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1.  Flavopiridol Pharmacogenetics: Clinical and Functional Evidence for the Role of SLCO1B1/OATP1B1 in Flavopiridol Disposition 
PLoS ONE  2010;5(11):e13792.
Background
Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy.
Methodology/Principal Findings
Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent.
Conclusions/Significance
Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy.
doi:10.1371/journal.pone.0013792
PMCID: PMC2967470  PMID: 21072184
2.  Part 4: Pharmacogenetic Variability in Anticancer Pharmacodynamic Drug Effects 
The Oncologist  2011;16(7):1006-1020.
This fourth part of a four-part series on pharmacogenetics focuses on pharmacodynamic variability and encompasses genetic variation in drug target genes such as those encoding thymidylate synthase, methylene tetrahydrofolate reductase, and ribonucleotide reductase. Potential implications and opportunities for patient and drug selection for genotype-driven anticancer therapy are outlined.
Learning Objectives
After completing this course, the reader will be able to: Identify genetic polymorphisms within pharmacodynamic candidate genes that are potential predictive markers for treatment outcome with anticancer drugs.Describe treatment selection considerations in patients with cancer who have genetic polymorphisms that could influence pharmacodynamic aspects of anticancer therapy.
This article is available for continuing medical education credit at CME.TheOncologist.com
Response to treatment with anticancer drugs is subject to wide interindividual variability. This variability is expressed not only as differences in severity and type of toxicity, but also as differences in effectiveness. Variability in the constitution of genes involved in the pharmacokinetic and pharmacodynamic pathways of anticancer drugs has been shown to possibly translate into differences in treatment outcome. The overall knowledge in the field of pharmacogenetics has tremendously increased over the last couple of years, and has thereby provided opportunities for patient-tailored anticancer therapy. In previous parts of this series, we described pharmacogenetic variability in anticancer phase I and phase II drug metabolism and drug transport. This fourth part of a four-part series of reviews is focused on pharmacodynamic variability and encompasses genetic variation in drug target genes such as those encoding thymidylate synthase, methylene tetrahydrofolate reductase, and ribonucleotide reductase. Furthermore, genetic variability in other pharmacodynamic candidate genes involved in response to anticancer drugs is discussed, including genes involved in DNA repair such as those encoding excision repair crosscomplementing group 1 and group 2, x-ray crosscomplementing group 1 and group 3, and breast cancer genes 1 and 2. Finally, somatic mutations in KRAS and the gene encoding epidermal growth factor receptor (EGFR) and implications for EGFR-targeted drugs are discussed. Potential implications and opportunities for patient and drug selection for genotype-driven anticancer therapy are outlined.
doi:10.1634/theoncologist.2010-0261
PMCID: PMC3228142  PMID: 21659612
Pharmacogenetics; Personalized medicine; Pharmacodynamics; Oncology; Anticancer drugs
3.  Three multiplex snapshot assays for SNP genotyping in candidate innate immune genes 
BMC Research Notes  2013;6:54.
Background
Innate immune system is the first line of research when studying immune response to diverse infections and autoimmune/inflammatory diseases. This immune response has been reported to be genetically diverse, due to polymorphisms coded by different genes. For this reason, our purpose was to develop a multiplex assay that allows the genotyping of candidate single nucleotide polymorphisms (SNPs) in innate immune genes.
Findings
We developed three multiplex PCR panels coupled with the minisequencing (SNaPshot) technique (multiplex PCR, multiplex primer extension, and capillary electrophoresis). The panels were tested in a sample set composed of 100 anonymous DNAs from healthy blood donors living in São Miguel Island (Azores, Portugal). Sixteen relevant SNPs among nine genes of the innate immune system – IL1α, IL1β, IL6, IL10, IL12RB1, TLR2, TLR4, TLR9 and CD14 – were genotyped and validated by direct sequencing, with the exception of one that was undetected by minisequencing. We suggest that these panels can be used in future studies for detection of risk gene variants in several populations and/or diseases.
Conclusions
In summary, we propose a multiplex assay that is able to identify the most frequent candidate SNPs in innate immune genes, using a medium scale genotyping platform. The assays can be used to evaluate the risk gene variants in populations of various geographic origins.
doi:10.1186/1756-0500-6-54
PMCID: PMC3626843  PMID: 23390954
Innate immune genes; Polymorphisms; SNaPshot multiplex PCR
4.  Genotyping of BCL11A and HBS1L-MYB SNPs associated with fetal haemoglobin levels: a SNaPshot minisequencing approach 
BMC Genomics  2014;15:108.
Background
B-thalassaemia and sickle cell disease (SCD) are two of the most common monogenic diseases that are found in many populations worldwide. In both disorders the clinical severity is highly variable, with the persistence of fetal haemoglobin (HbF) being one of the major ameliorating factors. HbF levels are affected by, amongst other factors, single nucleotide polymorphisms (SNPs) at the BCL11A gene and the HBS1L-MYB intergenic region, which are located outside the β-globin locus. For this reason, we developed two multiplex assays that allow the genotyping of SNPs at these two genomic regions which have been shown to be associated with variable HbF levels in different populations.
