Combining full-length gene assay and SpliceAI to interpret the splicing impact of all possible SPINK1 coding variants.

BACKGROUND: Single-nucleotide variants (SNVs) within gene coding sequences can significantly impact pre-mRNA splicing, bearing profound implications for pathogenic mechanisms and precision medicine. In this study, we aim to harness the well-established full-length gene splicing assay (FLGSA) in conjunction with SpliceAI to prospectively interpret the splicing effects of all potential coding SNVs within the four-exon SPINK1 gene, a gene associated with chronic pancreatitis. RESULTS: Our study began with a retrospective analysis of 27 SPINK1 coding SNVs previously assessed using FLGSA, proceeded with a prospective analysis of 35 new FLGSA-tested SPINK1 coding SNVs, followed by data extrapolation, and ended with further validation. In total, we analyzed 67 SPINK1 coding SNVs, which account for 9.3% of the 720 possible coding SNVs. Among these 67 FLGSA-analyzed SNVs, 12 were found to impact splicing. Through detailed comparison of FLGSA results and SpliceAI predictions, we inferred that the remaining 653 untested coding SNVs in the SPINK1 gene are unlikely to significantly affect splicing. Of the 12 splice-altering events, nine produced both normally spliced and aberrantly spliced transcripts, while the remaining three only generated aberrantly spliced transcripts. These splice-impacting SNVs were found solely in exons 1 and 2, notably at the first and/or last coding nucleotides of these exons. Among the 12 splice-altering events, 11 were missense variants (2.17% of 506 potential missense variants), and one was synonymous (0.61% of 164 potential synonymous variants). Notably, adjusting the SpliceAI cut-off to 0.30 instead of the conventional 0.20 would improve specificity without reducing sensitivity. CONCLUSIONS: By integrating FLGSA with SpliceAI, we have determined that less than 2% (1.67%) of all possible coding SNVs in SPINK1 significantly influence splicing outcomes. Our findings emphasize the critical importance of conducting splicing analysis within the broader genomic sequence context of the study gene and highlight the inherent uncertainties associated with intermediate SpliceAI scores (0.20 to 0.80). This study contributes to the field by being the first to prospectively interpret all potential coding SNVs in a disease-associated gene with a high degree of accuracy, representing a meaningful attempt at shifting from retrospective to prospective variant analysis in the era of exome and genome sequencing.

Pancreas-directed AAV8-hSPINK1 gene therapy safely and effectively protects against pancreatitis in mice.

OBJECTIVE: Currently, there is no cure for chronic pancreatitis (CP). Germline loss-of-function variants in SPINK1 (encoding trypsin inhibitor) are common in patients with CP and are associated with acute attacks and progression of the disease. This preclinical study was conducted to explore the potential of adeno-associated virus type 8 (AAV8)-mediated overexpression of human SPINK1 (hSPINK1) for pancreatitis therapy in mice. DESIGN: A capsid-optimised AAV8-mediated hSPINK1 expression vector (AAV8-hSPINK1) to target the pancreas was constructed. Mice were treated with AAV8-hSPINK1 by intraperitoneal injection. Pancreatic transduction efficiency and safety of AAV8-hSPINK1 were dynamically evaluated in infected mice. The effectiveness of AAV8-hSPINK1 on pancreatitis prevention and treatment was studied in three mouse models (caerulein-induced pancreatitis, pancreatic duct ligation and Spink1 c.194+2T>C mouse models). RESULTS: The constructed AAV8-hSPINK1 vector specifically and safely targeted the pancreas, had low organ tropism for the heart, lungs, spleen, liver and kidneys and had a high transduction efficiency (the optimal expression dose was 2×1011 vg/animal). The expression and efficacy of hSPINK1 peaked at 4 weeks after injection and remained at significant level for up to at least 8 weeks. In all three mouse models, a single dose of AAV8-hSPINK1 before disease onset significantly alleviated the severity of pancreatitis, reduced the progression of fibrosis, decreased the levels of apoptosis and autophagy in the pancreas and accelerated the pancreatitis recovery process. CONCLUSION: One-time injection of AAV8-hSPINK1 safely targets the pancreas with high transduction efficiency and effectively ameliorates pancreatitis phenotypes in mice. This approach is promising for the prevention and treatment of CP.

Pancreatitis affects gut microbiota via metabolites and inflammatory cytokines: an exploratory two-step Mendelian randomisation study.

