Novel mutations of the ATP7B gene among 109 Hungarian patients with Wilson's disease.

BACKGROUND/AIMS: Diagnosis of Wilson's disease may be difficult in patients presenting with liver disease and in asymptomatic siblings. The aim of the present study was to assess the impact of genetic testing for diagnosis of the disease in a large cohort (n=109) from Hungary. PATIENTS/METHODS: One hundred and nine patients with Wilson's disease were studied (65 men and 44 women; mean age at onset of symptoms: 20+/-9 years). Diagnosis of the disease was based on typical clinical and laboratory features (all had a Wilson's disease score of >or=4). H1069Q was assessed by the semi-nested polymerase chain reaction-based restriction fragment length polymorphism assay. H1069Q heterozygotes and H1069Q negative samples were then screened for mutations (on exons 6 to 20) by denaturating high-performance liquid chromatography and than sequenced on a genetic analyser. RESULTS: Twenty-three different mutations were found. H1069Q was the most frequent mutation in Hungary, detected in 77 patients (71%). Fourteen further known mutations were found by sequencing. We identified eight new mis-sense mutations not described before: N676I, S693Y, Y715H, M769L, W939C, P1273S, G1281D and G1341V. In 36/109 patients (33%) the diagnosis of Wilson's disease was established by adding mutational analysis. The Kayser-Fleischer ring was more frequent in H1069Q homozygous patients and their mean age at the time of diagnosis was higher than in patients heterozygous or negative for H1069Q. CONCLUSION: Eight novel mutations in addition to the 15 that are already known were found in Hungarian patients with Wilson's disease. Our results underline the importance and usefulness of genetic testing for patients presenting with liver disease and for family screening.

Bone disorders in experimentally induced liver disease in growing rats.

AIM: To investigate the change of bone parameters in a new model of experimentally induced liver cirrhosis and hepatocellular carcinoma (HCC) in growing rats. METHODS: Fischer-344 rats (n = 55) were used. Carbon tetrachloride (CCl(4)), phenobarbital (PB), and a single diethylnitrosamine (DEN) injection were used. Animals were killed at wk 8 and 16. Bone mineral content, femoral length, cortical index (quotient of cortical thickness and whole diameter) and ultimate bending load (F(max)) of the femora were determined. The results in animals treated with DEN+PB+CCl(4) (DPC, n = 21) were compared to those in untreated animals (UNT, n = 14) and in control group treated only with DEN+PB (DP, n = 20). RESULTS: Fatty liver and cirrhosis developed in each DPC-treated rat at wk 8 and HCC was presented at wk 16. No skeletal changes were found in this group at wk 8, but each parameter was lower (P<0.05 for each) at wk 16 in comparison to the control group. Neither fatty liver nor cirrhosis was observed in DP-treated animals at any time point. Femoral length and F(max) values were higher (P<0.05 for both) in DP-treated animals at wk 8 compared to the UNT controls. However, no difference was found at wk 16. CONCLUSION: Experimental liver cirrhosis and HCC are accompanied with inhibited skeletal growth, reduced bone mass, and decreased mechanical resistance in growing rats. Our results are in concordance with the data of other studies using different animal models. A novel finding is the transiently accelerated skeletal growth and bone strength after a 8-wk long phenobarbital treatment following diethylnitrosamine injection.

Increased nociceptin/orphanin FQ plasma levels in hepatocellular carcinoma.

AIM: The heptadecapeptide nociceptin alias orphanin FQ is the endogenous agonist of opioid receptor-like1 receptor. It is involved in modulation of pain and cognition. High blood level was reported in patients with acute and chronic pain, and in Wilson disease. An accidental observation led us to investigate nociceptin in hepatocellular carcinoma. METHODS: Plasma nociceptin level was measured by radioimmunoassay, aprotinin was used as protease inhibitor. Hepatocellular carcinoma was diagnosed by laboratory, ultrasound, other imaging, and confirmed by fine needle biopsy. Results were compared to healthy controls and patients with other chronic liver diseases. RESULTS: Although nociceptin levels were elevated in patients with Wilson disease (14.0+/-2.7 pg/mL, n=26), primary biliary cirrhosis (12.1+/-3.2 pg/mL, n=21) and liver cirrhosis (12.8+/-4.0 pg/mL, n=15) compared to the healthy controls (9.2+/-1.8 pg/mL, n=29, P<0.001 for each), in patients with hepatocellular carcinoma a ten-fold increase was found (105.9+/-14.4 pg/mL, n=29, P<0.0001). High plasma levels were found in each hepatocellular carcinoma patient including those with normal alpha fetoprotein and those with pain (104.9+/-14.9 pg/mL, n=12) and without (107.7+/-14.5 pg/mL, n=6). CONCLUSION: A very high nociceptin plasma level seems to be an indicator for hepatocellular carcinoma. Further research is needed to clarify the mechanism and clinical significance of this novel finding.