Results
Two multiplex assays based on the SNaPshot minisequencing approach were developed. The two assays can be used to simultaneous genotype twelve SNPs at the BCL11A gene and sixteen SNPs at HBS1L-MYB intergenic region which were shown to modify HbF levels. The different genotypes can be determined based on the position and the fluorescent colour of the peaks in a single electropherogram. DNA sequencing and restriction fragment length polymorphism (PCR-RFLP) assays were used to verify genotyping results obtained by SNaPshot minisequencing.
Conclusions
In summary, we propose two multiplex assays based on the SNaPshot minisequencing approach for the simultaneous identification of SNPs located at the BCL11A gene and HBS1L-MYB intergenic region which have an effect on HbF levels. The assays can be easily applied for accurate, time and cost efficient genotyping of the selected SNPs in various populations.
doi:10.1186/1471-2164-15-108
PMCID: PMC3922441  PMID: 24502199
BCL11A; HBS1L-MYB; HbF; Thalassaemia; SCD; SNaPshot minisequencing; Multiplex PCR; Polymorphisms
5.  ABCB1 4036A>G and 1236C>T Polymorphisms Affect Plasma Efavirenz Levels in South African HIV/AIDS Patients 
Frontiers in Genetics  2012;3:236.
The ABCB1 gene encodes P-glycoprotein, an ATP-dependent drug efflux pump, which is responsible for drug transport across extra- and intra-cellular membranes. The variability in the expression of ABCB1 may contribute to variable plasma efavirenz concentration which results in variability in the levels of suppression of the human immunodeficiency syndrome virus (HIV). The aim of the study was to evaluate the role of polymorphisms in ABCB1 gene on plasma efavirenz levels and treatment response in the form of change in viral load and CD-4 cell count in HIV/AIDS patients receiving efavirenz-containing highly active antiretroviral treatment regimens. Two hundred and eighty-two HIV-infected patients were recruited from Themba Lethu Clinic in Johannesburg and plasma efavirenz drug concentration levels were measured using LC-MS/MS. SNaPshot was used to genotype five known ABCB1 single nucleotide polymorphisms (SNPs). Genotype-phenotype correlations were computed. The ABCB1 4036A/G and 4036G/G genotypes were significantly associated with low plasma efavirenz concentrations (P = 0.0236), while the ABCB1 1236C/T and 1236T/T genotypes were associated with high efavirenz concentrations (P = 0.0282). A haplotype ABCB1 T-G-T-A is reported that is associated with significantly increased plasma efavirenz levels. This is the first report on 61A>G, 2677G>T/A, and 4036A>G SNPs in the South African population. ABCB1 plays a role in determining the plasma concentrations of efavirenz and should be taken into account in future design of assays for genotype-based dosing of efavirenz-containing regimens.
doi:10.3389/fgene.2012.00236
PMCID: PMC3488761  PMID: 23133441
ABCB1; efavirenz; HIV/AIDS; South Africa; pharmacogenetics
6.  Function-Altering SNPs in the Human Multidrug Transporter Gene ABCB1 Identified Using a Saccharomyces-Based Assay 
PLoS Genetics  2007;3(3):e39.
The human ABCB1 (MDR1)-encoded multidrug transporter P-glycoprotein (P-gp) plays a major role in disposition and efficacy of a broad range of drugs including anticancer agents. ABCB1 polymorphisms could therefore determine interindividual variability in resistance to these drugs. To test this hypothesis we developed a Saccharomyces-based assay for evaluating the functional significance of ABCB1 polymorphisms. The P-gp reference and nine variants carrying amino-acid–altering single nucleotide polymorphisms (SNPs) were tested on medium containing daunorubicin, doxorubicin, valinomycin, or actinomycin D, revealing SNPs that increased (M89T, L662R, R669C, and S1141T) or decreased (W1108R) drug resistance. The R669C allele's highly elevated resistance was compromised when in combination with W1108R. Protein level or subcellular location of each variant did not account for the observed phenotypes. The relative resistance profile of the variants differed with drug substrates. This study established a robust new methodology for identification of function-altering polymorphisms in human multidrug transporter genes, identified polymorphisms affecting P-gp function, and provided a step toward genotype-determined dosing of chemotherapeutics.
Author Summary
Patients often show varied drug responses ranging from lack of therapeutic efficacy to life-threatening adverse drug reactions. Drug therapy would be greatly improved if it were possible to predict individual drug sensitivity and tailor drugs to patients' genetic makeup. Like all other organisms, humans have a set of transporters and enzymes to detoxify and eliminate foreign molecules including drugs. Understanding the function of genetic variants in these proteins is a key goal toward personalized medicine. To that end, we examined the functional consequences of naturally occurring genetic variants in P-glycoprotein, the most versatile human multidrug transporter. A novel method was developed and employed that can identify function-altering variants in human transporters. This methodology was robust and powerful in that the functional effect of genetic variants can be directly assessed in yeast where all confounding variables in humans are excluded. Surprisingly, the majority of single amino acid substitutions were found to cause alterations in resistance to three tested anticancer agents. This study extends the impact of yeast-based medical research to a new niche, pharmacogenomics.
doi:10.1371/journal.pgen.0030039
PMCID: PMC1817653  PMID: 17352537
7.  Screening for 392 polymorphisms in 141 pharmacogenes 
Biomedical Reports  2014;2(4):463-476.
