Low-frequency Single Nucleotide Polymorphisms Research Articles

Plasmodium malariae structure and genetic diversity in sub-Saharan Africa determined from microsatellite variants and linked SNPs in orthologues of antimalarial resistance genes

Plasmodium malariae, a neglected human malaria parasite, contributes up to 10% of malaria infections in sub-Saharan Africa (sSA). Though P. malariae infection is considered clinically benign, it presents mostly as coinfections with the dominant P. falciparum. Completion of its reference genome has paved the way to further understand its biology and interactions with the human host, including responses to antimalarial interventions. We characterized 75 P. malariae isolates from seven endemic countries in sSA using highly divergent microsatellites. The P. malariae infections were highly diverse and five subpopulations from three ancestries (independent of origin of isolates) were determined. Sequences of 11 orthologous antimalarial resistance genes, identified low frequency single nucleotide polymorphisms (SNPs), strong linkage disequilibrium between loci that may be due to antimalarial drug selection. At least three sub-populations were detectable from a subset of denoised SNP data from mostly the mitochondrial cytochrome b coding region. This evidence of diversity and selection calls for including P. malariae in malaria genomic surveillance towards improved tools and strategies for malaria elimination.

Abstract 490: Single nucleotide variations within and around microRNA-binding sites

Abstract * equally contributed Grant #5P20GM103443 (NIGMS). Single nucleotide polymorphisms (SNPs) can either create or destroy microRNA binding sites. We analyzed 200 previously reported cancer associated SNPs within microRNA binding sites and found that more than 90% of them were surrounded by single and multiple low-frequency SNPs. The low-frequency SNPs were positioned within the expected microRNAs seed matching areas (58%), within the whole microRNA matching regions (71% incidence), and within the distance where they potentially can affect microRNA-mRNA interaction (36% incidence). We further analyzed the presence of SNPs within microRNA-binding sites in the 3'UTRs of mRNAs encoding the human V-set domain containing T-cell activation inhibitor 1 (VTCN1) and an AT-rich interaction domain 5B (ARID5B). In VTCN1 single SNPs were present in 36% of microRNAs seed matching areas, and two and more SNPs in 8% of microRNA seed matching sites. For ARID5B, single SNPs were present in 42%, and two and more SNPs in 26% of microRNA seed matching sites. In both VTCN1 and ARID5B, some microRNA seed matching areas harbored as many as 4 SNPs. The predicted binding site for microRNA-6870-5p (miR-6870) within the VTCN1 3'UTR consists of 11 uninterrupted and 2 additional Watson-Crick pairs. The length of mature miR-6870 is 19 bases, and the corresponding VTCN1 mRNA fragment harbors 6 nucleotide variations: rs758251859, rs1001277215, rs551576201, rs539444165, rs949692788, and indel rs9055595515. Hypothetically, the rs1001277215 minor allele (MA) eliminates the miR-6870-mRNA complementarity between the corresponding nucleotides. The rs551576201 MA weakens miR-6870-mRNA complementarity by creating a wobble G-U pair, the rs949692788 MA enhances miR-6870-mRNA complementarity by switching from a non-canonical G-U to canonical Watson-Crick A-U pair, and for indel rs9055595515, the absence of deletion preserves miR-6870-mRNA complementarity between CA and GU nucleotides. The probabilities of the “best” and “worst” matches between miR-6870 and VTCN1 mRNA are 0.009995% and 0.000006% correspondingly. The binding between miR-6870 and VTCN1mRNA may also be affected by variations between A and G within rs758251859 (MA frequency is unknown). As microRNA-binding efficacy depends on the mRNA sequences outside the target region, the indels rs35182629 (located 5 nucleotides upstream and covers 2 bases) and rs896747700 (located 1 nucleotide downstream and covers 1 base) may also impact miR-6870-mediated regulation of VTCN1 expression. The possible coincidence of hyper-functional or completely disabled microRNA-binding sites may result in significant phenotypic variations and predisposition to cancers. Citation Format: Amber Budmark*, Michael Catalano*, Tyrel Haley*, Brady Hicks*, Maria Koenen*, Thea Patrick*, Tyler Larson*, Tyler Wagner*, Clark Butler, Joshua Feiner, Rebecca Frick, Sierra Haage, James Miller, Mackayla Nohr, Dillon Stadlman, Dillon Turner, Sara Husher, Nicholas Woslum, Nathan Stadem, John Dosch, Tyler Fortuna, Chandler Fredrich, Elise Hadley, Brooklynn Oehlerking, Delayna Paulson, Cal Wiese, Paula Mazzer, Tim Mullican, Cynthia Anderson, Mark Larson, Elena Paryiskaya, Alexandra Kharazova, Paola Vermeer, Samuel Milanovich, Alexei Savinov, John Collins, Alexander Kofman. Single nucleotide variations within and around microRNA-binding sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 490.

