Pharmacogenetics and pharmacogenomics: role of mutational analysis in anti-cancer targeted therapy

The goal of cancer pharmacogenomics is to obtain benefit from personalized approaches of cancer treatment and prevention. Recent advances in genomic research have shed light on the crucial role of genetic variants, mainly involving genes encoding drug-metabolizing enzymes, drug transporters and targets, in driving different treatment responses among individuals, in terms of therapeutic efficacy and safety. Although a considerable amount of new targeted agents have been designed based on a finely understanding of molecular alterations in cancer, a wide gap between pharmacogenomic knowledge and clinical application still persists. This review focuses on the relevance of mutational analyses in predicting individual response to antitumor therapy, in order to improve the translational impact of genetic information on clinical practice.     

药物代谢酶特异性探针底物的研发与应用进展

人体内分布数百种结构多样、功能各异的药物代谢酶,其表达和功能易受遗传和环境因素的影响,导致其在不同人种、个体及组织 中的分布都不尽相同。特异性探针底物作为药物代谢酶活性表征的关键工具分子,其在新药发现、药-药相互作用评价、临床药理学及精准 医学检测等领域具有重要的应用价值。围绕人体分布的重要药物代谢酶,综述其特异性探针底物的设计研发及生物医学应用进展,以期为 药物代谢酶的精准检测及相关应用研究提供参考。     

Decoding variants in drug-metabolizing enzymes and transporters in solid tumor patients by whole-exome sequencing

BackgroundPharmacogenetics is involved in customizing therapy according to the genetic makeup of an individual, and is applicable for chemotherapy, radiotherapy as well as targeted therapy. Drug metabolizing enzymes (DMEs) involving both phase I, and phase II reactions are widely studied. Our study was involved in whole exome sequencing (WES) of cancer patients, followed by analysis for identifying key variations in DMEs, and associated transporters that have a potential impact on treatment outcome.MethodologyA total of 181 solid tumor patients at stage >/= III were subjected to WES by the SureSelect^XT Human All Exon V6 + UTR library preparation kit, and sequencing in the Illumina NextSeq 550 system. Bioinformatics analysis involved use of GATK pipeline, and the variants were further assessed for population frequency, functional impact with annovar insilico algorithms. Further variant information from significant DMEs, and transporters were extracted and analyzed with PharmGKB to assess level of evidence and infer their impact on the pathways involved in drug response.ResultsThe total study cohort of 181 solid tumor patients included 60 males, and 121 females respectively. Among DMEs, deleterious mutation in dihydropyrimidine dehydrogenase (DPYD; rs67376798), solute carrier organic anion transporter family member 1B1 (SLCO1B1*5), and cytochrome P450 2D6 (CYP2D6*10) associated with metabolism of anticancer drugs was detected to be in high frequency of 26%, 21% and 25% respectively.ConclusionOur analysis detected variations in both phase I and phase II DMEs, as well as associated transporter genes which has been documented to reduce drug efficacy, as well as cause grade 3 and 4 toxicity. Our study reiterates the significance of pharmacogenomics in stratifying patients for appropriate therapy regimen focused at better treatment outcome and quality of life.     

Human loci involved in drug biotransformation: worldwide genetic variation, population structure, and pharmacogenetic implications

Understanding the role of inheritance in individual variation in drug response is the focus of pharmacogenetics (PGx). A key part of this understanding is quantifying the role of genetic ancestry in this phenotypic outcome. To provide insight into the relationship between ethnicity and drug response, this study first infers the global distribution of PGx variation and defines its structure. Second, the study evaluates if geographic population structure stems from all PGx loci in general, or if structure is caused by specific genes. Lastly, we identify the genetic variants contributing the greatest proportion of such structure. Our study describes the global genetic structure of PGx loci across the 52 populations of the Human Genome Diversity Cell-Line Panel, the most inclusive set of human populations freely available for studies on human genetic variation. By analysing genetic variation at 1,001 single nucleotide polymorphisms (SNPs) involved in biotransformation of exogenous substances, we describe the between-populations PGx variation, as well geographical groupings of diversity. In addition, with discriminant analysis of principal component (DAPC), we infer how many and which groups of populations are supported by PGx variation, and identify which SNPs actually contribute to the PGx structure between such groups. Our results show that intergenic, synonymous and non-synonymous SNPs show similar levels of genetic variation across the globe. Conversely, loci coding for Cytochrome P450s (mainly metabolizing exogenous substances) show significantly higher levels of genetic diversity between populations than the other gene categories. Overall, genetic variation at PGx loci correlates with geographic distances between populations, and the apportionment of genetic variation is similar to that observed for the rest of the genome. In other words, the pattern of PGx variation has been mainly shaped by the demographic history of our species, as in the case of most of our genes. The population structure defined by PGx loci supports the presence of six genetic clusters reflecting geographic location of samples. In particular, the results of the DAPC analyses show that 27 SNPs substantially contribute to the first three discriminant functions. Among these SNPs, some, such as the intronic rs1403527 of NR1I2 and the non-synonymous rs699 of AGT, are known to be associated with specific drug responses. Their substantial variation between different groups of populations may have important implications for PGx practical applications.     

