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We previously reported that tacrolimus (TAC) trough blood concentrations for African American (AA) kidney allograft recipients were lower than those observed in white patients. Subtherapeutic TAC troughs may be associated with acute rejection (AR) and AR-associated allograft failure. This variation in TAC troughs is due, in part, to differences in the frequency of the cytochrome P450 CYP3A5*3 allele (rs776746, expresses nonfunctional enzyme) between white and AA recipients; however, even after accounting for this variant, variability in AA-associated troughs is significant. We conducted a genomewide association study of TAC troughs in AA kidney allograft recipients to search for additional genetic variation. We identified two additional CYP3A5 variants in AA recipients independently associated with TAC troughs: CYP3A5*6 (rs10264272) and CYP3A5*7 (rs41303343). All three variants and clinical factors account for 53.9% of the observed variance in troughs, with 19.8% of the variance coming from demographic and clinical factors including recipient age, glomerular filtration rate, anticytomegalovirus drug use, simultaneous pancreas-kidney transplant and antibody induction. There was no evidence of common genetic variants in AA recipients significantly influencing TAC troughs aside from the CYP3A gene. These results reveal that additional and possibly rare functional variants exist that account for the additional variation., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Inhaled short-acting beta-agonist (SABA) medication is commonly used in asthma patients to rapidly reverse airway obstruction and improve acute symptoms. We performed a genome-wide association study of SABA medication response using gene-based association tests. A linear mixed model approach was first used for single-nucleotide polymorphism associations, and the results were later combined using GATES to generate gene-based associations. Our results identified SPATA13-AS1 as being significantly associated with SABA bronchodilator response in 328 healthy African Americans. In replication, this gene was associated with SABA response among the two separate groups of African Americans with asthma (n=1073, P=0.011 and n=1968, P=0.014), 149 healthy African Americans (P=0.003) and 556 European Americans with asthma (P=0.041). SPATA13-AS1 was also associated with longitudinal SABA medication usage in the two separate groups of African Americans with asthma (n=658, P=0.047 and n=1968, P=0.025). Future studies are needed to delineate the precise mechanism by which SPATA13-AS1 may influence SABA response.

To develop an efficient strategy for mapping genetic factors associated with common diseases, we constructed linkage disequilibrium (LD) maps of human chromosomes 5, 7, 17, and X. These maps consist of common single nucleotide polymorphisms at an average intermarker distance of 100 kb. The genotype data from these markers in a panel of American samples of European descent were analyzed to produce blocks of markers in strong pair-wise LD. Power calculations were used to guide block definitions and predicted that high-level LD maps would be useful in initial genome scans for susceptibility alleles in case-control association studies of complex diseases. As anticipated, LD blocks on the X chromosome were larger and covered more of the chromosome than those found on the autosomes.

Extensive, new databases of single-nucleotide polymorphisms (SNPs) provide a powerful resource for disease gene discovery, and they will be even more useful as more frequency data become available. Interesting observed genomic patterns include SNP deserts (regions of low SNP incidence) and lengthy regions of linkage disequilibrium containing only a few haplotypes. A variety of genetic studies will benefit from SNP resources.

The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.

Background: The PCR-Invader assay is a robust, homogeneous assay that has been shown to be highly sensitive and specific in genotyping single-nucleotide polymorphism (SNP) markers. In this study, we introduce two changes to improve the assay: (a) we streamline the PCR-Invader method by assaying both alleles for each SNP in one reaction; and (b) we reduce the cost of the method by adopting fluorescence polarization (FP) as the detection method., Methods: PCR product was incubated with Invader oligonucleotide and two primary probes at 93 degrees C for 5 min. Signal probes corresponding to the cleaved flaps of the primary probes [labeled with fluorescein and 6-carboxytetramethylrhodamine (TAMRA) dye] and Cleavase VIII enzyme (a flap endonuclease) were then added to the mixture. This reaction mixture was incubated at 63 degrees C for 5 min. FP measurements were made with a fluorescence plate reader., Results: Eighty-eight individuals were genotyped across a panel of 10 SNPs, using PCR product as template, for a total of 880 genotypes. An average "no call" rate of 3.2% was observed after first round of experiments. PCR products were remade in those samples that failed to produce any genotype in the first round, and all gave clear-cut genotypes. When the genotypes determined by the PCR-Invader assay and template-directed dye-terminator incorporation assay with FP were compared, they were in 100% concordance for all SNP markers and experiments., Conclusions: The improvements introduced in this study make PCR-Invader assay simpler and more cost-effective, and therefore more suitable for high-throughput genotyping.

