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PubMed2001

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.

Genotype↗
PubMed2006

Comparison of PrASE and Pyrosequencing for SNP Genotyping.

BACKGROUND: There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers. RESULTS: The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated. CONCLUSION: The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.

Alleles↗
PubMed2006

DNA-based diagnosis of malignant osteopetrosis by whole-genome scan using a single-nucleotide polymorphism microarray: standardization of molecular investigations of genetic diseases due to consanguinity.

Malignant osteopetrosis, a severe disease causing early infantile death in humans, is caused by mutations in the TCIRG1, CLCN7, or OSTM1 genes. We have established the molecular basis of malignant osteopetrosis in a Chinese family by means of whole-genome scans based on high-density single-nucleotide polymorphism (SNP) microarrays. Because the parents were consanguineous, the disease-causing locus should be located in an autozygous chromosomal region. Mapping revealed that among the three possible causal loci, only the CLCN7 gene was located in an autozygous region. Mutational analysis of the CLCN7 gene showed that the proband was homozygous for a novel missense mutation, p.I261F. p.I261 is located in helix F of the chloride channel, near a critical site for gating of the channel. This mapping study prepares the ground for future mutation studies by decreasing the burden of completely sequencing all possible loci for this disease. This approach can be used to standardize molecular investigations of genetic diseases due to consanguinity to a whole-genome scan and subsequent sequencing of the mapped disease gene.

Asian People↗
PubMed2003

Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles.

We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes. Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers. The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA. The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions. The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate. BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate. This system permits the simultaneous heating and magnetic separation of 96 samples per assay. The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs.

Aldehyde Dehydrogenase↗
PubMed2006

Survey of microarray technologies suitable to elucidate transcriptional networks as exemplified by studying KRAB zinc finger gene families.

Current microarray systems are suitable to monitor genome-wide expression patterns, to detect single-nucleotide polymorphisms (SNP), to identify target genes of transcription factors and DNA-protein interaction sites thereof as well as to determine genomic sites that are modified by methylation of CpG islands. In this review, advantages and limitations of individual microarray technologies are presented as well as experiences from ongoing studies on KRAB zinc finger gene families are taken to exemplify how different microarray approaches are applicable to elucidate complex transcriptional networks of gene regulation. However, bioinformaticians should be aware that each microarray technology has limitations in its sensitivity and selectivity that has to be taken into account once data mining on comprehensive genome-wide microarray data is conducted. In many cases, microarray results are the initial step to identify target genes of interest and to study the molecular regulation of biological processes thereof followed and validated by complementary proteome, metabolome or toponome analysis. Thus, microarray technologies can be considered a reliable approach for determining gene functions that might be modulated by electromagnetic fields.

Gene Expression Profiling↗
PubMed2003

Set association analysis of SNP case-control and microarray data.

Common heritable diseases ("complex traits") are assumed to be due to multiple underlying susceptibility genes. While genetic mapping methods for Mendelian disorders have been very successful, the search for genes underlying complex traits has been difficult and often disappointing. One of the reasons may be that most current gene-mapping approaches are still based on conventional methodology of testing one or a few SNPs at a time. Here, we demonstrate a simple strategy that allows for the joint analysis of multiple disease-associated SNPs in different genomic regions. Our set-association method combines information over SNPs by forming sums of relevant single-marker statistics. As previously hypothesized, we show here that this approach successfully addresses the "curse of dimensionality" problem--too many variables should be estimated with a comparatively small number of observations. We also report results of simulation studies showing that our method furnishes unbiased and accurate significance levels. Power calculations demonstrate good power even in the presence of large numbers of nondisease associated SNPs. We extended our method to microarray expression data, where expression levels for large numbers of genes should be compared between two tissue types. In applications to such data, our approach turned out to be highly efficient.

Case-Control Studies↗
PubMed2004

Guidelines for incorporating non-perfectly matched oligonucleotides into target-specific hybridization probes for a DNA microarray.

