SPINK1 Overexpression Correlates with Hepatocellular Carcinoma Treatment Resistance Revealed by Single Cell RNA-Sequencing and Spatial Transcriptomics.

Low efficacy of treatments and chemoresistance are challenges in addressing refractory hepatocellular carcinoma (HCC). SPINK1, an oncogenic protein, is frequently overexpressed in many HCC cases. However, the impact of SPINK1 on HCC treatment resistance remains poorly understood. Here, we elucidate the functions of SPINK1 on HCC therapy resistance. Analysis of SPINK1 protein level reveals a correlation between elevated SPINK1 expression and unfavorable prognosis. Furthermore, intercellular variations in SPINK1 expression levels are observed. Subsequent examination of single cell RNA-sequencing data from two HCC cohorts further suggest that SPINK1-high cells exhibit heightened activity in drug metabolic pathways compared to SPINK1-low HCC cells. High SPINK1 expression is associated with reduced sensitivities to both chemotherapy drugs and targeted therapies. Moreover, spatial transcriptomics data indicate that elevated SPINK1 expression correlates with non-responsive phenotype during treatment with targeted therapy and immune checkpoint inhibitors. This is attributed to increased levels of drug metabolic regulators, especially CES2 and CYP3A5, in SPINK1-high cells. Experimental evidence further demonstrates that SPINK1 overexpression induces the expression of CES2 and CYP3A5, consequently promoting chemoresistance to sorafenib and oxaliplatin. In summary, our study unveils the predictive role of SPINK1 on HCC treatment resistance, identifying it as a potential therapeutic target for refractory HCC.

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.

Genetic Factors Associated With Adverse Pregnancy Outcomes in Chronic Pancreatitis.

INTRODUCTION: The effects of genetic factors on pregnancy outcomes in chronic pancreatitis (CP) patients remain unclear. We evaluated the impacts of clinical features and mutations in main CP-susceptibility genes ( SPINK1 , PRSS1 , CTRC , and CFTR ) on pregnancy outcomes in Chinese CP patients. METHODS: This was a prospective cohort study with 14-year follow-up. The sample comprised female CP patients with documented pregnancy and known genetic backgrounds. Adverse pregnancy outcomes were compared between patients with and without gene mutations. Univariate and multivariate analyses were performed to determine the impact factors for adverse pregnancy outcomes. RESULTS: Totally, 160 female CP patients with a pregnancy history were enrolled; 59.4% of patients carried pathogenic mutations in CP-susceptibility genes. Adverse pregnancy outcomes occurred in 38 patients (23.8%); the prevalence of adverse outcomes was significantly higher in those harboring gene mutations than those without (30.5% vs 13.8%, P = 0.015). Notably, the rates of preterm delivery (12.6% vs 3.1%, P = 0.036) and abortion (17.9% vs 4.6%, P = 0.013) were remarkably higher in patients with gene mutations (especially SPINK1 mutations) than those without. In multivariate analyses, both CP-susceptibility gene mutations (odds ratio, 2.52; P = 0.033) and SPINK1 mutations (odds ratio, 2.60; P = 0.037) significantly increased the risk of adverse pregnancy outcomes. Acute pain attack during pregnancy was another risk factor for adverse pregnancy outcomes. DISCUSSION: Pathogenic mutations in CP-susceptibility genes, especially SPINK1 , were independently related to adverse pregnancy outcomes in CP patients. Significant attention should be paid to pregnant females harboring CP-susceptibility gene mutations (ClinicalTrials.gov: NCT06055595).

Tumor microenvironment deconvolution identifies cell-type-independent aberrant DNA methylation and gene expression in prostate cancer.

