Genetic polymorphisms and mutations in drug metabolizing enzymes, transporters, receptors, and other drug targets (
Pharmacogenomics, which deals with heredity and response to drugs, is the scientific field that attempts to explain individual variability of drug responses and to search for the genetic basis of such variations or differences (Evans et al., 2001). The inter-individual variation in the rate of drug metabolism has been known for many years. Initially, the study of pharmacogenetics was only of academic interest, but today it is of major concern to the pharmaceutical industry as a means for documenting the metabolism of a new drug in development before registration. The knowledge of how a drug is metabolized and which enzymes are involved may help to predict drug-drug interactions and the rate at which individual patients may metabolize a specific drug. Such information is now required for registration by the U.S. Food and Drug Administration (FDA) and similar authorities (Salerno & Lesko, 2004a, 2004b). To improve drug safety, the FDA has started to update the labels and package inserts of previously approved drugs as new clinical and genetic evidence accrues (Frueh et al., 2008, Lesko, 2008).
The current important step is to incorporate pharmacogenomics data into routine clinical practice. As a means of implementing personalized medicine, it is critically important to understand the molecular mechanisms underlying inter-individual differences in the drug response, namely, pharmacological effect vs. side effect. The occurrence of personal variations in the response to a drug may result from many different causes, for example, genetic variations and expression levels of drug-targeted molecules, including membrane receptors, nuclear receptors, signal transduction components, and enzymes, as well as those of drug-metabolizing enzymes and drug transporters (Evans et al., 2001). Recently, tools such as next-generation sequencing technologies and genome-wide association studies (GWAS) have been used to uncover a number of variants that affect drug toxicity and efficacy as well as potential risk of diseases. The costs involved in carrying out GWAS and sequencing have been dropping dramatically, while providing data at an unprecedented rate. The GWAS approach has been applied for identifying genetic contributions to variations in drug response (The SEARCH Collaborative Group, 2008, Kamatani et al., 2010, Cooper et al., 2008, Schuldiner et al., 2009, Ge et al., 2009, Daly et al., 2009). As a result, there have been dramatic increases in our understanding of the mechanisms of drug action and of the genetic determinants responsible for variable responses to both rarely and widely used drugs, such as warfarin, tomoxifen, and clopidogrel.
Technologies are evolving to transform diagnostic devices for rapid genetic testing. Portable devices are being engineered for use in a range of settings to perform robust assays for the diagnosis of disease that will improve patient management, and result in greater convenience and speed to answer.The genetic diagnostics is a growing field that is gradually becoming more user-friendly with the introduction of portable devices and quicker nucleic acid detection. Successful genetic diagnostics require 4 major elements, such as rapid reaction, low cost, low energy consumption, and simple analysis (with minimal technical training and inclusion of controls but no off-instrument processing or reagent preparation). In this context, we decided to develop a point-of-care “POC” technology and to apply it to medical advances.
Development of personalized medicine including POC technology requires integration of various segments of biotechnology, clinical medicine, and pharmacology. A key requirement for advancing personalized medicine is the ability to rapidly and conveniently test for patients’ genetic polymorphisms and/or mutations. To address this urgent need, we have recently developed a rapid and cost-effective method, named Smart Amplification Process (SmartAmp), which enables us to detect genetic polymorphisms or mutations in target genes within 30 to 45 minutes under isothermal conditions that do not require DNA isolation and PCR amplification (Mitani et al., 2007, Mitani et al., 2009, Ishikawa et al., 2010, Aw et al., 2011, Ota et al., 2010, Lezhava et al., 2010, Toyoda et al., 2009, Aomori et al., 2009, Watanabe et al., 2007, Okada et al., 2010, Azuma et al., 2011). In this book chapter, we will present the technological development and clinical applications of the SmartAmp method.
2. SmartAmp method
The SmartAmp method was developed based on the principal concept that DNA amplification itself is the signal for detection of a genetic mutation or SNP. Differing from the widely-used PCR, the SmartAmp method is an isothermal DNA amplification reaction (Mitani et al., 2007, Mitani et al., 2009). In the SmartAmp method, the entire DNA amplification process requires five primers: turnback primer (TP), boost primer (BP), folding primer (FP), and two outer primers (OP1 and OP2) (Fig. 1). Primers are selected based on those algorithms considering the free energy, probability of base-pairing, product size range, optimal melting temperature, and product size range. The design of these primers contributes to the specificity of SmartAmp. In particular two primers (TP and FP) are critically important for the amplification process. The genomic sequence between the annealing sites of the TP and FP primers is the target region that will be amplified by the SmartAmp reaction. The other primers (BP, OP1, and OP2) are additionally employed to accelerate the process and enhance specificity.
2.1. Molecular mechanism underlying isothermal DNA amplification
In isothermal DNA amplification by the SmartAmp method, the initial step of copying a target sequence from the genomic DNA is a prerequisite. FP and TP hybridize the template genomic DNA. Next, both products primed for the FP and TP are detached from template genomic DNA by strand-displacing DNA polymerase, whose extensions are primed by OP1 and OP2. Single-stranded DNA products, thus displaced, become templates in the second step for the opposing FP and TP. These single stranded DNA products are generated by the strand-displacement activity of the DNA polymerase, being primed from the flanking region of OP primers adjacent to the target sequence. The resulting DNA products are referred to as “intermediate products”, IM1 and IM2, which play key roles in the subsequent amplification steps (Fig. 2).