Measurement of von Willebrand factor as the marker of endothelial dysfunction in vascular diseases.

BACKGROUND: von Willebrand factor is a blood glycoprotein that is required for normal hemostasis. Its level can be increased by endothelial cell damage. HYPOTHESIS: von Willebrand factor is a suitable marker of endothelial dysfunction. METHODS: von Willebrand factor activity was determined by ELISA in patients with acute coronary syndromes, acute stroke and chronic vascular diseases, and was compared with the values of healthy controls. RESULTS: von Willebrand factor activity of patients in each group was significantly higher (P<0.001) than that of the control group. The values of patients with acute coronary syndrome and acute stroke were significantly higher (P<0.05 and P<0.01, respectively) than those of patients with chronic vascular diseases. von Willebrand factor activity was significantly higher in patients with acute coronary syndrome and acute stroke (P<0.05 and P<0.01, respectively) on the sixth day than on admission. CONCLUSIONS: By measuring von Willebrand factor activity, a considerable, significant difference could be found between healthy people and chronic and acute vascular patients. The routine measurement of von Willebrand factor activity in vascular patients as an index of endothelial dysfunction may have clinical importance, because detection of this marker can be a noninvasive way of assisting diagnosis and indicating disease progression.

High serum osteoprotegerin and low RANKL in primary biliary cirrhosis.

BACKGROUND/AIMS: Osteoprotegerin is decoy receptor for osteoclast activating factor, RANKL, and impairs osteoclast funtion. Since osteoporosis is common in primary biliary cirrhosis (PBC), we investigated osteoprotegerin, RANKL and markers of bone turnover in PBC. METHODS: Serum osteoprotegerin, RANKL, osteocalcin (OC) and C-terminal cross-linking telopeptide of type I collagen (CTX-I) were measured by ELISA in 41 patients with PBC, 16 women with chronic hepatitis C (CHC), and as controls in 44 age-matched healthy and 74 post-menopausal osteopenic otherwise healthy women. RESULTS: Serum osteoprotegerin levels were higher in PBC patients (7.8+/-3.0 pmol/l) than in healthy controls (4.4+/-2.3 pmol/l) and osteopenic women (4.0+/-1.0 pmol/l, P<0.0001 for both). RANKL levels were lower in PBC (0.9+/-1.8 pmol/l, P<0.0001) than in healthy controls (1.3+/-0.5 pmol/l). In CHC both osteoprotegerin (9.7+/-4.2 pmol/l) and RANKL (3.2+/-4.7 pmol/l) were elevated compared to the control groups (P<0.0001, for both). There was a positive correlation between serum osteoprotegerin and OC, CTX-I and AST but not with bone mineral density in PBC. CONCLUSIONS: The mechanisms and role of elevated osteoprotegerin and low RANKL in PBC are unclear, but it might partly represent a compensatory mechanism to negative balance of bone remodeling. High OPG and RANKL levels found in CHC might suggest that inflammatory process in the liver could also contribute to the elevation of osteoprotegerin.

[Investigation of von Willebrand-factor as a marker of endothelial dysfunction in atherosclerotic patients]. / A von Willebrand-faktornak, mint az endothelium diszfunkció markerének vizsgálata érbetegekben.

OBJECTIVE: To determine von Willebrand-factor activity as the marker of endothelium dysfunction in vascular diseases and to compare it to healthy controls. METHODS: von Willebrand-factor activity was determined by enzyme-linked immunosorbant assay (ELISA) in patients with acute coronary syndromes (29 patients, 67 +/- 13 years), acute stroke (15 pts, 67 +/- 12 years) on admission, 2nd and 6th day; and chronic vascular diseases (56 pts, 67 +/- 10 years) and was compared to the values of healthy controls (23 persons, 36 +/- 12 years). RESULTS: von Willebrand-factor activity was significantly (p < 0.001) higher in all the measured vascular patients than in the control group. The values of acute patients were significantly (p < 0.05-0.001) higher than those of patients with chronic vascular diseases. In the hospital phase von Willebrand-factor activity in acute patients increased continuously and on day 6 was significantly (p < 0.05-0.01) higher than on admission. von Willebrand-factor activity was significantly (p < 0.05) higher in troponin positive patients with acute coronary syndromes compared to the troponin negative subjects. CONCLUSIONS: von Willebrand-factor was found to be a suitable marker of endothelial dysfunction. The higher von Willebrand-factor activity in patients with vascular diseases compared to the control group can be caused by the endothelial dysfunction and extensive atherosclerosis. The significantly higher von Willebrand-factor activity in acute disorders suggests the more severe endothelium dysfunction and could be related to the development of acute event through increased platelet adhesion and aggregation.