Sequence-specific oligonucleotide probes play a crucial role in hybridization techniques including PCR, DNA microarray and RNA interference. Once the entire genome becomes the search space for target genes/genomic sequences, however, cross-hybridization to non-target sequences becomes a problem. Large gene families with significant similarity among family members, such as the P450s, are particularly problematic. Additionally, accurate single nucleotide polymorphism (SNP) detection depends on probes that can distinguish between nearly identical sequences. Conventional oligonucleotide probes that are perfectly matched to target genes/genomic sequences are often unsuitable in such cases. Carefully designed mismatches can be used to decrease cross-hybridization potential, but implementing all possible mismatch probes is impractical. Our study provides guidelines for designing non-perfectly matched DNA probes to target DNA sequences as desired throughout the genome. These guidelines are based on the analysis of hybridization data between perfectly matched and non-perfectly matched DNA sequences (single-point or double-point mutated) calculated in silico. Large changes in hybridization temperature predicted by these guidelines for non-matched oligonucleotides fit independent experimental data very well. Applying the guidelines to find oligonucleotide microarray probes for P450 genes, we confirmed the ability of our point mutation method to differentiate the individual genes in terms of thermodynamic calculations of hybridization and sequence similarity.

Base Sequence↗
PubMed2004

Multiple sample amplification and genotyping integrated on a single electronic microarray.

We report a novel method that allows simultaneous in situ amplification and then genotyping of single nucleotide polymorphism (SNP) for multiple samples on a single electronic microarray. The locus coding for one of the common inherited thrombosis risk factors, Factor V Leiden (FVL), was chosen as a model system for SNP analysis. This method combines strand displacement amplification (SDA) with electrophoretic movement and concentration of DNA on electronic microarrays to provide a single platform for DNA amplification and analysis. The method includes: electronic anchoring of allele-specific SDA amplifiable primers (APs) and a nonamplifiable primer (NAP) to different electrodes, electronic hybridization of genomic DNA from different samples to those primers, in situ amplification of target DNA, and genotyping of FVL. Compared to previous anchored SDA methods, the addition of a NAP improves detection signals by at least 20-fold. The sensitivity of this method is dependent on the amplification time. Using this method, nine different genomic DNA samples with known FVL genotypes were amplified and correctly genotyped on a single electronic microarray without any contamination between samples. The present method could streamline development of nucleic acid-based assays in applications of molecular diagnostic, point-of-care testing, and forensic detection, which often require the capability to analyze multiple samples efficiently.

Base Sequence↗
PubMed2006

Long-range multilocus haplotype phasing of the MHC.

Haplotypes are a powerful tool for identifying the genetic basis of common complex diseases. Disease-association mapping requires molecular methods for haplotyping biallelic SNP variation and highly complex polymorphisms. We developed a method for phasing HLA-A, HLA-B, and HLA-DRB1 alleles on chromosome 6 in unrelated individuals. This method uses the highly polymorphic HLA-B locus to discriminate the two HLA haplotypes in heterozygous individuals and its ideal location 1.4 Mbp telomeric to HLA-DRB1 and 1.2 Mbp centromeric to HLA-A to capture 2-Mbp-long genomic DNA. Genomic DNA representing a single HLA-B-captured haplotype is genotyped for HLA-A and HLA-DRB1 alleles and linkage to HLA-B is established. Proof of principle was established in a large blinded study of phase-known samples. Availability of an efficient method for MHC haplotype phase determination will facilitate the mapping of causative MHC-resident genes in many human diseases and has the potential to be broadened to other polymorphic gene complexes.

Genes, MHC Class I↗
PubMed2004

Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles.

Single-nucleotide polymorphisms (SNPs) have the potential to be particularly useful as markers for monitoring of chimerism after stem cell transplantation (SCT) because they can be analyzed by accurate and robust methods. We used a two-phased minisequencing strategy for monitoring chimerism after SCT. First, informative SNPs with alleles differing between donor and recipient were identified using a multiplex microarray-based minisequencing system screening 51 SNPs to ensure that multiple informative SNPs were detected in each donor-recipient pair. Secondly, the development of chimerism was followed up after SCT by sensitive, quantitative analysis of individual informative SNPs by applying the minisequencing method in a microtiter plate format. Using this panel of SNPs, we identified multiple informative SNPs in nine unrelated and in 16 related donor-recipient pairs. Samples from nine of the donor-recipient pairs taken at time points ranging from 1 month to 8 years after transplantation were available for analysis. In these samples, we monitored the allelic ratios of two or three informative SNPs in individual minisequencing reactions. The results agreed well with the data obtained by microsatellite analysis. Thus, we conclude that the two-phased minisequencing strategy is a useful approach in the following up of patients after SCT.