BACKGROUND: Among men, prostate cancer (PCa) is the second most common cancer and the second leading cause of cancer death. Etiologic factors associated with both prostate carcinogenesis and somatic alterations in tumors are incompletely understood. While genetic variants associated with PCa have been identified, epigenetic alterations in PCa are relatively understudied. To date, DNA methylation (DNAm) and gene expression (GE) in PCa have been investigated; however, these studies did not correct for cell-type proportions of the tumor microenvironment (TME), which could confound results. METHODS: The data (GSE183040) consisted of DNAm and GE data from both tumor and adjacent non-tumor prostate tissue of 56 patients who underwent radical prostatectomies prior to any treatment. This study builds upon previous studies that examined methylation patterns and GE in PCa patients by using a novel tumor deconvolution approach to identify and correct for cell-type proportions of the TME in its epigenome-wide association study (EWAS) and differential expression analysis (DEA). RESULTS: The inclusion of cell-type proportions in EWASs and DEAs reduced the scope of significant alterations associated with PCa. We identified 2,093 significantly differentially methylated CpGs (DMC), and 51 genes associated with PCa, including PCA3, SPINK1, and AMACR. CONCLUSIONS: This work illustrates the importance of correcting for cell types of the TME when performing EWASs and DEAs on PCa samples, and establishes a more confounding-adverse methodology. We identified a more tumor-cell-specific set of altered genes and epigenetic marks that can be further investigated as potential biomarkers of disease or potential therapeutic targets.

PTEN, ERG, SPINK1, and TFF3 Status and Relationship in a Prostate Cancer Cohort from Jordanian Arab Population.

Background and Objectives: Prognostic biomarkers in prostate cancer (PCa) include PTEN, ERG, SPINK1, and TFF3. Their relationships and patterns of expression in PCa in developing countries, including Jordan, have not yet been investigated. Materials and Methods: A tissue microarray (TMA) of PCa patients was taken from paraffin-embedded tissue blocks for 130 patients. PTEN, ERG, SPINK1, and TFF3 expression profiles were examined using immunohistochemistry (IHC) and correlated with each other and other clinicopathological factors. Results: PTEN loss of any degree was observed in 42.9% of PCa cases. ERG and TFF3 were expressed in 59.3% and 46.5% of PCa cases, respectively. SPINK1 expression was observed in 6 out of 104 PCa cases (5.4%). Among all PCa cases (n = 104), 3.8% (n = 4) showed SPINK1+/ERG+ phenotype, 1.9% (n = 2) showed SPINK1+/ERG- phenotype, 56.7% (n = 59) showed SPINK1-/ERG+ phenotype, and 37.5% showed SPINK1-/ERG- phenotype (n = 39). Among ERG positive cases (n = 63), 6.3% were SPINK1 positive. Among SPINK1 positive cases (n = 6), 66.7% were ERG positive. SPINK1 expression was predominantly observed in a subgroup of cancers that expressed TFF3 (6/6). Additionally, a statistically significant loss of PTEN expression was observed from Gleason Score 6 (GS6) (Grade Group 1 (GG1)) to GS9-10 (GG5); (p-value 0.019). Conclusions: This is the first study to look at the status of the PTEN, ERG, SPINK1, and TFF3 genes in a Jordanian Arab population. Loss of PTEN has been linked to more aggressive prostate cancer with high GSs/GGs. SPINK1 expression was predominantly observed in a subgroup of cancers that expressed TFF3. Our results call for screening these biomarkers for grading and molecular subtyping of the disease.

SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma.

Tumor lineage plasticity, considered a hallmark of cancer, denotes the phenomenon in which tumor cells co-opt developmental pathways to attain cellular plasticity, enabling them to evade targeted therapeutic interventions. However, the underlying molecular events remain largely elusive. Our recent study identified CD133/Prom1 in hepatocellular carcinoma (HCC) tumors to mark proliferative tumor-propagating cells with cancer stem cell-like properties, that follow a dedifferentiation trajectory towards a more embryonic state. Here we show SPINK1 to strongly associate with CD133 + HCC, and tumor dedifferentiation. Enhanced transcriptional activity of SPINK1 is mediated by promoter binding of ELF3, which like CD133, is found to increase following 5-FU and cisplatin treatment; while targeted depletion of CD133 will reduce both ELF3 and SPINK1. Functionally, SPINK1 overexpression promotes tumor initiation, self-renewal, and chemoresistance by driving a deregulated EGFR-ERK-CDK4/6-E2F2 signaling axis to induce dedifferentiation of HCC cells into their ancestral lineages. Depleting SPINK1 function by neutralizing antibody treatment or in vivo lentivirus-mediated Spink1 knockdown dampens HCC cancer growth and their ability to resist chemotherapy. Targeting oncofetal SPINK1 may represent a promising therapeutic option for HCC treatment.

