{"paper_id":"8bdde1c2-4ee8-4e2c-8e34-63ca85bd8a2a","body_text":"Chronic pancreatitis (CP) is a progressive inflammatory disorder of the pancreas, which develops due to an interplay between environmental and genetic risk factors [ 1 ]. Research on the identification of the underlying genetic basis has been rapidly evolving; there are at least a dozen genes reported to date that may contribute to CP risk. Based on their function, these susceptibility genes and their alterations can be categorized into three distinct pathophysiological pathways [ 2 ]. Genetic variants in the so-called trypsin-dependent pathway alter premature intrapancreatic trypsinogen activation, and include the serine protease 1 and 2 ( PRSS1 ,  PRSS2 ) genes encoding human cationic and anionic trypsinogen, the serine protease inhibitor Kazal type 1 ( SPINK1 ) gene, the chymotrypsinogen C ( CTRC ) gene, and an inversion at the chymotrypsinogen B1-B2 ( CTRB1-CTRB2 ) locus [ 3 ]. Variants in the misfolding-dependent pathological pathway induce digestive enzyme misfolding and endoplasmic reticulum stress. Certain variants in  PRSS1 ,  CTRC , carboxypeptidase A1 ( CPA1 ), and carboxyl ester lipase ( CEL ) belong to this group [ 4 ]. Finally, variants in the ductal pathway of CP risk affect genes encoding different channels expressed predominantly in the pancreatic ductal epithelial cells. These include alterations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, the transient receptor potential cation channel subfamily V member 6 ( TRPV6)  gene, and the claudin 2—MORC family CW-type zinc finger 4 ( CLDN2-MORC4 ) locus [ 2 ]. More recently, protease-sensitive pancreatic lipase ( PNLIP ) variants, and the loss-of-function c.129+1G>A chymotrypsin like elastase 3B ( CELA3B ) variant have been reported to increase CP risk. The pathomechanism by which these variants promote the development of CP remains to be elucidated [ 5 ,  6 ].\nCFTR is a chloride (Cl - )/bicarbonate (HCO 3 - ) ion channel expressed in the secretory epithelia of airways, gastrointestinal tract, pancreas, reproductive organs, and exocrine glands [ 7 ]. In the pancreas it has a dual function; CFTR-mediated HCO 3 -  secretion drives the transepithelial fluid secretion in pancreatic ducts while maintaining the characteristic alkaline pH of the pancreatic juice. Mutations in  CFTR  that diminish the ion channel function and lead to impaired epithelial fluid transport cause cystic fibrosis (CF), the most common autosomal recessive disorder among European populations. When both  CFTR  alleles harbor severe loss-of-function mutations, CF with pancreatic insufficiency develops [ 8 ]. A severe mutation on one  CFTR  allele and a milder mutation on the other allele with some residual CFTR function may result in CF with pancreatic sufficiency or in CFTR-related disorders such as CP. Heterozygous carriers of  CFTR  mutations do not develop CF but exhibit increased risk for CP [ 8 ].\nThe Whitcomb laboratory proposed that certain  CFTR  mutations that are not associated with CF may be risk factors for CP by preferentially lowering the HCO 3 -  conductance and permeability of the CFTR channel (bicarbonate defective  CFTR  variants;  CFTR BD ) [ 9 ,  10 ]. Genetic and functional assays identified 9 such  CFTR BD  variants (p.R74Q, p.R75Q, p.R117H, p.R170H, p.L967S, p.L997F, p.D1152H, p.S1235R, and p.D1270N). Subsequent studies, however, failed to replicate the association of the relatively frequent variant p.R75Q with CP, raising doubt about the clinical relevance of the  CFTR BD  variants [ 11 ,  12 ]. To resolve this controversy, here we investigated the role of  CFTR BD  variants in CP by a meta-analytical approach.\n\nTwo authors independently performed a systematic search on June 7, 2022, in four databases (MEDLINE via Pubmed, Embase, Scopus, and Cochrane Library) using the following search key: ((CFTR related disorders) OR (CFTR RD) OR pancreatitis)) AND ((CFTR OR (cystic fibrosis transmembrane conductance regulator) AND (mutation* OR variant* OR polymorphism*)). To reduce the number of results in Scopus, the search was conducted within the ‘article title, abstract, keywords’ fields. Language or date restrictions were not applied. Citing (using MEDLINE via Pubmed and Google Scholar) and cited reference searches were conducted on June 23, 2022.