Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies

BACKGROUND: Pharmacogenetics is a scientific discipline that examines the genetic basis for individual variations in response to therapeutics. Pharmacogenetics promises to develop individualized medicines tailored to patients' genotypes. However, identifying and genotyping a vast number of genetic polymorphisms in large populations also pose a great challenge. APPROACH: This article reviews the recent technology development in mutation detection and genotyping with a focus on genotyping of single nucleotide polymorphisms (SNPs). CONTENT: Novel mutations/polymorphisms are commonly identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. With a large amount of public sequence information available, in silico SNP mapping has also emerged as a cost-efficient way for new polymorphism identification. Gel electrophoresis-based genotyping methods for known polymorphisms include PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing. Fluorescent dye-based genotyping technologies are emerging as high-throughput genotyping platforms, including oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan, and molecular beacon genotyping. Rolling circle amplification and Invader assays are able to genotype directly from genomic DNA without PCR amplification. DNA chip-based microarray and mass spectrometry genotyping technologies are the latest development in the genotyping arena. SUMMARY: Large-scale genotyping is crucial to the identification of the genetic make-ups that underlie the onset of diseases and individual variations in drug responses. Enabling technologies to identify genetic polymorphisms rapidly, accurately, and cost effectively will dramatically impact future drug and development processes.     

A new single nucleotide polymorphism genotyping method based on gap ligase chain reaction and a microsphere detection assay.

BACKGROUND: The establishment of new methods to detect human genetic variations, such as single nucleotide polymorphisms, is of importance in hereditary disease diagnosis and pharmacogenomics. Several single nucleotide polymorphism genotyping technologies have been presented in recent years. However, techniques which would allow accurate, fast and cheap allelic determination and multiple single nucleotide polymorphism detection in parallel are still in great need. METHODS: Here, we present a new genotyping technique based on gap ligase chain reaction and a fluorescent polystyrene microsphere measurement platform. We chose the human polymorphism rs3730386 as our candidate to establish this method. Probes for gap ligase chain reaction were designed to recognize the target alleles sensitively and specifically and to produce templates for amplifying the correspondent target fragments used in the following hybridization. The genotypes were finally determined by a hybridization process based on the Luminex fluorescent polystyrene microspheres measurement platform. RESULTS: This method was successfully applied for the detection of selected single nucleotide polymorphisms with high sensitivity and specificity. The genotypes were validated by DNA sequencing. CONCLUSIONS: The new genotyping method benefited from the high sensitivity and specificity of gap ligase chain reaction and the detection platform of Luminex. The method allows multiplex analysis of single nucleotide polymorphism, because 100 types of microspheres are available from Luminex.     

Applications of whole-genome high-density SNP genotyping

The technology to simultaneously genotype hundreds of thousands of single nucleotide polymorphisms in a single assay has only recently been developed. These advances have the potential to revolutionize our ability to identify disease-associated proteins and their corresponding pathways as drugable targets. Several strategies that can take advantage of extremely high-density, genome-wide single nucleotide polymorphism genotyping to hone in on pathogenic genetic variants will be discussed. In familial linkage studies, high-density single nucleotide polymorphism genotyping has already been proven to speed up mutation identification of Mendelian traits several fold. Many studies now report examining loss of heterozygosity and genomic amplifications on a whole-genome level. Genotyping hundreds of thousands of single nucleotide polymorphisms in a single set of assays now also allows for whole-genome association studies in complex, multigenic diseases. The technology of high-density single nucleotide polymorphism genotyping has emerged rapidly, leaving data analysis and bioinformatic challenges only partially met. In this review, the immediate applications and implications of the rapidly changing high-density, whole-genome single nucleotide polymorphism genotyping field on translational research will be described.     

TotalPlex gene amplification using bulging primers for pharmacogenetic analysis of acute lymphoblastic leukemia

