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Objective: Gain-of-function mutations in Na v 1.9 have been identified in three families with rare heritable pain disorders, and in patients with painful small-fibre neuropathy. Identification and functional assessment of new Na v 1.9 mutations will help to elucidate the phenotypic spectrum of Na v 1.9 channelopathies., Methods: Patients from a large family with early-onset pain symptoms were evaluated by clinical examination and genomic screening for mutations in SCN9A and SCN11A . Electrophysiological recordings and multistate modelling analysis were implemented for functional analyses., Results: A novel Na v 1.9 mutation, p.Arg222His, was identified in patients with early-onset pain in distal extremities including joints and gastrointestinal disturbances, but was absent from an asymptomatic blood relative. This mutation alters channel structure by substituting the highly conserved first arginine residue in transmembrane segment 4 (domain 1), the voltage sensor, with histidine. Voltage-clamp recordings demonstrate a hyperpolarising shift and acceleration of activation of the p.Arg222His mutant channel, which make it easier to open the channel. When expressed in dorsal root ganglion neurons, mutant p.Arg222His channels increase excitability via a depolarisation of resting potential and increased evoked firing., Conclusions: This study expands the spectrum of heritable pain disorders linked to gain-of-function mutations in Na v 1.9, strengthening human validation of this channel as a potential therapeutic target for pain., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Polycystic livers are seen in the rare inherited disorder isolated polycystic liver disease (PCLD) and are recognized as the most common extrarenal manifestation in autosomal dominant polycystic kidney disease. Hepatic cystogenesis is characterized by progressive proliferation of cholangiocytes, ultimately causing hepatomegaly. Genetically, polycystic liver disease is a heterogeneous disorder with incomplete penetrance and caused by mutations in PRKCSH, SEC63, PKD1, or PKD2. Genome-wide SNP typing and Sanger sequencing revealed no pathogenic variants in hitherto genes in an extended PCLD family. We performed whole-exome sequencing of DNA samples from two members. A heterozygous variant c.3562C > T located at a highly conserved amino acid position (p.R1188W) in the low density lipoprotein receptor-related protein 5 (LRP5) gene segregated with the disease (logarithm of odds score, 4.62) but was not observed in more than 1,000 unaffected individuals. Screening of LRP5 in a PCLD cohort identified three additional mutations in three unrelated families with polycystic livers (p.V454M, p.R1529S, and p.D1551N), again all undetected in controls. All variants were predicted to be damaging with profound structural effects on LRP5 protein domains. Liver cyst tissue and normal hepatic tissue samples from patients and controls showed abundant LRP5 expression by immunohistochemistry. Functional activity analyses indicated that mutant LRP5 led to reduced wingless signal activation. In conclusion, we demonstrate that germ-line LRP5 missense mutations are associated with hepatic cystogenesis. The findings presented in this study link the pathophysiology of PCLD to deregulation of the canonical wingless signaling pathway.

