Isolation of a Human Betaretrovirus from Patients with Primary Biliary Cholangitis.

A human betaretrovirus (HBRV) has been linked with the autoimmune liver disease, primary biliary cholangitis (PBC), and various cancers, including breast cancer and lymphoma. HBRV is closely related to the mouse mammary tumor virus, and represents the only exogenous betaretrovirus characterized in humans to date. Evidence of infection in patients with PBC has been demonstrated through the identification of proviral integration sites in lymphoid tissue, the major reservoir of infection, as well as biliary epithelium, which is the site of the disease process. Accordingly, we tested the hypothesis that patients with PBC harbor a transmissible betaretrovirus by co-cultivation of PBC patients' lymph node homogenates with the HS578T breast cancer line. Because of the low level of HBRV replication, betaretrovirus producing cells were subcloned to optimize viral isolation and production. Evidence of infection was provided by electron microscopy, RT-PCR, in situ hybridization, cloning of the HBRV proviral genome and demonstration of more than 3400 integration sites. Further evidence of viral transmissibility was demonstrated by infection of biliary epithelial cells. While HBRV did not show a preference for integration proximal to specific genomic features, analyses of common insertion sites revealed evidence of integration proximal to cancer associated genes. These studies demonstrate the isolation of HBRV with features similar to mouse mammary tumor virus and confirm that patients with PBC display evidence of a transmissible viral infection.

Trend level gene-gender interaction effect for the BDNF rs6265 variant on age of onset of psychosis.

A BDNF rs6265 [A/A] by gender by cannabis use interaction has been associated with age of onset of psychosis (AoP). We examined the gender and cannabis use-adjusted association between BDNF rs6265 [G>A] and AKT1 rs2494732 [T>C] and AoP. Data from 167 Caucasians on AoP and age at first regular cannabis use were collected. Kaplan-Meier and Cox regression analyses were conducted. A trend level gene-gender interaction effect was observed for the BDNF rs6265 A/A genotype, controlling for age at first regular cannabis use. Larger collaborative research projects are required to further investigate this effect.

Functional analysis of mutations in the ATP loop of the Wilson disease copper transporter, ATP7B.

Wilson disease (WND) is an autosomal recessive disorder resulting from mutation of ATP7B. Transport of copper by ATP7B from the trans-Golgi of hepatocytes into apical membrane-trafficked vesicles for excretion in the bile is the major means of copper elimination from the body. Although copper is an essential nutrient, homeostasis must be carefully maintained. If homeostasis is disrupted, copper can accumulate within the liver, kidney, cornea, and/or brain. The range of organs affected leads to clinical heterogeneity and difficulty in WND diagnosis. Sequencing of ATP7B is an important adjunct for diagnosis but has led to the discovery of many novel missense variants. Although prediction programs are available, functional characterization is essential for determining the consequence of novel variants. We have tested 12 missense variants localized to the ATP loop of ATP7B and compared three predictive programs (SIFT, PolyPhen, and Align-GVGD). We found p.L1043P, p.G1000R, p.G1101R, p.I1102T, p.V1239G, and p.D1267V deleterious; p.G1176E and p.G1287S intermediate; p.E1173G temperature sensitive; p.T991M and p.I1148T mild; and p.R1228T functioning as wild type. We found that SIFT most often agreed with functional data (92%), compared with PolyPhen (83%) and Align-GVGD (67%). We conclude that variants found to negatively affect function likely contribute to the WND phenotype in patients.

A minigene approach for analysis of ATP7B splice variants in patients with Wilson disease.

Wilson disease (WND) is an autosomal recessive condition that results in accumulation of copper in the liver and brain when a membrane bound copper transporter, ATP7B, is defective. ATP7B is expressed in hepatic, brain and kidney cells, and a defect can lead to liver, neurological and renal damage in WND patients. Presentation is variable with a broad range of age of onset and symptoms, and not all biochemical signs used in diagnosis are found in every patient. Therefore, diagnosis by mutation analysis is particularly important. To date, there are approximately 380 probable disease-causing variants in ATP7B, 33 of which are splice site variants that are predicted to affect splicing, based on their location. Few of these splice site variants have been analyzed in vivo. Some exonic variations also have the potential to affect splicing. The aim of this project was to use minigenes for transcript analysis. We have chosen exon 8 as our focus and have cloned a wild-type three-exon minigene into a mammalian expression vector. After transfection, extracted RNA was analyzed by reverse transcription PCR and accurate splicing was detected. This minigene will facilitate the analysis of the numerous potential splice variants identified in exon 8 of ATP7B, with the advantage that patient cell lines are not required for each variant.

