiari 1896）.Interestingly，early intracellular trypsinogen activation was noted consistently in several animal models of pancreatitis studied over the past few decades（Hofbauer et al.1998；Lerch and Gorelick 2000；Dawra et al.2011）.The discovery of PRSS1 c.365G＞A（p.R122H）association with hereditary pancreatitis further supported the trypsin-central paradigm of pancreatitis（Whitcomb et al.1996a）.This led pancreatitis researchers to investigate mechanisms of intracellular trypsinogen activation.Subsequent focus was thus on trypsin itself or other genes that regulate its activity.The major trypsin-central candidate genes studied in CP including TCP so far are:cationic trypsinogen（PRSS1，MIM276000），anionic trypsinogen（PRSS2，MIM601564），Serine Protease Inhibitor Kazal Type I（SPINK1，MIM167790），Cathepsin B（CTSB，MIM 116810）and Chymotrypsin C（CTRC，MIM601405）.
Trypsinogen genes（PRSS1 and PRSS2）
Several coding mutations in PRSS1（particularly p.A16V，p.N291 and p.R122H）are known to be associated with CP in Europeans（Whitcomb et al.1996a）.Ours and other groups have attempted to affirm this association in TCP.However，neither mutations in PRSS1 predicting susceptibility to CP in Western populations have been identified in TCP patients（Chandak et al.2002，2004）.Mutations in PRSS2 were investigated presuming the disease mechanism akin to that of PRSS1.Previous research by various groups ruled out any causal role for PRSS2 in ICP and TCP（Idris et al.2005；Chen et al.1999）.The protective role of p.G191R PRSS2 mutation，identified in Europeans（Witt et al.2006）has also not been replicated in Indians（Mahurkar et al.2009）.Additionally，no copy number mutations in PRSS1/PRSS2 have been found in TCP patients（Masson et al.2008a）.More recently，the sole genome wide association study（GWAS）in individuals of European descent identified a strong association of PRSS1-PRSS2 loci variant rs10273639 with both sporadic and alcohol-related CP（ACP）（Whitcomb et al.2012）.The results，however，could be replicated only with ACP in another European study（Derikx et al.2015）.It was argued that non-replication of results with non-alcoholic CP（NACP）was primarily due to mixture of patients with different etiologies in the GWAS.Interestingly，using TCP as a model of NACP，we have been able to replicate the association of rs10273639［OR（95%CI）=0.72（0.61-0.85）；P=3.5 × 10-5］（Paliwal et al.2016）.Moreover，this variant was found to be in perfect linkage disequilibrium（r2=1.0）with another regulatory variant rs4726576 which has been identified to be the causal variant（Boulling et al.2015）.Nevertheless，the frequency of the risk allele（AFC=0.27）was found to be significantly lower in Indians compared with the Europeans（CEU；AFc=0.59；P＜1 × 10-4）indicating a swap in major and minor allele between the two populations（Fig.11.4）.The same is reflected in the haplotype carrying the risk allele at rsl0272629/rs4726576 in PRSS1.The prevalence of PRSS1 lowering allele in Indians is consistent with other reports that the content of digestive enzymes such as trypsin，chymotrypsin etc.in pancreatic juice of Indians is 2-4 times lower compared with Europeans（Balakrishnan et al.1988）.It may thus be speculated that low levels of cationic trypsinogen undermines central role of trypsinogen in the pathophysiology of TCP.
Serine Protease Inhibitor Kazal Type I（SPINK1）
Fig.11.4 Risk allele frequency of PRSS1 rs10273639 in various groups/sub-populations including TCP patients and controls.TCP Tropical calcific pancreatitis，GIH Gujarati Indians in Houston，CEU Utah residents with ancestry from northern and western Europe，CHB Han Chinese in Beijing，China，JPT Japanese in Tokyo，Japan，YRI Yoruba in Ibadan，Nigeria
SPINK1 encodes the pancreatic secretory trypsin inhibitor（PSTI），also synthesized in acinar cells of the exocrine pancreas.It has long been considered as a line of defense against premature intrapancreatic activation of trypsin due to its ability to inhibit upto 20%of the potential trypsin activity.Following the initial discovery（Witt et al.2000），multiple studies worldwide have reported identification and association of several mutations in SPINK1 with various forms of chronic pancreatitis including TCP（Chandak et al.2002，2004；Bhatia et al.2002；Chowdhury et al.2002；Hassan et al.2002；Schneider et al.2002）.Most common of these is the c.101G＞A（p.N34S）variant.More recently the role of SPINK1 promoter variants in pathophysiology of ICP together with TCP has also been assessed（Boulling et al.2011）.A rare loss-of-function variant c.-142T＞C that disrupts the HNFl binding site thereupon leading to reduced SPINK1 expression was identified exclusively in two TCP patients.A second rare variant c.-215G＞T observed in three TCP patients however did not show any effect on SPINK1 expression.These results point to the fact that risk variants in SPINK1 promoter are rare and thus p.N34S SPINK1 remains the predominant risk predictor for TCP.
