J Korean Med Sci.  2004 Aug;19(4):541-546. 10.3346/jkms.2004.19.4.541.

Detection of YMDD Motif Mutants by Oligonucleotide Chips in Lamivudine-Untreated Patients with Chronic Hepatitis B Virus Infection

• Heo J
• Cho M
• Kim HH
• Shin YM
• Jang HJ
• Park HK
• Kim CM
• Kim GH
• Kang DH
• Song GA
• Yang US

• KMID: 2157683
• DOI: http://doi.org/10.3346/jkms.2004.19.4.541

Abstract

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.

Keyword

Hepatitis; Viral; Hepatitis B; Chronic; Lamivudine; Polymorphism (Genetics); YMDD Motif Mutants; Oligonucleotide Array Sequence Analysis

MeSH Terms

Adolescent
Adult
*Amino Acid Motifs
Chronic Disease
Female
Hepatitis B/*drug therapy
Hepatitis B virus/*genetics
Humans
Lamivudine/*therapeutic use
Male
Middle Aged
Mutation
*Oligonucleotide Array Sequence Analysis
Polymorphism, Restriction Fragment Length
Reverse Transcriptase Inhibitors/*therapeutic use
Sensitivity and Specificity
Sequence Analysis, DNA
Variation (Genetics)

Figure

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  Fig. 1 This schematic illustration shows the probe layout for the 17 mer oligonucleotide sequences noted in Table 1 and the interpretative results of oligonucleotide chips for YMDD motif mutation detection. The oligonucleotide probes are patterned as duplex arrays. ① Positive is mixture of HB552-WM, HB552-MV, HB552-MI1, HB552-MI2 and HB552-MI3 probes (Table 1), ② 522WM, ③ 552MI3, ④ 552MI2, ⑤ 552MI1 and ⑥ 552MV indicates HB552-WM, HB552-MV, HB552-MI1, HB552-MI2 and HB552-MI3 probes, respectively.

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  Fig. 2 Sensitivity and specificity of the detection of the YMDD mutants using oligonucleotide chip. (A) Detection limit of the YMDD mutants by oligonucleotide chip, (B) Detection capacity of mixture of wild-type and YMDD motif mutants. YMDD: YVDD represents ratios of YMDD and YVDD; YMDD:YIDD3 represents ratios of YMDD and YIDD3.

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  Fig. 3 Scanned images of oligonucleotide chip (A) wild type [YMDD], (B), (C), (D) YMDD motif mutation [YMDD+YIDD(I3)], slides of No. 24, 27 and 37, respectively.

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  Fig. 4 (A) YVDD RFLP analysis in 4% agarose gel does not show any band digested by the restriction enzyme ApaLI. M: 100 bp ladder. (B) YIDD RFLP analysis in 4% agarose gel shows DNA bands (arrows) digested by the restriction enzyme SspI in 3 YIDD (I3) mutants (sample Nos. 24, 27 and 37). M: B 100 bp ladder.

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  Fig. 5 The sequences of 290 bp DNA including codon 552 represent only the wild-type (ATG) in the samples with YIDD mutants (No. 24, 27 and 37).

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