New mutations in the Wilson disease gene, ATP7B: implications for molecular testing.

Wilson disease (WND), an autosomal recessive disorder of copper transport with a broad range of genotypic and phenotypic characteristics, results from mutations in the ATP7B gene. ATP7B encodes a copper transporting P-type ATPase involved in the transport of copper into the plasma protein ceruloplasmin, and for excretion of copper from the liver. Defects in ATP7B lead to copper storage in liver, brain and kidney. Mutation analysis was carried out on 300 WND patients of various origins, and new mutations not previously reported were identified: European white (p.L217X, c.918_931, c.1073delG, c.3082_3085delAAGAinsCG, p.V536A, p.S657R, p.A971V, p.T974M, p.Q1004P, p.D1164N, p.E1173G, p.I1230V, p.M1359I, c.2355+4A>G), Sephardic Jewish (p.Q286X), Filipino (p.G1149A), Lebanese (p.R1228T), Japanese (p.D1267V) and Taiwanese (p.A1328T). All but one missense variant have strong evidence for classification as disease-causing mutations. In the patients reported here, we also identified 20 nucleotide substitutions, six not previously reported, which cause silent amino acid changes or intronic changes. Documentation and characterization of all variants is essential for accurate DNA diagnosis in WND because of the wide range of clinical and biochemical variability.

Sequence variation in the ATP-binding domain of the Wilson disease transporter, ATP7B, affects copper transport in a yeast model system.

ATP7B is a copper transporting P-type ATPase defective in the autosomal recessive copper storage disorder, Wilson disease (WND). Functional assessment of variants helps to distinguish normal from disease-causing variants and provides information on important amino acid residues. A total of 11 missense variants of ATP7B, originally identified in WND patients, were examined for their capacity to functionally complement a yeast mutant strain in which the yeast gene ortholog, CCC2, was disrupted. Solution structures of ATP7B domains were used to predict the effects of each variant on ATP7B structure. Three variants lie within the copper-binding domain and eight within the ATP-binding domain of ATP7B. All three ATP7B variants within the copper-binding domain and four within the ATP-binding domain showed full complementation of the yeast ccc2 phenotype. For the remaining four located in the ATP-binding domain, p.Glu1064Lys and p.Val1106Asp were unable to complement the yeast ccc2 high-affinity iron uptake deficiency phenotype, apparently due to mislocalization and/or change in conformation of the variant protein. p.Leu1083Phe exhibited a temperature-sensitive phenotype with partial complementation at 30 degrees C and a severe deficit at 37 degrees C. p.Met1169Val only partially complemented the ccc2 phenotype at 30 degrees C and 37 degrees C. Therefore, four variant positions were identified as important for copper transport and as disease-causing changes. Since the yeast assay specifically evaluates copper transport function, variants with normal transport could be defective in some other aspect of ATP7B function, particularly trafficking in mammalian cells. Functional assessment is critical for reliable use of mutation analysis as an aid to diagnosis of this clinically variable condition.

Value of an enzymatic assay for the determination of serum ceruloplasmin.

The serum concentration of the copper protein ceruloplasmin has been an important diagnostic indicator of Wilson's disease (WD). It is widely quoted that 95% of people with WD have low serum ceruloplasmin concentrations. Current evidence suggests that a normal serum ceruloplasmin concentration is more common in patients with WD, particularly those with liver disease, perhaps in part because of the routine use of an immunologic assay. This assay might indicate a normal level of ceruloplasmin when the enzymatic activity is lower. Enzymatic activity is the biologically relevant parameter. We compared the immunologic measurement with the enzymatic assessment of oxidase activity in patients with liver or neurologic symptoms of unknown origin in whom WD was considered in the differential diagnosis. Although a strong correlation of ceruloplasmin protein concentration with oxidase activity was observed in controls, this was not the case for these patients. Twelve patients, presenting with various types of hepatic disease, demonstrated a weak correlation between ceruloplasmin protein concentration and oxidase activity. Ten percent of patients with neurologic symptoms ( n = 41) had low ceruloplasmin concentrations and oxidase activity, and another 8% had normal ceruloplasmin concentrations associated with low oxidase activity. Although the enzymatic method is preferred for its biologic relevance, ceruloplasmin analysis is not a reliable diagnostic parameter for the diagnosis of WD in patients with liver disease. An important use of the ceruloplasmin oxidase assay is in the follow-up of patients with WD. Ceruloplasmin oxidase activity was undetectable in sera from patients with WD who were undergoing long-term chelation therapy, suggesting an early sign of copper depletion and a need for subsequent monitoring for symptoms of copper deficiency.