Pharmacogenomics is the study of the association between inter-individual genetic differences and drug responses. Researches in pharmacogenomics have been performed in compliance with the use of several genotyping technologies. In this study, a total of 392 single-nucleotide polymorphisms (SNPs) located in 141 pharmacogenes, including 21 phase I, 13 phase II, 18 transporter and 5 modifier genes, were selected and genotyped in 150 subjects using the GoldenGate assay or the SNaPshot technique. These variants were in Hardy-Weinberg equilibrium (HWE) (P>0.05), except for 22 SNPs. Genotyping of the 392 SNPs revealed that the minor allele frequencies of 47 SNPs were <0.05, 105 SNPs were monomorphic and 22 variants were not in HWE. Also, based on previous studies, we predicted the association between the polymorphisms of certain pharmacogenes, such as cytochrome P450 2D6, cytochrome P450 2C9, vitamin K epoxide reductase complex, subunit 1, cytochrome P450 2C19, human leukocyte antigen, class I, B and thiopurine S-methyltransferase, and drug efficacy. In conclusion, our study demonstrated the allele distribution of SNPs in 141 pharmacogenes as determined by high-throughput screening. Our results may be helpful in developing personalized medicines by using pharmacogene polymorphisms.
doi:10.3892/br.2014.272
PMCID: PMC4051470  PMID: 24944790
gene screening; pharmacogene; single-nucleotide polymorphism
8.  GMFilter and SXTestPlate: software tools for improving the SNPlex™ genotyping system 
BMC Bioinformatics  2009;10:81.
Background
Genotyping of single-nucleotide polymorphisms (SNPs) is a fundamental technology in modern genetics. The SNPlex™ mid-throughput genotyping system (Applied Biosystems, Foster City, CA, USA) enables the multiplexed genotyping of up to 48 SNPs simultaneously in a single DNA sample. The high level of automation and the large amount of data produced in a high-throughput laboratory require advanced software tools for quality control and workflow management.
Results
We have developed two programs, which address two main aspects of quality control in a SNPlex™ genotyping environment: GMFilter improves the analysis of SNPlex™ plates by removing wells with a low overall signal intensity. It enables scientists to automatically process the raw data in a standardized way before analyzing a plate with the proprietary GeneMapper software from Applied Biosystems. SXTestPlate examines the genotype concordance of a SNPlex™ test plate, which was typed with a control SNP set. This program allows for regular quality control checks of a SNPlex™ genotyping platform. It is compatible to other genotyping methods as well.
Conclusion
GMFilter and SXTestPlate provide a valuable tool set for laboratories engaged in genotyping based on the SNPlex™ system. The programs enhance the analysis of SNPlex™ plates with the GeneMapper software and enable scientists to evaluate the performance of their genotyping platform.
doi:10.1186/1471-2105-10-81
PMCID: PMC2661053  PMID: 19267942
9.  Association analysis of nine candidate gene polymorphisms in Indian patients with type 2 diabetic retinopathy 
BMC Medical Genetics  2010;11:158.
Background
Diabetic retinopathy (DR) is classically defined as a microvasculopathy that primarily affects the small blood vessels of the inner retina as a complication of diabetes mellitus (DM).It is a multifactorial disease with a strong genetic component. The aim of this study is to investigate the association of a set of nine candidate genes with the development of diabetic retinopathy in a South Indian cohort who have type 2 diabetes mellitus (T2DM).
Methods
Seven candidate genes (RAGE, PEDF, AKR1B1, EPO, HTRA1, ICAM and HFE) were chosen based on reported association with DR in the literature. Two more, CFH and ARMS2, were chosen based on their roles in biological pathways previously implicated in DR. Fourteen single nucleotide polymorphisms (SNPs) and one dinucleotide repeat polymorphism, previously reported to show association with DR or other related diseases, were genotyped in 345 DR and 356 diabetic patients without retinopathy (DNR). The genes which showed positive association in this screening set were tested further in additional sets of 100 DR and 90 DNR additional patients from the Aravind Eye Hospital. Those which showed association in the secondary screen were subjected to a combined analysis with the 100 DR and 100 DNR subjects previously recruited and genotyped through the Sankara Nethralaya Hospital, India. Genotypes were evaluated using a combination of direct sequencing, TaqMan SNP genotyping, RFLP analysis, and SNaPshot PCR assays. Chi-square and Fisher exact tests were used to analyze the genotype and allele frequencies.
Results
Among the nine loci (15 polymorphisms) screened, SNP rs2070600 (G82S) in the RAGE gene, showed significant association with DR (allelic P = 0.016, dominant model P = 0.012), compared to DNR. SNP rs2070600 further showed significant association with DR in the confirmation cohort (P = 0.035, dominant model P = 0.032). Combining the two cohorts gave an allelic P < 0.003 and dominant P = 0.0013). Combined analysis with the Sankara Nethralaya cohort gave an allelic P = 0.0003 and dominant P = 0.00011 with an OR = 0.49 (0.34 - 0.70) for the minor allele. In HTRA1, rs11200638 (G>A), showed marginal significance with DR (P = 0.055) while rs10490924 in LOC387715 gave a P = 0.07. No statistical significance was observed for SNPs in the other 7 genes studied.