Alleles↗
PubMed2006

Cis- and trans-acting gene regulation is associated with osteoarthritis.

Osteoarthritis (OA) is a complex disease of the skeleton and is associated with aging. Both environmental and genetic factors contribute to its pathogenesis. We set out to identify novel genes associated with OA, concentrating on regulatory polymorphisms allowing for differential expression. Our strategy to identify differentially expressed genes included an initial transcriptome analysis of the peripheral blood mononuclear cells of six patients with OA and six age-matched healthy controls. These were screened for allelic expression imbalances and potentially regulatory single-nucleotide polymorphisms (SNPs) in the 5' regions of the genes. To establish disease association, disparate promoter SNP distributions correlating with the differential expression were tested on larger cohorts. Our approach yielded 26 candidate genes differentially expressed between patients and controls. Whereas BLP2 and CIAS1 seem to be trans-regulated, as the absence of allelic expression imbalances suggests, the presence of allelic imbalances confirms cis-regulatory mechanisms for RHOB and TXNDC3. Interestingly, on/off-switching suggests additional trans-regulation for TXNDC3. Moreover, we demonstrate for RHOB and TXNDC3 statistically significant associations between 5' SNPs and the disease that hint at regulatory functions. Investigating the respective genes functionally will not only shed light on the disease association but will also add to the understanding of the pathogenic processes involved in OA and may point out novel therapeutic approaches.

Adult↗
PubMed2003

Systems toxicology and the Chemical Effects in Biological Systems (CEBS) knowledge base.

The National Center for Toxicogenomics is developing the first public toxicogenomics knowledge base that combines molecular expression data sets from transcriptomics, proteomics, metabonomics, and conventional toxicology with metabolic, toxicologcal pathway, and gene regulatory network information relevant to environmental toxicology and human disease. It is called the Chemical Effects in Biological Systems (CEBS) knowledge base and is designed to meet the information needs of "systems toxicology," involving the study of perturbation by chemicals and stressors, monitoring changes in molecular expression and conventional toxicological parameters, and iteratively integrating biological response data to describe the functioning organism. Based upon functional genomics approaches used successfully in analyzing yeast gene expression data sets, relational and descriptive compendia will be assembled for toxicologically important genes, groups of genes, single nucleotide polymorphisms (SNPs), and mutant and knockout phenotypes. CEBS data sets will be fully documented in the experimental protocol and therefore searchable by compound, structure, toxicity end point, pathology and point, gene, gene group, SNP, pathway, and network as a function of dose, time, and the phenotype of the target tissue. A knowledge base is being developed by assimilating toxicological, biological, and chemical information from multiple public domain databases and by progressively refining that information about gene, protein, and metabolite expression for classes of chemicals and their biological effects in various species. By analogy to the GenBank database for genome sequences, researchers will globally query (or BLAST) CEBS using a transcriptome of a tissue of interest (or a list of outliers) to have the knowledge base return information on genes, groups of genes, metabolic and toxicological pathways, and contextually associated phenotypic information for compounds that display similar response profiles. With high-quality data content, CEBS will ultimately become a resource to support hypothesis-driven and discovery research that contributes effectively to drug safety and the improvement of risk assessments for chemicals in the environment. The CEBS development effort will span a decade or more.

Computational Biology↗
PubMed2003

Rapid detection of the known SNPs of CYP2C9 using oligonucleotide microarray.