Induction of drug-metabolizing enzymes and transporters in human bronchial epithelial cells by beclomethasone dipropionate

Inhaled steroids are the most potent anti-inflammatory therapy commonly used in bronchial asthma. There are, however, a small number of asthmatic patients who do not respond to inhaled steroid-treatment. The stimulation of metabolism and excretion of inhaled drugs at bronchial tissues might lead to a decrease in the effect of the drugs, although the molecular mechanism of this resistance is unclear. In this study, we found that beclomethasone dipropionate (BDP) stimulated the expression of mRNAs for uridine 5'-diphosphate glucuronosyl transferase 2B4 and 2B11, and transporters such as multidrug resistance P-glycoprotein, multidrug resistance-associated protein 1 and 2 in cultured bronchial epithelial cells. It is possible that the individual differences of expression of drug metabolizing enzymes and transporters and their enhancement with BDP are implicated in the individual differences of reactivity over steroid medical treatment.     

Pharmacogenetics in drug regulation: promise, potential and pitfalls

Pharmacogenetic factors operate at pharmacokinetic as well as pharmacodynamic levels-the two components of the dose-response curve of a drug. Polymorphisms in drug metabolizing enzymes, transporters and/or pharmacological targets of drugs may profoundly influence the dose-response relationship between individuals. For some drugs, although retrospective data from case studies suggests that these polymorphisms are frequently associated with adverse drug reactions or failure of efficacy, the clinical utility of such data remains unproven. There is, therefore, an urgent need for prospective data to determine whether pre-treatment genotyping can improve therapy. Various regulatory guidelines already recommend exploration of the role of genetic factors when investigating a drug for its pharmacokinetics, pharmacodynamics, dose-response relationship and drug interaction potential. Arising from the global heterogeneity in the frequency of variant alleles, regulatory guidelines also require the sponsors to provide additional information, usually pharmacogenetic bridging data, to determine whether data from one ethnic population can be extrapolated to another. At present, sponsors explore pharmacogenetic influences in early clinical pharmacokinetic studies but rarely do they carry the findings forward when designing dose-response studies or pivotal studies. When appropriate, regulatory authorities include genotype-specific recommendations in the prescribing information. Sometimes, this may include the need to adjust a dose in some genotypes under specific circumstances. Detailed references to pharmacogenetics in prescribing information and pharmacogenetically based prescribing in routine therapeutics will require robust prospective data from well-designed studies. With greater integration of pharmacogenetics in drug development, regulatory authorities expect to receive more detailed genetic data. This is likely to complicate the drug evaluation process as well as result in complex prescribing information. Genotype-specific dosing regimens will have to be more precise and marketing strategies more prudent. However, not all variations in drug responses are related to pharmacogenetic polymorphisms. Drug response can be modulated by a number of non-genetic factors, especially co-medications and presence of concurrent diseases. Inappropriate prescribing frequently compounds the complexity introduced by these two important non-genetic factors. Unless prescribers adhere to the prescribing information, much of the benefits of pharmacogenetics will be squandered. Discovering highly predictive genotype-phenotype associations during drug development and demonstrating their clinical validity and utility in well-designed prospective clinical trials will no doubt better define the role of pharmacogenetics in future clinical practice. In the meantime, prescribing should comply with the information provided while pharmacogenetic research is deservedly supported by all concerned but without unrealistic expectations.     

