Digital Genotyping of Macrosatellites and Multicopy Genes Reveals Novel Biological Functions Associated with Copy Number Variation of Large Tandem Repeats

Abstract

Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in modulating both genomic and phenotypic diversity.

Linkage disequilibrium between single nucleotide polymorphisms and hypermutable loci

“…Due to their high mutability, tandem repeats can mutate away 57 from linkage with surrounding SNPs, and therefore SNP association studies are not 58 expected to pick up all of the heritability caused by hypermutable variants. Studies 59 using large numbers of tandem repeat loci have shown that tandem repeat variants are 60 usually very weakly linked with surrounding SNPs [4][5][6]. These studies highlight how 61 SNP data can be uninformative about hypermutable loci, supporting the hypothesis 62 that hypermutable loci are sources of missing heritability.…”

“…Recent advances in sequencing technology [42,43,45] and tandem repeat 387 genotyping [6,44,46,47] provide hope that some hypermutable elements will be included 388 in future studies of genetic heritability and genetic disease. Nevertheless, some of the often caused by expansion [14,18].…”

Copy number variation of the REXO1L1 gene cluster; euchromatic deletion variant or susceptibility factor?

“…Understanding the possible role of this variation in gastrointestinal impairment and viral infection would be aided by more extensive testing of copy number in affected cohorts and controls with accurate quantitative techniques such as digital copy number estimation 1,6 or massively parallel sequencing. 10 Phase and haploid copy number could also be established in multigenerational families including those…”

“…If not due to dosage, any possible effects of a deletion would require alternative mechanisms such as enhancer adoption by genes flanking the REXO1L1 cluster 7 or the release of other genes from epigenetic control. 6 …”

0309 Systematic profiling of short tandem repeats in the cattle genome

“…Indeed, STR has become an essential marker for mapping quantitative trait loci (QTL) due to their high variability and easy amplification by PCR (Georges et al 1993;Lipkin et al 1998;Van Tassell et al 2000;Ashwell et al 2001;Schnabel et al 2005). Furthermore, the exploration of the relationship between STRs with nearby SNPs can further help explain the results from SNP-based genome wide association studies (Brahmachary et al 2014). Although previous studies have reported that most STRs can be tagged by SNPs (McClure et al 2012(McClure et al , 2013, these conclusions were mainly based on a rather limited number of known microsatellites.…”