Background & Aims: Heterozygous germline mutations in PRKCSH cause autosomal dominant polycystic liver disease (PCLD), but it is not clear how they lead to cyst formation. We investigated whether mutations in cyst epithelial cells and corresponding loss of the PRKCSH gene product (hepatocystin) contributed to cyst development., Methods: Liver cyst material was collected through laparoscopic cyst fenestration from 8 patients with PCLD who had a heterozygous germline mutation in PRKCSH. Tissue sections from 71 cysts (2-14 per patient) were obtained for hepatocystin staining and mutation analysis. Cyst epithelium was acquired using laser microdissection; DNA was isolated and analyzed for loss of heterozygosity (LOH) and somatic mutations using restriction analysis and sequencing. Common single nucleotide polymorphisms (SNPs) in a 70-kilobase region surrounding the germline mutation were used to determine variations in the genomic region with LOH., Results: The wild-type allele of PRKCSH was lost (LOH) in 76% of cysts (54/71). Hepatocystin was not detected in cyst epithelia with LOH, whereas heterozygous cysts still expressed hepatocystin. The variation observed in the LOH region analysis indicates that cysts develop independently. We also detected somatic mutations in PRKCSH in 17% (2/12) of the cysts without LOH. Trans-heterozygous mutations in SEC63 were not observed., Conclusions: Among patients with PCLD who have a heterozygous germline mutation in PRKCSH, we found secondary, somatic mutations (second hits) in more than 76% of the liver cyst epithelia. PCLD is recessive at the cellular level, and loss of functional PRKCSH is an important step in cystogenesis., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background and Aim: Isolated polycystic liver disease (PCLD) is characterized by the presence of multiple cysts in the liver in the absence of polycystic kidneys. The clinical profile of PCLD is poorly defined and we set up a study for the clinical characteristics of PCLD., Methods: We collected clinical data on 188 PCLD patients (defined as >10 liver cysts) from five tertiary referral centres, and 137 patients were selected for the purpose of this study. We performed molecular analysis of the PCLD associated genes PRKCSH and SEC63 in 91 patients., Results: A total of 118 (86%) patients were female. The majority of patients (88%) had >20 cysts. The median age at diagnosis was 47 years (range 23-84). 37 (41%) patients carried a mutation. Clinical symptoms at presentation were present in 111 (84%) patients. γ-glutamyl transferase was elevated to 1.4 times upper limit of normal (interquartile range 1.0-2.7). The presence of a mutation and female gender predicted a more severe course: female patients were 9 years younger at the time of diagnosis (47 years; range 23-84) and 91% had symptoms (P<0.01); likewise, mutation carriers were younger at presentation (39 years; range 35-48) and 95% of this cohort had symptoms (P<0.01). During follow-up [median 8.2 years (range 0-35)], 10% of untreated and 51% of treated patients developed complications. Mortality in this cohort was 8%, but only 2% died of PCLD-related causes. 58% of patients were treated a median of 2 years (range 0-25) after diagnosis., Conclusion: Symptomatic PCLD patients are mainly females. Females and mutation carriers were younger at diagnosis and had a more severe course of disease., (© 2010 John Wiley & Sons A/S.)

Polycystic liver disease (PCLD) is characterized by intralobular bile duct cysts in the liver. It is caused by mutations in PRKCSH, encoding hepatocystin, and SEC63, encoding Sec63p. The main goals of this study were to screen for novel mutations and to analyze mutations for effects on protein structure and function. We screened 464 subjects including 76 probands by direct sequencing or conformation-sensitive capillary electrophoresis. We analyzed the effects of all known and novel mutations using a combination of splice site recognition, evolutionary conservation, secondary and tertiary structure predictions, PolyPhen, and pMut and sift. We identified a total of 26 novel mutations in PRKCSH (n = 14) and SEC63 (n = 12), including four splice site mutations, eight insertions/ deletions, six non-sense mutations, and eight missense mutations. Out of 48 PCLD mutations, 13 were predicted to affect splicing. Most mutations were located in highly conserved regions and homology modeling for two domains of Sec63p showed severe effects of the residue substitutions. In conclusion, we identified 26 novel mutations associated with PCLD and we provide in silico analysis in order to delineate the role of these mutations.

Chronic pancreatitis (CP) is a clinical situation with persisting inflammation leading to destruction of the pancreas ensuing endocrine and exocrine failure. There are 4 subtypes: hereditary, idiopathic, alcoholic and tropical pancreatitis. Genetic factors can explain a significant proportion of CP cases. The PRSS1 gene, encoding cationic trypsinogen, was found to be correlated with hereditary CP. This signalled the extensive search for other candidate genes within the trypsin pathway. Genes like SPINK1 and CTRC are associated with CP and should be considered as important contributing factors rather than causative. The search for candidate genes not part of the trypsin pathway has been less successful and the only gene consistently associated with CP is the Cystic Fibrosis Transmembrane Regulator. In this review we will discuss the various CP subtypes in relation to the respective genetic variants. This review will also address the implications of genetic testing in daily clinical practise., (2010 Elsevier Ltd. All rights reserved.)

