TAGster: efficient selection of LD tag SNPs in single or multiple populations

Genetic association studies increasingly rely on the use of linkage disequilibrium (LD) tag SNPs to reduce genotyping costs. We developed a software package TAGster to select, evaluate and visualize LD tag SNPs both for single and multiple populations. We implement several strategies to improve the efficiency of current LD tag SNP selection algorithms: (1) we modify the tag SNP selection procedure of Carlson et al. to improve selection efficiency and further generalize it to multiple populations. (2) We propose a redundant SNP elimination step to speed up the exhaustive tag SNP search algorithm proposed by Qin et al. (3) We present an additional multiple population tag SNP selection algorithm based on the framework of Howie et al., but using our modified exhaustive search procedure. We evaluate these methods using resequenced candidate gene data from the Environmental Genome Project and show improvements in both computational and tagging efficiency. AVAILABILITY: The software Package TAGster is freely available at http://www.niehs.nih.gov/research/resources/software/tagster/     

118 DNA structure-induced genetic instability in mammals

Naturally occurring repetitive DNA sequences can adopt alternative (i.e. non-B) DNA secondary structures, and often co-localize with chromosomal breakpoint “hotspots,” implicating non-B DNA in translocation-related cancer etiology. We have found that sequences capable of adopting H-DNA and Z-DNA structures are intrinsically mutagenic in mammals. For example, an endogenous H-DNA-forming sequence from the human c-MYC promoter and a model Z-DNA-forming CpG repeat induced genetic instability in mammalian cells, largely in the form of deletions resulting from DNA double-strand breaks (Wang & Vasquez, 2004; Wang et al., 2006). Characterization of the mutants revealed microhomologies at the breakpoints, consistent with a microhomology-mediated end-joining repair of the double-strand breaks (Kha et al., 2010). We have constructed transgenic mutation-reporter mice containing these human H-DNA- and Z-DNA-forming sequences to determine their effects on genomic instability in a chromosomal context in a living organism (Wang et al., 2008). Initial results suggest that both H-DNA- and Z-DNA-forming sequences induced genetic instability in mice, suggesting that these non-B DNA structures represent endogenous sources of genetic instability and may contribute to disease etiology and evolution. Our current studies are designed to determine the mechanisms of DNA structure-induced genetic instability in mammals; the roles of helicases, polymerases, and repair enzymes in H-DNA and Z-DNA-induced genetic instability will be discussed.     

Allele frequency matching between SNPs reveals an excess of linkage disequilibrium in genic regions of the human genome

Significant interest has emerged in mapping genetic susceptibility for complex traits through whole-genome association studies. These studies rely on the extent of association, i.e., linkage disequilibrium (LD), between single nucleotide polymorphisms (SNPs) across the human genome. LD describes the nonrandom association between SNP pairs and can be used as a metric when designing maximally informative panels of SNPs for association studies in human populations. Using data from the 1.58 million SNPs genotyped by Perlegen, we explored the allele frequency dependence of the LD statistic r(2) both empirically and theoretically. We show that average r(2) values between SNPs unmatched for allele frequency are always limited to much less than 1 (theoretical approximately 0.46 to 0.57 for this dataset). Frequency matching of SNP pairs provides a more sensitive measure for assessing the average decay of LD and generates average r(2) values across nearly the entire informative range (from 0 to 0.89 through 0.95). Additionally, we analyzed the extent of perfect LD (r(2) = 1.0) using frequency-matched SNPs and found significant differences in the extent of LD in genic regions versus intergenic regions. The SNP pairs exhibiting perfect LD showed a significant bias for derived, nonancestral alleles, providing evidence for positive natural selection in the human genome.     