Genetic polymorphism among patients with acute lymphoblastic leukemia (ALL) is an important factor in the effectiveness and toxicity of anti-leukemic drugs. Genotyping of various polymorphisms that impact the outcome of anti-leukemic drug therapy (pharmacogenetics) presents an attractive approach for developing individualized therapy. We developed an easy and accurate method of analyzing multiple genes using a small amount of DNA, which we termed TotalPlex amplification. We used 16 pairs of specific bulging specific primers (SBS primers) for simultaneous amplification of 16 loci in a single PCR tube. Sixteen single nucleotide polymorphisms (SNPs) (CYP3A4*1B A>G, CYP3A5*3 G>A, GSTP1 313 A>G, GSTM1 deletion, GSTT1 deletion, MDR1 exon 21 G>T/A, MDR1 exon 26 C>T, MTHFR 677 C>T, MTHFR 1298 A>C, NR3C1 1088 A>G, RFC 80 G>A, TPMT 238 G>C, TPMT 460 G>A, TPMT 719 A>G, VDR intron 8 G>A, VDR FokI T>C) that have been implicated in the pharmacogenetics of ALL therapy were analyzed by TotalPlex amplification and SNP genotyping. We successfully amplified specific gene fragments using 16 pairs of primers in one PCR reaction tube with minimal spurious amplification products using TotalPlex amplification coupled to a multiplexed bead array detection system. The genotypes of 16 loci from 34 different genomic DNA (gDNA) samples derived using the TotalPlex system were consistent with the results of several standard genotyping methods, including automatic sequencing, PCR restriction fragment length polymorphism (RFLP) analysis, PCR, and allele-specific PCR (AS-PCR). Thus, the TotalPlex system represents a useful method of amplification that can improve the time, cost, and sample size required for high-throughput pharmacogenetic analysis of SNPs.     

The use of real-time PCR methods in DNA sequence variation analysis

BACKGROUND: Real-time (RT) PCR methods for discovering and genotyping single nucleotide polymorphisms (SNPs) are becoming increasingly important in various fields of biological sciences. SNP genotyping is widely used to perform genetic association studies aimed at characterising the genetic factors underlying inherited traits. The detection and quantification of somatic mutations is an important tool for investigating the genetic causes of tumorigenesis. In infectious disease diagnostics there is an increasing emphasis placed on genotyping variation within the genomes of pathogenic organisms in order to distinguish between strains. METHODS: There are several platforms and methods available to the researcher wishing to undertake SNP analysis using real-time PCR methods. These use fluorescent technologies for discriminating between the alternate alleles of a polymorphism. There are several real-time PCR platforms currently on the market. Two of the key technical challenges are allele discrimination and allele quantification. CONCLUSIONS: Applications of this technology include SNP genotyping, the sensitive detection of somatic mutations and infectious disease subtyping.     

Application of oligonucleotide arrays to high-content genetic analysis

The scope of single nucleotide polymorphism genotyping for genetic association studies has expanded recently from the use of relatively small numbers of candidate genes and markers, to include hypothesis-free, whole-genome approaches using hundreds of thousands of polymorphisms. The ability to perform such large-scale association studies has been dependant on the development of highly parallel and cost-effective genotyping platforms, of which those based on oligonucleotide arrays have proved to be the most scalable and widely adopted. It is to be expected that the new array-based genotyping methods will not only greatly expand the scope of genetic studies, but, as further content is added to arrays, will also form part of an integrated set of DNA, RNA and proteomic analyses enabling the detailed, multilayered study of complex disease-linked phenotypes.     

Competitive amplification and unspecific amplification in polymerase chain reaction with confronting two-pair primers

Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is an inexpensive, time-saving genotyping method that is applicable for most single nucleotide polymorphisms. To date, we have applied PCR-CTPP successfully for the genotyping of more than 30 polymorphisms. This paper demonstrates the differences in DNA amplification among different annealing temperatures of PCR-CTPP with given melting temperatures for four primers. The NQO1 C609T (Pro187Ser) polymorphism was used as an example. Two sets of four primers were applied for PCR-CTPP; the first set with different melting temperatures (Tms), and the second with similar Tms. The comparisons with one-pair primer PCR (allele-specific PCR) revealed that PCR-CTPP amplified DNA more specifically than allele-specific PCR. The primers with different Tms caused competitive DNA amplification for heterozygous genotype. Four primers with similar Tms amplified both alleles unspecifically at a lower annealing temperature, while the same DNA samples were correctly genotyped under an optimal annealing temperature. These findings are unique for PCR-CTPP, and important characteristics when the primers and annealing temperatures in PCR-CTPP are designed. The knowledge of these characteristics will extend the applicability of PCR-CTPP for polymorphism genotyping.     

Performance of whole-genome amplified DNA isolated from serum and plasma on high-density single nucleotide polymorphism arrays