Conclusions
This study confirms significant association of one polymorphism only (rs2070600 in RAGE) with DR in an Indian population which had T2DM.
doi:10.1186/1471-2350-11-158
PMCID: PMC2994838  PMID: 21067572
10.  Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus 
Background
The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus.
Method
This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors.
Result
Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA.
Conclusion
The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories. Keywords: single nucleotide polymorphisms, genotyping, plant pathology, viruses, microbial forensics, Single base primer extension, SNaPshot Multiplex Kit
doi:10.1186/2041-2223-3-10
PMCID: PMC3488013  PMID: 22594601
11.  Gene polymorphisms of ABC transporters are associated with clinical outcomes in children with acute lymphoblastic leukemia 
Introduction
Genetic variability affects clinical outcome in pediatric acute lymphocytic leukemia (ALL) patients. Evaluating gene polymorphisms in ABC transporters could help identify relapse risk and predict outcome.
Material and methods
The SNaPshot SNP technique was used to analyze single-nucleotide polymorphisms (SNPs) in the multidrug transporter 1 (MDR1), multidrug resistance associated proteins (MRP1, MRP2) and breast cancer resistance protein (BCRP) genes of 82 pediatric ALL patients. The association between the SNPs with risk of all events and death as well as with survival was evaluated by the univariate Cox proportional hazard model.
Results
The BCRP G34A SNP was the only SNP significantly associated with ALL. Risk factors included pre-treatment WBC counts and post-treatment peripheral and bone marrow leukemic cell counts. We found no association between MDR1 SNPs with these factors. The BCRP C421A C/A and C/C genotypes were significantly associated with low pre-treatment WBC counts while MRP2 G1249A G/G was significantly associated with low levels of post-treatment peripheral and bone marrow leukemic cells. A combination of C1236T, G1249A and/or G34A SNPs was significantly associated with lower EFS and OS.
Conclusions
Polymorphisms associated with risk of ALL and clinical outcome may be potential biomarkers to predict clinical outcome and improve prognosis in childhood ALL.
doi:10.5114/aoms.2012.30290
PMCID: PMC3460503  PMID: 23056078
acute lymphoblastic leukemia; single nucleotide polymorphism; survival; outcome
12.  Impact of the CYP3A5, CYP3A4, COMT, IL-10 and POR Genetic Polymorphisms on Tacrolimus Metabolism in Chinese Renal Transplant Recipients 
PLoS ONE  2014;9(1):e86206.
Tacrolimus is a widely used immunosuppressive drug for preventing the rejection of solid organ transplants. The efficacy of tacrolimus shows considerable variability, which might be related to genetic variation among recipients. We conducted a retrospective study of 240 Chinese renal transplant recipients receiving tacrolimus as immunosuppressive drug. The retrospective data of all patients were collected for 40 days after transplantation. Seventeen SNPs of CYP3A5, CYP3A4, COMT, IL-10 and POR were identified by the SNaPshot assay. Tacrolimus blood concentrations were obtained on days 1–3, days 6–8 and days 12–14 after transplantation, as well as during the period of the predefined therapeutic concentration range. Kruskal–Wallis test was used to examine the effect of genetic variation on the tacrolimus concentration/dose ratio (C0/D) at different time points. Chi-square test was used to compare the proportions of patients who achieved the target C0 range in the different genotypic groups at weeks 1, 2, 3 and 4 after transplantation. After correction for multiple testing, there was a significant association of C0/D with CYP3A5*3, CYP3A4*1G and CYP3A4 rs4646437 T>C at different time points after transplantation. The proportion of patients in the IL-10 rs1800871-TT group who achieved the target C0 range was greater (p = 0.004) compared to the IL-10 rs1800871-CT and IL-10 rs1800871-CC groups at week 3 after transplantation. CYP3A5*3, CYP3A4 *1G, CYP3A4 rs4646437 T>C and IL-10 rs1800871 C>T might be potential polymorphisms affecting the interindividual variability in tacrolimus metabolism among Chinese renal transplant recipients.
doi:10.1371/journal.pone.0086206
PMCID: PMC3897654  PMID: 24465960
13.  The association between Toll-like receptor 2 single-nucleotide polymorphisms and hepatocellular carcinoma susceptibility 
BMC Cancer  2012;12:57.
Background
Toll-like receptors (TLR) are key innate immunity receptors participating in an immune response. Growing evidence suggests that mutations of TLR2/TLR9 gene are associated with the progress of cancers. The present study aimed to investigate the temporal relationship of single nucleotide polymorphisms (SNP) of TLR2/TLR9 and the risk of hepatocellular carcinoma (HCC).
Methods
In this single center-based case-control study, SNaPshot method was used to genotype sequence variants of TLR2 and TLR9 in 211 patients with HCC and 232 subjects as controls.