A novel role for Greatwall kinase in recovery from DNA damage

Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression (Halazonetis et al., 2008; Motoyama and Naka, 2004; Zhou and Elledge, 2000). The "turn off" process of the DDR upon satisfaction of DNA repair, also known as "checkpoint recovery", involves deactivation of DDR elements, but the mechanism is poorly understood. Greatwall kinase (Gwl) has been identified as a key element in the G2/M transition (Archambault et al., 2007; Jackson, 2006; Zhao et al., 2008; Yu et al., 2004; Yu et al., 2006; Zhao et al., 2006) and helps maintain M phase through inhibition of PP2A/B55δ (Burgess et al., 2010; Castilho et al., 2009; Goldberg, 2010; Lorca et al., 2010; Vigneron et al., 2009), the principal phosphatase for Cdk-phosphorylated substrates. Here we show that Gwl also promotes recovery from DNA damage and is itself directly inhibited by the DNA damage response (DDR). In Xenopus egg extracts, immunodepletion of Gwl increased the DDR to damaged DNA, whereas addition of wild type, but not kinase dead Gwl, inhibited the DDR. The removal of damaged DNA from egg extracts leads to recovery from checkpoint arrest and entry into mitosis, a process impaired by Gwl depletion and enhanced by Gwl over-expression. Moreover, activation of Cdk1 after the removal of damaged DNA is regulated by Gwl. Collectively, these results defines Gwl as a new regulator of the DDR, which plays an important role in recovery from DNA     

Assessment of LD matrix measures for the analysis of biological pathway association

Complex diseases will have multiple functional sites, and it will be invaluable to understand the cross-locus interaction in terms of linkage disequilibrium (LD) between those sites (epistasis) in addition to the haplotype-LD effects. We investigated the statistical properties of a class of matrix-based statistics to assess this epistasis. These statistical methods include two LD contrast tests (Zaykin et al., 2006) and partial least squares regression (Wang et al., 2008). To estimate Type 1 error rates and power, we simulated multiple two-variant disease models using the SIMLA software package. SIMLA allows for the joint action of up to two disease genes in the simulated data with all possible multiplicative interaction effects between them. Our goal was to detect an interaction between multiple disease-causing variants by means of their linkage disequilibrium (LD) patterns with other markers. We measured the effects of marginal disease effect size, haplotype LD, disease prevalence and minor allele frequency have on cross-locus interaction (epistasis). In the setting of strong allele effects and strong interaction, the correlation between the two disease genes was weak (r=0.2). In a complex system with multiple correlations (both marginal and interaction), it was difficult to determine the source of a significant result. Despite these complications, the partial least squares and modified LD contrast methods maintained adequate power to detect the epistatic effects; however, for many of the analyses we often could not separate interaction from a strong marginal effect. While we did not exhaust the entire parameter space of possible models, we do provide guidance on the effects that population parameters have on cross-locus interaction.     

Coding variants in human double-strand break DNA repair genes

DNA repair is essential for the maintenance of genomic integrity. Consequently, altered repair capacity may impact individual health in such areas as aging and susceptibility to certain diseases. Defects in some DNA repair genes, for example, have been shown to increase cancer risk, accelerate aging and impair neurological functions. Now that over 115 genes directly involved in human DNA repair have been characterized at the DNA sequence level, the identification of single nucleotide polymorphisms (SNPs) in DNA repair genes is becoming a reality. This information will likely lead to the identification of alleles, or combinations of alleles that affect disease predisposition. This communication summarizes SNPs identified to date in the coding region of 24 human double-strand break repair (DSBR) genes. SNP data for four of these genes were obtained by screening at least 100 individuals in our laboratory. For each SNP, the codon number, amino acid substitution, allele frequency and population information is supplied.     

Acetylation of Werner protein at K1127 and K1117 is important for nuclear trafficking and DNA repair

Werner syndrome is a rare autosomal recessive disorder where Werner (WRN) gene is mutated. Being a nucleolar protein, during DNA damage, WRN translocates at the damage site where its catalytic function is required in DNA repair. Several studies have indicated that WRN acetylation may modulate WRN trafficking and catalytic function (Blander et al., 2002; Lozada et al., 2014). Among the six acetylation sites in WRN protein identified by mass-spectrometry analysis (Li et al., 2010) we here explore the role of acetylation sites in C-terminal of WRN (K1127, K1117, K1389, K1413) because the C- terminal domain is the hub for protein- protein interaction and DNA binding activity (Brosh et al. [4]; Muftuoglu et al., 2008; Huang et al., 2006). To explore their functional activity, we created mutations in these sites by changing the acetylation residue lysine (K) to a non-acetylation residue arginine (R) and expressed them in WRN mutant cell lines. We observed that K1127R and K1117R mutants are sensitive to the DNA damaging agents etoposide and mitomycin C and display deficient DNA repair. Importantly, deacetylation of WRN by SIRT1 (Mammalian Sir2) is necessary for restoration of WRN localization at nucleoli after completion of DNA repair. Among all putative acetylation sites, K1127R, K1117R and the double mutant K1127R/K1117R showed significantly delayed re-entry to the nucleolus after damage recovery, even when SIRT1 is overexpressed. These mutants showed partial interaction with SIRT1 compared to WT WRN. Thus, our results suggest that K1127 and K1117 are the major sites of acetylation, necessary for DNA repair. These results elucidate the mechanism by which SIRT1 regulates WRN trafficking via these acetylation sites during DNA damage.     