Molecular Networks of Platinum Drugs and Their Interaction with microRNAs in Cancer.

The precise mechanism of resistance to anti-cancer drugs such as platinum drugs is not fully revealed. To reveal the mechanism of drug resistance, the molecular networks of anti-cancer drugs such as cisplatin, carboplatin, oxaliplatin, and arsenic trioxide were analyzed in several types of cancers. Since diffuse-type stomach adenocarcinoma, which has epithelial-mesenchymal transition (EMT)-like characteristics, is more malignant than intestinal-type stomach adenocarcinoma, the gene expression and molecular networks in diffuse- and intestinal-type stomach adenocarcinomas were analyzed. Analysis of carboplatin revealed the causal network in diffuse large B-cell lymphoma. The upstream regulators of the molecular networks of cisplatin-treated lung adenocarcinoma included the anti-cancer drug trichostatin A (TSA), a histone deacetylase inhibitor. The upstream regulator analysis of cisplatin revealed an increase in FAS, BTG2, SESN1, and CDKN1A, and the involvement of the tumor microenvironment pathway. The molecular networks were predicted to interact with several microRNAs, which may contribute to the identification of new drug targets for drug-resistant cancer. Analysis of oxaliplatin, a platinum drug, revealed that the SPINK1 pancreatic cancer pathway is inactivated in ischemic cardiomyopathy. The study showed the importance of the molecular networks of anti-cancer drugs and tumor microenvironment in the treatment of cancer resistant to anti-cancer drugs.

Genetic Investigations in Turkish Idiopathic Pancreatitis Patients Show Unique Characteristics.

BACKGROUND/AIMS: Pancreatitis is one of the leading causes of digestive system-related hospital admissions, and it has a genetic background in a considerable portion of the patients. In this study, we aimed to investigate the genetic risk factors of idiopathic pancreatitis in Turkish patients and the contribution of copy number variations to the pathogenesis. MATERIALS AND METHODS: Idiopathic pancreatitis is defined as failure to detect risk factors despite comprehensive clinical assessments. Next-generation sequencing and multiple ligand-dependent probe amplification of PRSS1, SPINK1, CTRC, and CFTR were performed. For further genotype-phenotype correlations, patients were also questioned for the age of onset, family history, and pancreatic divisum. RESULTS: A total of 68 idiopathic pancreatitis cases were enrolled. Variants with potential clinical significance of PRSS1 were identified in 13.4%, SPINK1 in 6.3%, CTRC in 4.7%, and CFTR in 26.5% of the patients. No copy number variants were seen in any of these genes. At least 7.4% of the participants had complex genetic etiology involving 2 genes. CONCLUSIONS: At least 42.6% of the participants had a potential genetic risk factor. Five novel genetic variants were identified, and distinctive genetic risk factors of Turkish population were shown. The results showed that genetic etiology was frequent in pancreatitis and it was even more prominent in patients with early-onset disease. Considering that genetic risk factors may be informative for decisionmaking in the treatment options in addition to providing extensive prognostic value and familial genetic consultation; clinicians need to be more eager to offer genetic tests to pancreatitis patients.

Clinical and Genetic Description of Hereditary Chronic Pancreatitis in Pakistani Children.

BACKGROUND/AIMS: The purpose of this study was to identify the spectrum and frequency of pathogenic variants as well as the clinical and genetic insight of hereditary chronic pancreatitis in Pakistani children. MATERIALS AND METHODS: The deoxyribonucleic acid of affected probands of 44 unrelated Pakistani families, having hereditary chronic pancreatitis-affected children, were subjected to massive parallel sequencing for candidate reported genes (SPINK1, PRSS1, CFTR, CPA1, CTRC, CBS, AGL, PHKB, and LPL). Data were analyzed using different bioinformatics tools for the variants and in-silico analysis. All the identified variants were validated by direct sequencing of the targeted exons in the probands and their parents. RESULTS: There were 50 patients included in this study with confirmed hereditary chronic pancreatitis. Nine known mutations in SPINK1, PRSS1, CFTR, CTRC, CBS, and AGL genes, and 10 novel variants in LPL, CFTR, CTR, and PHKB genes were identified. The identified variants were found in heterozygous, compound heterozygous, and trans-heterozygous forms, with rare allele frequency in the normal population. The novel variants were [c.378C>T(p.Lys126Asn) and c.719G>A(p.Arg240Gln) in CTRC, c.586-3C>A and c.763A>G(p.Arg255Gly) in CPA1, c.1160_1161insT(p.Lys387Asnfs*26), c.784C>T(p.Gln262*), c.1139+1G>A, c.175G>A(p.Gly59Arg) in LPL, c.388C>G(p.leu130val) in CFTR, and c.2327G>A(p.Arg776His in PHKB)]. The phenotypic characteristics were variable and correlated with the relevant variant. CONCLUSIONS: The genetic composition plays a significant role in the predisposition of hereditary chronic pancreatitis. The clinical presentation varies with the genetic determinant involved. This information would help in building up a diagnostic algorithm for our population that can be used for genetic screening services in affected cohorts.

