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To investigate the genetic etiology of anophthalmia and microphthalmia, we used exome sequencing in a Caucasian female with unilateral microphthalmia and coloboma, bilateral optic nerve hypoplasia, ventricular and atrial septal defects, and growth delays. We found two sequence variants in SALL4 - c.[575C>A], predicting p.(Ala192Glu), that was paternally inherited, and c.[2053G>C], predicting p.(Asp685His), that was maternally inherited. Haploinsufficiency for SALL4 due to nonsense or frameshift mutations has been associated with acro-renal ocular syndrome that is characterized by eye defects including Duane anomaly and coloboma, in addition to radial ray malformations and renal abnormalities. Our report is the first description of structural eye defects associated with two missense variants in SALL4 inherited in trans; the absence of reported findings in both parents suggests that both sequence variants are hypomorphic mutations and that both are needed for the ocular phenotype. SALL4 is expressed in the developing lens and regulates BMP4, leading us to speculate that altered BMP4 expression was responsible for the eye defects, but we could not demonstrate altered BMP4 expression in vitro after using small interfering RNAs (siRNAs) to reduce SALL4 expression. We conclude that SALL4 hypomorphic variants may influence eye development.

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.)

Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome(TM) (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

Wnt signaling, which encompasses multiple biochemical pathways that regulate neural development downstream of extracellular Wnt glycoprotein ligands, has been suggested to contribute to major psychiatric disorders including autism spectrum disorders (ASD). We used next-generation sequencing and Sequenom genotyping technologies to resequence 10 Wnt signaling pathway genes in 198 ASD patients and 240 matched controls. Results for single-nucleotide polymorphisms (SNPs) of interest were confirmed in a second set of 91 ASD and 144 control samples. We found a significantly increased burden of extremely rare missense variants predicted to be deleterious by PolyPhen-2, distributed across seven genes in the ASD sample (3.5% in ASD vs 0.8% in controls; Fisher's exact test, odds ratio (OR)=4.37, P=0.04). We also found a missense variant in WNT1 (S88R) that was overrepresented in the ASD sample (8 A/T in 267 ASD (minor allele frequency (MAF)=1.69%) vs 1 A/T in 377 controls (MAF=0.13%), OR=13.0, Fisher's exact test, P=0.0048; OR=8.2 and P=0.053 after correction for population stratification). Functional analysis revealed that WNT1-S88R is more active than wild-type WNT1 in assays for the Wnt/β-catenin signaling pathway. Our findings of a higher burden in ASD of rare missense variants distributed across 7 of 10 Wnt signaling pathway genes tested, and of a functional variant at the WNT1 locus associated with ASD, support that dysfunction of this pathway contributes to ASD susceptibility. Given recent findings of common molecular mechanisms in ASD, schizophrenia and affective disorders, these loci merit scrutiny in other psychiatric conditions as well.

Prior studies have demonstrated associations between beta-adrenergic receptor (βAR) polymorphisms and left ventricular dysfunction-an important cause of allograft nonutilization for transplantation. We hypothesized that βAR polymorphisms predispose donor hearts to LV dysfunction after brain death. A total of 1043 organ donors managed from 2001-2006 were initially studied. The following βAR single nucleotide polymorphisms were genotyped: β1AR 1165C/G (Arg389Gly), β1AR 145A/G (Ser49Gly), β2AR 46G/A (Gly16Arg) and β2AR 79C/G (Gln27Glu). In multivariable regression analyses, the β2AR46 SNP was significantly associated with LV systolic dysfunction, with each minor allele additively decreasing the odds for LV ejection fraction <50%. The β1AR1165 and β2AR46 SNPs were associated with higher dopamine requirement during the donor management period: donors with the GG and AA genotypes had ORs of 2.64 (95% CI 1.52-4.57) and 2.70 (1.07-2.74) respectively for requiring >10 μg/kg/min of dopamine compared to those with the CC and GG genotypes. However, no significant associations were found between βAR SNPs and cardiac dysfunction in 364 donors managed from 2007-2008, perhaps due to changes in donor management, lack of power in this validation cohort, or the absence of a true association. βAR polymorphisms may be associated with cardiac dysfunction after brain death, but these relationships require further study in independent donor cohorts., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

