Hepatic cyst penetration of cefazolin in patients receiving aspiration sclerotherapy.

BACKGROUND: Hepatic cyst infection is a potentially severe complication in cystic disease. Treatment demands effective antibiotic concentrations within the infected cyst. OBJECTIVES: The aim of this study was to use elective hepatic cyst drainage as a unique pharmacokinetic model to investigate whether cefazolin, a first-generation cephalosporin, is able to penetrate hepatic cysts. PATIENTS AND METHODS: Patients scheduled to undergo percutaneous aspiration sclerotherapy of a symptomatic non-infected, non-neoplastic hepatic cyst were eligible for this study. All participants received a single perioperative prophylactic dose of cefazolin (1000 mg, intravenously). We collected blood and cyst fluid samples to determine total and unbound cefazolin concentrations using HPLC. The primary outcome was hepatic cyst penetration, expressed as the ratio (%) of unbound concentration of cefazolin in cyst fluid to plasma (both in mg/L). RESULTS: We included eight patients [male = 25%, median age = 60 years (IQR 54-75), median estimated glomerular filtration rate = 97 mL/min/1.73 m(2) (IQR 67-102) and median serum albumin = 40 g/L (IQR 37-40)]. We detected low concentrations of unbound cefazolin in cyst fluid (≤1.0 mg/L). The median plasma unbound cefazolin peak level (immediately after cefazolin administration) was 36.6 mg/L (IQR 23.7-54.1) and the level at the time of cyst fluid aspiration was 16.1 mg/L (IQR 13.0-20.1). In total, the hepatic cyst penetration of free cefazolin was only 2.2% (IQR 0.7-5.2). CONCLUSIONS: We developed a study model to investigate the penetration of antibiotics into hepatic cysts. Cefazolin did not reach adequate intracystic concentrations. Future studies should explore alternatives.

Secondary and tertiary structure modeling reveals effects of novel mutations in polycystic liver disease genes PRKCSH and SEC63.

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.

Genetic polymorphism in UDP-glucuronosyltransferase 2B7 and colorectal cancer risk.

Colorectal cancer (CRC) is one of the most common malignancies in the Western world. CRC is strongly associated with lifestyle factors. Susceptibility to CRC may be partly due to deficient detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes result in variations in detoxification activities, which might influence the levels of carcinogens in the gastrointestinal tract, influencing the risk for CRC. To determine whether a genetic polymorphism in the detoxification enzyme UDP-glucuronosyltransferase 2B7 (UGT2B7) predisposes to CRC, 411 Caucasian patients with sporadic CRC and 600 Caucasian controls recruited from the same geographic area were genotyped for the functional UGT2B7 H268Y polymorphism. DNA was isolated and tested by a dual-color real-time polymerase chain reaction assay. Overall, no differences in genotype distributions between patients with CRC and controls were observed. When analyzed with respect to tumor location, a shift from the UGT2B7*I *2 into the UGT2B7*2*2 genotype was seen in patients with proximal CRC (OR 1.80, 95% CI 1.11-2.89). In the male patient subpopulation an even stronger association was observed (*1*1 + *1*2 vs. *2*2: OR 2.17, 95% CI 1.11-4.04; *1*2 vs. *2*2: OR 2.19, 95% CI 1.10-4.37). No associations with respect to tumor stage were seen. In conclusion, the frequency of the UGT2B7*2*2 genotype is higher in CRC patients with proximal location of the tumor, especially in males, which suggests that this genotype is associated with an increased risk for proximal CRC.

Detoxification enzyme polymorphisms are not involved in duodenal adenomatosis in familial adenomatous polyposis.