[ATP7B gene mutations in Hungarian patients with Wilson disease--case reports to illustrate the diverse clinical presentations]. / ATP7B génmutációk magyarországi Wilson-kóros betegekben. Esetismertetések a betegség változatos klinikai megjelenésének bemutatására.

ATP7B gene mutations were examined in 70 Wilson patients from Hungary. 11 different mutations were found. In Hungary, similarly to other Central-Eastern European countries, the H1069Q was the most the frequent mutation, detected in 51 patients (73%) by semi-nested polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) assay. 10 further mutations have been found by sequencing as follows: P767P-fs, R778G, K844K-fs, I857T, R969Q, T977M, E1064K, M769L, Y715H and P1273S. These latter three mutations have not been described before. Among the 11 mutations there are five, which have been published only in patients of Turkish, Italian or Albanian origin. It might be the genetic consequence of the 150 years long occupation of Hungary in the 16th and 17th century by Turks. The genotype-phenotype analysis showed that the Kayser-Fleischer ring was more frequent (10/12 = 83%), and the age at the diagnosis was higher in H1069Q homozygous patients than in compound heterozygous or negative patients. Diverse clinical presentation of the disease was demonstrated by case reports giving messages for the practitioners. The gene mutation analysis is of particular importance in siblings of the index patient, since the detection of two mutant allels confirm the diagnosis of the disease even in absence of symptoms. The clinical manifestation of the disease can be preceded by the treatment.

Vitamin D receptor, oestrogen receptor-alpha gene and interleukin-1 receptor antagonist gene polymorphisms in Hungarian patients with primary biliary cirrhosis.

BACKGROUND: Genetic factors have been implicated in the pathogenesis of osteoporosis, a common disorder in primary biliary cirrhosis. Oestrogen receptor-alpha gene, vitamin D receptor gene and interleukin-1 receptor antagonist gene are all attractive candidates for osteoporosis susceptibility. We investigated the polymorphisms of the above genes and bone disease in Hungarian patients with primary biliary cirrhosis. PATIENTS AND METHODS: Thirty-three female patients with primary biliary cirrhosis were enrolled (age range, 39-72 years; anti-mitochondrial antibody M2 positive, stage II-IV). Eighty-four healthy and 76 osteoporotic age matched female subjects served as controls. Vitamin D receptor BsmI, interleukin-1 receptor antagonist gene variable- number tandem repeat and oestrogen receptor-alpha PvuII and XbaI polymorphisms were determined. Bone mineral density was measured by dual energy X-ray absorptiometry (XR26, Norland) in lumbar spine and femoral neck. RESULTS: The genotype frequency of vitamin D receptor BsmI (BB, 57.5%; Bb, 33.3%; bb, 9.1%) and oestrogen receptor-alpha PvuII (PP, 18.2%; Pp, 75.6%; pp, 6.2%) and XbaI (XX, 9.1%; Xx, 90.9%; xx, 0%) of the primary biliary cirrhosis patients was different from that of the healthy and osteoporotic control groups (P < 0.03 for each). Osteoporosis (t score < -2.5) was present in 42.4% of the patients. Osteoporotic primary biliary cirrhosis patients were older and had a longer disease history (P = 0.01 for both). No association was found between the polymorphisms and bone mineral density values at either position. CONCLUSIONS: We confirmed previous findings concerning the higher frequency of vitamin D receptor BsmI BB genotype in patients with primary biliary cirrhosis. The oestrogen receptor-alpha PvuII and XbaI Pp and Xx genotypes were more frequent in primary biliary cirrhosis patients, while interleukin-1 receptor antagonist gene variable-number tandem repeat polymorphism was not different. Since none of the polymorphisms was associated with bone mineral density, it is unlikely that these polymorphisms are essential in predicting bone mineral density in primary biliary cirrhosis.

Elevated plasma nociceptin level in patients with Wilson disease.

Plasma level of nociceptin, the endogenous agonist of orphanin FQ/ORL1 receptor was found to be significantly elevated in Wilson disease patients (13.98+/-2.44pg/ml, p<0.001, n=20) compared to age-matched healthy controls (9.18+/-1.63pg/ml, n=25). Wilson disease is an autosomal recessive disorder of copper metabolism caused by mutation of the gene ATP7B leading to toxic copper accumulation in the liver and other organs such as brain, kidney and cornea. Measurements were performed by 125I-radioimmunoassay. Neither sex differences nor correlation between plasma nociceptin levels and liver function test results were found. It is suggested that elevated plasma nociceptin level found in Wilson disease patients is due to inhibition of nociceptin-inactivating Zn-metallopeptidases (aminopeptidase N (APN) and endopeptidase 24.15) by the toxic copper deposits in liver and/or brain.