Predicting Duodenal Cancer Risk in Patients with Familial Adenomatous Polyposis Using Machine Learning Model.

BACKGROUND/AIMS: The aim of this study was to both classify data of familial adenomatous polyposis patients with and without duode- nal cancer and to identify important genes that may be related to duodenal cancer by XGboost model. MATERIALS AND METHODS: The current study was performed using expression profile data from a series of duodenal samples from familial adenomatous polyposis patients to explore variations in the familial adenomatous polyposis duodenal adenoma-carcinoma sequence. The expression profiles obtained from cancerous, adenomatous, and normal tissues of 12 familial adenomatous polyposis patients with duodenal cancer and the tissues of 12 familial adenomatous polyposis patients without duodenal cancer were compared. The ElasticNet approach was utilized for the feature selection. Using 5-fold cross-validation, one of the machine learning approaches, XGboost, was utilized to classify duodenal cancer. Accuracy, balanced accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and F1 score performance metrics were assessed for model performance. RESULTS: According to the variable importance obtained from the modeling, ADH1C, DEFA5, CPS1, SPP1, DMBT1, VCAN-AS1, APOB genes (cancer vs. adenoma); LOC399753, APOA4, MIR548X, and ADH1C genes (adenoma vs. adenoma); SNORD123, CEACAM6, SNORD78, ANXA10, SPINK1, and CPS1 (normal vs. adenoma) genes can be used as predictive biomarkers. CONCLUSIONS: The proposed model used in this study shows that the aforementioned genes can forecast the risk of duodenal cancer in patients with familial adenomatous polyposis. More comprehensive analyses should be performed in the future to assess the reliability of the genes determined.

Spectrum of PRSS1, SPINK1, CTRC, CFTR, and CPA1 Gene Variants in Chronic Pancreatitis Patients in Russia.

The aim of the study was to define the spectrum of genetic risk factors of chronic pancreatitis (CP) development in patients living in the European part of the Russian Federation. Materials and Methods: The study group included 105 patients with CP, with the age of the disease onset under 40 years old (the average age of onset was 26.9 years). The control group consisted of 76 persons without clinical signs of pancreatitis. The diagnosis of chronic pancreatitis in patients was made on the basis of clinical manifestations and the results of laboratory and instrumental investigations. Genetic examination of patients was conducted using the next-generation sequencing (NGS) technology and included targeted sequencing of all exons and exon-intron boundaries of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes. The genotyping of the rs61734659 locus of the PRSS2 gene was also conducted. Results: Genetic risk factors of the CP development were found in 61% of patients. Pathogenic and likely-pathogenic variants associated with the risk of CP development were identified in the following genes: CTRC (37.1% of patients), CFTR (18.1%), SPINK1 (8.6%), PRSS1 (8.6%), and CPA1 (6.7%). The frequent gene variants in Russian patients with CP were as follows: CTRC gene - c.180C>T (rs497078), c.760C>T (rs121909293), c.738_761del24 (rs746224507); cumulative odds ratio (OR) for all risk alleles was 1.848 (95% CI: 1.054-3.243); CFTR gene - c.3485G>T (rs1800120), c.1521_1523delCTT (p.Phe508del, rs113993960), and c.650A>G (rs121909046); OR=2.432 (95% CI: 1.066-5.553). In the SPINK1, PRSS1, and CPA1 genes, pathogenic variants were found only in the group of patients with CP. The frequent variants of the SPINK1 gene include c.101A>G (p.Asn34Ser, rs17107315) and c.194+2T>C (rs148954387); of the PRSS1 gene - c.86A>T (p.Asn29Ile, rs111033566); of the CPA1 gene - c.586-30C>T (rs782335525) and c.696+23_696+24delGG. The OR for the CP development for the c.180TT genotype (rs497078) CTRC according to the recessive model (TT vs. CT+CC) was 7.05 (95% CI: 0.86-263, p=0.011). In the CTRC gene, the variant c.493+49G>C (rs6679763) appeared to be benign, the c.493+51C>A (rs10803384) variant was frequently detected among both the diseased and healthy persons and did not demonstrate a protective effect. The protective factor c.571G>A (p.Gly191Arg, rs61734659) of the PRSS2 gene was detected only in the group of healthy individuals and confirmed its protective role. 12.4% of the patients with CP had risk factors in 2 or 3 genes. Conclusion: Sequencing of the coding regions of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes allowed to identify genetic risk factors of the CP development in 61% of cases. Determining the genetic cause of CP helps to predict the disease course, perform preventive measures in the proband's relatives, and facilitate a personalized treatment of the patient in future.

