Lamivudine resistance-associated gene mutations
Related Terms
3TC, acquired immune deficiency syndrome, AIDS, antiretroviral therapy, DNA polymerase, Epivir?, Epivir? resistance-associated gene mutations, genotypes, HBV, HBV genotype, hepatitis B virus infection, HIV-1, human immunodeficiency virus infection, lamivudine, mutation, NRTI, nucleoside analog reverse transcriptase inhibitor, PCR, polymerase chain reaction, polymerase gene, resistance, reverse transcription, sequencing, YMDD motif.
Background
Genes (deoxyribonucleic acid/DNA) are considered the building blocks of life because they provide instructions for all the cells in the body. Genes, which are located inside the cells (in the nucleus), control an organism's development and functions by instructing cells to make new molecules (i.e., proteins). DNA is a long, thread-like molecule made up of large numbers of nucleotides. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information. Nucleotides are molecules composed of a nitrogen containing base, a 5-carbon sugar, and one or more phosphate groups. Long strands of nucleotides form nucleic acids. A permanent variation in a DNA sequence of a gene is called a mutation. Ribonucleic acids (RNA) are a single chain of nucleotides that contains ribose as its sugar (ribonucleotides), which determines the protein synthesis and transmission of genetic information.
Hepatitis B virus (HBV): HBV or hepatitis DNA virus causes a form of liver infection known as hepatitis B, which is a major health problem leading to around one million deaths annually worldwide. The prevalence of HBV infection varies around the world, with high rates of infection in sub-Saharan Africa, Asia, the Amazon, and the southern parts of Central and Eastern Europe. The chronic (long-term) HBV infection may present without any symptoms in some people; but more often, it causes severe complications, such as liver failure, liver cancer, liver cirrhosis (scarring), or even death. The HBV is transmitted through contact with bodily fluids, such as blood and semen, from an infected individual. HBV is also transmitted through women to their babies during childbirth. When the infection is passed from mother to fetus, it is called vertical transmission.
Structurally, an HBV particle (virion) consists of a central core particle containing the viral genome and an outer lipid (fat)-based envelope with embedded proteins. The genome is the total genetic material of an organism that carries information for its development and function.
The HBV viral genome is made of circular double-stranded DNA and the enzyme DNA polymerase. The double-stranded DNA has one strand that is of full length (minus strand), forming a complete circle, and the second strand (positive strand) is shorter and tends to vary in length. The viral genome carries four genes, namely C, X, P, and S. Gene C provides instructions (encodes) for the making of core protein; gene P encodes for the enzyme DNA polymerase, and gene S encodes for surface antigen (HBsAg). An antigen is a substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune system response in the body. The genome is surrounded by the core antigens HBcAg and HBeAg. The HBeAg is detected in the blood only when the HBV viruses are present. Hence, measurement of HBeAg is used as a marker to monitor the effectiveness of HBV treatment, as well as to estimate the ability of an infected individual to spread the virus to other people.
HBV is one of the few viruses that employ reverse transcription in the presence of DNA polymerase as part of its replication (multiplication) process. Reverse transcription is a process of synthesis of DNA under the direction of RNA. HBV has been classified into eight genotypes (A-H) based on the differences in the genome sequences. The HBV genotypes have distinct geographical distributions and are associated with virus replication, virus variations (variations in the gene sequence), disease progression, and the choice of drugs.
Human immunodeficiency virus (HIV): HIV is a retrovirus that causes acquired immune deficiency syndrome (AIDS), a debilitating and fatal disease of the human immune system. Retroviruses are a group of viruses that contain RNA and replicate by the reverse transcription process. HIV-1 and HIV-2 are the two strains, or variations, of HIV known to exist. HIV-1 virus is highly infectious (virulent), relatively easily transmitted, and is the cause of most HIV infections globally. HIV-2 is less transmittable and is largely confined to West Africa. Currently, the worldwide disease prevalence is more than 38 million HIV infections, and 95% of these HIV infected persons are in developing countries, generally in sub-Saharan Africa and Southeast Asia.