BACKGROUND: Patients with familial adenomatous polyposis (FAP) are at high risk of developing duodenal adenomas and carcinomas. Besides germline mutations in the adenomatous polyposis coli (APC) gene, additional factors may influence the age of onset and number of duodenal adenomas. This study compared the genotype distributions of duodenal detoxification enzyme isoforms in patients with FAP and controls. METHODS: The study included 85 patients with FAP and 218 healthy age- and sex-matched controls. Genotyping of all participants using polymerase chain reaction was performed to detect polymorphisms in isoforms of uridine 5'-diphosphate glucuronosyltransferases (UGTs) and glutathione S-transferases (GSTs): UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A10, UGT2B4, UGT2B7, UGT2B15, GSTA1, GSTP1, GSTM1 and GSTT1. RESULTS: The variant genotypes of UGT1A3 were less common in patients with FAP than in controls (odds ratio 0.39 (95 per cent confidence interval 0.22 to 0.67)). There were no associations between FAP and the other polymorphic genes. The polymorphisms investigated had no predictive value for the severity of duodenal adenomatosis in patients with FAP. CONCLUSION: Although the variant genotypes of UGT1A3 were less common in patients with FAP than in those without, this did not modulate the severity of duodenal adenomatosis.

Prevalence of hepatitis C in the general population in the Netherlands.

BACKGROUND: Chronic hepatitis C virus (HCV) is transmitted by blood-blood contact and this leads to high HCV prevalence in risk populations such as haemophilia patients and intravenous drug users. The prevalence in the general Dutch population is unknown, although it appears to be very low in screened blood donors (0.0169%). AIM: The objective of this study is to estimate the prevalence of HCV in a general population sample living in an urbanized region in the Netherlands. METHODS: We randomly selected 2200 EDTA blood samples that had been submitted for analysis of biochemical parameters to a regional servicing laboratory for general practitioners (SHO, Arnhem/Nijmegen, the Netherlands). HCV antibody testing was performed using a three-step approach. For initial screening, an enzyme immunoassay (Bioelisa HCV 4.0, Biokit, Spain) was used. Positive samples were subjected to a second, microparticle enzyme-linked immunoassay (AxSYM HCV version 3.0, Abbott laboratories, IL , USA). Genotypes were determined by Line Probe Assay. RESULTS: A total of four persons (two females, two males) (0.2%) tested positive for HCV antibodies. The average OD/cut-off ratio of the screening assay was 2.9 (range 1.0 to 7.3) and serological findings were confirmed using a specific second immunoassay. HCV RNA (genotype 1b) was found in the sera of two persons. CONCLUSION: The HCV prevalence in our sample of the Dutch population was 0.2% which accords with earlier estimates from prevalence studies in the Netherlands.

Polymorphisms of UDP-glucuronosyltransferase 1A7 are not involved in pancreatic diseases.

BACKGROUND: Xenobiotic mediated cellular injury is thought to play a major role in the pathogenesis of pancreatic diseases. Genetic variations that reduce the expression or activity of detoxifying phase II biotransformation enzymes such as the UDP-glucuronosyltransferases might be important in this respect. Recently, a UGT1A7 low detoxification activity allele, UGT1A7*3, has been linked to pancreatic cancer and alcoholic chronic pancreatitis. OBJECTIVE: To investigate whether UGT1A7 polymorphisms contribute to the risk of pancreatitis and pancreatic cancer. METHODS: Genetic polymorphisms in the UGT1A7 gene were assessed in a large cohort of patients with different types of pancreatitis and pancreatic cancer originating from the Czech Republic (n = 93), Germany (n = 638), Netherlands (n = 136), and Switzerland (n = 106), and in healthy (n = 1409) and alcoholic (n = 123) controls from the same populations. Polymorphisms were determined by melting curve analysis using fluorescence resonance energy transfer probes. In addition, 229 Dutch subjects were analysed by restriction fragment length polymorphism. RESULTS: The frequencies of UGT1A7 genotypes did not differ between patients with acute or chronic pancreatitis or pancreatic adenocarcinoma and alcoholic and healthy controls. CONCLUSIONS: The data suggest that, in contrast to earlier studies, UGT1A7 polymorphisms do not predispose patients to the development of pancreatic cancer and pancreatitis.

[From gene to disease; primary erythermalgia--a neuropathic disease as a consequence of mutations in a sodium pump gene]. / Van gen naar ziekte; de neuropathische ziekte 'primaire erythermalgie' als gevolg van mutaties in een natriumpompgen.

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.

Long-term olive oil-based parenteral nutrition sustains innate immune function in home patients without active underlying disease.