Previous studies have observed relationships between pancreatitis and gut microbiota; however, specific changes in gut microbiota abundance and underlying mechanisms in pancreatitis remain unknown. Metabolites are important for gut microbiota to fulfil their biological functions, and changes in the metabolic and immune environments are closely linked to changes in microbiota abundance. We aimed to clarify the mechanisms of gut-pancreas interactions and explore the possible role of metabolites and the immune system. To this end, we conducted two-sample Mendelian randomisation (MR) analysis to evaluate the casual links between four different types of pancreatitis and gut microbiota, metabolites, and inflammatory cytokines. A two-step MR analysis was conducted to further evaluate the probable mediating pathways involving metabolites and inflammatory cytokines in the causal relationship between pancreatitis and gut microbiota. In total, six potential mediators were identified in the causal relationship between pancreatitis and gut microbiota. Nineteen species of gut microbiota and seven inflammatory cytokines were genetically associated with the four types of pancreatitis. Metabolites involved in glucose and amino acid metabolisms were genetically associated with chronic pancreatitis, and those involved in lipid metabolism were genetically associated with acute pancreatitis. Our study identified alterations in the gut microbiota, metabolites, and inflammatory cytokines in pancreatitis at the genetic level and found six potential mediators of the pancreas-gut axis, which may provide insights into the precise diagnosis of pancreatitis and treatment interventions for gut microbiota to prevent the exacerbation of pancreatitis. Future studies could elucidate the mechanism underlying the association between pancreatitis and the gut microbiota.

Exploring the enigmatic association between PNLIP variants and risk of chronic pancreatitis in a large Chinese cohort.

BACKGROUND & AIMS: Protease-sensitive PNLIP variants were recently associated with chronic pancreatitis (CP) in European populations. The pathological mechanism yet remains elusive. Herein, we performed a comprehensive genetic and functional analysis of PNLIP variants found in a large Chinese cohort, aiming to further unravel the enigmatic association of PNLIP variants with CP. METHODS: All coding and flanking intronic regions of the PNLIP gene were analyzed for rare variants by targeted next-generation sequencing in 1082 Chinese CP patients and 1196 controls. All novel missense variants were subject to analysis of secretion, lipase activity, and proteolytic degradation. One variant was further analyzed for its potential to misfold and induce endoplasmic reticulum (ER) stress. p.F300L, the most common PNLIP variant associated with CP, was used as a control. RESULTS: We identified 12 rare heterozygous PNLIP variants, with 10 being novel. The variant carrier frequency did not differ between the groups. Of them, only the variant p.A433T found in a single patient was considered pathologically relevant. p.A433T exhibited increased susceptibility to proteolytic degradation, which was much milder than p.F300L. Interestingly, both variants exhibited an increased tendency to misfold, leading to intracellular retention as insoluble aggregates, reduced secretion, and elevated ER stress. CONCLUSIONS: Our genetic and functional analysis of PNLIP variants identified in a Chinese CP cohort suggests that the p.A433T variant and the previously identified p.F300L variant are not only protease-sensitive but also may be potentially proteotoxic. Mouse studies of the PNLIP p.F300L and p.A433T variants are needed to clarify their role in CP.

Expanding ACMG variant classification guidelines into a general framework.

BACKGROUND: The American College of Medical Genetics and Genomics (ACMG)-recommended five variant classification categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign) have been widely used in medical genetics. However, these guidelines are fundamentally constrained in practice owing to their focus upon Mendelian disease genes and their dichotomous classification of variants as being either causal or not. Herein, we attempt to expand the ACMG guidelines into a general variant classification framework that takes into account not only the continuum of clinical phenotypes, but also the continuum of the variants' genetic effects, and the different pathological roles of the implicated genes. MAIN BODY: As a disease model, we employed chronic pancreatitis (CP), which manifests clinically as a spectrum from monogenic to multifactorial. Bearing in mind that any general conceptual proposal should be based upon sound data, we focused our analysis on the four most extensively studied CP genes, PRSS1, CFTR, SPINK1 and CTRC. Based upon several cross-gene and cross-variant comparisons, we first assigned the different genes to two distinct categories in terms of disease causation: CP-causing (PRSS1 and SPINK1) and CP-predisposing (CFTR and CTRC). We then employed two new classificatory categories, "predisposing" and "likely predisposing", to replace ACMG's "pathogenic" and "likely pathogenic" categories in the context of CP-predisposing genes, thereby classifying all pathologically relevant variants in these genes as "predisposing". In the case of CP-causing genes, the two new classificatory categories served to extend the five ACMG categories whilst two thresholds (allele frequency and functional) were introduced to discriminate "pathogenic" from "predisposing" variants. CONCLUSION: Employing CP as a disease model, we expand ACMG guidelines into a five-category classification system (predisposing, likely predisposing, uncertain significance, likely benign, and benign) and a seven-category classification system (pathogenic, likely pathogenic, predisposing, likely predisposing, uncertain significance, likely benign, and benign) in the context of disease-predisposing and disease-causing genes, respectively. Taken together, the two systems constitute a general variant classification framework that, in principle, should span the entire spectrum of variants in any disease-related gene. The maximal compliance of our five-category and seven-category classification systems with the ACMG guidelines ought to facilitate their practical application.

