a structural variation reference for medical and population genetics

Sequencing the DNA or mRNA of multiple individuals of one or more species (that is, population-scale sequencing) aims to identify genetic variation at a population level to address. In this study, we developed gnomAD-SV, a sequence-resolved reference for SVs from 14,891 genomes. African and African American samples exhibited the greatest genetic diversity and their common SVs were in weaker linkage disequilibrium with nearby short variants than Europeans, whereas East Asians featured the highest levels of homozygosity (Fig. Sebat, J. et al. Third, we estimated that 3.8% of the general population (95% confidence interval of 3.24.6%) carries at least one very large (1 Mb) rare autosomal SV, roughly half of which (45.2%) were balanced or complex (Fig. Although previous studies have attempted to compute similar scores using large CNVs detected by microarray and exome sequencing29,30, or to correlate deletions with pLI18, no gene-level metrics comparable to LOEUF exist for SVs at WGS resolution. 1h, Extended Data Fig. Collins, R.L., Brand, H., Karczewski, K.J. CAS Genet. 49, 3645 (2016). ClassifyCNV: a tool for clinical annotation of copy-number variants We evaluated the quality of gnomAD-SV with seven orthogonal analyses detailed in Supplementary Table 4, Supplementary Figs. Soc. 5 Rearrangement size is a primary determinant of allele frequency for most classes of SVs. We've expanded the catalog of clinically relevant structural variants (SV) in dbVar by adding 57,520 ClinVar records. Towards population-scale long-read sequencing | Nature Reviews Genetics Multi-platform discovery of haplotype-resolved structural variation in human genomes. Constrained and unconstrained includes the least and most constrained 15% of all genes based on LOEUF4, respectively. be, Samples with outlier autosome ploidy estimates typically contained somatic or mosaic chromosomal abnormalities, such as somatic aneuploidy of chromosome 1 (chr1) (b) or chromosome 8 (e), or large focal somatic or mosaic CNVs on chromosome 3 (c) and chromosome 7 (d). A structural variation reference for medical and population genetics. and M.E.T. As orthogonal support for these trends, we identified an inverse correlation between APS and SNV constraint across all functional categories of SVs, which was consistent with our observed depletion of rare, functional SVs in constrained genes (Extended Data Fig. Each algorithm captures unique characteristics of SVs; hence has its merits in specific SV types and size regimes. All other authors declare no competing interests. a, Proportion of singleton SVs in five SV size bins for each class of biallelic SVs considered in this study. contributed to the production and quality control of the gnomAD dataset. Cell 176, 663675.e19 (2019). An open resource of structural variation for medical and population ; U01MH105669 to M.J.D., B.N. Mol. White diamonds indicate medians. A structural variation reference for medical and population genetics | Nature Article Open Access Published: 27 May 2020 A structural variation reference for medical and population genetics. When restricted to rare (allele frequency < 1%) SVs, we observed a mean of 10.2 altered genes per genome (5.5 pLoF, 3.4 copy-gain, and 1.3 IED). Google Scholar. SVs are DNA rearrangements that involve at least 50 nucleotides1. PubMed a, SV classes catalogued in this study. Following family-based analyses using 970 parentchild trios for quality assessments, we pruned all first-degree relatives from the cohort, retaining 12,653 unrelated genomes for subsequent analyses. Taking advantage of the sample size and resolution of gnomAD-SV, we evaluated whether SNVs associated with human traits might be in linkage disequilibrium with SVs not directly genotyped in GWAS. Reference variants - Reference variants (analogous to rs identifiers in dbSNP - e.g., rs1872633) do not currently exist for structural variants. Nat. A structural variation reference for medical and population Chiang, C. et al. 4d, Supplementary Fig. (PDF) Author Correction: A structural variation reference for medical Beroukhim, R. et al. & Koonin, E. V. Selection in the evolution of gene duplications. Individuals presenting with DDs are often referred for diagnostic testing with chromosomal microarrays (CMAs) to identify large copy-number variants (CNVs) and/or with single-gene, gene-panel . A structural variation reference for medical and population genetics. We identified 15,634 common SVs (allele frequency >1%) in strong linkage disequilibrium (R20.8) with at least one common short variant (Supplementary Fig. Ruderfer, D. M. et al. b, HardyWeinberg equilibrium (HWE) metrics for all biallelic SVs localized to autosomes. Google Scholar. Am. For comparison, variants that did not pass post hoc site-level filters (not pass) are also shown in purple. R.L.C., H.B., K.J.K., X.Z., J.A., L.C.F., A.V.K., L.D.G., H.W., N.A.W., M.S., A.OD.