Cathepsin B（CTSB）
Human cathepsin B，a member of the peptidase Cl family，is a 339 amino acid long cysteine protease.It predominantly localizes to the lysosomes and plays an pivotal role in intracellular degradation and turnover of proteins.About three decades ago，a role for lysosomal enzymes was speculated in the pathophysiology of pancreatitis（Steer et al.1984）.This idea stemmed from an earlier observation wherein cathepsin B was shown to be capable of activating trypsinogen（Greenbaum and Hirshkowitz 1961）.Results from several subsequent studies further supported the thought（Kukor et al.2002；Szilagyi et al.2001）.Further，evidence also existed that supramaximal stimulation directs redistribution of lysosomal enzymes leading to their co-localization with digestive enzyme zymogens within intra-acinar cytoplasmic vacuoles（Saluja et al.1987）.Above observations presented CTSB as a strong candidate gene for pancreatitis.Indeed，in a study involving 306 TCP patients and 330 controls，we revealed a unique association of p.L26V CTSB polymorphism with TCP（OR=2.09，95%CI:1.55-2.81；P=0.013）（Mahurkar et al.2006）.Besides p.L26V variant，polymorphism p.S53G also had a significantly dissimilar distribution in p.N34S SPINK1 carriers and non-carriers（Mahurkar et al.2006）.These variants reside in the pro-peptide region and were proposed to lead to CTSB mis-localization to zymogen granules thus resulting in premature trypsinogen activation.However，the functional validation is still awaited.Lately，a study with moderate sample size of 150 cases and 150 controls from North India failed to replicate the previous association（Singh et al.2014）.The minor allele frequency of 0.33%in controls observed in this study is surprisingly much lesser than that of 30%reported earlier，which raises doubt on the veracity of the results.
Chymotrypsin C（CTRC）
Human chymotrypsin C，a 268 amino acid long serine protease that is secreted from the pancreas and has a chymotrypsin-like protease activity is shown to regulate trypsin（ogen）activity with high specificity in calcium-dependent manner（Nemoda and Sahin-Tóth 2006）.A fewrecent reports have documented that in humans，CTRC proteolytically regulates auto-activation of trypsinogens via two independent and seemingly conflicting mechanisms（Nemoda and Sahin-T6th 2006；Szmola and Sahin-Tóth 2007）.Considering the focal role of premature intracellular activation of cationic trypsinogen in pancreatitis，impairment of the CTRC-dependent regulation of trypsinogen was hypothesized to be critical in the pathophysiology of CP.Two studies involving individuals of Indian origin reported the association of CTRC variants with CP including TCP（Rosendahl et al.2008；Derikx et al.2009）.Nevertheless，both the studies focused on specific region of the gene in a small number of subjects.Our study（Paliwal et al.2013）in a large，ethnically matched case-control cohort，predominantly comprising TCP patients，had interesting observations.Unlike European CP patients wherein c.738_761de124（p.K247_R254de1）and c.760C＞T（p.R254W）were the key mutations，non-synonymous variants c.217G＞A（p.A73T）［OR（95%CI）=9.48（2.19-41.03）；P=2.5 × 10-4］and c.703G＞A（p.V2351）［OR（95%CI）=7.60（2.52-25.71）；P=1 × 10-5］were the major risk predictors for TCP.Functionally，p.A73T exhibits its pathogenicity by eliciting ER stress whereas p.V235I variant reduced the activity and secretion of the protein（Szmola and Sahin-Tóth 2010；Beer et al.2013）.In addition，for synonymous variant c.180C＞T heterozygous subjects had an increased risk of TCP compared with the individuals harboring wild allele［OR（95%CI）=2.44（1.81-3.30）；P=2.9× 10-9］and the risk magnified manifold for individuals homozygous for risk allele［OR（95%CI）=10.09（2.98-34.22）；P=5.7 × 10-6］.Interestingly，the repertiore of CTRC mutations identified in TCP patients was altogether different from that reported in Western CP patients，providing further stress to the earlier evidence of genetic and mutational heterogeneity in TCP.