The Trouble with MEAM2: Implications of Pseudogenes on Species Delimitation in the Globally Invasive Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species Complex.

Molecular species identification using suboptimal PCR primers can over-estimate species diversity due to coamplification of nuclear mitochondrial (NUMT) DNA/pseudogenes. For the agriculturally important whitefly Bemisia tabaci cryptic pest species complex, species identification depends primarily on characterization of the mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene. The lack of robust PCR primers for the mtDNA COI gene can undermine correct species identification which in turn compromises management strategies. This problem is identified in the B. tabaci Africa/Middle East/Asia Minor clade which comprises the globally invasive Mediterranean (MED) and Middle East Asia Minor I (MEAM1) species, Middle East Asia Minor 2 (MEAM2), and the Indian Ocean (IO) species. Initially identified from the Indian Ocean island of Réunion, MEAM2 has since been reported from Japan, Peru, Turkey and Iraq. We identified MEAM2 individuals from a Peruvian population via Sanger sequencing of the mtDNA COI gene. In attempting to characterize the MEAM2 mitogenome, we instead characterized mitogenomes of MEAM1. We also report on the mitogenomes of MED, AUS, and IO thereby increasing genomic resources for members of this complex. Gene synteny (i.e., same gene composition and orientation) was observed with published B. tabaci cryptic species mitogenomes. Pseudogene fragments matching MEAM2 partial mtDNA COI gene exhibited low frequency single nucleotide polymorphisms that matched low copy number DNA fragments (<3%) of MEAM1 genomes, whereas presence of internal stop codons, loss of expected stop codons and poor primer annealing sites, all suggested MEAM2 as a pseudogene artifact and so not a real species.

High density genome wide genotyping-by-sequencing and association identifies common and low frequency SNPs, and novel candidate genes influencing cow milk traits.

High-throughput sequencing technologies have increased the ability to detect sequence variations for complex trait improvement. A high throughput genome wide genotyping-by-sequencing (GBS) method was used to generate 515,787 single nucleotide polymorphisms (SNPs), from which 76,355 SNPs with call rates >85% and minor allele frequency ≥1.5% were used in genome wide association study (GWAS) of 44 milk traits in 1,246 Canadian Holstein cows. GWAS was accomplished with a mixed linear model procedure implementing the additive and dominant models. A strong signal within the centromeric region of bovine chromosome 14 was associated with test day fat percentage. Several SNPs were associated with eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, CLA:9c11t and gamma linolenic acid. Most of the significant SNPs for 44 traits studied are novel and located in intergenic regions or introns of genes. Novel potential candidate genes for milk traits or mammary gland functions include ERCC6, TONSL, NPAS2, ACER3, ITGB4, GGT6, ACOX3, MECR, ADAM12, ACHE, LRRC14, FUK, NPRL3, EVL, SLCO3A1, PSMA4, FTO, ADCK5, PP1R16A and TEP1. Our study further demonstrates the utility of the GBS approach for identifying population-specific SNPs for use in improvement of complex dairy traits.