Inhibition of mouse trypsin isoforms by SPINK1 and effect of human pancreatitis-associated mutations.

Serine protease inhibitor Kazal type 1 (SPINK1) is a trypsin-selective inhibitor protein secreted by the exocrine pancreas. Loss-of-function SPINK1 mutations predispose to chronic pancreatitis through either reduced expression, secretion, or impaired trypsin inhibition. In this study, we aimed to characterize the inhibitory activity of mouse SPINK1 against cationic (T7) and anionic (T8, T9, T20) mouse trypsin isoforms. Kinetic measurements with a peptide substrate, and digestion experiments with ß-casein indicated that the catalytic activity of all mouse trypsins is comparable. Human SPINK1 and its mouse ortholog inhibited mouse trypsins with comparable efficiency (KD range 0.7-2.2 pM), with the sole exception of T7 trypsin, which was inhibited less effectively by the human inhibitor (KD 21.9 pM). Characterization of four chronic pancreatitis-associated human SPINK1 mutations in the context of the mouse inhibitor revealed that the reactive-loop mutations R42N (human K41N) and I43M (human I42M) impaired SPINK1 binding to trypsin (KD 60 nM and 47.5 pM, respectively), whereas mutations D35S (human N34S) and A56S (human P55S) had no impact on trypsin inhibition. Our results confirmed that high-affinity trypsin inhibition by SPINK1 is conserved in the mouse, and the functional consequences of human pancreatitis-associated SPINK1 mutations can be replicated in the mouse inhibitor.

Associations between Etiologic or Prognostic Tumor Tissue Markers and Neighborhood Contextual Factors in Male Health Professionals Diagnosed with Prostate Cancer.

BACKGROUND: There is growing evidence that unfavorable neighborhood contexts may influence prostate cancer progression. Whether these associations may be explained in part by differences in tumor-level somatic alterations remain unclear. METHODS: Data on tumor markers (PTEN, p53, ERG, and SPINK1) were obtained from 1,157 participants with prostate cancer in the Health Professionals Follow-up Study. Neighborhood greenness, socioeconomic status, and the income Index of Concentration at Extremes were obtained from satellite and census data and linked to participants' address at diagnosis and at study enrollment. Exposures were scaled to an interquartile range and modeled as tertiles. Bivariate associations between tertiles of neighborhood factors and tumor markers were assessed in covariate adjusted logistic regression models to estimate ORs and 95% confidence intervals. RESULTS: There was no association between any of the neighborhood contextual factors and PTEN, p53, ERG, or SPINK1 in bivariate or multivariable adjusted models. Results were generally consistent when modeling exposure using exposure at diagnosis or at study enrollment. CONCLUSIONS: In this multilevel study of men with prostate cancer, we found no evidence of associations between neighborhood context and tumor tissue markers. IMPACT: Our results provide some of the first empirical data in support of the hypothesis that prostate cancer risk conferred by tumor tissue markers may arise independently of underlying neighborhood context. Prospective studies in more diverse populations are needed to confirm these findings.