2.3.2 Trypsin-Independent Pathways
Carboxypeptidase Al（CPA 1）
Biochemical data presented in a recent study led to the proposition that CTRC is likely a physiological co-activator of pro-carboxypeptidase Al（proCPAl）and pro-carboxypeptidase A2（proCPA2）（Szmola et al.2011）.Next to trypsinogens，proCPAl is the most abundant protein in pancreatic juice comprising over 10%of the total protein.In view of this，a large multinational collaborative study lately analyzed the involvement of CPA1in chronic pancreatitis.The genetic and functional data demonstrated the global role of CPA1variants in the pathogenesis of CP including TCP（Witt et al.2013）.However，the spectrum of mutations observed in different populations was heterogeneous.In TCP patients of Indian origin，three novel non-synonymous variants（p.D32H，p.R169H and p.Y308H）identified were present solely in patients whereas the frequency of p.A208T was similar between patients and controls.Apparent activities of p.D32H，p.R169H and p.Y308H variants were 79%，24%and 3%respectively of the wild protein，whereas their respective relative secretion levels were 75%，23%and 17%of the native protein.This supports the earlier presumption that the mutational spectrum in various CP-associated genes is different in TCP than in other types of CP in the Western world.(https://www.daowen.com)
Pancreatic Stone Protein（Regla）
Pancreatic calcification occurs in majority of CP cases.Human Reg protein accounts for nearly 10-14%of total protein content in pancreatic juice.It was previously identified as a major component of protein matrix of calculi in patients with ACP and hence termed as pancreatic stone protein（PSP）（De Caro et al.1979）.Conflicting reports exist for its role in the process of calcification.In vitro observations that it is able to delay the crystal nucleation process as well as inhibit the growth of preformed calcium carbonate crystals led to the proposition that PSP could act as a crystal growth inhibitor in the normally supersaturated pancreatic juice（Multigner et al.1983）.Prior reports of reduced PSP concentration in the pancreatic juice of CP patients supported this hypothesis（Giorgi et al.1989）.However，later reports challenged the idea by demonstrating that PSP does not interact specifically with calcium carbonate crystals（De Reggi et al.1998）.Further，it was argued that pancreatic juice of CP patients instead may have higher levels of smaller PSP isoforms like pancreatic thread protein（PTP）that tend to precipitate because they are insoluble at the physiological pH（Cerini et al.1999）.These observations made ReglA an interesting candidate gene.The fact that Reg proteins are also associated with pancreatic islet regeneration，diabetogenesis and amelioration of surgical diabetes（Watanabe et al.1994）in animal models compelled earlier researchers to look at its role in FCDP（Boonyasrisawat et al.2002；Hawrami et al.1997）.These studies conducted on a limited number of samples，however，could not gather any evidence in support.Our study on relatively larger sample size also failed to detect any association with TCP or FCPD suggesting that polymorphisms in Reg1A are unlikely to contribute to the pathogenesis of pancreaticolithogenesis in TCP（Mahurkar et al.2007）.
Glycoprotein 2（GP2）
Several preliminary studies in various species reported that a single glycoprotein of 75-92 kDa represents bulk（＞25-30%）of the zymogen granule membrane protein（MacDonald and Ronzio 1972；Paquet et al.1982；LeBel and Beattie 1984a，b）.This glycoprotein，GP2，was identified as an integral membrane.It is attached to the cytoplasmic leaflet of the membrane via a glycosyl phosphatidylinositol linkage and is susceptible to release from the membrane by the enzyme phosphatidylinositol-specific phospholipase C（Tsujii-Hayashi et al.2002）.GP2 has been reported to be the most abundant protein in the pancreatic acinar cells and is not detected in the endocrine pancreas.Almost 50%of the protein is soluble in granule content and is derived from the membrane-bound precursor as a result of shedding after arrival the zymogen granule（Havinga et al.1985）.A form of the molecule is also released into the pancreatic juice where it undergoes coversion into a stable protein aggregate（Rindler and Hoops 1990）.Considering the fact that intra-ductal plug formation is one of the early events in the pathophysiology of CP and GP2 is a major constituent of these plugs（Freedman et al.1993），it was speculated that variations in GP2 may possibly influence the risk of duct obstruction and hence chronic pancreatitis.To address this question we screened exons 3 and 9 of the GP2 gene in TCP patients and identified two variants of which the variant c.1275A＞G showed a disease predisposing effect that was in contrast to the observations made in French white patients（Masson et al.2010）.A recent study has shown that c.1275A＞G variant significantly reduces the rate of exon 9 inclusion and hence the last 116 amino acids are substituted by 15 new amino acids.Consequently there is a decrease in the ratio of full-length transcript:total transcript compared with that derived from the wild type（Boulling et al.2010）.These changes may lead to structural alterations and hence compromise the function of the protein.
