Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment.

Abstract

To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV to tyrosine-valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-aspartate-aspartate (YIDD).
Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration, and annealing temperature of primers were systematically examined. Pair primers specific to rtL180M and rtM204V were selected for YVDD detection. Primer specific to rtM204I with an additional 3'-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment.
We could detect as little as 0.001%-0.00001% of mutant viruses coexisting in 10(8)-10(9) copies of wild-type HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAg-negative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations.
We developed a highly sensitive and specific assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment.

Full text

Page 1

methionine-aspartate-aspartate; Mutant-specifi c primer
Lee CZ, Lee HS, Huang GT, Yang PM, Sheu JC. Detection
of YMDD mutation using mutant-specifi c primers in chronic
hepatitis B patients before and after lamivudine treatment.
World J Gastroenterol 2006; 12(33): 5301-5305
http://www.wjgnet.com/1007-9327/12/5301.asp
INTRODUCTION
Hepatitis B virus (HBV) is one of the most common
infectious diseases in the world. More than 300 million
people worldwide are estimated to have chronic HBV
infection. Ten percent of these patients will die as a direct
consequence of persistent viral infection[1]. Nucleoside
analogue therapy allows safe, long-term suppression of
HBV and is a major milestone in the treatment of chronic
hepatitis B. Lamivudine, the fi rst of these agents approved
worldwide, effectively suppresses viral replication, reduces
disease activity, improves liver histology, and delays clinical
progression[2-4]. However, the development of lamivudine
resistant mutations occurs in 14%-32% of patients
after 1 year of therapy[5,6]. The longer the treatment is
continued, the more frequently resistance is seen (65% at
5 years)[7]. Lamivudine-resistant HBV is associated with
mutations of the YMDD motif in the polymerase gene.
The key mutations are the substitutions of methionine
at the rtM204 (domain C) to either isoleucine (rtM204I,
YIDD variant) or valine (rtM204V, YVDD variant)[8,9].
The rtM204V variant is almost always accompanied by an
additional rtL180M mutation in the domain B[10,11].
Several assays, including DNA sequencing[12], restriction
fragment length polymorphism (RFLP)[13,14], peptide
nucleic acid (PNA) mediated PCR clamping[15], line probe
assay[16,17], and oligonucleotide microarray[18,19], have been
developed to detect YMDD mutations. The best sensitivity
achieved so far was 104 copies of variant in the mixture of
109 copies of the wild-type virus and 101 in 105 of wild-
type[15].
In this study, we developed a simple but highly sensitive
and specifi c assay using mutant-specifi c primers to detect
lamivudine-resistant mutations. We applied this method
to detect YMDD mutants in chronic hepatitis B patients
before and after lamivudine treatment.
VIRAL HEPATITIS
Detection of YMDD mutation using mutant-specific primers
in chronic hepatitis B patients before and after lamivudine
treatment
Cha-Ze Lee, Hsuan-Shu Lee, Guan-Tarn Huang, Pei-Ming Yang, Jin-Chuan Sheu
www.wjgnet.com
Cha-Ze Lee, Hsuan-Shu Lee, Guan-Tarn Huang, Pei-Ming
Yang, Jin-Chuan Sheu, Department of Internal Medicine,
National Taiwan University Hospital, Taipei, Taiwan, China
Correspondence to: Cha-Ze Lee, MD, PhD, Departments of
Internal Medicine, National Taiwan University Hospital, No. 7,
Chung-Shan South Road, Taipei 10016, Taiwan,
China. czlee@ha.mc.ntu.edu.tw
Telephone: +886-2-23123456-7243 Fax: +886-2-2381723
Received: 2005-10-27 Accepted: 2005-11-18
Abstract
AIM: To develop a PCR assay using mutant-specific
primers to detect mutation of tyrosine-methionine-
aspartate-aspartate (YMDD) motif of HBV to tyrosine-
valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-
aspartate-aspartate (YIDD).
METHODS: Cloned wild-type and mutant HBV
sequences were used as templates to test the sensitivity
and specificity of the assay. A variety of primer
construction, primer concentration, dNTP concentration,
and annealing temperature of primers were
systematically examined. Pair primers specifi c to rtL180M
and rtM204V were selected for YVDD detection. Primer
specifi c to rtM204I with an additional 3’-penultimate base
mismatched to both the mutant and wild-type sequence
was selected for YIDD detection. We applied this assay
to study YMDD mutants in 28 chronic hepatitis B patients
before and after lamivudine treatment.