Classification of PRSS1 variants responsible for chronic pancreatitis: An expert perspective from the Franco-Chinese GREPAN study group.

BACKGROUND: PRSS1 was the first reported chronic pancreatitis (CP) gene. The existence of both gain-of-function (GoF) and gain-of-proteotoxicity (GoP) pathological PRSS1 variants, together with the fact that PRSS1 variants have been identified in CP subtypes spanning the range from monogenic to multifactorial, has made the classification of PRSS1 variants very challenging. METHODS: All currently reported PRSS1 variants (derived primarily from two databases) were manually reviewed with respect to their clinical genetics, functional analysis and population allele frequency. They were classified by variant type and pathological mechanism within the framework of our recently proposed ACMG/AMP guidelines-based seven-category system. RESULTS: The total number of distinct germline PRSS1 variants included for analysis was 100, comprising 3 copy number variants (CNVs), 12 5' and 3' variants, 19 intronic variants, 5 nonsense variants, 1 frameshift deletion variant, 6 synonymous variants, 1 in-frame duplication, 3 gene conversions and 50 missense variants. Based upon a combination of clinical genetic and functional analysis, population data and in silico analysis, we classified 26 variants (all 3 CNVs, the in-frame duplication, all 3 gene conversions and 19 missense) as "pathogenic", 3 variants (missense) as "likely pathogenic", 5 variants (four missense and one promoter) as "predisposing", 13 variants (all missense) as "unknown significance", 2 variants (missense) as "likely benign", and all remaining 51 variants as "benign". CONCLUSIONS: We describe an expert classification of the 100 PRSS1 variants reported to date. The results have immediate implications for reclassifying many ClinVar-registered PRSS1 variants as well as providing optimal guidelines/standards for reporting PRSS1 variants.

Translation and validation of a Chinese version of the pancreatitis quality of life instrument (PANQOLI) in patients with chronic pancreatitis.

PURPOSE: Chronic pancreatitis (CP) is a chronic fibroinflammatory pancreatic disease that severely impacts patients' quality of life (QoL). The Pancreatitis Quality of Life Instrument (PANQOLI) is an 18-item measure specifically designed to assess QoL amongst patients with CP. This study aimed to develop a Chinese version of PANQOLI and assess its reliability and validity in the Chinese CP cohort. METHODS: Translation was performed according to forward-backwards translation steps and transcultural adaptation. Five hundred Mandarin Chinese-speaking patients with CP were enrolled, 250 for the exploratory factor analysis (EFA) and 250 for the confirmatory factor analysis (CFA). Item analysis, reliability analysis (internal consistency, split-half reliability, test-retest reliability), and validity analysis (content validity, construct validity, and convergent validity) were performed. RESULTS: Item analysis of the Chinese version of PANQOLI revealed that the absolute t values of all items were > 3. Reliability analysis showed that Cronbach's α coefficient was 0.868, split-half coefficient was 0.934, and intraclass correlation coefficient was 0.859, demonstrating excellent reliability. For content validity, item level content validity index (I-CVI) ranged from 0.8 to 1.0, and average of I-CVI scores across all items (S-CVI/Ave) was 0.91. In construct validity analysis, EFA produced four dimensions after rotation, and results of CFA showed χ2/df = 2.346, comparative fit index (CFI) = 0.929, Tucker-Lewis index (TLI) = 0.915, and root-mean-square error of approximation (RMSEA) = 0.074. The analysis of convergent validity indicated that the Chinese version of PANQOLI was moderately correlated with the physical (r = 0.436, P < 0.001) and mental component summary (r = 0.518, P < 0.001) of the 36-Item Short Form Health Survey. CONCLUSION: The Chinese version of PANQOLI appears to be culturally appropriate, reliable, and valid for assessing the QoL amongst Chinese patients with CP.