An analysis of a case-control study of rhabdomyolysis was conducted to screen for previously unrecognized cytochrome P450 enzyme (CYP) 2C8 inhibitors that may cause other clinically important drug-drug interactions. Medication use in cases of rhabdomyolysis using cerivastatin (n = 72) was compared with that in controls using atorvastatin (n = 287) for the period 1998-2001. The use of clopidogrel was strongly associated with rhabdomyolysis (odds ratio (OR) 29.6; 95% confidence interval (CI), 6.1-143). In a replication effort that used the US Food and Drug Administration (FDA) Adverse Event Reporting System (AERS), it was found that clopidogrel was used more commonly in patients with rhabdomyolysis receiving cerivastatin (17%) than in those receiving atorvastatin (0%, OR infinity; 95% CI = 5.2-infinity). Several medications were tested in vitro for their potential to cause drug-drug interactions. Clopidogrel, rosiglitazone, and montelukast were the most potent inhibitors of cerivastatin metabolism. Clopidogrel and its metabolites also inhibited cerivastatin metabolism in human hepatocytes. These epidemiological and in vitro findings suggest that clopidogrel may cause clinically important, dose-dependent drug-drug interactions with other medications metabolized by CYP2C8.

Multidrug and toxin extrusion 2 (MATE2-K (SLC47A2)), a polyspecific organic cation exporter, facilitates the renal elimination of the antidiabetes drug metformin. In this study, we characterized genetic variants of MATE2-K, determined their association with metformin response, and elucidated their impact by means of a comparative protein structure model. Four nonsynonymous variants and four variants in the MATE2-K basal promoter region were identified from ethnically diverse populations. Two nonsynonymous variants-c.485C>T and c.1177G>A-were shown to be associated with significantly lower metformin uptake and reduction in protein expression levels. MATE2-K basal promoter haplotypes containing the most common variant, g.-130G>A (>26% allele frequency), were associated with a significant increase in luciferase activities and reduced binding to the transcriptional repressor myeloid zinc finger 1 (MZF-1). Patients with diabetes who were homozygous for g.-130A had a significantly poorer response to metformin treatment, assessed as relative change in glycated hemoglobin (HbA1c) (-0.027 (-0.076, 0.033)), as compared with carriers of the reference allele, g.-130G (-0.15 (-0.17, -0.13)) (P=0.002). Our study showed that MATE2-K plays a role in the antidiabetes response to metformin.

Little is known about how genetic variations in enhancers influence drug response. In this study, we investigated whether nucleotide variations in enhancers that regulate drug transporters can alter their expression levels. Using comparative genomics and liver-specific transcription factor binding site (TFBS) analyses, we identified evolutionary conserved regions (ECRs) surrounding nine liver membrane transporters that interact with commonly used pharmaceuticals. The top 50 ECRs were screened for enhancer activity in vivo, of which five--located around ABCB11, SLC10A1, SLCO1B1, SLCO1A2, and SLC47A1--exhibited significant enhancer activity. Common variants identified in a large ethnically diverse cohort (n = 272) were assayed for differential enhancer activity, and three variants were found to have significant effects on reporter activity as compared with the reference allele. In addition, one variant was associated with reduced SLCO1A2 mRNA expression levels in human liver tissues, and another was associated with increased methotrexate (MTX) clearance in patients. This work provides a general model for the rapid characterization of liver enhancers and identifies associations between enhancer variants and drug response.