RESULTS: We could detect as little as 0.001%-0.00001%
of mutant viruses coexisting in 108-109 copies of wild-
type HBV using this assay. YMDD mutants were detected
in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAg-
negative patients before lamivudine treatment. After
treatment, two more patients in HBeAg-positive patients
and seven more patients in HBeAg-negative patients
developed YMDD mutations.
CONCLUSION: We developed a highly sensitive and
specifi c assay for detecting YMDD mutants. This assay
can be applied to monitor chronic hepatitis B patients
before and during lamivudine treatment.
© 2006 The WJG Press. All rights reserved.
Key words: Hepatitis B virus; Lamivudine; Tyrosine-
PO Box 2345, Beijing 100023, China World J Gastroenterol 2006 September 7; 12(33): 5301-5305
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MATERIALS AND METHODS
Clinical samples and method for extraction of HBV DNA
Serum samples were collected from 28 chronic hepatitis
B patients who had histologically confirmed cirrhosis
and had received lamivudine treatment for 12 to 48
mo. Informed consent was obtained from all patients
according to the ethical guidelines of the 1975 Declaration
of Helsinki. HBV DNA was extracted from serum using a
DNA extraction kit (Qiagen, Hilden, Germany).
Subcloning of the HBV gene
Sera with wide-type HBV DNA, YVDD or YIDD mutants
were amplifi ed by PCR with a primer set (sense primer: 5’
-GATGTGTCTGCGGCGTTTTA-3’, antisense primer: 5’
-CAGCAAAGCCCAAAAGACCCAC-3’). The obtained
PCR products (625bp, containing site codons rt180 and
rt204) were then cloned into pOSI-T vector (GeneMark,
Taichung, Taiwan) using the standard method.
Detection of YMDD mutation using PCR with mutant-
specifi c primers
We used cloned YVDD, YIDD and wild-type HBV
genomes as templates to determine the optimal conditions
of mutant-specifi c PCR. A variety of primer construction
(Table 1), primer concentration, dNTP concentration
and annealing temperature of primers were systematically
examined (data not shown). The primers finally selected
were listed with boldface in Table 1. The single-step PCR
for YVDD detection was carried out with the primers
YVDD669 and YVDD758R in a 25 L reaction volume
containing 10 mmol/L Tris HCl, pH 8.3, 50 mmol/L KCl,
2.0 mmol/L MgCl2, 0.03 mmol/L of each dNTP, 2.5 pmol
of each primer, 1.5 units of Taq polymerase (Viogene,
Taipei, Taiwan). The PCR condition included initial
denaturation for 3 min at 94℃, 48 cycles of amplifi cation
with denaturation at 94℃ for 30 s, primer annealing at 57℃
for 30 s, extension of primer at 72℃ for 25 s, and final
extension at 72℃ for 1 min. The nest-PCR was performed
with HBV376 and HBV1021R as outer primers. The
fi rst PCR was conducted in a 20 L reaction volume with
0.0375 mmol/L dNTP and 1 pmol of each outer primer.
The PCR condition was initial denaturation for 3 min at
94℃, 30 cycles of amplifi cation with denaturation at 94℃
for 30 s, primer annealing at 53℃ for 30 s, extension of
primer at 72℃ for 50 s, and final extension at 72℃ for
2 min. One microliter of the first reaction was used for
the second PCR. The second PCR condition was the same
as the single-step PCR except the annealing temperature
was 55℃. The single-step PCR for YIDD detection was
carried out with primers HBV379 and YIDDR2T in the
same reaction condition as for YVDD detection except
the annealing temperature was 59℃. The fi rst PCR of the
nest-PCR for YIDD detection was performed in the same
condition as for YVDD detection. One microliter of the
fi rst reaction was used for the second PCR. The second
PCR was carried out in a 25 L reaction volume with 0.03
mmol/L of each dNTP, 2.5 pmol of each primer (HBV485
and YIDDR2T) and 0.1U of Perfect Match PCR enhancer
(Stratagene, La Jolla, California, USA), 1.5 units of Taq
polymerase. The reaction condition was initial one cycle of
www.wjgnet.com
94℃ for 5 min, 62℃ for 5 min and 72℃ for 30 s, 48 cycles
of amplifi cation with denaturation at 94℃ for 30 s, primer
annealing at 62℃ for 30 s, extension of primer at 72℃
for 30 s, and fi nal extension at 72℃ for 1 min. The PCR
products were subjected to electrophoresis in a 3% agarose
gel.