Sequencing XMET genes to promote genotype-guided risk assessment and precision medicine

High-throughput next generation sequencing(NGS) is a shotgun approach applied in a parallel fashion by which the genome is fragmented and sequenced through small pieces and then analyzed either by aligning to a known reference genome or by de novo assembly without reference genome. This technology has led researchers to conduct an explosion of sequencing related projects in multidisciplinary fields of science. However, due to the limitations of sequencing-based chemistry, length of sequencing reads and the complexity of genes, it is difficult to determine the sequences of some portions of the human genome, leaving gaps in genomic data that frustrate further analysis. Particularly, some complex genes are difficult to be accurately sequenced or mapped because they contain high GC-content and/or low complexity regions, and complicated pseudogenes, such as the genes encoding xenobiotic metabolizing enzymes and transporters(XMETs). The genetic variants in XMET genes are critical to predicate interindividual variability in drug efficacy, drug safety and susceptibility to environmental toxicity. We summarized and discussed challenges, wet-lab methods, and bioinformatics algorithms in sequencing "complex" XMET genes, which may provide insightful information in the application of NGS technology for implementation in toxicogenomics and pharmacogenomics.     

In vitro and in vivo assessment of herb drug interactions

Herbal products contain several chemicals that are metabolized by phase 1 and phase 2 pathways and also serve as substrates for certain transporters. Due to their interaction with these enzymes and transporters there is a potential for alteration in the activity of drug metabolizing enzymes and transporters in presence of herbal components. Induction and inhibition of drug metabolizing enzymes and transporters by herbal component has been documented in several in vitro studies. While these studies offer a system to determine the potential for a herbal component to alter the pharmacokinetics of a drug, they cannot always be used to predict the magnitude of any potential effect in vivo. In vivo studies are the ultimate way to determine the clinical importance of herb drug interactions. However, lack of content uniformity and lack of documentation of the bioavailability of herbal components makes even in vivo human studies difficult to interpret as the effect may be product specific. It appears that St. John's wort extract is probably one of the most important herbal product that increases the metabolism and decreases the efficacy of several drugs. Milk thistle on the other hand appears to have minimal effect on phase 1 pathways and limited data exists for phase 2 pathways and transporter activity in vivo. Further systematic studies are necessary to assess the significance of herb drug interactions.     

Clines, clusters, and the effect of study design on the inference of human population structure

Previously, we observed that without using prior information about individual sampling locations, a clustering algorithm applied to multilocus genotypes from worldwide human populations produced genetic clusters largely coincident with major geographic regions. It has been argued, however, that the degree of clustering is diminished by use of samples with greater uniformity in geographic distribution, and that the clusters we identified were a consequence of uneven sampling along genetic clines. Expanding our earlier dataset from 377 to 993 markers, we systematically examine the influence of several study design variables—sample size, number of loci, number of clusters, assumptions about correlations in allele frequencies across populations, and the geographic dispersion of the sample—on the “clusteredness” of individuals. With all other variables held constant, geographic dispersion is seen to have comparatively little effect on the degree of clustering. Examination of the relationship between genetic and geographic distance supports a view in which the clusters arise not as an artifact of the sampling scheme, but from small discontinuous jumps in genetic distance for most population pairs on opposite sides of geographic barriers, in comparison with genetic distance for pairs on the same side. Thus, analysis of the 993-locus dataset corroborates our earlier results: if enough markers are used with a sufficiently large worldwide sample, individuals can be partitioned into genetic clusters that match major geographic subdivisions of the globe, with some individuals from intermediate geographic locations having mixed membership in the clusters that correspond to neighboring regions.     

Strong and weak plasma response to dietary carotenoids identified by cluster analysis and linked to beta-carotene 15,15'-monooxygenase 1 single nucleotide polymorphisms

The mechanisms as well the genetics underlying the bioavailability and metabolism of carotenoids in humans remain unclear. To begin to address these questions, we used cluster analysis to examine individual temporal responses of plasma carotenoids from a controlled-diet study of subjects who consumed carotenoid-rich beverages. Treatments, given daily for 3 weeks, were watermelon juice at two levels (20-mg lycopene, 2.5-mg β-carotene, n=23 and 40-mg lycopene, 5-mg β-carotene, n=12) and tomato juice (18-mg lycopene, 0.6-mg β-carotene, n=10). Cluster analysis revealed distinct groups of subjects differing in the temporal response of plasma carotenoids and provided the basis for classifying subjects as strong responders or weak responders for β-carotene, lycopene, phytoene and phytofluene. Individuals who were strong or weak responders for one carotenoid were not necessarily strong or weak responders for another carotenoid. Furthermore, individual responsiveness was associated with genetic variants of the carotenoid metabolizing enzyme β-carotene 15,15'-monooxygenase 1. These results support the concept that individuals absorb or metabolize carotenoids differently across time and suggest that bioavailability of carotenoids may involve specific genetic variants of β-carotene 15,15'-monooxygenase 1.     

