Most pathway analyses place constraints on pathway size: small pathways can exhibit false positive associations due to large single-gene or single-SNP effects [24], whereas large pathways are more likely to show association by chance alone [22]. The most common minimum threshold for pathway size appears to be ten genes [4, 6, 13, 25]. It is important for analysts to note that this threshold may exclude highly-specific and potentially-informative functional sets, including those involving protein complexes and DNA sequence motifs. Frequently-used maximum thresholds for pathway size include 100 genes [4] and 200 genes [6, 25]. Notably, in the latter two studies, upper limits of 300 genes [6] and 400 genes [25] did not alter the results. However, larger pathways are relatively rare and often derive their size from being more general in scope; thus, their exclusion may not significantly affect analyses or downstream biological interpretation. Overall, investigators should consider their study goals when applying such thresholds and should evaluate results in that context. While future efforts might develop size-dependent statistical corrections, at present the reporting of pathway size and related summary statistics (e.g. [26]) alongside association data can aid interpretation.