-L., A.B., R.M., G.T., K.M.L., C.S., N.G., C.C., L.M., K.D.T., H.J.L., S.S.R., W.P., Y.-D.I.C., J.I.R., C.N., A.P., E.L., S.G., B.M.N., S.K., M.J.D., E.B., D.G.M. As national biobanks, human disease association studies, and clinical genetic testing are increasingly reliant on whole-genome sequencing, population references for small variants ( i.e. Each category was compared to neutral variation (APS=0) using a one-tailed t-test. A substantial and confusing variation exists in handling of baseline covariates in randomized controlled trials: a review of trials published in leading medical journals . The proportion of singleton variants has been established as a proxy for strength of selection6; however, this metric is confounded for SVs given the strong correlation between allele frequency and SV size, among other factors. Nature 489, 5774 (2012). The ExAC browser: displaying reference data information from over 60 000 exomes. Colour codes are consistent between a, c, eh, and between b and d. Although SVs alter more nucleotides per genome than SNVs and short insertion/deletion variants (indels; <50 bp)1, surprisingly little is known about their mutational spectra on a global scale. This final set of high-quality SVs corresponded to a median of 7,439 SVs per genome, or more than twice the number of variants per genome captured by previous WGS-based SV studies such as the 1000 Genomes Project (3,441 SVs per genome from approximately 7 coverage WGS), which underscores the benefits of high-coverage WGS and improved multi-algorithm ensemble methods for SV discovery. Opin. The average genome in gnomAD-SV contained a mean of 179.8 genes altered by biallelic SVs (144.3 pLoF, 24.3 copy-gain, and 11.2 IED), of which 11.6 were predicted to be completely inactivated by homozygous pLoF (Fig. contributed to the analysis of data. and 15,708 whole-genome sequences from 141,456 unrelated humans sequenced as part of various disease-specific and population genetic studies. Trends Genet. 612, Supplementary Table 4 and Supplementary Note1, but we highlight just a few here to demonstrate that gnomAD-SV conforms to many fundamental principles of population genetics, including Mendelian segregation, genotype distributions, and linkage disequilibrium. Top, the GWAS signal among genotyped SNVs in the UK Biobank, coloured by strength of linkage disequilibrium (Pearsons R2 value) with the 336-bp deletion identified in gnomAD-SV. ADS Deviation from HWE was assessed using a chi-square goodness-of-fit test with one degree of freedom. Structural variants (SVs) rearrange large segments of DNA 1 and can have profound consequences in evolution and human disease 2,3.As national biobanks, disease-association studies, and clinical genetic testing have grown increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD) 4 have become integral in the interpretation of single . 8 An extremely complex SV involving 49 breakpoints and seven chromosomes. Cell 149, 525537 (2012). Mastering variation: variance components and personalised medicine PLoS Genet. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. Although this imperfect method extrapolates from data pooled across unrelated individuals, we previously demonstrated comparable rates from molecularly validated observations in 519 quartet families20. Pearsons correlation coefficient (R2) is provided. Therefore, the weighted mean of the four samples was used as a study-wide long-read WGS confirmation rate, weighting the confirmation rate of each sample based on the square root of its long-read WGS sequencing depth. Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples. Provided by the Springer Nature SharedIt content-sharing initiative, Journal of Neurodevelopmental Disorders (2023). Correspondence to Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. was supported by Desmond and Ann Heathwood. We also uncovered modest selection against noncoding SVs in cis-regulatory elements, although selection against protein-truncating SVs was stronger than all noncoding effects. Google Scholar. Am. These comparisons confirm that selection against most classes of gene-altering SVs mirrors patterns observed for short variants18,30. The diversity of SVs in humans is therefore far greater than has been widely