The TNFAIP3 locus at 6q23, encoding A20, has been associated with multiple autoimmune diseases (AIDs). In this study, we sequence the coding portions of the gene to identify contributing causal polymorphisms that may explain some of the observed associations. A collection of 123 individuals from the Multiple Autoimmune Disease Genetics Consortium (MADGC) collection, each with multiple AIDs (mean=2.2 confirmed diagnoses), and 397 unrelated healthy controls were used for initial sequencing. A total of 32 polymorphisms were identified in the sequencing experiments, including 16 novel and 11 coding variants. Association testing in the entire MADGC collection (1,008 Caucasians with one or more AIDs and 770 unaffected family controls) revealed association of a novel intronic insertion-deletion polymorphism with rheumatoid arthritis (RA) (odds ratio (OR)=2.48, P=0.041). Genotyping of the most common coding polymorphism, rs2230926, in the MADGC collection and additional control individuals revealed a significant association with Sjögren's syndrome (OR=3.38, P=0.038), Crohn's disease (OR=2.25, P=0.041), psoriasis (OR=0.037, P=0.036) and RA (OR=1.9, P=0.025). Finally, haplotype and additional testing of polymorphisms revealed that cases were enriched for 5' and 3' untranslated region variants (one-sided P-value=0.04), but not specifically for common (>2% minor allele frequency), rare, exonic, intronic, non-synonymous or synonymous variants.

Background: Peripheral arterial disease (PAD) is associated with significant morbidity and mortality, and has a higher prevalence in African Americans than Caucasians. Ankle-arm index (AAI) is the ratio of systolic blood pressure in the leg to that in the arm, and, when low, is a marker of PAD., Methods: The authors used an admixture mapping approach to search for genetic loci associated with low AAI. Using data from 1040 African American participants in the observational, population based Health, Aging, and Body Composition Study who were genotyped at 1322 single nucleotide polymorphisms (SNPs) that are informative for African versus European ancestry and span the entire genome, we estimated genetic ancestry in each chromosomal region and then tested the association between AAI and genetic ancestry at each locus., Results: The authors found a region of chromosome 11 that reaches its peak between 80 and 82 Mb associated with low AAI (p<0.001 for rs12289502 and rs9665943, both within this region). 753 African American participants in the observational, population based Cardiovascular Health Study were genotyped at rs9665943 to test the reproducibility of this association, and this association was also statistically significant (odds ratio (OR) for homozygous African genotype 1.59, 95% confidence interval (CI) 1.12 to 2.27). Another candidate SNP (rs1042602) in the same genomic region was tested in both populations, and was also found to be significantly associated with low AAI in both populations (OR for homozygous African genotype 1.89, 95% CI 1.29 to 2.76)., Conclusion: This study identifies a novel region of chromosome 11 representing an area with a potential candidate gene associated with PAD in African Americans.

To elucidate the mechanism of inducing translationally controlled tumor protein (TCTP) in stress adaptation of adenophorean nematodes, the complete coding sequence of TCTP of the infective-stage larvae of Trichinella pseudospiralis was characterized. Two cDNA clones with different 3' untranslated region were identified. Tp-TCTP contained an open reading frame of 534 bp encoding 177 residues. The gene with five introns was expressed as histidine-tagged fusion protein having a molecular mass of 17.5 kDa. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that TCTP RNA was not accumulated when the infective-stage larvae were heat-shocked for 1 h at 45 or 60 degrees C. Using enzyme-linked immunosorbent assay and antiserum against the fusion protein, the expression of TCTP was found to be up-regulated at the translational level. The data suggest that translational regulation of TCTP may play an important role in the early heat-stress adaptation of the trichinellid. Cluster analysis demonstrated that the TCTP sequence of T. pseudospiralis is closely related to that of T. spiralis, but is diverged from the secernentean species.

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.

