Wilson disease (WD) is an autosomal recessive disorder of copper transport which map to chromosome 13q14.3. In pursuit of the WD gene, we developed yeast artificial chromosome and cosmid contigs, and microsatellite markers which span the WD gene region. Linkage disequilibrium and haplotype analysis of 115 WD families confined the disease locus to a single marker interval. A candidate cDNA clone was mapped to this interval which, as shown in the accompanying paper, is very likely the WD gene. Our haplotype and mutation analyses predict that approximately half of all WD mutations will be rare in the American and Russian populations.

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A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefiuness of in situ hybridization in the study of pathogenesis of HBV infection.HBV infection is a major public health problem, with an estimated 200 million persistently infected people worldwide (1)(2)(3). HBV is a DNA virus that infects only humans and certain nonhuman primates and causes a wide spectrum of acute and chronic liver disease (4). Recent evidence further suggests that this virus is involved in the etiology of hepatocellular carcinoma (4-13). In infected individuals, the virion and DNA-free particles of hepatitis B surface antigen (HBsAg) circulate in blood, and viral antigen has been found in most body fluids, including bile (4, 14-16). However, the study of the pathobiology of HBV infection is impeded by the current inability to propagate HBV in cell culture. The availability of cloned HBV DNA now permits us to investigate by in situ hybridization the presence and quantity of viral copies at the cellular level and their correlation with histologic lesions. This paper presents findings indicating that HBV DNA in virus-infected liver is detectable not only in hepatocytes, as previously reported by Gowans et al. (17), but also in bile duct epithelium and vascular elements, thereby identifying new and unsuspected target cells for HBV. Cellular DNA Extraction. PLC/PRF/5 cells in a confluent monolayer were trypsinized and washed once with phosphatebuffered saline. The cells were lysed at room temperature by incubation in 10 mM Tris HCI, pH 7.4/1 mM EDTA/100 mM NaCl/1% sodium dodecyl sulfate. After addition of proteinase K (Boehringer Mannheim) to a final concentration of 100 ,g/ ml, the lysate was incubated at 37°C for 12-16 hr. The solution was then extracted twice with buffer-saturated phenol/chloroform/isoamyl alcohol, 25:24:1 (vol/vol), and once with chloroform/isoamyl alcohol, 24:1. The nucleic acids were precipitated with 2 vol of ethanol. The precipitate was dissolved in 10 mM Tris'HCl, pH 7.4/1 mM EDTA/100 mM NaCl, treated with DNase-free RNase (Millipore) at 100 ,ug/ml for 3 hr at 370C, and extracted and precipitated as described above. The number of HBV DNA copies per Alexander cell was determined by dot blot analysis (22) with 32P-labeled HBV DNA as a probe (specific activity, 1-2 x 109 cpm/,ug) and a known amount of cloned HBV DNA or of DNA extracted from PLC/ PRF/5 cells. The hybridization was quantitated by liquid scintillation counting. The number of four copies of HBV DNA per Alexander cell was calculated by assuming a cellular DNA content of 6 pg. MATERIALS AND METHODSIn 6685The publication costs of this article were defrayed in part by page charge ...

This study reports 12 novel mutations of the Wilson disease (WD) gene which have been detected by the molecular analysis of 29 patients of Mediterranean descent carrying uncommon chromosomal haplotypes at the WD locus. These mutations include two nonsense, one splice site and nine missense. The missense mutations lie in regions of the WD gene critical for its function, such as the transmembrane region, the transduction domain and the ATP loop and ATP-binding domain, indicating that they are disease-causing mutations. These new findings improve our knowledge for the role played by functional domains on the ATP7B function.

The high endemicity of hepatitis B virus (HBV) infection and liver disease in Sardinia led us to assess the occurrence of HBV DNA in 1,411 sera of two selected groups of hepatitis B surface antigen (HBsAg)- negative blood donors: 793 with abnormal serum alanine aminotransferase (ALT) and 618 with normal serum ALT values (determined during routine testing of their blood donation). HBV DNA sequences were detected by dot-blot hybridization in 68 of 793 subjects (9%) with abnormal ALT but only in three of 618 subjects (0.5%) with normal ALT. HBV-core antibody (anti-HBc) was detected in 338 of 793 subjects (43%) with abnormal ALT as well as in 125 of 618 subjects (20.2%) with normal ALT. Among the 71 subjects positive for serum HBV DNA, 22 (31%) were positive for anti- HBc, while 49 (69%) were negative for all serologic markers of HBV infection. Thus, a high frequency of anti-HBc in apparently healthy HBsAg-negative individuals and a high prevalence of serum HBV DNA in the absence of immunologic markers of HBV infection suggest the existence of genetic variants of HBV that may be responsible for some of the presumed NANB hepatitis encountered in Sardinia and possibly other areas of high endemicity for HBV.