Transcription Factor 7-Like 2（TCF7L2）
TCP patients often progress to developing secondary diabetes also known as FCPD.Interestingly，FCPD patients present characteristics of both，type 1（T1D）and type 2（T2D）diabetes.An early study from our group proposed that the nature and mechanism of diabetes in these patients can be inferred by investigating a known genetic risk factor for T1D or T2D（Mahurkar et al.2008）.Therefore，two polymorphisms（rs7903146 and rs 12255372）in transcription factor 7 like protein 2（TCF7L2，MIM602228）that were reported to be strongly associated with T2D were genotyped in TCP and FCPD patients.Although neither polymorphism showed an independent association with FCPD，the data indicated that these polymorphisms might interact with SPINKI and CTSB mutations and result in FCPD（Mahurkar et al.2008）.
Claudin 2（CLDN2）
The CP GWAS，in addition to rs10273639 in PRSS1，also reported the association of two other common variants namely rs12688220 and rs7057398（Whitcomb et al.2012）.These variants lie in the CLDN2-MORC4 loci on X chromosome.Similar to PRSS1 rs10273639，the European replication study could not establish the association of these two SNPs with NACP（Derikx et al.2015）.We addressed this inconsistency by genotyping a large case-control cohort（Paliwal et al.2016）.We observed a strong association of both，rs12688220［OR（95%CI）=1.54（1.35-1.75）；P=1.22 × 10-10］and rs7057398［OR（95%CI）=1.50（1.29-1.75）；P=1.44 × 10-7］with TCP.Noticeably，although the major alleles for these variants are same in different populations，their frequencies vary considerably.For instance，the frequency of the proposed risk（T）allele at rs12688220 in Indians（AFT=0.48）is nearly double compared with other Asian（AFT=0.24-0.28；P＜1 × 10-4）or European（AFT=35；P=7× 10-4）populations（Fig.11.5）.Similarly，for rs7057398，the risk allele（C）frequency is highest in Indians（AFc=0.52）compared with other Asian or European population（AFc=0.28-0.35；P=6 × 10-3）（Fig.11.5）.Although the GWAS reported altered localisation of CLDN2 to the baso-lateral membrane of acinar cells in patients carrying high-risk genotype at rs12688220，the exact mechanism is still unclear.A recent report attributed half of pancreatic injury during pancreatitis to NFkB-mediated inflammatory pathways（Dawra et al.2011）.CLDN2 promoter harbors binding site for NFkB（Sakaguchi et al.2002）.CLDN2 expression is shown to correlate with inflammation and increases under conditions of stress and injury by regulation through different cytokines（Suzuki et al.2011）.Thus，it is likely that these variants might be involved in the altered inflammatory response.However，further investigation is needed to confirm its role in the etiopathogenesis of chronic pancreatitis.
Fig.11.5 Risk allele frequency of CLDN2-MORC4 loci variants（rs12688220 and rs7057398）in various groups/sub-populations including TCP patients and controls.TCP Tropical calcific pancreatitis，GIH Gujarati Indians in Houston，CEU Utah residents with ancestry from northern and western Europe，CHB Han Chinese in Beijing，China，JPT Japanese in Tokyo，Japan，YRI Yoruba in Ibadan，Nigeria
Carboxyl-Ester Lipase（CEL）
CEL is a non-specific lipolytic enzyme capable of hydrolyzing a variety of lipid substrates and aids in their digestion and absorption.It is a major constituent of pancreatic juice.Mutation in CEL lead to maturity-onset diabetes of the young and pancreatic exocrine dysfunction（Raeder et al.2006）.CEL-HYB is a fusion gene between carboxyl ester lipase（CEL）and its identical pseudo gene（CELP）arranged in tandem.CEL-CELP genic constellation is one of the good examples for possible occurrence of non-allelic homologous recombination（NAHR）.The product of NAHR event at this region named CEL-HYB is indeed reported to be associated with chronic pancreatitis in European populations（Fjeld et al.2014）.Expression of CEL-HYB in cellular models showed reduced lipolytic activity，impaired secretion，prominent intra acinar accumulation and induced autophagy.Our recent study from three Asian populations including Indians failed to detect this recombination allele and replicate the association（Zou et al.2016）.We，instead found an alternative NAHR allele CELHYB2（Fig.11.6）with the breakpoint region spanning 239bp between intron9-exon10.The CEL-HYB2 that we observed in the Indian cohort corresponds to the minor allele of CELHYB2（CEL-HYB2b）and is also not associated with TCP.The non-association of CEL-HYB2a/b could be because of the fact that its transcribed mRNA significantly undergoes non-sense mediated decay owing to the absence of truncated protein and its toxic effects.
Fig.11.6 A representative gel image showing presence of CEL-HYB2b allele in an individual（lane 12）.L，1 Kb ladder