Prevalence and Risk Factors of Germline Pathogenic Variants in Pancreatic Ductal Adenocarcinoma.

PURPOSE: The genetic attribution for pancreatic ductal adenocarcinoma (PDAC) has been reported as 5%-10%. However, the incidence of germline pathogenic variants (PVs) in Korean PDAC patients has not been thoroughly investigated. Therefore, we studied to identify the risk factors and prevalence of PV for future treatment strategies in PDAC. MATERIALS AND METHODS: Total of 300 (155 male) patients with a median age of 65 years (range, 33 to 90 years) were enrolled in National Cancer Center in Korea. Cancer predisposition genes, clinicopathologic characteristics, and family history of cancer were analyzed. RESULTS: PVs were detected in 20 patients (6.7%, median age 65) in ATM (n=7, 31.8%), BRCA1 (n=3, 13.6%), BRCA2 (n=3), and RAD51D (n=3). Each one patient showed TP53, PALB2, PMS2, RAD50, MSH3, and SPINK1 PV. Among them, two likely PVs were in ATM and RAD51D, respectively. Family history of various types of cancer including pancreatic cancer (n=4) were found in 12 patients. Three patients with ATM PVs and a patient with three germline PVs (BRCA2, MSH3, and RAD51D) had first-degree relatives with pancreatic cancer. Familial pancreatic cancer history and PVs detection had a significant association (4/20, 20% vs. 16/264, 5.7%; p=0.035). CONCLUSION: Our study demonstrated that germline PVs in ATM, BRCA1, BRCA2, and RAD51D are most frequent in Korean PDAC patients and it is comparable to those of different ethnic groups. Although this study did not show guidelines for germline predisposition gene testing in patients with PDAC in Korea, it would be emphasized the need for germline testing for all PDAC patients.

[Influence of environmental factors and polymorphic loci rs6580502 of the SPINK1 gene, rs10273639 of the PRSS1 gene, rs213950 of the CFTR gene on the risk of developing acute alcoholic-alimentary pancreatitis].

The study of genetic and environmental factors on the risk of acute alcoholic-alimentary pancreatitis (AАAР) is especially relevant to interpret individual links of pathogenesis, to reduce the incidence by eliminating the impact of harmful factors and improve the quality of life of the population through the introduction of optimal nutrition, and a healthy lifestyle, which is especially important for carriers of risk genotypes. The aim of the research was to study the influence of environmental factors and polymorphic loci rs6580502 of the SPINK1 gene, rs10273639 of the PRSS1 gene, rs213950 of the CFTR gene on the risk of АAР. Material and methods. Blood DNA samples obtained from 547 patients with AАAР and 573 healthy individuals were used as the material for the study. The groups were comparable by sex and age. All participants were assessed qualitatively and quantitatively for risk factors, smoking and alcohol consumption, the frequency, quantity and regularity of intake of various types of foods, as well as the size and number of portions eaten. Genomic DNA was isolated by the standard phenol-chloroform extraction method, multiplex genotyping of SNPs was performed on a MALDI-TOF MassARRAY-4 genetic analyzer. Results. It was found that the T/T genotype (p=0.0012) of the rs6580502 SPINK1 was associated with an increased risk of AAAP, and the T allele (p=0.0001) and C/T and T/T genotypes (p=0.0001) of the rs10273639 PRSS1, A allele (p=0.01) and A/G and A/A genotypes (p=0.0006) of the rs213950 CFTR were associated with an decreased risk of the disease. The revealed effects of polymorphic loci of candidate genes were enhanced by the effect of alcohol consumption. The risk of AAAP was reduced by fat intake of less than 89 g per day in carriers of the A/G-A/A CFTR genotypes (rs213950), consumption of fresh vegetables and fruits of more than 27 g per day in carriers of the T/C-T/T PRSS1 genotypes (rs10273639), protein intake of more 84 g per day in carriers of T/C-T/T PRSS1 rs10273639 and A/G-A/A CFTR rs213950. The most significant models of gene-environment interactions included risk factors: deficiency in the diet of protein, fresh vegetables and fruits, smoking, and polymorphic variants of the PRSS1 (rs10273639) and SPINK (rs6580502) genes. Conclusion. In order to prevent the development of AAAP, carriers of risk genotypes of candidate genes need not only to exclude or significantly reduce alcohol consumption (in terms of volume, frequency and duration); but also carriers of the A/G-A/A CFTR genotypes (rs213950) need to balance the diet by reducing fat intake to less than 89 g per day and increasing protein intake to more than 84 g per day; carriers of the T/C-T/T PRSS1 (rs10273639) genotypes should increase their intake of fresh vegetables and fruits over 27 g/day and protein over 84 g/day.