Objective: To assess genetic, clinical and morphological characteristics of hereditary pancreatitis, a rare type of chronic pancreatitis with an early onset of symptoms, which is, among others, caused by mutations in the PRSS1 gene., Design: Observational cohort study., Method: The study population consisted of 496 patients (27,375 person-years) who were referred to Radboud University Nijmegen Medical Centre for molecular diagnosis of hereditary pancreatitis during period 2000 to 2007. 61 patients with a positive family history of hereditary pancreatitis were selected. Analysis for PRSS1 gene mutations was performed by complete sequence analysis of the exons. All patients received a structured questionnaire., Results: From 25 families 61 patients were included (2,047 person-years). PRSS1 mutations were detected in 52 patients (85.2%): p.R122H (67.2%), p.N29I (14.8%), p.E79K (1.6%), p.N29T (1.6%). In the 40 patients whose clinical data were known the median age at diagnosis was 10.5 years (range: 0-42 years). Pain was reported in 28 (70% of 40 patients in whom all information was complete). 27 patients (67.5%) were admitted to the hospital once or more due to the attacks of pancreatitis. Exocrine and endocrine dysfunction was seen in 6 patients (15%). 24 patients (60%) had undergone a surgical intervention, 10 of whom had undergone a pancreaticojejunostomy. A family history of pancreatic carcinoma was found in 5 patients (12.5%)., Conclusion: The percentage of PRSS1 mutation was high (85.2%) among this Dutch population that was selected on basis of a positive family history of hereditary pancreatitis. Most patients had no chronic pain.

Gain-of-function mutations of Na(V)1.7 have been shown to produce two distinct disorders: Na(V)1.7 mutations that enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; Na(V)1.7 mutations that impair inactivation produce a different, nonoverlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular, and perimandibular pain. Here we report a novel Na(V)1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4-S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (-7 mV) the voltage dependence of activation, slows deactivation, and enhances ramp responses, as observed in Na(V)1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage dependence of fast inactivation, slows fast inactivation, and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a Na(V)1.7 mutant with biophysical characteristics common to PEPD (impaired fast inactivation) and IEM (hyperpolarized activation, slow deactivation, and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.

Autosomal dominant polycystic liver disease (PCLD) is characterized by multiple liver cysts and is caused by mutations in PRKCSH (hepatocystin). Mechanisms of cystogenesis are unknown, but previous studies have shown that hepatocystin is secreted in vitro. The goal of this study was to determine the fate of hepatocystin in vivo. Using immunoprecipitation, we determined that mutant hepatocystin is secreted from both apical and basolateral cell surface of MDCK cells stably transfected with mutant hepatocystin. Analysis of 60 cyst fluid samples from polycystic livers using Western blot, MALDI-TOF MS or nLC-MS/MS did not detect hepatocystin in liver cyst fluid. We did identify 163 ubiquitous serum proteins. No paracrine or autocrine factors were recognized. Although cyst fluids vary greatly in protein concentration, a PCLD specific protein pattern was not established. In conclusion, hepatocystin is not secreted in PCLD liver cyst fluid, suggesting that mutant hepatocystin is either not produced or degraded intracellularly. PCLD cysts develop from intralobular bile ductules and cyst fluid mainly contains common serum proteins comparable to that of other polycystic diseases.

Mutations in nucleotide-binding oligomerization domain-2 (NOD2), leading to defective recognition of bacterial peptidoglycans, are associated with Crohn's disease. The underlying mechanism that results in increased inflammation in the guts of the patients bearing NOD2 mutations is still unclear. We hypothesized that NOD2 engagement leads to cross-tolerance to stimulation of Toll-like receptors (TLR), and we investigated whether patients with Crohn's disease who bear NOD2 mutations display a disturbed NOD2/TLR cross-tolerance. Peripheral blood mononuclear cells preincubated with NOD2 ligands were specifically down-regulated for the production of tumour necrosis factor-alpha (TNF-alpha) induced by the TLR4 ligand lipopolysaccharide, as well as by intestinal microorganisms, whereas the production of anti-inflammatory cytokines was not modulated. While in cells isolated from patients with Crohn's disease with the wild-type NOD2 allele, the NOD2 engagement led to a similar cross-tolerance to TLR4-dependent stimulation of TNF-alpha, the cross-tolerance between NOD2 and TLR4 was absent in the cells of five patients homozygous for the 3020insC NOD2 mutation, leading to uninhibited release of TNF-alpha by TLR4 ligands and intestinal bacteria. In conclusion, we propose the absence of NOD2/TLR4 cross-tolerance as a central mechanism for the increased susceptibility to Crohn's disease in individuals with NOD2 mutations.

