Gene-panel and whole-exome analyses are now standard methodologies for mutation detection in Mendelian disease. However, the diagnostic yield achieved is at best 50%, leaving the genetic basis for disease unsolved in many individuals. New approaches are thus needed to narrow the diagnostic gap. Whole-genome sequencing is one potential strategy, but it currently has variant-interpretation challenges, particularly for non-coding changes. In this study we focus on transcriptome analysis, specifically total RNA sequencing (RNA-seq), by using monogenetic neuromuscular disorders as proof of principle. We examined a cohort of 25 exome and/or panel “negative” cases and provided genetic resolution in 36% (9/25). Causative mutations were identified in coding and non-coding exons, as well as in intronic regions, and the mutational pathomechanisms included transcriptional repression, exon skipping, and intron inclusion. We address a key barrier of transcriptome-based diagnostics: the need for source material with disease-representative expression patterns. We establish that blood-based RNA-seq is not adequate for neuromuscular diagnostics, whereas myotubes generated by transdifferentiation from an individual’s fibroblasts accurately reflect the muscle transcriptome and faithfully reveal disease-causing mutations. Our work confirms that RNA-seq can greatly improve diagnostic yield in genetically unresolved cases of Mendelian disease, defines strengths and challenges of the technology, and demonstrates the suitability of cell models for RNA-based diagnostics. Our data set the stage for development of RNA-seq as a powerful clinical diagnostic tool that can be applied to the large population of individuals with undiagnosed, rare diseases and provide a framework for establishing minimally invasive strategies for doing so.

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Developmental signal transduction pathways act diversely, with context-dependent roles across systems and disease types. Glioblastomas (GBMs), which are the poorest prognosis primary brain cancers, strongly resemble developmental systems, but these growth processes have not been exploited therapeutically, likely in part due to the extreme cellular and genetic heterogeneity observed in these tumors. The role of Wnt/βcatenin signaling in GBM stem cell (GSC) renewal and fate decisions remains controversial. Here, we report context-specific actions of Wnt/ βcatenin signaling in directing cellular fate specification and renewal. A subset of primary GBM-derived stem cells requires Wnt proteins for self-renewal, and this subset specifically relies on Wnt/βcatenin signaling for enhanced tumor burden in xenograft models. In an orthotopic Wnt reporter model, Wnt hi GBM cells (which exhibit high levels of βcatenin signaling) are a faster-cycling, highly self-renewing stem cell pool. In contrast, Wnt lo cells (with low levels of signaling) are slower cycling and have decreased self-renewing potential. Dual inhibition of Wnt/βcatenin and Notch signaling in GSCs that express high levels of the proneural transcription factor ASCL1 leads to robust neuronal differentiation and inhibits clonogenic potential. Our work identifies new contexts for Wnt modulation for targeting stem cell differentiation and self-renewal in GBM heterogeneity, which deserve further exploration therapeutically.

Endemic species flocks inhabiting ancient lakes, oceanic islands and other long-lived isolated habitats are often interpreted as adaptive radiations. Yet molecular evidence for directional selection during species flocks radiation is scarce. Using partial transcriptomes of 64 species of Lake Baikal (Siberia, Russia) endemic amphipods and two nonendemic outgroups, we report a revised phylogeny of this species flock and analyse evidence for positive selection within the endemic lineages. We confirm two independent invasions of amphipods into Baikal and demonstrate that several morphological features of Baikal amphipods, such as body armour and reduction in appendages and sensory organs, evolved in several lineages in parallel. Radiation of Baikal amphipods has been characterized by short phylogenetic branches and frequent episodes of positive selection which tended to be more frequent in the early phase of the second invasion of amphipods into Baikal when the most intensive diversification occurred. Notably, signatures of positive selection are frequent in genes encoding mitochondrial membrane proteins with electron transfer chain and ATP synthesis functionality. In particular, subunits of both the membrane and substrate-level ATP synthases show evidence of positive selection in the plankton species Macrohectopus branickii, possibly indicating adaptation to active plankton lifestyle and to survival under conditions of low temperature and high hydrostatic pressures known to affect membranes functioning. Other functional categories represented among genes likely to be under positive selection include Ca-binding muscle-related proteins, possibly indicating adaptation to Ca-deficient low mineralization Baikal waters.

The fitness landscape of a locus, the array of fitnesses conferred by its alleles, can be affected by allele replacements at other loci, in the presence of epistatic interactions between loci. In a pair of diverging homologous proteins, the initially high probability that an amino acid replacement in one of them will make it more similar to the other declines with time, implying that the fitness landscapes of homologous sites diverge. Here, we use data on within-population non-synonymous polymorphisms and on amino acid replacements between species to study the dynamics, after an amino acid replacement, of the fitness of the ancestral amino acid, and show that selection against its restoration increases with time. This effect can be owing to increase of fitness conferred by the new amino acid occupying the site, and/or to decline of fitness conferred by the replaced amino acid. We show that the fitness conferred by the replaced amino acid rapidly declines, reaching a new lower steady-state level after approximately 20 per cent of amino acids in the protein get replaced. Therefore, amino acid replacements in evolving proteins are routinely involved in negative epistatic interactions with currently absent amino acids, and chisel off the unused parts of the fitness landscape.

Populations of different species vary in the amounts of genetic diversity they possess. Nucleotide diversity π, the fraction of nucleotides that are different between two randomly chosen genotypes, has been known to range in eukaryotes between 0.0001 in Lynx lynx and 0.16 in Caenorhabditis brenneri. Here, we report the results of a comparative analysis of 24 haploid genotypes (12 from the United States and 12 from European Russia) of a split-gill fungus Schizophyllum commune. The diversity at synonymous sites is 0.20 in the American population of S. commune and 0.13 in the Russian population. This exceptionally high level of nucleotide diversity also leads to extreme amino acid diversity of protein-coding genes. Using whole-genome resequencing of 2 parental and 17 offspring haploid genotypes, we estimate that the mutation rate in S. commune is high, at 2.0 × 10−8 (95% CI: 1.1 × 10−8 to 4.1 × 10−8) per nucleotide per generation. Therefore, the high diversity of S. commune is primarily determined by its elevated mutation rate, although high effective population size likely also plays a role. Small genome size, ease of cultivation and completion of the life cycle in the laboratory, free-living haploid life stages and exceptionally high variability of S. commune make it a promising model organism for population, quantitative, and evolutionary genetics.

Sexual reproduction is almost ubiquitous among extant eukaryotes. As most asexual lineages are short-lived, abandoning sex is commonly regarded as an evolutionary dead end. Still, putative anciently asexual lineages challenge this view. One of the most striking examples are bdelloid rotifers, microscopic freshwater invertebrates believed to have completely abandoned sexual reproduction tens of Myr ago. Here, we compare whole genomes of 11 wild-caught individuals of the bdelloid rotifer Adineta vaga and present evidence that some patterns in its genetic variation are incompatible with strict clonality and lack of genetic exchange. These patterns include genotype proportions close to Hardy-Weinberg expectations within loci, lack of linkage disequilibrium between distant loci, incongruent haplotype phylogenies across the genome, and evidence for hybridization between divergent lineages. Analysis of triallelic sites independently corroborates these findings. Our results provide evidence for interindividual genetic exchange and recombination in A. vaga, a species previously thought to be anciently asexual.

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