HBV DNA quantifi cation and Sequencing
HBV DNA quantification was performed using the
Lightycler real-time PCR assay[20]. PCR amplification of
the HBV genome was carried out using the same primers
as subcloning of HBV DNA described above. PCR-
amplified HBV DNA was purified and sequenced using
specifi c sequencing primers and a commercial sequencing
Kit (ABI Prism Dye Terminator Cycle Sequencing, Perkin
Elmer, Warrington, UK). The sequencing reactions were
analyzed on an automatic DNA sequencer.
RESULTS
We assessed the sensitivity and specifi city of this method
using mutant type and wild-type HBV plasmid. For
single-step PCR, the detection limit was 104 copies for
YVDD variant or 8 × 104 copies for YIDD variant (data
not shown). For nest-PCR, the detection limit was up
to 101 copies for both variants (Figure 1A, B). Wild-
type HBV, even up to 1010 copies, was not detected using
these mutant-specific primers for both single-step PCR
(data not shown) and nest-PCR (Figure 1A, B). We then
tested the mixture of wild-type and mutant HBV using
nest-PCR. Various copies of wild-type plasmid (107-1010
copies) were mixed with YVDD variant at the indicated
concentrations (101-104 copies). In the presence of 108
wild-type sequences, a total of 101 copies of both variants
could be detected (Figure 1A, B). A total of 104 copies
of YVDD variants or 103 copies of YIDD variants were
detected in the mixture of 109 copies of the wild-type
sequences (Figure 1A, B). Thus, the detection limit was
Table 1 DNA sequence of the primers tested for detection of
YMDD mutation
1Primers
2DNA sequenceNuceotide
YVDD758
YVDD758R 5’-CCCAAAACCACATCATCCAC (T)
YVDD669 5’-GGGCCTCAGTCCGTTTCTCA (T)
YIDD5’-CTGTTTGGCTTTCAGTTATATT (G) 739-760 Sense
YIDD2G5’-GTTTGGCTTTCAGTTATAGT (TG)
YIDD2A5’-GTTTGGCTTTCAGTTATAAT (TG)
YIDD2C5’-GTTTGGCTTTCAGTTATACT (TG)
YIDDR 5’-CCCCCAAAACCACATCATCA (C)
YIDDR2T 5’-CCCCCAAAACCACATCATTA (CC) 760-745 Antisense
YIDDR2A5’-CCCCCAAAACCACATCATAA (CC) 760-745 Antisense
YIDDR2G5’-CCCCCAAAACCACATCATGA (CC) 760-745 Antisense
HBV376 5’-GATGTGTCTGCGGCGTTTTA
HBV4855’-CCAGGAACATCAACCACCAG
HBV1021R 5’-CAGCAAAGCCCAAAAGACCCAC 1021-1000 Antisense
5’-CACTGTTTGGCTTTCAGTTATG (A) 737-758 Sense
758-739 Antisense
650-669 Sense
741-760 Sense
741-760 Sense
741-760 Sense
760-745 Antisense
376-395 Sense
485-504 Sense
1 The primers finally selected are indicated in boldface; 2 The engineered
mismatched nucleotides are underlined; the wild-type nucleotides are shown
in parenthesis.
5302 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol September 7, 2006 Volume 12 Number 33

Page 3

0.0001%-0.00001%.
We then applied this method to 28 chronic hepatitis B
patients who had been treated with lamivudine for 12 to
48 mo (Table 2). Before treatment, there were 12 patients
with positive HBeAg and 16 patients with negative HBeAg
and positive anti-HBe. Among 12 HBeAg-positive patients,
four were free of YMDD mutant, seven with YIDD
mutant, and one with YVDD mutant. One developed
HBeAg sero-conversion after treatment. Among 4 patients
with initial negative YMDD mutant, two developed
mixed mutation, and two remained free from YMDD
mutation. Among 7 patients with initial YIDD mutant,
three developed mixed mutations, and four remained with
YIDD mutation. The patient with initial YVDD mutant
developed mixed mutation. Among 16 HBeAg-negative
patients, before treatment, twelve were free of YMDD
mutation, two with YIDD mutation, and two with mixed
mutation. Among 12 patients with initial negative YMDD
mutant, fi ve developed YVDD mutation, one developed
YIDD mutation, one developed mixed mutation, and
five remained free of YMDD mutation after treatment.
