Rapid diagnosis of Wilson disease by a 28-mutation panel: real-time amplification refractory mutation system in diagnosing acute Wilsonian liver failure.

BACKGROUND: Wilson disease is one of the commonest inherited and potentially fatal yet treatable liver disorders. About 5-27% patients present with acute liver failure and require prompt chelation therapy and life-saving liver transplantation. Diagnosis during acute liver failure is particularly difficult with short time allowance. Direct molecular diagnosis remains the most decisive tool but is often hindered by demanding techniques and numerous mutations. We developed a one-step, 3-h, reproducible, and accurate real-time amplification refractory mutation system which can simultaneously detect 28 ATP7B mutations. METHODS: Primers were designed to complement the mutant sequence at the 3' end. The mutations were p.S105X, p.Q511X, p.R616Q, p.S693P, p.S693C, p.R778L, p.A874V, p.T888P, p.R919G, p.T935M, p.P992L, p.M1025R, p.D1047V, p.I1148T, p.R1156H, p.T1178A, p.V1216M, p.P1273Q, p.G1281C, p.R1320S, p.V1334D, p.V176SfsX28, p.G869EfsX4, IVS3+1G>T, IVS4-1G>C, IVS4-5T>G, IVS6+9A>G, and IVS9+5G>T. Reaction was performed using QuantiTect SYBR Green PCR Master Mix on an Applied Biosystems StepOne thermal cycler. Values of the threshold cycle were compared between normal and mutant alleles. RESULTS: Primers of all mutations were highly specific with absence of wild-type amplification. All the results were validated by direct DNA sequencing. CONCLUSIONS: This rapid and cost-efficient method allows wide mutation coverage, rendering the SYBR-green assay feasible and attractive for large-scale routine application.

EUS in the management of uninvestigated dyspepsia.

BACKGROUND: Management of dyspepsia often involves multiple diagnostic tests. By virtue of luminal and extraluminal imaging, EUS may be a satisfactory single test for the evaluation of patients with dyspepsia. METHODS: Patients with uninvestigated dyspepsia of more than 4 weeks duration were recruited. All underwent transabdominal US followed by EUS and EGD to look for both luminal and extraluminal lesions that could account for the dyspeptic symptoms. Each of these studies was performed by an independent operator blinded to the results of the other examinations. Patients were followed for 1 year to monitor clinical outcome. RESULTS: Two hundred patients were recruited. Compared with EGD, EUS had accuracies of 92% to 100% for the diagnosis of different luminal lesions. All gastric ulcers and cancers were detected by EUS, but 3 duodenal ulcers were missed. EUS also identified 48 pancreaticobiliary lesions, 23 of which were detected by US, that could account for the dyspeptic symptoms. Other extraintestinal lesions including mediastinal malignant lymphadenopathy and a dissecting aortic aneurysm were also diagnosed by EUS. Through the identification of extraluminal lesions, staging of tumors, and exclusion of pancreaticobiliary diseases, EUS altered clinical management in 50 patients (25%). CONCLUSION: EUS is a useful one-step investigation in patients with dyspepsia.

Occult HBV infection in cryptogenic liver cirrhosis in an area with high prevalence of HBV infection.

OBJECTIVES: Hepatitis B surface antigen (HBsAg) is often used as the serological marker to screen for hepatitis B virus (HBV) infection in the investigation of liver cirrhosis. In Hong Kong, where HBV infection is endemic, some patients may have persistent viral infection after the loss of HBsAg. We aimed to investigate 1) the prevalence of occult HBV infection in cryptogenic liver cirrhosis in Hong Kong and 2) the role of HBV "a" determinant mutations among these patients. METHODS: Twenty-eight patients with cryptogenic liver cirrhosis (group I), 49 subjects with no liver disease (group II), and 26 patients with HBV-related cirrhosis (group III) were studied. HBV DNA was determined by the cross-linking assay (sensitivity = 0.5 mEq/ml) and polymerase chain reaction (PCR). Occult HBV infection was defined as HBV DNA detectable by PCR among patients with negative HBsAg. RESULTS: Eighty-nine percent and 92% of patients in groups I and II, respectively, had positive anti-HBs and/or anti-hepatitis B core. Nine (32%), no (0%), and 14 (54%) patients in groups I, II, and III, respectively, had detectable HBV DNA by PCR (group I vs group II, p < 0.001; group I vs group III, p = 0.36). Four patients in group I had HBV DNA detectable by the cross-linking assay (median = 5.98 mEq/ml, range = 3.1-8.01). "a" determinant mutations were detected in two patients in group I (K122N and G145R, C125A) and one patient in group II (1126N). CONCLUSIONS: Occult HBV infection is common among patients with cryptogenic liver cirrhosis, and it cannot be explained by mutations in the HBV "a" determinant.