A simple and accurate SNP scoring strategy based on typeIIS restriction endonuclease cleavage and matrix-assisted laser desorption/ionization mass spectrometry.

BACKGROUND: We describe the development of a novel matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-based single nucleotide polymorphism (SNP) scoring strategy, termed Restriction Fragment Mass Polymorphism (RFMP) that is suitable for genotyping variations in a simple, accurate, and high-throughput manner. The assay is based on polymerase chain reaction (PCR) amplification and mass measurement of oligonucleotides containing a polymorphic base, to which a typeIIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products leads to excision of oligonucleotide fragments representing base variation of the polymorphic site whose masses were determined by MALDI-TOF MS. RESULTS: The assay represents an improvement over previous methods because it relies on the direct mass determination of PCR products rather than on an indirect analysis, where a base-extended or fluorescent report tag is interpreted. The RFMP strategy is simple and straightforward, requiring one restriction digestion reaction following target amplification in a single vessel. With this technology, genotypes are generated with a high call rate (99.6%) and high accuracy (99.8%) as determined by independent sequencing. CONCLUSION: The simplicity, accuracy and amenability to high-throughput screening analysis should make the RFMP assay suitable for large-scale genotype association study as well as clinical genotyping in laboratories.

De Novo Superinfection of Hepatitis B Virus in an Anti-HBs Positive Patient with Recurrent Hepatitis C Following Liver Transplantation.

A 60-year-old woman with end stage liver cirrhosis caused by genotype 2 hepatitis C virus (HCV) infection received an orthotopic liver transplantation (OLT). The patient was negative for the hepatitis B surface antigen (HBsAg) and positive for the anti-hepatitis B surface antibody (anti-HBs) prior to and one and a half months following the OLT. Due to reactivation of hepatitis C, treatment with interferon-alpha and Ribavirin started two months following the OLT and resulted in a sustained virological response. We performed a liver biopsy because a biochemical response was not achieved. Surprisingly, liver pathology showed HBsAg-positive hepatocytes with a lobular hepatitis feature, which had been negative in the liver biopsy specimen obtained one and a half months post-OLT. High titers of both HBsAg and HBeAg were detected, while anti-HBs antibodies were not found. Tests for IgM anti-hepatitis B core antibody and anti-delta virus antibodies were negative. The serum HBV DNA titer was over 1×10(7) copies/mL. A sequencing analysis showed no mutation in the "a" determinant region, but revealed a mixture of wild and mutant strains at an overlapping region of the S and P genes (S codon 213 (Leu/Ile); P codons 221 (Phe/Tyr) and 222 (Ala/Thr)). These findings suggest that de novo hepatitis B can develop in patients with HCV infection during the post-OLT period despite the presence of protective anti-HBs.

A YIDD Mutation in a Case of Recurrent Hepatitis B after Liver Transplantation Induced by an S-escape Mutant.

A 47-year-old woman underwent orthotopic liver transplantation (OLT) for hepatitis B virus (HBV)-related end-stage liver cirrhosis. The patient received hepatitis B immunoglobulin prophylaxis after OLT. Despite the protective level of the serum anti-hepatitis-B surface antibody, HBV recurred at 22 months post-OLT and induced subacute hepatic failure. The pre-OLT HBV genome contained a complex mutation pattern in overlapping frame regions of the surface (S) and polymerase (P) genes, which is the same mutation pattern as seen in post-OLT HBV DNA. G145R and K141R mutations in the "a" determinant were detected only in the post-OLT sample. Clevudine (30 mg once daily) was administered for recurrent hepatitis B. Hepatitis B was reactivated with a flare-up, and a M204I mutation (YIDD mutant type) appeared with a higher viral load at 9 months after clevudine treatment. We report here a case of a YIDD mutation that developed in recurrent hepatitis B after OLT induced by an S-escape mutant.