AIM: Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. Individuals with mutant enzymes may risk serious side effects under routine therapy with certain drugs metabolized by CYP2C9. In order to facilitate the detection of the known SNPs of CYP2C9, an allele-specific oligonucleotide (ASO) based microarray was made. METHODS: An oligonucleotide microarray was made to facilitate the SNP (single nucleotide polymorphism) screening and was applied for the detection of CYP2C9 polymorphism in 62 high blood pressure (HBP) patients who received Irbesartan for treatment. Part of the genotyping results was confirmed by direct sequencing. And the relation between CYP2C9 polymorphism and therapeutic outcome of Irbesartan was statistically analyzed. RESULTS: Heterozygous alleles of CYP2C9*1/*3 were found in 7 out of 62 subjects. No mutant alleles of CYP2C9*2, *4 and *5 and no homozygous mutant alleles were detected. The 7 heterozygous CYP2C9*1/*3 and 13 random wild type DNA samples were subjected to direct sequencing with purified PCR products and same genotyping results were obtained with the 20 DNA samples. There was no significant difference in the odds of effectiveness of Irbesartan between the wild type (normal) group and CYP2C9*1/*3 (mutant) group (P>0.05). CONCLUSION: The oligonucleotide microarray made in this study is a reliable assay for detecting the CYP2C9 known alleles and the heterozygous CYP2C9*1/*3 has no significant effects on the therapeutic outcome of Irbesartan.

Alleles↗
PubMed2002

"A system biology" approach to bioinformatics and functional genomics in complex human diseases: arthritis.

Human and other annotated genome sequences have facilitated generation of vast amounts of correlative data, from human/animal genetics, normal and disease-affected tissues from complex diseases such as arthritis using gene/protein chips and SNP analysis. These data sets include genes/proteins whose functions are partially known at the cellular level or may be completely unknown (e.g. ESTs). Thus, genomic research has transformed molecular biology from "data poor" to "data rich" science, allowing further division into subpopulations of subcellular fractions, which are often given an "-omic" suffix. These disciplines have to converge at a systemic level to examine the structure and dynamics of cellular and organismal function. The challenge of characterizing ESTs linked to complex diseases is like interpreting sharp images on a blurred background and therefore requires a multidimensional screen for functional genomics ("functionomics") in tissues, mice and zebra fish model, which intertwines various approaches and readouts to study development and homeostasis of a system. In summary, the post-genomic era of functionomics will facilitate to narrow the bridge between correlative data and causative data by quaint hypothesis-driven research using a system approach integrating "intercoms" of interacting and interdependent disciplines forming a unified whole as described in this review for Arthritis.

Animals↗
PubMed2006

Coverage and characteristics of the Affymetrix GeneChip Human Mapping 100K SNP set.

Improvements in technology have made it possible to conduct genome-wide association mapping at costs within reach of academic investigators, and experiments are currently being conducted with a variety of high-throughput platforms. To provide an appropriate context for interpreting results of such studies, we summarize here results of an investigation of one of the first of these technologies to be publicly available, the Affymetrix GeneChip Human Mapping 100K set of single nucleotide polymorphisms (SNPs). In a systematic analysis of the pattern and distribution of SNPs in the Mapping 100K set, we find that SNPs in this set are undersampled from coding regions (both nonsynonymous and synonymous) and oversampled from regions outside genes, relative to SNPs in the overall HapMap database. In addition, we utilize a novel multilocus linkage disequilibrium (LD) coefficient based on information content (analogous to the information content scores commonly used for linkage mapping) that is equivalent to the familiar measure r2 in the special case of two loci. Using this approach, we are able to summarize for any subset of markers, such as the Affymetrix Mapping 100K set, the information available for association mapping in that subset, relative to the information available in the full set of markers included in the HapMap, and highlight circumstances in which this multilocus measure of LD provides substantial additional insight about the haplotype structure in a region over pairwise measures of LD.

Chromosome Mapping↗
PubMed2000

Microchip electrophoresis: a method for high-speed SNP detection.

As a trial practical application, we have applied optimized microfabricated electrophoresis devices, combined with enzymatic mutation detection methods, to the determination of single nucleotide polymorphism (SNP) sites in the p53 suppressor gene. Using clinical samples, we have achieved robust assays with quality factors as good as conventional electrophoresis in approximately 100 s. This is 10 and 50 times faster than capillary and slab gel electro-phoresis, respectively. The method was highly accurate with an average error of mutation site measurement of only +/-5 bp. No clean-up of the digestion mixtures was needed prior to injection. This greatly simplifies sample handling relative to capillary instruments, which is important for high-throughput screening applications. Following identification, absolute mutation determination of the screened samples was achieved in a second microdevice optimized for four-color DNA sequencing. Total run time was 25 min in this second device and sequencing data were in full agreement with ABI Prism 377 sequencing runs which required 3.5 h. The tandem application of microdevices for location then full characterization of SNPs appears to confirm many of the improvements claimed for future application of microdevices in practical scaled screening for mutational analysis.