Results
Two synonymous SNPs in the exon of TLR2 were closely associated with risk of HCC. Compared with those carrying wild-type homozygous genotypes (T/T), risk of HCC decreased significantly in individuals carrying the heterozygous genotypes (C/T) of the rs3804099 (adjusted odds ratio (OR), 0.493, 95% CI 0.331 - 0.736, P < 0.01) and rs3804100 (adjusted OR, 0.509, 95% CI 0.342 - 0.759, P < 0.01). There was no significant association found in two TLR9 SNPs concerning the risk of HCC. The haplotype TT for TLR2 was associated significantly with the decreased risk of HCC (OR 0.524, 95% CI 0.394 - 0.697, P = 0.000). Inversely, the risk of HCC increased significantly in patients with the haplotype CC (OR 2.743, 95% CI 1.915 - 3.930, P = 0.000).
Conclusions
These results suggested that TLR2 rs3804099 C/T and rs3804100 C/T polymorphisms were closely associated with HCC. In addition, the haplotypes composed of these two TLR2 synonymous SNPs have stronger effects on the susceptibility of HCC.
doi:10.1186/1471-2407-12-57
PMCID: PMC3311588  PMID: 22309608
14.  Comprehensive SNP-chip for retinitis pigmentosa-Leber congenital amaurosis diagnosis: new mutations and detection of mutational founder effects 
Fast and efficient high-throughput techniques are essential for the molecular diagnosis of highly heterogeneous hereditary diseases, such as retinitis pigmentosa (RP). We had previously approached RP genetic testing by devising a chip based on co-segregation analysis for the autosomal recessive forms. In this study, we aimed to design a diagnostic tool for all the known genes (40 up to now) responsible for the autosomal dominant and recessive RP and Leber congenital amaurosis (LCA). This new chip analyzes 240 single nucleotide polymorphisms (SNPs) (6 per gene) on a high-throughput genotyping platform (SNPlex, Applied Biosystems), and genetic diagnosis is based on the co-segregation analysis of SNP haplotypes in independent families. In a single genotyping step, the number of RP candidates to be screened for mutations is considerably reduced, and in the most informative families, all the candidates are ruled out at once. In a panel of RP Spanish pedigrees, the disease chip became a crucial tool for selecting those suitable for genome-wide RP gene search, and saved the burdensome direct mutational screening of every known RP gene. In a large adRP family, the chip allowed ruling out of all but the causative gene, and identification of an unreported null mutation (E181X) in PRPF31. Finally, on the basis of the conservation of the SNP haplotype linked to this pathogenic variant, we propose that the E181X mutation spread through a cohort of geographically isolated families by a founder effect.
doi:10.1038/ejhg.2009.114
PMCID: PMC2987167  PMID: 19584904
co-segregation; SNP genotyping; RP; LCA; mutation; founder effect
15.  Distinct Cancer-Specific Survival in Metastatic Prostate Cancer Patients Classified by a Panel of Single Nucleotide Polymorphisms of Cancer-Associated Genes 
Genes & Cancer  2013;4(1-2):54-60.
Individual genetic variations may have a significant influence on the survival of metastatic prostate cancer (PCa) patients. We aimed to identify target genes and their variations involved in the survival of PCa patients using a single nucleotide polymorphism (SNP) panel. A total of 185 PCa patients with bone metastasis at the initial diagnosis were analyzed. Germline DNA in each patient was genotyped using a cancer SNP panel that contained 1,421 SNPs in 408 cancer-related genes. SNPs associated with survival were screened by a log-rank test. Fourteen SNPs in 6 genes, XRCC4, PMS1, GATA3, IL13, CASP8, and IGF1, were identified to have a statistically significant association with cancer-specific survival. The cancer-specific survival times of patients grouped according to the number of risk genotypes of 6 SNPs selected from the 14 SNPs differed significantly (0-1 v. 2-3 v. 4-6 risk genotypes; P = 7.20 × 10−8). The high-risk group was independently associated with survival in a multivariate analysis that included conventional clinicopathological variables (P = 0.0060). We identified 14 candidate SNPs in 6 cancer-related genes, which were associated with poor survival in patients with metastatic PCa. A panel of SNPs may help predict the survival of those patients.
doi:10.1177/1947601913481354
PMCID: PMC3743152  PMID: 23946871
prostate cancer; bone metastasis; survival; single nucleotide polymorphism
16.  Part 2: Pharmacogenetic Variability in Drug Transport and Phase I Anticancer Drug Metabolism 
The Oncologist  2011;16(6):820-834.
This second part of a four-part series deals with pharmacogenetic variability in drug transport and anticancer phase I drug metabolism, and emphasizes opportunities for patient-tailored pharmacotherapy based on the current knowledge in the field of pharmacogenetics in oncology.
Learning Objectives
After completing this course, the reader will be able to: List currently identified candidate genes involved in phase I metabolism that are potential pharmacogenetic markers in anticancer therapy.Describe the general effect on standard treatment of allelic variants of the candidate genes and the implications for individualized treatment.