\nThe present work is reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement ( S1 Checklist ) [ 13 ]. The protocol of the meta-analysis was registered in advance in the PROSPERO database under the registration number CRD42020163218.\nThe study selection process was completed by two authors using a reference management program (Endnote X7.5; Clarivate Analytics, Philadelphia, PA). Genetic association case-control studies with adequately defined CP patients [ 14 ] and controls investigating some or all previously reported  CFTR BD  variants (p.R74Q, p.R75Q, p.R117H, p.R170H, p.L967S, p.L997F, p.D1152H, p.S1235R, and p.D1270N) were included. Studies (i) analyzing autoimmune, hereditary, or familial chronic pancreatitis; (ii) with overlapping cohorts; and (iii) without proper allele frequency or genotype distribution data were excluded.\nEligible original studies were subjected to data collection onto a pre-defined Excel sheet by two authors independently. The following data were extracted: first author, publication year, cohort ethnicity, range and mean age of participants, etiology of CP, number of cases and controls, genotyping method, allele frequencies of  CFTR BD  variants, and  SPINK1  p.N34S carrier status. In some cases, allele frequencies were calculated from the reported genotype distribution.\nQuality of the included studies was assessed by two authors independently using the modified version of the Newcastle-Ottawa Scale (NOS) and by calculating the Hardy-Weinberg Equilibrium with the χ 2  test ( S1 Table ).\nDiscrepancies during search, selection, data extraction, and quality evaluation between authors were resolved by the corresponding author or by mutual agreement.\nCombined or individual effects of  CFTR BD  variants were assessed by calculating pooled odds ratios (OR) with 95% confidence intervals (CI) using the random-effects model with Der-Simonian Laird estimation. Results were displayed on forest plots. To determine the cumulative effect of  CFTR BD  variants, studies investigating all nine variants were included. Heterogeneity between studies was investigated with the I 2  (p≥0.1) and χ 2 tests, interpretation of results was based on the Cochrane Handbook for Systematic Reviews of Interventions version 6.3 [ 15 ]. Sensitivity analysis was carried out by repeating the quantitative synthesis while leaving out one study at a time (leave-one-out method). Where the number of included studies allowed, funnel plots were generated to rule out publication bias and the small study effect was estimated by Egger’s test. The effect of compound heterozygosity for  CFTR BD  and  SPINK1  p.N34S variants was assessed by Fisher’s exact test. Statistical analyses were performed with the Stata 15 (Stata Corp) program.\n\nThe comprehensive systematic search and selection process identified 22 case-control studies that reported on some or all of the 9  CFTR BD  variants and met the inclusion criteria for quantitative synthesis ( Fig 1 ) [ 9 ,  11 ,  12 ,  16 – 34 ]. We noted significant geographic/ethnic differences in the distribution of the  CFTR BD  variants. In the cohorts of European origin or ancestry, the overall allele frequency of all  CFTR BD  variants was 6.1% (174/2862) in patients and 3.6% (130/3592) in controls, whereas  CFTR BD  variants were nearly absent in the Indian and East-Asian cohorts ( S2  and  S3  Tables). Although  CFTR BD  variants were relatively common in an African American cohort, there was no difference between their allelic distribution in patients (10/464, 2.2%) and controls (10/476, 2.1%, OR = 1.03; 95% CI 0.42–2.49; p = 0.95). To avoid the potentially confounding effect of these geographic/ethnic disparities, we focused our analysis on cohorts of European origin. All  CFTR BD  variants were reported in the heterozygous state with the sole exception of the p.R75Q variant, which was also detected in the homozygous state in 3 patients and 1 control. Hence, our analyses considered allele frequencies only.\nThe aggregate analysis of the 9  CFTR BD  variants (p.R74Q, p.R75Q, p.R117H, p.R170H, p.L967S, p.L997F, p.D1152H, p.S1235R, and p.D1270N) showed significant association with CP (OR = 2.31, 95% CI = 1.17–4.56) ( Fig 2 ). When analyzed individually, conclusive results were obtained for 3  CFTR BD  variants, p.R75Q, p.R117H, and p.L967S. The most common variant p.R75Q showed no association with CP (OR = 1.12, 95% CI = 0.89–1.40) ( Fig 3A ). In contrast, variants p.R117H and p.L967S were significantly overrepresented in CP cases relative to controls (OR = 3.16, 95% CI = 1.94–5.14, and OR = 3.88, 95% CI = 1.32–11.47, respectively) ( Fig 3B and 3C ). Individual analysis of the remaining 6  CFTR BD  variants (p.R74Q, p.R170H, p.L997F, p.D1152H, p.S1235R, and p.D1270N) gave inconclusive results due to their low frequency in the studied cohorts ( S1 – S4  Figs). However, a pooled analysis of these 6 variants showed significant enrichment in CP cases versus controls (OR = 2.08, 95% CI = 1.38–3.13) ( Fig 4 ).