Gabriel A. Bien‐Willner

Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC.

SOX9 is a temporal and tissue-specific transcription factor involved in male sexual development and bone formation. Haploinsufficiency of SOX9 is known to cause campomelic dysplasia (CD). CD cases without SOX9 coding region mutations have been described in association with translocations that have breakpoints mapping as far as 932 kb upstream from the gene. These rearrangements suggest that position effects acting from a great distance regulate SOX9 gene expression. Studies of one such case (900 kb upstream to SOX9) have led to the delineation of a potential 2.1 kb cis-acting regulatory element 1.1 Mb upstream of SOX9, termed SOX9cre1. We investigated the role of this putative regulator in SOX9 expression. SOX9cre1 increases the activity of a minimal SOX9 promoter in reporter constructs in a dose-dependent and tissue-specific manner, consistent with an enhancer role. In silico studies identify a putative binding site within SOX9cre1 for GLI1, a downstream mediator of sonic hedgehog (SHH). Furthermore, the stimulation of primary human chondrocyte cells in culture with SHH increases endogenous SOX9 expression 3-fold. Electrophoresis mobility shift assay (EMSA) studies that demonstrate physical interactions between the GLI1 transcription factor and a putative binding site within SOX9cre1, as well as experiments in which reporter constructs are co-transfected with GLI1, suggest a direct interaction between GLI1 and SOX9cre1. GLI1-SOX9cre1 interactions are verified in chromatin immunoprecipitation experiments. These data support a direct molecular link between the Hh signaling pathway and SOX9 regulation, wherein SHH stimulates SOX9 through its mediator GLI1, and are consistent with a mechanism of SOX9 regulation through distal chromatin interactions.

Cornelia de Lange syndrome (CdLS) is a multisystem congenital anomaly disorder characterized by prenatal and postnatal growth retardation, developmental delay, distinctive facial dysmorphism, limb malformations, and multiple organ defects. Mutations in the NIPBL gene have been discovered recently as a major etiology for this syndrome, and were detected in 27-56% of patients. Two groups have found significant differences in the severity or penetrance of some phenotypes between mutation positive and mutation negative patients. Different clinical features have also been described among patients with missense versus truncating mutations. In this study, we identified 13 NIPBL mutations in 28 unrelated Polish CdLS patients (46.4%), 11 were novel. Mutation positive patients were more severely affected in comparison to mutation negative individuals with respect to weight, height, and mean head circumference at birth, facial dysmorphism and speech impairment. Analyses of combined data from this and the two previous studies revealed that the degree of growth, developmental delay and limb defects showed significant differences between patients with and without mutations and between patients with missense and truncating mutations, whereas only a portion of these features differed significantly in any individual study. Furthermore, bioinformatic analyses of the NIPBL protein revealed several novel domains, which may give further clues about potential functions of this protein.