Proteome-Wide Mendelian Randomization Identifies Causal Links Between Blood Proteins and Acute Pancreatitis.

BACKGROUND & AIMS: Acute pancreatitis (AP) is a complex disease and the leading cause of gastrointestinal disease-related hospital admissions. Few therapeutic options exist for AP prevention. Blood proteins with causal evidence may represent promising drug targets, but few have been causally linked with AP. Our objective was to identify blood proteins linked with AP by combining genome-wide association meta-analysis and proteome-wide Mendelian randomization (MR) studies. METHODS: We performed a genome-wide association meta-analysis totalling 10,630 patients with AP and 844,679 controls and a series of inverse-variance weighted MR analyses using cis-acting variants on 4719 blood proteins from the deCODE study (N = 35,559) and 4979 blood proteins from the Fenland study (N = 10,708). RESULTS: The meta-analysis identified genome-wide significant variants (P <5 × 10-8) at 5 loci (ABCG5/8, TWIST2, SPINK1, PRSS2 and MORC4). The proteome-wide MR analyses identified 68 unique blood proteins that may causally be associated with AP, including 29 proteins validated in both data sets. Functional annotation of these proteins confirmed expression of many proteins in metabolic tissues responsible for digestion and energy metabolism, such as the esophagus, adipose tissue, and liver as well as acinar cells of the pancreas. Genetic colocalization and investigations into the druggable genome also identified potential drug targets for AP. CONCLUSIONS: This large genome-wide association study meta-analysis for AP identified new variants linked with AP as well as several blood proteins that may be causally associated with AP. This study provides new information on the genetic architecture of this disease and identified pathways related to AP, which may be further explored as possible therapeutic targets for AP.

The Coexistence of TRPV6 Variants With Other Pancreatitis-Associated Genes Affects Pediatric-Onset Pancreatitis.

OBJECTIVES: Recently, a genetic risk for chronic pancreatitis (CP) was found to be conferred by pathogenic variants in the transient receptor potential cation channel, subfamily V, member 6 ( TRPV6 ). Interestingly, 20%-57% of patients with functionally defective TRPV6 variants have other susceptibility genes such as cationic trypsinogen, serine protease inhibitor Kazal type 1, chymotrypsin C, cystic fibrosis transmembrane conductance regulator, and carboxypeptidase A1. In this study, we focused on pediatric patients with acute recurrent pancreatitis or CP with at least 1 variant in these 5 genes and investigated the presence of coexisting TRPV6 mutations. METHODS: Ninety Japanese pediatric patients (median age at first onset, 8.0 years) who had at least 1 variant of these 5 genes were enrolled in this study. DNA samples were extracted for analysis from peripheral blood leukocytes. Coding regions of TRPV6 were screened by Sanger sequencing. RESULTS: Regardless of functional defects or non-defects in TRPV6 variants, 14 of the 90 patients (15.6%) were trans-heterozygous for TRPV6 variants [p.A18S (n = 3), p.C197R (n = 3), p.I223T (n = 3), p.D324N (n = 4), p.M418V (n = 3), p.V540F (n = 1), p.A606T (n = 1), and p.M721T (n = 3)] and the 5 susceptibility genes noted above. Of these variants, p.D324N, p.V540F, and p.A606T are associated with pancreatitis. Three patients had the ancestral haplotype [p.C197R + p.M418V + p.M721T]. CONCLUSIONS: Overall, 4 of 90 patients (4.4%) had the coexistence of clearly pathogenic TRPV6 variants with pancreatitis-associated variants. The cumulative accumulation of these genetic factors may contribute to the development of pancreatitis at a young age.

Identification of Kazal Inhibitor Scaffolds with Identical Canonical Binding Loops and Their Effects on Binding Properties.