Defining genetic variation associated with complex human diseases requires standards based on high-quality DNA from well-characterized patients. With the development of robust technologies for whole-genome amplification, sample repositories such as serum banks now represent a potentially valuable source of DNA for both genomic studies and clinical diagnostics. We assessed the performance of whole-genome amplified DNA (wgaDNA) derived from stored serum/plasma on high-density single nucleotide polymorphism arrays. Neither storage time nor usage history affected either DNA extraction or whole-genome amplification yields; however, samples that were thawed and refrozen showed significantly lower call rates (73.9 +/- 7.8%) than samples that were never thawed (92.0 +/- 3.3%) (P < 0.001). Genotype call rates did not differ significantly (P = 0.13) between wgaDNA from never-thawed serum/plasma (92.9 +/- 2.6%) and genomic DNA (97.5 +/- 0.3%) isolated from whole blood. Approximately 400,000 genotypes were consistent between wgaDNA and genomic DNA, but the overall discordance rate of 4.4 +/- 3.8% reflected an average of 11,110 +/- 9502 genotyping errors per sample. No distinct patterns of chromosomal clustering were observed for single nucleotide polymorphisms showing discordant genotypes or homozygote conversion. Because the effects of genotyping errors on whole-genome studies are not well defined, we recommend caution when applying wgaDNA from serum/plasma to high-density single nucleotide polymorphism arrays in addition to the use of stringent quality control requirements for the resulting genotype data.     

Rolling-circle amplification in DNA diagnostics: the power of simplicity

Due to its robustness and simplicity, the rolling replication of circular DNA probes holds a distinct position in DNA diagnostics among other isothermal methods of target, probe or signal amplification. Major rolling-circle amplification approaches to DNA detection via posthybridization probe/signal turn-by-turn enhancement are briefly overviewed here with an emphasis on the new concepts and latest progress in the field, including the single-molecule and single-mutation detection assays as exemplary applications. Underlying mechanisms, current controversies and principal advantages of rolling-circle amplification are also considered. Possible future directions for the further advancement of this diagnostic methodology are outlined.     

Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms

The typing of a single nucleotide polymorphism with DNA probes is sometimes problematic because of the limited discriminating power of long DNA probes. As an alternative to existing assays, we have developed a real-time PCR assay for the genotyping of single nucleotide polymorphisms using short peptide nucleic acid (PNA) molecular beacons. A single nucleotide polymorphism in exon 6 of the XPD gene was chosen as the model system. The genotyping experiments were performed in the ABI 7700 using beacons labeled with either fluorescein or JOE, and in the Lightcycler using a fluorescein labeled beacon. QSY-7 was used as the quencher in all the beacons. The result of the genotyping was the same on both instruments and was in agreement with a previously performed RFLP genotyping of 79 samples. The length of PNA molecular beacons is significantly shorter than that of TaqMan or Lightcycler probes, making probe design and genotype discrimination easier.     

Technologies for detecting genetic polymorphisms in pharmacogenomics.

BACKGROUND: Pharmacogenomics is an emerging scientific discipline examining the genetic basis for individual variations in response to therapeutics. METHODS AND RESULTS: Genetic polymorphisms are a major cause of individual differences in drug response. Metabolic phenotyping can be accomplished by administering a probe drug or substrate and measuring the metabolites and clinical outcomes. However, this approach tends to be labor intensive and requires repeated sample collection from the individual being tested. Alternatively, genotyping allows determination of individual DNA sequence differences for a particular trait. Commonly used genotyping methods include gel electrophoresis-based techniques, such as polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism analysis, multiplex PCR, and allele-specific amplification. Fluorescent dye-based high-throughput genotyping procedures are increasing in popularity, including oligonucleotide ligation assay, direct heterozygote sequencing, and TaqMan (Perkin Elmer, Foster City, CA) allelic discrimination. High-density chip array and mass spectrometry technologies are the newest advances in the genotyping field, but their wide application is yet to be developed. Novel mutations/polymorphisms also can be identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. CONCLUSIONS: Rapid and accurate detection of genetic polymorphisms has great potential for application to drug development, animal toxicity studies, improvement of human clinical trials, and postmarket monitoring surveillance for drug efficacy and toxicity.     

Genotyping single nucleotide polymorphisms by MALDI mass spectrometry in clinical applications

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become one of the most powerful and widely applied technologies for SNP scoring and determination of allele frequencies in the post-genome sequencing era. Although different strategies for allele discrimination combined with MALDI were devised, in practice only primer extension methods are nowadays routinely used. This combination enables the rapid, quantitative, and direct detection of several genetic markers simultaneously in a broad variety of biological samples. In the field of molecular diagnostics, MALDI has been applied to the discovery of genetic markers, that are associated with a phenotype like a disease susceptibility or drug response, as well as an alternative means for diagnostic testing of a range of diseases for which the responsible mutations are already known. It is one of the first techniques with which whole genome scans based on single nucleotide polymorphisms were carried out. It is equally well suited for pathogen identification and the detection of emerging mutant strains as well as for the characterization of the genetic identity and quantitative trait loci mapping in farm animals. MALDI can also be used as a detection platform for a range of novel applications that are more demanding than standard SNP genotyping such as mutation/polymorphism discovery, molecular haplotyping, analysis of DNA methylation, and expression profiling. This review gives an introduction to the application of mass spectrometry for DNA analysis, and provides an overview of most studies using SNPs as genetic markers and MALDI mass spectrometric detection that are related to clinical applications and molecular diagnostics. Further, it aims to show specialized applications that might lead to diagnostic applications in the future. It does not speculate on whether this methodology will ever reach the diagnostic market.     