Status quo of the public's knowledge of probiotics based on video-sharing platforms.

BACKGROUND: Probiotics have been deemed multipotent and unprecedentedly applied in the health field recently. However, there are challenges in promoting credible and reliable resources while avoiding misinformation regarding probiotics for the public. METHODS: This study analysed 400 eligible probiotic-related videos selected from YouTube, and the three most popular video-sharing platforms (Bilibili, Weibo and TikTok) in China. Video retrieval was performed on September 5th, 2022. GQS and tailored DISCERN tool assess each video's quality, usage, and reliability. A comparative analysis of videos from different sources was carried out. RESULTS: The identity distribution of probiotic video-producers was predominantly experts (n = 202, 50.50%), followed by amateurs (n = 161, 40.25%) and health-related institutions (n = 37, 9.25%). The videos' content category mainly discussed the function of probiotics (n = 120, 30%), the way to choose suitable products (n = 81, 20.25%), and the methods for taking probiotics (n = 71, 17.75%).The overall quality of videos was moderate (3/5 point) assessed by GQS, while the usage (1/6 point) and reliability (2/5 point) detailing probiotics assessed by tailored DISCERN tool were poor. The attitude of probiotic video-producers was primarily positive (n = 323, 80.75%), followed by neutral (n = 52, 13.00%) and negative (n = 25, 6.25%) (P < 0.001). CONCLUSIONS: The current study showed that videos on social media platforms publicise important information including the concepts, usage, and precautions of probiotics to the public. But the overall quality of uploaded videos about probiotics was unsatisfactory. More efforts are needed to improve the higher-quality content of probiotic-related online videos and better propagate probiotic knowledge to the public in the future.

Trypsinogen (PRSS1 and PRSS2) gene dosage correlates with pancreatitis risk across genetic and transgenic studies: a systematic review and re-analysis.

Trypsinogen (PRSS1, PRSS2) copy number gains and regulatory variants have both been proposed to elevate pancreatitis risk through a gene dosage effect (i.e., by increasing the expression of wild-type protein). However, to date, their impact on pancreatitis risk has not been thoroughly evaluated whilst the underlying pathogenic mechanisms remain to be explicitly investigated in mouse models. Genetic studies of the rare trypsinogen duplication and triplication copy number variants (CNVs), and the common rs10273639C variant, were collated from PubMed and/or ClinVar. Mouse studies that analyzed the influence of a transgenically expressed wild-type human PRSS1 or PRSS2 gene on the development of pancreatitis were identified from PubMed. The genetic effects of the different risk genotypes, in terms of odds ratios, were calculated wherever appropriate. The genetic effects of the rare trypsinogen duplication and triplication CNVs were also evaluated by reference to their associated disease subtypes. We demonstrate a positive correlation between increased trypsinogen gene dosage and pancreatitis risk in the context of the rare duplication and triplication CNVs, and between the level of trypsinogen expression and disease risk in the context of the heterozygous and homozygous rs10273639C-tagged genotypes. We retrospectively identify three mouse transgenic studies that are informative in relation to the pathogenic mechanism underlying the trypsinogen gene dosage effect in pancreatitis. Trypsinogen gene dosage correlates with pancreatitis risk across genetic and transgenic studies, highlighting the fundamental role of dysregulated expression of wild-type trypsinogen in the etiology of pancreatitis. Specifically downregulating trypsinogen expression in the pancreas may serve as a potential therapeutic and/or prevention strategy for pancreatitis.

The Impacts of Genetic and Environmental Factors on the Progression of Chronic Pancreatitis.