BACKGROUND & AIMS: It remains unclear whether impaired host defenses contribute to the increased risk for infectious complications seen in patients on home parenteral nutrition (HPN). The aim of this study was to compare the innate immune function of patients on olive oil-based HPN with that of healthy controls. METHODS: Innate immune functions and (anti-)oxidant balance were studied in 20 patients on olive oil-based HPN without an active underlying immune-mediated disease (Clinoleic(®), ≥ 6 months; >3 times/week), and 21 age- and sex-matched healthy controls. RESULTS: Neutrophils of patients and controls had a similar capacity to eliminate Streptococcus pneumoniae. Also, levels of activation markers (CD66b, CD11b, CD62L) in granulocytes and monocytes, phorbol ester- and zymosan-induced neutrophil oxygen radical production were not different between patients and controls. No differences in (anti-)oxidant status were found, except for higher concentrations of oxidized glutathione and lower plasma selenium and vitamin C in patients compared to controls. CONCLUSION: Compromised innate immune function does not seem to explain the increased risk for infectious complications in HPN patients using olive oil-based lipid emulsions.

Nav1.7-related small fiber neuropathy: impaired slow-inactivation and DRG neuron hyperexcitability.

OBJECTIVES: Although small fiber neuropathy (SFN) often occurs without apparent cause, the molecular etiology of idiopathic SFN (I-SFN) has remained enigmatic. Sodium channel Na(v)1.7 is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons and their small-diameter peripheral axons. We recently reported the presence of Na(v)1.7 variants that produce gain-of-function changes in channel properties in 28% of patients with painful I-SFN and demonstrated impaired slow-inactivation in one of these mutations after expression within HEK293 cells. Here we show that the I739V Na(v)1.7 variant in a patient with biopsy-confirmed I-SFN impairs slow-inactivation within DRG neurons and increases their excitability. METHODS: A patient with SFN symptoms including pain, and no identifiable underlying cause, was evaluated by skin biopsy, quantitative sensory testing, nerve conduction studies, screening of genomic DNA for variants in SCN9A, and functional analysis. RESULTS: Voltage-clamp analysis following expression within DRG neurons revealed that the Na(v)1.7/I739V substitution impairs slow-inactivation, depolarizing the midpoint (V(1/2)) by 5.6 mV, and increasing the noninactivating component at 10 mV from 16.5% to 22.2%. Expression of I739V channels within DRG neurons rendered these cells hyperexcitable, reducing current threshold and increasing the frequency of firing evoked by graded suprathreshold stimuli. CONCLUSIONS: These observations provide support, from a patient with biopsy-confirmed SFN, for the suggestion that functional variants of Na(v)1.7 that impair slow-inactivation can produce DRG neuron hyperexcitability that contributes to pain in SFN. Na(v)1.7 channelopathy-associated SFN should be considered in the differential diagnosis of cases of SFN in which no other cause is found.

Catechol-O-methyltransferase (COMT) gene variants and pain in chronic pancreatitis.

BACKGROUND: Pain is the major symptom of chronic pancreatitis. The role of genetics in pancreatic pain is unclear. Catechol-O-methyltransferase (COMT) regulates enkephalin levels and influences pain perception. The COMT gene contains functional polymorphisms that have been found to influence human pain perception. The aim of our study was to investigate COMT single-nucleotide polymorphisms (SNP s) and diplotypes in chronic pancreatitis patients and healthy controls. METHODS: We genotyped four COMT gene SNP s: c.1-98A>G (rs6269), c.186C>T (p.=) (rs4633), c.408C>G (p.=) (rs4818) and c.472G>A (p.Val158Met) (rs4680) using a dual-colour discrimination assay in 240 chronic pancreatitis patients and 445 controls. We generated five diplotypes with a frequency >0.5% and compared prevalence between patients and controls. RESULTS: There was no significant association between the SNPs in the COMT gene and chronic pancreatitis. The diplotype ATCA÷ACCG was more prevalent in controls compared with patients (OR 0.48, 95% CI 0.24 to 0.93, p=0.03) where the most common diplotype GCGG ÷ATCA served as reference. However, after correction for multiple testing, this is not a significant difference. The distribution of other diplotypes was not significantly different between patients and controls. CONCLUSION: COMT SNP s and diplotypes are not associated with chronic pancreatitis. As a consequence, our results do not support a significant role for the COMT gene in chronic pancreatitis.