Application of a BRAF pyrosequencing assay for mutation detection and copy number analysis in malignant melanoma

Mutations in the BRAF gene are found in the majority of cutaneous malignant melanomas and subsets of other tumors. These mutations lead to constitutive activation of BRAF with increased downstream ERK (extracellular signal-regulated kinase) signaling; therefore, the development of RAF kinase inhibitors for targeted therapy is being actively pursued. A methodology that allows sensitive, cost-effective, high-throughput analysis of BRAF mutations will be needed to triage patients for specific molecular-based therapies. Pyrosequencing is a high-throughput, sequencing-by-synthesis method that is particularly useful for analysis of single nucleotide polymorphisms or hotspot mutations. Mutational analysis of BRAF is highly amenable to pyrosequencing because the majority of mutations in this gene localize to codons 600 and 601 and consist of single or dinucleotide substitutions. In this study, DNAs from a panel of melanocyte cell lines, melanoma cell lines, and melanoma tumors were used to validate a pyrosequencing assay to detect BRAF mutations. The assay demonstrates high accuracy and precision for detecting common and variant exon 15 BRAF mutations. Further, comparison of pyrosequencing data with 100K single nucleotide polymorphism microarray data allows characterization of BRAF amplification events that may accompany BRAF mutation. Pyro-sequencing serves as an excellent platform for BRAF genotyping of tumors from patients entering clinical trial.     

Pyrosequencing: history, biochemistry and future

BACKGROUND: Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. METHODS: The technique is built on a 4-enzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand. Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. Mutation detection and single-nucleotide polymorphism genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. In order to expand the field for pyrosequencing, the read length needs to be improved. CONCLUSIONS: Th pyrosequencing system is based on an enzymatic system. There are different current and future applications of this technique.     

Genotyping of multiple single nucleotide polymorphisms with hyperbranched rolling circle amplification and microarray

BackgroundThe emerging role of single nucleotide polymorphisms (SNPs) in clinical diagnostics and studies has created a need for simple and high-throughput genotyping methods. Previously, we developed a 3-dimensional polyacrylamide gel-based microarray (3-D microarray) of PCR-product. This method can detect single SNP locus from multiple DNA samples on one chip.MethodsHyperbranched rolling circle amplification (HRCA) was used to recognize different SNP loci and amplify the fragments from genomic samples. Different HRCA products were used to fabricate the 3-D microarray, and dual-color fluorescent probes were used to detect signals.ResultsThis assay was applied to genotype 2 SNP loci from a set of 6 genomic DNA samples on one chip. Universal acryl-modified primer and one pair of dual-color fluorescent probes were used for all sample detection to reduce the cost. We demonstrate that this assay can detect 10 ng genomic DNA.ConclusionsCombination of HRCA and 3-D microarray allows parallel discrimination of different alleles from different samples on a single chip. It is a feasible method for high-throughput mutation analysis and disease diagnosis.     

Microelectronic array system for molecular diagnostic genotyping: Nanogen NanoChip 400 and molecular biology workstation

Hundreds of gene mutations responsible for Mendelian disorders are currently tested in the clinical laboratory for pre- and postnatal diagnosis, carrier screening and presymptomatic testing. Since human genetic research is currently focused on determining the etiology of complex diseases, including heart disease, diabetes and neuropsychiatric traits, laboratorians will genotype increasing numbers of clinically relevant loci in the future. This will require accurate, high-throughput and cost-effective genotyping platforms, such as the DNA microarray. The Nanogen NanoChip platforms employ hybridization-based technology, using fluorescent detection and electronic control of the target or probe, to obtain clear genotype signal relative to background, and increased flexibility relative to similar chip-based single nucleotide polymorphism genotyping platforms. The scope of this review is intended to describe the operating principle, chips and instrumentation, analyte-specific reagents, published assay protocols, assay development, and clinical use of the NanoChip platforms. It is beyond the scope of this review to describe the use of NanoChip platforms in basic research, and to compare it against all available clinical single nucleotide polymorphism genotyping applications and platforms.     