\nOR, odds ratio; CI, confidence interval.\nA,  p.R75Q;  B,  p.R117H;  C,  p.L967S. OR, odds ratio; CI, confidence interval.\nOR, odds ratio; CI, confidence interval.\nNo substantial heterogeneity was observed among studies. Sensitivity analysis (leave-one-out method) revealed a significant impact of the largest cohort study conducted by Larusch et al. (2014) on the summary OR values in case of three variants; omitting this study resulted in loss of significance in case of the p.L967S and p.S1235R variants, while the calculated risk became significant in case of the p.L997F variant.\nAssessment of the Hardy-Weinberg equilibrium in control subjects for the individual  CFTR BD  variants revealed no deviations in the included studies ( S1 Table ). Based on the modified Newcastle-Ottawa Scale, all studies met the excellent-quality criteria ( S1 Table ). Funnel plots were generated for the p.R75Q, p.R117H, and p.D1152H variants ( S4 Fig ).\n\nThe pathogenic significance of  CFTR  mutations has been established not only in the development of CF, but also in CFTR-related disorders such as congenital absence of the vas deferens, chronic sinopulmonary disorders, and CP. Observations that heterozygous and compound heterozygous  CFTR  mutations are linked with CP were first reported in the late 1990s [ 35 ,  36 ]. Large cohort analyses confirmed the initial findings and determined an increased risk for heterozygous carriers of the severe p.F508del mutation (OR~2.5), and the mild p.R117H mutation (OR~4) [ 9 ,  11 ,  12 ,  18 – 20 ,  37 ]. Compound heterozygous carriers of a severe and a mild  CFTR  variants are at increased risk of developing CP and such constellations can be considered causative [ 8 ,  12 ]. It has been proposed that a subset of 9  CFTR  variants somewhat selectively reduces the permeability of CFTR to HCO 3-  and thereby increases risk for CP [ 9 ,  10 ]. According to the CFTR2 database, when combined with a CF-causing allele, these  CFTR BD  variants do not cause CF or have variable clinical effects [ 38 ].\nThe aim of the present study was to determine the risk of CP in heterozygous  CFTR BD  carriers using meta-analysis of published case-control studies. First, we investigated the association of CP with all 9  CFTR BD  variants combined, based on the assumption that these variants act via the same pathomechanism. We found that  CFTR BD  variants increased CP risk by about 2.3-fold, as estimated by the odds ratio. Next, individual analysis of the 9  CFTR BD  variants was performed. Two variants, p.R117H and p.L967S, showed significant association with CP with moderate effect sizes (OR~3.2 and 3.9, respectively). The CFTR2 database reports the p.R117H variant as a mild CF-associated mutation with variable clinical consequences influenced by the length of the poly-T tract in intron 8. The variant acts as CF-causing when found  in cis  with the T5 tract [ 38 ]. When combined with a T7 tract  in cis  and a CF-causing variant  in trans , some subjects develop CF while others do not. Notwithstanding the poly-T tract length of p.R117H carriers, CF patients are likely to remain pancreatic sufficient. With respect to CP, it seems that p.R117H increases disease risk regardless of the poly-T tract status [ 9 ,  11 ]. Unfortunately, due to the limited data on the intron 8 poly-T tract in the studies included in our meta-analysis, we could not investigate this relationship further. Mechanistically, the p.R117H is an outlier among the  CFTR BD  variants, as it significantly reduces Cl -  transport, while the other 8 variants have minimal or no impact on this CFTR function (see  Fig 1B  in [ 9 ]). A recent study demonstrated that mutation p.R117H impairs channel gating due to the loss of a hydrogen bond between the side chain of Arg117 and the backbone carbonyl group of Glu1124 [ 39 ].