Hairy math: insights into hair-follicle spacing and orientation

Hair follicles in the skin have a characteristic spacing and orientation. Two recent papers (Sick et al., 2006 and Wang et al., 2006) report the use of contrasting mathematical models and experimental manipulations to gain insight into the mechanisms underlying patterns of hair-follicle distribution and orientation.     

Certainly can't live without this: SIRT6

Cellular metabolic rates might regulate aging by impinging on genomic stability through the DNA repair pathways. A new study published in Cell (Mostoslavsky et al., 2006) reports that deficiency in one of the mammalian Sir2 homologs, SIRT6, results in genome instability through the DNA base excision repair pathway and leads to aging-associated degenerative phenotypes.     

Assessment of linkage disequilibrium in potato genome with single nucleotide polymorphism markers

The extent of linkage disequilibrium (LD) is an important factor in designing association mapping experiments. Unlike other plant species that have been analyzed so far for the extent of LD, cultivated potato (Solanum tuberosum L.), an outcrossing species, is a highly heterozygous autotetraploid. The favored genotypes of modern cultivars are maintained by vegetative propagation through tubers. As a first step in the LD analysis, we surveyed both coding and noncoding regions of 66 DNA fragments from 47 accessions for single nucleotide polymorphism (SNP). In the process, we combined information from the potato SNP database with experimental SNP detection. The total length of all analyzed fragments was >25 kb, and the number of screened sequence bases reached almost 1.4 million. Average nucleotide polymorphism (=11.5x10(-3)) and diversity (pi=14.6x10(-3)) was high compared to the other plant species. The overall Tajima's D value (0.5) was not significant, but indicates a deficit of low-frequency alleles relative to expectation. To eliminate the possibility that an elevated D value occurs due to population subdivision, we assessed the population structure with probabilistic statistics. The analysis did not reveal any significant subdivision, indicating a relatively homogenous population structure. However, the analysis of individual fragments revealed the presence of subgroups in the fragment closely linked to the R1 resistance gene. Data pooled from all fragments show relatively fast decay of LD in the short range (r2=0.208 at 1 kb) but slow decay afterward (r2=0.137 at approximately 70 kb). The estimate from our data indicates that LD in potato declines below 0.10 at a distance of approximately 10 cM. We speculate that two conflicting factors play a vital role in shaping LD in potato: the outcrossing mating type and the very limited number of meiotic generations.     

ATP-dependent chromatin remodeling complex SWI/SNF in cardiogenesis and cardiac progenitor cell development

The recent identification of cardiac progenitor cells (CPCs) provides a new paradigm for studying and treating heart disease.To realize the full potential of CPCs for therapeutic purposes,it is essenti...     

Clue to a New Deafness Gene: A Large Chinese Nonsyndromic Hearing Loss Family Linked to DFNA4

Hereditary hearing loss is one of the most common neurosensory defects in humans.Approximately 70％ of cases are nonsyndromic and could be inherited in autosomal dominant,autosomal recessive,mitochondrial,X-linked,and Y-linked manners (Wang et al.,2004;Alford,2011).The autosomal dominant type,comprising 15％-20％ of nonsyndromic hearing loss,is monogenic and genetically heterogeneous.Since the first dominant deafness locus (DFNA1) was identified in 1992,a total of 64 DFNA loci have been mapped (DFNA1-DFNA64),and 27 corresponding genes have been identified (http://hereditaryhearingloss.org).Previous studies have revealed that one deafness locus can be linked to more than one gene (Bayazit and Yilmaz,2006),and the question "one locus,how many genes?" was first raised about a decade ago (Van-Hauwe et al.,1999).So far,several loci,including DFNA2 and DFNA3,have been shown to be related to one or more genes,showing high genetic heterogeneity in hereditary hearing loss (Grifa et al.,1999;Goldstein and Lalwani,2002;Yan et al.,2011).     