Background: A recent study reported that the c.30T>A (p.Cys10Ter; rs2043211) variant, in the CARD8 (TUCAN) gene, is associated with Crohn's disease (CD). The aim of this study was to analyze the frequency of p.C10X in 3 independent European (IBD) cohorts from Germany, Hungary, and the Netherlands., Methods: We included a European IBD cohort of 921 patients and compared the p.C10X genotype frequency to 832 healthy controls. The 3 study populations analyzed were: (1) Germany [CD, n = 317; ulcerative colitis (UC), n = 180], (2) Hungary (CD, n = 149; UC, n = 119), and (3) the Netherlands (CD, n = 156). Subtyping analysis was performed in respect to NOD2 variants (p.Arg702Trp, p.Gly908Arg, c.3020insC) and to clinical characteristics. Ethnically matched controls were included (German, n = 413; Hungarian, n = 202; Dutch, n = 217)., Results: We observed no significant difference in p.C10X genotype frequency in either patients with CD or patients with UC compared with controls in all 3 cohorts. Conversely to the initial association study, we found a trend toward lower frequencies of the suggestive risk wild type in CD from the Netherlands compared with controls (P = 0.14). We found neither evidence for genetic interactions between p.C10X and NOD2 nor the C10X variant to be associated with a CD or UC phenotype., Conclusions: Analyzing 3 independent European IBD cohorts, we found no evidence that the C10X variant in CARD8 confers susceptibility for CD.

Primary erythermalgia is a rare autosomal dominant inherited disorder characterized by recurrent attacks of red, warm and painful burning extremities. The gene involved in primary erythermalgia, SCN9A, encodes for a voltage dependent sodium channel alpha subunit (NaV1.7). NaV1.7 is located in dorsal root ganglions and in nociceptive peripheral neurons. It has been hypothesized that mutations lead to a gain of function and hyperexcitability of peripheral sensory neurons contributing to symptoms in primary erythermalgia.

Primary erythermalgia is a rare disorder characterized by recurrent attacks of red, warm and painful hands, and/or feet. We previously localized the gene for primary erythermalgia to a 7.94 cM region on chromosome 2q. Recently, Yang et al identified two missense mutations of the sodium channel alpha subunit SCN9A in patients with erythermalgia. The presence of voltage-gated sodium channels in sensory neurons is thought to play a crucial role in several chronic painful neuropathies. We examined four different families and two sporadic cases and detected missense sequence variants in SCN9A to be present in primary erythermalgia patients. A total of five of six mutations were located in highly conserved regions. One family with autosomal dominantly inherited erythermalgia was double heterozygous for two separate SCN9A mutations. These data establish primary erythermalgia as a neuropathic disorder and offers hope for treatment of this incapacitating painful disorder.

Purpose: Autosomal dominant hereditary diffuse gastric cancer (HDGC) is caused by germ-line E-cadherin (CDH1) gene mutations. Early detection of cancer in carriers is difficult because HDGC escapes endoscopic detection. We hypothesized that the glucose metabolism is enhanced in HDGC and that this can be detected with [18F]fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET)., Experimental Design and Results: An asymptomatic twenty-eight year-old female was seen at our outpatient clinic because of a request for screening on HDGC. Her father and younger sister died of diffuse gastric cancer, at the ages of 52 and 27, respectively. Mutational analysis of the CDH1 gene in this patient demonstrated a novel heterozygous splice-site mutation in exon 8 (1135delACGGTAATinsTTAGA). Upper gastrointestinal endoscopies revealed no macroscopic abnormalities, but one of the 40 random biopsy specimens showed well-differentiated signet-cell carcinoma. A FDG-PET scan demonstrated two spots of FDG accumulation, one located in the proximal part of the stomach and the second in the region of the pylorus. A total gastrectomy was performed and microscopic examination showed focal localization of intramucosal adenocarcinoma of the signet-cell type in the cardiac and antrum area. Most notably, the localization of the FDG accumulation matched the localization of the carcinoma., Conclusions: We present an asymptomatic patient from a HDGC family carrying a novel CDH1 mutation in whom FDG-PET scanning facilitated early detection of HDGC. This calls for further investigation of the role of FDG-PET scan as a screening modality in HDGC.