Pharmacogenetics in Primary Care: The Promise of Personalized Medicine and the Reality of Racial Profiling

Many anticipate that expanding knowledge of genetic variations associated with disease risk and medication response will revolutionize clinical medicine, making possible genetically based Personalized Medicine where health care can be tailored to individuals, based on their genome scans. Pharmacogenetics has received especially strong interest, with many pharmaceutical developers avidly working to identify genetic variations associated with individual differences in drug response. While clinical applications of emerging genetic knowledge are becoming increasingly available, genetic tests for drug selection are not as yet widely accessible, and many primary care clinicians are unprepared to interpret genetic information. We conducted interviews with 58 primary care clinicians, exploring how they integrate emerging pharmacogenetic concepts into their practices. We found that in their current practices, pharmacogenetic innovations have not led to individually tailored treatment, but instead have encouraged use of essentialized racial/ethnic identity as a proxy for genetic heritage. Current manifestations of Personalized Medicine appear to be reinforcing entrenched notions of inherent biological differences between racial groups, and promoting the belief that racial profiling in health care is supported by cutting-edge scientific authority. Our findings raise concern for how pharmacogenetic innovations will actually affect diverse populations, and how unbiased treatment can be assured.     

History shaped the geographic distribution of genomic admixture on the island of Puerto Rico

Contemporary genetic variation among Latin Americans human groups reflects population migrations shaped by complex historical, social and economic factors. Consequently, admixture patterns may vary by geographic regions ranging from countries to neighborhoods. We examined the geographic variation of admixture across the island of Puerto Rico and the degree to which it could be explained by historic and social events. We analyzed a census-based sample of 642 Puerto Rican individuals that were genotyped for 93 ancestry informative markers (AIMs) to estimate African, European and Native American ancestry. Socioeconomic status (SES) data and geographic location were obtained for each individual. There was significant geographic variation of ancestry across the island. In particular, African ancestry demonstrated a decreasing East to West gradient that was partially explained by historical factors linked to the colonial sugar plantation system. SES also demonstrated a parallel decreasing cline from East to West. However, at a local level, SES and African ancestry were negatively correlated. European ancestry was strongly negatively correlated with African ancestry and therefore showed patterns complementary to African ancestry. By contrast, Native American ancestry showed little variation across the island and across individuals and appears to have played little social role historically. The observed geographic distributions of SES and genetic variation relate to historical social events and mating patterns, and have substantial implications for the design of studies in the recently admixed Puerto Rican population. More generally, our results demonstrate the importance of incorporating social and geographic data with genetics when studying contemporary admixed populations.     

Assessing cell-specific effects of genetic variations using tRNA microarrays

Background

Short-read resequencing of genomes produces abundant information of the genetic variation of individuals. Due to their numerous nature, these variants are rarely exhaustively validated. Furthermore, low levels of undetected variant miscalling will have a systematic and disproportionate impact on the interpretation of individual genome sequence information, especially should these also be carried through into in reference databases of genomic variation.

Results

We find that sequence variation from short-read sequence data is subject to recurrent-yet-intermittent miscalling that occurs in a sequence intrinsic manner and is very sensitive to sequence read length. The miscalls arise from difficulties aligning short reads to redundant genomic regions, where the rate of sequencing error approaches the sequence diversity between redundant regions. We find the resultant miscalled variants to be sensitive to small sequence variations between genomes, and thereby are often intrinsic to an individual, pedigree, strain or human ethnic group. In human exome sequences, we identify 2–300 recurrent false positive variants per individual, almost all of which are present in public databases of human genomic variation. From the exomes of non-reference strains of inbred mice, we identify 3–5000 recurrent false positive variants per mouse – the number of which increasing with greater distance between an individual mouse strain and the reference C57BL6 mouse genome. We show that recurrently miscalled variants may be reproduced for a given genome from repeated simulation rounds of read resampling, realignment and recalling. As such, it is possible to identify more than two-thirds of false positive variation from only ten rounds of simulation.

Conclusion

Identification and removal of recurrent false positive variants from specific individual variant sets will improve overall data quality. Variant miscalls arising are highly sequence intrinsic and are often specific to an individual, pedigree or ethnicity. Further, read length is a strong determinant of whether given false variants will be called for any given genome – which has profound significance for cohort studies that pool datasets collected and sequenced at different points in time.