MutLalpha: at the cutting edge of mismatch repair

The mismatch repair process corrects errors in newly synthesized DNA. In this issue, Modrich and colleagues (Kadyrov et al., 2006) show that a component of the human mismatch repair machinery, MutLalpha, has endonuclease activity. MutLalpha introduces single-strand breaks near the mismatch and thus generates new entry points for the exonuclease EXOI to degrade the strand containing the mismatch.     

Estimation of the extent of linkage disequilibrium in seven regions of the porcine genome

Linkage disequilibrium (LD) refers to the correlation among neighboring alleles, reflecting non-random patterns of association between alleles at (nearby) loci. A better understanding of LD in the porcine genome is of direct relevance for identification of genes and mutations with a certain effect on the traits of interest. Here, 215 SNPs in seven genomic regions were genotyped in individuals of three breeds. Pairwise linkage disequilibrium was calculated for all marker pairs. To estimate the extent of LD, all pairwise LD values were plotted against the distance between the markers. Based on SNP markers in four genomic regions analyzed in three panels from populations of Large White, Dutch Landrace, and Meishan origin, useful LD is estimated to extend for approximately 40 to 60 kb in the porcine genome.     

Linkage disequilibrium grouping of single nucleotide polymorphisms (SNPs) reflecting haplotype phylogeny for efficient selection of tag SNPs

Single nucleotide polymorphisms (SNPs) have been proposed to be grouped into haplotype blocks harboring a limited number of haplotypes. Within each block, the portion of haplotypes is expected to be tagged by a selected subset of SNPs; however, none of the proposed selection algorithms have been definitive. To address this issue, we developed a tag SNP selection algorithm based on grouping of SNPs by the linkage disequilibrium (LD) coefficient r(2) and examined five genes in three ethnic populations--the Japanese, African Americans, and Caucasians. Additionally, we investigated ethnic diversity by characterizing 979 SNPs distributed throughout the genome. Our algorithm could spare 60% of SNPs required for genotyping and limit the imprecision in allele-frequency estimation of nontag SNPs to 2% on average. We discovered the presence of a mosaic pattern of LD plots within a conventionally inferred haplotype block. This emerged because multiple groups of SNPs with strong intragroup LD were mingled in their physical positions. The pattern of LD plots showed some similarity, but the details of tag SNPs were not entirely concordant among three populations. Consequently, our algorithm utilizing LD grouping allows selection of a more faithful set of tag SNPs than do previous algorithms utilizing haplotype blocks.     

Horizontal gene transfer in microbial genome evolution

Horizontal gene transfer is the collective name for processes that permit the exchange of DNA among organisms of different species. Only recently has it been recognized as a significant contribution to inter-organismal gene exchange. Traditionally, it was thought that microorganisms evolved clonally, passing genes from mother to daughter cells with little or no exchange of DNA among diverse species. Studies of microbial genomes, however, have shown that genomes contain genes that are closely related to a number of different prokaryotes, sometimes to phylogenetically very distantly related ones. (Doolittle et al., 1990, J. Mol. Evol. 31, 383-388; Karlin et al., 1997, J. Bacteriol. 179, 3899-3913; Karlin et al., 1998, Annu. Rev. Genet. 32, 185-225; Lawrence and Ochman, 1998, Proc. Natl. Acad. Sci. USA 95, 9413-9417; Rivera et al., 1998, Proc. Natl. Acad. Sci. USA 95, 6239-6244; Campbell, 2000, Theor. Popul. Biol. 57 71-77; Doolittle, 2000, Sci. Am. 282, 90-95; Ochman and Jones, 2000, Embo. J. 19, 6637-6643; Boucher et al. 2001, Curr. Opin., Microbiol. 4, 285-289; Wang et al., 2001, Mol. Biol. Evol. 18, 792-800). Whereas prokaryotic and eukaryotic evolution was once reconstructed from a single 16S ribosomal RNA (rRNA) gene, the analysis of complete genomes is beginning to yield a different picture of microbial evolution, one that is wrought with the lateral movement of genes across vast phylogenetic distances. (Lane et al., 1988, Methods Enzymol. 167, 138-144; Lake and Rivera, 1996, Proc. Natl. Acad. Sci. USA 91, 2880-2881; Lake et al., 1999, Science 283, 2027-2028).