The formation of those intermediate products (IM1 ad IM2) is the rate-limiting step in SmartAmp-based isothermal DNA amplification. IM1 has the TP sequence at the 5’ end and the FP complementary sequence at the 3’ end; and IM2 is complementary to IM1 (Fig. 3). The initial self-priming site on IM1 is the 3’-end of the FP sequence of IM1. Concatenated products of IM1 are synthesized by an elongation process termed pathway A. The characteristic feature of the products of pathway A is that the free 5’ and 3’ ends carry TP and its complementary sequence, forming long double stranded hairpin DNA. The initial self-priming elongation site on IM2 is located at the 3’ end of the TP sequence of IM2. Long concatenated DNA products are synthesized as in pathway A, but end products in pathway B are different. The long-hairpin DNA products of pathway B carry FP and its complementary sequence at the free 5’ and 3’ ends respectively. There is another elongation pathway which starts from the 3’ end of a free TP-primer that hybridizes to the looping structure of the TP complementary sequence, which is located at the intermediate region of the long products of pathway A. Thus, concatenated DNA products are formed in the SmartAmp reaction. The resulting DNA products could be detected by conventional agarose gel electrophoresis, where DNA ladder patterns represented the formation of concatenated DNA products (Mitani et al., 2007) (Fig. 3).
2.2. Molecular mechanism underlying SNP detection
To ensure the high fidelity of SNP detection by the SmartAmp method, exponential amplification of mis-primed DNA must be suppressed. In the original SmartAmp method, this was achieved by adding either the mismatch binding protein (MutS)
The SmartAmp method utilizes
2.3. Example of SNP detection by SmartAmp method
Clinical application of SmartAmp to practical SNP detection should be evaluated with clinical samples (either blood or genomic DNA) according to the principle of amplification versus non-amplification as compared to threshold values. The amount of DNA-intercalating SYBR Green I dye during the reaction can be monitored in a real-time PCR system (e.g., Mx3000P), and thereby SNP typing can be determined by referring to the intensity of fluorescence.
Each SmartAmp2 reaction is performed in a 25 μl-volume tube at 60˚C. The standard reaction mixture contains 3.2 μM each of TP and FP, 0.4 μM each of OP1 and OP2, 1.6 μM BP, 1.4 mM dNTPs, 5% dimethyl sulfoxide (DMSO), 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% Tween 20, SYBR Green I (1/100,000-diluted), 40 units of
3. Clinical applications of SmartAmp method
Hitherto, we have proven that the SmartAmp method is capable of detecting SNPs in drug transporter genes (
3.1. Thioprine toxicity and genetic polymorphisms in
Thiopurines are effective immunosuppressants and anticancer agents used for treating childhood acute lymphoblastic leukemia, acute myeloblastic leukemia, autoimmune disease, rheumatoid arthritis, and inflammatory bowel diseases. The intracellular accumulation of such active metabolites as 6-thioguanine nucleotides (6-TGN), however, causes dose-limiting hematopoietic toxicity (Weinshilboum & Sladek, 1980). TPMT deficiency has been reported to exacerbate thiopurine toxicity (Fig. 6).
The enzyme TPMT operates in the main inactivation pathway for thiopurine drugs. The
3.2. SNP 2269G>A (Glu757Lys) in
ABCC4gene and thiopurine toxicity
For largely unknown reasons, there are subsets of Japanese patients who suffer from dose-limiting hematopoietic toxicity, but are not
Unlike the situation for
Accumulating evidence strongly suggests that genetic polymorphisms in drug metabolizing enzymes, transporters, receptors, and other drug targets (e.g., toxicity targets) are linked to inter-individual differences in the efficacy and toxicity of many medications. The genetic polymorphisms of drug metabolizing enzymes and transporters have been studied in many laboratories worldwide. In fact, efforts to discover and characterize gene polymorphisms resulted in new diagnostic tests for discriminating between different gene alleles and better strategies for pharmacotherapy.
To realize the promise of individualized medicine, however, genetic diagnosis should be further integrated with therapy for selecting drugs and treatments as well as for monitoring results. It is also critically important to reduce the cost of genetic diagnosis. Technologies are evolving to transform diagnostic devices for rapid genetic testing. Portable devices are being engineered for use in a range of settings to perform robust assays for the diagnosis of disease that will improve patient management, and result in greater convenience and speed to answer. Indeed, the POC diagnostics is a growing field that is gradually becoming more user-friendly with the introduction of portable devices and quicker nucleic acid detection.
The isothermal amplification technologies have a potential to cover different applications. A key requirement for the advancing personalized medicine resides in the ability of rapidly and conveniently testing patients’ genetic polymorphisms and/or mutations. With this respect, isothermal nucleic acid amplification technologies, including the SmartAmp method, are expected to translate into less complex and less expensive instrumentation.
The authors thank Prof. John D. Schuetz (St. Jude Children’s Hospital, Memphis, TN, USA) and Prof. Akira Andoh (Shiga University of Medical Science, Otsu, Japan) for their fruitful discussion about genetic polymorphisms of human ABCC4 gene. Our thanks go to Drs. Alexander Lezhava and Wanping Aw (Omics Science Center, RIKEN) for their generous support in SmartAmp-based genotyping experiments. The authors’ study was supported by a Japan Science and Technology Agency (JST) research project named “Development of the world’s fastest SNP detection system” (to TI) and Research Grant for RIKEN Omics Science Center from the Ministry of Education, Culture, Sports, Science and Technology (to YH).