appreciated, as is their influence on genome structure and function. The outlier points marked in red and blue correspond to the samples highlighted in be. Zhao, X., Weber, A. M. & Mills, R. E. A recurrence-based approach for validating structural variation using long-read sequencing technology. Nat. Broadly, these results followed trends we observed for protein-coding SVs, which we interpreted as evidence for weak but widespread selection against CNVs altering most classes of annotated regulatory elements. Author Correction: A structural variation reference for medical and Common SVs in linkage disequilibrium with GWAS variants were enriched for genic SVs across multiple functional categories (Supplementary Table 6), and included candidate SVs such as a deletion of a thyroid enhancer in the first intron of ATP6V0D1 at a hypothyroidism-associated locus34 (Extended Data Fig. Expectations and blind spots for structural variation detection from developed tools and methods that enabled the scientific discoveries herein. Categories with fewer than tenSVs are not shown. Ryan L. Collins & Harrison Brand & Konrad J. Karczewski & Xuefang Zhao & Jessica Alfldi & Laurent C. Francioli & Amit V. Khera & Chelsea Lowther & Laura D. Gauthier & Harold Wang & Nicholas A. Watts , 2020. SVs per category listed in Supplementary Table 9. If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 47 (D1), D1005D1012 (2019). An analytical framework for whole-genome sequence association studies and its implications for autism spectrum disorder. B.M.N. A structural variation reference for medical and population genetics Article As national biobanks, disease-association studies, and clinical genetic testing have grown increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD)4 have become integral in the interpretation of single . INTRODUCTION. In the current release, we catalogued at least one pLoF or copy-gain variant for 36.9% and 23.7% of all autosomal genes, respectively, and 490 genes with at least one homozygous pLoF SV (Fig. and M.E.T. P values were computed using a two-sided t-test and were Bonferroni-adjusted for 21 comparisons. 6 Most SVs within genes appear under negative selection. 41, 4452 (2016). Extended Data Fig. N. Engl. The Lancet, and the British Medical Journal between January 1, 2007 and June 30, 2007. 1 Detection of chromosome-scale dosage alterations. Structural variants (SVs) rearrange large segments of DNA and can have profound consequences in evolution and human disease. PubMed Genet. 2 Benchmarking the technical qualities of the gnomAD-SV callset. 14, 125138 (2013). There are a handful of examples of SVs with strong noncoding effects, although they are scarce in humans and model organisms31,32. Accurate detection of large, repeat-mediated CNVs is thus crucial for WGS-based diagnostic testing as chromosomal microarray is the recommended first-tier diagnostic screen at present for unexplained developmental disorders37. Nature, 581 (7809), 444-451. https://doi.org/10.1038/s41586-020-2287-8 Readers' Seniority PhD / Post grad / Masters / Doc 235 54% Researcher 154 35% Chaisson, M. J. P. et al. MathSciNet 6f). M.E.T has received research support from Levo Therapeutics. ADS Structural variants (SVs) rearrange large segments of DNA1 and can have profound consequences in evolution and human disease2,3. We use cookies to ensure that we give you the best experience on our website. We thank T. Hefferon of the NIH National Center for Biotechnology Information for his help hosting gnomAD-SV on dbVar. Extended Data Fig. Google Scholar. We found strong correlations between natural selection against damaging SNVs and rare SVs that disrupt or duplicate protein-coding sequence, which suggests that genes that are highly intolerant to loss-of-function are also sensitive to increased dosage6. Copy-number variation is a form of structural genetic variation that involves a gain or loss of DNA segments. A structural variation reference for medical and population genetics. PubMedGoogle Scholar. Genome Biol. 44, 390397 (2012). Nature 550, 204213 (2017). Sudmant, P. H. et al. Our analyses also demonstrate that gene-altering effects of SVs beyond pLoF are remarkably similar to the mutational constraints of SNVs, and that SNV constraint metrics are not specific to haploinsufficiency but underlie a general intolerance to alterations of both gene dosage and structure. Epub 2020 May 27. Intergenic SVs (light colours; n=206,954) exhibited reduced singleton proportions when compared to all SVs (dark colours; n=335,470) of the same size and class. Version Item Date Summary; 2: 1721.1/135670.2: 