This unit describes two methods of genotyping DNA sequences containing known nucleotide variations. The first protocol describes a colorimetric method for genotyping DNA samples amplified by the polymerase chain reaction (PCR) using an oligonucleotide ligation assay (OLA). The second protocol describes the ligase chain reaction (LCR), a method for simultaneously amplifying and genotyping genomic DNA samples. A Support Protocol describes the preparation of modified biotin- and digoxigenin-labeled oligonucleotide primers.

Allelic association between pairs of loci is derived in terms of the association probability rho as a function of recombination theta, effective population size N, linear systematic pressure v, and time t, predicting both rho(rt), the decrease of association from founders and rho(ct), the increase by genetic drift, with rho(t) = rho(rt) + rho(ct). These results conform to the Malecot equation, with time replaced by distance on the genetic map, or on the physical map if recombination in the region is uniform. Earlier evidence suggested that rho is less sensitive to variations in marker allele frequencies than alternative metrics for which there is no probability theory. This robustness is confirmed for six alternatives in eight samples. In none of these 48 tests was the residual variance as small as for rho. Overall, efficiency was less than 80% for all alternatives, and less than 30% for two of them. Efficiency of alternatives did not increase when information was estimated simultaneously. The swept radius within which substantial values of rho are conserved lies between 385 and 893 kb, but deviation of parameters between measures is enormously significant. The large effort now being devoted to allelic association has little value unless the rho metric with the strongest theoretical basis and least sensitivity to marker allele frequencies is used for mapping of marker association and localization of disease loci.

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.)

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.

When the temperature and viscosity of the solvent is held constant, the degree of fluorescence polarization (FP) detected when a fluorescent dye is excited by plane polarized light depends mostly on the molecular weight of the dye molecule. By monitoring the FP of a fluorescent dye molecule, one can detect significant changes in the molecular weight of a fluorescent molecule without separation or purification. The 5'-nuclease (TaqMan) assay is a robust single nucleotide polymorphism genotyping method where an allele-specific probe that binds to a perfectly complementary target is cleaved by the 5'-nuclease activity of Taq DNA polymerase. Because the TaqMan probe is labeled with a fluorescent dye, it has high FP value when intact but a low FP value after cleavage. In this study, we compared the results of the 5'-nuclease assay based on standard fluorescence intensity readings and FP readings when genotyping 90 individuals with 20 single nucleotide polymorphisms. Our results show that FP is just as robust and reliable as the standard fluorescence detection method. Use of FP detection makes it possible to reduce the cost of TaqMan probes by abrogating the need for a fluorescence quencher.

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.

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.

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.)

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.

Hundreds of thousands of candidate single nucleotide polymorphisms (SNPs) are being identified as part of the human genome project. For these markers to be useful in any study their allele frequencies in the study population must be known, or much effort will be wasted when markers with the wrong characteristics are selected for studies involving large-scale genotyping of SNP markers. Because allele frequency estimations by genotyping a representative sample of a population is a costly endeavour, an obvious strategy to reduce the cost is to identify accurate and efficient approaches to allele frequency estimation using DNA pools. There are several allele frequency estimation approaches currently in use, including PCR-RFLP analysis, DNA sequencing, the Taqman assay and kinetic PCR. All these approaches give reasonably good allele frequency estimates. Accurate and efficient allele frequency estimation in DNA pools will reduce the cost and effort needed in genetic and association studies and opens up ways to perform pharmacogenomic and evolutionary studies efficiently.