The other four patients, two with YIDD mutant, two
with mixed mutation, remained with the same mutation.
Conventional sequencing of post-treatment sera showed
11 cases of wild-type HBV, 8 cases of YVDD mutation,
and 9 cases of YIDD mutation. Among 11 cases of wild-
Figure 1 Sensitivity and specifi city of the YMDD mutant detection by mutant-specifi c PCR. A: Various copies of wild-type sequence, YVDD mutant sequence, and mix of
both sequences at the indicated concentrations. The left most lane contains molecular weight marker in base pair unit. The detection limit was 101 copies of YVDD mutant in
108 copies of wild-type HBV or 104 copies of YVDD mutant in 109 copies of wild-type HBV; B: Various copies of wild-type sequence, YIDD mutant sequence, and mix of both
sequences at the indicated concentrations. The left most lane contains molecular weight marker in base pair unit. The detection limit was 101 copies of YIDD mutant in 108
copies of wild-type HBV or 103 copies of YIDD mutant in 109 copies of wild-type HBV.
104
103
102
101102
102102102
101
101
101
101
104
109
109
1010
106
107
108
109
107
108
109
1010
YVDD
200 bp
100 bp
Wild-type
104
103
102
101102
102102102
101
101
101
101
103
109
109
1010
106
107
108
109
107
108
109
1010
YIDD
Wild-type
300 bp
200 bp
AB
Table 2 Clinical Characteristics of 28 patients with chronic hepatitis B before and after long-term treatment of lamivudine
Before treatment After treatment
No.AST/ALT (IU/L) HBeAg/anti-HBe
1HBV-DNA
2Nest-PCR AST/ALT (IU/L)HBeAg/anti-HBe
1HBV-DNA Nest-PCR
3Sequencing
1
2
3
61/62
31/43
85/86
-/+
+/-
-/+
39.93
3574.29
348.66
-29/16
29/54
20/11
-/+
+/-
-/+
0.25
4400.65
0.20
V
M
-
V
V
W
V
-
445/90-/+ 8.50-23/22-/+ 0.03-W
574/75-/+ 256.99I63/38-/+ 0.01II
6 82/163-/+1596.12-38/64-/+3356.38VV
737/43+/- 13.07-25/39+/- 0.26-W
826/62-/+ 49.16-24/40-/+ 0.03-W
9 82/102-/+3313.93- 29/17-/+ 0.60VW
1040/59+/- 546.76I24/28 -/+ 0.11MI
11117/232+/- 49.50 I26/27+/- 346.96MI
12
13
14
46/61
72/107
155/159
-/+
-/+
+/-
47.40
5.37
2325.13
- 24/32
66/92
22/14
-/+
-/+
+/-
0.06
2389.09
0.10
V
M
M
V
V
W
M
I
15
16
65/54
133/157
-/+
+/-
1461.13
7943.81
-
I
42/29
73/64
-/+
+/+
0.12
2.05
V
I
W
I
17107/146-/+ 23.13M151/232-/+ 59.94MV
18 35/48 +/-1666.59-22/21 +/- 0.15-W
19247/183+/-9336.17I 27/14+/- 0.01IW
20
21
22
23
24
45/56
109/186
173/335
51/81
43/68
-/+
-/+
+/-
-/+
+/-
219.58
61.27
11.98
1403.11
365.07
-
-
-
I
-
28/38
30/48
29/31
28/42
22/27
-/+
-/+
+/-
-/+
+/-
0.03
2.91
0.03
303.09
1.28
-W
I
V
I
V
M
M
I
V
25 45/57+/-7530.63I30/25 +/-6228.83II
26
27
130/286
64/91
-/+
+/-
126.67
4700.63
-
I
21/32
52/73
-/+
+/-
1.46
6.12
I
I
I
I
28 88/164 -/+ 0.38-30/38-/+ 0.07-W
1 Unit (105copies/mL); 2 M: YVDD + YIDD, V: YVDD, I: YIDD, -: YVDD and YIDD not detectable; 3 W: wild-type, V: YVDD, I: YIDD.