2022-09-19T17:19:38Z: Metadata changed: Verified or entered author name and department authority metadata. Finally, we identified very large (over one megabase), rare SVs in 3.9% of samples, and estimate that 0.13% of individuals may carry an SV that meets the existing criteria for clinically important incidental findings7. In principle, any SV can result in predicted loss-of-function (pLoF), either by deleting coding nucleotides or altering open-reading frames. We also documented unresolved non-reference breakends (BNDs), but they were excluded from all analyses as low-quality variants. By virtue of their size and abundance, SVs represent an important mutational force that shape genome evolution and function2,3, and contribute to germline and somatic diseases9,10,11. Copy number variation and evolution in humans and chimpanzees. We found that the precision of gnomAD-SV was comparable to our previous study of 519 autism quartets that attained a 97% molecular validation rate for all de novo SV predictions20: in gnomAD, analyses of 970 parentchild trios indicated a median Mendelian violation rate of 3.8% and a heterozygous de novo rate of 3.0%. 1). Genet. Detecting cryptic clinically relevant structural variation in exome f, Relationship of APS and constraint against pLoF SNVs4. Population-scale genotyping of structural variation in the era of long Audano, P. A. et al. Therefore, we developed a new metric, adjusted proportion of singletons (APS), to account for SV class, size, genomic context, and other technical covariates (Extended Data Fig. By submitting a comment you agree to abide by our Terms and Community Guidelines. 5b). K.J.K. This corrects the article "A structural variation reference for medical and population genetics" in Nature, volume 581 on page 444. Residual deviation from APS=0 is maintained when considering all SVs, owing to APS being intentionally calibrated to intergenic SVs as a proxy for neutral variation. Given that short-read WGS is blind to a disproportionate fraction of repeat-mediated SVs and small insertions by comparison to long-read methods, this study certainly underestimates the true mutation rates within such hypermutable regions. Huddleston, J. et al. You can access the newly added data through study nstd102. Genet. Kondrashov, F. A., Rogozin, I. Extended Data Fig. Test statistics, SV counts, and Pvalues are provided in Supplementary Table 6. b, Example locus at 16q22.1, where we identified a 336-bp deletion in strong linkage disequilibrium with SNVs significantly associated with hypothyroidism in the UK Biobank34. Individual points are outlier samples at least three standard deviations away from the cohort-wide mean. R.L.C., H.B., K.J.K., X.Z., J.A., L.C.F, C.L., A.OD.-L., E.V., H.J.L., J.I.R, M.J.D., D.G.M. Commun. 19, 192203 (2017). As short-read WGS is rapidly becoming the predominant technology in large-scale human disease studies, and will probably displace conventional methods for diagnostic screening, there is a mounting need for comparable references of SVs across global populations. Article Comprehensive evaluation of structural variation detection algorithms for whole genome sequencing. One category of disease-associated SVs, recurrent CNVs mediated by homologous segmental duplications known as genomic disorders, are particularly important because they collectively represent a common cause of developmental disorders37. e, The median genome contained 7,439 SVs. Structural variations have attracted remarkable attention in both evolutionary and medical studies over the last two decades. SVs can cause a wide range of genetic diseases and are prevalent in rare developmental disorders (DDs). Most custom scripts used in the production and/or analysis of the gnomAD-SV dataset are publicly available via GitHub (https://github.com/talkowski-lab/gnomad-sv-pipeline). Dennis, M. Y. 7, 256276 (1975). Xie, J. et al. Counts of SVs per category for both a and b are listed in Supplementary Table 9. a, Enrichments for pLoF consequences among rare and singleton SVs across SV classes. We performed extensive in silico confirmation of 19,316 SVs predicted from short-read WGS using matched long-read WGS from four samples21,23, finding a 94.0% confirmation rate with breakpoint-level read evidence, and revealing that 59.8% of breakpoint coordinates were accurate within a single nucleotide of the long-read data. Google Scholar. Thank you for visiting nature.com. Hurles, M. E., Dermitzakis, E. T. & Tyler-Smith, C. The functional impact of structural variation in humans. 