Molecular diagnostic issues

HBV GENETIC ORGANISATION

The genetic organization of HBV is extremely compact with part of the core and all of the surface genes (blue) overlapping the polymerase gene (light blue). Consequently, mutations affecting one gene product may affect the other. The polymerase is subdivided into functional domains: terminal protein (tp), spacer (sd), reverse transcriptase (rt) and RNAseH. The active site of rt (YMDD, dark blue) and adjacent sequences are the target of mutations conferring resistance to antiviral drugs. The core protein constitutes the viral capsid, while the surface antigen constitutes the envelope of the virus upon anchoring in a lipid membrane of cellular origin. Other important features are not represented here for sake of simplicity: the pregenomic RNA used as template for viral DNA replication, the X open reading frame whose function is unknown as well as regulatory elements necessary for viral gene expression and viral DNA replication. Binding sites for primers used in diagnostics are shown in green.

MOLECULAR TOOLS TO IDENTIFY MUTATIONS

The identification of mutations can only be done after polymerase chain reaction (PCR) amplification of rt with DNA extracted from the plasma of infected patients. Primers used in our laboratory are depicted in green on the HBV genome displayed above.

Precise identification of mutations can be done by reverse blotting hybridization against a panel of probes (for known mutations, high sensitivity of detection), or by DNA sequencing (for known as well as for unknown mutations) provided they represent at least 20% of the whole population. This level of detection is sufficient for routine diagnostics. Once the DNA sequence is determined, it is translated into its corresponding amino acid stretch. Mutations can then be identified after alignment of the amino acid sequence against reference HBV protein databases. This method, used in our laboratory, is illustrated below by sequencing chromatograms of two samples from the same patient, either before (upper strand) or after failure treatment with telbivudine (lower strand). The G/T transversion indicated by the blue shade leads to the M204I mutation known to confer resistance to this drug.