Lee CZ et al. YMDD mutation detection with mutant-specifi c primers 5303
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Page 4

type HBV, eight were wild-type, two were YVDD, one
was YIDD, and one was mixed mutation by nest-PCR.
Among 8 cases of YVDD mutation, four were YVDD
and four were mixed mutation by nest-PCR. Among 9
cases of YIDD mutation, six were YIDD and three were
mixed mutation by nest-PCR (Table 2). Overall, mutant-
specifi c PCR agreed with sequencing on YMDD mutation
detection, but was more sensitive in detecting additional
mutations.
DISCUSSION
Taq DNA polymerases extend mismatches much less
effi ciently than correct matches. Depending on the nature
of the mismatch composition, the elongation efficiency
of the mismatched duplex can vary from 0.0001% to 0.1%
of the elongation efficiency of the perfectly matched
duplex[21]. Reduced dNTP and primer concentration can
increase the specificity of mutant-specific PCR[22]. We
have lowered 2-4 fold the dNTP and primer concentration
of conventional PCR and compensated for the reduced
efficiency by increasing amplification cycles. With this
modifi ed PCR condition, we found that in the presence of
106 to 107 copies of wild-type HBV DNA, a single 3’-end
mismatch primer still showed a false positive result (data
not shown). For the YVDD mutation, rtL180M mutation is
almost always accompanied by an additional the rtM204V
mutation[10,11]. We therefore selected pair primers with 3’
-end specifi c to rtL180M and rtM204V to detect YVDD
mutation. For YIDD mutation detection, we designed
primers containing a mutant-specifi c 3’-terminal base and
a 3’-penultimate base mismatched to both the mutant and
wild-type sequence. We checked 3 forward (YIDD2G,
YIDD2A, and YIDD2C) and 3 reverse (YIDDR2T,
YIDDR2A, and YIDDR2C) primer constructions.
The YIDDR2T primer had the greatest specificity and
sensitivity in detecting YIDD mutation. Besides, we add
Perfect Match PCR enhancer to increase the specificity
of the assay[23]. The present assay had a detection limit of
0.0001%-0.00001%, which was better than that of PNA
mediated PCR clamping (0.001%-0.0001%)[15] and that of
RFLP assay (1%-10%)[13]. However, the latter two methods
have the advantage of detecting both wild-type and mutant
HBV simultaneously in one reaction. The choice of assay
should depend on the context of the application.
Hepatitis fl are can occur during lamivudine treatment
because of the emergence of YMDD mutants. The serum
titer of the mutant viruses during hepatitis flare is well
above 105 copies/mL[24]. The single-step PCR in this study
only detected YMDD mutation at copy number greater
than 104, and can be applied in this clinical context. In fact,
we have applied single-step PCR to check two cases of
hepatitis flare during lamivudine treatment. It correlated
well with HBV DNA quantifi cation and sequencing (data
not shown).
Because HBV employs reverse transcription to copy its
genome, mutant viral genomes are found frequently. Under
conditions of high levels of viral replication, it is likely that
HBV mutations conferring resistance to lamivudine may
pre-exist. Indeed, pre-existing YMDD mutants have been
demonstrated in some lamivudine-naive asymptomatic
HBV carriers[25] or chronic hepatitis B patients[15]. The
patients with pre-existing YMDD mutants were all positive
for anti-HBe antibody[15,25]. In this study, twelve patients
were shown to have pre-existing YMDD mutants, and
eight of them were HBeAg-positive. Seven cases with pre-
existing mutation had a serum HBV DNA level greater
than 108 copies/mL. However there were also cases with
a high level of HBV DNA but without a pre-existing
mutation. We suspect that with more sensitive detecting
methods, more cases of pre-existing mutation will be
demonstrated. The patients with a pre-existing mutation
had persistent mutation or developed mixed mutation
during treatment, but only five of them had a poor
virological response. Whether patients with a pre-existing
lamivudine-resistant mutation should be treated with other
nucleoside analogues requires further prospective studies.
In conclusion, we have developed a sensitive and
specifi c method using mutant-specifi c primers to detect the
YMDD mutation. This PCR assay might be an effective
procedure for monitoring chronic hepatitis B patients
treated with lamivudine or screening them before starting
this anti-viral therapy.
ACKNOWLEDGMENTS
We thank Ms. Hsiao-Mann Lin for her laboratory work.
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