Familial hypobetalipoproteinemia (FHBL) is an apparently autosomal dominant disorder of lipid metabolism characterized by less than fifth percentile age- and sex-specific levels of apolipoprotein beta (apobeta) and low-density lipoprotein-cholesterol. In a minority of cases, FHBL is due to truncation-producing mutations in the apobeta gene on chromosome 2p23-24. Previously, we reported on a four-generation FHBL kindred in which we had ruled out linkage of the trait to the apobeta gene. To locate other loci containing genes for low apobeta levels in the kindred, a genomewide search was conducted. Regions on 3p21.1-22 with two-point LOD scores >1.5 were identified. Additional markers were typed in the region of these signals. Two-point LOD scores in the region of D3S2407 increased to 3.35 at O = 0. GENEHUNTER confirmed this finding with an nonparametric multipoint LOD score of 7.5 (P=.0004). Additional model-free analyses were conducted with the square root of the apobeta level as the phenotype. Results from the Loki and SOLAR programs further confirmed linkage of FHBL to 3p21.1-22. Weaker linkage to a region near D19S916 was also indicated by Loki and SOLAR. Thus, a heretofore unidentified genetic susceptibility locus for FHBL may reside on chromosome 3.

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.

Soluble guanylate cyclase (sGC), the receptor for nitric oxide, is a heterodimer consisting of alpha and beta subunits. We investigated the mRNA species for the alpha(1) subunit in human brain, heart, artery and immortalized B-lymphocytes. Three mRNA species were identified in these tissues. The major mRNA species contained the full expression sequence of the alpha(1) subunit. Two other types of mRNA were detected in which 5' sequences were deleted by splicing (506-590 and 412-590). Each of these deletions included the predicted translation start site, indicating that translation of these two alternatively spliced RNA species does not result in the production of full-length alpha(1) subunits. The relative amounts of the two mRNA species with deletions of the translation start site differed significantly between cell lines of immortalized B-lymphocytes from different individuals. sGC enzymic activity was significantly decreased in cellular extracts from cell lines with high proportions of mRNA species containing the deletion 506-590 when compared with extracts from cell lines that contained mostly mRNA without this deletion.

High-throughput genotyping approaches are being developed to meet the demands of pharmacogenomnics, where numerous individuals are studied with thousands of single nucleotide polymorphism (SNP) markers. All non-gel-based genotyping approaches achieve allelic discrimination by one of four mechanisms: allele-specific hybridisation, allele-specific primer extension, allele-specific oligonucleotide ligation and allele-specific cleavage of a flap probe. By combining one of these allelic discrimination mechanisms with either a homogeneous or solid-phase reaction format and a detection method such as fluorescence intensity, fluorescence polarisation or mass spectrometry, a number of viable high-throughput genotyping methods have been developed and are being readied for routine use. With the biochemistry for robust genotyping in place, good engineering solutions are needed to make high-throughput genotyping a reality.

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.

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.

Darier's disease (DD) is a rare, dominantly inherited disorder that affects the skin producing a variety of types of lesion. Close examination of lesional DD skin shows the presence of abnormal keratinization (epidermal differentiation) and acantholysis (loss of cohesion) of keratinocytes. A number of clinical studies have described the co-occurrence of various neurological and psychiatric symptoms with DD, including mood disorders, epilepsy, mental retardation and a slowly progressive encephalopathy. A single locus for DD has been mapped to chromosome 12q23-q24.1, and a variety of missense, nonsense, frameshift and splicing mutations in the ATP2A2 gene have been described recently in families with DD. This gene encodes the sarcoplasmic/endoplasmic reticulum calcium-pumping ATPase SERCA2, which has a central role in intra-cellular calcium signalling. In this study, we performed mutation analysis on ATP2A2 in 19 unrelated DD patients, of whom 10 had neuropsychiatric phenotypes. We identified and verified 17 novel mutations predicting conservative and non-conservative amino acid changes, potential premature translation terminations and potential altered splicing. Our findings confirm that mutations in ATP2A2 are associated with DD. In neuropsychiatric cases, there was a non-random clustering of mutations in the 3' end of the gene ( P = 0.01), and a predominance of the missense type (70% versus 38% in DD patients). This supports the hypothesis that the DD gene has pleiotropic effects in brain and that mutations in SERCA2 are implicated in the pathogenesis of neuropsychiatric disorders.