BACKGROUND & AIMS: Both environmental factors, such as alcohol consumption and smoking, and genetic factors are strongly associated with the risk of developing chronic pancreatitis (CP). However, comprehensive understanding of their impacts on the progression of CP remains elusive. METHODS: A prospective cohort study was performed on a large cohort of CP patients with known genetic backgrounds. The cumulative incidence of pancreatic insufficiency after the onset of CP was analyzed using Kaplan-Meier survival curves. Multivariate Cox proportional hazards regression analysis also was performed. RESULTS: A total of 798 patients were enrolled in the study and followed up for 10.5 years. Rare pathogenic genotypes in the SPINK1, PRSS1, CTRC, or CFTR genes were identified in 410 (51.4%) patients. The development of pancreatic insufficiency was significantly earlier in patients with a history of smoking and/or alcohol consumption in both the positive (P < .001) and negative (P = .001) gene mutation groups. However, the development of pancreatic insufficiency did not differ significantly between patients with and without gene mutations despite alcohol and/or smoking status, with P values of .064 and .115, respectively. Multivariate Cox regression analysis showed that age at onset of CP (hazard ratio, [HR], 1.02; P < .001) and alcohol consumption (HR, 1.86; P < .001) were independent risk factors for the development of diabetes, while male sex (HR, 1.84; P = .022) and smoking (HR, 1.56; P = .028) were predictors of steatorrhea. CONCLUSIONS: Although rare pathogenic mutations in the 4 major susceptibility genes for CP were not correlated significantly with the development of pancreatic insufficiency, environmental factors (either alcohol consumption or smoking) significantly accelerated disease progression (ClinicalTrials.gov: NCT04574297).

Association between sedation and small neoplasm detection during diagnostic esophagogastroduodenoscopy: a propensity score-matched retrospective study.

BACKGROUND: Esophagogastroduodenoscopy (EGD) is fundamental for detecting upper gastrointestinal (GI) neoplasms. However, the impact of sedation on small neoplasm detection during EGD has not been evaluated. The aim of this study was to investigate whether EGD with sedation could improve small upper GI neoplasm detection. METHODS: This propensity score-matched retrospective study analyzed the medical records of outpatients undergoing diagnostic EGD at a large tertiary center between January 2013 and December 2018. The primary outcome was the detection rate of small upper GI neoplasms (≤10 mm). The secondary outcomes were biopsy rate and small neoplasms in different anatomic subsites. RESULTS: After propensity score matching, 20,052 patients undergoing diagnostic EGD with or without propofol sedation were identified. A higher detection rate of small upper GI neoplasms was observed in the sedation group (2.80% vs. 2.02%; p < .001). In particular, the detection rate of small cancers in the sedation group was 3-fold higher than that in the no-sedation group (0.16% vs. 0.05%; p = .023). Small neoplasms were more likely identified at the gastric antrum (1.60% vs. 1.09%; p = .002) and angulus (0.66% vs. 0.45%; p = .044) in the sedation group. In addition, endoscopists were more likely to take biopsies when performing sedated EGD (41.4% vs. 36.4%, p < .001), and a higher biopsy rate was associated with an increased detection rate of small neoplasms. CONCLUSIONS: Sedation was significantly associated with a higher detection rate of small upper GI neoplasms and might be recommended for improving the quality of EGD.

Homozygosity of short VNTR lengths in the CEL gene may confer susceptibility to idiopathic chronic pancreatitis.

OBJECTIVE: The carboxyl-ester lipase (CEL) gene contains a variable number of tandem repeats (VNTR) region. It remains unclear whether the number of repeats in the CEL VNTR is related to the risk of pancreatic diseases. The aim of this study was to investigate whether CEL VNTR length is associated with idiopathic chronic pancreatitis (ICP), alcoholic chronic pancreatitis (ACP), or pancreatic cancer in a cohort of Chinese patients. METHODS: CEL VNTRs were genotyped in patients diagnosed with ICP (n = 771), ACP (n = 222), or pancreatic cancer (n = 263), and in healthy controls (n = 927). CEL VNTR lengths were determined using a screening method combining PCR and DNA fragment analysis. RESULTS: Overall, the CEL VNTR lengths ranged from 5 to 22 repeats, with the 16-repeat allele ('normal' size, N) accounting for 73.82% of all observed alleles. The VNTR allele frequencies and genotype distributions were not significantly different between healthy controls and patients with ACP or pancreatic cancer. For the ICP group, allele frequencies did not differ significantly from the controls, while the frequency of the SS genotype (homozygosity for 5-15 repeats) was significantly higher in the patients (4.67%) than in the controls (1.94%) (p = 0.0014; OR = 2.47; 95% CI = 1.39-4.39). CONCLUSIONS: There were no associations between the CEL VNTR length and ACP or pancreatic cancer. However, homozygosity for short VNTR lengths may confer susceptibility to ICP.

SPINK1 mutations and risk of pancreatic cancer in a Chinese cohort.