Abstract 16: Impact of germline TP53 mutations and polymorphisms in women with premenopausal breast cancer

Abstract Aim: Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome that confers a high lifetime risk of developing malignancies, including pre-menopausal breast cancer. Although many families with classic LFS phenotypes harbor germline TP53 mutations, approximately 25% do not. We observe a high frequency of polymorphic TP53 variants in LFS patients not found to harbor deleterious mutations. We sought to determine if the spectrum of TP53 variants in pre-menopausal breast cancer patients influences cancer risk. Methods: Germline DNA was obtained from whole blood of 90 ethnically diverse patients diagnosed with pre-menopausal breast cancer, as well as patients diagnosed with breast cancer in addition to another primary malignancy. The median age at first cancer diagnosis was 35 years. Sanger sequencing of exons 2-11, and flanking intron regions of the TP53 gene was performed. HapMap population frequencies for TP53 polymorphisms were used as reference values. Results: Five of 90 patients in our study were found to harbor known deleterious mutations in TP53, consistent with LFS. The most commonly observed single nucleotide polymorphism (SNP) across all samples was the c.215C&amp;gt;G (p.Pro72Arg) in exon 4 (rs1042522). This polymorphism is a common variant that has G allele frequencies of 76.7%, 51.1% and 33.1% in European, Asian (Han Chinese) and Sub-Saharan African populations, respectively. We observe a G allele frequency of 66.0% in our study group. There was no significant difference in age at cancer diagnosis between any of the three genotypic groups (C/C: 33 years; C/G: 34 years: G/G: 36 years). Three additional low-frequency SNPs in TP53 were observed: the exon 4: c.108G&amp;gt;A (p.Pro36Pro) variant was found in 2 patients, exon 6: c.639A&amp;gt;G (p.Arg213Arg) was found in 9 patients and intron 9: c.993+12T&amp;gt;C was found in 4 patients. There was no observed distinct signature of TP53 polymorphism spectrum in patients who developed multiple primary malignancies. Discussion: This study represents a genetic survey of TP53 in pre-menopausal breast cancer patients of diverse ethnicities. TP53 polymorphisms alone were not sufficient to make clinical predictions about age at breast cancer diagnosis. The TP53 polymorphism, rs1042522, was observed in a large proportion of patients in this study, consistent with its reported frequencies in SNP databases. Parallel studies of CpG methylation of the TP53 promoter and shore regions flanking the locus are being conducted to investigate alternate hypotheses linking TP53 and risk of early-onset breast cancer in the absence of germline TP53 mutations. Citation Format: Nardin Samuel, Ana Novokmet, Thomas J. Hudson, David Malkin. Impact of germline TP53 mutations and polymorphisms in women with premenopausal breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 16. doi:10.1158/1538-7445.CANSUSC14-16

No evidence for mutations in NLRP7, NLRP2 or KHDC3L in women with unexplained recurrent pregnancy loss or infertility.