UGT1A7 polymorphisms in chronic pancreatitis: an example of genotyping pitfalls.

UDP-glucuronosyltransferases (UGT) catalyze the glucuronidation of various compounds and thus inactivate toxic substrates. Genetic variations reducing the activity of UGT1A7 have been associated with various gastrointestinal cancers. Most recently, the UGT1A7*3 allele has been reported as a significant risk factor for pancreatic disorders, but we could not confirm these data. This study focused on the possible causes for the noted discrepancy. UGT1A7 genotypes were assessed in 37 samples, which were previously analyzed for UGT1A7 polymorphisms by others. We determined genotypes by melting curve analysis and by DNA sequencing. Additionally, we produced UGT1A7*1 and *3 constructs with or without a mutation at position - 57 of UGT1A7 and analyzed various combinations of these constructs. In 14/37 samples UGT1A7 genotyping results differed. The discrepancy could be explained by polymerase chain reaction bias owing to an unbalanced allelic amplification which was caused by a -57T>G variant located within the sequence of the chosen primer template in previous studies. Our findings indicate that most of the previously reported genetic associations between UGT1A7 and gastrointestinal cancers are based on primer-dependent genotyping errors.

Genetic heterogeneity and exclusion of a modifying locus at 2q in a family with autosomal dominant primary erythermalgia.

BACKGROUND: Primary erythermalgia is a rare disorder characterized by recurrent attacks of red, warm and painful hands and/or feet. In a previous study we reported localization of a gene for primary erythermalgia to a 7.94-cM region on chromosome 2q. A recent study reported voltage-gated sodium channel gene SCN9a sequence variants in a family and a single individual with primary erythermalgia. OBJECTIVES: To describe the clinical characteristics of a large three-generation family with primary erythermalgia and to test for genetic linkage to chromosome 2q. METHODS: We collected clinical data of a 10-member three-generation family with autosomal dominant primary erythermalgia. In addition, we performed linkage analysis and searched for SCN9a variants using a restriction fragment length polymorphism assay. RESULTS: We established the diagnosis of autosomal dominant primary erythermalgia in six of 10 family members. We excluded linkage to chromosome 2q and could not detect SCN9A variants in this family. CONCLUSIONS: In this family with autosomal dominant primary erythermalgia, exclusion of linkage to chromosome 2q is strongly suggestive for genetic heterogeneity.

Genetic polymorphisms in UDP-glucuronosyltransferases and glutathione S-transferases and colorectal cancer risk.

Colorectal cancer (CRC) is one of the most common malignancies in the Western world showing an increasing incidence, and has been associated with genetic and lifestyle factors. Individual susceptibility to CRC may be due partly to variations in detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes may result in variations in detoxification activities, which subsequently might influence the levels of toxic/carcinogenic compounds, and this may influence the risk for CRC. To determine whether genetic polymorphisms in detoxification enzymes predispose to the development of CRC, 371 patients with sporadic CRC and 415 healthy controls were genotyped for polymorphisms in the important detoxification enzymes UDP-glucuronosyltransferase UGT1A1, UGT1A6, UGT1A7 and UGT1A8, and glutathione S-transferase GSTA1, GSTM1, GSTP1 and GSTT1. Patients and controls were all of Caucasian origin. DNA was isolated from either blood or tissue and tested by polymerase chain reaction followed by restriction fragment length polymorphism analyses. Logistic regression analyses showed significant age- and gender-adjusted risks for CRC associated with variant genotypes of UGT1A6 [OR 1.5, 95% (confidence interval) CI 1.03-2.3] and UGT1A7 (OR 2.4, 95% CI 1.3-4.6), whereas no associations were found between CRC and the other polymorphic genes as mentioned above. In conclusion, the data suggest that the presence of variant UGT1A6 and UGT1A7 genotypes with expected reduced enzyme activities, might enhance susceptibility to CRC.