A simultaneous LightCycler detection assay for five genetic polymorphisms influencing drug sensitivity

OBJECTIVES: The routine detection of polymorphisms affecting drug sensitivity in patients before treatment is important in the identification of drug responders or nonresponders, and patients at increased risk of drug toxicity. Here, we present an assay for the simultaneous and rapid genotyping of five polymorphisms influencing drug sensitivity. DESIGN AND METHODS: We used a hybridization probe assay on the LightCycler to detect five single nucleotide polymorphisms (SNPs): INPP1 (973C>A), ADRB2 (R16G and Q27E), HTR2A (102T>C), and mtDNA (1555A>G). Two fluorescent labeled hybridization probes were designed for the simultaneous detection of the five SNPs and detection of the variant alleles was performed by melting curve analysis. RESULTS: All five SNPs were detected with a single thermocycle protocol within 40 min. The genotypes determined in this assay were identical to those obtained with conventional PCR and restriction fragment length polymorphism analysis. CONCLUSIONS: To our knowledge, we report here for the first time a method for simultaneous detection of five SNPs, on a single thermocycle protocol by the LightCycler. This method is rapid, highly sensitive, and high-throughput, and is thus suitable for routine clinical use and large-scale epidemiologic studies.     

江西汉族人群NRG3基因rs17101655 T→G单核苷酸多态性与ITP的关联性研究

目的：运用TaqMan探针法对NRG3基因rs17101655 T→G单核苷酸多态性进行基因分型,探讨rs17101655 T→G单核苷酸多态性与原发免疫性血小板减少症（ITP）的关联性,为该病的发病机制提供重要线索。方法针对NRG3基因rs17101655位点T→G多态性,设计1对PCR引物和TaqMan探针,通过实时荧光PCR扩增进行基因分型;采用病例-对照研究,对中国汉族江西地区200例ITP和200例健康对照人群进行基因分型,比较两组人群基因型分布及频率差异。结果Rs17101655位点T→G多态性的基因型和等位基因频率分布在ITP病例组和对照组中分布差异无统计学意义（P〉0.05）。结论 TaqMan探针法可快速、高效地对单核苷酸多态性进行基因分型,本研究结果显示NRG3基因rs17101655位点不作为中国汉族人群ITP的易感位点。     

Simultaneous genotyping of alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 by single‐strand conformation polymorphism analysis

Alcohol dehydrogenase (ADH; EC 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3) have important roles in the elimination of ingested ethanol. These enzymes have polymorphisms resulting from single‐point mutations that cause kinetic differences in their respective enzyme activities. Simultaneous observation of these enzymes would be useful in investigating the association between these enzyme polymorphisms and alcohol‐related problems. In this study amplified genomic DNA was amplified from nail clippings with two sets of primers for ADH2 and ALDH2 genes, respectively, in a micro test tube and the accuracy of the amplification was verified by direct sequencing. The PCR products were separated into four distinct bands by single‐strand conformation polymorphism analysis. This genotyping method is fast, accurate, reliable and inexpensive, and requires the same amount of template DNA as non‐simultaneous methods. In other words, the required amount of template DNA for this method is only half that required for the separate genotyping of ADH2 and ALDH2.     

Accurate and rapid "multiplex heteroduplexing" method for genotyping key enzymes involved in folate/homocysteine metabolism

BACKGROUND: Hyperhomocysteinemia, which is often associated with low folate status, is an independent risk factor for cardiovascular diseases and several other pathologies. The four most common functional polymorphisms in genes involved in folate/homocysteine metabolism are methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MS) A2756G, and cystathionine beta-synthase (CBS) 844ins68. The pathogenic impact of these variants is under active investigation in many laboratories. However, conventional genotyping methods, mostly using PCR followed by restriction enzyme digestion, often are compromised by partial fragment digestion. There is, therefore, a need to develop more reliable approaches to genotyping the above polymorphisms that may be applied in large-scale studies. METHODS: Sequence-specific heteroduplex generators for each of the MTHFR and MS single nucleotide polymorphisms were generated by site-directed mutagenesis. These were subcloned into a single construct, pHcyHG-1, which could be multiplexed with a simple PCR amplification across the CBS 844ins68 polymorphic site to generate composite genotype-specific banding patterns from individual genomic DNA samples that could be electrophoretically resolved. RESULTS: The "multiplex heteroduplexing" method yielded unambiguous MTHFR, MS, and CBS genotypes in a single-tube reaction that could be analyzed in a single gel run. CONCLUSIONS: This method permits unambiguous genotyping of the four most common functional variants of enzymes involved in folate/homocysteine metabolism. It is rapid, reproducible, and inexpensive, and requires no special preparative or analytic facilities; consequently, it will facilitate large-scale studies of the genetic basis of hyperhomocysteinemia and the many pathologies that have been associated with this phenotype.