Are mutations in NLRP2/7 (NACHT, LRR and PYD domains-containing protein 2/7) or KHDC3L (KH Domain Containing 3 Like) associated with recurrent pregnancy loss (RPL) or infertility? We found no evidence for mutations in NLRP2/7 or KHDC3L in unexplained RPL or infertility. Mutations in NLRP7 and KHDC3L are known to cause biparental hydatidiform moles (BiHMs), a rare form of pregnancy loss. NLRP2, while not associated with the BiHM pathology, is known to cause recurrent Beckwith Weidemann Syndrome (BWS). Ninety-four patients with well characterized, unexplained infertility were recruited over a 9-year period from three IVF clinics in Sweden. Blood samples from 24 patients with 3 or more consecutive miscarriages of unknown etiology were provided by the Recurrent Miscarriage Clinic at St Mary's Hospital, London, UK. Patients were recruited into both cohorts following extensive clinical studies. Genomic DNA was isolated from peripheral blood and subject to Sanger sequencing of NLRP2, NLRP7 and KHDC3L. Sequence electropherograms were analyzed by Sequencher v5.0 software and variants compared with those observed in the 1000 Genomes, single nucleotide polymorphism database (dbSNP) and HapMap databases. Functional effects of non-synonymous variants were predicted using Polyphen-2 and sorting intolerant from tolerant (SIFT). No disease-causing mutations were identified in NLRP2, NLRP7 and KHDC3L in our cohorts of unexplained infertility and RPL. Due to the limited patient size, it is difficult to conclude if the low frequency single nucleotide polymorphisms observed in the present study are causative of the phenotype. The design of the present study therefore is only capable of detecting highly penetrant mutations. The present study supports the hypothesis that mutations in NLRP7 and KHDC3L are specific for the BiHM phenotype and do not play a role in other adverse reproductive outcomes. Furthermore, to date, mutations in NLRP2 have only been associated with the imprinting disorder BWS in offspring and there is no evidence for a role in molar pregnancies, RPL or unexplained infertility. This study was funded by the following sources: Estonian Ministry of Education and Research (Grant SF0180044s09), Enterprise Estonia (Grant EU30020); Mentored Resident research project (Department of Obstetrics and Gynecology, Baylor College of Medicine); Imperial NIHR Biomedical Research Centre; Grant Number C06RR029965 from the National Center for Research Resources (NCCR; NIH). No competing interests declared.

Abstract 3333: In vitro-based biochemical studies of erbb2 gene variation to address racial disparities in breast cancer mortality

Abstract According to a 2012 study by the U.S. Center for Disease Control and Prevention (CDC), African-American women had a 41% higher rate of breast cancer mortality during 2005-2009 than did Caucasian women. Although overall cancer mortality rates have declined significantly in the U.S. since the early 1990s, the black-white mortality gap has been steadily growing. The underlying causes of this racial disparity are likely diverse, and include factors such as lower quality of care and reduced access to mammographic screening. However, more aggressive tumor biology may also contribute significantly to racial disparity in breast cancer outcomes. Recently, a low frequency single nucleotide polymorphism (SNP) of the HER2 receptor tyrosine kinase, W452C, which occurs predominantly in African-American women (about 10% frequency), was significantly correlated with breast cancer. This study reported that tumors from patients harboring W452C do not amplify the erbB2 (HER2) gene or overexpress the protein, suggesting that this variant may contribute to breast cancer development through a mechanism distinct from the amplification common to HER2-positive breast cancer patients. Understanding the structural and functional differences associated with W452C will allow us to identify new strategies for customized breast cancer treatments for African American women. To address the role of the W452C SNP in breast cancer we applied a cell-free in vitro reconstitution system that uses nanolipoprotein particles (NLPs) to solubilize and support functional membrane proteins. Cell-free produced wild type HER2 and W452C were tested for tyrosine phosphorylation as well as specific binding to therapeutic anti-HER2 monoclonal antibodies trastuzumab and pertuzumab. Our results showed comparable tyrosine phosphorylation levels for both wild type and W452C HER2, suggesting that the variant itself might not alone be a driver of cancer. We observed a decreased binding affinity of trastuzumab for W452C compared to wild type HER2, suggesting that W452-positive patients might not respond to trastuzumab treatment. On the other hand, we found that W452C has a higher affinity for pertuzumab. Overall, our studies suggest that the W452C variant may be differentially sensitive to clinically pertinent therapeutic agents. Further characterization of this variant will be important for the development of more effective and precise therapeutic interventions. This in turn could lead to targeted measures that help address racial disparity problems in breast cancer mortality. Citation Format: Wei He, Matthew Saldana, Tiffany Scharadin, Steven Hoang-Phou, Denise Trans, Dennis Chang, Kermit Carraway, Paul Henderson, Matthew A. Coleman. In vitro-based biochemical studies of erbb2 gene variation to address racial disparities in breast cancer mortality. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3333. doi:10.1158/1538-7445.AM2014-3333

Rare variant analysis of blood pressure phenotypes in the Genetic Analysis Workshop 18 whole genome sequencing data using sequence kernel association test.

