Genetic variation among individuals within a population is often observed by phenotypic changes that in some cases manifest as a disease or disorder. There are some instances where a disease or disorder only manifests as an adverse reaction to an external stimulus such as a pharmaceutical drug or other chemicals. The association studies of adverse drug reactions (ADRs) and patients' genetic makeup hold the promise of greatly reducing the ADR-related morbidity and mortality by genetic testing. There are several known links between ADR and genetic variants.
Fluorouracil (5-FU) has been used as a chemotherapy agent in the treatment of patients with breast, colorectal, lung and other cancers for several decades. Side-effects of 5-FU include diarrhea, stomatitis, mucositis, neurotoxicity, and in some cases, death. These are largely due to genetic inability to metabolize the drug. Dihydropyrimidine dehydrogenase (DPD), an enzyme encoded by the DPD gene, is responsible for the elimination of ˜80% of the standard dose of 5-FU. DPD deficiency due to DPD gene mutations has been associated with severe 5-FU toxicity. It has been found that ˜3-5% of patients harbor at least a partial DPD deficiency. The most common mutation associated with DPD deficiency is IVS14+1 G>A, a G>A base change at the splice recognition sequence of intron 14 (known as DPD*2A). This single-nucleotide variation leads to exon skipping and results in a 165-bp deletion in the DPD mRNA. A homozygote DPD*2A genotype results in complete deficiency, while the heterozygous DPD*2A genotype results in partial deficiency of DPD. The pre-therapeutic detection of this metabolic dysfunction could prevent severe side-effects of 5-FU by administering alternative treatments.
Similarly, the linkage of CYP2C19 genotype with clinical outcomes among clopidogrel-treated acute coronary syndrome (ACS) patients, particularly those undergoing percutaneous coronary intervention (PCI) has been reported. Clopidogrel is a commonly prescribed oral, antiplatelet agent used to inhibit blood clots in coronary artery disease, peripheral vascular disease, and cerebrovascular disease. As a thienopyridine prodrug, clopidogrel requires hepatic biotransformation to form an active metabolite. The hepatic CYP2C19 enzyme is one of CYP450 superfamily members that is involved in the metabolism of clopidogrel. The CYP2C19 gene is highly polymorphic with over 25 known variant alleles. The most common CYP2C19 loss-of-function allele is CYP2C19*2 (c.681G>A; rs4244285), with allele frequencies of ˜15% in Caucasians and Africans, and 29-35% in Asians. However, current genetic testing methods for loss of function generally require expensive instruments and long turnaround time.
Another example is the discovery of the strong linkage between carbamazepine-induced Steven Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) and a specific allele of human leukocyte antigen (HLA), HLA-B*1502. Carbamazepine is the first-line drug for treatment of patients with epilepsy, neuropathic pain and bipolar disorder. However, carbamazepine therapy may cause dangerous or even fatal skin reactions, such as SJS and TEN in patients with the HLA-B*1502 allelic variation. Prevalence of the HLA-B*1502 allele in Asian populations, except in Japanese and Koreans, is much higher than in Caucasian and African populations. Some studies revealed that the negative predictive value of HLA-B*1502 test for carbamazepine-induced SJS could be close to 100%. HLA-B gene is highly polymorphic with more than 2000 unique alleles, which complicates the determination of a specific allele. Sanger sequencing is considered to be the gold standard for HLA typing. However, the phase ambiguity often causes problems for SBT in identification of allele combinations. Next-generation sequencing (NGS) technologies are able to obtain the sequence of a single DNA molecule and may rule out the phase ambiguity, but drawbacks of the current NGS methods include the short read length, high workloads for library preparation and lengthy processing time. A recent report employed loop-mediated isothermal amplification (LAMP) for HLA-B*1502 screening (Cheng, et al. Clin. Chem. 2009, 55, 1568.). Unfortunately, the specificity of these methods is not sufficient to distinguish HLA-B*1502 from quite a few other alleles that are not rare in some populations. The false assays will rule out the patients with very low SJS risk from the effective carbamazepine therapy, and increase the treatment cost. Since the cost-effectiveness of HLA-B*1502 screening is highly dependent on the accuracy and cost of the genetic test, there is an urgent need for a new assay that is more specific and less costly.
The US Food and Drug Administration (FDA) and other regulatory bodies have required labelling of several drugs that may have adverse effects in certain genotypes. For Example a requirement for fluorouracil (5-FU)-based drugs is to contain a warning for hypersensitivity in some individuals. In another example, the US Food and Drug Administration issued an alert in 2007 that recommends screening of the HLA-B*1502 allele before starting treatment with carbamazepine for patients with ancestry from areas in which the specific allele is present. Similarly, the boxed warning on the clopidogrel label recommends consideration of alternative antiplatelet therapy in poor metabolizers with ACS or PCI.
Commercial kits for genotyping tests are available for some genetic variants and are used to predict an individual's tolerance or intolerance of a drug. The testing platforms used include Sanger sequencing and restriction fragment length polymorphism (RFLP) analysis. The assays based on these platforms are either time-consuming or costly, and therefore, less suitable for on-demand testing. Commercial kit based on real-time PCR is also available unfortunately, the specificity of these methods is not sufficient to distinguish complex allelic variants from quite a few other alleles that are not rare in some populations. Since the cost-effectiveness of screening is highly dependent on the accuracy and cost of the genetic test, there is an urgent need for a new assay that is more specific and less costly.