Transduction‐Specific ATLAS Reveals a Cohort of Highly Active L 1 Retrotransposons in Human Populations

L ong IN terspersed E lement‐1 ( LINE ‐1 or L 1) retrotransposons are the only autonomously active transposable elements in the human genome. The average human genome contains ∼80–100 active L1s, but only a subset of these L1s are highly active or ‘hot’. Human L1s are closely related in sequence, making it difficult to decipher progenitor/offspring relationships using traditional phylogenetic methods. However, L1 m RNA s can sometimes bypass their own polyadenylation signal and instead utilize fortuitous polyadenylation signals in 3′ flanking genomic DNA . Retrotransposition of the resultant m RNA s then results in lineage specific sequence “tags” (i.e., 3′ transductions) that mark the descendants of active L1 progenitors. Here, we developed a method (Transduction‐Specific Amplification Typing of L1 Active Subfamilies or TS ‐ ATLAS ) that exploits L1 3′ transductions to identify active L1 lineages in a genome‐wide context. TS ‐ ATLAS enabled the characterization of a putative active progenitor of one L1 lineage that includes the disease causing L1 insertion L1 RP , and the identification of new retrotransposition events within two other “hot” L1 lineages. Intriguingly, the analysis of the newly discovered transduction lineage members suggests that L1 polyadenylation, even within a lineage, is highly stochastic. Thus, TS ‐ ATLAS provides a new tool to explore the dynamics of L1 lineage evolution and retrotransposon biology. Long INterspersed Element‐1 (L1) retrotransposons are the only independently mobile elements in the human genome. We developed Transduction‐Specific Amplification Typing of L1 Active Subfamilies (TS‐ATLAS), which utilizes L1‐transduced genomic sequences, to identify a subset of highly active L1s genome‐wide. TS‐ATLAS enabled the characterization of the putative progenitor of an active disease‐causing L1 lineage, and identified new retrotransposition events within two other “hot” L1 lineages.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98809/1/humu22327.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/98809/2/humu22327-sup-0001-si.pd

Somatic Mutations Detected in Parkinson Disease Could Affect Genes With a Role in Synaptic and Neuronal Processes

The role of somatic mutations in complex diseases, including neurodevelopmental and neurodegenerative disorders, is becoming increasingly clear. However, to date, no study has shown their relation to Parkinson disease's phenotype. To explore the relevance of embryonic somatic mutations in sporadic Parkinson disease, we performed whole-exome sequencing in blood and four brain regions of ten patients. We identified 59 candidate somatic single nucleotide variants (sSNVs) through sensitive calling and a careful filtering strategy (COSMOS). We validated 27 of them with amplicon-based ultra-deep sequencing, with a 70% validation rate for the highest-confidence variants. The identified sSNVs are in genes with synaptic functions that are co-expressed with genes previously associated with Parkinson disease. Most of the sSNVs were only called in blood but were also found in the brain tissues with ultra-deep amplicon sequencing, demonstrating the strength of multi-tissue sampling designs

IAP Display: A Simple Method to Identify Mouse Strain Specific IAP Insertions

Intracisternal A-type particle (IAP) elements are high copy number long terminal repeat (LTR) rodent retrotransposons. Some IAP elements can transpose, and are responsible for ~12% of spontaneous mouse mutations. Inbred mouse strains show variation in genomic IAP distribution, contributing to inter-strain genetic variability. Additionally IAP elements can influence the transcriptional regulation of neighbouring genes through their strong LTR promoter, effecting phenotypic variation. This genetic and phenotypic variability can translate into experimental variability between mouse strains. For example, it has been demonstrated that strain-specific genetic/epigenetic factors can interact to yield variable responses to drugs. Therefore, in experimental contexts it is essential to unequivocally identify mouse strains. Recently it was estimated that any two inbred strains share only ~40% of their IAP insertions. Of the remaining 60%, some insertions will be strain specific, fixed during inbreeding. These fixed insertions can be exploited as genetic markers to identify inbred strains, if they can be identified simply and efficiently. Here, we report the development of a PCR-based system allowing direct acquisition of strain-specific IAP insertions. In a pilot study, we identified 21 IAP loci, genotyped IAP insertions at 9 loci, and discovered two strain-specific insertions that could reliably identify these strains

Increased somatic mutation burdens in normal human cells due to defective DNA polymerases.

Funder: Wellcome PhD StudentshipFunder: Jean Shank/Pathological Society Intermediate FellowshipFunder: Wellcome Clinical PhD fellowshipMutation accumulation in somatic cells contributes to cancer development and is proposed as a cause of aging. DNA polymerases Pol ε and Pol δ replicate DNA during cell division. However, in some cancers, defective proofreading due to acquired POLE/POLD1 exonuclease domain mutations causes markedly elevated somatic mutation burdens with distinctive mutational signatures. Germline POLE/POLD1 mutations cause familial cancer predisposition. Here, we sequenced normal tissue and tumor DNA from individuals with germline POLE/POLD1 mutations. Increased mutation burdens with characteristic mutational signatures were found in normal adult somatic cell types, during early embryogenesis and in sperm. Thus human physiology can tolerate ubiquitously elevated mutation burdens. Except for increased cancer risk, individuals with germline POLE/POLD1 mutations do not exhibit overt features of premature aging. These results do not support a model in which all features of aging are attributable to widespread cell malfunction directly resulting from somatic mutation burdens accrued during life

Meiosis and beyond – understanding the mechanistic and evolutionary processes shaping the germline genome

The separation of germ cell populations from the soma is part of the evolutionary transition to multicellularity. Only genetic information present in the germ cells will be inherited by future generations, and any molecular processes affecting the germline genome are therefore likely to be passed on. Despite its prevalence across taxonomic kingdoms, we are only starting to understand details of the underlying micro‐evolutionary processes occurring at the germline genome level. These include segregation, recombination, mutation and selection and can occur at any stage during germline differentiation and mitotic germline proliferation to meiosis and post‐meiotic gamete maturation. Selection acting on germ cells at any stage from the diploid germ cell to the haploid gametes may cause significant deviations from Mendelian inheritance and may be more widespread than previously assumed. The mechanisms that affect and potentially alter the genomic sequence and allele frequencies in the germline are pivotal to our understanding of heritability. With the rise of new sequencing technologies, we are now able to address some of these unanswered questions. In this review, we comment on the most recent developments in this field and identify current gaps in our knowledge

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts

Common clonal origin of conventional T cells and induced regulatory T cells in breast cancer patients

Regulatory CD4+ T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4+ cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.

Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year