Background: Although chronic pancreatitis is associated with risk factors such as alcoholism, hyperparathyroidism, and hypertriglyceridaemia, little is known of the actual aetiology of the disease. It is thought that inappropriate activation of trypsinogen causes pancreatitis, and indeed in cases of hereditary pancreatitis mutations of cationic trypsinogen (PRSS1) have been described. As serine protease inhibitor Kazal type 1 (SPINK1) is a potent natural inhibitor of pancreatic trypsin activity, we hypothesised that SPINK1 mutations would be more common than expected among an unselected cohort of adult chronic pancreatitis patients., Aims: To detect the prevalence of SPINK1 mutations in a cohort of chronic pancreatitis patients., Methods: DNA was isolated from a cohort of 115 adult patients with chronic pancreatitis of alcoholic (n=72), hereditary (n=10), idiopathic (n=24), and miscellaneous (n=9) origin. We performed mutational analysis for two PRSS1 mutations (R122H, N29I) and four specific SPINK1 gene mutations (M1T, L14P, N34S, P55S) and compared the results with a control group of 120 healthy Dutch subjects., Results: In six of the 10 patients that fulfilled the criteria for hereditary pancreatitis, but in none of the control subjects, mutations in the PRSS1 gene were found. In 14 patients we detected a SPINK1 mutation. Eleven patients were heterozygous for the N34S mutation and sequencing confirmed the homozygous state of N34S in a brother and sister. Two patients carried the P55S mutation, one as a compound heterozygote with N34S. The M1T and L14P SPINK1 mutations were not found in our cohort. The N34S mutation was detected in only two of 120 controls, while the P55S, M1T, and L14P mutations were absent in the same group. Patients with the N34S allele had a later onset of disease than those with PRSS1 gene mutations but earlier onset compared with the mutation negative group., Conclusion: Identification of SPINK1 mutations in 12.2% of patients with adult alcoholic and idiopathic chronic pancreatitis suggests an important role for SPINK1 as a predisposing factor in adult chronic pancreatitis.

Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) is an autosomal recessive inflammatory disorder characterised by recurrent episode of fever associated with lymphadenopathy, abdominal distress, joint involvement and skin lesions. We recently demonstrated that mutations in the mevalonate kinase gene (MVK) are associated with HIDS. Direct DNA sequencing was done to screen the entire coding region of MVK in 25 unrelated patients with HIDS. Mutations were detected in the coding region of the gene including 11 missense mutations, one deletion, the absence of expression of one allele, as well as three novel polymorphisms. Seven of these mutations are novel. The large majority of the patients were compound heterozygotes for two mutations. Of these, V377I (G-->A) is the most common mutation occurring in 20 unrelated patients and was found to be associated with I268T in six patients. Mutations were associated with a decrease of mevalonate kinase (MK) (ATP:mevalonate 5-phosphotransferase, EC 2.7.I.36) enzymatic activity but not as profound as in mevalonic aciduria, a syndrome also caused by a deficient activity of MK. In HIDS the mutations are located all along the protein which is different from mevalonic aciduria where MK mutations are mainly clustered to a same region of the protein. On the basis of this study, we propose that the diagnostic screen of MVK in HIDS should be first directed on V377I and I268T mutations. Three patients are also described to illustrate the genotypic and phenotypic overlap with mevalonic aciduria.

Hereditary pancreatitis is an autosomal dominant form of chronic pancreatitis. It presents with recurrent attacks of acute pancreatitis, usually starting in early childhood. The attacks may vary from mild abdominal pain to pancreatic necrosis, splenic vein thrombosis, pseudocysts and death. Ultimately chronic pancreatitis ensues with unrelenting pain, calcifications, endocrine and exocrine dysfunction. The penetrance is estimated at 80%. With the use of genetic linkage analysis the gene for hereditary pancreatitis was placed on the long arm of chromosome 7 (7q35). Mutational analysis identified cationic trypsinogen as the disease gene. Cationic trypsinogen mutations are thought to result in resistance of this molecule to autolysis.