Volker Keim

Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis.

Chronic pancreatitis is a persistent inflammatory disease of the pancreas, in which the digestive protease trypsin has a fundamental pathogenetic role. Here we have analyzed the gene encoding the trypsin-degrading enzyme chymotrypsin C (CTRC) in German subjects with idiopathic or hereditary chronic pancreatitis. Two alterations in this gene, p.R254W and p.K247_R254del, were significantly overrepresented in the pancreatitis group, being present in 30 of 901 (3.3%) affected individuals but only 21 of 2,804 (0.7%) controls (odds ratio (OR) = 4.6; confidence interval (CI) = 2.6–8.0; P = 1.3 × 10−7). A replication study identified these two variants in 10 of 348 (2.9%) individuals with alcoholic chronic pancreatitis but only 3 of 432 (0.7%) subjects with alcoholic liver disease (OR = 4.2; CI = 1.2–15.5; P = 0.02). CTRC variants were also found in 10 of 71 (14.1%) Indian subjects with tropical pancreatitis but only 1 of 84 (1.2%) healthy controls (OR = 13.6; CI = 1.7–109.2; P = 0.0028). Functional analysis of the CTRC variants showed impaired activity and/or reduced secretion. The results indicate that loss-of-function alterations in CTRC predispose to pancreatitis by diminishing its protective trypsin-degrading activity.

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 × 10−8). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Variants in CPA1 are strongly associated with early onset chronic pancreatitis

Chronic pancreatitis is an inflammatory disorder of the pancreas. We analyzed CPA1, encoding carboxypeptidase A1, in subjects with nonalcoholic chronic pancreatitis (cases) and controls in a German discovery set and three replication sets. Functionally impaired variants were present in 29/944 (3.1%) German cases and 5/3,938 (0.1%) controls (odds ratio (OR) = 24.9, P = 1.5 × 10−16). The association was strongest in subjects aged ≤10 years (9.7%; OR = 84.0, P = 4.1 × 10−24). In the replication sets, defective CPA1 variants were present in 8/600 (1.3%) cases and 9/2,432 (0.4%) controls from Europe (P = 0.01), 5/230 (2.2%) cases and 0/264 controls from India (P = 0.02) and 5/247 (2.0%) cases and 0/341 controls from Japan (P = 0.013). The mechanism by which CPA1 variants confer increased pancreatitis risk may involve misfolding-induced endoplasmic reticulum stress rather than elevated trypsin activity, as is seen with other genetic risk factors for this disease.

Mutations of the cystic fibrosis gene, but not cationic trypsinogen gene, are associated with recurrent or chronic idiopathic pancreatitis

OBJECTIVE:We investigated whether mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and cationic trypsinogen gene are associated with recurrent acute, or chronic idiopathic pancreatitis.METHODS:Twenty patients with idiopathic pancreatitis (11 women, nine men; mean age, 30 yr) were studied for the presence of a CFTR mutation by screening the genomic DNA for more than 30 mutations and variants in the CFTR gene. Selected mutations of the cationic trypsinogen gene were screened by Afl III restriction digestion or by a mutation-specific polymerase chain reaction (PCR). In each patient exons 1, 2, and 3 of the cationic trypsinogen gene were sequenced. Patients with a CFTR mutation underwent evaluation of further functional electrophysiological test (intestinal current measurement).RESULTS:No mutation of the cationic trypsinogen gene was detected. A CFTR mutation was detected in 6/20 (30.0%) patients. Three patients (15.0%) had a cystic fibrosis (CF) mutation on one chromosome (ΔF508, I336K, Y1092X), which is known to cause phenotypical severe cystic fibrosis. One patient was heterozygous for the 5T allele. In addition, two possibly predisposing CFTR variants (R75Q, 1716G→A) were detected on four patients, one of these being a compound heterozygous for the missense mutation I336K and R75Q. No other family member (maternal I336K; paternal R75Q; sister I1336K) developed pancreatitis. An intestinal current measurement in rectum samples of patients with a CFTR mutation revealed no CF-typical constellations.CONCLUSIONS:CFTR mutations are associated with recurrent acute, or chronic idiopathic pancreatitis, whereas mutations of the cationic trypsinogen mutation do not appear to be a frequent pathogenetic factor.