372, 22352242 (2015). c, Linkage disequilibrium between SVs and SNVs or indels for 23,706 common (allele frequency >1%) SVs represented as cross-population maximum R2 values after excluding repetitive and low-complexity regions (see Supplementary Fig. From these analyses, we determined that SVs represent more than 25% of all rare protein-truncating events per genome, emphasizing the unrealized potential of routine SV detection in WGS studies. PubMed ; P01HD068250 to H.B.) Publication Type: Journal Article: Year of Publication: 2020: Authors: Google Scholar. Bars reflect 95% confidence intervals from 100-fold bootstrapping. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in g, Histogram representation of the data from f. Essentially all samples conformed to canonical sex chromosome ploidies. Here, we used the Watterson estimator26 to project a mean mutation rate of 0.29 de novo SVs (95% confidence interval 0.130.44) per generation in regions of the genome accessible to short-read WGS, or roughly one new SV every 28 live births, with mutation rates varying markedly by SV class (Fig. The deletion lies amidst histone mark peaks commonly found at active enhancers (H3K27ac and H3K4me1) based on publicly available chromatin data from adult thyroid samples, a phenotype-relevant tissue48. Article Expected Alu, SVA and LINE1 mobile element insertion peaks are marked at approximately 300 bp, 2.1 kb and 6 kb, respectively. J. Hum. is a founder of Maze Therapeutics. Genetic effects on gene expression across human tissues. PubMed Article Categories surpassing Bonferroni-corrected significance for 32 comparisons are indicated with dark shaded points. P values calculated using a two-tailed paired two-sample t-test for the 14 categories from a. c, d, Spearman correlations between sequence conservation and APS for noncoding deletions (n=143,353) (c) and duplications (n=30,052) (d). 1a). Curr. Dark, medium and light-grey background shading indicates the range of copy number estimates for 90%, 99% and 99.9% of all gnomAD-SV samples, respectively, and the medium grey line indicates the median copy number estimate across all samples. A structural variation reference for medical and population genetics contributed equally to this study. More recently, exotic species of complex SVs have been discovered that involve two or more distinct SV signatures in a single mutational event interleaved on the same allele, and can range from CNV-flanked inversions to rare instances of localized chromosome shattering, such as chromothripsis13,14. A structural variation reference for medical and population genetics For c, d and f, points and vertical bars represent 95% confidence intervals from 100-fold bootstrapping, respectively. Single and triple asterisks correspond to nominal (P<0.05) and Bonferroni-corrected (P<0.0083) significance thresholds from a two-sided Fishers exact test, respectively. 3 Division of Medical Sciences, Harvard Medical School, Boston, MA, USA. 1, Supplementary Figs. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. As national biobanks, disease-association studies, and clinical genetic testing have grown increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD) have . We also benchmarked carrier rates for several categories of clinically relevant variants in gnomAD-SV. ADS Nat. Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries. Origins and functional impact of copy number variation in the human genome. Popul. J. A central challenge to these efforts will be the uniform analysis and interpretation of all variation accessible to WGS, particularly SVs, which are frequently invoked as a source of added value offered by WGS. This file contains Supplementary Figures 1-24, Supplementary Notes, Supplementary Methods, and additional references. Genome Biol. Redin, C. et al. Correction to: Nature 10.1038/s41586-020-2287-8Published online 27 May 2020 In this Article, author Marquis P. Vawter was missing from the Genome Aggregation Database Consortium list. 6b, Extended Data Fig. Although the proportion of singletons varied by SV class, it was strongly dependent on SV size across all classes, which suggests that the amount of DNA rearranged is a key determinant of selection against most SVs (Fig. A structural variation reference for medical and population genetics Most foundational assumptions about human genetic variation were consistent between SVs and short variants in gnomAD, most notably that SVs segregate stably on haplotypes in the population and experience selection commensurate with their predicted biological consequences. Nonetheless, 92.7% of all known autosomal protein-coding nucleotides are not localized to