\nAssociation of the p.L967S variant with CP was mainly driven by a single study with the largest cohort [ 9 ], as determined by the sensitivity analysis. Since the variant was found only once in two other studies each, the overall confidence regarding the effect size of the demonstrated disease association remains tempered. The CFTR2 database indicates that the p.L967S variant has varying clinical consequences. When combined with another CF-causing variant, it may or may not cause CF. Patients with CF who have this variant are likely to be pancreatic sufficient [ 38 ].\nIn contrast to the p.R117H and p.L967S variants, variant p.R75Q was not associated with CP. According to the CFTR2 database, this variant does not cause CF when combined with a CF-causing variant [ 38 ]. Earlier studies suggested that p.R75Q elevates CP risk in subjects transheterozygous for the  SPINK1  p.N34S mutation [ 10 ,  40 ]. We were unable to perform a rigorous test of this assumption, because of the very low number of transheterozygotes in controls. We considered comparing the detected and expected number of transheterozygous CP patients, however, ethnic/geographic differences in the carrier frequency of the individual variants makes the prediction of the expected number unreliable. Furthermore, we note that there are no other examples in the genetics of CP when a variant would confer no disease risk but it would act pathogenic when combined with another risk variant. To date, all identified CP-associated genetic variants seem to be independent risk factors whose effects become multiplied in carriers of multiple variants. Finally, there is no known mechanistic link between the SPINK1 and CFTR proteins, which might suggest a direct interaction.\nDue to the relatively rare occurrence of the remaining 6  CFTR BD  variants (p.R74Q, p.R170H, p.L997F, p.D1152H, p.S1235R, and p.D1270N), individual analyses were inconclusive, although all variants showed a trend toward disease association. When we calculated their combined effect size, we found significant association with CP (OR~2.1). Variants p.D1152H and p.D1270N are listed in the CFTR2 database as having a variable clinical effect [ 38 ]. CF patients with these variants are likely to be pancreatic sufficient. The database lists p.R170H, p.L997F, and p.S1235R as non-CF causing variants. There is no entry for p.R74Q, however, another variant that affects the same position, p.R74W, is associated with a variable clinical phenotype. Taken together, it appears that this group of heterozygous  CFTR  variants increases CP risk modestly, but the effect sizes of the individual variants cannot be determined with confidence until more data becomes available.\nFinally, it is curious to note that genetic effect size correlates poorly with the reported functional defects in the  CFTR BD  variants. Thus, variant p.R75Q, which does not alter CP risk, had a significant impact on HCO 3 -  permeability and conductance (see  Fig 1E and 1F  in [ 9 ]). In contrast, variant p.L967S, which showed the highest OR in our analysis, had the smallest impact on ion permeability. We also note that the published functional analysis was performed under somewhat artificial conditions, with CFTR variants as well as WNK1 and SPAK overexpressed in transfected HEK 293T cells. It is possible, even likely, that the  CFTR BD  variants behave somewhat differently in their native environment, which might explain these discrepancies.\nTaken together, our meta-analysis confirmed that with the sole exception of p.R75Q,  CFTR BD  variants increase the risk for CP by approximately 2-4-fold. The limitation of this meta-analysis is the relatively small cohort size in many of the included studies, which likely precluded detection of some of the rare variants. Furthermore, due to the limited data available, no subgroup analyses regarding CP etiology could be performed.\n\n(PDF)\nClick here for additional data file.\nA,  p.R74Q;  B , p.R170H. OR, odds ratio; CI, confidence interval.\n(PPTX)\nClick here for additional data file.\nA,  p.L997F;  B,  p.D1152H. OR, odds ratio; CI, confidence interval.\n(PPTX)\nClick here for additional data file.\nA,  p.S1235R;  B,  p.D1270N. OR, odds ratio; CI, confidence interval.\n(PPTX)\nClick here for additional data file.\nA,  p.R75Q;  B,  p.R117H;  C,  p.D1152H.\n(PPTX)\nClick here for additional data file.\n(DOCX)\nClick here for additional data file.\n(DOCX)\nClick here for additional data file.\n(DOCX)\nClick here for additional data file.","source_license":"CC-BY-4.0","license_restricted":false}