HIV is transmitted through exposure to blood and blood products (i.e., by sharing hypodermic needles and accidental needle sticks), and also though contact with semen and female genital secretions. HIV may also be transmitted from women to their babies during childbirth (vertical transmission).
Structurally, the HIV viral particle has a central nucleocapsid that contains two copies of RNA, along with core proteins and reverse transcriptase enzymes. Surrounding this is the viral envelope made of cellular lipids (fats) and viral envelope proteins.
Lamivudine: Lamivudine is an antiviral drug, commonly used to treat HBV infections and HIV-1/2 infections. Lamivudine basically inhibits the reverse transcription process and is called a nucleoside analog reverse transcriptase inhibitor (NaRTI or NRTI) because it is very similar (analog) to naturally occurring deoxynucleotides, which are required to produce viral DNA. During the viral replication process, the NRTI gets incorporated instead of a nucleotide from the supply in the cell, which in turn stops the DNA chain from growing. This disrupts the DNA synthesis process.
Lamivudine does not cure HBV or HIV-1 infections and neither does it prevent the spread of these infections to other people. However, it can achieve a significant decrease in viral replication, thereby reducing the amount of viruses in the blood (viral load) to very low levels. It also reduces disease activity and delays progression of the disease. The viral load is used to assess the infectivity and disease status in a viral-infected individual, and also helps to monitor treatment in the infected individuals. Epivir? (also known as 3TC) is a brand name for lamivudine, used to treat HIV in combination with other antiretroviral drugs. Epivir-HBV? is used in the treatment of HBV infections in combination with other antiretroviral drugs, or rarely, used alone (monotherapy).
Lamivudine resistance: Long-term monotherapy with lamivudine may not result in complete suppression of viral replication, which may lead to diminished treatment response in HBV and HIV-1 infections. This is as a result of emergence of mutations on the HBV and HIV-1 viral genes, which in turn leads to viral infections that are resistant to the drug lamivudine. Resistance to lamivudine has also been reported where these drugs are used in the treatment of HBV recurrence in patients with progressive liver damage (cirrhosis), to prevent HBV re-infection of the liver after transplantation, and in HIV-1-infected patients with concurrent HBV infection. Transplantation is a surgical procedure by which a tissue or an organ is removed and replaced by a corresponding part, either from another part of the body or from another individual. Lamivudine resistance may also develop when it is used to treat HBV and HIV concurrently, which should not be done.
The lamivudine resistance is related to mutations in the gene of polymerase enzyme at the tyrosine-methionine-aspartate-aspartate (YMDD) motif. Motif of a gene refers to the three-dimensional structure of a gene product, such as protein with known function. Detection of lamivudine-resistant mutations on the viral genes may be useful for determination of the need for alternative drug therapy in these viral infections.
Methods
General: Lamivudine is an antiviral drug used to treat hepatitis B virus (HBV) infection and human immunodeficiency virus type-1 (HIV-1) infection. Long-term monotherapy with lamivudine may not completely suppress the viral replication, leading to diminished treatment response in patients. This drug-resistance is a result of the emergence of mutations in the HBV and HIV-1 viral genes. A mutation refers to permanent variation in a DNA sequence of a gene.
Lamivudine resistance in HBV is due to mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of gene coding for polymerase enzyme. Motif of a gene refers to the three-dimensional structure of a gene product, such as protein with known function. Polymerase enzyme viral DNA replicate. The two most common mutations on the YMDD motif include methionine to isoleucine change at position 204 (rtM204I) and methionine to valine at position 204 (rtM204V); the latter is often accompanied with compensatory leucine to methionine change at position 180 (rtL80 M). Methionine, isoleucine, and valine are amino acids, the building blocks of proteins. Detection of lamivudine-resistant mutations on the viral genes may be useful to determine the need for alternative drug therapy in these viral infections.
Methods available to detect and quantify (measure the amount of) lamivudine drug-resistant mutations in viral genes include polymerase chain reaction (PCR), followed by DNA sequencing or real-time PCR. Initially, the DNA is isolated and purified (extraction of DNA) from the sample of interest (blood) to get a clean DNA, which is used as a template (serves as a pattern for synthesis).