DNA Mutational Analysis↗
PubMed2005

Preparation and analysis of cSNP chip on hepatocellular carcinoma-related genes.

BACKGROUND: The understanding of cSNPs of cancer-related genes harboring in high frequency loss regions of tumor chromosomes can advance the disclosure of genetic and variant mechanisms of tumorigenesis, and the investigation of cancer susceptibility. In preparing a gene chip for detecting polymorphisms on coding region of genes in hepatocellular carcinoma tissues, some cSNPs are of interest for their potential links with phenotype. METHODS: The genes harboring in loss regions with high frequency of hepatocellular carcinoma (HCC) were selected, the related information of cSNP sequences was obtained from the SNP database (dbSNP) of the National Center for Biotechnology Information (NCBI). Then appropriate primers and oligonucleotide probes were designed according to the SNP sites, and a gene chip for the detection of SNPs was constructed. The chip included 48 cSNPs of 25 hepatocellular carcinoma-related genes. The PCR products labeled by Dig-dUTP were hybridized with the cSNP chip. RESULTS: The sensitivity, influence by probe concentration, and reiteration of the chip were detected, with a high sensitivity of 6X10(-3) ng/mul. The signal of hybridization was reduced with a lower concentration of probe. Seven polymorphisms of caspase 9 (rs2308941)C-->T and DOK2(rs2242241) T-->G, 6 of polymorphisms of EGFL3 (rs947345)A -->G, caspase 9 ( rs2308938) C-->G and PHGDH(rs1801955)T-->A, 5 of polymorphisms of E2F2(rs3218170) G-->A,4 of polymorphisms of MUTYH(rs1140507)T-->C and BNIP3L(rs1055806)G-->T, and 1 of polymorphism of TNFRSF1B (rs1061622)T-->G were detected by the chip in the tissues of 10 HCC. Samples of caspase 9 (rs2308941G) and (rs2308941A) were verified by PCR-SSCP and sequencing. CONCLUSION: The cSNP chip of hepatocellular carcinoma-related genes can accelerate the discovery of polymorphic markers on hepatocellular carcinoma.

Adult↗
PubMed2005

Genomewide single nucleotide polymorphism microarray mapping in basal cell carcinomas unveils uniparental disomy as a key somatic event.

Basal cell carcinoma is the most common human cancer with increasing incidence reported worldwide. Despite the aberrant signaling role of the Hedgehog pathway, little is known about the genetic mechanisms underlying basal cell carcinomas. Towards a better understanding of global genetic events, we have employed the Affymetrix Mapping 10K single nucleotide polymorphism (SNP) microarray technique for "fingerprinting" genomewide allelic imbalance in 14 basal cell carcinoma-blood pair samples. This rapid high-resolution SNP genotyping technique has revealed a somatic recombination event-uniparental disomy, leading to a loss of heterozygosity (LOH), as a key alternative genetic mechanism to allelic imbalances in basal cell carcinomas. A highly conserved LOH region at 9q21-q31 was found in 13 of 14 (93%) basal cell carcinomas. Further statistical and fluorescence in situ hybridization analyses confirmed that the 9q LOH was a result of uniparental disomy in 5 of 13 (38%) basal cell carcinomas. De novo mutations in the Patched 1 gene (PTCH) were found in 9 of 13 (69%) basal cell carcinomas with 9q LOH. A second important locus, containing LOH at 6q23-q27 was found in 5 of 14 (36%) basal cell carcinomas, suggesting that the presence of an additional putative tumor suppressor gene may be contributing to basal cell carcinoma development. This study shows that the rate of 9q LOH in basal cell carcinomas has been previously underestimated. Furthermore, we provide the first evidence that uniparental disomy due to somatic recombination constitutes one of the mechanisms of LOH in basal cell carcinoma tumorigenesis.

Aged↗