There is a concerted effort by a number of public and private groups to identify a large set of human single-nucleotide polymorphisms (SNPs). As of March 2001, 2.84 million SNPs have been deposited in the public database, dbSNP, at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/). The 2.84 million SNPs can be grouped into 1.65 million non-redundant SNPs. As part of the International SNP Map Working Group, we recently published a high-density SNP map of the human genome consisting of 1.42 million SNPs (ref. 3). In addition, numerous SNPs are maintained in proprietary databases. Our survey of more than 1,200 SNPs indicates that more than 80% of TSC and Washington University candidate SNPs are polymorphic and that approximately 50% of the candidate SNPs from these two sources are common SNPs (with minor allele frequency of > or =20%) in any given population.

Comparison of different metrics, using three large samples of haplotypes from different populations, demonstrates that rho is the most efficient measure of association between pairs of single nucleotide polymorphisms (SNPs). Pairwise data can be modeled, using composite likelihood, to describe the decline in linkage disequilibrium with distance (the Malecot model). The evidence from more isolated populations (Finland, Sardinia) suggests that linkage disequilibrium extends to 427-893 kb but, even in samples representative of large heterogeneous populations, such as CEPH, the extent is 385 kb or greater. This suggests that isolated populations are not essential for linkage disequilibrium mapping of common diseases with SNPs. The in parameter of the Malecot model (recombination and time), evaluated at each SNP, indicates regions of the genome with extensive and less extensive disequilibrium (low and high values of in respectively). When plotted against the physical map, the regions with extensive and less extensive linkage disequilibrium may correspond to recombination cold and hot spots. This is discussed in relation to the Xq25 cytogenetic band and the HFE gene region., (Copyright 2001 Wiley-Liss, Inc.)

We describe a map of 1.42 million single nucleotide polymorphisms (SNPs) distributed throughout the human genome, providing an average density on available sequence of one SNP every 1.9 kilobases. These SNPs were primarily discovered by two projects: The SNP Consortium and the analysis of clone overlaps by the International Human Genome Sequencing Consortium. The map integrates all publicly available SNPs with described genes and other genomic features. We estimate that 60,000 SNPs fall within exon (coding and untranslated regions), and 85% of exons are within 5 kb of the nearest SNP. Nucleotide diversity varies greatly across the genome, in a manner broadly consistent with a standard population genetic model of human history. This high-density SNP map provides a public resource for defining haplotype variation across the genome, and should help to identify biomedically important genes for diagnosis and therapy.

While scanning for single-nucleotide polymorphisms (SNPs) in the human Xq25-q28 region of CEPH families, we found six long "deserts" of low SNP incidence representing 28% of the investigated genome. One was 1.66 Mb in length. To determine whether these SNP deserts were due to reduced input of mutations or to recent coalescent events such as bottlenecks or selective sweeps, comparative sequence was determined from a female orangutan. The mean divergence was 2.9% and was not reduced in deserts compared with nondesert regions. Thus, the best explanation for the SNP deserts is recent coalescent events in humans. These events are the cause of substantial variation in human noncoding SNP incidence. In addition, the mutational spectrum in humans and orangutans was estimated as 63% AG (and CT), 17% AC (and GT), 8% CG, 4% AT, and 8% insertion/deletions. The average lifetime of a SNP destined to become fixed for a new allele between these species was estimated as 284,000 years., (Copyright 2001 Academic Press.)