Polymerase chain reaction (PCR): PCR is an efficient and sensitive laboratory technique to amplify (by replication) a specific sequence of DNA into billions of copies in the presence of sequence-specific oligonucleotide primers and a DNA polymerase enzyme. Oligonucleotide primer is a sequence of nucleotides, usually of 20-50 bases, that is complementary to a specific DNA sequence and serves as a starting point for DNA replication. DNA polymerase is an enzyme that synthesizes new DNA strands using preexisting DNA strands as template, thereby assisting in DNA replication. A radioactive molecule (radionucleotide) is also incorporated into the PCR product during the amplification process (radiolabeling of PCR). This assists in visualizing the PCR products at a later stage.
DNA sequencing: DNA sequencing is a process in which the precise sequence of nucleotides in a sample of DNA is determined. Nucleotides are building blocks of DNA in a sequence and are made of nitrogenous bases, sugars, and phosphate. DNA sequencing as a scanning method for mutations has become very competitive, with the development of automated DNA sequencers, Taq DNA polymerase, and newer detection methods. Taq DNA polymerase is an enzyme that synthesizes new DNA strands using preexisting DNA strands as a template. The method used for DNA sequencing is usually by Sanger's method (dideoxy or chain termination method).
The initial step of Sanger's method involves extraction of high-quality DNA from the sample of interest, followed by polymerase chain reaction (PCR) in the presence of fluorescent labeled (attached) dideoxynucleotide triphosphate (ddNTP). The synthetic nucleotides known as ddNTP's are somewhat structurally different from regular nucleotides and function as DNA chain terminators (stopper) during synthesis of a DNA sequence. The end reaction product is a set of DNA sequences differing in length by one nucleotide, and the last nucleotide base in each sequence is the unique fluorescent labeled ddNTP.
The reaction products are run on the electrophoresis, which separates the DNA fragments based on their size. During this process, the fluorescent signals produced by the labeled nucleotides are detected by the fluorescent detection systems, thereby identifying the nucleotide base. These fluorescent signals are fed and analyzed by a computer, giving out the exact sequence of DNA. The whole process is automated and the resultant DNA sequence is compared with other sequences by various computer programs, thereby spotting the mutations in the sample DNA sequence.
Conventional DNA sequencing is labor-intensive, time-consuming, and expensive. The development of the automated DNA sequencer (as described above) with newer detection methods (fluorescent detection) has become competitive in detecting gene mutations. Other advantages with direct DNA sequencing (automated) is the complete information it provides in a single experiment, such as the type of the mutation and exact location of mutation on the DNA sequence.
Real-time PCR: The lamivudine-resistant mutation analysis is performed on a real-time PCR platform (e.g. LightCycler?, TaqMan? etc.) with fluorescence detection systems using hybridization probes and the melting curve analysis technique.
Real-time PCR (RT-PCR), also known as real-time quantitative PCR (RTq-PCR), is a polymerase chain reaction (PCR)-based laboratory technique that enables both detection and quantification of a specific sequence in a DNA sample simultaneously. This follows the general principle of PCR with a key feature, i.e., the amplification or increase in the amount of DNA that can be viewed and quantified as it accumulates in the reaction in real time (at the same time during which the process occurs) after each round of the amplification cycle. The advantages of RT-PCR over conventional PCR are due to its fast, efficient, accurate analysis and quantitation in real time. RT-PCR is fully automated with the fluorescent detection system against the use of radioactive molecules and gel electrophoresis.
The amplified reaction products of PCR (amplicon) can be detected and measured by two commonly used methods. One method follows the use of fluorescent dyes (e.g. SYBR green) during the reaction process. Another method uses a fluorescent reporter probe, which is a modified, single-stranded oligonucleotide probe that fluoresces (emits fluorescence light) when it gets paired with a complementary DNA template (hybridization). A probe is a nucleic acid sequence used to detect complementary nucleic acid sequences on the target DNA.
