Effects of sample size on differential gene expression, rank order and prediction accuracy of a gene signature.

Top differentially expressed gene lists are often inconsistent between studies and it has been suggested that small sample sizes contribute to lack of reproducibility and poor prediction accuracy in discriminative models. We considered sex differences (69♂, 65 ♀) in 134 human skeletal muscle biopsies using DNA microarray. The full dataset and subsamples (n = 10 (5 ♂, 5 ♀) to n = 120 (60 ♂, 60 ♀)) thereof were used to assess the effect of sample size on the differential expression of single genes, gene rank order and prediction accuracy. Using our full dataset (n = 134), we identified 717 differentially expressed transcripts (p<0.0001) and we were able predict sex with ~90% accuracy, both within our dataset and on external datasets. Both p-values and rank order of top differentially expressed genes became more variable using smaller subsamples. For example, at n = 10 (5 ♂, 5 ♀), no gene was considered differentially expressed at p<0.0001 and prediction accuracy was ~50% (no better than chance). We found that sample size clearly affects microarray analysis results; small sample sizes result in unstable gene lists and poor prediction accuracy. We anticipate this will apply to other phenotypes, in addition to sex.

Central nervous system inflammation induces muscle atrophy via activation of the hypothalamic-pituitary-adrenal axis.

Skeletal muscle catabolism is a co-morbidity of many chronic diseases and is the result of systemic inflammation. Although direct inflammatory cytokine action on muscle promotes atrophy, nonmuscle sites of action for inflammatory mediators are less well described. We demonstrate that central nervous system (CNS)-delimited interleukin 1ß (IL-1ß) signaling alone can evoke a catabolic program in muscle, rapidly inducing atrophy. This effect is dependent on hypothalamic-pituitary-adrenal (HPA) axis activation, as CNS IL-1ß-induced atrophy is abrogated by adrenalectomy. Furthermore, we identified a glucocorticoid-responsive gene expression pattern conserved in models of acute and chronic inflammatory muscle atrophy. In contrast with studies suggesting that the direct action of inflammatory cytokines on muscle is sufficient to induce catabolism, adrenalectomy also blocks the atrophy program in response to systemic inflammation, demonstrating that glucocorticoids are requisite for this process. Additionally, circulating levels of glucocorticoids equivalent to those produced under inflammatory conditions are sufficient to cause profound muscle wasting. Together, these data suggest that a significant component of inflammation-induced muscle catabolism occurs indirectly via a relay in the CNS.