Sequence kernel association test (SKAT) has become one of the most commonly used nonburden tests for analyzing rare variants. Performance of burden tests depends on the weighting of rare and common variants when collapsing them in a genomic region. Using the systolic and diastolic blood pressure phenotypes of 142 unrelated individuals in the Genetic Analysis Workshop 18 data, we investigated whether performance of SKAT also depends on the weighting scheme. We analyzed the entire sequencing data for all 200 replications using 3 weighting schemes: equal weighting, Madsen-Browning weighting, and SKAT default linear weighting. We considered two options: all single-nucleotide polymorphisms (SNPs) and only low-frequency SNPs. A SKAT default weighting scheme (which heavily downweights common variants) performed better for the genes in which causal SNPs are mostly rare. This SKAT default weighting scheme behaved similarly to other weighting schemes after eliminating all common SNPs. In contrast, the equal weighting scheme performed the best for MAP4 and FLT3, both of which included a common variant with a large effect. However, SKAT with all 3 weighting schemes performed poorly. Overall power across all causal genes was about 0.05, which was almost identical to the type I error rate. This poor performance is partly due to a small sample size because of the need to analyze only unrelated individuals. Because a half of causal SNPs were not found in the annotation file based on the 1000 Genomes Project, we suspect that performance was also affected by our use of incomplete annotation information.

SAT0012 Estimating heritability of response to treatment with anti-TNF biologic agents using linear mixed models

BackgroundThe effectiveness of treatment with anti-TNF inhibitors in rheumatoid arthritis (RA) is variable with a significant minority of patients experiencing poor response. Ideally clinicians would like to identify which patients...

104 Probing DNA shape on molecular and genomic scales

Linear DNA sequence encodes its three-dimensional structure. The resulting DNA shape gives DNA unique physical and chemical properties that are explored by DNA-binding proteins (Rohs et al., 2009). Probing DNA shape, therefore, helps to reveal the origin of specificity in DNA-protein recognition. We have developed a Monte Carlo (MC) method for all-atom DNA structure prediction (Joshi et al., 2007). With key components such as parallelization and functionality to handle chemical modifications added, the revamped MC method can efficiently recover the DNA shape of any DNA sequence. To embrace the genomic era, we have trained a high-throughput (HT) prediction method on 2121 independent MC simulations (Slattery et al., 2011). Applying this method on whole genome DNA sequences of 66 Drosophila individuals, we found that among single nucleotide polymorphisms (SNPs) within the immediate vicinity of in vivo transcription factor (TF) binding sites, low-frequency SNPs tend to change the DNA minor groove width more than high-frequency SNPs. This indicates that shape-changing mutations are detrimental to TF binding and in vivo function. We also examined the dependency of DNase I cleavage events on DNA shape using our HT method. Our results show that the rate of DNase I cleavage closely tracks the width of the minor groove. MC predictions for chemically modified bases further demonstrate that cytosine methylation enhances cleavage directly adjacent to CpG dinucleotides through narrowing of the minor groove. In summary, with tools on hand to address biological questions on both molecular and genomic scales, our work provides new insights into gene regulation and evolution.

Abstract 64: Fine mapping and in silico analysis of the 8q24.21 region for glioma identifies a low-frequency risk variant insight into etiological basis of glioma.

