Bottleneck and selection in the germline and maternal age influence transmission of mitochondrial DNA in human pedigrees.

Heteroplasmy-the presence of multiple mitochondrial DNA (mtDNA) haplotypes in an individual-can lead to numerous mitochondrial diseases. The presentation of such diseases depends on the frequency of the heteroplasmic variant in tissues, which, in turn, depends on the dynamics of mtDNA transmissions during germline and somatic development. Thus, understanding and predicting these dynamics between generations and within individuals is medically relevant. Here, we study patterns of heteroplasmy in 2 tissues from each of 345 humans in 96 multigenerational families, each with, at least, 2 siblings (a total of 249 mother-child transmissions). This experimental design has allowed us to estimate the timing of mtDNA mutations, drift, and selection with unprecedented precision. Our results are remarkably concordant between 2 complementary population-genetic approaches. We find evidence for a severe germline bottleneck (7-10 mtDNA segregating units) that occurs independently in different oocyte lineages from the same mother, while somatic bottlenecks are less severe. We demonstrate that divergence between mother and offspring increases with the mother's age at childbirth, likely due to continued drift of heteroplasmy frequencies in oocytes under meiotic arrest. We show that this period is also accompanied by mutation accumulation leading to more de novo mutations in children born to older mothers. We show that heteroplasmic variants at intermediate frequencies can segregate for many generations in the human population, despite the strong germline bottleneck. We show that selection acts during germline development to keep the frequency of putatively deleterious variants from rising. Our findings have important applications for clinical genetics and genetic counseling

Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

Several studies have revealed that aquaporins play a role in tumor progression and invasion. In breast carcinomas, high levels of aquaporin 5 (AQP5), a membrane protein involved in water transport, have been linked to increased cell proliferation and migration, thus facilitating tumor progression. Despite the potential role of AQP5 in mammary oncogenesis, the mechanisms controlling mammary AQP5 expression are poorly understood. In other tissues, AQP5 expression has been correlated with its promoter methylation, yet, very little is known about AQP5 promoter methylation in the mammary gland. In this work, we used the mouse mammary gland cell line EpH4, in which we controlled AQP5 expression via the steroid hormone dexamethasone (Dex) to further investigate mechanisms regulating AQP5 expression. In this system, we observed a rapid drop of AQP5 mRNA levels with a delay of several hours in AQP5 protein, suggesting transcriptional control of AQP5 levels. Yet, AQP5 expression was independent of its promoter methylation, or to the presence of negative glucocorticoid receptor elements (nGREs) in its imminent promoter region, but was rather influenced by the cell proliferative state or cell density. We conclude that AQP5 promoter methylation is not a universal mechanism for AQP5 regulation and varies on cell and tissue type

Streamlined analysis of duplex sequencing data with Du Novo

Duplex sequencing was originally developed to detect rare nucleotide polymorphisms normally obscured by the noise of high-throughput sequencing. Here we describe a new, streamlined, reference-free approach for the analysis of duplex sequencing data. We show the approach performs well on simulated data and precisely reproduces previously published results and apply it to a newly produced dataset, enabling us to type low-frequency variants in human mitochondrial DNA. Finally, we provide all necessary tools as stand-alone components as well as integrate them into the Galaxy platform. All analyses performed in this manuscript can be repeated exactly as described at http://usegalaxy.org/duplex. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13059-016-1039-4) contains supplementary material, which is available to authorized users

Family reunion via error correction: an efficient analysis of duplex sequencing data

Background Duplex sequencing is the most accurate approach for identification of sequence variants present at very low frequencies. Its power comes from pooling together multiple descendants of both strands of original DNA molecules, which allows distinguishing true nucleotide substitutions from PCR amplification and sequencing artifacts. This strategy comes at a cost—sequencing the same molecule multiple times increases dynamic range but significantly diminishes coverage, making whole genome duplex sequencing prohibitively expensive. Furthermore, every duplex experiment produces a substantial proportion of singleton reads that cannot be used in the analysis and are thrown away. Results In this paper we demonstrate that a significant fraction of these reads contains PCR or sequencing errors within duplex tags. Correction of such errors allows “reuniting” these reads with their respective families increasing the output of the method and making it more cost effective. Conclusions We combine an error correction strategy with a number of algorithmic improvements in a new version of the duplex analysis software, Du Novo 2.0. It is written in Python, C, AWK, and Bash. It is open source and readily available through Galaxy, Bioconda, and Github: https://github.com/galaxyproject/dunovo