OBJECTIVE: The relationship between SPINK1 and pancreatic cancer (PC) remains controversial. The current study aimed to determine the effect of SPINK1 mutations on PC development among patients with chronic pancreatitis (CP). METHODS: This is a prospective observational study including a large cohort of 965 CP patients with 11-year follow-up. Patients' demographic characteristics and clinical CP outcomes were documented in detail. Genetic testing was performed. The effect of SPINK1 mutations on the clinical development of PC was explored using Cox proportional hazards regression. Subgroup analyses conducted included the consideration of gender, onset age of CP (early- and late-onset), etiologies of CP, smoking, and alcoholic drinking status. RESULTS: PC was diagnosed in 2.5% (24/965) of patients, and the cumulative incidence rates were 0.2%, 0.8%, and 1.5% at 3, 5, and 10 years since the onset of CP, respectively. In this cohort, SPINK1 c.194+2T > C was the most common variant with a proportion of 39.1%. And the risk of PC development varied marginally between patients with and without SPINK1 mutations (Cox HR 0.39(0.14-1.04), P = 0.059). In the subgroup analyses, patients carrying SPINK1 mutations had a significantly lower risk of PC (Cox HR 0.18(0.04-0.80), P = 0.025) in the non-smoking group. SPINK1 mutations showed no significant effect in the other subgroups considered. CONCLUSIONS: CP patients harboring SPINK1 mutations do not have an elevated risk of PC development compared to mutation-negative CP patients. On the contrary, SPINK1 mutations may be a protective factor in non-smoking patients with CP.

Use of artificial intelligence for detection of gastric lesions by magnetically controlled capsule endoscopy.

BACKGROUND AND AIMS: Magnetically controlled capsule endoscopy (MCE) has become an efficient diagnostic modality for gastric diseases. We developed a novel automatic gastric lesion detection system to assist in diagnosis and reduce inter-physician variations. This study aimed to evaluate the diagnostic capability of the computer-aided detection system for MCE images. METHODS: We developed a novel automatic gastric lesion detection system based on a convolutional neural network (CNN) and faster region-based convolutional neural network (RCNN). A total of 1,023,955 MCE images from 797 patients were used to train and test the system. These images were divided into 7 categories (erosion, polyp, ulcer, submucosal tumor, xanthoma, normal mucosa, and invalid images). The primary endpoint was the sensitivity of the system. RESULTS: The system detected gastric focal lesions with 96.2% sensitivity (95% confidence interval [CI], 95.7%-96.5%), 76.2% specificity (95% CI, 75.97%-76.3%), 16.0% positive predictive value (95% CI, 15.7%-16.3%), 99.7% negative predictive value (95% CI, 99.74%-99.79%), and 77.1% accuracy (95% CI, 76.9%-77.3%) (sensitivity was 99.3% for erosions; 96.5% for polyps; 89.3% for ulcers; 87.2% for submucosal tumors; 90.6% for xanthomas; 67.8% for normal; and 96.1% for invalid images). Analysis of the receiver operating characteristic curve showed that the area under the curve for all positive images was 0.84. Image processing time was 44 milliseconds per image for the system and 0.38 ± 0.29 seconds per image for clinicians (P < .001). The kappa value of 2 times repeated reads was 1. CONCLUSIONS: The CNN faster-RCNN-based diagnostic program system showed good performance in diagnosing gastric focal lesions in MCE images.

Prevalence and Risk Factors for Osteopathy in Chronic Pancreatitis.

BACKGROUND AND AIMS: The high incidence of osteopathy among patients with chronic pancreatitis (CP) has garnered increased attention over recent years. The aims of this study were to assess the prevalence and risk factors for osteopathy in Chinese patients with CP. METHODS: This was a cross-sectional study of CP patients from a large center in China; patients were recruited between 31 January 2017 and 31 January 2018. Bone density and laboratory tests, including bone-related biochemical, inflammatory, and hormone parameters, were assessed prospectively. Differences between patients with and without osteopathy were analyzed. Logistic regression analysis was used to investigate associations between variables. RESULTS: In total, 104 CP patients were enrolled in this study (68.3% idiopathic and 31.7% alcoholic). According to the M-ANNHEIM classification, 87.5% of the patients were at an early stage (0-II). Osteopenia was diagnosed in 30.8% of patients and osteoporosis in 5.8%; thus, a total of 36.5% of patients presented with osteopathy. In multivariate analysis, the independent risk factors for osteopathy in CP patients were age (OR = 1.04; 95% CI = 1.00-1.08; P = 0.030), BMI (OR = 0.72; 95% CI = 0.58-0.89; P = 0.003), and PTH (OR = 0.96; 95% CI = 0.93-1.00; P = 0.022). CONCLUSIONS: This study is the first to report the prevalence of osteopathy in Chinese patients with CP. It found that age and low BMI are significant risk factors for osteopathy. Low PTH (but within the normal range) showed a weak association with osteopathy, which warrants further exploration.