Kazal inhibitors hold high potential as scaffolds for therapeutic molecules, taking advantage of the easily exchangeable canonical binding loop. Different Kazal inhibitor backbones have been suggested to be therapeutically useful, but the impact of different Kazal-like scaffolds on binding properties is still largely unknown. Here, we identified trypsin-targeting human serine protease inhibitor Kazal type 1 (SPINK1) homologues in different mammalian species that cluster in two P2-P1 combinations, implying the coevolution of these residues. We generated loop exchange variants of human SPINK1 for comparison with Kazal inhibitors from related species. Using comprehensive biophysical characterization of the inhibitor-enzyme interactions, we found not only affinity but also pH resistance to be highly backbone-dependent. Differences are mostly observed in complex stability, which varies by over one order of magnitude. We provide clear evidence for high backbone dependency within the Kazal family. Hence, when designing Kazal inhibitor-based therapeutic molecules, testing different backbones after optimizing the canonical binding loop can be beneficial and may result in increased affinity, complex stability, specificity, and pH resistance.

Hereditary Pancreatitis Related to SPINK-1 Mutation. Is There an Increased Risk of Developing Pancreatic Cancer?

It is thought that many of the idiopathic pancreatitis could have a genetic base. Approximately 50% of them correspond to CFTR (cystic fibrosis transmembrane conductance regulator gene) and SPINK-1 (serine protease inhibitor Kazal type 1) mutations. A recent study compares patients with acute pancreatitis and SPINK-1 mutation with patients with idiopathic acute pancreatitis. The study highlights a 12-fold increased risk of developing pancreatic cancer with SPINK-1 mutation versus the control group. Nonetheless, authors conclude that only specific pN34s mutation is related to pancreatic cancer. This relation is controversial, and international consensus guidelines for the follow-up in chronic pancreatitis with pancreatic cancer still do not recommend follow-up in SPINK-1 p. N34S mutation. We believe that developing prospective studies in which subgroups of patients with SPINK-1 mutation benefit from closer follow-ups would be necessary.

Loss of KLK4::KLKP1 pseudogene expression by RNA chromogenic in-situ hybridization is associated with PTEN loss and increased risk of biochemical recurrence in a cohort of middle eastern men with prostate cancer.

BACKGROUND: KLK4::KLKP1 fusion is a recently described pseudogene that is enriched in prostate cancer (PCa). This new biomarker has not been characterized in the Middle Eastern population. OBJECTIVE: To establish the incidence and prognostic value of KLK4::KLKP1 fusion in a cohort of Middle Eastern men with PCa and explore the relationship of this marker to other relevant biomarkers (PTEN, ERG, SPINK1). DESIGN, SETTING, AND PARTICIPANTS: We interrogated a cohort of 340 Middle Eastern men with localized PCa treated by radical prostatectomy between 2005 and 2015. KLK4::KLKP1 fusion status was assessed by RNA Chromogenic in situ hybridization (CISH) and correlated to pathological and clinical parameters. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: RNA-CISH expression of KLK4::KLKP1 was correlated with prognostic factors, ERG, PTEN, and SPINK1 expression, and biochemical recurrence (BCR) following prostatectomy. RESULTS AND LIMITATIONS: 51.7% of patient samples showed positive KLK4::KLKP1 expression; more commonly in cores of PCa (38%) versus non-cancer (20.6%) (p < 0.0001) and in lower Gleason Grade Group tumors (1-3) vs (4-5). KLK4::KLKP1 expression positively correlated with ERG positivity and inversely associated with PTEN loss. No significant association was found with SPINK1 expression, seminal vesicle invasion, positive surgical margin, pathological stage, or patient age (< 50 or ≥ 50). The association between PTEN loss and BCR increased when combined with KLK4::KLKP1 negativity (HR 2.31, CI 1.03-5.20, p = 0.042). CONCLUSIONS: KLK4::KLKP1 expression is more common in this cohort of Middle Eastern men than has been reported in North American men. It is associated with ERG positivity and inversely correlated with PTEN loss. In isolation, KLK4::KLKP1 expression was not significantly associated with clinical outcome or pathological parameters. However, its expression is associated with certain molecular subtypes (ERG-positive, PTEN-intact) and as we demonstrate may help further stratify the risk of recurrence within these groups.