This article is available for continuing medical education credit at CME.TheOncologist.com
Equivalent drug doses in anticancer chemotherapy may lead to wide interpatient variability in drug response reflected by differences in treatment response or in severity of adverse drug reactions. Differences in the pharmacokinetic (PK) and pharmacodynamic (PD) behavior of a drug contribute to variation in treatment outcome among patients. An important factor responsible for this variability is genetic polymorphism in genes that are involved in PK/PD processes, including drug transporters, phase I and II metabolizing enzymes, and drug targets, and other genes that interfere with drug response. In order to achieve personalized pharmacotherapy, drug dosing and treatment selection based on genotype might help to increase treatment efficacy while reducing unnecessary toxicity.
We present a series of four reviews about pharmacogenetic variability in anticancer drug treatment. This is the second review in the series and is focused on genetic variability in genes encoding drug transporters (ABCB1 and ABCG2) and phase I drug-metabolizing enzymes (CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, DPYD, CDA and BLMH) and their associations with anticancer drug treatment outcome. Based on the literature reviewed, opportunities for patient-tailored anticancer therapy are presented.
doi:10.1634/theoncologist.2010-0259
PMCID: PMC3228220  PMID: 21632461
Pharmacogenetics; Drug transport; Phase I metabolism; Personalized medicine; Oncology; Anticancer drugs
17.  DNA repair gene variants are associated with an increased risk of myelodysplastic syndromes in a Czech population 
Background
Interactions between genetic variants and risk factors in myelodysplastic syndromes are poorly understood. In this case–control study, we analyzed 1 421 single nucleotide polymorphisms in 408 genes involved in cancer-related pathways in 198 patients and 292 controls.
Methods
The Illumina SNP Cancer Panel was used for genotyping of samples. The chi-squared, p-values, odds ratios and upper and lower limits of the 95% confidence interval were calculated for all the SNPs that passed the quality control filtering.
Results
Gene-based analysis showed nine candidate single nucleotide polymorphisms significantly associated with the disease susceptibility (q-value < 0.05). Four of these polymorphisms were located in oxidative damage/DNA repair genes (LIG1, RAD52, MSH3 and GPX3), which may play important roles in the pathobiology of myelodysplastic syndromes. Two of nine candidate polymorphisms were located in transmembrane transporters (ABCB1 and SLC4A2), contributing to individual variability in drug responses and patient prognoses. Moreover, the variations in the ROS1 and STK6 genes were associated with the overall survival of patients.
Conclusions
Our association study identified genetic variants in Czech population that may serve as potential markers for myelodysplastic syndromes.
doi:10.1186/1756-8722-6-9
PMCID: PMC3556100  PMID: 23339595
Myelodysplastic syndromes; SNP; DNA repair; Association study
18.  Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry-Based Single Nucleotide Polymorphism Genotyping Assay Using iPLEX Gold Technology for Identification of Mycobacterium tuberculosis Complex Species and Lineages▿ 
Journal of Clinical Microbiology  2011;49(9):3292-3299.
The major goal of the present study was to investigate the potential use of a novel single nucleotide polymorphism (SNP) genotyping technology, called iPLEX Gold (Sequenom), for the simultaneous analysis of 16 SNPs that have been previously validated as useful for identification of Mycobacterium tuberculosis complex (MTBC) species and classification of MTBC isolates into distinct genetic lineages, known as principal genetic groups (PGGs) and SNP cluster groups (SCGs). In this context, we developed a 16-plex iPLEX assay based on an allele-specific-primer single-base-extension reaction using the iPLEX Gold kit (Sequenom), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis on the commercially available Sequenom MassARRAY platform. This assay was tested on a panel of 55 well-characterized MTBC strains that were also genotyped for the same loci using the previously reported SNaPshot assay, as well as 10 non-MTBC mycobacteria and 4 bacteria not belonging to the genus Mycobacterium. All MTBC samples were successfully analyzed with the iPLEX assay, which yielded clear allelic data for 99.9% of the SNPs (879 out of 880). No false-positive results were obtained with the negative controls. Compared to the SNaPshot assay, the newly developed 16-plex iPLEX assay produced fully concordant results that allowed reliable differentiation of MTBC species and recognition of lineages, thus demonstrating its potential value in diagnostic, epidemiological, and evolutionary applications. Compared to the SNaPshot approach, the implementation of the iPLEX technology could offer a higher throughput and could be a more flexible and cost-effective option for microbiology laboratories.
doi:10.1128/JCM.00744-11
PMCID: PMC3165613  PMID: 21734028
19.  Association Analysis of IL-17A and IL-17F Polymorphisms in Chinese Han Women with Breast Cancer 
PLoS ONE  2012;7(3):e34400.
Background
Research into the etiology of breast cancer has recently focused on the role of the immunity and inflammation. The proinflammatory cytokines IL-17A and IL-17F can mediate inflammation and cancer. To evaluate the influences of IL-17A and IL-17F gene polymorphisms on the risk of sporadic breast cancer, a case-control study was conducted in Chinese Han women.