     

Human and animal hepatocytes in vitro with extrapolation in vivo

Human and animal hepatocytes are now being used as an in vitro technique to aid drug discovery by predicting the in vivo metabolic pathways of drugs or new chemical entities (NCEs), identifying drug-metabolizing enzymes and predicting their in vivo induction. Because of the difficulty of establishing whether the cytotoxic susceptibility of human hepatocytes to xenobiotics/drugs in vitro could be used to predict in vivo human hepatotoxicity, a comparison of the susceptibility of the hepatocytes of human and animal models to six chemical classes of drugs/xenobiotics in vitro have been related to their in vivo hepatotoxicity and the corresponding activity of their metabolizing enzymes. This study showed that the cytotoxic effectiveness of 16 halobenzenes towards rat hepatocytes in vitro using higher doses and short incubation times correlated well with rat hepatotoxic effectiveness in vivo with lower doses/longer times. The hepatic/hepatocyte xenobiotic metabolizing enzyme activities of various animal species and human have been reviewed for use by veterinarians and research scientists. Where possible, recommendations have been made regarding which animal hepatocyte model is most applicable for modeling the susceptibility to xenobiotic induced hepatotoxicity of those humans with slow versus rapid metabolizing enzyme polymorphisms. These recommendations are based on the best human fit for animal drug/xenobiotic metabolizing enzymes in terms of activity, kinetics and substrate/inhibitor specificity. The use of human hepatocytes from slow versus rapid metabolizing individuals for drug metabolism/cytotoxicity studies; and the research use of freshly isolated rat hepatocytes and "Accelerated Cytotoxicity Mechanism Screening" (ACMS) techniques for identifying drug/xenobiotic reactive metabolites are also described. Using these techniques the molecular hepatocytotoxic mechanisms found in vitro for seven classes of xenobiotics/drugs were found to be similar to the rat hepatotoxic mechanisms reported in vivo.     

The legacy of pharmacogenetics and potential applications

Some 40 years of pharmacogenetic research indicates that knowledge of human genetic diversity is essential to a broader understanding of variation in human drug response, and suggests that drug therapy tailored to the genetic characteristics of the individual may be a realistic goal. Aided by new technologies, molecular studies of genetic polymorphisms of many human enzymes, receptors, and other proteins indicate that only a limited number of important protein variants account for the diversity in drug response, raising the prospect that these variants may be cataloged relatively soon for many human populations. The next great challenge of pharmacogenetics is to pin down the cellular location and effect of these variant proteins on the pathways and networks that govern individual variation in responses to drugs and other exogenous chemicals. In this paper, we will discuss some the current challenges to progress in pharmacogenetics and newer strategies that might be used to improve prospects of drug design and personalized therapy.     

Personal receptor repertoires: olfaction as a model

ABSTRACT: BACKGROUND: Information on nucleotide diversity along completely sequenced human genomes has increased tremendously over the last few years. This makes it possible to reassess the diversity status of distinct receptor proteins in different human individuals. To this end, we focused on the complete inventory of human olfactory receptor coding regions as a model for personal receptor repertoires. RESULTS: By performing data-mining from public and private sources we scored genetic variations in 413 intact OR loci, for which one or more individuals had an intact open reading frame. Using 1000 Genomes Project haplotypes, we identified a total of 4069 full-length polypeptide variants encoded by these OR loci, average of ~10 per locus, constituting a lower limit for the effective human OR repertoire. Each individual is found to harbor as many as 600 OR allelic variants, ~50% higher than the locus count. Because OR neuronal expression is allelically excluded, this has direct effect on smell perception diversity of the species. We further identified 244 OR segregating pseudogenes (SPGs), loci showing both intact and pseudogene forms in the population, twenty-six of which are annotatively "resurrected" from a pseudogene status in the reference genome. Using a custom SNP microarray we validated 150 SPGs in a cohort of 468 individuals, with every individual genome averaging 36 disrupted sequence variations, 15 in homozygote form. Finally, we generated a multi-source compendium of 63 OR loci harboring deletion Copy Number Variations (CNVs). Our combined data suggest that 271 of the 413 intact OR loci (66%) are affected by nonfunctional SNPs/indels and/or CNVs. CONCLUSIONS: These results portray a case of unusually high genetic diversity, and suggest that individual humans have a highly personalized inventory of functional olfactory receptors, a conclusion that might apply to other receptor multigene families.