One of the fruits of the Human Genome Project is the discovery of millions of DNA sequence variants in the human genome. The majority of these variants are single nucleotide polymorphisms (SNPs). A dense set of SNP markers opens up the possibility of studying the genetic basis of complex diseases by population approaches. In all study designs, a large number of individuals must be genotyped with a large number of markers. In this review, the current status of SNP genotyping is discussed in terms of the mechanisms of allelic discrimination, the reaction formats, and the detection modalities. A number of genotyping methods currently in use are described to illustrate the approaches being taken. Although no single genotyping method is ideally suited for all applications, a number of good genotyping methods are available to meet the needs of many study designs. The challenges for SNP genotyping in the near future include increasing the speed of assay development, reducing the cost of the assays, and performing multiple assays in parallel. Judging from the accelerated pace of new method development, it is hopeful that an ideal SNP genotyping method will be developed soon.

Linkage disequilibrium (LD), or the non-random association of alleles, is poorly understood in the human genome. Population genetic theory suggests that LD is determined by the age of the markers, population history, recombination rate, selection and genetic drift. Despite the uncertainties in determining the relative contributions of these factors, some groups have argued that LD is a simple function of distance between markers. Disease-gene mapping studies and a simulation study gave differing predictions on the degree of LD in isolated and general populations. In view of the discrepancies between theory and experimental observations, we constructed a high-density SNP map of the Xq25-Xq28 region and analysed the male genotypes and haplotypes across this region for LD in three populations. The populations included an outbred European sample (CEPH males) and isolated population samples from Finland and Sardinia. We found two extended regions of strong LD bracketed by regions with no evidence for LD in all three samples. Haplotype analysis showed a paucity of haplotypes in regions of strong LD. Our results suggest that, in this region of the X chromosome, LD is not a monotonic function of the distance between markers, but is more a property of the particular location in the human genome.

A high-density single-nucleotide polymorphism (SNP) map was developed for Xq25-q28 using a targeted approach to SNP discovery. This high-density map includes 217 new SNP markers, and 117 are informative in the CEPH parent population with >20% minor allele frequency. The average distance between SNP markers is 100 kb in the targeted regions. This is the densest genetic map of Xq25-q28 to date. The SNP markers are presented in order by their distance in megabases along the X chromosome, and the markers from the current genetic map are placed using the same scale to produce an integrated map of the region.

Single-nucleotide polymorphisms (SNPs) are the most abundant form of human genetic variation and a resource for mapping complex genetic traits. The large volume of data produced by high-throughput sequencing projects is a rich and largely untapped source of SNPs (refs 2, 3, 4, 5). We present here a unified approach to the discovery of variations in genetic sequence data of arbitrary DNA sources. We propose to use the rapidly emerging genomic sequence as a template on which to layer often unmapped, fragmentary sequence data and to use base quality values to discern true allelic variations from sequencing errors. By taking advantage of the genomic sequence we are able to use simpler yet more accurate methods for sequence organization: fragment clustering, paralogue identification and multiple alignment. We analyse these sequences with a novel, Bayesian inference engine, POLYBAYES, to calculate the probability that a given site is polymorphic. Rigorous treatment of base quality permits completely automated evaluation of the full length of all sequences, without limitations on alignment depth. We demonstrate this approach by accurate SNP predictions in human ESTs aligned to finished and working-draft quality genomic sequences, a data set representative of the typical challenges of sequence-based SNP discovery.

A great deal of time and money is currently being invested in the production of large libraries of single nucleotide polymorphisms (SNPs) - variations of one nucleotide between the DNA sequence of individuals. This review compares and contrasts the available sources of SNP data, and describes the rationale behind the SNP mapping efforts, from the study of common diseases to unraveling an individual's response to medication.