Clinical characterization of patients with hereditary pancreatitis and mutations in the cationic trypsinogen gene

PURPOSE We determined the clinical manifestations of hereditary pancreatitis in nearly 30 families. PATIENTS AND METHODS The two trypsinogen mutations N29I and R122H were identified in a group of 550 patients with chronic pancreatitis of unclear origin. The following criteria were used to characterize the severity of chronic pancreatitis (one point each): calcifications, cysts, dilation of the pancreatic duct, diabetes, hospital treatment, and operation. Stages were defined as stage 0 (no points), stage 1 (one to two points), stage 2 (three to four points), and stage 3 (more than four points). Smoking and drinking habits were also recorded. RESULTS Six families with the N29I mutation (25 subjects with the mutation) and 21 families with the R122H mutation (76 subjects with the mutation) were identified. The median ages for the onset of disease were 11 years in N29I and 10 years in R122H patients. The severity of chronic pancreatitis and symptoms were similar for both mutations. About 26% (n = 26) of the 101 subjects carrying a mutation were asymptomatic, and 42% (n = 42) had mild disease (stage 1). Twenty-nine percent (n = 29) had moderate disease (stage 2), and only 4% (n = 4) had severe disease (stage 3). CONCLUSIONS Symptoms of patients with the N29I or R122H trypsinogen mutation were generally similar. The majority of subjects with trypsinogen mutations had mild disease or was asymptomatic.

CFTR, SPINK1, CTRC and PRSS1 variants in chronic pancreatitis: is the role of mutated CFTR overestimated?

Objective In chronic pancreatitis (CP), alterations in several genes have so far been described, but only small cohorts have been extensively investigated for all predisposing genes. Design 660 patients with idiopathic or hereditary CP and up to 1758 controls were enrolled. PRSS1, SPINK1 and CTRC were analysed by DNA sequencing, and cystic fibrosis transmembrane conductance regulator (CFTR) by melting curve analysis. Results Frequencies of CFTR variants p.R75Q, p.I148T, 5T-allele and p.E528E were comparable in patients and controls. We identified 103 CFTR variants, which represents a 2.7-fold risk increase (p<0.0001). Severe cystic fibrosis (CF)-causing variants increased the risk of developing CP 2.9-fold, and mild CF-causing variants 4.5-fold (p<0.0001 for both). Combined CF-causing variants increased CP risk 3.4-fold (p<0.0001), while non-CF-causing variants displayed a 1.5-fold over-representation in patients (p=0.14). CFTR compound heterozygous status with variant classes CF-causing severe and mild represented an OR of 16.1 (p<0.0001). Notably, only 9/660 (1.4%) patients were compound heterozygotes in this category. Trans-heterozygosity increased CP risk, with an OR of 38.7, with 43/660 (6.5%) patients and 3/1667 (0.2%) controls being trans-heterozygous (p<0.0001). Conclusions Accumulation of CFTR variants in CP is less pronounced than reported previously, with ORs between 2.7 and 4.5. Only CF-causing variants reached statistical significance. Compound and trans-heterozygosity is an overt risk factor for the development of CP, but the number of CFTR compound heterozygotes in particular is rather low. In summary, the study demonstrates the complexity of genetic interactions in CP and a minor influence of CFTR alterations in CP development.

MUTATIONS OF THE CATIONIC TRYPSINOGEN IN HEREDITARY PANCREATITIS

Hereditary pancreatitis (OMIM 167800) is thought to be associated with a mutation of the exon 3 of cationic trypsinogen (Nature Genet (1996): 14:141–145). This paper reports sequence data of two independent families suffering from this disease. PCR amplificates from leukocyte or buccal swab DNA showed no mutation of exon 3 of cationic trypsinogen. Instead, in exon 2, an A‐to‐T tranversion was found that led to the substitution of Asn by Ile in the sixth amino acid of the active trypsin. In exons 4 and 5, silent mutations were found. In the other expressed trypsinogens, several homozygous alterations not associated to hereditary pancreatitis were identified. As a model of pathogenesis, we hypothesize that mutation of trypsinogen in exon 2 could lead to premature cleavage of the activation peptide of trypsinogen or to altered intracellular transport. Hum Mutat 12:39–43, 1998.

Individual patient data meta-analysis of controlled attenuation parameter (CAP) technology for assessing steatosis