Plasma extracellular vesicle long RNA profiling identifies a diagnostic signature for the detection of pancreatic ductal adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is difficult to diagnose at resectable stage. Recent studies have suggested that extracellular vesicles (EVs) contain long RNAs. The aim of this study was to develop a diagnostic (d-)signature for the detection of PDAC based on EV long RNA (exLR) profiling. DESIGN: We conducted a case-control study with 501 participants, including 284 patients with PDAC, 100 patients with chronic pancreatitis (CP) and 117 healthy subjects. The exLR profile of plasma samples was analysed by exLR sequencing. The d-signature was identified using a support vector machine algorithm and a training cohort (n=188) and was validated using an internal validation cohort (n=135) and an external validation cohort (n=178). RESULTS: We developed a d-signature that comprised eight exLRs, including FGA, KRT19, HIST1H2BK, ITIH2, MARCH2, CLDN1, MAL2 and TIMP1, for PDAC detection. The d-signature showed high accuracy, with an area under the receiver operating characteristic curve (AUC) of 0.960, 0.950 and 0.936 in the training, internal validation and external validation cohort, respectively. The d-signature was able to identify resectable stage I/II cancer with an AUC of 0.949 in the combined three cohorts. In addition, the d-signature showed superior performance to carbohydrate antigen 19-9 in distinguishing PDAC from CP (AUC 0.931 vs 0.873, p=0.028). CONCLUSION: This study is the first to characterise the plasma exLR profile in PDAC and to report an exLR signature for the detection of pancreatic cancer. This signature may improve the prognosis of patients who would have otherwise missed the curative treatment window.

TRPV6 variants confer susceptibility to chronic pancreatitis in the Chinese population.

Chronic pancreatitis (CP) is a progressive fibroinflammatory syndrome of the pancreatic tissue caused by genetic and environmental factors. Previously reported susceptibility genes in CP explain less than half of the apparent heritability. To uncover novel pathogenic mechanisms, we initially performed low-coverage whole-genome sequencing on 464 Chinese CP patients and 504 controls. The transient receptor potential cation channel, Subfamily V, Member 6 (TRPV6) gene was found to be significantly associated with CP after a burden test of aggregated rare nonsynonymous variants with a combined annotation dependent depletion score > 20 (p = .020). In the replication stage, we analyzed the entire coding sequence and exon/intron boundaries of the TPRV6 gene by Sanger sequencing in another 205 patients with CP and 105 controls. Integration of the findings from the two stages resulted in the identification of 25 TRPV6 variants: 1 rare nonsense variant, 20 rare missense variants, and 4 common missense variants. Loss-of-function variants, as determined by intracellular Ca2+ concentration in transfected HEK293T cells, were significantly overrepresented in patients as compared to controls (9/669 [1.35%] vs. 1/609 [0.16%]; odds ratio = 8.29; p = .022). This study provides evidence suggesting that TRPV6 is a novel susceptibility gene for CP.

Toward a clinical diagnostic pipeline for SPINK1 intronic variants.

BACKGROUND: The clinical significance of SPINK1 intronic variants in chronic pancreatitis has been previously assessed by various approaches including a cell culture-based full-length gene assay. A close correlation between the results of this assay and in silico splicing prediction was apparent. However, until now, a clinical diagnostic pipeline specifically designed to classify SPINK1 intronic variants accurately and efficiently has been lacking. Herein, we present just such a pipeline and explore its efficacy and potential utility in potentiating the classification of newly described SPINK1 intronic variants. RESULTS: We confirm a close correlation between in silico splicing prediction and results from the cell culture-based full-length gene assay in the context of three recently reported pathogenic SPINK1 intronic variants. We then integrated in silico splicing prediction and the full-length gene assay into a stepwise approach and tested its utility in the classification of two novel datasets of SPINK1 intronic variants. The first dataset comprised 16 deep intronic variants identified in 52 genetically unexplained Chinese chronic pancreatitis patients by sequencing the entire intronic sequence of the SPINK1 gene. The second dataset comprised five novel rare proximal intronic variants identified through the routine analysis of the SPINK1 gene in French pancreatitis patients. Employing a minor allele frequency of > 5% as a population frequency filter, 6 of the 16 deep intronic variants were immediately classified as benign. In silico prediction of the remaining ten deep intronic variants and the five rare proximal intronic variants with respect to their likely impact on splice site selection suggested that only one proximal intronic variant, c.194 + 5G > A, was likely to be of functional significance. Employing the cell culture-based full-length gene assay, we functionally analyzed c.194 + 5G > A, together with seven predicted non-functional variants, thereby validating their predicted effects on splicing in all cases. CONCLUSIONS: We demonstrated the accuracy and efficiency of in silico prediction in combination with the cell culture-based full-length gene assay for the classification of SPINK1 intronic variants. Based upon these findings, we propose an operational pipeline for classifying SPINK1 intronic variants in the clinical diagnostic setting.