simple- or low-copy repeats, and therefore we expect that the catalogues of SVs accessible to short-read WGS across large populations like gnomAD-SV will capture a majority of the most interpretable gene-disrupting SVs in humans. Submitted by ja607 on Sat, 10/03/2020 - 22:31. As genomic medicine advances towards diagnostic screening at sequence resolution, computational methods for variant discovery from WGS and population references for interpretation will become indispensable. A plethora of SV detection methods has been developed utilizing read depth, discordant read pairs, split reads and soft-clipped read information of short-read sequencing data. PubMed An open resource of structural variation for medical and population Article Human Alu elements are known to frequently act as enhancers, and the sentinel hypothyroidism SNV from the UK Biobank GWAS is a significant expression-modifying variant (that is, eQTL) for ATP6V0D1 and other nearby genes across many tissues, which indicates that the hypothyroidism risk haplotype modifies expression of ATP6V0D1 and/or other genes, potentially through the deletion of an intronic enhancer4,49. A structural variation reference for medical and population genetics A structural variation reference for medical and population genetics Authors: Ryan L Collins Dana-Farber Cancer Institute Harrison Brand Harvard Medical School Konrad J Karczewski Stanford. ADS PubMed Central Google Scholar. Nature 538, 265269 (2016). A.V.K. Structural variants (SVs) rearrange the linear and three-dimensional organization of the genome, which can have profound consequences in evolution, diversity, and disease. These and other benchmarking approaches suggested that gnomAD-SV was sufficiently sensitive and specific to be used as a reference dataset for most applications in human genomics. Colours correspond to predicted functional consequence. ad, Counts of SVs per genome across a variety of parameters, corresponding to median counts of total SVs (a), homozygous SVs (b), rare SVs (c) and singleton SVs (d). Choi, S. H. et al. A fundamental question in human genetics is the degree to which natural selection acts on coding and noncoding loci. 6c). MESA and the MESA SHARe project are conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with MESA investigators. PDF Article A structural variation reference for medical and population The NHGRI-EBI GWAS Catalog of published genome-wide association studies, targeted arrays and summary statistics 2019. PubMed Central and the Simons Foundation for Autism Research Initiative (SFARI #573206 to M.E.T.). Google Scholar. 86, 749764 (2010). 9, 3391 (2018). We also observed that 86% of SVs were in HardyWeinberg equilibrium, and common SVs were in strong linkage disequilibrium with nearby SNVs or indels (median peak R2=0.85). 1a) and 11 subclasses of complex SVs22 (Fig. is a member of the scientific advisory board at Deep Genomics and consultant for Camp4 Therapeutics, Takeda Pharmaceutical, and Biogen. Michael E. Talkowski. In general, the effects of noncoding deletions appeared stronger than noncoding duplications, and CNVs predicted to delete or duplicate entire elements were under stronger selection than partial element disruption (Fig. They further suggest that SNV-derived constraint metrics such as LOEUF capture a general correspondence between haploinsufficiency and triplosensitivity for a large fraction of genes in the genome. a, Circos representation of all 49 breakpoints and seven chromosomes involved in this SV. Cell 171, 710722.e12 (2017). Sign up for the Nature Briefing: Translational Research newsletter top stories in biotechnology, drug discovery and pharma. 15, 565574 (2013). BMC Genomics 19, 867 (2018). a, Counts of SVs evaluated per sample in this analysis. 18). Google Scholar. 10, 1784 (2019). This pipeline integrated four orthogonal evidence types to capture SVs across the size and allele frequency spectra, including six classes of canonical SVs (Fig. , SNVs & indels) in protein-coding . Genet. d, Correlation of allele frequency(AF) for 37,907 common SVs captured by both the 1000 Genomes Project and gnomAD-SV1. B., Wolf, Y. I. Many important genetic diseases, including cancer (1, 2), autism (), Alzheimer's disease (), Parkinson's disease (), amyotrophic lateral sclerosis (), heart failure (), neurodevelopmental disorders () and autoimmune diseases were found to be associated . High Paying Remote Jobs - Part-time, Does Whole Life Insurance Premium Increase With Age, What Type Of Bond Joins Nucleotides To Each Other, Sunset Drive Apartments, Home Team Bbq Mount Pleasant Menu, Articles A