Low LDL cholesterol and apoB levels in plasma cosegregate with mutations of apoB in some kindreds with familial hypobetalipoproteinemia. Approximately 35 apoB mutations, many specifying apoB truncations, have been described. Based on the centile nomenclature where the full-length nature apoB consisting of 4536 amino acids is designated as apoB-100, only those truncations of apoB >25% of normal length are detectable in plasma. Previously, we reported on five unrelated kindreds with familial hypobetalipoproteinemia in whom although no apoB truncations were detectable in plasma, low apoB levels were nevertheless linked to the apoB gene. In one of those kindreds, we reported a donor splice site mutation in intron 5 (specifying apoB- 4). We now describe a nonsense mutation in exon 10 (apoB-9) in two of the other unrelated families. Both the apoB-4 and apoB-9 mutations have been reported by others in unrelated families. Recurrent mutations of apoB-40 and apoB-55 also have been reported, suggesting that recurrent mutations of apoB may account for an appreciable proportion of familial hypobetalipoproteinemia kindreds. To test this hypothesis, we searched for four apoB mutations whose products are not detected in plasma including the apoB-4, apoB-9, and two other previously reported mutations in exons 21 and 25. We studied three groups with plasma cholesterols <130 mg/dl in whom no apoB truncations were detected in plasma: a) 28 FHBL probands from St. Louis, b) 151 individual St. Louisians, and c) 28 individual Sicilians. One subject from the 28 kindreds and two subjects among 151 hypobeta individuals from St. Louis harbored the exon 10 mutation. None of the other mutations were detected. Thus, among hypobeta lipoproteinemic subjects without any detectable apoB truncations in plasma, <5% had an apoB truncation-producing mutation. As only about 0.5% of hypobeta lipoproteinemic subjects have plasma-detectable apoB truncations, our data suggest that the known apoB truncations account for only a small proportion of hypocholesterolemia.

A new method for DNA diagnostics based on template-directed primer extension and detection by fluorescence polarization is described. In this method, amplified genomic DNA containing a polymorphic locus is incubated with oligonucleotide primers (designed to hybridize to the DNA template adjacent to the polymorphic site) in the presence of allele-specific dye-labeled dideoxyribonucleoside triphosphates and a commercially available modified Taq DNA polymerase. The primer is extended by the dye-terminator specific for the allele present on the template, increasing approximately 10-fold the molecular weight of the fluorophore. At the end of the reaction, the fluorescence polarization of the two dye-terminators in the reaction mixture are analyzed directly without separation or purification. This homogeneous DNA diagnostic method is shown to be highly sensitive and specific and is suitable for automated genotyping of large number of samples. [The data shown in Figure 3 are available as an online supplement at.]

Single-nucleotide polymorphisms (SNPs) are the most frequently found DNA sequence variations in the human genome. It has been argued that a dense set of SNP markers can be used to identify genetic factors associated with complex disease traits. Because all high-throughput genotyping methods require precise sequence knowledge of the SNPs, any SNP discovery approach must involve both the determination of DNA sequence and allele frequencies. Furthermore, high-throughput genotyping also requires a genomic DNA amplification step, making it necessary to develop sequence-tagged sites (STSs) that amplify only the DNA fragment containing the SNP and nothing else from the rest of the genome. In this report, we demonstrate the utility of a SNP-screening approach that yields the DNA sequence and allele frequency information while screening out duplications with minimal cost and effort. Our approach is based on the use of a homozygous complete hydatidiform mole (CHM) as the reference. With this homozygous reference, one can identify and estimate the allele frequencies of common SNPs with a pooled DNA-sequencing approach (rather than having to sequence numerous individuals as is commonly done). More importantly, the CHM reference is preferable to a single individual reference because it reveals readily any duplicated regions of the genome amplified by the PCR assay before the duplicated sequences are found in GenBank. This approach reduces the cost of SNP discovery by 60% and eliminates the costly development of SNP markers that cannot be amplified uniquely from the genome.