The real-time PCR, along with the monitoring and quantification of DNA synthesis, also determines the melting point of the amplicon at the end of amplification reactions. Each double-stranded DNA has its own specific melting temperature, which is the temperature at which 50% of the DNA becomes single-stranded. After PCR amplification, a 'melt curve' is generated by raising the temperature by a fraction of a degree and the change in the fluorescence is measured. All the PCR products for a particular primer should have the same melting temperature and constant emission of fluorescence. Any single mismatch between the labeled probe and amplicon will significantly reduce the melting temperature and so also the fluorescence intensity, which is visualized and analyzed with a suitable computer program. Hence, the melting curve technique may be utilized to detect and analyze the mismatch base pairs, which are representative of gene variations associated with lamivudine-resistance. The whole process is automated, and the assay is analyzed within 45 minutes.
Research
Currently, research is underway to identify newer mutations associated with lamivudine drug-resistance, as well as cross-resistance with other antiretroviral drugs in relation to clinical outcomes of hepatitis B virus (HBV) infections and human immunodeficiency virus (HIV) infections. Drug resistance refers to the ability of an organism or virus, such as HBV or HIV, to overcome the effects of a drug given to destroy it, resulting in a diminished treatment response to the drug, and cross-resistance refers to development of resistance to other drugs, which are similar to the main drug.
Newer mutations in relation to lamivudine-resistance in HBV genes are being identified. These mutations may not be identical among the different genotypes (genetic make-up) of HBV as a result of virus replication (multiplication) and virus variations. Thus, large studies are in progress to understand these mutations in association with each HBV genotype.
Implications
General: Lamivudine is an antiviral drug used to treat hepatitis B virus (HBV) infection and human immunodeficiency virus type-1 (HIV-1) infection. Long-term monotherapy with lamivudine may not completely suppress the viral replication, leading to diminished treatment response in patients. This drug-resistance is a result of the emergence of mutations in the HBV and HIV-1 viral genes. A mutation refers to a permanent variation in a DNA sequence of a gene.
Combination antiviral therapy: The rate of lamivudine-resistance increases with increased duration of treatment, particularly if used as a monotherapy to treat HBV infections. Studies have indicated that about 14-32% of patients develop lamivudine-resistance after one year of therapy, 38% after two years, 53-76% after three years, and 65-70% after five years of lamivudine monotherapy. Hence, early detection of lamivudine-resistance and the type of mutations, as well as the combination of lamivudine with other antiretroviral drugs delays the onset of drug resistance, resulting in better management of HBV and HIV infections.
Cross-resistance: Cross-resistance refers to the development of resistance in other drugs, which are similar to the main drug. Resistance to lamivudine has been observed where these drugs are used in the treatment of HBV recurrence with progressive liver damage (cirrhosis), to prevent HBV re-infection of the liver after transplantation, and in HBV infections co-infected with HIV-1. Drug resistance has also been reported among patients who have not received lamivudine treatment. In such patients, lamivudine resistance increases the potential of cross-resistance to other antiretroviral drugs (e.g. entecavir, only used for HBV), which are similar to lamivudine, thereby limiting treatment options. Thus, evaluation of HBV infections in such cases for lamivudine resistance by detection of drug-resistant mutations and planning the treatment accordingly may help prevent complications, thereby increasing the effectiveness of the treatment.
Vertical transmission: HBV and HIV infections may be passed on from the mother to the child during pregnancy, delivery or breastfeeding (vertical transmission). In such patients, it is of importance to treat these infections efficiently, so as to prevent transmission effectively. Lamivudine resistance causes a major drawback in effective treatment of HBV infections. Thus, early detection of lamivudine resistance may help in better therapeutic management, by utilizing alternative antiviral drugs in combination forms of treatment, thereby preventing prenatal transmission of viral infections.
Limitations
The disadvantage with use of DNA sequencing to detect mutations is that it requires very high-quality DNA. It is also expensive, although this is being overcome by the automation. Another major drawback with this method is that only about 400 bp (base pair) of sequencing data can be generated by DNA sequencing in a series of experiments, thereby increasing the cost and time
Future research
It has been observed that variations in the gene sequence (mutations) associated with lamivudine resistance may exist even in newly acquired lamivudine-resistant hepatitis B virus (HBV) infections. Further research is required to understand the preexisting HBV mutations and their clinical outcomes with lamivudine therapy alone or in combination with other antiviral drugs.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
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