BACKGROUND & AIMS The prevalence of fatty liver underscores the need for non-invasive characterization of steatosis, such as the ultrasound based controlled attenuation parameter (CAP). Despite good diagnostic accuracy, clinical use of CAP is limited due to uncertainty regarding optimal cut-offs and the influence of covariates. We therefore conducted an individual patient data meta-analysis. METHODS A review of the literature identified studies containing histology verified CAP data (M probe, vibration controlled transient elastography with FibroScan®) for grading of steatosis (S0-S3). Receiver operating characteristic analysis after correcting for center effects was used as well as mixed models to test the impact of covariates on CAP. The primary outcome was establishing CAP cut-offs for distinguishing steatosis grades. RESULTS Data from 19/21 eligible papers were provided, comprising 3830/3968 (97%) of patients. Considering data overlap and exclusion criteria, 2735 patients were included in the final analysis (37% hepatitis B, 36% hepatitis C, 20% NAFLD/NASH, 7% other). Steatosis distribution was 51%/27%/16%/6% for S0/S1/S2/S3. CAP values in dB/m (95% CI) were influenced by several covariates with an estimated shift of 10 (4.5-17) for NAFLD/NASH patients, 10 (3.5-16) for diabetics and 4.4 (3.8-5.0) per BMI unit. Areas under the curves were 0.823 (0.809-0.837) and 0.865 (0.850-0.880) respectively. Optimal cut-offs were 248 (237-261) and 268 (257-284) for those above S0 and S1 respectively. CONCLUSIONS CAP provides a standardized non-invasive measure of hepatic steatosis. Prevalence, etiology, diabetes, and BMI deserve consideration when interpreting CAP. Longitudinal data are needed to demonstrate how CAP relates to clinical outcomes. LAY SUMMARY There is an increase in fatty liver for patients with chronic liver disease, linked to the epidemic of the obesity. Invasive liver biopsies are considered the best means of diagnosing fatty liver. The ultrasound based controlled attenuation parameter (CAP) can be used instead, but factors such as the underlying disease, BMI and diabetes must be taken into account. Registration: Prospero CRD42015027238.

Mutational screening of patients with nonalcoholic chronic pancreatitis: identification of further trypsinogen variants

OBJECTIVES:Mutations of the cationic trypsinogen (CT) and the serine protease inhibitor, Kazal type 1 (SPINK 1) are associated with chronic pancreatitis. After mutational screening of a cohort of patients with nonalcoholic chronic pancreatitis, we report three novel variants of the trypsinogen molecule and the clinical characteristics of the carriers.METHODS:The coding region of the exon 2 and 3 of the CT gene of 523 patients with chronic nonalcoholic pancreatitis (108 patients with suspected hereditary pancreatitis (HP) and 415 patients with “idiopathic” pancreatitis [IP]) and 82 controls was analyzed after polymerase chain reaction amplification. Clinical characteristics were obtained by questioning the patients and their relatives and physicians. HP was suspected when two members of a family had chronic pancreatitis. A restriction digestion was used to analyze the N34S mutation SPINK 1.RESULTS:The mutation R122H of the cationic trypsinogen was found in 21 index patients, N29I in six index patients, and A16V and D22G in one index patient, all from HP families. The N34S mutation of SPINK 1 was found in two index patients with a family history of HP. In three patients, the novel point mutations L104P, R116C, and C139F of the cationic trypsinogen were found. A clear autosomally dominant inheritance of chronic pancreatitis was not present in these families. In 75 index patients from HP families (69.4%), no mutation could be found. The SPINK 1-mutation N34S was detected in only one patient carrying a CT mutation, and was found in 68 (16.4%) of patients with IP.CONCLUSIONS:The R122H and N29I mutations of CT are the most common disease-associated mutations in HP; the N34S mutation of SPINK 1 is the most frequent genetic risk factor associated with IP. The CT gene carries several variations that could be associated with chronic pancreatitis. To avoid overestimating the pathogenetic impact of novel trypsinogen variants, a detailed clinical characterization of all patients with early onset chronic pancreatitis is mandatory.

Acoustic radiation force impulse imaging (ARFI) for non-invasive detection of liver fibrosis: examination standards and evaluation of interlobe differences in healthy subjects and chronic liver disease.

Abstract Objective. Acoustic radiation force impulse imaging (ARFI) is a non-invasive method for the quantification of liver stiffness. We aimed to develop standards for the measuring procedure and studied the impact of different measuring sites. Materials and Methods. ARFI was tested in a tissue phantom and in 50 healthy volunteers. In addition, 116 patients with chronic liver disease underwent ARFI. The results were compared with histological staging (non-viral liver disease) and transient elastography (hepatitis C). ARFI diagnostic performance was evaluated with receiver operating characteristic curves. Results. ARFI results were not normally distributed in >20% of cases. Deep inspiration significantly increased ARFI values by 13% (p < 0.05). The mean shear-wave velocity in healthy individuals was 1.28 ± 0.19 m/s in the left liver lobe and 1.15 ± 0.17 m/s in the right liver lobe (p < 0.001). Similarly, in 79/116 patients with chronic liver disease a significant difference of shear-wave velocity between both liver lobes was detected. The histological staging correlated with ARFI results of the biopsy site (r = 0.661, p < 0.001) in non-viral liver disease (n = 47). The mean shear-wave velocity in cases with F1 and F2 fibrosis was increased in the left compared with the right liver lobe (2.1 ± 0.73 m/s vs. 1.75 ± 0.89 m/s, p = 0.041). Similar results were obtained in patients with hepatitis C (n = 69). Conclusion. Our study strengthens the necessity for definition of examination standards and demonstrates the usefulness of ARFI in non-viral liver disease. Interlobe variations of liver stiffness demand further investigation.