Methodology and Principal Findings
We genotyped three single-nucleotide polymorphisms (SNPs) in IL-17A (rs2275913, rs3819025 and rs3748067) and five SNPs in IL-17F (rs7771511, rs9382084, rs12203582, rs1266828 and rs763780) to determine the haplotypes in 491 women with breast cancer and 502 healthy individuals. The genotypes were determined using the SNaPshot technique. The differences in the genotypic distribution between breast cancer patients and healthy controls were analyzed with the Chi-square test for trends. For rs2275913 in IL-17A, the frequency of the AA genotype was higher in patients than controls (P = 0.0016). The clinical features analysis demonstrated significant associations between IL-17 SNPs and tumor protein 53 (P53), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her-2) and triple-negative (ER-/PR-/Her-2-) status. In addition, the haplotype analysis indicated that the frequency of the haplotype Ars2275913Grs3819025Grs3748067, located in the IL-17A linkage disequilibrium (LD) block, was higher in patients than in controls (P = 0.0471 after correction for multiple testing).
Conclusions and Significance
Our results suggested that SNPs in IL-17A but not IL-17F were associated with the risk of breast cancer. Both IL-17A and IL-17F gene polymorphisms may provide valuable information for predicting the prognosis of breast cancer in Chinese women.
doi:10.1371/journal.pone.0034400
PMCID: PMC3312906  PMID: 22461912
20.  SNP high-throughput screening in grapevine using the SNPlex™ genotyping system 
BMC Plant Biology  2008;8:12.
Background
Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP) discovery and genotyping in grapevine (Vitis vinifera L.). However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs) thus providing a valuable source for high-throughput genotyping methods.
Results
Herein we report the first application of the SNPlex™ genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA) methods were used for preparation of genomic DNA for the SNPlex assay.
Conclusion
Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA), is a good solution for future applications in well-equipped laboratories.
doi:10.1186/1471-2229-8-12
PMCID: PMC2268689  PMID: 18226250
21.  High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology 
BMC Genomics  2007;8:424.
Background
Single-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes.
Results
In order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape (π = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlex™ genotyping technology in a sample of grapevine genotypes and segregating progenies.
Conclusion
These results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlex™ genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlex™ high throughput genotyping technology provide a powerful tool for grapevine genetic analysis.
doi:10.1186/1471-2164-8-424
PMCID: PMC2212664  PMID: 18021442
22.  Breast cancer resistance protein (ABCG2) and drug disposition: intestinal expression, polymorphisms and sulfasalazine as an in vivo probe 
Pharmacogenetics and genomics  2008;18(5):439-448.
Breast cancer resistance protein (BCRP) is an efflux transporter expressed in tissues that act as barriers to drug entry. Given that single nucleotide polymorphisms (SNPs) in the ABCG2 gene encoding BCRP are common, the possibility exists that these genetic variants may be a determinant of interindividual variability in drug response. The objective of this study is to confirm the human BCRP-mediated transport of sulfasalazine in vitro, evaluate interindividual variation in BCRP expression in human intestine and to determine the role of ABCG2 SNPs to drug disposition in healthy patients using sulfasalazine as a novel in vivo probe. To evaluate these objectives, pinch biopsies were obtained from 18 patients undergoing esophagogastro–duodenoscopy or colonoscopy for determination of BCRP expression in relation to genotype. Wild-type and variant BCRP were expressed in a heterologous expression system to evaluate the effect of SNPs on cell-surface trafficking. A total of 17 healthy individuals participated in a clinical investigation to determine the effect of BCRP SNPs on sulfasalazine pharmacokinetics. In vitro, the cell surface protein expression of the common BCRP 421 C>A variant was reduced in comparison with the wild-type control. Intestinal biopsy samples revealed that BCRP protein and mRNA expression did not significantly differ between patients with 34GG/421CC versus patients with 34GG/421CA genotypes. Remarkably, in subjects with 34GG/421CA genotype, sulfasalazine area under the concentration-time curve was 2.4-fold greater compared with 34GG/421CC subjects (P<0.05). This study links commonly occurring SNPs in BCRP with significantly increased oral sulfasalazine plasma exposure in humans. Accordingly, sulfasalazine may prove to have utility as in vivo probe for assessing the clinical impact of BCRP for the disposition and efficacy of drugs.
doi:10.1097/FPC.0b013e3282f974dc
PMCID: PMC4043148  PMID: 18408567
breast cancer resistance protein/ATP binding cassette; drug transport; member 2; pharmacogenetics; pharmacokinetics; subfamily G (WHITE); sulfasalazine
23.  Mining of haplotype-based expressed sequence tag single nucleotide polymorphisms in citrus 
BMC Genomics  2013;14:746.
Background
Single nucleotide polymorphisms (SNPs), the most abundant variations in a genome, have been widely used in various studies. Detection and characterization of citrus haplotype-based expressed sequence tag (EST) SNPs will greatly facilitate further utilization of these gene-based resources.
