MicroRNAs are small, non-coding RNAs that influence gene regulatory networks by post-transcriptional regulation of specific messenger RNA targets. MicroRNA expression is dysregulated in human malignancies, frequently leading to loss of expression of certain microRNAs. We report that expression of hsa-miR-342, a microRNA encoded in an intron of the gene EVL, is commonly suppressed in human colorectal cancer. The expression of hsa-miR-342 is coordinated with that of EVL and our results indicate that the mechanism of silencing is CpG island methylation upstream of EVL. We found methylation at the EVL/hsa-miR-342 locus in 86% of colorectal adenocarcinomas and in 67% of adenomas, indicating that it is an early event in colorectal carcinogenesis. In addition, we observed a higher frequency of methylation (56%) in histologically normal colorectal mucosa from individuals with concurrent cancer compared to mucosa from individuals without colorectal cancer (12%), suggesting the existence of a 'field defect' involving methylated EVL/hsa-miR-342. Furthermore, reconstitution of hsa-miR-342 in the colorectal cancer cell line HT-29 induced apoptosis, suggesting that this microRNA could function as a proapoptotic tumor suppressor. In aggregate, these results support a novel mechanism for silencing intronic microRNAs in cancer by epigenetic alterations of cognate host genes.

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations.

Escape from X inactivation results in expression of genes embedded within inactive chromatin, suggesting the existence of boundary elements between domains. We report that the 5' end of Jarid1c, a mouse escape gene adjacent to an inactivated gene, binds CTCF, displays high levels of histone H3 acetylation, and functions as a CTCF-dependent chromatin insulator. CpG island methylation at Jarid1c was very low during development and virtually absent at the CTCF sites, signifying that CTCF may influence DNA methylation and chromatin modifications. CTCF binding sites were also present at the 5' end of two other escape genes, mouse Eif2s3x and human EIF2S3, each adjacent to an inactivated gene, but not at genes embedded within large escape domains. Thus, CTCF was specifically bound to transition regions, suggesting a role in maintaining both X inactivation and escape domains. Furthermore, the evolution of X chromosome domains appears to be associated with repositioning of chromatin boundary elements.

Toward the goal of generating a mouse medulloblastoma model with increased tumor incidence, we developed a homozygous version of our ND2:SmoA1 model. Medulloblastomas form in 94% of homozygous Smo/Smo mice by 2 months of age. Tumor formation is, thus, predictable by age, before the symptomatic appearance of larger lesions. This high incidence and early onset of tumors is ideal for preclinical studies because mice can be enrolled before symptom onset and with a greater latency period before late-stage disease. Smo/Smo tumors also display leptomeningeal dissemination of neoplastic cells to the brain and spine, which occurs in many human cases. Despite an extended proliferation of granule neuron precursors (GNP) in the postnatal external granular layer (EGL), the internal granular layer formed normally in Smo/Smo mice and tumor formation occurred only in localized foci on the superficial surface of the molecular layer. Thus, tumor formation is not simply the result of over proliferation of GNPs within the EGL. Moreover, Smo/Smo medulloblastomas were transplantable and serially passaged in vivo, demonstrating the aggressiveness of tumor cells and their transformation beyond a hyperplastic state. The Smo/Smo model is the first mouse medulloblastoma model to show leptomeningeal spread. The adherence to human pathology, high incidence, and early onset of tumors thus make Smo/Smo mice an efficient model for preclinical studies.

mac25, the subject of this report, was selected by the differential display of mRNA method in a search for genes overexpressed in senescent human mammary epithelial cells. mac25 had previously been cloned as a discrete gene, preferentially expressed in normal, leptomeningial cells compared with meningioma tumors. mac25 is another member of the insulin growth factor-binding protein (IGFBP)

Purpose:The short arm of chromosome 16 is rich in segmental duplications, predisposing this region of the genome to a number of recurrent rearrangements. Genomic imbalances of an approximately 600-kb region in 16p11.2 (29.5-30.1 Mb) have been associated with autism, intellectual disability, congenital anomalies, and schizophrenia. However, a separate, distal 200-kb region in 16p11.2 (28.7-28.9 Mb) that includes the SH2B1 gene has been recently associated with isolated obesity. The purpose of this study was to better define the phenotype of this recurrent SH2B1-containing microdeletion in a cohort of phenotypically abnormal patients not selected for obesity. Methods: Array comparative hybridization was performed on a total of 23,084 patients in a clinical setting for a variety of indications, most commonly developmental delay. Results: Deletions of the SH2B1-containing region were identified in 31 patients. The deletion is enriched in the patient population when compared with controls (P ϭ 0.003), with both inherited and de novo events. Detailed clinical information was available for six patients, who all had developmental delays of varying severity.Body mass index was Ն95th percentile in four of six patients, supporting the previously described association with obesity. The reciprocal duplication, found in 17 patients, does not seem to be significantly enriched in our patient population compared with controls. Conclusions: Deletions of the 16p11.2 SH2B1-containing region are pathogenic and are associated with developmental delay in addition to obesity. Genet Med 2010:12(10):641-647.

Ubiquitination of murine cyclin E is triggered by phosphorylation on threonine 393. Cyclin E(T393A) knockin mice exhibited increased cyclin E stability, but no phenotypic abnormalities. Importantly, loss of the p53 pathway exacerbated the effect of the T393A mutation. Thus, in p21(-/-) cells the T393A mutation had an exaggerated effect on cyclin E abundance and its associated kinase activity, which caused abnormal cell cycle progression, and genetic instability involving chromosome breaks and translocations. Moreover, cyclin E(T393A) acted synergistically with p53 deficiency to accelerate tumorigenesis in cyclin E(T393A) p53(-/-) mice; Ras more readily transformed cyclin E(T393A) p53(-/-) cells than p53(-/-) cells in vitro; and cyclin E(T393A) mice had a greatly increased susceptibility to Ras-induced lung cancer.

Pediatric fibroblastic/myofibroblastic lesions are a relatively common group of tumors with varying morphologies, for which the molecular mechanisms are becoming increasingly well characterized. Congenital infantile fibrosarcoma (CIFS), perhaps the most well studied of these lesions is characterized by a recurrent ETV6-NTRK3 gene fusion. However, a notable subset of locally aggressive congenital/infantile soft tissue lesions with similar morphologic features to CIFS, have not to-date, shown evidence of any canonical molecular aberration. We describe 6 patients with mesenchymal tumors composed of infiltrative fibroblastic/myofibroblastic tumor cells and showing a morphologic spectrum of features much analogous to that previously described in CIFS but without ETV6 fusion transcripts. These tumors lacked a uniform immunoprofile, but showed variable expression of CD34, S100, smooth muscle actin, and CD30. All patients first developed a mass in infancy (≤2 months of age). Using next-generation DNA sequencing, TMP3-NTRK1 fusions were identified in 4 cases, an LMNA-NTRK1 fusion in one case, and a variant EML4-NTRK3 fusion in one case. Similar to infantile fibrosarcoma, these tumors were locally aggressive (with local recurrences if incompletely excised) and rarely metastasized (lung metastases in one patient). Proper identification of these tumors including investigation for NTRK family gene rearrangements is essential for diagnostic accuracy, as well as for clinical management decisions. Given the morbidity associated with radical resection of large soft tissue tumors, children with unresectable, recurrent, and/or metastatic disease may benefit from treatment with NTRK targeted therapies.