Abstract Glioma comprises ∼40% of all primary brain tumors and most are associated with a poor prognosis irrespective of clinical care, with the most common type of glioma, glioblastoma (GBM), having a median overall survival of only ˜15 months. A number of glioma subtypes defined in part by malignancy grade (e.g., pilocytic astrocytomas WHO grade 1, diffuse “low-grade” gliomas WHO grade 2, anaplastic gliomas WHO grade 3, and GBM WHO grade 4) can be distinguished. Accumulating data indicate that these subtypes have different molecular genetic profiles possibly resulting from different etiologic pathways, which might be shared by different tumor subtypes or be type specific. We have previously demonstrated using a genome-wide association study (GWAS) strategy that genetic variation at 5p15.33, 8q24.21, 9p21.3, 11q23.3 and 20q13.33 influences the risk of glioma. We have sought to fine-map the location of the functional basis of the 8q24.21 association using data from four different non-overlapping GWASs of glioma, comprising a total of 4,147 glioma cases and 7,435 controls. To improve marker density across the 800kb region of association, we imputed genotypes using data from the 1000 Genomes Project and Hapmap3 data sets and high-density (80×) sequencing data generated on 250 CEU individuals. This combination of high-density genotyping and imputation allowed a wide and deep examination of SNPs with minor allele frequency (MAF) &amp;gt; 0.01 in this region. Coupled with these studies we performed a bioinformatic analysis of the most strongly associated variant to provide insight into the functional basis for the association. The region contained a number of single nucleotide polymorphisms (SNPs) showing a clearly stronger association signal than the original tagSNP rs4295627, notably the low frequency SNP rs55705857 (MAF=0.05 in the European population) which was sufficient to capture the 8q24.21 association (P= 2.24 x10-38). This 8q24 association is primarily for low-grade glioma, stronger for 1p/19q deletion oligodendroglioma and is associated with IDH mutation glioma. Functional annotation showed rs55705857 maps to a highly-evolutionarily conserved sequence predicted to impact on OCT1 binding. Glioma incidence varies between countries and is much lower in individuals of African/Asian descent than European. It is possible that these differences reflect differences in genetic predisposition. In this regard it intriguing that the rs55705857 is monomorphic in Asians, and has a MAF 30 times lower in Africans compared to Europeans. Citation Format: Victor Enciso-mora, Yanhong Liu, Fay Hosking, Richard Houlston, Melissa Bondy. Fine mapping and in silico analysis of the 8q24.21 region for glioma identifies a low-frequency risk variant insight into etiological basis of glioma. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 64.

Deep Resequencing Unveils Genetic Architecture of ADIPOQ and Identifies a Novel Low-Frequency Variant Strongly Associated With Adiponectin Variation

Increased adiponectin levels have been shown to be associated with a lower risk of type 2 diabetes. To understand the relations between genetic variation at the adiponectin-encoding gene, ADIPOQ, and adiponectin levels, and subsequently its role in disease, we conducted a deep resequencing experiment of ADIPOQ in 14,002 subjects, including 12,514 Europeans, 594 African Americans, and 567 Indian Asians. We identified 296 single nucleotide polymorphisms (SNPs), including 30 amino acid changes, and carried out association analyses in a subset of 3,665 subjects from two independent studies. We confirmed multiple genome-wide association study findings and identified a novel association between a low-frequency SNP (rs17366653) and adiponectin levels (P = 2.2E–17). We show that seven SNPs exert independent effects on adiponectin levels. Together, they explained 6% of adiponectin variation in our samples. We subsequently assessed association between these SNPs and type 2 diabetes in the Genetics of Diabetes Audit and Research in Tayside Scotland (GO-DARTS) study, comprised of 5,145 case and 6,374 control subjects. No evidence of association with type 2 diabetes was found, but we were also unable to exclude the possibility of substantial effects (e.g., odds ratio 95% CI for rs7366653 [0.91–1.58]). Further investigation by large-scale and well-powered Mendelian randomization studies is warranted.

