In primates and other animals, reverse transcription of mRNA followed by genomic integration creates retroduplications. Expressed retroduplications are either “retrogenes” coding for functioning proteins, or expressed “processed pseudogenes,” which can function as noncoding RNAs. To date, little is known about the variation in retroduplications in terms of their presence or absence across individuals in the human population. We have developed new methodologies that allow us to identify “novel” retroduplications (i.e., those not present in the reference genome), to find their insertion points, and to genotype them. Using these methods, we catalogued and analyzed 174 retroduplication variants in almost one thousand humans, which were sequenced as part of Phase 1 of The 1000 Genomes Project Consortium. The accuracy of our data set was corroborated by (1) multiple lines of sequencing evidence for retroduplication (e.g., depth of coverage in exons vs. introns), (2) experimental validation, and (3) the fact that we can reconstruct a correct phylogenetic tree of human subpopulations based solely on retroduplications. We also show that parent genes of retroduplication variants tend to be expressed at the M-to-G1 transition in the cell cycle and that M-to-G1 expressed genes have more copies of fixed retroduplications than genes expressed at other times. These findings suggest that cell division is coupled to retrotransposition and, perhaps, is even a requirement for it.

Because phenotypic innovations must be genetically heritable for biological evolution to proceed, it is natural to consider new mutation events as well as standing genetic variation as sources for their birth. Previous research has identified a number of single-nucleotide polymorphisms that underlie a subset of adaptive traits in organisms. However, another well-known class of variation, genomic structural variation, could have even greater potential to produce adaptive phenotypes, due to the variety of possible types of alterations (deletions, insertions, duplications, among others) at different genomic positions and with variable lengths. It is from these dramatic genomic alterations, and selection on their phenotypic consequences, that adaptations leading to biological diversification could be derived. In this review, using studies in humans and other mammals, we highlight examples of how phenotypic variation from structural variants might become adaptive in populations and potentially enable biological diversification. Phenotypic change arising from structural variants will be described according to their immediate effect on organismal metabolic processes, immunological response and physical features. Study of population dynamics of segregating structural variation can therefore provide a window into understanding current and historical biological diversification.

Genomic deletions provide a powerful loss-of-function model in noncoding regions to assess the role of purifying selection on genetic variation. Regulatory element function is characterized by nonuniform tissue and cell type activity, necessarily linking the study of fitness consequences from regulatory variants to their corresponding cellular activity. We generated a callset of deletions from genomes in the Alzheimer's Disease Neuroimaging Initiative (ADNI) and used deletions from The 1000 Genomes Project Consortium (1000GP) in order to examine whether purifying selection preserves noncoding sites of chromatin accessibility marked by DNase I hypersensitivity (DHS), histone modification (enhancer, transcribed, Polycomb-repressed, heterochromatin), and chromatin loop anchors. To examine this in a cellular activity-aware manner, we developed a statistical method, pleiotropy ratio score (PlyRS), which calculates a correlation-adjusted count of “cellular pleiotropy” for each noncoding base pair by analyzing shared regulatory annotations across tissues and cell types. By comparing real deletion PlyRS values to simulations in a length-matched framework and by using genomic covariates in analyses, we found that purifying selection acts to preserve both DHS and enhancer noncoding sites. However, we did not find evidence of purifying selection for noncoding transcribed, Polycomb-repressed, or heterochromatin sites beyond that of the noncoding background. Additionally, we found evidence that purifying selection is acting on chromatin loop integrity by preserving colocalized CTCF binding sites. At regions of DHS, enhancer, and CTCF within chromatin loop anchors, we found evidence that both sites of activity specific to a particular tissue or cell type and sites of cellularly pleiotropic activity are preserved by selection.

purifying selection | genomic deletions | noncoding regulatory elements | cellular pleiotropy L arge-scale sequencing studies have provided tremendous 1 insight into biological function and human disease, with 2 statistical signatures of natural selection serving as a primary 3 identifying feature. The classic example is the analysis of 4 selective constraints on protein coding genes evident from the 5 depletion of missense or nonsense genetic variants. These ad-6 vances, however, are not directly translatable to the analysis 7 of noncoding DNA, which has increasingly become a focus 8 of human genetics research. Functional genomic studies have 9 revealed numerous regions of regulatory activity marked by 10 chromatin accessibility and histone modification (1, 2). Associ-11 ation signals for common human phenotypes are dramatically 12 enriched in these regulatory regions of the genome (3), show-13 casing the importance of specialized cellular function. In 14 contrast to protein-coding sequences, the function of regula-15 tory sequences is not determined by triplet codon structure 16 thereby providing no obvious analog to protein-truncating 17 single nucleotide variants (SNVs) to identify loss of function. 18 This ambiguity of the mutational consequences of individual 19 nucleotides within regulatory sequences complicates the ability 20 to study their function through the lens of purifying natural 21 selection. Previous work focusing on SNVs within noncoding 22 regions developed sophisticated genetic models that relied on 23 functional proxies such as transcription factor binding sites, 24 nucleotide conservation across species, or machine learning (4-25 May 19, 2020 | vol. XXX | no. XX | 1-9 42 effects (what we term "cellular pleiotropy", i.e. the same regula-43 tory element locus is active in more than one tissue or cell-type) 44 might be expected to be, on average, more deleterious (i.e. 45 fitness-reducing) than deletions that remove cell-type-specific 46 sites, since any changes at the DNA level to these regulators 47 potentially affects multiple tissues/cell-types simultaneously. 48 Another possibility is that since tissue/cell-type-specific reg-49 ulation is what enables widespread cellular diversity, these 50 regulatory elements must be under strong selective constraint 51 to preserve their specialized biological function. These two 52 potential modes of selection preserving regulation of cellular 53 activity are not mutually exclusive, as selection may be oper-54 ating to remove overlapping deletions to preserve the utility 55 of both types of regulators. Prior work has provided sug-56 gestive evidence that tissue activity count is a contributor 57 to selective constraint in regulatory sequences (10, 16, 17). 58 Studying purifying selection on noncoding deletions is thus 59 inherently tied to the cellular activity of corresponding deleted 60 regulatory sequences. To address this, we have developed a 61 statistical method, Pleiotropy Ratio Score (PlyRS), to quantify 62 the amount of tissue/cell-type activity...