Results
In this paper, haplotype-based SNPs were mined out of publicly available citrus expressed sequence tags (ESTs) from different citrus cultivars (genotypes) individually and collectively for comparison. There were a total of 567,297 ESTs belonging to 27 cultivars in varying numbers and consequentially yielding different numbers of haplotype-based quality SNPs. Sweet orange (SO) had the most (213,830) ESTs, generating 11,182 quality SNPs in 3,327 out of 4,228 usable contigs. Summed from all the individually mining results, a total of 25,417 quality SNPs were discovered – 15,010 (59.1%) were transitions (AG and CT), 9,114 (35.9%) were transversions (AC, GT, CG, and AT), and 1,293 (5.0%) were insertion/deletions (indels). A vast majority of SNP-containing contigs consisted of only 2 haplotypes, as expected, but the percentages of 2 haplotype contigs varied widely in these citrus cultivars. BLAST of the 25,417 25-mer SNP oligos to the Clementine reference genome scaffolds revealed 2,947 SNPs had “no hits found”, 19,943 had 1 unique hit / alignment, 1,571 had one hit and 2+ alignments per hit, and 956 had 2+ hits and 1+ alignment per hit. Of the total 24,293 scaffold hits, 23,955 (98.6%) were on the main scaffolds 1 to 9, and only 338 were on 87 minor scaffolds. Most alignments had 100% (25/25) or 96% (24/25) nucleotide identities, accounting for 93% of all the alignments. Considering almost all the nucleotide discrepancies in the 24/25 alignments were at the SNP sites, it served well as in silico validation of these SNPs, in addition to and consistent with the rate (81%) validated by sequencing and SNaPshot assay.
Conclusions
High-quality EST-SNPs from different citrus genotypes were detected, and compared to estimate the heterozygosity of each genome. All the SNP oligo sequences were aligned with the Clementine citrus genome to determine their distribution and uniqueness and for in silico validation, in addition to SNaPshot and sequencing validation of selected SNPs.
doi:10.1186/1471-2164-14-746
PMCID: PMC3827885  PMID: 24175923
Haplotype; Heterozygosity; Polymorphism; Transition; Transversion; Insertion/deletion; Non-synonymous; Synonymous
24.  A miRNA Binding Site Single-Nucleotide Polymorphism in the 3′-UTR Region of the IL23R Gene Is Associated with Breast Cancer 
PLoS ONE  2012;7(12):e49823.
Background
Research into the etiology of breast cancer has recently focused on the role of the immunity and inflammation. Interleukin-23 and its receptor (IL23R) guide T cells towards the Th17 phenotype. IL23R single nucleotide polymorphisms (SNPs) have been shown to be associated with digestive system cancers. To evaluate the influences of IL23R gene polymorphisms on the risk of sporadic breast cancer, a case-control study was conducted in Chinese Han women.
Methodology and Principal Findings
We genotyped two tag SNPs (rs10889677 in the 3′-UTR region and nonsynonymous variants rs1884444 in exon 2) in IL23R gene of 491 breast cancer patients and 502 matched healthy controls. The genotypes were determined using the SNaPshot technique. The differences in the genotypic distribution between breast cancer patients and healthy controls were analyzed with the Chi-square test for trends. For rs10889677 in IL23R, the frequencies of the AA genotype and the A allele were statistical significant higher in breast cancer patients than in controls (P = 0.0084 and P = 0.0171, respectively), whereas the C allele was associated with an earlier age of breast cancer onset (50.6 years for AA, 48.7 years for AC and 46.0 years for CC (P = 0.0114)) in case-only study. The clinical features analysis demonstrated significant associations between rs1884444 in IL23R and human epidermal growth factor receptor 2 (Her-2) and tumor size status.
Conclusions and Significance
Our results suggest that a miRNA binding site SNP in the 3′-UTR region of the IL23R gene may be associated with the risk of breast cancer and contribute to the early development of breast cancer in Chinese women.
doi:10.1371/journal.pone.0049823
PMCID: PMC3519811  PMID: 23239971
25.  Identification and Genotyping of Mycobacterium tuberculosis Complex Species by Use of a SNaPshot Minisequencing-Based Assay▿  
Journal of Clinical Microbiology  2010;48(5):1758-1766.
The aim of the present study was to investigate the use of the SNaPshot minisequencing method for the identification of Mycobacterium tuberculosis complex (MTBC) isolates to the species level and for further genotyping of M. tuberculosis isolates. We developed an innovative strategy based on two multiplex allele-specific minisequencing assays that allowed detection of eight species-specific and eight lineage-specific single nucleotide polymorphisms (SNPs). Each assay consisted of an eightplex PCR amplification, followed by an eightplex minisequencing reaction with the SNaPshot multiplex kit (Applied Biosystems) and, finally, analysis of the extension products by capillary electrophoresis. The whole strategy was developed with a panel of 56 MTBC strains and 15 negative controls. All MTBC strains tested except one M. africanum clinical isolate were accurately identified to the species level, and all M. tuberculosis isolates were successfully further genotyped. This two-step strategy based on SNaPshot minisequencing allows the simultaneous differentiation of closely related members of the MTBC, the distinction between principal genetic groups, and the characterization of M. tuberculosis isolates into one of the seven prominent SNP cluster groups (SCGs) and could be a useful tool for diagnostic and epidemiological purposes.
doi:10.1128/JCM.02255-09
PMCID: PMC2863856  PMID: 20220173

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