Synthetic Associations Are Unlikely to Account for Many Common Disease Genome-Wide Association Signals

The goal of human disease genetics is to connect genetic variation with disease risk, but the optimal study design for gene mapping varies widely with the underlying genetic architecture of the disease. Family based linkage studies made the identification of genetic defects that directly cause rare single gene diseases, like cystic fibrosis and sickle cell disease, routine. By contrast, the goal of identifying the genes that explain the heritability of complex diseases has remained elusive because they are affected by a host of genetic and environmental factors. More recently, however, genome-wide association studies (GWAS) have identified many common variants associated with complex traits. In some cases these studies have provided valuable insight into disease pathogenesis [1]–[3] but each associated variant often confers only a modest increase in risk (odds ratios [ORs] typically range from 1.1 to 1.5). One consequence of these small effects is that, even in aggregate, these discoveries fail to explain most of the heritability of complex disease. Identifying sources of this missing heritability is one of the most active areas of complex disease genetics research [4], and the relative contribution of rare ( 5%) causal variants remains unknown. GWAS have deeply probed the role of common variation by exploiting the fact that a subset of single nucleotide polymorphisms (SNPs) can act as proxies for (or “tag”) the majority of common SNPs. This efficiency has allowed inexpensive microarrays, which directly genotype half a million SNPs, to indirectly capture >80% of common variation [5]. While this design underpins the success of the GWAS approach, it also presents a drawback: associations to rare and low frequency SNPs are typically missed because they do not have a strongly correlated tag on GWAS chips. Dickson et al. [6], in a recent simulation-based experiment, argue that common variant associations arising from GWAS may actually reflect multiple low frequency causal variants rather than a single common causal variant (Figure 1). They demonstrate that “synthetic associations,” where a cluster of low-frequency, highly penetrant mutations occurs stochastically more frequently with one allele than the other at a common SNP, can cause association signals at these common variants. If synthetic associations were widespread they could in theory explain an appreciable fraction of the missing heritability. Furthermore, the proportion of GWAS signals attributable to synthetic associations has profound implications for the design of GWAS follow-up studies. Therefore, while Dickson et al. argue that synthetic associations are an “obvious theoretical possibility,” it is worthwhile to broadly assess, in light of other theoretical and empirical evidence, the prevalence of synthetic associations in complex human disease. Figure 1 SNPs in the NOD2 region as an example of synthetic association.

DNA Suspension Arrays: Silencing Discrete Artifacts for High-Sensitivity Applications

Detection of low frequency single nucleotide polymorphisms (SNPs) has important implications in early screening for tumorgenesis, genetic disorders and pathogen drug resistance. Nucleic acid arrays are a powerful tool for genome-scale SNP analysis, but detection of low-frequency SNPs in a mixed population on an array is problematic. We demonstrate a model assay for HIV-1 drug resistance mutations, wherein ligase discrimination products are collected on a suspension array. In developing this system, we discovered that signal from multiple polymorphisms was obscured by two discrete hybridization artifacts. Specifically: 1) tethering of unligated probes on the template DNA elicited false signal and 2) unpredictable probe secondary structures impaired probe capture and suppressed legitimate signal from the array. Two sets of oligonucleotides were used to disrupt these structures; one to displace unligated reporter labels from the bead-bound species and another to occupy sequences which interfered with array hybridization. This artifact silencing system resulted in a mean 21-fold increased sensitivity for 29 minority variants of 17 codons in our model assay for mutations most commonly associated with HIV-1 drug resistance. Furthermore, since the artifacts we characterized are not unique to our system, their specific inhibition might improve the quality of data from solid-state microarrays as well as from the growing number of multiple analyte suspension arrays relying on sequence-specific nucleic acid target capture.