Evaluating the Evidence for Transmission Distortion in Human Pedigrees

Children of a heterozygous parent are expected to carry either allele with equal probability. Exceptions can occur, however, due to meiotic drive, competition among gametes, or viability selection, which we collectively term “transmission distortion” (TD). Although there are several well-characterized examples of these phenomena, their existence in humans remains unknown. We therefore performed a genome-wide scan for TD by applying the transmission disequilibrium test (TDT) genome-wide to three large sets of human pedigrees of European descent: the Framingham Heart Study (FHS), a founder population of European origin (HUTT), and a subset of the Autism Genetic Resource Exchange (AGRE). Genotyping error is an important confounder in this type of analysis. In FHS and HUTT, despite extensive quality control, we did not find sufficient evidence to exclude genotyping error in the strongest signals. In AGRE, however, many signals extended across multiple SNPs, a pattern highly unlikely to arise from genotyping error. We identified several candidate regions in this data set, notably a locus in 10q26.13 displaying a genome-wide significant TDT in combined female and male transmissions and a signature of recent positive selection, as well as a paternal TD signal in 6p21.1, the same region in which a significant TD signal was previously observed in 30 European males. Neither region replicated in FHS, however, and the paternal signal was not visible in sperm competition assays or as allelic imbalance in sperm. In maternal transmissions, we detected no strong signals near centromeres or telomeres, the regions predicted to be most susceptible to female-specific meiotic drive, but we found a significant enrichment of top signals among genes involved in cell junctions. These results illustrate both the potential benefits and the challenges of using the TDT to study transmission distortion and provide candidates for investigation in future studies

Immune Response Associated Gene Signatures in Aortic Dissection Compared to Aortic Aneurysm

Background: Thoracic aortic dissections (TAD) are life-threatening events mostly requiring immediate surgical treatment. Although dissections mainly occur independently of thoracic aortic aneurysms (TAA), both share a high comorbidity. There are several indications for an involvement of the immune system in the development of TAD, just as in TAA. Nevertheless, specific disease-relevant genes, biomolecular processes, and immune-specific phenotypes remain unknown. Methods: RNA from isolated aortic smooth muscle cells from TAD (n = 4), TAA (n = 3), and control patients were analyzed using microarray-based technologies. Additionally, three publicly available bulk RNA-seq studies of TAD (n = 23) and controls (n = 17) and one single-cell RNA-seq study of TAA (n = 8) and controls (n = 3) were analyzed. Differentially expressed genes were identified and used to identify affected pathways in TAD. Five selected genes were validated by quantitative real-time polymerase chain reaction (PCR). Results: We identified 37 genes that were significantly dysregulated in at least three TAD studies—24 of them were not shown to be associated with TAD, yet. Gene ontology analysis showed that immune response was significantly affected. Five of the genes (CCL2, RNASE2, HAVCR2, CXCL8, and IL6R) were revealed as core genes that affect immune response in TAD. We compared the gene expression of those genes to TAA and found that CXCL8, IL6R, and potentially also CCL2 were upregulated in TAD. Conclusions: The identified immune-related genes showed TAD-specificity, independent of possible pre-existing comorbidities like TAA. So, these genes represent potential biomarkers and therapeutic targets linked to the immune response in acute TAD. Additionally, we identified a set of differentially expressed genes that represents a resource for further studies

Crossovers are associated with mutation and biased gene conversion at recombination hotspots

Meiosis is a potentially important source of germline mutations, as sites of meiotic recombination experience recurrent double-strand breaks (DSBs). However, evidence for a local mutagenic effect of recombination from population sequence data has been equivocal, likely because mutation is only one of several forces shaping sequence variation. By sequencing large numbers of single crossover molecules obtained from human sperm for two recombination hotspots, we find direct evidence that recombination is mutagenic: Crossovers carry more de novo mutations than nonrecombinant DNA molecules analyzed for the same donors and hotspots. The observed mutations were primarily CG to TA transitions, with a higher frequency of transitions at CpG than non-CpGs sites. This enrichment of mutations at CpG sites at hotspots could predominate in methylated regions involving frequent single-stranded DNA processing as part of DSB repair. In addition, our data set provides evidence that GC alleles are preferentially transmitted during crossing over, opposing mutation, and shows that GC-biased gene conversion (gBGC) predominates over mutation in the sequence evolution of hotspots. These findings are consistent with the idea that gBGC could be an adaptation to counteract the mutational load of recombination

High Satellite Repeat Turnover in Great Apes Studied with Short- and Long-Read Technologies.

Satellite repeats are a structural component of centromeres and telomeres, and in some instances, their divergence is known to drive speciation. Due to their highly repetitive nature, satellite sequences have been understudied and underrepresented in genome assemblies. To investigate their turnover in great apes, we studied satellite repeats of unit sizes up to 50 bp in human, chimpanzee, bonobo, gorilla, and Sumatran and Bornean orangutans, using unassembled short and long sequencing reads. The density of satellite repeats, as identified from accurate short reads (Illumina), varied greatly among great ape genomes. These were dominated by a handful of abundant repeated motifs, frequently shared among species, which formed two groups: 1) the (AATGG)n repeat (critical for heat shock response) and its derivatives; and 2) subtelomeric 32-mers involved in telomeric metabolism. Using the densities of abundant repeats, individuals could be classified into species. However, clustering did not reproduce the accepted species phylogeny, suggesting rapid repeat evolution. Several abundant repeats were enriched in males versus females; using Y chromosome assemblies or Fluorescent In Situ Hybridization, we validated their location on the Y. Finally, applying a novel computational tool, we identified many satellite repeats completely embedded within long Oxford Nanopore and Pacific Biosciences reads. Such repeats were up to 59 kb in length and consisted of perfect repeats interspersed with other similar sequences. Our results based on sequencing reads generated with three different technologies provide the first detailed characterization of great ape satellite repeats, and open new avenues for exploring their functions