Targeted capture and massively parallel sequencing of 12 human exomes.

Genome-wide association studies suggest that common genetic variants explain only a modest fraction of heritable risk for common diseases, raising the question of whether rare variants account for a significant fraction of unexplained heritability. Although DNA sequencing costs have fallen markedly, they remain far from what is necessary for rare and novel variants to be routinely identified at a genome-wide scale in large cohorts. We have therefore sought to develop second-generation methods for targeted sequencing of all protein-coding regions ('exomes'), to reduce costs while enriching for discovery of highly penetrant variants. Here we report on the targeted capture and massively parallel sequencing of the exomes of 12 humans. These include eight HapMap individuals representing three populations, and four unrelated individuals with a rare dominantly inherited disorder, Freeman-Sheldon syndrome (FSS). We demonstrate the sensitive and specific identification of rare and common variants in over 300 megabases of coding sequence. Using FSS as a proof-of-concept, we show that candidate genes for Mendelian disorders can be identified by exome sequencing of a small number of unrelated, affected individuals. This strategy may be extendable to diseases with more complex genetics through larger sample sizes and appropriate weighting of non-synonymous variants by predicted functional impact.

Muscle stem cells contribute to myofibres in sedentary adult mice.

Skeletal muscle is essential for mobility, stability and whole body metabolism, and muscle loss, for instance, during sarcopenia, has profound consequences. Satellite cells (muscle stem cells) have been hypothesized, but not yet demonstrated, to contribute to muscle homeostasis and a decline in their contribution to myofibre homeostasis to play a part in sarcopenia. To test their role in muscle maintenance, we genetically labelled and ablated satellite cells in adult sedentary mice. We demonstrate via genetic lineage experiments that, even in the absence of injury, satellite cells contribute to myofibres in all adult muscles, although the extent and timing differs. However, genetic ablation experiments showed that satellite cells are not globally required to maintain myofibre cross-sectional area of uninjured adult muscle.

Transiently active Wnt/ß-catenin signaling is not required but must be silenced for stem cell function during muscle regeneration.

Adult muscle's exceptional capacity for regeneration is mediated by muscle stem cells, termed satellite cells. As with many stem cells, Wnt/ß-catenin signaling has been proposed to be critical in satellite cells during regeneration. Using new genetic reagents, we explicitly test in vivo whether Wnt/ß-catenin signaling is necessary and sufficient within satellite cells and their derivatives for regeneration. We find that signaling is transiently active in transit-amplifying myoblasts, but is not required for regeneration or satellite cell self-renewal. Instead, downregulation of transiently activated ß-catenin is important to limit the regenerative response, as continuous regeneration is deleterious. Wnt/ß-catenin activation in adult satellite cells may simply be a vestige of their developmental lineage, in which ß-catenin signaling is critical for fetal myogenesis. In the adult, surprisingly, we show that it is not activation but rather silencing of Wnt/ß-catenin signaling that is important for muscle regeneration.