Multiple Rare Variants as a Cause of a Common Phenotype: Several Different Lactase Persistence Associated Alleles in a Single Ethnic Group

Persistence of intestinal lactase into adulthood allows humans to use milk from other mammals as a source of food and water. This genetic trait has arisen by convergent evolution and the derived alleles of at least three different single nucleotide polymorphisms (-13910C>T, -13915T>G, -14010G>C) are associated with lactase persistence in different populations. Each allele occurs on an extended haplotype, consistent with positive directional selection. The SNPs are located in an 'enhancer' sequence in an intron of a neighboring gene (MCM6) and modulate lactase transcription in vitro. However, a number of lactase persistent individuals carry none of these alleles, but other low-frequency single nucleotide polymorphisms have been observed in the same region. Here we examine a cohort of 107 milk-drinking Somali camel-herders from Ethiopia. Eight polymorphic sites are identified in the enhancer. -13915*G and -13907*G (a previously reported candidate) are each significantly associated with lactase persistence. A new allele, -14009*G, has borderline association with lactase persistence, but loses significance after correction for multiple testing. Sequence diversity of the enhancer is significantly higher in the lactase persistent members of this and a second cohort compared with non-persistent members of the two groups (P = 7.7 x 10(-9) and 1.0 x 10(-3)). By comparing other loci, we show that this difference is not due to population sub-structure, demonstrating that increased diversity can accompany selection. This contrasts with the well-documented observation that positive selection decreases diversity by driving up the frequency of a single advantageous allele, and has implications for association studies.

The Promoter Region of theMDR1Gene Is Largely Invariant, but Different Single Nucleotide Polymorphism Haplotypes Affect MDR1 Promoter Activity Differently in Different Cell Lines

The MDR1 multidrug transporter represents one of the better characterized drug transporters that play an important role in protecting the body against xenobiotic insults. Single nucleotide polymorphisms (SNPs) and SNP haplotypes within this gene have been variously associated with differences in MDR1 expression/function, drug response as well as disease susceptibility. Nonetheless, the effect of polymorphisms at the MDR1 promoter region on its promoter activity remains less characterized. Through the examination of approximately 1.5 kilobases of MDR1 promoter region from five populations, including the Chinese, Malays, Indians, European Americans, and African Americans, we identified eight low-frequency SNPs, of which only two were polymorphic in at least four of the five populations examined. The other SNPs are mainly population-specific, the majority of which occur only in the African-American population. Recapitulation of the various combinations of SNP haplotypes in vitro in promoter-reporter assays revealed a few notable trends. The African and European American-specific haplotypes tended to result in enhanced MDR1 promoter activity only in the human embryonic kidney (HEK) 293 cell line. Haplotype GCTAACC, which occurs at variable frequencies in all the populations examined, with Asians having much lower frequencies (<2%) compared with the European Americans/African Americans (>4%), affected MDR1 promoter activity differently in different cell lines. Compared with the commonest haplotype, GCTA-ACC haplotype resulted in a significant decrease in MDR1 promoter activity in HeLa cells (P < 0.05) but a significant increase in the same promoter activity in HEK293 cells (P < 0.05). These results suggest that the MDR1 promoter region is largely invariant but that different haplotypes have differential effects on the MDR1 promoter activity in different cell lines.

The dilemma of screening for antibodies against low‐frequency human platelet antigens

The dilemma of screening for antibodies against low‐frequency human platelet antigens

SNP Discovery in Pooled Samples With Mismatch Repair Detection

A targeted discovery effort is required to identify low frequency single nucleotide polymorphisms (SNPs) in human coding and regulatory regions. We here describe combining mismatch repair detection (MRD) with dideoxy terminator sequencing to detect SNPs in pooled DNA samples. MRD enriches for variant alleles in the pooled sample, and sequencing determines the nature of the variants. By using a genomic DNA pool as a template, approximately 100 fragments were amplified and subsequently combined and subjected en masse to the MRD procedure. The variant-enriched pool from this one MRD reaction is enriched for the population variants of all the tested fragments. Each fragment was amplified from the variant-enriched pool and sequenced, allowing the discovery of alleles with frequencies as low as 1% in the initial population. Our results support that MRD-based SNP discovery can be used for large-scale discovery of SNPs at low frequencies in a population.