A homogeneous detection mechanism based on fluorescence resonance energy transfer (FRET) has been developed for two DNA diagnostic tests. In the template-directed dye-terminator incorporation (TDI) assay, a donor dye-labeled primer is extended by DNA polymerase using allele-specific, acceptor dye-labeled ddNTPs. In the dye-labeled oligonucleotide ligation (DOL) assay, a donor dye-labeled common probe is joined to an allele-specific, acceptor dye-labeled probe by DNA ligase. Once the donor and acceptor dyes become part of a new molecule, intramolecular FRET is observed over background intermolecular FRET. The rise in FRET, therefore, can be used as an index for allele-specific ddNTP incorporation or probe ligation. Real time monitoring of FRET greatly increases the sensitivity and reliability of these assays. Change in FRET can also be measured by end-point reading when appropriate controls are included in the experiment. FRET detection proves to be a robust method in homogeneous DNA diagnostic assays.

We report a 49-member four-generation kindred in which 11 members express familial hypobetalipoproteinemia (FHBL). In other kindreds, various truncated apoB species cosegregate with the FHBL phenotype. In contrast, no truncated apoB proteins were found by immunoblotting of plasma samples in this kindred. Previous linkage analysis showed strong linkage of FHBL to apoB markers. Nucleotide sequence analysis demonstrated a 665 + 1 G&#95;T transition in the splice donor site of intron 5. This probably alters the accuracy and efficiency of mRNA splicing leading to the extremely low apoB levels in plasma. In addition, we detected four novel polymorphisms in the apoB gene.

Establishing the pattern in peak heights within local sequence contexts improves the accuracy of base calling and the identification of DNA sequence variations in dye-terminator cycle sequencing. We have systematically examined pairs of sequence-tagged sites (STSs) that vary at only a single nucleotide to determine how base changes influence the peak heights of neighboring bases in sequencing traces generated by two recently commercialized dye-terminator chemistries, the dichloro-rhodamine (dRhodamine) and the energy transfer (BigDye) terminators. For sequencing traces generated with the dRhodamine terminators, the peak height of a particular base in 28 of 64 possible 3-base windows (44%) can be predicted by knowing just one or two bases 5' to the base in question. For those generated with the BigDye terminators, the peak height of a particular base in 23 of 64 possible 3-base windows (36%) can be predicted by knowing the local sequence context. When the peak heights are binned slightly differently, 75% (48 out of 64 cases) of the base peaks generated by both dRhodamine and BigDye terminators fall in the middle half, confirming that the peak patterns of these two new dye terminator chemistries are much more even than those found in the original rhodamine dye terminator sequences.

An efficient strategy to develop a dense set of single-nucleotide polymorphism (SNP) markers is to take advantage of the human genome sequencing effort currently under way. Our approach is based on the fact that bacterial artificial chromosomes (BACs) and P1-based artificial chromosomes (PACs) used in long-range sequencing projects come from diploid libraries. If the overlapping clones sequenced are from different lineages, one is comparing the sequences from 2 homologous chromosomes in the overlapping region. We have analyzed in detail every SNP identified while sequencing three sets of overlapping clones found on chromosome 5p15.2, 7q21-7q22, and 13q12-13q13. In the 200.6 kb of DNA sequence analyzed in these overlaps, 153 SNPs were identified. Computer analysis for repetitive elements and suitability for STS development yielded 44 STSs containing 68 SNPs for further study. All 68 SNPs were confirmed to be present in at least one of the three (Caucasian, African-American, Hispanic) populations studied. Furthermore, 42 of the SNPs tested (62%) were informative in at least one population, 32 (47%) were informative in two or more populations, and 23 (34%) were informative in all three populations. These results clearly indicate that developing SNP markers from overlapping genomic sequence is highly efficient and cost effective, requiring only the two simple steps of developing STSs around the known SNPs and characterizing them in the appropriate populations.