Altered diversity and composition of gut microbiota in Chinese patients with chronic pancreatitis.

BACKGROUND/OBJECTIVES: Gut microbiota alterations in chronic pancreatitis (CP) are seldomly described systematically. It is unknown whether pancreatic exocrine insufficiency (PEI) and different etiologies in patients with CP are associated with gut microbiota dysbiosis. METHODS: The fecal microbiota of 69 healthy controls (HCs) and 71 patients with CP were compared to investigate gut microbiome alterations in CP and the relationship among gut microbiome dysbiosis, PEI and different etiologies. Fecal microbiomes were analyzed through 16S ribosomal RNA gene profiling, based on next-generation sequencing. Pancreatic exocrine function was evaluated by determining fecal elastase 1 activity. RESULTS: Patients with CP showed gut microbiota dysbiosis with decreased diversity and richness, and taxa-composition changes. On the phylum level, the gut microbiome of the CP group showed lower Firmicutes and Actinobacteria abundances than the HC group and higher Proteobacteria abundances. The abundances of Escherichia-Shigella and other genera were high in gut microbiomes in the CP group, whereas that of Faecalibacterium was low. Kyoto Encyclopedia of Genes and Genomes pathways (lipopolysaccharide biosynthesis and bacterial invasion of epithelial cells) were predicted to be enriched in the CP group. Among the top 5 phyla and 8 genera (in terms of abundance), only Fusobacteria and Eubacterium rectale group showed significant differences between CP patients, with or without PEI. Correlation analysis showed that Bifidobacterium and Lachnoclostridium correlated positively with fecal elastase 1 (r = 0.2616 and 0.2486, respectively, P < 0.05). CONCLUSIONS: The current findings indicate that patients with CP have gut microbiota dysbiosis that is partly affected by pancreatic exocrine function.

Characterization of CEL-DUP2: Complete duplication of the carboxyl ester lipase gene is unlikely to influence risk of chronic pancreatitis.

BACKGROUND/OBJECTIVES: Carboxyl ester lipase is a pancreatic enzyme encoded by CEL, an extremely polymorphic human gene. Pathogenic variants of CEL either increases the risk for chronic pancreatitis (CP) or cause MODY8, a syndrome of pancreatic exocrine and endocrine dysfunction. Here, we aimed to characterize a novel duplication allele of CEL (CEL-DUP2) and to investigate whether it associates with CP or pancreatic cancer. METHODS: The structure of CEL-DUP2 was determined by a combination of Sanger sequencing, DNA fragment analysis, multiplex ligation-dependent probe amplification and whole-genome sequencing. We developed assays for screening of CEL-DUP2 and analyzed cohorts of idiopathic CP, alcoholic CP and pancreatic cancer. CEL protein expression was analyzed by immunohistochemistry. RESULTS: CEL-DUP2 consists of an extra copy of the complete CEL gene. The allele has probably arisen from non-allelic, homologous recombination involving the adjacent pseudogene of CEL. We found no association between CEL-DUP2 carrier frequency and CP in cohorts from France (cases/controls: 2.5%/2.4%; P = 1.0), China (10.3%/8.1%; P = 0.08) or Germany (1.6%/2.3%; P = 0.62). Similarly, no association with disease was observed in alcohol-induced pancreatitis (Germany: 3.2%/2.3%; P = 0.51) or pancreatic cancer (Norway; 2.5%/3.2%; P = 0.77). Notably, the carrier frequency of CEL-DUP2 was more than three-fold higher in Chinese compared with Europeans. CEL protein expression was similar in tissues from CEL-DUP2 carriers and controls. CONCLUSIONS: Our results support the contention that the number of CEL alleles does not influence the risk of pancreatic exocrine disease. Rather, the pathogenic CEL variants identified so far involve exon 11 sequence changes that substantially alter the protein's tail region.